CANCER THERAPY
Volume 2 Number 2 December 2004
CANCER THERAPY FREE ACCESS www.cancer-therapy.org
!!!!!!!!!!!!!!!!!!!!!!!! ! Editor
Teni Boulikas Ph. D., CEO Regulon Inc. 715 North Shoreline Blvd. Mountain View, California, 94043 USA Tel: 650-968-1129 Fax: 650-567-9082 E-mail: teni@regulon.org
Teni Boulikas Ph. D., CEO, Regulon AE. Gregoriou Afxentiou 7 Alimos, Athens, 17455 Greece Tel: +30-210-9853849 Fax: +30-210-9858453 E-mail: teni@regulon.org
!!!!!!!!!!!!!!!!!!!!!!!! ! Assistant to the Editor Maria Vougiouka B.Sc., Gregoriou Afxentiou 7 Alimos, Athens, 17455 Greece Tel: +30-210-9858454 Fax: +30-210-9858453 E-mail: maria@cancer-therapy.org
!!!!!!!!!!!!!!!!!!!!!!!! ! Editorial Board
Ablin, Richard J., Ph.D., Arizona Cancer Center, University of Arizona, USA Armand, Jean Pierre, M.D. Ph.D., European Organization for Research and Treatment of Cancer (EORTC), Belgium Aurelian, Laure, Ph.D., University of Maryland School of Medicine, USA Berdel, Wolfgang E, M.D., University Hospitals, Germany Bertino, Joseph R., M.D., Cancer Institute of New Jersey, USA Beyan Cengiz, M.D.,!Gulhane Military Medical Academy, Turkey Bottomley, Andrew, Ph.D., European Organization for Research and Treatment of Cancer Data Center (EORTC), Belgium Bouros, Demosthenes, M.D., University Hospital of Alexandroupolis. Greece Cabanillas, Fernando, M.D, The University of Texas M. D. Anderson Cancer Center, USA Castiglione, Monica, MHA, SIAK/IBCSG Coordinating Center, Switzerland Chou, Kuo-Chen, Ph.D., D.Sc., Pharmacia Upjohn, USA Chu, Kent-Man, M.D., University of Hong Kong Medical Center, Queen Mary Hospital, Hong Kong, China Chung, Leland W.K, Ph.D., Winship Cancer Institute,
USA Coukos, George, M.D., Ph.D., Hospital of the University of Pennsylvania, USA Darzynkiewicz, Zbigniew, M.D., Ph.D., New York Medical College, USA Devarajan, Prasad M.D., Cincinnati Children's Hospital, USA Der Channing, J. Ph.D, Lineberger Comprehensive Cancer Center, USA Dritschilo, Anatoly, M.D., Georgetown University Hospital, USA Duesberg, Peter H., Ph.D, University of California at Berkeley, USA El-Deiry, Wafik S. M.D., Ph.D., Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, USA Federico, Massimo, M.D. Università di Modena e Reggio Emilia, Italy Fiebig, Heiner H, Albert-Ludwigs-Universität, Germany Fine, Howard A., M.D., National Cancer Institute, USA Frustaci, Sergio, M.D., Centro di Riferimento Oncologico di Aviano, Italy Georgoulias, Vassilis, M.D., Ph.D., University General Hospital of Heraklion, Greece Giordano, Antonio, M.D., Ph.D., Sbarro Institute for Cancer Research and Molecular Medicine, Temple University, USA Greene, Frederick Leslie, M.D., Carolinas Medical
Center, USA Gridelli, Cesare M.D., Azienda Ospedaliera, "S.G.Moscati", Italy Hengge, Ulrich, M.D., Heinrich-Heine-University Duesseldorf, Germany Huber, Christian M.D., Johannes-GutenbergUniversity, Germany Hunt, Kelly, M.D., The University of Texas M. D. Anderson Cancer Center, USA Kamen, Barton A., M.D. Ph.D, Cancer Institute of New Jersey, USA Kaptan, Kürsat, M.D., Gülhane Military Medicine Academy, Turkey Kazuma, Ohyashiki, M.D., Ph.D., Tokyo Medical University, Japan Kinsella, Timothy J. M.D., The research Institute of University Hospitals in Cleveland, USA Kmiec, Eric B, Ph.D., University of Delaware, USA Kosmidis Paris, M.D., "Hygeia" Hospital, Athens, Greece Koukourakis Michael, M.D., Democritus University of Thrace, Greece Kroemer, Guido, M.D. Ph.D., Institut Gustave Roussy, France Kurzrock, Razelle, M.D., F.A.C.P., M. D. Anderson Cancer Center, USA Leung, Thomas Wai-Tong M.D., Chinese University of Hong Kong, China Levin, Mark M.D., Sister Regina Lynch Regional Cancer Center, Holy Name Hospital, USA Lichtor, Terry M.D., Ph.D., Rush Medical College, USA Liebermann, Dan A., Ph.D., Temple Univ. School of Medicine, USA Lipps, Hans J, Ph.D., Universität Witten/Herdecke, Germany Lokeshwar, Balakrishna L., Ph.D., University of Miami School of Medicine, USA Mackiewicz, Andrzej, M.D., Ph.D., University School of Medical Sciences (USOMS) at Great Poland Cancer Center, Poland Marin, Jose J. G., Ph.D., University of Salamanca, Spain McMasters, Kelly M., M.D., Ph.D., University of Louisville, J. Graham Brown Cancer Center, USA Morishita, Ryuichi, M.D., Ph.D., Osaka University, Japan Mukhtar, Hasan Ph.D., University of Wisconsin, USA Norris, James Scott, Ph.D., Medical University of South Carolina, USA Palu, Giorgio, M.D., University of Padova, Medical School, Italy
Park, Jae-Gahb, M.D., Ph.D., Seoul National University College of Medicine, Korea Perez-Soler, Roman M.D., The Albert Einstein Cancer Center, USA Peters, Godefridus J., Ph.D., VU University Medical Center (VUMC), The Netherlands Poon, Ronnie Tung-Ping, M.D., Queen Mary Hospital, Hong Kong, China Possinger, Kurt-Werner, M.D., Humboldt University, Germany Rainov G Nikolai M.D., D.Sc., The University of Liverpool. UK Randall, E Harris, M.D., Ph.D., The Ohio State University, USA Ravaioli Alberto, M.D. Ospedale Infermi, Italy Remick, Scot, C. M.D., University Hospitals of Cleveland, USA Rhim, Johng S M.D., Uniformed Services University of Health Sciences, USA Schadendorf, Dirk, M.D., Universitäts-Hautklinik Mannheim, Germany Schmitt, Manfred, Ph.D., Universität München, Klinikum rechts der Isar, Germany Schuller, Hildegard M., D.V.M., Ph.D., University of Tennessee, USA Slaga, Thomas J., Ph.D., AMC Cancer Research Center (UICC International Directory of Cancer Institutes and Organisations), USA Soloway, Mark S., M.D., University of Miami School of Medicine, USA Srivastava, Sudhir, Ph.D., MPH, MS, Division of Cancer Prevention, National Cancer Institute, USA Stefanadis, Christodoulos, M.D., University of Athens, Medical School, Greece, Stein, Gary S Ph.D., University Of Massachusetts, USA Tirelli, Umberto, National Cancer Institute, Italy Todo, Tomoki, M.D., Ph.D., The University of Tokyo, Japan van der Burg, Sjoerd H, Leiden University Medical Center, The Netherlands Wadhwa Renu, Ph. D., Nat. Inst. of Advan. Indust. Sci. and Technol. (AIST), Japan Waldman, Scott A. M.D., Ph.D., USA Walker, Todd Ph.D., Charles Sturt University, Australia Watson, Dennis K. Ph.D., Medical University of South Carolina, Hollings Cancer Center, USA Waxman, David J., Ph.D., Boston University, USA Weinstein, Bernard I., M.D., D.Sci (Hon.), Columbia University, USA
!!!!!!!!!!!!!!!!!!!!!!!! ! Associate Board Members Chen, Zhong, M.D, Ph.D, National Institute of Deafness and other Communication Disorders, National Institutes of Health, USA Dietrich Pierre Yves, Hopitaux Universitaires de GenFve Switzerland Jeschke Marc G, M.D., Ph.D. Universität Erlangen-Nürnberg. Germany Limacher Jean-Marc, MD Hôpitaux Universitaires de Strasbourg, France Los Marek J, M.D., Ph.D. University of Manitoba, USA Mazda Osam, M.D., Ph.D. Kyoto Prefectural University of Medicine, Japan Merlin Jean-Louis, Ph.D Centre Alexis Vautrin, National Cancer Institute University Henri Poincaré France Okada Takashi, M.D., Ph.D. Jichi Medical School Japan Pisa Pavel, M.D, Ph.D. Karolinska Hospital, Sweden
Squiban Patrick, MD Transgene SA France Tsuchida Masanori, M.D, Ph.D Niigata University Graduate School of Medical and Dental Sciences Japan Ulutin, Cuneyt, M.D., Gulhane Military Medicine Academy, Turkey Xu Ruian, Ph.D., The University of Hong Kong, Hong Kong
!!!!!!!!!!!!!!!!!!!!!!!! ! For submission of manuscripts and inquiries: Editorial Office Teni Boulikas, Ph.D./ Maria Vougiouka, B.Sc. Gregoriou Afxentiou 7 Alimos, Athens 17455 Greece Tel: +30-210-985-8454 Fax: +30-210-985-8453 and electronically to maria@cancer-therapy.org
Instructions to authors: Cancer Therapy FREE ACCESS www.cancer-therapy.org
Scope This journal, bridging various fields is one of the most rapid with free access at www.cancer-therapy.org. The scope of Cancer Therapy is to rapidly publish original and in-depth review articles on cancer embracing all fields from molecular mechanisms to results on clinical trials. Articles (both invited and submitted) review or report novel findings of importance to a general audience in cancer therapy, molecular medicine, gene discovery, and molecular biology with emphasis to molecular mechanisms and clinical applications. The journal will accept papers on all aspects of cancer, at the clinical, preclinical or cell culture stage on chemotherapy and new experimental drugs, gene discovery, cancer immunotherapy, DNA vaccines, use of DNA regulatory elements in gene transfer, cell therapy and drug discovery related to cancer therapy. The authors are encouraged to elaborate on the molecular mechanisms that govern a cancer therapy approach. To make the publication attractive authors are encouraged to include color figures.
Type of articles Both review articles and original research articles will be considered. Original research articles should contain a generous introduction in addition to experimental data. The articles contain information important to a general audience as the volume is addressed to researches outside the field. There is no limit on the length of the articles provided that the subject is interesting to a general audience and covers exhaustively a field. The typical length of each manuscript is 12-60 manuscript pages (approximately 420 printed pages) plus Figures and Tables. Free of Charge publication, Complimentary reprints & Subscriptions There are no charges for color figures or page numbers. Corresponding authors get a one-year free subscription (hard copy) plus 25 reprints free of charge. The free subscription can be renewed for additional years by having one paper per year accepted for publication. Sections of the manuscript Each manuscript should have a Title, Authors, Affiliation, Corresponding Author (with Tel, Fax, and Email), Summary, and Introduction; review articles are subdivided into headings I, II, III, etc. (starting with I. Introduction) and subdivided into A, B, C, etc. You can further subdivide into 1, 2, 3, etc. Research articles are divided into Summary; I. Introduction; II. Results; III Discussion; Acknowledgments IV. Materials and Methods and References. Please include in your text citations the name of authors and year in parenthesis; for three or more authors use: (name of first author et al, with year); for two authors please use both names. Please delete hidden text for references. In the reference list, please, type references with year and Journal in boldface and provide full title of the article such as:
Buschle M, Schmidt W, Berger M, Schaffner G, Kurzbauer R, Killisch I, Tiedemann J-K, Trska B, Kirlappos H, Mechtler K, Schilcher F, Gabler C, and Birnstiel ML (1998) Chemically defined, cell-free cancer vaccines: use of tumor antigen-derived peptides or polyepitope proteins for vaccination. Gene Ther Mol Biol 1, 309-321. Please use Microsoft Word, font “Times” (Mac users) or “Times New Roman” (PC users) and insert Greek or other characters using the “Insert/Symbol” function in the Microsoft Word rather than simple conversion to font “Symbol”. Please boldface Figure 1, 2, 3 etc. as well as Table 1, 2, etc. throughout the text. Please provide the highest quality of prints of your Figures; whenever possible, please provide in addition an electronic version of your figures (optional). Corresponding authors are kindly requested to provide a color (or black/white) head photo of themselves (preferably 4x5 cm or any size), as we shall include these in the publication.
Submission and reviewing Peer reviewing is by members of the Editorial Board and external referees. Please suggest 2-3 reviewers providing their electronic addresses, mailing addresses and telephone/fax numbers. Authors are being sent page proofs. Cancer Therapy (Volume 1, 2003) is published on high quality paper with excellent reproduction of color figures and electronically.
Reviewing is completed within 5-15 days from receiving the manuscript. Articles accepted without revisions (i.e., review articles) will be published online (www.cancertherapy.org) in approximately 1 month following submission. Please submit an electronic version of full text and figures preferably in jpeg format. The electronic version of the figures will be used for the rapid reviewing process. High quality prints or photograph of the figures and the original with one copy should be sent via express mail to the Editorial Office. Editorial Office Teni Boulikas, Ph.D./ Maria Vougiouka, B.Sc. Gregoriou Afxentiou 7 Alimos, Athens 17455 Greece Tel: +30-210-985-8454 Fax: +30-210-985-8453 and electronically to maria@cancer-therapy.org The free electronic access to articles published in "Cancer Therapy" to a big general audience, the attractive journal title, the speed of the reviewing process, the no-charges for page numbers or color figure reproduction, the 25 complimentary reprints, the rapid electronic publication, the embracing of many fields in cancer, the anticipated high quality in depth reviews and first rate research articles and most important, the eminent members of the Editorial Board being assembled are prognostic factors of a big success for the newly established journal.
Table of contents Cancer Therapy Vol 2 number 2, December 2004 Pages
Type of Article
Article title
Authors (corresponding author is in boldface)
239-244
Research Article
Pre-treatment with a non-therapeutic dose of cisplatin increases solid tumour response to liposomal-p53 gene therapy- An in vivo study
Jason C. Steel, Wouter H. J. Kalle, Daniel J. Dingwall, Heather M.A. Cavanagh, and Mark A. Burton
245-262
Review Article
Carbonic anhydrase IX (CA IX) as a potential target for cancer therapy
Silvia Pastorekov! and Jan Z!vada
263-270
Review Article
Calcium signalling and tumorigenesis
Juan A. Rosado, Pedro C. Redondo, José A. Pariente, Ginés M. Salido
271-278
Review Article
Integrins and the cellular radiation response
Emil Lisiak and Nils Cordes
279-290
Review Article
Potential application of desmopressin as a perioperative adjuvant in cancer surgery. Biological effects, antitumor properties and clinical usefulness
Daniel E. Gomez, Giselle V. Ripoll, Santiago Gir"n and Daniel F. Alonso
291-296
Research Article
A pilot clinical study of a combination of docetaxel and doxifluridine for the treatment of advanced/recurrent gastric cancer with prior chemotherapy
Hajime Kase, Naoyasu Saito, Natsuki Tokura, Naohiro Washizawa, Makoto Kikuchi and Kazuo Kobayashi
297-304
Review Article
Promiscuous and specific anti-cancer drugs: combatting biological complexity with complex therapy
Maria C. Shoshan and Stig Linder
305-316
Review Article
The current management of primary ovarian cancer: a review
Anish Bali, Karina Reynolds
317-328
Review Article
Receptor tyrosine kinases as targets for cancer therapy
Francesca De Bacco, Michela Fassetta and Andrea Rasola
329-344
Research Article
Imaging of pancreatic cancer: a promise for early diagnosis through targeted strategies
Zdravka Medarova, Anna Moore
345-352
Review Article
Kerstin Maaser, Andreas P. Sutter, and The peripheral benzodiazepine Hans Scherübl receptor: A target for innovative diagnostic and therapeutic approaches in gastrointestinal oncology
353-364
Research
A single monoclonal antinuclear
Ananthsrinivas R. Chakilam, Santhosh
Article
Pabba, Dmitry Mongayt, LeonidZ. autoantibody with nucleosomerestricted specificity inhibits growth of Iakoubov, Vladimir P. Torchilin diverse human tumors in nude mice
365-374
Review Article
The multidisciplinary management of pain and palliative care in cancer patients: a review
Frank E. Mott, Carl Chakmakjian and Joel Marcus
375-388
Research Article
Reversal of immune suppression following vaccination with recombinant vaccinia virus expressing IL-2 in an orthotopic murine model of head and neck squamous cell carcinoma
Santanu Dasgupta, Malaya BhattacharyaChatterjee, Bert W. O’Malley, Jr., Sunil K. Chatterjee
389-402
Research Article
Development of a tetracyclineinducible system for expression of the Ca 2+ permeable TRPL channel and the killing of prostate cancer cells
Prasit Ruengrairatanaroje, Michael Hahn, Helen M. Brereton, Lei Zhang, Mario Froscio, Tadija Petronijevic and Gregory J. Barritt
403-414
Review Article
Cellular drug resistance in lung cancer
Jürgen Mattern
415-422
Research Article
Salted meat consumption and risk of squamous cell carcinoma of the oesophagus: a case-control study in Uruguay
Eduardo De Stefani, Paolo Boffetta, Pelayo Correa, Hugo Deneo-Pellegrini, Mar£a Mendilaharsu and Alvaro L. Ronco
423-428
Case Report
Case report on combined radiation myelopathy and intramedullary metastases
Robbert JHA Tersteeg, Sherif Y El Sharouni, Henk B Kal, Gerard H Jansen, Petra M De Jong, Jacobus S. Straver
429-440
Research Article
Genistein induces apoptosis via mitochondrial damage in acute lymphoblastic leukemia T-cell lines
Fayth K. Yoshimura
441-454
Review Article
How should a screening programme for prostate cancer be designed? A case of aiming at a moving target
Gabriel Sandblom, Eberhard Varenhorst
455-462
Review Article
Thermal ablation of liver tumors
Charles R. Scoggins, John F. Gleason Jr, Robert C. G. Martin, Farid J. Kehdy, Julie R. Hutchinson, and Kelly M. McMasters
463-468
Research Article
Genetic variation in the P2X7 apoptosis purinoreceptor correlated with anti – nuclear and cytoskeleton autoantibodies induction in nasopharyngeal carcinoma
Majida Jalbout, Noureddine Bouaouina, and Lotfi Chouchane
469-474
Review Article
High dose rate endobronchial brachytherapy for the management of non-small cell lung cancer withan endobronchial or peribronchial component
Jiade J. Lu, Yadvindera S. Bains, Aaron H. Wolfson, Elio Donna, Alfred H. Brandon, William A. Raub, Arnold M. Markoe
475-500
Review Article
Complementary alternative medicine for cancer: a review of effectiveness and safety
Ursula Werneke, David Ladenheim, Tim McCarthy
501-510
Research Article
Inhibition of telomerase improves chemosensitivity in cisplatin resistant ovarian cancer cells
Brenda L. Shoup, Nancy E. Lowell and Patricia A. Kruk
511-518
Review Article
Current treatment strategies in locally advanced non-small cell lung cancer
H. Cuneyt Ulutin and Gorkem Aksu
519-524
Review Article
Skull base chondrosarcoma
William M. Mendenhall, Stephen B. Lewis, Douglas B. Villaret, and Nancy P. Mendenhall
525-532
Research Article
Lymphokine-activated killer (LAK) cell activity is not a continuously inducible activity: implications for LAK cell and IL-2 immunotherapy
David T. Harris
533-548
Research Article
Abscopal regression of subcutaneously Bertil R.R. Persson, Catrin Baureus Koch, Gustav Grafstrรถm, Crister Ceberg implanted N29 rat glioma after and Leif Salford treatment of the contra-lateral tumours with pulsed electric fields (PEF) or radiation therapy (RT) and their combinations (PEF+RT)
549-552
Case Report
Pregnancy post choriocarcinoma treatment
Branka Nikolic
553-560
Research Article
Occult node metastasis in cervical cancer
Sumonmal Manusirivithaya, Sumalee Siriaungkul, Surapan Khunamornpong, Sunida Rewsuwan, Siriwan Tangjitgamol, Manit Sripramote, Jatupol Srisomboon, Somnuek Jesadapatarakul
561-570
Review Article
Fas ligand gene transfer for cancer therapy
Jaime F. Modiano, Angela R. Lamerato-Kozicki, Cristan M. Jubala, David Coffey, Michelle Borakove, Jerome Schaack, Donald Bellgrau
571-574
Case Report
Primary peritoneal malignant mixed mullerian tumor (MMMT): a case report
Marcelo Carraro Nascimento, Poh See Choo, Judy Bligh, Andreas Obermair
575-578
Case Report
Complete remission of an unusual location of metastatic gestational trophoblastic neoplasia GTN:a case report
Nadereh Behtash, Malihe Hasanzadeh, Parviz Hanjani
Cancer Therapy Vol 2, page 239 Cancer Therapy Vol 2, 239-244, 2004
Pre-treatment with a non-therapeutic dose of cisplatin increases solid tumour response to liposomal-p53 gene therapy- An in vivo study Research Article
Jason C. Steel*, Wouter H. J. Kalle, Daniel J. Dingwall, Heather M.A. Cavanagh, and Mark A. Burton School of Biomedical Science, Charles Sturt University, P.O. Box 588, Wagga Wagga 2678, Australia
__________________________________________________________________________________ *Correspondence: Jason C. Steel; School of Biomedical Science, Charles Sturt University, P.O. Box 588, Wagga Wagga 2678, Australia; Telephone: (612) 69332958; Fax: (612) 69332587; E-mail: jsteel@csu.edu.au Key Words: liposomes, cisplatin, gene therapy, p53, chemotherapy Abbreviations: dimethyldioctadecyl ammonium bromide, (DDAB); Dioleoyl-L-phosphatidylethanolamine, (DOPE); phosphate buffered saline, (PBS); QIAprep! Spin Miniprep Kit, (QIAGEN); salivary adenocarcinoma, (CSU-SA1); small unilamellar vesicles, (SUV); sodium dodecyl sulfate, (SDS) Received: 1 June 2004; Revised: 22 July 2004 Accepted: 23 July 2004; electronically published: July 2004
Summary Successful liposomal-mediated gene therapy is often limited by poor transfection efficiencies. One method previously shown to increase the efficiency of liposomal gene delivery is through the administration of a nontherapeutic dose of the chemotherapeutic drug cisplatin prior to lipofection. The currents study aims to utilise this method to deliver lipoplexes containing the p53 tumour suppressor gene with the aim of increasing therapeutic effect of the p53 gene on a solid tumour in vivo. Rats, implanted with solid salivary adenocarcinomas, were pretreated with a low dose of cisplatin seven days prior to liposomal mediated p53 treatment. Following treatment with p53, tumour growth, p53 expression and levels of apoptosis were examined and compared to animals treated with p53 without cisplatin pre-treatment and a saline control. Tumours that had been pre-treated with cisplatin prior to p53-lipofection were significantly smaller than both the saline control and the non-cisplatin treated tumours. Saline treated tumours increased in size by an average of 164% over a 96-hour period compared to 64% and 101% for the cisplatin and non-cisplatin p53-liposome treated tumours. The cisplatin pre-treated tumours resulted in significantly higher levels of apoptosis surrounding the treatment site and exhibited prolonged p53 expression when compared to the non-cisplatin pre-treated tumours. The results suggest that the use of cisplatin to pre-sensitise tumours to lipofection has significant benefits when used in conjunction with p53. colchicines, mechlorethamine (nitrogen mustard) or paclitaxel (Son and Huang, 1996; Son, 1997, 1999; Nair et al, 2002). The use of chemotherapeutic agents to increase the transfection efficiency of liposomal gene therapy may have significant benefits in gene therapy protocols to treat cancer, as these patients may be already receiving chemotherapy as part of their current treatment. Genetic alteration to the p53 tumour suppressor gene is the most commonly observed abnormality in human cancers, making it an ideal target for gene therapy trials (Fisher, 2001). p53 plays a significant role in cell cycle regulation and the induction of apoptosis in response to DNA damage. Its mutation has been implicated with poor clinical prognosis for various types of malignancies as well as the induction of chemo-resistance (Rahko et al,
I. Introduction Liposomal vectors have a number of advantages over viral vectors. They have been shown to have the ability to transfect cells without the need for specific receptors, are able to carry large DNA molecules and generally display low levels of cytotoxicity and immunogenicity (Rolland, 1998). The low levels of cytotoxicity and immunogenicity make liposomal vectors generally safer for human gene therapy studies. Possibly the greatest drawback to the use of liposomal vectors is found in their lack of transfection efficiency when compared to viral vectors (Audouy et al, 2002). One method found to increase the transfection efficiency of liposomal vectors is to pre-treat the target cells with chemotherapeutic agents, such as cisplatin,
239
Steel et al: CDDP increases response to liposomal-p53 cancer gene therapy cisplatin, which exhibited no significant anti-tumour effect, or signs of toxicity was chosen for the p53 study.
2003). A number of studies have previously shown that the reintroduction of p53 via gene therapy can result in a return of chemo-sensitivity in cells which were previously chemo-resistant to cisplatin (Miyake et al, 1998; Weinrib et al, 2001). These cells treated with cisplatin following p53 gene therapy exhibited increased levels of apoptosis and tumour reductions compared to either treatment alone. The current study differs from these previous studies by using a non-therapeutic dose of cisplatin seven days prior to liposomal delivered p53 gene therapy. The function of the cisplatin is to sensitise the tumour to increased lipofection rather than induce a therapeutic response. Any tumour reduction would occur as a result of increased transfection of the p53 tumour suppressor gene. In this study we examine whether the pre-treatment of a tumour, in vivo, with cisplatin can result in both an increased anti-tumoural response as well as increased levels of p53 transfection.
E. Preparation of liposomes Liposomes consisting of small unilamellar vesicles (SUV) were prepared by the injection of an ethanoic solution of lipids into an aqueous solution as previously described (Campbell, 1995). Briefly, eight milligrams of dimethyldioctadecyl ammonium bromide (DDAB) and four milligrams of Dioleoyl-Lphosphatidylethanolamine (DOPE) were dissolved in 1.0mL absolute ethanol to give mass ratios of 2:1 of DDAB:DOPE. 50 µl of the ethanoic lipid mix was rapidly injected (over 0.5s) into vortexing water. This method results in spontaneous rearrangement of the lipids into SUV. The liposome mix was filtered through 0.2 µm polycarbonate filters to size liposomes to a maximum diameter of 0.2 µm.
F. Preparation of p53-liposome complexes The p53-liposome complexes were prepared by adding 35 µg of p53 plasmid to 35nmol of DDAB:DOPE liposomes made up to a total volume of 150 µl with saline. The formulation was mixed gently and incubated at room temperature for 15-30 minutes to allow complexing of DNA to the liposomes. Plasmidliposome complexes were made on the day of the treatment and kept on ice until 5 minutes before use, at which point they were they were warmed to 37°C for injection.
II. Materials and methods A. p53 DNA preperation A 1.85 kb XbaI-bound human p53 cDNA cloned into the polylinker of the pRcCMV eukaryotic expression vector was kindly provided by The Children’s Medical Research Institute, Sydney, Australia. The plasmid was amplified in JM109 E.Coli cells and purified with QIAprep™ Spin Miniprep Kit (QIAGEN). Quantification of plasmid was performed by gel electrophoresis prior to binding with liposomes (Sambrook & Russell 2000).
G. Injection protocol On day six (post-tumour implantation) a dose of 1mg/kg of cisplatin was injected intra-peritoneally into the treatment group of DA rats (6 animals per treatment group). This was the dose of cisplatin shown not to produce a tumour response from the dose determination study. On day 13, the treatment and control groups received two 150 µl intratumoural injections of either saline (for saline control) or p53-liposome complexes (for p53 control and treatment groups). The injections were performed over a 3 second period and the needle was left in the tumour for up to 5 minutes to prevent back-flushing of the treatment. The animals were sacrificed 96 hours later and their tumours examined histologically for signs of apoptosis.
B. Animals Syngeneic DA rats were housed 3-5 per cage, sex segregated, in temperature and humidity controlled rooms. The lighting conditions were based off a 12 hour light/dark cycle. Food and tap water were given ad libitum through wire roofed plastic cages. For experimental work, all animals were over 10 weeks old and were randomised by weight and sex into designated groups (six animals per group). All experiments were conducted with the approval of the Charles Sturt University Animal Care and Ethics Committee.
H. p53 expression detection For this study, tumours were examined for p53 expression 12, 36, or 96 hours post-treatment. Tumours from 4 animals per time point were analysed for each of the treatments. The tumour tissue was homogenised in phosphate buffered saline (PBS), lysed with 2% sodium dodecyl sulfate (SDS) and pelleted by centrifugation (12 000 g, 10 min). A 50µl aliquot of the resulting supernatant was added to 50µl of sample buffer (0.05M Tris (pH 6.8), 3% SDS, 20% Glycerol, 6% 2-Mercaptoethanol and 0.001% Bromophenol blue) and boiled for 3 minutes. The resulting mix was loaded on a SDS/Polyacrylamide gel (3% stacking, 12% running gel) and electrophoresed at 15mA for 10 hours. The protein was transferred to a nitrocellulose membrane using established techniques (Napoli et al, 1992). The blotted proteins were immunologically probed using a human monoclonal anti-p53 antibody (1:1,000 (vol/vol); Pab-1801, Santa Cruz) and an anti-goat immunoglobulin G conjugated with alkaline phosphatase (1:1,000 (vol/vol); Sigma). The membrane was then washed and developed colorimetrically with FAST Red TR/Naphthol AS-MX (4-chloro-2-methylbenzene diazonium/3hydroxy-2-naphthoic acid 2,4-dimethylanilide phosphate; Sigma). The membrane was scanned, and density analysis was performed with the Sigma Gel program (SPSS, Inc.). All densitometry readings were normalized to nanograms of total protein loaded onto the gel.
C. Tumour implantation Solid tumours were established from a transplantable rat salivary adenocarcinoma (CSU-SA1). CSU-SA1 has been shown to have a mutation to the p53 tumour suppressor gene (unpublished results). The tumours were grown on the lateral aspect of the hind limbs of DA rats. A small incision was made through the skin and a 1mm3 piece of healthy tumour was implanted subcutaneously. Tumour growth was assessed daily using calibrated vernier callipers and expressed as the product of the minimal and maximal axises of the tumour. This method has been used by this and other groups (Burton et al, 1990; Napoli et al, 1992; Walker et al, 1996).
D. Cisplatin dose determination A preliminary study was performed to determine a nontherapeutic dose of cisplatin. Five animals per group were implanted with tumours, as above. Six days post-implantation of the tumour tissues the animals were treated with a single intraperitoneal injection of either 5mg/kg or 1mg/kg of cisplatin. The animals were examined at 24-hour intervals for the next 5 days for tumour growth as well as signs of cytotoxicity. The dose of
240
Cancer Therapy Vol 2, page 241 1mg/kg dose of cisplatin was chosen for the presensitisation studies.
I. Histology Tumours were excised from the hind limb and fixed in 10% neutral buffered formalin for a minimum of 48 hours before being wax-embedded. Serial sections were cut and stained with Haematoxylin and Eosin. Sections were examined for histological signs of apoptosis, namely the presence of apoptotic bodies. The level of apoptosis at the treatment site of the tumour was determined as the average number of apoptotic bodies per 1000 cells taken from 10 sections of the tumour. This was repeated for each of the 6 tumours per group. Slides were examined randomly to avoid microscopist bias.
B. In vivo tumour response In vivo experiments were designed to test the efficiency of p53-liposome complexes on the growth of CSU-SA1 cells following a non-therapeutic dose of cisplatin. A dose of 1mg/kg of cisplatin was delivered i.p. into the treatment group seven days prior to liposome transfection. Following p53 lipofection, the cisplatin treated group was compared to non-cisplatin treated groups (saline control, empty liposomes and p53-liposome control) for differences in growth kinetics (Figure 2). Both the p53-liposome control and the cisplatin treated p53-liposome group caused significant tumour growth retardation (p<0.05) when compared to the saline and liposome controls. The saline and the liposome treated tumours increased in size by greater than 164% over a 96hour period compared to 64% and 101% for the cisplatin and non-cisplatin p53-liposome treated tumours over the same time period. The cisplatin p53-liposome treated tumours showed a maximum therapeutic response 24-48 hours post p53-liposome injection. During this period these tumours increased in size by only 2% compared to 29% for saline and 27% for the non-cisplatin treated tumours. At the 48-hour time point the cisplatin treated tumours were significantly smaller than either of the noncisplatin treated tumours (p<0.05). The tumours of the cisplatin treated tumours remained significantly smaller than the non-cisplatin tumours for the remainder of the study (p<0.05). By the 72-hour time point the liposome delivered p53 treated tumours without cisplatin, were exhibiting significant anti-tumour effect when compared to the saline control however these tumours remained larger than the cisplatin pre-treated tumours.
J. Statistical evaluation Statistical evaluation of the differences between each of the treatments was determined using the one-way analysis of variance test followed by the Student-Kewman-Keuls multiple comparison procedure. Statistical analysis was performed at 95% confidence level (p=0.05).
III. Results A. Cisplatin dose determination This experiment was designed to determine the dose of cisplatin to be used for pre-sensitisation in the p53 tumour response assays. Two doses of cisplatin were examined, 1mg/kg and 5mg/kg delivered intra-peritoneally (Figure 1). The 5mg/kg dose resulted in a significant antitumour response with tumours exhibiting regression in size (p<0.01). The mean size of the tumours (at the completion of the study) treated with the 5mg/kg was 25mm2 compared to the saline treated control tumours that were over 200mm2. Four of the five rats given 5mg/kg cisplatin also exhibited some signs of toxicity. Animals receiving 1mg/kg exhibited no signs of toxicity and no significant reduction in tumour size or tumour growth when compared to the saline treated control (p>0.05). The
Figure 1. Tumour growth curve for cisplatin dose determination. Five rats per group were treated with a single intra-peritoneal injection of saline, cisplatin at 5mg/kg or cisplatin at 1mg/kg on day 6. Tumour growth was evaluated daily. Each time point is the average size of 5 tumours +/- the SD of the mean.
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Figure 2. Tumour growth curve following treatment with p53 on liposomes (lp/p53), saline or empty liposomes. Four treatment groups were evaluated: a saline control, a liposome control, a lp/p53 control and cisplatin pre-treated lp/p53 (cis+lp/p53). The cisplatin pretreated tumours were given a dose of cisplatin at 1mg/kg seven days prior to treatment with lp/p53. Each time point represents the average tumour size increase of 6 tumours +/- the SD of the mean. * indicates where cis+lp/p53 treated tumours are significantly smaller than other treatments.
C. Protein expression
D. Histological analysis
p53 protein expression was determined by SDSPAGE followed by â&#x20AC;&#x153;Western Blottingâ&#x20AC;?. A human specific p53 antibody (not cross reactive with rat p53) was used to detect the p53 expressed in the tumour (Figure 3). As human p53 was used in this experiment, all p53 detected with this antibody was as a direct result of liposome transfection. Three time points were chosen in which 4 tumours per group were analysed for p53 expression. The first analysis was performed 12 hours post p53-liposome treatment, the second 36 hours post treatment and the final 96 hours post treatment (Table 1). The level of p53 expression 12 hours post treatment does not reveal any significant difference in the protein expression levels between the cisplatin and non-cisplatin treated tumours. By the 36 hour time point a significant difference in the level of p53 protein expression between the cisplatin and non-cisplatin treated groups could be noted (p<0.05), with the cisplatin treated group producing 76% more p53 protein than the non-cisplatin treated tumours. This trend increased by the 96 hour time point where the cisplatin treated tumours were producing 8-fold more p53 protein than the non-cisplatin treated tumours. Whilst there was no difference in the maximum levels of p53 expression between cisplatin and non-cisplatin treated groups there was a significant difference in the levels of p53 over time. p53 expression for both the cisplatin and non cisplatin treated tumours fell significantly over the time course of the experiment, however the cisplatin treated tumours continued to express p53 at higher levels than non cisplatin treated tumours for the course of the experiment.
As apoptosis is one of the primary ways in which p53 functions to induce tumour suppression, increased levels of apoptosis is an indicator of the functionality and presence of the p53 in this rat model. Histological evaluation of the tumours revealed the presence of significantly more apoptotic bodies around the p53 treatment site than that of the saline control tumours (p<0.05) (Figure 4). At the treatment site, p53 treated tumours showed levels of apoptosis more than 50-fold greater than the level of apoptosis in the saline control tumours, however this high level of apoptosis was limited to areas close to the injection site and the needle tract (data not shown). Apoptosis levels at a distance to this site were not significantly different to the saline control. Table 1. p53 protein expression following treatment with lp/p53 with (cis+Lp/p53) and without (Lp/p53) cisplatin pre-treatment or a saline control. Protein determination was examined immunologically using a human p53 specific antibody, which did not cross react with rat p53, followed by western blotting. Each time point represents the average western blot band intensity of four tumours +/the SD of the mean. Treatment Cis+Lp/p53 Lp/p53 Saline control
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12 hours 10653 +/471 11140 +/383 4 +/- 3
36 hours 6595 +/- 389
96 hours 2281 +/- 286
3748 +/- 147
285 +/- 67
3 +/- 1
7 +/- 3
Cancer Therapy Vol 2, page 243
Figure 3. Western blot of human p53 expression within rat tumours following treatment with human p53, with or without cisplatin pretreatment, and saline. Lanes 1, 2, 3 and 7, 8, 9 represent the expression of p53 following cisplatin treatment 12, 36 and 96 hours respectively. Lanes 4, 5, 6 and 10, 11, 12 represent the expression of p53 without cisplatin treatment and lanes 13, 14, represents the p53 expression following treatment with a saline control.
Figure 4. Apoptotic bodies/1000 cells following treatment with lp/p53 (with or without cisplatin pre-treatment) or saline. The cisplatin pre-treated tumours were given a dose of cisplatin at 1mg/kg seven days prior to treatment with lp/p53. Animals were sacrificed 96 hours post-treatment and the tumours examined histologically for the numbers of apoptotic bodies present surrounding the treatment site. Each bar represents the average number of apoptotic bodies present in ten sections per tumour +/- the SD of the mean. Six animals were used for each treatment.
liposome p53 treatment. This correlated to a time point in which the cisplatin treated tumours were expressing over 75% more p53 protein than that of the non-cisplatin pretreated tumours. Interesting both the cisplatin and noncisplatin treated tumours exhibited the highest level of p53 protein expression after the first 12 hours at which point there was no statistical difference between the expression levels. However this time point correlated with no significant growth reduction for either p53 treated group when compared to the saline controls. At this early posttreatment stage the p53 protein would be building up within cells prior to apoptosis so it is reasonable to have little anti-tumour effect at this point. By the completion of the study (96-hours post-treatment) the level of p53 expression in both the cisplatin and non-cisplatin pretreated tumours had fallen significantly. The cisplatin pretreated tumours however had significantly higher levels of p53 protein detected indicating that these tumours exhibited prolonged expression. At this time point the cisplatin pre-treated tumours also displayed significantly higher levels of apoptosis. While the exact mechanism for the increased liposomal efficiency is unknown, we believe that the ability of the cisplatin to cause cross-linking of the DNA may induce a number of biological changes occur to the cell to aid in the repair of the DNA damage or to induce apoptosis. One of these changes known to occur is an increase in intracellular trafficking and its associated modification to membrane transport (Son & Huang 1994). The changes in intracellular trafficking may aid in the
The restriction of apoptosis to areas close to the injection site was not however a result of the lack of functional status of the p53 but as a consequence of limited treatment distribution as a result of the use of liposomes. (Nomura et al, 1997). On comparing the cisplatin pre-treated tumours with those not pre-treated with cisplatin we found that there was a statistical difference between the numbers of apoptotic bodies found following p53 lipofection (p<0.05), with an average of 129 apoptotic cells/ 1000 cells +/- 15 for the cisplatin treated tumours and 85 apoptotic cells/ 1000 +/- 7 for non-cisplatin treated tumours. Once again apoptosis was primary limited to the injection site and needle tract indicating that the cisplatin pre-treatment did not increase the distribution of the liposomes throughout the tumour.
IV. Discussion The primary aim of this study was to attempt to increase the efficiency of liposomal vectors by the pretreatment of the tumour with a low dose of the chemotherapeutic drug cisplatin (prior to lipofection) such that a greater anti-tumoural response could be achieved. We showed that this was indeed possible with animals pretreated with the cisplatin having 36% smaller tumours after lipofection with p53 compared to those animals without cisplatin pre-treatment. These animals showed a characteristic pattern of tumour response in which maximum anti-tumoural effects occurred 24-48 hours post
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Steel et al: CDDP increases response to liposomal-p53 cancer gene therapy Miyake H, Hara I, Gohji K, Yamanaka K, Arakawa S, and Kamidono S (1998) Enhancement of chemosensitivity in human bladder cancer cells by adenoviral-mediated p53 gene transfer. Anticancer Res 18, 3087 Nair R, Rodgers J, and Schwarz L (2002) Enhancement of transgene expression by combining glucocorticoids and antimitotic agents during transient transfection using DNAcationic liposomes. Mol Ther 5, 455 Napoli S, Burton M, Martins I, Chen Y, Codde J, and Gray B (1992) Dose response and toxicity of doxorubicin microspheres in a rat tumor model. Anticancer Drugs 3, 47 Nomura T, Nakajima S, Kawabata K, Yamashita F, Takakura Y, and Hashida M (1997) Intratumoural pharmacokinetics and in vivo gene expression of naked plasmid DNA and its cationic liposome complexes after direct gene transfer. Cancer Res 57, 2681 Rahko E, Blanco G, Soini Y, Bloigu R, and Jukkola A (2003) A mutant TP53 gene status is associated with a poor prognosis and anthracycline-resistance in breast cancer patients. Eur J Cancer 39, 447 Rolland JA (1998) From genes to gene medicines, Recent advances in nonviral gene delivery. Crit. Rev. Ther. Drug Carrier Syst. 15, 143 Sambrook J. and Russell D (2000) Molecular Cloning, a Laboratory Manual 3rd Edition (New York, Cold Spring Harbor Laboratory Press) Son K (1997) Cisplatin-based interferon gamma gene therapy of murine ovarian carcinoma. Cancer Gene Ther. 4, 391 Son K (1999) Chemical toxicants activate murine ovarian ascitic tumor cells for in situ lipofection. Drug Deliv. 6, 75 Son K, and Huang L (1994) Exposure of human ovarian carcinoma to cisplatin transiently sensitizes the tumor cells for liposome-mediated gene transfer. Proc Natl Acad Sci U S A 91, 12669 Son K, and Huang L (1996) Factors influencing the drug sensitization of human tumor cells for in situ lipofection. Gene Ther 3, 630 Walker T, White J, Esdale W, Burton M, and De C. E (1996) Tumour cells surviving in vivo cisplatin chemotherapy display elevated c-myc expression. Br J Cancer 73, 610 Weinrib L, Li J, Donovan J, Huang D, and Liu F (2001) Cisplatin chemotherapy plus adenoviral p53 gene therapy in EBVpositive and -negative nasopharyngeal carcinoma. Cancer Gene Ther 8, 352 Zabner J, Fasbender AJ Moninger T Poellinger KA and Welsh MJ (1995) Cellular and molecular barriers to gene transfer by a cationic lipid. J Biol Chem 270, 8997
escape of the liposomal vectors from the endosome (a limiting factors to successful liposomal transfection: (Rolland 1998)) or increase the efficiency of transport of the liposomes from the cytoplasm into the nucleus (another limiting factor to lipofection: (Zabner et al. 1995)). The altering of intracellular trafficking may also be responsible for the prolonged protein expression and the resulting prolonged tumour response, this however is yet to be confirmed. The use of liposomes as a gene vector for the treatment of cancer has been shown to be a safe clinical option resulting in little toxicity. Whilst the use of liposomes in vitro have shown efficient transfection rates, this has not transcribed into in vivo studies which have shown low transfection efficiencies. This study has shown that the transfection efficiencies of liposomes can be increased through the use of cisplatin resulting in increase tumour response when combined with the tumour suppressor gene p53. The use of cisplatin as a chemotherapeutic agent is often limited by a build-up of chemo-resistance. The return of functional p53 following cisplatin administration would not only induce tumour suppression but also return chemo-sensitivity allowing an increased effectiveness of cisplatin as a chemotherapeutic agent. A successful treatment regime may involve the pre-treatment of tumour cells with a dose of cisplatin, followed by liposomal delivered p53, followed by a second dose of cisplatin and a second dose of p53 and so on, with each cycle of treatment positively influencing the next treatment.
References Audouy S, de Leij L, Hoekstra D, and Molema G (2002) In vivo characteristics of cationic liposomes as delivery vectors for gene therapy. Pharm Res 19, 1599 Burton M, Jones C, Trotter J, Gray B, and Codde J (1990) Efficacy of ion-exchange resins for anti-tumour drug delivery. Regional Cancer Treatments. 3, 36 Campbell M (1995) Lipofection reagents prepared by a simple ethanol injection technique. Biotechniques 18, 1027 Fisher D (2001) The p53 tumor suppressor, critical regulator of life & death in cancer. Apoptosis 6, 7 Kigawa J, Sato S, Shimada M, Kanamori Y, Itamochi H, and Terakawa N (2002) Effect of p53 gene transfer and cisplatin in a peritonitis carcinomatosa model with p53-deficient ovarian cancer cells. Gynecol Oncol 84, 210
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Cancer Therapy Vol 2, page 245 Cancer Therapy Vol 2, 245-262, 2004
Carbonic anhydrase IX (CA IX) as a potential target for cancer therapy Review Article
Silvia Pastorekov!1 and Jan Z!vada2 1
Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, D!bravsk" cesta 9, 845 05 Bratislava, Slovak Republic 2 Institute of Molecular Genetics, Czech Academy of Sciences, Flemingovo n"m. 2, 166 37 Prague, Czech Republic
__________________________________________________________________________________ *Correspondence: J. Z"vada; Phone: +(420) 220 183 294; Fax: +(420) 233 320 702; e-mail: zavada@img.cas.cz Key words: carbonic anhydrase IX, MN, G250, cell adhesion, pH regulation, hypoxia, cancer detection, prognosis, therapy, sulfonamides, monoclonal antibodies Abbreviations: anion exchanger, (AE); carbonic anhydrase, (CA); cell adhesion molecule, (CAM); clear cell RCC, (ccRCC); glucose trasporter, (GLUT); hypoxia inducible factor, (HIF); hypoxia response element, (HRE); immunohistochemistry, (IHC); renal cell carcinoma, (RCC); vascular endothelial growth factor, (VEGF); von Hippel Lindau, (VHL) Received: 15 July 2004; Accepted: 27 July 2004; electronically published: July 2004
Summary CA IX is a catalytically active plasma membrane isoform of carbonic anhydrase, which normally controls differentiation of gastric mucosa. Its abnormal expression is strongly associated with tumors, where it is often regulated by hypoxia. There are several reasons to consider CA IX as a suitable target molecule for cancer therapy: (i) it is expressed ectopically in various commonly occurring carcinomas, which are rather resistant to conventional therapy; (ii) the antigen is exposed on the cell surface; (iii) normal expression of CA IX is restricted to the luminal epithelia of the alimentary tract, with limited accessibility to immune cells, antibodies and many drugs. Here we summarize the advance achieved by several groups of scientists, who displayed a remarkable originality of mind and proposed various strategies of CA IX-targeted therapy. No breakthrough has been reported yet, but there is a continuous progress, which stimulates optimism for the future experimentation. We also present currently available data on CA IX protein, its regulation and its role in normal tissues and tumors and point out where we see the main gaps in our knowledge. It is our belief that proper comprehension of CA IX functioning at the molecular level is likely to bring about decisive improvements for its therapeutic use.
I. Introduction
II. Classification
Modern diagnostic approaches and therapeutic anticancer strategies stem from enormous research and technical advances, which improved our understanding of cancer hallmarks and clarified underlying molecular networks (Hanahan and Weinberg, 2000). These strategies principally focus on targeting molecules that are relevant for particular type of tumor and relate to its phenotypic characteristics. Spectrum of potential targets includes regulatory genes and proteins, whose functional modulation may affect tumor development, and cancerassociated molecules, whose expression pattern per se provides a means to recognize and destroy malignant cells. Carbonic anhydrase IX (CA IX) is one of the recently emerged targets with distribution and molecular attributes offering various opportunities for clinical utilization (Table 1).
CA IX belongs to a carbonic anhydrase family of enzymes that use a zinc-activated hydroxide mechanism to catalyze the reversible conversion of carbon dioxide to carbonic acid in the net reaction CO2 + H2O ! HCO3- + H+ (Christianson and Cox, 1999; Supuran, 2004). Via this catalytic activity carbonic anhydrases either supply bicarbonate for biosynthetic reactions and ion transport across the membranes or consume produced/transported bicarbonate. Their proper performance is essential for various physiological processes in virtually all living organisms. Mammalian "-CAs exist in at least 15 isoforms (Figure 1), which can be divided according to various criteria to intracellular (CA I-III, VA, VB, VII, VIII, X, XI, XIII) and extracellular (CA IV, VI, IX, XII, XIV), catalytically active (CAI-VII, IX, XII-XIV) and inactive (CA VIII, X, XI), wide-spread (CA II, IV, VB, XII, XIV) and restricted to few tissues (CA I, III, VA, VI,
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Table 1. SWOT analysis of CA IX for diagnostic and therapeutic usefulness
• • • • • • • • • • • • • • • • • • • • • • • •
Strengths almost exclusive ectopic expression in tumors relationship to hypoxia and prognosis cell surface localization availability of specific monoclonal antibodies for detection internalizing monoclonal antibodies for targeting very low level of soluble antigen to interfere with targeting very limited expression in normal tissues low targeting of normal epithelia through intact basal membranes low extravasation of antibodies/drugs from the normal vascular network Weaknesses expression in areas distant from blood vessels function in cancer poorly understood presence in some normal tissues very low level of soluble antigen for current diagnostic methods enzyme inhibitors of insufficient specificity toward CA IX heterogeneous intratumoral expression Opportunities routine detection of hypoxic cancers detection of RCC cells in peripheral blood in vivo imaging of tumors via specific peptides/sulfonamides/antibodies antibody-mediated selective targeting of tumor cells hypoxia-regulated expression of therapeutic genes functional inhibition via peptides and/or specific sulfonamides monitoring of cancer development via detection of soluble CA IX Threats unsatisfactory efficiency and escape of some tumor cells from CA IX targeted treatment (need for combined approaches) undesired side effects overweighting the benefit
Figure 1. Mammalian CA isoforms, schematic illustration of their domain composition, enzyme activity and subcellular localization. Cytoplasmic and mitochondrial CAs consist only of the CA domain, secreted CA contains a short C-terminal extension, membrane-associated CAs in addition have a transmembrane anchor and, except for CA IV, also the cytoplasmic tail. CA IX is the only carbonic anhydrase with an N-terminal proteoglycan-like sequence, engaged in cell-to-cell adhesion.
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Cancer Therapy Vol 2, page 247 VII). Different combinations of these properties create a diversity allowing each isoform fulfill a unique role in a specific physiological context. Active CAs are generally expressed in differentiated cells and play important roles in respiration, bone resorption, production of gastric acid and different body fluids, renal and testicular acidification and other biological processes. Loss or deregulated activity of certain isozymes has been implicated in several diseases including glaucoma, osteopetrosis, oedema from heart and renal failure, neurological and neuromuscular disorders etc. (Pastorekova et al, 2004). However, despite many efforts, no consistent link to cancer had been found before the identification of CA IX.
MN protein and prediction of its domain composition. However, due to an error in the 5â&#x20AC;&#x2122; region of cDNA sequence, the N-terminal portion of MN protein was wrongly assumed to contain helix-loop-helix motif, what was corrected in the subsequent paper dealing with a genomic structure of MN (Opavsky et al, 1996). According to the correct sequence, MN cDNA codes for 459 amino acid (aa) protein with 414 aa N-terminal extracellular part linked through the 20 aa hydrophobic transmembrane region (TM) with 25 aa C-terminal intracellular tail (IC). Extracellular part is composed of 37 aa signal peptide, 59 aa region with similarity to keratan sulfate-binding domain of a large proteoglycan aggrecan (PG) and a 257 aa carbonic anhydrase domain (CA). Under non-reducing conditions, monomeric 58/54 kDa MN protein assembles into trimers reminding thus virus surface glycoproteins, what could at least partially explain its insertion into the viral envelope (Pastorekova et al, 1992). Linear illustration of the monomeric MN protein with indicated positions of important aa residues and a simplified scheme of its putative modular structure designed according to available experimental data are shown on Figure 2. CA domain of MN protein displays a significant identity with extracellular carbonic anhydrases, particularly with the secreted CA VI (40.8%). It also contains all three conserved histidine residues that coordinate catalytic zinc and are therefore crucial for the enzyme activity. Based on the homology with the carbonic anhydrases and because at that time it was the ninth isoform identified within the "-CA family, MN protein was renamed as carbonic anhydrase IX (Hewett-Emmett and Tashian, 1996). Exon-intron structure of the CA9 genomic region that encodes the CA domain is analogous to other mammalian carbonic anhydrase genes, further supporting its membership in "-CA family. Exons related to additional domains/regions of CA IX were probably acquired by exon shuffling. In particular the PG region is absent from the other "-CAs and represents a unique feature of CA IX. Interestingly, extracellular parts of distant CA relatives, namely two human receptor protein tyrosine phosphatases (RPTP#/$) and a soluble rat derivative phosphacan, contain inactive carbonic anhydrase domain and are expressed in the form of proteoglycan in the nervous system (Krueger and Saito, 1992; Barnea et al, 1994). Acatalytic CA domain of RPTP# forms a deep and wide pocket and functions as a ligand-binding receptor site that mediates an interaction of RPTP# with a neuronal cell adhesion molecule contactin (Peles et al, 1995). Via this interaction, RPTP# can modulate glial-neuronal cell crosstalk and influence differentiation, adhesion and motility of neurons. This example indicates that structurally similar domain of classical CAs may potentially play a dual role of enzyme and receptor, what could be especially relevant for the transmembrane isoforms (including CA IX), which are evolutionarily older and more closely related to RPTPs than the intracellular CAs (Hewett-Emmet and Tashian, 1996).
III. History The discovery of CA IX was in a way unintentional and unexpected. Our initial aim was to reveal envelope proteins of hypothetical human defective retrovirus by formation of phenotypically mixed virions (pseudotypes) of vesicular stomatitis virus (VSV). Indeed, the pseudotypes with predicted properties were produced in a cell line MaTu, reportedly derived from a mammary tumor (Z"vada et al, 1972). The extracts from MaTu cells, metabolically labeled with 35S-methionine, contain a 58 kDa protein immunoprecipitated with various human and animal sera. This protein turned out to be transmissible via cell-to-cell contact to certain other human cells, e.g. HeLa, derived from cervical carcinoma, in spite of the fact that no morphologically distinct virions could be detected (Z"vada and Zavadova, 1991). In an effort to elucidate the identity of the enigmatic MaTu agent, we produced monoclonal antibodies (Mabs) which enabled us to distinguish between two components, MN and MX (Pastorekova et al, 1992). Cell membrane protein, designated MN, formed a â&#x20AC;&#x153;twinâ&#x20AC;? band of 54 and 58 kDa on Western blots and was detected by MAb M75. This protein could be assembled into VSV virions, but it was not transmissible. The second, cytoplasmic protein MX reacted with the M67 antibody, formed a single band of 58 kDa and was transmissible, but did not integrate into the VSV pseudotype. Importantly, the MN protein was found to be tumorassociated. Its expression was inducible by high density of cells, correlated with the tumorigenicity of HeLa x fibroblast hybrids and was expressed in several types of human carcinomas, but not in corresponding normal tissues. It was also absent from human placenta and embryos (Z"vada et al, 1993). The transmissible component MX was later identified as a nucleoprotein of lymphocytic choriomengitis virus (LCMV), presumably a human strain (Reiserova et al, 1999). Further on, we concentrated on identification and analysis of MN, which after its sequencing was identified as a cellular protein and is now named as CA IX.
IV. Molecular properties MN cDNA was isolated by immunoscreening of cDNA expression library derived from MX-infected HeLa cells using M75 MAb (Pastorek et al, 1994). Sequence analysis allowed for deduction of the primary structure of
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Pastorekov! and Z!vada: Carbonic anhydrase IX as a potential target for cancer therapy Figure 2. Modular structure of CA IX deduced from the sequence and experimental data. Domain composition: SP–signal peptide, PGproteoglycan-like segment, CA– carbonic anhydrase domain, TM– transmembrane anchor, IC– intracytoplasmic tail. CA IX forms homotrimers linked with disulfidic bonds mediated by cystine residues. The CA catalytic centre contains three histidine residues, which are located on the bottom of an active site cavity and bind one Zn2+ ion.
V. CA IX alias G250
VI. Distribution in tissues
Several years after decoding the molecular identity of CA IX and resolution of its cDNA and genomic sequences, Grabmaier et al, (2000) revealed a full homology between CA IX and G250 antigen, which had been independently investigated for its close link to renal cell carcinomas (RCC). G250 antigen was detected on the surface of renal carcinoma cells with the monoclonal antibody G250 generated against cell homogenates from the primary RCC lesions (Oosterwijk et al, 1986). In contrast to M75 MAb, which recognizes both native as well as denatured CA IX and is useful for all immunodetection methods including immunoblotting and routine immunohistochemistry (IHC) on paraffinembedded tissues, G250 MAb is clearly directed against a conformational epitope and thus can be used in IHC on living cells or frozen sections only and does not work in immunoblotting. This property of G250 MAb apparently complicated cloning of the corresponding cDNA and accounted for a delayed molecular characterization of G250 antigen. Nevertheless, G250 MAb has become an important imaging and targeting tool as described below. Because G250 had been long considered as an RCCspecific antigen, it was mostly studied in relationship to RCC (Mulders et al, 2003). In between, M75 helped to uncover CA IX expression in a broad array of different tissues and to understand its regulation.
CA IX has a distinctive expression pattern: it is naturally expressed in few normal tissues, but its ectopic expression is induced in a wide spectrum of human tumors (Figure 3). The most abundant expression of CA IX was found in the normal mucosa of the stomach and gallbladder (Pastorekova et al, 1997). Lower levels are expressed in the intestinal epithelium, where it is confined to the cryptal areas composed of cells with high proliferation capacity (Saarnio et al, 1998a). Noteworthy, amount of CA IX progressively decreases with increasing distance from the stomach toward the rectum. Other normal tissues that display weak expression of CA IX include epithelia of pancreatic ducts, male reproductive organs and lining cells of body cavity (Kivela et al, 2000; Karhumaa et al, 2001; Ivanov et al, 2001). Almost perfectly complementary pattern can be seen when looking at the distribution of CA IX in the cancer tissues. CA IX is ectopically expressed at relatively high levels and with a high prevalence in tumors, whose normal counterparts do not contain this protein. These comprise carcinomas of the cervix uteri, esophagus, kidney, lung, breast and many other tumors (Liao et al, 1994, Turner et al, 1997, Liao et al, 1997, McKiernan et al, 1997, Vermylen et al, 1999, Bartosova et al, 2002). The opposite expression is evident also in tissues with high natural CA IX expression, such as stomach and gallbladder, which
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Figure 3. Distribution of CA IX in tissues with examples of IHC staining of RCC and papilloma coli. Most of the normal tissues do not contain any CA IX. On the other hand, it is strongly expressed in the gastric and duodenal mucosa. In the tumors, its expression is mostly ectopic. Photomicrographs: Expression of CA IX in papilloma coli (top) and in renal cell carcinoma (bottom). Immunoperoxidase stainig of paraffin sections with M75 antibody and DAB.
lose or reduce CA IX upon conversion to carcinomas (Saarnio et al, 2001; Leppilampi et al, 2003). In the colonic epithelium, CA IX is present normally in the deep crypts and abnormally in the superficial adenomas and carcinomas, with the most intense staining seen in tumors with mucinous component (Saarnio et al, 1998b). Especially striking is very high proportion of CA IXpositive specimens among the cervical, renal and lung cancers. In the cervical cancer, CA IX immunoreactivity with M75 can be observed in virtually all cervical carcinomas and the majority of cervical intraepithelial neoplasia (Liao et al, 1994). Diffuse CA IX-positive staining signal in normal cervical tissues is found only in concurrent presence of dysplasia or carcinoma and therefore it can be useful as an early diagnostic indicator of cervical neoplasia in Pap smears (Liao and Stanbridge, 1996). In the kidney cancer, CA IX protein expression is selectively linked with the most frequent carcinomas of renal clear cell type (ccRCC). High levels of CA IX are seen in primary, cystic and metastatic ccRCCs, but not in benign lesions (Liao et al, 1997). Simultaneous study using RT PCR has independently proven CA9 mRNA expression in RCC and its absence in benign renal tissue (McKiernan et al, 1997). These findings were further explored into the development of sensitive enhanced RT PCR assay for the detection of RCC cells circulating in the peripheral blood of renal cancer patients (McKiernan et al, 1999). In the lung cancer, CA IX is not found in preneoplastic lesions, but is readily present in malignant tumors (Vermylen et al, 1999). Some normally looking bronchial and alveolar epithelia in close vicinity to the tumors contain CA IX positive cells, whereas all other normal lung specimens sampled at a distance from the tumor are negative.
On the basis of the clear-cut division between the tissues with normal and ectopic expression of CA IX as well as on the predominant association of CA IX with different types of tumors, CA IX was proposed as a promising tumor biomarker and further studies strongly supported this view.
VII. Regulation of expression Unusual distribution of CA IX raised questions about mechanisms that control its differential expression in normal versus tumor cells and contribute to its frequent presence in many tumor types. Experimental evidence that the increased cell density can influence CA IX expression through the promoter activation redirected the attention to a transcriptional regulation of CA9 gene (Lieskovska et al, 1999). CA9 promoter analyzed under conditions of high cell density was shown to possess five regulatory regions containing several cis-acting elements (Kaluz et al, 1999). Two regions adjacent to the transcription initiation site bind AP-1 and SP transcription factors and their synergy is needed for the basic transcriptional activation of CA9 gene (Kaluzova et al, 2001). The most important regulatory element of CA9 promoter is localized on the antisense strand between the SP-1 binding site and the transcription start at position -10/-3 and consists of the nucleotide sequence 5â&#x20AC;&#x2122;-TACGTGCA-3â&#x20AC;&#x2122; corresponding to a hypoxia response element (HRE) (Wykoff et al, 2000). HRE element is recognized by HIF-1 transcription factor, which assembles under hypoxic conditions from constitutive # subunit and oxygen-regulated "-subunit. In normoxia, HIF-1" is modified by proline hydroxylases at two conserved proline residues in the central oxygendependent degradation domain and subsequently degraded
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Pastorekov! and Z!vada: Carbonic anhydrase IX as a potential target for cancer therapy via pVHL tumor suppressor protein (Epstein et al, 2001). Moreover, its transcriptional activity in blocked by factor inhibiting HIF (FIH)-mediated hydroxylation of arginine residue in C-terminal transactivation domain (Mahon et al, 2001). When oxygen level is reduced and the cell is exposed to hypoxic or anoxic conditions, HIF-1" escapes hydroxylation, accumulates and following dimerization with HIF-1# becomes transcriptionally active. Presence of the functional HRE element thus makes CA9 a transcriptional target of HIF-1 and places it among the genes regulated by hypoxia (Semenza, 2003). HRE element is utilized also in a density-induced transcription of CA9, but under these conditions it requires cooperation with juxtaposed SP-1 binding site. Activation of CA9 transcription by increased cell density, which is linked with pericellular hypoxia, involves PI3 kinase pathway and subhypoxic levels of HIF-1" (Kaluz et al, 2002). Since the level of HIF-1" is controlled by pVHL, it is not surprising that the expression of the wild type VHL transgene can suppress the transcription of CA9 mRNA in the normoxic cells and that VHL deletion or inactivating mutation leads to release of CA9 transcription (Ivanov et al, 1998). Loss of functional pVHL is linked with a majority of clear cell renal cell carcinomas and provides explanation for frequent presence of CA IX in ccRCC. Onset of CA IX expression is an early event occurring in morphologically normal single cells within the renal tubules of patients with VHL disease and therefore provides a robust system for the identification of early foci of VHL inactivation (Mandriota et al, 2002). In addition, transcription of CA9 gene can be modulated by methylation of CpG dinucleotides within the promoter region. In RCC cell lines and in tumors, expression of CA IX is associated with hypomethylation (Cho et al, 2000, 2002). No relationship between CA9 gene expression and promoter methylation was observed in RCC/normal kidney paired specimens by Grabmaier et al, (2002), but it could be missed because the authors did not take into account VHL status and/or presence of hypoxia. Indeed, Ashida et al, (2002) showed that the expression of CA IX in cells with VHL mutation does not occur without hypomethylation of the promoter, particularly CpG sites at positions -74 and -6 with respect to the transcription start, and they proposed that VHL and methylation cooperate in the regulation of CA IX. Methylation of -74 CpG site can also influence the expression of CA IX in the carcinoma cell lines of a different origin than RCC, where it seems to represent adverse factor modifying transcriptional response to cell density (Jakubickova et al, submitted). It is possible that CA IX expression is regulated also at higher stages of the biosynthetic trail, similarly to some other hypoxia-induced genes. There are several supportive indications, including the presence of consensus phosphorylation sites in the intracytoplasmic tail, which might affect functional performance of CA IX, and the shedding of soluble CA IX, which might control the amount of the plasma-membrane associated protein (Z"vada et al, 2003). Of course, these assumptions require thorough investigations. So far, it has been clearly shown that the posttranslational stability of CA IX protein is very high (with a half life corresponding to approximately 38 h)
and allows for its long persistence in reoxygenated cells (Rafajova et al, 2004). Such extended presence of CA IX on the surface of the post-hypoxic cells may have important implications for its intratumoral distribution.
VIII. Intratumoral expression pattern The highest level of CA IX expression in vitro is achieved upon mutual aid of high cell density and hypoxia. These conditions are readily found in vivo in solid tumor tissues as consequences of physiological barriers that limit expansive growth of tumor mass. Aberrant formation and functional defects of tumor vasculature cause decreased delivery of oxygen and insufficient removal of metabolic waste exposing distant tumor cell populations to hypoxia and low pH. Depending on severity of these stresses, the affected cells respond by a number of phenotypic changes that may involve necrosis and apoptosis, cessation of the cell cycle, or adaptation enabled by the metabolic shift to anaerobic glycolysis and induction of neoangiogenesis. These changes result from transcriptional activities of HIF, which besides CA IX induces spectrum of functionally relevant genes, including VEGF (vascular endothelial growth factor) and GLUT-1 (glucose transporter) (Semenza, 2003). Hypoxia-triggered architectural and phenotypic rearrangements of tumor tissue finally convert into development of necrotic areas surrounded by the zones of surviving hypoxic cells, which adapted to stress and acquired aggressive behavior. These cells are more inclined to produce metastases and are generally refractive to conventional anticancer treatment modalities. Accordingly, the presence of the hypoxic tumors correlates with poor cancer prognosis (Wouters et al, 2002). CA IX expression pattern in vivo clearly mirrors a distribution of hypoxic areas. The protein is localized in the perinecrotic regions of various solid tumors, including carcinomas of the breast, skin, ovary, cervix uteri, head and neck, lung, bladder (Wykoff et al, 2000, 2001; Chia et al, 2001, Giatromanolaki et al, 2001; Koukourakis et al, 2001; Loncaster et al, 2001; Olive et al, 2001). According to the measurements in head and neck carcinomas, CA IX expression starts at a distance of 40-140 Âľm (median 80 Âľm) from a blood vessel and continues toward necrosis (Beasley et al, 2001). Similar spatial relationship of CA IX to microvessels is found in bladder and lung cancer (Turner et al, 2002, Swinson et al, 2003). When compared to the distribution of HIF-1" and chemical marker of hypoxia EF5 assessed in an independent study (Vukovic et al, 2001), CA IX expression begins more distantly than HIF-1", but more closely than EF5. This might suggest that CA IX induction requires lower oxygen levels than HIF-1" and that it occurs in a perinecrotic zone, which is larger than the zone labeled by EF5. In support of this assumption, Olive et al, (2001) found that CA IX staining extends beyond the regions binding another chemical marker pimonidazole in cervical carcinomas. Moreover, they demonstrated that CA IX-expressing cells isolated from tumor xenografts are viable, clonogenic and resistant to killing by ionizing radiation. These important findings indicate that at least a fraction of the tumor cells that
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Cancer Therapy Vol 2, page 251 express CA IX is intermediate in oxygenation and may represent potential source of metastases. Nevertheless, the intratumoral distribution of CA IX suggests that hypoxia is not the only factor driving its expression. Immunohistochemical studies often refer to certain proportion of tumors that do not show signs of hypoxia (such as the presence of necrotic areas, expression of HIF-1", VEGF, and/or GLUT-1, incorporation of pimonidazole), but still do express CA IX and vice versa, some tumors with apparent hypoxic regions and absence of CA IX (Wykoff et al, 2001, Chia et al, 2001, Swinson et al, 2003). For instance, about 50% of the non-small cell lung carcinoma cases with focal or extensive necrosis do not stain for CA IX and about one fifth of CA IX positive cases do not contain HIF-1" (Giatromanolaki et al, 2001). Among 12 genes induced by hypoxia, CA9 had the greatest magnitude of induction and was induced in the highest number of cell lines (Lal et al, 2001). Coexpression of CA IX with c-ErbB2, EGFR, and MUC-1 indicates possible regulatory involvement of oncogenic pathways (Giatromanolaki et al, 2001; Bartosova et al, 2002), but these observations require further proof.
two markers into a single parameter allows for stratification of patients into low, intermediate and high risk groups, significantly predicts survival and can displace histological grade (Bui et al, 2004). It is not clear, whether the expression of CA IX in RCC is predominantly a function of VHL gene mutation or whether it reflects also other factors, nevertheless, it seems to be the most significant molecular marker described in kidney cancer so far (Pantuck et al, 2003).
X. Functions A. pH regulation Ectopic expression in tumors, hypoxia-related distribution pattern and correlation with prognosis favor functional involvement of CA IX in cancer development. CA IX is a highly active enzyme with a catalytic performance and proton transfer rate similar to prototype CA II isoform (Wingo et al, 2001). Its activity can be efficiently inhibited by sulfonamides, particularly with certain derivatives that show some selectivity toward CA IX when compared to other isoenzymes (Vullo et al, 2003, Ilies et al, 2003, Abbate et al, 2004, Vullo et al, 2004, Casey et al, 2004). Similarly as other active CA isoforms, CA IX has been proposed to play a role in pH regulation. This proposal seems meaningful especially in relationship to anaerobic tumor metabolism that generates excess of acidic products, such as lactic acid and H+, which have to be extruded from the cell interior to maintain the neutral intracellular pH and protect the cells from death. The extrusion of metabolic waste and its poor clearance by inadequate tumor vasculature creates acidic extracellular microenvironment that is more permissive for tumor cell growth and invasion (reviewed in Stubbs et al, 2000). However, lactic acid is not the only source of acidosis and the studies of intratumoral physiological parameters indicate significant contribution of CO2 (Helmlinger et al, 1997, Helmlinger et al, 2002). A role for CA IX in this process appears to involve catalytic conversion of CO2 to bicarbonate and proton at the extracellular side of the plasma membrane and facilitation of the bicarbonate transport to the cell cytoplasm. In analogy to another extracellular isoenzyme CA IV, which physically interacts with bicarbonate transporters such as anion exchangers (AE) to form a transport metabolon in differentiated cells (Sterling et al, 2002), CA IX may directly cooperate with AE in tumor cells and help to neutralize their intracellular space. At the same time, the protons produced by CA IX from hydration of CO2 may remain outside and improve acidosis of microenvironment (Figure 4). Our recent experimental data clearly fit within this concept (Svastova et al, submitted). It is well known, that the acidic extracellular milieu induces production of growth factors, increases genomic instability, perturbs cell-cell adhesion, and facilitates tumor spread and metastasis (Stubbs et al, 2000). Evidence for CA IX as a causal factor of tumor acidosis may thus support its functional involvement in these processes.
IX. Relationship to prognosis There are several studies showing positive correlation between CA IX expression and poor prognosis. In the breast tumors, CA IX is associated with necrosis and high grade of ductal carcinomas in situ (Wykoff et al, 2001), negative estrogen receptor status (Span et al, 2003), higher relapse rate and worse overall survival of patients with invasive carcinomas (Chia et al, 2001). In the head and neck cancer, CA IX expression correlates with necrosis, high microvascular density and advanced stage (Beasley et al, 2001), and with poor complete response rate to chemoradiotherapy and poor local relapse free survival (Koukourakis et al, 2001). In the non-small cell lung cancer, CA IX is a significant factor of poor prognosis independent of angiogenesis (Giatromanolaki et al, 2001) and its stromal expression is associated with advanced tumor stage (Swinson et al, 2003). In bladder cancer patients treated by radical radiotherapy with carbogen and nicotinamide, CA IX expression is significantly linked with worse cause-specific and overall survivals (Hoskin et al, 2003). In the carcinomas of the cervix, extent of CA IX expression correlates with electrode measurements of tumor oxygen and with overall survival and metastasis-free survival after radiation therapy (Loncaster et al, 2001). Additional paper describes lack of these correlations in cervical cancer (Hedley et al, 2003). The authors discuss intratumoral heterogeneity, influence by factors other than hypoxia and technical differences in staining procedures as potential causes of discrepancies and call for further studies. Quite a different situation with respect to CA IX prognostic relationships is evident in renal cell carcinomas, where overall expression of CA IX decreases with progressing stage, grade and development of metastases, and lower CA IX staining (cutoff 85%) is independently associated with poor survival in advanced RCC (Bui et al, 2003). In addition, CA IX staining is inversely correlated with Ki-67 and combination of these 251
Pastorekov! and Z!vada: Carbonic anhydrase IX as a potential target for cancer therapy of cells to non-adhesive solid support (Z"vada et al, 2000). This activity resides in the N-terminal end of the molecule, in the proteoglycan-like domain. The adhesion site of CA IX overlaps with the epitope for M75 monoclonal antibody â&#x20AC;&#x201C; PGEEDLP, since M75 blocks adhesion of cells to the immobilized CA IX protein (Figure 5). The PG region of CA IX contains three identical repeats of the motif GEEDLP and four modified repetitions. Also an oligopeptide AITFNAQYA identified with the use of a phage display library of random heptapeptides and synthesized with addition of alanine on both ends competed both with binding of CA IX to the M75 antibody and to the cell surface receptor. The amplification of the M75 epitope in the PG region is also reflected by the capacity of CA IX molecules to bind 2-3 times more of M75 IgG molecules than of any of three monoclonal antibodies V10, V12 and VII20 with epitopes in the CA domain (Z"vada et al, MS in preparation). These CA domain-specific monoclonal antibodies prepared by Zatovicova et al, (2003) represent important tools for study of structure-function relationships in CA IX molecule and allow for sensitive detection of soluble CA IX that is described below. A remarkable feature of the PG segment of the CA IX molecule is a high content of dicarboxylic aa (24 D + E out of total 59 residues) and at the same time, a low content of basic aa (4 R + K). We observed that the acidic character of the PG is reflected by easy dissociation of CA IX from the complex formed with the M75 antibody or with the cell surface receptor already at slightly acidic pH. This property might facilitate release of cells from the tumors acidified by the products of hypoxic metabolism. The cells might then attach elsewhere in the organism where the pH is neutral or slightly basic, and start a metastatic growth (Z"vada et al, MS in preparation). Moreover, CA IX appears to play a role in intercellular adhesion. In polarized epithelial MDCK cells transfected with the human CA9 cDNA, CA IX protein co-
Figure 4. Proposed involvement of CA IX in the pH regulation in tumors illustrated on a model that is based on the formation of a transport metabolon composed of anion exchanger (AE) and carbonic anhydrases (in analogy to CA IV-AE-CA II metabolon described by Sterling et al., 2002). CA IX as an extracellular component of the metabolon hydrates carbon dioxide and provides bicarbonate anions to AE, which transports them to the cytoplasm in exchange for chloride anions. At the intracellular side, CA II converts bicarbonate to carbon dioxide, which diffuses out through the plasma membrane. Extracellular CA IX activity also generates protons that contribute to acidification of external pH, whereas cytoplasmic CA II activity allows for consumption of intracellular protons and contributes to neutralization of internal pH.
B. Cell-to-cell adhesion Besides its enzyme activity, CA IX is also a cell adhesion molecule (CAM), which can mediate attachment
Figure 5. CA IX-mediated cell adhesion to a solid support. On a hydrophobic plastic, human cells (eg. CGL1) attach only on the areas coated with purified CA IX (A). Treatment of the area with CA IX-specific M75 antibody prevents cell adhesion (B), whereas M67 antibody directed to an unrelated antigen has no effect on cell adhesion (C). Reproduced from Z"vada et al, 2000 with kind permission from British Journal of Cancer.
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Cancer Therapy Vol 2, page 253 localizes with a key adhesion molecule E cadherin and destabilizes E cadherin-mediated cell-cell contacts via a mechanism that involves competitive interaction with #catenin (Svastova et al, 2003). This capability of CA IX is reminiscent of some oncoproteins (EGFR, c-ErbB2, MUC-1) and makes it a candidate contributor to tumorinvasion that is known to require a diminished intercellular adhesion. In contrast to cadherins, which are homophilic cell adhesion molecules, CA IX is a heterophilic molecule (Z"vada et al, 2000 and MS in preparation). We propose that CA IX, being a CAM, could mediate the communication between cells and transmit signals, but there is no conclusive evidence for this so far. Certain transmembrane proteins with inactive carbonic anhydrase domains termed RPTP (receptor-type protein tyrosine phosphatase) serve as signaling molecules and are involved in differentiation of embryonic brains (Tashian et al, 2000). In accord with the proposal, CA IX expression in normal human epithelia displays polarized distribution confined to basolateral plasma membranes engaged in contacts with the neighboring cells and with the basal membrane (Pastorekova et al, 1997). In the stomach and in large bile ducts CA IX is strongly expressed in differentiated cells, whereas in the intestine its presence is restricted to the bottom of Lieberkuhn crypts, where the cells are intensely dividing and co-express the Ki-67 protein (Saarnio et al, 1998). While the cells migrate to the top of the crypts and gradually differentiate, they are losing the CA IX protein. These observations suggest that in normal tissues CA IX plays a dual role: in the stomach it supports differentiation and in the intestine it is connected with the proliferation of the cells. Consistently with the situation in man, mice with the targeted disruption of Car9 gene develop hyperplastic stomach mucosa with a reduced number of glandular pepsinogenproducing cells and increased number of surface mucusproducing cells (Gut et al, 2003). This finding demonstrates that the mouse CA IX protein is involved in the control of differentiation and proliferation of epithelial cell lineages in the stomach mucosa during the stomach morphogenesis. No remarkable phenotypic alterations were detected in the intestines of the knock-out mice.
sCA IX is rapidly cleared from the blood, but until now it has not been shown whether this is due to absorption in unknown deposits, degradation or excretion in urine. One of the tumors, for which detection of sCA IX might be feasible, is bladder carcinoma. Therein, CA IX expression is associated with the luminal surface that is in a direct contact with urine (Turner et al, 2002) and therefore the shedding of sCA IX deserves further investigation. From the viewpoint of the targeted therapy it is a favorable observation, since higher concentrations of the soluble target molecules could interfere with the drug; however, it is less favorable for diagnostics.
XI.Therapeutic approaches For several reasons explained above, CA IX appears to be suitable as a target molecule for cancer therapy. Indeed, several groups of scientists realized this fact and they are attempting to develop â&#x20AC;&#x153;magic bulletsâ&#x20AC;&#x153;, which would be able to destroy tumor cells without causing any unwelcome side effects. Several ingenious strategies were designed and tested (Figure 6). This concentrated attack has been to a certain extent successful, as seen from more than 60 papers dealing with CA IX-targeted therapy. Most of the tested strategies indeed proved effective in in vitro experiments or in immunodeficient animals heterotransplanted with human tumors. Some of the therapies met criteria for testing in phase I and II in humans, various clinical phase II trials were completed and phase III trial in RCC patients with minimal residual disease after tumor nephrectomy is in preparation as a part of the clinical program of Wilex company. We believe that more of basic knowledge on how CA IX operates at the molecular level is needed, including the signaling pathways used by CA IX in the tumors. Quite possibly, these pathways are not the same in different tumors. Intratumoral heterogeneity of the cells is another problem, which may require suitable complementary therapy.
A. Radioimmunotherapy A majority of clinical trials have concentrated on radioimmunotherapy of renal cell carcinoma (Mulders et al, 2003). Monoclonal antibodies labeled with 131I have the advantage that they can be used both in diagnostic and therapeutic formats. For the human use, chimeric antibodies containing the Fc fragment derived from human IgG and variable parts of Fab fragments from the mouse G250 antibody were constructed and termed cG250. Chimeric antibodies allow repeated application, without raising human anti-mouse antibodies (HAMA), which would preclude repeated use of the Mab in humans (Oosterwijk and Debruyne, 1995). The 131I-cG250 proved to be very suitable for scintigraphic imaging of RCC in the patients with a very high specificity and extremely low background, enabling detection of very small tumors (less than 1 cm). RCC patients with primary tumors and metastases were injected with diagnostic doses of 131I-cG250 and examined by scintigraphy. Afterwards, their tumors were surgically removed, together with biopsies of normal
C. Soluble CA IX protein Most of the complete CA IX is integrated in the cell membrane as a trimer composed of 54 and 58 kDa monomeric molecules linked together with disulfidic bonds. Body fluids and TC media contain a soluble form sCA IX consisting of 50 and 54 kDa polypeptides (Z"vada et al, 2003). It has been proposed that sCA IX may be derived from the complete molecule by the proteolytic cleavage of the extracellular domain from TM and IC by membrane-associated proteases. While TC fluids of permanent cell lines or of primary human tumor explants contain a relatively high concentration of sCA IX (20-50 ng/ml), blood and urine of RCC patients contain extremely low levels of the antigen, of the order of 5-100 pg/ml. This is about 1000 times less than the levels of certain cancer markers, such as CEA (carcinoembryonic antigen) or PSA (prostate soluble antigen). It seems that in human body, 253
Pastorekov! and Z!vada: Carbonic anhydrase IX as a potential target for cancer therapy
Figure 6. Therapeutic strategies exploiting the tumor-associated expression and plasma membrane localization of CA IX. There are several variants of â&#x20AC;&#x153;magic bulletsâ&#x20AC;?: (a) CA IX-specific antibody serves for targeted delivery of toxins, drugs or radionuclides to tumor cells. A combination with cytokines potentiates the inhibitory effect of antibody molecules; (b) bispecific antibody molecules directed to tumor antigen and to cytotoxic cells ensure killing of the cancer cells; (c) sulfonamides (derivatives designed so as to exert maximum specificity for CA IX) will upset the pH-regulatory system of tumor cells; (d) cell-to-cell binding mediated by CA IX could be inhibited by the soluble form of CA IX receptors (yet hypothetical) or by receptor-mimicking small molecules; (e) recombinant therapeutic gene, which is under the regulatory control of CA IX promoter sequence, containing HRE. Under hypoxia, HIF binds to HRE and switches on the synthesis of the therapeutic protein which can convert an innocuous pro-drug into the toxic product, killing the tumor (Jaffar et al, 2001).
The Wilex company is now advertising that their 131Ilabeled product WX-G250 RIT is intended for the future treatment of biliary cancers (cholangiocarcinoma, gall bladder carcinoma), which are sensitive to radiation. There are other interesting papers worth mentioning: a mathematical model for antibody clearance from the patients has been produced (Loh et al, 1998); to reduce the radioactive burden to the organism, a two-step treatment comprised the application of bivalent antibody with one arm derived from G250 and the other directed to a chelate binding radioactive indium. The isotope was given after the clearance of unbound antibody (Kranenborg et al, 1998). For the scintigraphic imaging, the cG250 labeled with 99mTc (using 3 different methods of labeling) was superior to 131I-labeled antibody (Steffens et al, 1999b).
tissues, and distribution of the radioactivity was determined. Radiolabeled cG250 showed excellent tumor targeting, with only very low radioactivity in normal tissues. In vivo selectivity of the antibody was among the highest ever reported. Metastases were also strongly radiolabeled. Calculation showed that suitable doses of radioactive cG250 could achieve killing of all tumor cells, without seriously damaging any normal tissue. Scintigraphic images show no accumulation of radioactivity in the stomach, which is normally expressing high levels of the CA IX antigen (a fact not known at the time of this work). The most disappointing observation was a high heterogeneity of intratumoral distribution of 131 I-cG250. This fact proved also later as a main drawback for radioimmunotherapy of RCC (simultaneously with a relatively high radioresistance of RCC). The problem of intratumoral heterogenous distribution of therapeutic antibody could not be solved even by fractionated application (Steffens et al, 1999).
B. Chimeric and bispecific antibodies Non-radioactive MAbs were also tested. To activate antibody-dependent effector mechanisms, they were used in combination with cytokines or they were modified so as 254
Cancer Therapy Vol 2, page 255 to amplify their complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). There are two in vitro studies on killing RCC cells with human mononuclear cells from peripheral blood of healthy donors, activated with a combination of cG250 antibody and IL-2 (Surfus et al, 1996; Liu et al, 2002). Both of these groups are suggesting that the combination immunotherapy with cG250 and cytokines such as IL-2 shows promise in the treatment of RCC. Established xenografts of human RCC in nu-nu mice were treated with G250 in combination with IF + TNF. This cocktail proved to be therapeutically more efficient than each of the components alone (Van Dijk et al, 1994). A chimeric bispecific antibody G250/anti-CD3 was designed to bind simulatenously cytotoxic lymphocytes and RCC cells and induce a T-cell mediated cytolysis of tumor (Luiten et al, 1996a). An alternative way to target cytotoxic cells to the tumor was a chimeric G250 with the Fc fragment of human IgE. Such a construct, upon binding RCC cells, attracted mast cells, which in turn destroyed the tumor (Luiten et al, 1996b; 1997). A bispecific monoclonal antibody directed to CA IX antigen and to cellular membrane-bound complement regulator CD55 enhanced binding of complement C3 and increased the lysis of RCC cells (Blok et al, 1998). However, until now there were no reports on clinical testing of these ingenious engineered antibodies. The Wilex company is advertising the product Rencarex® (WX-250), which is a chimeric antibody with 25% content of Fab derived from mouse G250 and the rest 75% from human IgG. This product has passed the phase II testing in 36 patients with metastatic RCC, with a partial success. Its mechanism of action is ADCC (antibody dependent cell cytotoxicity).
polyclonal “third antibodies“ (Ab3), reactive with human RCC cells carrying the CA IX antigen. Indeed, these sera, when injected into nu-nu mice simultaneously with the live RCC cells protected them against development of the tumors. The response was quite spectacular – in certain variants it was a 100% protection. The treatment with Ab3 was highly efficient even in the mice with established human RCC xenografts (Uemura et al. 1995). Let us hope that analogous experiments in the patients will prove as effective as in the mice. Dendritic cell vaccine is an oligopeptide capable of binding dendritic cells and so to induce a specific cytotoxic lymphocyte response directed towards RCC cells. Vissers et al, (1999) analyzed the sequence of CA IX protein for potential HLA-A2.1 binding peptides using a computer program and found 60 candidate oligopeptides. They immunized with each of them transgenic mice expressing human HLA-A2.1 and found that four of them indeed developed cytotoxic T-lymphocytes (CTLs), capable to lyse transgenic mouse cells expressing human CA IX protein as well as HLA-A2.1. One of these oligopeptides (HLSTAFARV) was able to bind human A2.1 dendritic cells in vitro. After cocultivation with autologous CD8-positive T cells and after their restimulation, peptide-specific human CTL were obtained. They possessed the capacity to destroy target cells expressing the CA IX protein. Hopefully, this is one of the ways towards immunotherapy of RCC. Interestingly, the same CD4 (+) T cells in the context of HLA-DR molecules recognized this sequence also in the naturally processed CA IX protein (Vissers et al, 2002). This finding supports the view that peptide-based vaccines indeed could raise the cytotoxic cell response in the patients. Other vaccines derived from the sequence of CA IX were tested using mouse RCC cells transfected with human CA9 cDNA. Three nonapeptides were designed so as to be compatible both with murine H-2Kd as well as with human HLA-A24 histocompatibility antigens. One of the peptides was identified as a potential vaccine effective both in mouse and human organisms (Shimizu et al, 2003). Ringhoffer et al, (2004) have proposed a peptide vaccine combining four antigens which are frequently expressed in renal cell carcinoma: MAGE-1, MAGE-3, CA IX and PRAME. Tumor-specific expression of at least one of these T-cell activating antigens was detected in all of 41 RCC patients examined, 80% of these patients expressed two or more tumor-associated antigens simultaneously. An alternative method of vaccination is a culture of monocytes, derived from peripheral blood mononuclear cells (PBMC) transduced with the adenovirus-CA9 vector. This system has several advantages: PBMC are easy to obtain from the blood and they express the whole CA IX protein (Mukouyama et al, 2004). Chimeric protein vaccine was constructed from a part of CA IX protein and a part of GM-CSF (granulocyte/macrophage colony stimulating factor). Tso et al, (2001) produced the fusion gene and inserted it into a baculovirus expression vector system. Fusion protein of Mr 66,000 was expressed in insect Sf9 cells and purified
C. Anti-cancer vaccines There exist occasional reports on “spontaneous” regression in renal cell carcinoma, sometimes even resulting in disappearence of metastatic cancer. RCC is believed to be a relatively immunogenic tumor (Vissers et al, 1999). Vaccination is one of potential approaches towards immunotherapy of tumors carrying suitable antigens like CA IX. This might afford a life-long active immunity, repeatedly destroying newly emerging tumor recurrences. Anti-idiotype vaccines represent one possible solution. Their advantages are that a large amount of the immunizing antigen can be produced repeatedly, and that the antigen is in fact not perfectly identical with the original tumor antigen. The patient’s organism is probably tolerant to the antigen like CA IX, which is recognized as “self“, and does not respond to it immunologically. On the other hand, an anti-idiotype vaccine is somewhat different from the original antigen and can conceivably induce formation of antibodies reacting with the immunizing antigen and also cross-reacting with the original tumor. From the mice immunized with G250, six hybridomas were obtained, producing “second“ antibodies (Ab2) reactive with the G250 antibody molecules (Uemura et al. 1994a,b). These “second“ antibodies mimicked the CA IX antigen and they were used for immunization of another group of mice. Their sera after immunization contained 255
Pastorekov! and Z!vada: Carbonic anhydrase IX as a potential target for cancer therapy by affinity chromatography. The protein retained both antigenic activity of CA IX and immunity-stimulating GM-CSF activities. It proved to be a potent immunostimulant, inducing T-helper cell-supported antitumor response.
linked Fab fragments of the G250 antibody. Mice with tumors produced by mouse renal carcinoma cells transfected with human CA9 cDNA were injected with these immunomagnetoliposomes (or with control liposomes). After an interval, they were exposed to oscillating magnetic field, which induced vibrations of the magnetic particles. As a consequence, the temperature of the tumors increased to 43째C. This resulted in temporary growth arrest. Control tumors continued growing.
D. Antifection One of the key problems of developing new cancer treatment methods is a specific and efficient delivery of therapeutic genes into tumor cells, without transfecting normal tissues. Now a technique termed antifection could provide a solution. In principle it is very simple: therapeutic genes (or vectors) are chemically conjugated with monoclonal antibody directed to a suitable tumor antigen, eg., CA IX. This antibody-vector conjugate can attach to the tumor cell, and is subsequently internalized. Finally, the antifected gene is expressed. This idea indeed works, as shown by D체rrbach et al, (1999), who conjugated CA IX-specific MAb G250 with an IL-2 vector and demonstrated its internalization and prolonged expression in mouse cells previously transfected with a human CA 9 vector. Initially, this technique was only modestly efficient, but several further refinements (Deas et al, 2002) brought about an increased delivery and expression of the reporter gene.
G. Targeted oncolytic viruses For years, virologists have been thinking of specific oncolysis caused by viruses. Initial experiments were not successful, since the cytopathogenic viruses used (Sindbis, vaccinia) were infectious for both normal and tumor cells. One of possible solutions is retargeting the virus by bispecific scFv with one arm directed against the viral surface antigen (adenovirus knob) and the other against a tumor-associated antigen (CA IX). This virus-antibody complex binds preferably CA IX-expressing tumor cells and eventually destroys them (Jongmans et al, 2003). However, the next virus generation is again a plain adenovirus with no preference for tumor cells. Another strategy of producing oncolytic viruses was designed by Lim et al, (2004). They constructed a recombinant virus consisting of the CA9 promoter and complete coding sequence of adenovirus. This virus could replicate only in CA IX-expressing cells, which contained relevant transcription factors. At least 100 times higher MOI of such virus was needed for inhibiting CA IXnegative cells than for cells expressing CA IX. Moreover, the viral construct delayed growth of HeLa tumors in nude mice.
E. Genetically engineered cytotoxic cells Another strategy of the gene therapy is unlimited supply of cytotoxic lymphocytes (CTL) specifically destroying the tumor without damaging normal tissues. Moreover, these CTL should preferably be MHCindependent because (a) tumor cells often do not express MHC; (b) cytotoxic cells should be applicable for all patients carrying the same tumor (or group of tumors) and not tailored for each patient individually with his own combination of MHC antigens. Even this goal appears to be feasible. Weijtens et al, (1996) have engineered CTL armed with a fusion protein scFv/$. The single chain antibody scFv was derived from variable parts of anti-CA IX antibody G250; it has been fused with high affinity Fc($)RI, which can function in a CD3-independent manner. Such CTLs can specifically lyse target cells via their scFv/$ chimeric receptor independent of MHC. They produce cytokines in response to binding with the target cells and like normal CTL, they recycle their scFv/$ dictated lytic activity. In these artificial CTLs, a co-regulatory function of CD2, CD3, CD11a/CD18 and of other molecules was demonstrated (Weijtens et al, 1998). The authors also examined quantitative dependences between the density of T cell chimeric receptor scFv/$ and CA IX antigen density on the target cells and their effects on the CTL-mediated cytolysis and production of IL-2 and TNF (Weijtens et al, 2000).
XII. Future prospects There are also several theoretical possibilities to target CA IX function for the anticancer therapeutic purposes and/or utilize the mode of its regulation. These possibilities are based on a solid rationale but certainly require experimental proof of principle. CA IX-specific sulfonamides as efficient enzyme inhibitors might potentially reduce CA IX-mediated extracellular acidification of the tumor microenvironment and neutralization of the intracellular pH, thereby decreasing invasion propensity and/or survival of tumor cells. Sulfonamide inhibitors might be also applied together with the conventional chemotherapeutic drugs to improve their uptake and efficiency. This idea is based on the experiences that intracellular accumulation of weakly electrolytic drugs and their therapeutic effects depend on the pH gradient across the plasma membrane (Gerweck, 1998; Raghunand et al, 1999; Stubbs et al, 2000). Indirect arguments in favor of the above assumptions reside in observations that in vitro invasion of tumor cells and in vivo tumor growth in xenotrasplanted animals can be diminished by a non-selective CA inhibitor acetazolamide (Teicher et al, 1993; Parkkila et al, 2000), and that different derivatives of CA inhibitors can retard tumor cell growth in culture even in nanomolar concentrations (Casini et al, 2002). However, the mechanism of
F. Immunomagnetic hyperthermia Another ingenious and original method of cancer therapy targeted to CA IX-positive tumor cells has been designed (Shinkai et al, 2001). Submicron magnetic particles were enclosed in liposomes with covalently
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sulfonamide action and possible involvement of CA IX in those studies remains unclear. . An important step toward the elucidation of the phenotypic consequences of CA IX inhibition has been recently made by the synthesis of the first CA IX-selective, membrane-impermeable sulfonamides (Casey et al, 2004; Pastorekova et al, 2004). Function-blocking antibodies might represent alternative CA IX-targeted therapeutic tools, although at this point their potential usefulness is merely a matter of speculation based on the analogy with antibodies against some other cell surface molecules, such as EGFR and cErbB2. Nevertheless, series of CA IX-specific monoclonal antibodies is available (Zatovicova et al, 2003) and awaits evaluation of possible anticancer effects. HRE-driven gene therapy is a strategy designed to hit hypoxic tumor cells and cause their selective destruction (Dachs et al, 1997). This strategy principally utilizes HIF-1 regulated expression of the conditionally cytotoxic gene, which is cloned behind the HRE-elementcontaining promoter derived from the hypoxia-inducible gene. Obviously, the degree of the selectivity allowing for differential expression between well-oxygenated and hypoxic tumor cells depends on the magnitude of hypoxic induction, which is in turn largely affected by the regulatory context of the HRE element within the corresponding promoter. In different hypoxia-induced genes, position of HRE can range from several dozens to more than thousand nucleotides upstream of the start of transcription. Furthermore, some genes, such as VEGF and GLUT-1, can be significantly induced by the alternative pathways and are also expressed under normoxia, what may partially compromise the therapeutic employment of their promoters. In this respect, CA9 promoter raises a particular hope because its HRE element is localized just in front of the transcription initiation site and its transcriptional control by hypoxia is tighter compared to the other HRE-regulated genes (Lal et al, 2001; Rafajova et al, 2004).
XIII. Where information gaps?
we
see
the
•
What are the principles determining onset of expression in the course of carcinogenesis, quantity of the protein and percentage of CA IX-positive cells, why CA IX is expressed in tumor stroma? What are the phenotypic consequences of CA IX expression in different tumors?
XIV. Conclusion This review essentially supports the view that CA IX could be useful for targeted cancer therapy. More than sixty papers have reported a partial success, at least in vitro or in animal experiments. What is needed is the amplification of the therapeutic effects. We are showing that several entirely different approaches have been designed. The survey suggests that CA IX study is quite stimulating not only to mount the researchers’ imagination, but also to prompt clinicians’ interests for practical improvements of the management of cancer patients.
Acknowledgements The research of the authors is supported by Bayer Healthcare, the Academy of Sciences of the Czech Republic (project No. K5011112), the Slovak Scientific Grant Agency (VEGA – 2/3055/23), the Science and Technology Assistance Agency (APVT–51–005802) and by the 6th Framework Program of the European Commission (EUROXY). The authors wish to thank Dr. Juraj Kopacek (Institute of Virology, Bratislava) for the help with the graphical illustrations and to Dr. Eva Sloncova (Institute of Molecular Genetics, Prague) for the photomicrographs with IHC staining.
References Abbate F, Casini A, Owa T, Scozzafava A, Supuran CT (2004) Carbonic anhydrase inhibitors: E7070, a sulfonamide anticancer agent, potently inhibits cytosolic isozymes I and II, and transmembrane, tumor-associated isozyme IX. Bioorg Med Chem Lett 14, 217-23 Ashida S, Nishimori I, Tanimura M, Onishi S, and Shuin T (2002) Effects of von Hippel-Lindau gene mutation and methylation status on expression of transmembrane carbonic anhydrases in renal cell carcinoma. J Cancer Res Clin Oncol 128, 561-568. Barnea G, Grumet M, Milev P, Silvennoinen O, Levy JB, Sap J, Schlessinger J (1994) Receptor tyrosine phosphatase beta is expressed in the form of proteoglycan and binds to the extracellular matrix protein tenascin. J Biol Chem 269, 14349-52 Bartosova M, Parkkila S, Pohlodek K, Karttunen TJ, Galbavy S, Mucha V, Harris AL, Pastorek J, Pastorekova S (2002) Expression of carbonic anhydrase IX in breast is associated with malignant tissues and is related to overexpression of cerbB2. J Pathol 197, 314-321 Beasley NJP, Wykoff CC, Watson PH, Leek R, Turley H, Gatter K, Pastorek J, Cox GJ, Ratcliffe P, and Harris AL (2001) Carbonic anhydrase IX, an endogenous hypoxia marker, expression in head and neck squamous cell carcinoma and its relationship to hypoxia, necrosis and microvessel density. Cancer Res 61, 5262-5267. Blok VT, Daha MR, Tijsma O, Harris CL, Morgan BP, Fleuren GJ, Gorter A (1998) A bispecific monoclonal antibody
main
In spite of the increasing number of scientists and clinicians interested in CA IX, there are some fundamental questions which, when answered, could provide useful leads for drug development. As we see it, these are as follows: • What are the cell surface receptors/ligands binding CA IX? • Are the binding partners the same in normal stomach, in the intestines and in various tumors? • What signals (if any) are transmitted between CA IX and the interacting proteins and what are the downstream targets/effectors? • Which are additional regulatory pathways upstream of CA IX and what is their biological significance? • What are the mechanisms underlying intratumoral and intertumoral heterogeneity of CA IX distribution?
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Pastorekov! and Z!vada: Carbonic anhydrase IX as a potential target for cancer therapy directed against both membrane-bound complement regulator CD55 and the renal tumor-associated antigen G250 enhances C3 deposition and tumor cell lysis by complement. J Immunol 160, 3437-3443 Bui MH, Seligson D, Han KR, Pantuck AJ, Dorey FJ, Huang Y, Horvath S, Leibovich BC, Chopra S, Liao SY, Stanbridge E, Lerman MI, Palotie A, Figlin RA, Belldegrun AS (2003) Carbonic anhydrase IX is an independent predictor of survival in advanced renal clear cell carcinoma: implications for prognosis and therapy. Clin Cancer Res 9, 802-11 Bui MH, Visapaa H, Seligson D, Kim H, Han KR, Huang Y, Horvath S, Stanbridge EJ, Palotie A, Figlin RA, Belldegrun AS (2004) Prognostic value of carbonic anhydrase IX and KI67 as predictors of survival for renal clear cell carcinoma. J Urol 171, 2461-6. Casey JR, Morgan PE, Vullo D, Scozzafava A, Mastrolorenzo A, Supuran CT (2004) Carbonic anhydrase inhibitors. Design of selective, membrane-impermeant inhibitors targeting the human tumor-associated isozyme IX. J Med Chem 47, 2337-47. Casini A, Scozzafava A, Mastrolorenzo A, and Supuran CT (2002) Sulfonamides and sulfonylated derivatives as anticancer agents. Current Cancer Drug Targets 2, 55-75. Chia SK, Wykoff CC, Watson PH, Han C, Leek RD, Pastorek J, Gatter KC, Ratcliffe P, and Harris AL (2001) Prognostic significance of a novel hypoxia-regulated marker, carbonic anhydrase IX, in invasive breast carcinoma. J Clin Oncol 19, 3660-3668. Cho M, Grabmaier K, Kitahori Y, Hiasa Y, Nakagawa Y, Uemura H, Hirao Y, Ohnishi T, Yoshikawa K, Ooesterwijk E (2000) Activation of the MN/CA9 gene is associated with hypomethylation in human renal cell carcinoma cell lines. Mol Carcinog 27, 184-9 Christianson DW, Cox JD (1999) Catalysis by metal-activated hydroxide in zinc and manganese metalloenzymes. Annu Rev Biochem 68, 33-57. Dachs GU, Patterson AV, Firth JD, Ratcliffe PJ, Townsend KM, Stratford IJ, Harris AL (1997) Targeting gene expression to hypoxic tumour cells. Nature Med 3, 515-520. Deas O, Angevin E, Cherbonnier C, Senik A, Charpentier B, Levillain JP, Oosterwijk E, Hirsch F, Durrbach A (2002) In vivo-targeted gene delivery using antibody-based nonviral vector. Hum Gene Ther 13, 1101-1114 Durrbach A, Angevin E, Poncet P, Rouleau M, Chavanel G, Chapel A, Thierry D, Gorter A, Hirsch R, Charpentier B, Senik A, Hirsch F (1999) Antibody-mediated endocytosis of G250 tumor-associated antigen allows targeted gene transfer to human renal cell carcinoma in vitro. Cancer Gene Ther 6, 564-571 Epstein ACR, Gleadle JM, McNeil LA, Hewitson KS, O’Rourke J, Mole DR, Mukherji M, Metzen E, Wilson MI, Dhanda A, Tian YM, Masson N, Hamilton DL, Jaakkola P, Barstead R, Hodgkin J, Maxwell PH, Pugh CW, Schofield CJ, Ratclife PJ (2001) C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 107, 43-54. Gerweck LE (1998) Tumor pH: implications for treatment and novel drug design. Semin Radiat Oncol 8(3):176-82 Giatromanolaki A, Koukourakis MI, Sivridis E, Pastorek J, Wykoff CC, Gatter KC, and Harris AL (2001) Expression of hypoxia-inducible carbonic anhydrase-9 relates to angiogenic pathways and independently to poor outcome in non-small cell lung cancer. Cancer Res 61, 7992-7998. Grabmaier K, de Weijert M, Uemura H, Schalken J, Oosterwijk E (2002) Renal cell carcinoma-associated G250 methylation and expression: in vivo and in vitro studies. Urology 60, 357-62 Grabmaier K, Vissers JL, De Weijert MC, Oosterwijk-Wakka
JC, Van Bokhoven A, Brakenhoff RH, Noessner E, Mulders PA, Merkx G, Figdor CG, Adema GJ, Oosterwijk E (2000) Molecular cloning and immunogenicity of renal cell carcinoma-associated antigen G250. Int J Cancer 85, 865870 Gut MO, Parkkila S, Vernerova Z, Rohde E, Z"vada J, Hocker M, Pastorek J, Karttunen T, Gibadulinova A. Zavadova Z, Knobeloch K-P, Wiedermann B, Svoboda J, Horak I, Pastorekova S (2002) Gastric hyperplasia in mice with tageted disruption of the carbonic anhydrase gene Car9. Gastroenterology 123, 1889-1903 Hanahan D, and Weinberg RA (2000) The hallmarks of cancer. Cell 100, 57-70. Hedley D, Pintilie M, Woo J, Morrison A, Birle D, Fyles A, Milosevic M, Hill R (2003) Carbonic anhydrase IX expression, hypoxia, and prognosis in patients with uterine cervical carcinomas. Clin Cancer Res 9, 5666-74 Helmlinger G, Sckell A, Dellian M, Forbes NS and Jain RK (2002) Acid production in glycolysis-impaired tumors provides new insights into tumor metabolism. Clin Cancer Res 8, 1284-1291. Helmlinger G, Yuan F, Dellian M, and Jain RK (1997) Interstitial pH and pO2 gradients in solid tumors in vivo: high-resolution measurements reveal a lack of correlation. Nature Med 3, 177-182. Hewett-Emmett D, Tashian RE (1996) Functional diversity, conservation, and convergence in the evolution of the alpha-, beta-, and gamma-carbonic anhydrase gene families. Mol Phylogenet Evol 5, 50-77 Hoskin PJ, Sibtain A, Daley FM, Wilson GD (2003) GLUT1 and CAIX as intrinsic markers of hypoxia in bladder cancer: relationship with vascularity and proliferation as predictors of outcome of ARCON. Br J Cancer 89, 1290-1297 Ilies MA, Vullo D, Pastorek J, Scozzafava A, Ilies M, Caproiu MT, Pastorekova S, Supuran CT (2003) Carbonic anhydrase inhibitors. Inhibition of tumor-associated isozyme IX with halogenosulfanilamide and halogenophenylaminobenzolamide derivatives. J Med Chem 46, 2187-2196 Ivanov S, Liao SY, Ivanova A, Danilkovitch-Miagkova A, Tarasova T, Weirich G, Merrill MJ, Proescholdt MA, Oldfield EH, Lee J, Z"vada J, Waheed A, Sly W, Lerman MI, Stanbridge EJ (2001) Expression of hypoxia-inducible cellsurface transmembrane carbonic anhydrases in human cancer. Am J Pathol 158, 905-919 Ivanov SV, Kuzmin I, Wei MH, Pack S, Geil L, Johnson BE, Stanbridge EJ, and Lerman MI (1998) Down-regulation of transmembrane carbonic anhydrases in renal cell carcinoma cell lines by wild-type von Hippel-Lindau transgenes. Proc Natl Acad Sci USA 95,12596–12601. Jaffar M, Williams KJ, Stratford IJ (2001) Bioreductive and gene therapy approaches to hypoxic diseases. Advanced Drug Delivery Reviews 53, 217–228. Jongmans W, Van Den Oudenalder K, Tiemessen DM, Molkenboer J, Willemsen R, Mulders PF, Oosterwijk E (2003) Targeting of adenovirus to human renal cell carcinoma cells. Urology 62, 559-565 Kaluz S, Kaluzova M, Chrastina A, Olive Pl, Pastorekova S, Pastorek J, Lerman MI, Stanbridge EJ (2002) Lowered oxygen tension induces expression of the hypoxia marker MN/Carbonic anhydrase IX in the absence of hypoxiainducible factor 1" stabilization: a role for phosphatidylinositol 3’-kinase. Cancer Res 62, 4469-4477 Kaluz S, Kaluzova M, Opavsky R, Pastorekova S, Gibadulinova A, Dequiedt F, Kettmann R, Pastorek J (1999) Transcriptional regulation of the MN/CA9 gene coding for the tumor-associated carbonic anhydrase IX. Identification and characterization of a proximal silencer element. J Biol Chem 274, 32588-32595
258
Cancer Therapy Vol 2, page 259 Kaluzova M, Pastorekova S, Svastova E, Pastorek J, Stanbridge EJ, Kaluz S (2001) Characterization of the MN/CA 9 promoter proximal region: a role for specificity protein (SP) and activator protein 1 (AP1) factors. Biochem J 359, 669677 Karhumaa P, Kaunisto K, Parkkila S, Waheed A, Pastorekova S, Pastorek J, Sly WS, Rajaniemi H (2001) Expression of the transmembrane carbonic anhydrases, CA IX and CA XII, in the human male excurrent ducts. Mol Hum Reprod 7, 611616 Kivela AJ, Parkkila S, Saarnio J, Karttunen TJ, Kivela J, Parkkila AK, Pastorekova S, Pastorek J, Waheed A, Sly WS, and Rajaniemi H (2000) Expression of transmembrane carbonic anhydrase isoenzymes IX and XII in normal human pancreas and pancreatic tumours. Histochem Cell Biol 114, 197-204. Koukourakis MI, Giatromanolaki A, Sivridis E, Simopoulos K, Pastorek J, Wykoff CC, Gatter KC, and Harris AL (2001). Hypoxia-regulated carbonic anhydrase-9 (CA9) relates to poor vascularization and resistance of squamous cell head and neck cancer to chemoradiotherapy. Clin Cancer Res 7, 3399-3403. Kranenborg MH, Boerman OC. Oosterwijk-Wakka JC, deWeijert MC, Corstens FH, Oosterwijk E (1998) Two-step radio-immunotargeting of renal-cell carcinoma xenografts in nude mice with anti-renal-cell-carcinoma x anti-DTPA bispecific monoclonal antibodies. Int J Cancer 75, 74-80 Krueger NX, Saito H (1992) A human transmembrane proteintyrosine-phosphatase, PTP zeta, is expressed in brain and has an N-terminal receptor domain homologous to carbonic anhydrases. Proc Natl Acad Sci U S A 89, 7417-21 Lal A, Peters H, StCroix B, Haroon ZA et al, (2001) Transcriptional response to hypoxia in human tumors. J Natl Cancer Inst 93, 1337-1343 Leppilampi M, Saarnio J, Tuomo J, Kivela J, Pastorekova S, Pastorek J, Waheed A, Sly WS, Parkkila S (2003) Carbonic anhydrase isoenzymes IX and XII in gastric Tumors. World J Gastroenterol 9, 1398-1403 Liao SY, Aurelio ON, Jan K, Z"vada J, Stanbridge EJ, (1997) Identification of the MN/CA9 protein as a reliable diagnostic biomarker of clear cell carcinoma of the kidney. Cancer Res 57, 2827-2831 Liao SY, Brewer C, Z"vada J, Pastorek J, Pastorekova S, Manetta A, Berman ML, DiSaia PJ, Stanbridge EJ (1994) Identification of the MN antigen as a diagnostic biomarker of cervical intraepithelial squamous and glandular neoplasia and cervical carcinomas. Amer J Pathol 145, 598-609 Liao SY, Stanbridge EJ (1996) Expression of the MN antigen in cervical Papanicolaou smears is an early diagnostics biomarker of cervical dysplasia. Cancer Epidemiol Biomarkers Prev 5, 549-557 Lieskovska J, Opavsky R, Zacikova L, Glasova M, Pastorek J, Pastorekova S (1999) Study of in vitro conditions modulating expression of MN/CA IX protein in human cell lines derived from cervical carcinoma. Neoplasma 46, 17-24 Lim HY, Ahn M, Chung HC, Gardner TA, Kao C, Lee SJ, Kim SJ (2004) Tumor-specific gene therapy for uterine cervical cancer using MN/CA9-directed replication-competent adenovirus. Cancer Gene Ther [May 28, Epub ahead of print] Liu Z, Smyth FE, Renner C, Lee F-T, Oosterwijk E, Scott AM (2002) Anti-renal cell carcinoma chimeric antibody G250: cytokine ehancement of in vitro antibody-dependent cellular cytotoxicity. Cancer Immunol Immunother 51, 171-177 Loh A, Sgouros G, O'Donoghue JA, Deland D, Puri D, Capitelli P, Humm JL, Larson SM, Old LJ, Divgi CR (1998) Pharmacokinetic model of iodine-131-G250 antibody in renal cell carcinoma patients. J Nucl Med 39, 484-489 Loncaster JA, Harris AL, Davidson SE, Logue JP, Hunter RD,
Wykoff CC, Pastorek J, Ratcliffe P, Stratford IJ, and West CML (2001) Carbonic anhydrase IX expression, a potential new intrinsic marker of hypoxia: correlations with tumour oxygen measurements and prognosis in locally advanced carcinoma of the cervix. Cancer Res 61, 6394-6399. Luiten RM, Coney LR, Fleuren GJ, Warnaar SO, Litvinov SV (1996) Generation of chimeric bispecific G250/anti-CD3 monoclonal antibody, a tool to combat renal cell carcinoma. Brit J Cancer 74, 735-744 Luiten RM, Fleuren GJ, Warnaar SO, Litvinov SV (1996) Target-specific activation of mast cells by immunoglobulin E reactive with a renal cell carcinoma-associated antigen. Lab Invest 74, 467-475 Luiten RM, Warnaar SO, Schuurman J, Pasmans SG, Latour S, Daeron M, Fleuren GJ, Litvinov SV (1997) Chimeric immunoglobulin E reactive with tumor-associated antigen activates human Fc epsilon RI bearing cells. Hum Antibodies 8, 169-180 Mahon PC, Hirota K, Semenza GL (2001) FIH-1: a novel protein that interacts with HIF-1" and VHL to mediate repression of HIF-1 transcriptional activity. Genes Dev 15, 2675-86. Mandriota SJ, Turner KJ, Davies DR, Murray PG, Morgan NV, Sowter HM, Wykoff CC, Maher ER, Harris AL, Ratcliffe PJ, and Maxwell PH (2002) HIF activation identifies early lesions in VHL kidneys: evidence for site-specific tumor suppressor function in the nephron. Cancer Cell 1, 459-468 McKiernan JM, Buttyan R, Bander NH, De la Taille A, Stifelman MD, Emanuel ER, Bagiella E, Rubin MA, Katz AE, Olsson CA, Sawczuk IS (1999) The detection of renal carcinoma cells in the peripheral blood with an enhanced reverse transcriptase-polymerase chain reaction assay for MN/CA9. Cancer 86, 492-497 McKiernan JM, Buttyan R, Bander NH, Stifelman MD, Katz AE, Chen MW, Olsson CA, Sawczuk IS (1997) Expression of the tumor-associated gene MN: A potential biomarker for human renal cell carcinoma. Cancer Res 57, 2362-2365 Mukouyama H, Janzen NK, Hernandez JM, Lam RC, Wang AY, Figlin RA, Belldegrun AS, Zeng G (2004) Generation of kidney cancer-specific antitumor immune responses using peripheral blood monocytes transduced with a recombinant adenovirus encoding carbonic anhydrase 9. Clin Cancer Res 10, 1421-1429 Mulders P, Bleumer I, Oosterwijk E (2003) Tumor antigens and markers in renal cell carcinoma. Urol Clin North Am 30, 455-65 Olive PL, Aquino-Parsons C, MacPhail SH, Laio SY, Raleigh JA, Lerman MI, and Stanbridge EJ (2001) Carbonic anhydrase 9 as an endogenous marker for hypoxic cells in cervical cancer. Cancer Res 61, 8924-8929. Oosterwijk E, Debruyne FM (1995) Radiolabeled monoclonal antibody G250 in renal-cell carcinoma. World J Urol 13, 186-190 Oosterwijk E, Ruiter DJ, Hoedemaeker PJ, Pauwels EKJ, Jonas U, Zwartendijk J, Warnaar SO (1986) Monoclonal antibody G250 recognizes determinant present in renal cell carcinoma and absent from normal kidney. Int J Cancer 38, 489-494 Opavsky R, Pastorekova S, Zelnik V, Gibadulinova A, Stanbridge EJ, Z"vada J, Kettmann R, Pastorek J, (1996) Human MN/CA9 gene, a novel member of the carbonic anhydrase family: structure and exon to protein domain relationships. Genomics 33, 480-487 Pantuck AJ, Zeng G, Belldegrun AS, Figlin RA (2003) Pathobiology, prognosis, and targeted therapy for renal cell carcinoma: exploiting the hypoxia-induced pathway. Clin Cancer Res 9, 4641-52 Parkkila S, Rajaniemi H, Parkkila AK, Kivela J, Waheed A, Pastorekova S, Pastorek J, and Sly WS (2000) Carbonic anhydrase inhibitor suppresses invasion of renal cancer cells
259
Pastorekov! and Z!vada: Carbonic anhydrase IX as a potential target for cancer therapy in vitro. Proc Natl Acad Sci USA 97, 2220â&#x20AC;&#x201C;2224. Pastorek J, Pastorekova S, Callebaut I, Mornon J-P, Zelnik V, Opavsky R, Zatovicova M, Liao S, Portetelle D, Stanbridge E, Z"vada J, Burny A, Kettmann R (1994) Cloning and characterization of MN, a human tumor-associated protein with a domain homologous to carbonic anhydrase and putative helix-loop-helix DNA binding segment. Oncogene 9, 2877-2888 Pastorekova S, Parkkila S, Parkkila AK, Opavsky R, Zelnik V, Saarnio J, Pastorek J (1997) Carbonic anhydrase IX, MN/CA IX: Analysis of stomach complementary DNA sequence and expression in human and rat alimentary tracts. Gastroenterology 112, 398-408 Pastorekova S, Parkkila S, Pastorek J and Supuran CT (2004) Carbonic anhydrases: Current state of the art, therapeutic applications and future prospects. J Enz Inhib Med Chem, 19, 199-229 Pastorekova S, Zavadova Z, Kostal M, Babusikova O, Z"vada J. (1992) A novel quasi-viral agent, MaTu, is a two-component system. Virology 187, 620-626 Peles E, Nativ M, Campbell PL, Sakurai T, Martinez R, Lev S, Clary DO, Schilling J, Barnea G, Plowman GD, et al (1995) The carbonic anhydrase domain of receptor tyrosine phosphatase beta is a functional ligand for the axonal cell recognition molecule contactin. Cell 82, 251-60 Rafajova M, Zatovicova M, Kettmann R, Pastorek J, Pastorekova S (2004) Induction by hypoxia combined with low glucose or low bicarbonate and high posttranslational stability upon reoxygenation contribute to carbonic anhydrase IX expression in cancer cells. Int J Oncol 24, 995-1004. Raghunand N, He X, van Sluis R, Mahoney B, Baggett B, Taylor CW, Paine-Murrieta G, Roe D, Bhujwalla ZM, Gillies RJ (1999) Enhancement of chemotherapy by manipulation of tumour pH.Br J Cancer 80,1005-11 Reiserova L, Kaluzova M, Kaluz S, Willis AC, Z"vada J, Zavodska E, Zavadova Z, Ciampor F, Pastorek J, Pastorekova S (1999) Identification of MaTu-MX agent as a new strain of lymphocytic choriomeningitis virus (LCMV) and serological indication of horizontal spread of LCMV in human population. Virology 257, 73-83 Ringhoffer M, Muller CR, Schenk A, Kirsche H, Schmitt M, Greiner J, Gschwend JE (2004) Simultaneous expression of T-cell activating antigens in renal cell carcinoma: implications for specific immunotherapy. J Urol 171, 24562460 Saarnio J, Parkkila S, Parkkila AK, Haukipuro K, Pastorekova S, Pastorek J, Kairaluoma MI, Karttunen TJ (1998b) Immunohistochemical study of colorectal tumors for expression of a novel transmembrane carbonic anhydrase, MN/CA IX, with potential value as a marker of cell proliferation. Am J Pathol 153, 279-285 Saarnio J, Parkkila S, Parkkila AK, Pastorekova S, Haukipuro K. Pastorek J, Juvonen T, Karttunen TJ (2001) Transmebrane carbonic anhydrase, MN/CA IX, is a potential biomarker for biliary tumors. J Hepatol 35, 643-649 Saarnio J, Parkkila S, Parkkila AK, Waheed A, Casey MC, Zhou XY, Pastorekova S, Pastorek J, Karttunen T, Haukipuro K, Kairaluoma MI, Sly WS (1998a) Immunochemistry of carbonic anhydrase isozyme IX (MN/CA IX) in human gut reveals polarized expression in the epithelial cells with the highest proliferative capacity. J Histochem Cytochem 46, 497-504 Semenza GL (2003) Targeting HIF-1 for cancer therapy. Nat Rev Cancer 3, 721-32 Shimizu K, Uemura H, Yoshikawa M, Yoshida K, Hirao Y, Iwashima K, Saga S, Yoshikawa K (2003) Induction of antigen specific cellular immunity by vaccination with peptides from MN/CA IX in renal cell carcinoma. Oncol
Rep 10, 1307-1311 Shinkai M, Le B, Honda H, Yoshikawa K, Shimizu K, Saga S, Wakabayashi T, Yoshida J, Kobayashi T (2001) Targeting hyperthermia for renal cell carcinoma using human MN antigen-specific magnetoliposomes. Jpn J Cancer Res 92, 1138-1145 Span PN, Bussink J, Manders P, Beex LV, Sweep CG (2003) Carbonic anhydrase-9 expression levels and prognosis in human breast cancer: association with treatment outcome. Br J Cancer 89, 271-6. Steffens MG, Oosterwijk E, Kraneneborg MH, Manders JM, Debruyne FM, Corstens FH, Boerman OC (1999) In vivo and in vitro characterizations of three 99mTc-labeled monoclonal antibody G250 preparations. J Nucl Med 40, 829-836 Steffens MG, Oosterwijk-Wakka JC, Zegwaart-Hagemeier NE, Boerman OC, Debruyne F.M, Corstens FH, Oosterwijk E (1999) Immunohistochemical analysis of tumor antigen saturation following injection of monoclonal antibody G250. Anticancer Res 19, 1197-2000 Sterling D, Alvarez BV, and Casey JR (2002) The extracellular component of a transport metabolon. Extracellular loop 4 of the human AE1 Cl-/HCO3+ exchanger binds carbonic anhydrase IV. J Biol Chem 277, 25239-46. Stubbs M, McSheehy PMJ, Griffiths JR, Bashford CL (2000) Causes and consequences of tumor acidity and implications for treatment. Mol Med Today 6, 15-19. Supuran CT (2004) Carbonic anhydrases: Catalytic mechanisms, distribution and physiological roles. In: Carbonic Anhydrase: Its Inhibitors and Activators. CT Supuran, A Scozzafava, J Conway (eds), pp. 1-24, CRC Press Boca Raton-London-New York-Washington D.C. Surfus JE, Hank JA, Oosterwijk E, Welt S, Lindstrom MJ, Albertini MR, Schiller JH, Sondel PM (1996) Anti-renal-cell carcinoma chimeric antibody G250 facilitates antibodydependent cellular cytotoxicity with in vitro and in vivo interleukin-2-activated effectors. J Immunother Emphasis Tumor Immunol 19, 184-191 Svastova E, Zilka N, Zatovicova M, Gibadulinova A, Ciampor F, Pastorek J, Pastorekova S. (2003) Carbonic anhydrase IX reduces E-cadherin-mediated adhesion of MDCK cells via interaction with b-catenin. Exp Cell Res 290, 332-345 Swinson DE, Jones JL, Richardson D, Wykoff C, Turley H, Pastorek J, Taub N, Harris AL, O'Byrne KJ (2003) Carbonic anhydrase IX expression, a novel surrogate marker of tumor hypoxia, is associated with a poor prognosis in non-smallcell lung cancer. J Clin Oncol 21, 473-82 Tashian RE, Hewett-Emmett D, Carter N, Bergenhem NCH (2000) Carbonic anhydrase (CA)-related proteins (CA-RPs), and transmembrane proteins with CA or CA-RP domains. In: The Carbonic Anhydrases, New Horizons. Chegwidden WR, Carter ND, Edwards YH (eds), pp. 105-120. Basel: Birkhäuser Verlag Teicher BA, Liu SD, Liu LT, Holden SA, Herman TS (1993) A carbonic anhydrase inhibitor as a potential modulator of cancer therapies. Anticancer Res 13, 1549-1556 Tso CL, Zisman A, Pantuck A, Calilliw R, Hernandez JM, Paik S, Nguyen D, Gitlitz B, Shintaku PI, de Kernion J, Figlin R, Belldegrun A (2001) Induction of G250-targeted and T-cellmediated antitumor activity against renal cell carcinoma using a chimeric fusion protein consisting of G250 and granulocyte/monocyte-colony stimulating factor. Cancer Res 61, 7925-7933 Turner JR, Odze RD, Crum CP, Resnick MB (1997) MN antigen expression in normal, preneoplastic, and neoplastic esophagus: A clinicopathological study of a new cancerassociated biomarker. Hum Pathol 28, 740-744
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Cancer Therapy Vol 2, page 261 Turner KJ, Crew JP, Wykoff CC, Watson PH, Poulsom R, Pastorek J, Ratcliffe PJ, Cranston D and Harris AL (2002) The hypoxia-inducible genes VEGF and CA9 are differentially regulated in superficial vs invasive bladder cancer. Br J Cancer 86, 1276-82. Uemura H, Beniers AJ, Okajima E, Debruyne FMJ, Oosterwijk E (1994) Vaccination with anti-idiotype antibodies mimicking a renal cell carcinoma-associated antigen induces tumor immunity. Int J Cancer 58, 555-561 Uemura H, Okajima E, Debruyne FMJ, Oosterwijk E (1995) Anti-tumor effect induced by vaccination with anti-idiotype antibodies in 7 mice with established human renal cell carcinoma xenografts. Urol Oncol 1, 73-79 Uemura H, Okajima E. Debruyne FMJ, Oosterwijk E (1994) Internal image anti-idiotype antibodies related to renal-cell carcinoma-associated antigen G250. Int J Cancer 56, 609614 Van Dijk J, Uemura H, Beniers AJ, Peelen WP, Zegveld ST, Fleuren GJ, Warnaar SO, Oosterwijk E (1994) Therapeutic effect of monoclonal antibody G250, interferons and tumor necrosis factor, in mice with renal-cell carcinoma xenografts. Int J Cancer 56, 262-268 Vermylen P, Roufosse C, Burny A, Verhest A, Bosschaerts T, Pastorekova S,Ninane V, Sculier J-P (1999) Carbonic anhydrase IX antigen differentiates between preneoplastic lesions in non-small-cell lung carcinoma. Eur Respir J 14, 806-811 Vissers JL, De Vries IJ, Schreurs MW, Engelen LP, Oosterwijk E, Figdor CG, Adema GJ (1999) The renal cell carcinomaassociated antigen G250 encodes a human leukocyte antigen (HLA)-A2.1-restricted epitope recognized by cytotoxic T lymphocytes. Cancer Res 59, 5554-5559 Vissers JL, De Vries JM, Engelen LP, Scharenborg NM, Molkenboer J, Figdor CG, Oosterwijk E, Adema GJ (2002) Renal cell carcinoma-associated antigen G250 encodes naturally processed epitope presented by human leukocyte antigen-DR molecules to CD4+ T lymphocytes. Int J Cancer 100, 441-444 Vukovic V, Haugland HK, Nicklee T, Morrison AJ and Hedley DW (2001) Hypoxia-inducible factor-1" is an intrinsic marker for hypoxia in cervical cancer xenografts. Cancer Res 61, 7394-7398 Vullo D, Franchi M, Gallori E, Pastorek J, Scozzafava A, Pastorekova S, and Supuran C (2003) Carbonic anhydrase inhibitors. Inhibition of the tumor-associated isozyme IX with aromatic and heterocyclic sulfonamides. Bioorg Med Chem Let 13, 1005-1009. Vullo D, Scozzafava A, Pastorekova S, Pastorek J, Supuran CT (2004) Carbonic anhydrase inhibitors: inhibition of the tumor-associated isozyme IX with fluorine-containing sulfonamides. The first subnanomolar CA IX inhibitor discovered. Bioorg Med Chem Lett 14, 2351-6. Weijtens ME, Hart EH, Bolhuis RL (2000) Functional balance between T cell chimeric receptor density and tumor associated antigen density: CTL mediated cytolysis and lymphokine production. Gene Ther 7, 35-42
Weijtens ME, Willemsen RA, Van-Krimpen BA, Bolhuis RL (1998) Chimeric scFv/gamma receptor-mediated T-cell lysis of tumor cells is coregulated by adhesion and accesory molecules. Int J Cancer 77, 181-187 Weijtens MEM, Willemsen RA, Valerio D, Stam K, Bolhus RL (1996) Single chain Ig/gamma gene-redirected human T lymphocytes produce cytokines, specifically lyse tumor cells, and recycle their lytic capacity. J Immunol 157, 836-843 Wingo T, Tu C, Laipis PJ, Silverman DN (2001) The catalytic properties of human carbonic anhydrase IX. Biochem Biophys Res Commun 288, 666-669 Wouters BG, Weppler SA, Koritzinski M, Landuyt W, Nuyts S, Theys J, Chiu RK, and Lambin P (2002) Hypoxia as a target for combined modality treatments. Eur J Cancer 38, 240257 Wykoff CC, Beasley N, Watson PH, Campo L, Chia SK, English R, Pastorek J, Sly WS, Ratcliffe P, and Harris AL (2001) Expression of the hypoxia-inducible and tumor-associated carbonic anhydrases in ductal carcinoma in situ of the breast. Am J Pathol 158, 1011-1019. Wykoff CC, Beasley NJ, Watson PH, Turner KJ, Pastorek J, Sibtain A, Wilson GD, Turley H, Talks KL, Maxwell PH, Pugh CW, Ratcliffe PJ, Harris AL (2000) Hypoxia-inducible expression of tumor-associated carbonic anhydrases. Cancer Res 60, 7075-7083 Zatovicova M, Tarabkova K, Svastova E, Gibadulinova A, Mucha V, Jakubickova L, Biesova Z, Rafajova M, Ortova Gut M, Parkkila S, Parkkila A-K, Waheed A, Sly WS, Horak I, Pastorek J, Pastorekova S (2003) Monoclonal antibodies generated in CA IX-deficient mice recognize different domains of tumor-associated hypoxia-induced carbonic anhydrase IX. J Immunol Methods 282, 117-134 Z"vada J, Zavadova Z (1991) An unusual transmissible agent â&#x20AC;&#x201C; MaTu. Arch Virol 118, 189-197 Z"vada J, Zavadova Z, Machon O, Kutinova L, Nemeckova S, Opavsky R, Pastorek J (1997) Transient transformation of mammalian cells by MN protein, a tumor-associated cell adhesion molecule with carbonic anhydrase activity. Int J Oncol 10, 857-863 Z"vada J, Zavadova Z, Malir A, Kocent A (1972) VSV pseudotype produced in cell line derived from human mammary carcinoma. Nature New Biol 240, 124-125 Z"vada J, Zavadova Z, Pastorek J, Biesova Z, Jezek J, Velek J (2000) Human tumour-associated cell adhesion protein MN/CA IX: Identification of M75 epitope and of the region mediating cell adhesion. Br J Cancer 82, 1808-1813 Z"vada J, Zavadova Z, Pastorekova S, Ciampor F, Pastorek J, Zelnik V (1993) Expression of MaTu-MN protein in human tumor cultures and in clinical specimens. Int J Cancer 54, 268-274 Z"vada J, Zavadova Z, Zatovicova M, Hyrsl, Kawaciuk I (2003) Soluble form of carbonic anhydrase IX (CA IX) in the serum and urine of renal carcinoma patients. Br J Cancer 89,10671071
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Cancer Therapy Vol 2, page 263 Cancer Therapy Vol 2, 263-270, 2004
Calcium signalling and tumorigenesis Review Article
Juan A. Rosado*, Pedro C. Redondo, JosĂŠ A. Pariente, GinĂŠs M. Salido Department of Physiology, University of Extremadura, C!ceres, Spain
__________________________________________________________________________________ *Correspondence: Dr. Juan A. Rosado, Faculty of Veterinary Medicine, Department of Physiology, University of Extremadura. Av. Universidad s/n, 10071 C!ceres. Spain; Phone: +34 927257154; Fax: +34 927257154; e-mail: jarosado@unex.es Key words: calcium, tumorigenesis Abbreviations: calcium-sensing receptors, (CaR); cytosolic calcium concentration, ([Ca2+]c); calcium concentration in the stores, ([Ca2+]s); mitochondrial calcium concentration, ([Ca2+]m); nuclear calcium concentration, ([Ca2+]n); calcium influx factor, (CIF); 1)2diacylglicerol, (DAG); endoplasmic reticulum, (ER); G protein-coupled receptors, (GPCR); inositol trisphosphate, (IP3); nicotinic acidâ&#x20AC;&#x201C; adenine dinucleotide phosphate, (NAADP); phosphatidylinositol 4)5-bisphosphate, (PIP2); protein kinase C, (PKC); Phospholipase C, (PLC); plasma membrane Ca2+ ATPase, (PMCA); receptor-operated channels, (ROC); sarcoendoplasmic reticulum Ca2+ ATPase, (SERCA); second messenger-operated channels, (SMOC); store-operated channels, (SOC); 2)5-di(tert-butyl)-1)4-benzohydroquinone, (TBHQ); transient receptor potential channelsf, (TRPC); voltage-operated Ca2+ channels, (VOC) Received: 20 July 2004; Accepted: 02 August 2004; electronically published: August 2004
Summary 2+
Ca is a ubiquitous and versatile intracellular messenger that regulates many different cellular processes such as contraction, secretion, fertilisation and proliferation. Cells increase the cytosolic Ca2+ concentration by releasing Ca2+ from internal stores or by opening Ca2+ channels in the plasma membrane to allow extracellular Ca2+ to enter. Several pumps and exchangers are responsible for returning the elevated levels of cytosolic Ca2+ back to the resting state when the stimulus is terminated. The mitochondrion also plays an important role in that it is involved in the removal process by taking Ca2+ up from the cytosol, which might then be slowly released. The Ca2+ signalling systems are constantly being remodelled in both health and disease, and a disorder in Ca2+ homeostasis has been reported in tumoral cells. This review summarises the mechanisms that regulate intracellular Ca2+ homeostasis and the alterations in the Ca2+ transport systems that are involved in the development of tumorigenesis.
intracellular stores and/or the entry of extracellular Ca2+ across the plasma membrane. Immediately after agonist stimulation several mechanisms, mainly pumps and exchangers, remove Ca2+ from the cytosol, pumping this ion outside the cell or into intracellular stores, to restore the resting [Ca2+]c (Petersen, 2002; Berridge et al, 2003; Schulz and Krause, 2004). The physiological implications of Ca2+ range from the role in muscle (Mc Carron et al, 2004; Morales et al, 2004) or secretory tissues (Salido et al, 1999; Raraty et al, 2000) to neural transmission (Yamashita and Sugioka, 1998; Braet et al, 2004). Additionally, Ca2+ is an intracellular messenger for cellular growth and proliferation (Short et al, 1993). Accumulating evidence suggests that altered cytosolic Ca2+ homeostasis might be involved with excessive cell proliferation, a hallmark of tumorigenesis.
I. Introduction Calcium is a chemical element widely distributed in nature, whose physiological relevance was demonstrated by Ringer in 1883. The presence of calcium in the extracellular medium was necessary for muscle contraction. One century later it was shown that in response to a signal ionised calcium (Ca2+) is released to the cytoplasm from an intracellular calcium store (Streb et al, 1983). In resting conditions, eukaryotic cells are surrounded by an extracellular medium containing a concentration of Ca2+ ([Ca2+]o) of 1.2 mM and the Ca2+ concentration into the main intracellular stores ([Ca2+]s) ranges from 0.1 to 1 mM. By contrast, the Ca2+ concentrations in the cytoplasm ([Ca2+]c), mitochondria ([Ca2+]m) and nucleus ([Ca2+]n) are about 100 nM (Petersen, 2002; Berridge et al, 2003). When an agonist binds to a specific receptor site on the outside of a cell membrane, a transduction process occurs, creating an intracellular signal. In many cases, the signal is a rise in [Ca2+]c. The increase in [Ca2+]c is induced by the release of compartmentalised Ca2+ from 263
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II. Mechanisms of Ca2+ signalling
(PIP2) by phospholipase Cb (PLCb) after the occupation of G-protein coupled receptors or by growth factor receptors coupled to PLCg(Bernstein et al, 1992; Kim et al, 2000) At least three IP3 receptor isoforms have been described, that appear as homo- and heterotetramers (Monkawa et al, 1995; Vermassen et al, 2004). Structurally, IP3 receptors have three different domains: a N-terminus cytoplasmic domain with an IP3 binding site, a regulatory central domain, which binds allosteric effectors such as Ca2+ or ATP (Mikoshiba, 1993; Sato et al, 2004), and a transmembrane region near the C terminus composed of six-spanning domains, involved in the formation of a tetrameric complex (Sayers et al, 1997;
A. Calcium release from intracellular stores Ca2+ release from the intracellular compartments is regulated by channels located in the membrane of the stores, such as the IP3 receptor, the ryanodine receptor, the NAADP receptor and the sphingosine 1-phosphate receptor (Figure 1; Berridge et al, 2000).
1. The inositol trisphosphate (IP3) receptor The molecule that regulate the opening of this channel is inositol 1,4,5 trisphosphate (IP3), generated by the hydrolysis of phosphatidylinositol 4,5-bisphosphate
Figure 1. Mechanisms of calcium signalling. Cell stimulation by agonists activate the formation of second messengers that induce the release of Ca2+ stored in the endoplasmic/sarcoplasmic reticulum (ER/SR) through the IP3 receptor (IP3R), the ryanodine receptor (RyR), the NAADP receptor (NAADPR) or the sphingosine 1-phosphate binding site (S1P-R). In addition, agonists stimulate the entry of extracellular Ca2+ through plasma membrane channels. Most of the cytosolic Ca2+ is bound to buffers, and only a small percentage binds to effectors and activates cellular functions. Ca2+ removal is mediated by various pumps and exchangers, including the Na+/Ca2+ exchanger (NCX) and the plasma membrane Ca2+ ATPase (PMCA) that extrude Ca2+ from the cytosol and the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) that pumps Ca2+ back to the ER/SR. During the Ca2+ signal mitochondria sequester Ca2+ through an uniporter that might be then released slowly into the cytoplasm through the NCX or the permeability transition pore (PTP).
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Cancer Therapy Vol 2, page 265 Hamada et al, 2003). Cytosolic Ca2+ exerts a dual effect on the activity of the IP3 receptor, so that low Ca2+ concentrations (<300 nM) are stimulatory while higher concentrations are either inhibitory or, after IP3 binding, do not alter IP3 receptor activity (Bootman and Lipp, 1999; Berridge et al, 2000).
(SOC). VOCs are Ca2+ permeable channels that are briefly activated by changes in the membrane potential (Tsien et al, 1995). These channels are mainly found in excitable cells, where they open in response to membrane depolarisations to allow Ca2+ to enter the cell (McCleskey, 1994). ROC belong to a heterogeneous family of channels that are specially relevant in secretory cells and neurones. ROCs are activated by a number of cellular agonists inducing a rapid Ca2+ entry indicative of a direct coupling between the receptor and a Ca2+ permeable channel (Sage, 1992). SMOC are Ca2+ channels activated by a second messenger, such as inositol phosphate or Ca2+ itself (Sage, 1992). These channels have been described mainly in non excitable cells. In endothelial cells, a Ca2+ channel activated by Ca2+ and inositol 1,3,4,5-tetrakisphosphate has been found (L端ckhoff and Clapham, 1992). In human platelets, thrombin activates a store-independent (noncapacitative) Ca2+ entry, which is mediated by PKC (Rosado and Sage, 2000a). In addition, some transient receptor potential channels (TRPC) have been reported to be activated by DAG analogues in different non-excitable cells (Ma et al, 2000). The major mechanism for Ca2+ entry in non-excitable cells is store-operated Ca2+ entry through SOC, controlled by the filling state of the intracellular Ca2+ stores. It is not yet clear how store depletion is communicated to the plasma membrane but a number of hypotheses have been suggested that can be divided into those which propose a role for a diffusible messenger and those which propose a direct interaction between proteins in the ER and PM (conformational coupling). Diffusible messengers include cyclic GMP, small GTP-binding proteins, a product of cytochrome P450, tyrosine kinases and a yet unknown Ca2+ influx factor (CIF; Parekh and Penner, 1997). Alternatively the conformational coupling model suggests an interaction between the IP3 receptor (IP3R) in the membrane of the ER and a Ca2+ channel in the PM (Berridge, 1995). The conformational coupling has recently received support from studies that propose a secretion-like coupling for the activation of SMCE (Patterson et al, 1999; Rosado et al, 2000a; Redondo et al, 2003). The secretion-like coupling appears as an integrative model where messenger molecules and the actin cytoskeleton interact to facilitate a physical and reversible coupling between elements in the ER and PM. Consistent with this proteins of the Ras family and tyrosine kinases, initially considered as members of the diffusible messenger hypothesis for the activation of SMCE, are essential for actin reorganisation induced by store depletion (Rosado and Sage, 2000b; Rosado et al, 2000b) and proteins classically involved in exocytosis, such as SNAP-25 appear to be involved in Ca2+ entry (Yao et al, 1999; Redondo et al, 2004) . In addition, remodelling of the cytoskeletal cortical barrier has been suggested to facilitate the activation of SMCE by CIF (Xie et al, 2002). The secretion-like coupling model for the activation of SMCE proposes a direct interaction between the IP3Rs
2. The ryanodine receptor Ryanodine receptors were described in the skeletal muscle as a tetrameric channel responsible for sarcoplasmic reticulum Ca2+ release (Pessah et al, 1985). As for the IP3 receptors, there are also three ryanodine receptor isoforms. These channels are regulated by several chemical signals, such as cyclic ADP ribose, Ca2+ itself, caffeine, or the neutral plant alkaloid ryanodine (McPherson and Campbell, 1993). In skeletal muscle, ryanodine receptors are activated by direct coupling to L-type Ca2+ channels facilitated by the proximity of the T tubules and the sarcoplasmic reticulum (McPherson and Campbell, 1993).
3. NAADP receptor The discovery of Ca2+ release by NAADP in sea urchin eggs (Lee and Aarhus, 1995) described a new mechanism for Ca2+ mobilisation. NAADP receptors are likely located on Ca2+ stores distinct from the endoplasmic reticulum, which have been identified as acidic organelles (Yamasaki et al, 2004). The activation of these channel evokes complex changes in [Ca2+]c, which involve crosstalk with the IP3 and ryanodine receptors (Patel, 2004). NAADP initiates the activation of Ca2+-induced Ca2+ release from IP3- and cyclic ADP ribose-sensitive stores, leading to an oscillatory pattern (Santella et al, 2000; Churchill and Galione, 2001). A complex cross-talk between NAADP, IP3 and ryanodine receptors has been described in pancreatic acinar cells, where acetylcholinestimulated local Ca2+ responses, which are triggered via IP3 receptors, are converted into global responses by the presence of NAADP or cyclic ADP ribose, and Ca2+ mobilisation stimulated by cholecystokinin, mediated by converging pathway, involving cyclic ADP ribose and NAADP, is potentiated by IP3 (Cancela et al, 1999).
4. Sphingosine 1-phosphate receptor Sphingosine 1-phosphate is a second messenger that releases Ca2+ from the endoplasmic reticulum by possibly binding to a sphingolipid Ca2+-release-mediating protein on the membrane of the endoplasmic reticulum (Mao et al, 1996). Alternatively, this lipid might activate ryanodine receptors, since sphingosine 1-phosphate enhances ryanodine binding to its receptor and Ca2+ release by sphingosine 1-phosphate is prevented by ryanodine receptor blockers (Dettbarn et al, 1995).
B. Calcium entry In excitable cells, such as neurones and muscle cells, Ca2+ entry mostly occurs through voltage-operated Ca2+ channels (VOC); however, in non-excitable cells, where VOC are not present Ca2+ influx mainly occurs through receptor-operated channels (ROC), second messengeroperated channels (SMOC) or store-operated channels
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Rosado et al: Calcium signalling in cancer in the ER and a Ca2+ channel in the PM. The involvement of IP3Rs in SMCE has widely been confirmed (Irvine, 1990; Mikoshiba, 1997), and the role of mammalian homologues of the Drosophila TRPC has received support from studies that demonstrate coupling between TRPCs and IP3Rs in transfected cells (Kiselyov et al, 1998; Vazquez et al, 2001) and in cells naturally expressing TRPCs (Rosado and Sage, 2000c; Rosado et al, 2002). In this model, the actin cytoskeleton plays a dual role as for secretion, both as a cortical barrier preventing constitutive coupling and also supporting the transport of portions of the ER towards the PM (Rosado and Sage, 2001).
isoforms in humans: PMCA1-4, although its number is increased by the existence of alternative splice variants (Strehler and Zacharias, 2001). The structure of the PMCA consists of ten transmembrane domains and five extracellular regions, with the NH2 and COOH termini located in the cytosolic site of the membrane (Guerini, 1998; Strehler and Zacharias, 2001). PMCA activity is regulated by several messenger molecules including Ca2+/calmodulin, protein tyrosine kinases, PIP2, protein serine/threonine kinases and by proteases like calpain (Strehler and Zacharias, 2001; Pariente et al, 2003) and agonists might either increase or inhibit the PMCA activity by activating these intracellular pathways (Rosado and Sage, 2000d; Pariente et al, 2001). On the other hand, the Na+/Ca2+ exchanger is a bidirectional electrogenic ion transporter that couples the movement of Na+ in one direction with the transport of Ca2+ in the opposite direction. The Na+/Ca2+ exchanger modulates [Ca2+]c by either removing Ca2+ from the cytosol (forward mode) or by transporting Ca2+ inside the cell (reverse mode). Three different Na+/Ca2+ exchangers have been described. Two of them are electrogenic, the K+-independent Na+/Ca2+ exchanger, which catalyses the countertransport of either 3 or 4 Na+ for 1 Ca2+ (Blaustein and Lederer, 1999) and the K+-dependent Na+/Ca2+ exchanger, which catalyses the exchange of 4 Na+ by 1 Ca2+ and 1 K+ (Dong et al, 2001). In addition, an electroneutral Na+/Ca2+ exchanger has been described in mitochondria (Matsuda et al, 1997).
C. Mechanisms for calcium removal from the cytosol Ca2+ removal from the cytosol is carried out by several Ca2+ pumps and exchangers which reintroduce Ca2+ into the internal stores or extrude it out of the cell (Meldolesi and Pozzan, 1998).
1. Calcium reuptake into internal stores Ca2+ uptake into the intracellular stores mostly occurs against a concentration gradient, since [Ca2+]c is lower than [Ca2+]s which at rest is in a high-micromolar to lowmillimolar concentration range. Released Ca2+ is returned to the stores by the sarcoendoplasmic reticulum Ca2+ ATPase (Figure 1; SERCA). There are three different SERCA genes, and additional isoform subtypes are generated by alternative splicing (Exton, 1997). SERCA has a high affinity for Ca2+ (0.1-0.4 ÂľM), which suggests that SERCA is likely to be activated by an increase in [Ca2+]c and inhibited by an increase in [Ca2+]s (Carafoli, 1992). Several pharmacological tools, such as thapsigargin, 2,5-di(tert-butyl)-1,4-benzohydroquinone (TBHQ) and cyclopiazonic acid, have been developed to investigate the role of SERCA in Ca2+ signalling. Among them, the most widely used is thapsigargin, which binds to all SERCAs although with different affinity (Cavallini et al, 1995) and causes an irreversible inhibition of their activity by blocking the ATPase in the Ca2+-free state (Wictome et al, 1992). A similar effect is induced with TBHQ, although with lower potency, and some isoforms seems to be insensitive to this inhibitor, which has been used to identify distinct intracellular Ca2+ stores (Cavallini et al, 1995) that, in turn, activate different Ca2+ entry mechanisms (Rosado et al, 2004).
3. Role of mitochondria in Ca2+ signalling Mitochondria are a relevant component of the Ca2+ signalling machinery. Localised in the vicinity of the Ca2+ channels, mitochondria sequesters Ca2+ coming from intracellular stores or the extracellular medium modulating the Ca2+ signals (Figure 1; Gonzalez and Salido, 2001; Parekh, 2003). Ca2+ entry into mitochondria is regulated by a high capacity and low affinity uniporter that transports Ca2+ down a electrochemical gradient. This uniporter requires local high [Ca2+]c to function suggesting that mitochondria should be close to the Ca2+ channels (Berridge et al, 2000; Pariente et al, 2003). Efflux of Ca2+ occurs by two different exchangers that countertransport Ca2+ for either Na+ or H+, or through a permeability transition pore that have two different states, a reversible low conductance state, that allow mitochondria to participate in Ca2+ signalling and an irreversible high conductance state that collapses the mitochondrial membrane potential leading to the activation of apoptosis (Ichas et al, 1997; Berridge et al, 2000). Mitochondrial Ca2+ accumulation has a dual role in cell function, an universal role that consist in the activation of mitochondrial enzymes involved in the generation of ATP, and a more specific role involved in the modulation of the spatio-temporal aspects of Ca2+ signalling (Camello-Almaraz et al, 2002; Brini, 2003; Gonzalez et al, 2003; Parekh, 2003).
2. Calcium extrusion across the plasma membrane Perhaps the major mechanism for the removal of cytosolic Ca2+ is the extrusion of Ca2+ to the extracellular medium against a concentration gradient. Ca2+ efflux is mainly carried out by two different transporters, the plasma membrane Ca2+ ATPase (PMCA) and the Na+/Ca2+ exchanger (Figure 1). The PMCA is an ATPase highly sensitive to vanadate and lanthanum (Pedersen and Carafolli, 1987; Pariente et al, 1999; Lajas et al, 2001). Molecular biology studies revealed the expression of at least four PMCA
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4. Ca2+ binding proteins
extracellular Ca2+ entry has been shown to be necessary for the induction of neoplastic transformation and cell treatment with calcium channel blockers, such as lanthanum or nifedipine, reduce their transformation (Smith et al, 1986). In addition, the sustained Ca2+ store depletion and prevention of the refilling via SMCE influences the rate of cellular protein synthesis and reduces cell proliferation in experimental cancer (Palakurthi et al, 2000). These results indicate that cell transformation requires the influx of external Ca2+ and in most cases it has been shown to accelerate Ca2+ entry; therefore, reduction of this mechanism might reduce the progression of the tumour and might be one of the targets of cancer therapy. This is the case of the human prostate cancer cells, where blockade of the Ca2+ entry by using TH-1177, extend the mean life span in mice inoculated with these cells by 38 % without obvious cytotoxicity (Haverstic et al, 2000). In addition, a potential antitumor action for another Ca2+ channel blocker, the dihydropyridine amlodipine, has recently been described (Yoshida et al, 2004). Amlodipine has been reported to reduce the tumour growth and increase the survival of mice inoculated with human epidermoid carcinoma A431 cells (Yoshida et al, 2004). Several alterations have also been observed in the function of the mechanisms involved in Ca2+ removal from the cytosol, which indicate that this transporters might be involved in the development of neoplasia. Recent studies have reported that an altered expression of the PMCA might occur in tumorigenesis. The expression of PMCA 1 in MCF-7 and MDA-MB-231 breast cancer cells is significantly increased compared to that in normal cells (Lee et al, 2002). This effect might be a cause of the cell transformation or a response to this stimuli since PMCA expression has been shown to be lower in skin and lung fibroblast transformed by treatment with SV40 (Reisner et al, 1997). An interesting issue that is still unclear is how cancer cells avoid apoptosis. It has been reported that early preneoplastic cells are highly susceptible to apoptosis, whereas later preneoplastic cells are quite resistant (Preston et al, 1997). Although several hypothesis have been proposed to explain the resistance to apoptosis, a plausible mechanism involving Ca2+ signalling has been presented, in which reduced Ca2+ levels in the ER were observed in early preneoplastic cells that undergo apoptosis compared to a higher level of stored Ca2+ in the ER in late preneoplastic cells. The mechanism by which these cells accumulated different amount of Ca2+ in the ER lies on the different activation of SMCE. In late preneoplastic cells, less susceptible to apoptosis and therefore with a great potential to become transformed, Ca2+ entry is increased compared to that found in cells in the early steps of neoplastic transformation (Preston et al, 1997). The role of Ca2+ entry in apoptosis is supported by a more recent report suggesting that a reduced SMCE correlates with the development of apoptosis (Jayadev et al, 1999). In the last decade, the scientists have considerably advanced in the cancer therapy, trying to apply genetic therapy to stop the development of several cancer types.
2+
It is well known that approximately 98-99 % of Ca ions in the cytosol are bound by buffer molecules, including Ca2+ binding proteins (Neher and Augustine, 1992; Mogami et al, 1999). There is a variety of Ca2+binding proteins involved in Ca2+ signalling either acting as Ca2+ sensors, effectors or buffers that either initiate, execute or terminate Ca2+-dependent functions. Most of Ca2+-binding molecules inside the cell are Ca2+ buffer proteins that maintain a low [Ca2+]c at rest, including calretinin, calbindin and parvalbumin in the cytosol and calsequestrin and calreticulin in the intracellular stores.
III. Ca2+ signalling in cancer Cancer is a disease characterised by a complete deregulation of the cell proliferation cycle. It is well known that cell exposure to external agents, such as some heavy metals (Krantz and Dorevicht, 2004), virus infection (Shah et al, 2004) or radiations, increases the number of cancer incident. In addition, intracellular signals can also induce tumorigenesis. Among the different cancer types and locations, lung, skin or prostate cancer in men and breast cancer in women appear as the more common neoplasia. To understand the mechanism involved in gene deregulation, we have to investigate the signalling mechanisms that induce the activation of transcription factors (Arbiser, 2004), where Ca2+ signals are among the most important pathways involved in tumorigenesis (Lipskaia and LomprĂŠ, 2004). A number of studies have reported alterations in Ca2+ homeostasis in tumoral cells, ranging from those that affect the [Ca2+]o to those that involve dysfunction of one or several intracellular Ca2+ mobilising mechanism. Since cancer is predominantly a disease of disordered balance between proliferation, differentiation and apoptosis, an altered function of the extracellular calciumsensing receptors (CaR) might contribute to the progression of the neoplastic disease. Parathyroid hyperplasias as well as colon carcinoma have been shown to be correlated with an altered expression of CaR (MartÂŁn-Salvago et al, 2003; Rodland, 2004), leading to loss of the growth suppressing effects of elevated extracellular Ca2+. CaR dysfunction has been reported to be involved in cancer progression and its activation has been linked to an increased expression and secretion of parathyroid hormone-related peptide, a promoter of the metastatic progress in bone as well as a causal factor involved in hypercalcemia of malignancy (Rodland, 2004). The mechanisms involved in the regulation of [Ca2+]c are also either involved or altered in tumoral cells. In the last years a number of studies have provided information about the transduction mechanisms involved in Ca2+ entry induced by mitogenic factors, binding either to tyrosine kinase receptors or to G protein coupled receptors. Among the early events induced by mitogens Ca2+ mobilisation and specially Ca2+ entry are widespread signals (Munaron, 2002), which supports the involvement of Ca2+ signalling in the development of neoplastic transformation. In preneoplastic mouse JB6 epidermal cells 267
Rosado et al: Calcium signalling in cancer Exton JH (1997) New developments in phospholipase D. J Biol Chem 272, 15579-15582. Gonzalez A, Granados MP, Salido GM and Pariente JA. (2003) Changes in mitochondrial activity evoked by cholecystokinin in isolated mouse pancreatic acinar cells. Cell Signal 15, 1039-1048. Gonzalez A, Salido GM (2001) Participation of mitochondria in calcium signalling in the exocrine pancreas. J Physiol Biochem 57, 331-339. Guerini D (1998) The significance of the isoforms of plasma membrane calcium ATPase. Cell Tissue Res 292, 191-197. Hamada K, Terauchi A and Mikoshiba K (2003) Threedimensional rearrangements within inositol 1,4,5trisphosphate receptor by calcium. J Biol Chem 278, 5288152889. Haverstick DM, Heady TN, Macdonald TL and Gray LS. (2000) Inhibition of human prostate cancer proliferation in vitro and in a mouse model by a compound synthesized to block Ca2+ entry. Cancer Res 60, 1002-1008. Ichas F, Jouaville LS and Mazat JP (1997) Mitochondria are excitable organelles capable of generating and conveying electrical and calcium signals. Cell 89, 1145-1153. Irvine RF (1990) 'Quantal' Ca2+ release and the control of Ca2+ entry by inositol phosphates--a possible mechanism. FEBS Lett 263, 5-9. Jayadev S, Petranka JG, Cheran SK, Biermann JA, Barrett JC and Murphy E (1999) Reduced capacitative calcium entry correlates with vesicle accumulation and apoptosis. J Biol Chem 274, 8261-8268. Kim MJ, Kim E, Ryu SH and Suh PG (2000) The mechanism of phospholipase C-gamma1 regulation. Exp Mol Med 32, 101-109. Kiseljov K, Xu X, Mozhayeva G, Kuo T, Pessah I, Mignery G, Zhu X, Birnbaumer L and Muallem S (1998) Functional interaction between InsP3 receptors and store-operated Htrp3 channels. Nature 396, 478-482. Krantz A, Dorevitch S (2004) Metal exposure and common chronic diseases: a guide for the clinician. Dis Mon 50, 220262. Lajas AI, Sierra V, Camello PJ, Salido GM and Pariente JA (2001) Vanadate inhibits the calcium extrusion in rat pancreatic acinar cells. Cell Signal 13, 451-456. Lee HC and Aarhus R (1995) A derivative of NADP mobilizes calcium stores insensitive to inositol trisphosphate and cyclic ADP-ribose. J Biol Chem 270, 2152-2157. Lee WJ, Roberts-Thomson SJ, Holman NA, May FJ, Lehrbach GM and Monteith GR (2002) Expression of plasma membrane calcium pump isoform mRNAs in breast cancer cell lines. Cell Signal 14, 1015-1022. Lipskaia L and Lompre AM. (2004) Alteration in temporal kinetics of Ca2+ signaling and control of growth and proliferation. Biol Cell 96, 55-68. Lückhoff A and Clapham DE (1992) Inositol 1,3,4,5tetrakisphosphate activities an endothelial Ca2+-permeable channel. Nature 335, 356-358. Ma HT, Patterson RL, van Rossum DB, Birnbaumer L, Mikoshiba K and Gill DL (2000) Requirement of the inositol trisphosphate receptor for activation of store-operated Ca2+ channels. Science 287, 1647-1651. Mao C, Kim SH, Almenoff JS, Rudner XL, Kearney DM and Kindman LA (1996) Molecular cloning and characterization of SCaMPER, a sphingolipid Ca2+ release-mediating protein from endoplasmic reticulum. Proc Natl Acad Sci USA 93, 1993–1996. Martin-Salvago M, Villar-Rodriguez JL, Palma-Alvarez A, Beato-Moreno A and Galera-Davidson H. (2003) Decreased expression of calcium receptor in parathyroid tissue in patients with hyperparathyroidism secondary to chronic renal
At present, there is not treatment that can totally sure the cancer elimination, and quite often they found another problem, cancer cells can leave the main focus, travel around the body and proliferate in other substrate, the so called metastatic cells. The current therapies usually have severe side effects in the patients. However, the recent observations provide reasons to be optimistic and raise the possibility that directed synthesis of novel and specific Ca2+ channel blockers or inhibitors of Ca2+ mobilising mechanisms could be useful in the therapy of cancer by minimising effects on normal organs and cells and by maximising the cytostatic efficacy for the neoplastic tissue of clinical concern.
References Arbiser JL (2004) Molecular regulation of angiogenesis and tumorigenesis by signal transduction pathways: evidence of predictable and reproducible patterns of synergy in diverse neoplasms. Semin Cancer Biol 14, 81-91. Berridge MJ (1995) Capacitative calcium entry. Biochem J 312, 1-11. Berridge MJ, Bootman MD and Roderick HL (2003) Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev 4, 517-529. Berridge MJ, Lipp P and Bootman MD (2000) The versatility and universatility of calcium signalling. Nat Rev 1, 11-21. Berstein G, Blank JL, Smrcka AV, Higashijima T, Sternweis PC, Exton JH and Ross E M. (1992) Reconstitution of agoniststimulated phosphatidylinositol 4,5-bisphosphate hydrolysis using purified m1 muscarinic receptor, Gq/11 and phospholipase C-! 1. J Biol Chem 267, 8081-8088. Blaustein MP and Lederer WJ (1999) Sodium/Calcium Exchange: Its Physiological Implications. Physiol Rev 79, 763-854. Bootman MD and Lipp P (1999) Calcium signalling: Ringing changes to the “bell-shaped curve”. Curr Biol 9, 876-878. Braet K, Cabooter L, Paemeleire K and Leybaert L (2004) Calcium signal communication in the central nervous system. Biol Cell 96, 79-91. Brini M (2003) Ca2+ signalling in mitochondria: mechanism and role in physiology and pathology. Cell Calcium 34, 399-405. Camello-Almaraz C, Salido GM, Pariente JA and Camello PJ (2002) Role of mitochondria in Ca2+ oscillation and shape of Ca2+ signals in pancreatic acinar cells. Biochem Pharmacol 63, 283-292. Cancela JM, Churchill GC and Galione A (1999) Coordination of agonist-induced Ca2+-signalling patterns by NAADP in pancreatic acinar cells. Nature 398, 74-76. Carafoli E (1992) Calcium pump of the plasma membrane. Physiol Rev 71, 283-292. Cavallini L, Coassin M and Alexandre A (1995) Two classes of agonist-sensitive Ca2+ stores in platelets, as identified by their differential sensitivity to 2,5-di-(tert-butyl)-1,4benzohydroquinone and thapsigargin. Biochem J 310, 449452. Churchill GC and Galione A (2001) NAADP induces Ca2+ oscillations via a two-pool mechanism by priming IP3- and cADPR-sensitive Ca2+ stores. EMBO J 20, 2666-2671. Dettbarn C, Betto R, Salviati G, Sabbadini R and Palade P (1995) Involvement of ryanodine receptors in sphingosylphosphorylcholine-induced calcium release from brain microsomes. Brain Res 669, 79–85. Dong, H, Light PE, French RJ and Lytton J (2001) Electrophysiological characterization and ionic stoichiometry of the rat brain K+-dependent Na+-Ca2+ exchanger, NCKX2. J Biol Chem 276, 25919-25928.
268
Cancer Therapy Vol 2, page 269 failure. Endocr Pathol 14, 61-70. Matsuda T, Takuma K and Baba A (1997) Na+-Ca2+ exchanger: physiology and pharmacology. Jpn J Pharmacol 74, 1-20. McCarron JG, Bradley KN, MacMillan D, Chalmers S and Muir TC. (2004) The sarcoplasmic reticulum, Ca2+ trapping and wave mechanisms in smooth muscle. News Physiol Sci 19, 138-147. McCleskey EW (1994) Calcium channels: cellular roles and molecular mechanisms. Curr Opinion Neurobiol 4, 304312. McPherson PS and Campbell KP (1993) The ryanodine receptor/Ca2+ release channel. J Biol Chem 268, 1376513768. Meldolesi J and Pozzan T (1998) The endoplasmic reticulum Ca2+ store: a view from the lumen. Trends Biochem Sci 23, 10-14. Mikoshiba K (1993) Inositol 1,4,5-trisphosphate receptor. Trends Pharmacol Sci 14, 86-89. Mikoshiba K (1997) The InsP3 receptor and intracellular Ca2+ signaling. Curr Opin Neurobiol 7, 339-345. Mogami H, Gardner J, Gerasimenko OV, Camello P, Petersen OH and Tepikin AV (1999) Calcium binding capacity of the cytosol and endoplasmic reticulum of mouse pancreatic acinar cells. J Physiol 518, 463-467. Monkawa T, Miyawaki A, Sugiyama T, Yoneshima H, Yamamoto-Hino M, Furuichi T, Saruta T, Hasegawa M and Mikoshiba K (1995) Heterotetrameric complex formation of inositol 1,4,5-trisphosphate receptor subunits. J Biol Chem 270, 14700-14704. Morales S, Camello PJ, Alcon S, Salido GM, Mawe G and Pozo MJ (2004) Coactivation of capacitative calcium entry and Ltype calcium channels in guinea pig gallbladder. Am J Physiol Gastrointest Liver Physiol 286, 1090-1100. Munaron L (2002) Calcium signalling and control of cell proliferation by tyrosine kinase receptors. Int J Mol Med 10, 671-676. Neher E and Augustine GJ (1992) calcium gradients and buffers in bovine chromaffin cells. J Physiol 450, 273-301. Palakurthi SS, Fluckiger R, Aktas H, Changolkar AK, Shahsafaei A, Harneit S, Kilic E and Halperin JA. (2000) Inhibition of translation initiation mediates the anticancer effect of the n-3 polyunsaturated fatty acid eicosapentaenoic acid. Cancer Res 60, 2919-2925. Parekh AB (2003) Mitochondrial regulation of intracellular Ca2+ signaling: more than just simple Ca2+ buffers. News Physiol Sci 18, 252-256. Parekh AB and Penner R (1997) Store depletion and calcium influx. Physiol Rev 77, 901-930. Pariente JA, Camello C, Camello PJ and Salido GM (2001) Release of calcium from mitochondrial and nonmitochondrial intracellular stores in mouse pancreatic acinar cells by hydrogen peroxide. J Membr Biol 179, 2735. Pariente JA, Lajas AI, Pozo MJ, Camello PJ and Salido GM (1999) Oxidizing effects of vanadate on calcium mobilization and amylase release in rat pancreatic acinar cells. Biochem Pharmacol 58, 77-84. Pariente JA, Redondo PC, Granados MP, Lajas AI, Gonzalez A, Rosado JA and Salido GM (2003) Calcium signaling in nonexcitable cells. E. C. Qua. L. 1, 29-43. Patel S (2004) NAADP-induced Ca2+ Release - a new signalling pathway. Biol Cell 96, 19-28. Patterson RL, van Rossum DB and Gill DL (1999) Storeoperated Ca2+ entry: evidence for a secretion-like coupling model. Cell 98, 487-499. Pedersen PL and Carafoli E (1987) Ion motive ATPase. I. Ubiquity, properties and significance for cell function. Trends Biochem Sci 12, 146-150.
Pessah IN, Waterhouse AL and Casida JE (1985) The calciumryanodine receptor complex of skeletal and cardiac muscle. Biochem Biophys Res Commun 128, 449-456. Petersen OH (2002) Calcium signal compartmentalization. Biol Res 35, 177-182. Preston GA, Barrett JC, Biermann JA and Murphy E (1997) Effects of alterations in calcium homeostasis on apoptosis during neoplastic progression. Cancer Res 57, 537-542. Raraty M, Ward J, Erdemli G, Vaillant C, Neoptolemos JP, Sutton R, Petersen OH. (2000) Calcium-dependent enzyme activation and vacuole formation in the apical granular region of pancreatic acinar cells. Proc Natl Acad Sci USA 97, 13126-13131. Redondo PC, Harper AG, Salido GM, Pariente JA, Sage SO and Rosado JA (2004) A role for SNAP-25 but not VAMPs in store-mediated Ca2+ entry in human platelets. J Physiol 558, 99-109. Redondo PC, Lajas AI, Salido GM, Gonzalez A, Rosado JA and Pariente JA (2003) Evidence for secretion-like coupling involving pp60src in the activation and maintenance of storemediated Ca2+ entry in mouse pancreatic acinar cells. Biochem J 370, 255-263. Reisner PD, Brandt PC and Vanaman TC. (1997) Analysis of plasma membrane Ca2+-ATPase expression in control and SV40-transformed human fibroblasts. Cell Calcium 21, 5362. Ringer S (1883) A further contribution regarding the influence of the different constituents of the blood and the contraction of the heart. J Physiol 4, 29-43. Rodland KD (2004) The role of the calcium-sensing receptor in cancer. Cell Calcium 35, 291-295. Rosado JA and Sage SO (2000a) Protein kinase C activates noncapacitative calcium entry in human platelets. J Physiol 529, 159-169. Rosado JA and Sage SO (2000b) Farnesylcysteine analogues inhibit store-regulated Ca2+ entry in human platelets: evidence for involvement of small GTP-binding proteins and actin cytoskeleton. Biochem J 347, 183-192. Rosado JA and Sage SO (2000c) Coupling between inositol 1,4,5-trisphosphate receptors and human transient receptor potential channel 1 when intracellular Ca2+ stores are depleted. Biochem J 350, 631-635. Rosado JA and Sage SO (2000d) Regulation of plasma membrane Ca2+-ATPase by small GTPases and phosphoinositides in human platelets. J Biol Chem 275, 19529-19535. Rosado JA and Sage SO (2001) Activation of store-mediated calcium entry by secretion-like coupling between the inositol 1,4,5-trisphosphate receptor type II and human transient receptor potential (hTrp1) channels in human platelets. Biochem J 356, 191-198. Rosado JA, Brownlow SL and Sage SO (2002) Endogenously expressed Trp1 is involved in store-mediated Ca2+ entry by conformational coupling in human platelets. J Biol Chem 277, 42157-42163. Rosado JA, Graves D and Sage SO (2000b) Tyrosine kinases activate store-mediated Ca2+ entry in human platelets through the reorganization of the actin cytoskeleton. Biochem J 351, 429-437. Rosado JA, Jenner S and Sage SO (2000a) A role for the actin cytoskeleton in the initiation and maintenance of storemediated calcium entry in human platelets. Evidence for conformational coupling. J Biol Chem 275, 7527-7533. Rosado JA, Lopez JJ, Harper AG, Harper MT, Redondo PC, Pariente JA, Sage SO and Salido GM (2004) Two Pathways for Store-mediated Calcium Entry Differentially Dependent on the Actin Cytoskeleton in Human Platelets. J Biol Chem 279, 29231-29235.
269
Rosado et al: Calcium signalling in cancer Sage SO (1992) Three routes for receptor-mediated Ca2+ entry. Curr Biol 2, 312-314. Salido GM, Singh J and Camello PJ (1999) Growth factors and the exocrine pancreas. Ars Pharmaceutica 40, 81-85. Santella L, Kyozuka K, Genazzani AA, De Riso L and Carafoli E (2000) Nicotinic acid adenine dinucleotide phosphateinduced Ca2+ release. Interactions among distinct Ca2+ mobilizing mechanisms in starfish oocytes. J Biol Chem 275, 8301-8306. Sato C, Hamada K, Ogura T, Miyazawa A, Iwasaki K, Hiroaki Y, Tani K, Terauchi A, Fujiyoshi Y and Mikoshiba K (2004) Inositol 1,4,5-trisphosphate receptor contains multiple cavities and L-shaped ligand-binding domains. J Mol Biol 336, 155-164. Sayers LG, Miyawaki A, Muto A, Takeshita H, Yamamoto A, Michikawa T, Furuichi T and Mikoshiba K (1997) Intracellular targeting and homotetramer formation of a truncated inositol 1,4,5-trisphosphate receptor-green fluorescent protein chimera in Xenopus laevis oocytes: evidence for the involvement of the transmembrane spanning domain in endoplasmic reticulum targeting and homotetramer complex formation. Biochem J 323, 273-280. Schulz I and Krause E (2004) Inositol 1,4,5-trisphosphate and its co-players in the concert of Ca2+ signalling-new faces in the line up. Curr Mol Medicine 4, 313-322. Shah KV, Galloway DA, knowles WA and Viscidi RP (2004) Simian virus 40 (SV 40) and human cancer: a review of the serological data. Rev Med Virol 14, 231-239. Short AD, Bian J, Ghosh TK, Waldron RT, Rybak SL and Gill DL (1993) Intracellular Ca2+ pool content is linked to control of cell growth. Proc Natl Acad Sci USA 90, 4986-4990. Smith BM, Gindhart TD and Colburn NH (1986) Extracellular calcium requirement for promotion of transformation in JB6 cells. Cancer Res 1986 46, 701-706. Streb H, Irvine RF, Berridge MJ and Schulz I (1983) Release of Ca2+ from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-1,4,5-trisphosphate. Nature 306, 6769. Strehler EE and Zacharias DA (2001) Role of alternative splicing in generating isoform diversity among plasma membrane calcium pumps. Physiol Rev 81, 21-50. Tsien RW, Lipscombe D, Madison D, Bley K and Fox A (1995) Reflections on Ca2+-channel diversity, 1988-1994. Trends Neurosci 18, 52-54. Vazquez G, Lievremont JP, Bird G and Putney JW Jr (2001) Human Trp3 forms both inositol trisphosphate receptor-
dependent and receptor-independent store-operated cation channels in DT40 avian B lymphocytes. Proc Natl Acad Sci USA 98, 11777-11782. Vermassen E, Parys JB and Mauger JP (2004) Subcellular distribution of the inositol 1,4,5-trisphosphate receptors: functional relevance and molecular determinants. Biol Cell 96, 3-17. Wictome M, Henderson I, Lee AG and East JM (1992) Mechanism of inhibition of the calcium pump of sarcoplasmic reticulum by thapsigargin. Biochem J 283, 525-529. Xie Q, Zhang Y, Zhai C and Bonanno JA (2002) Calcium influx factor from cytochrome P-450 metabolism and secretion-like coupling mechanisms for capacitative calcium entry in corneal endothelial cells. J Biol Chem 277, 16559-16566. Yamasaki M, Masgrau R, Morgan AJ, Churchill GC, Patel S, Ashcroft SJ and Galione A (2004) Organelle selection determines agonist-specific Ca2+ signals in pancreatic acinar and ! cells. J Biol Chem 279, 7234-7240. Yamashita M, Sugioka M (1998) Calcium Mobilization Systems During Neurogenesis. News Physiol Sci 13, 75-79. Yao Y, Ferrer-Montiel AV, Montal M and Tsien RY (1999) Activation of store-operated Ca2+ current in Xenopus oocytes requires SNAP-25 but not a diffusible messenger. Cell 98, 475-485. Yoshida J, Ishibashi T and Nishio M. (2004) Antitumor effects of amlodipine, a Ca2+ channel blocker, on human epidermoid carcinoma A431 cells in vitro and in vivo. Eur J Pharmacol 492, 103-112.
Juan A. Rosado
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Integrins and the cellular radiation response Review Article
Emil Lisiak and Nils Cordes* Bundeswehr Institute of Radiobiology, Neuherbergstrasse 11, 80937 Munich, Germany
__________________________________________________________________________________ *Correspondence: Dr. Nils Cordes, M.D., Bundeswehr Institute of Radiobiology, Neuherbergstrasse 11, 80937 Munich, Germany; Phone: ++89 – 3168 – 3634; Fax: ++89 – 3168 – 2635; E-mail: cordes@radiation-biology.de Key words: Integrins, radiosensitivity, ECM, tumor cells Abbreviations: cell adhesion-mediated radioresistance, (CAM-RR); cell adhesion-mediated drug resistance, (CAM-DR); extracellular matrix, (ECM); focal adhesion kinase, (FAK); integrin-linked kinase, (ILK); ionizing radiation, (IR); mitogen-activated protein kinase, (MAPK); phosphatidylinositol-3- kinase, (PI3K); protein kinase B/AKT, (AKT) Received: 28 July 2004; Accepted: 12 August 2004; electronically published: August 2004
Summary Resistance of tumor cells against ionizing radiation or cytotoxic drugs is a widespread phenomenon. In addition to specific genetic alterations, microenvironmental components such as soluble growth factors and cytokines, and extracellular matrix (ECM) proteins serve as critical determinants of the cellular response to genotoxic injury. Based on recent observations, engagement of cells with ECM proteins via the integrin family of cell adhesion molecules modulates the cellular radiation survival and growth response. In dependence on cell type and cellular context, identification of specific differences between integrin signaling and integrin-growth factor receptor crosstalk may provide new facettes for innovative oncological therapies and important molecular insights into oncogenic transformation and anchorage-independent growth. 2000; Barcellos-Hoff, 2001; Henning et al, 2004). In addition to microenvironmental factors related to the vasculature or oxygen, the extracellular matrix (ECM) has been found to be a strong modifier of the reaction of transformed and normal cells to ionizing radiation (Rose et al, 1999; Cordes and Meineke, 2003; Cordes, 2004; Cordes and van Beuningen, 2004). Attachment of cells to ECM components triggers biochemical- and mechanicalinduced signaling for stimulation of cytoplasmic protein kinases, growth factor receptors, ion channels, and organization of the actin cytoskeleton. Cell-matrix binding is primarily mediated by integrins, a family of 18 ! and 8 ß transmembrane glycoproteins (Hynes, 2002). To date, 24 different !ßheterodimers were identified. Most integrins recognize several ECM proteins and individual matrix proteins bind to several integrins. Activation of integrin-mediated intracellular pathways regulate signaling elements in proliferation, survival, migration, tissue organization and many more (Assoian, 1997; Giancotti and Ruoslahti, 1999; Schwartz, 2001; Watt, 2002; Zahir and Weaver, 2004; Brakebusch and Fässler, 2003). Integrin signaling and assembly of the cytoskeleton are intimately linked. As integrins bind to ECM, they associate with cytoskeletal and signaling complexes to form focal adhesions (Burridge, 1988). In this manner integrins serve as structural integrators between the ECM and the
I. Introduction During the past decade enormous progress has been made in understanding the molecular differences between normal and cancer cells. Cancer cells have defects in regulatory circuits that govern cell survival, proliferation, differentiation, and homeostasis (Jacks and Weinberg, 2002). Consequently, these defects facilitate improved insensitivity to anti-growth factors or cytotoxic agents such as ionizing radiation (IR) or drugs, evasion of programmed cell death, ability of unlimited replicative potential, relative self-sufficiency in growth signals, undirected invasion into normal tissue and formation of metastasis (Hanahan and Weinberg, 2000). Many proteins and genes are known to be involved in these cancer phenomena and modification of these processes seem critical for the development of new anti-cancer strategies. In preclinical and clinical trials, some of the new molecularly targeted treatments based on either single administration of protein kinase inhibitors or blockade of growth factor receptors or administration of these compounds in combination with chemo- or radiotherapy have indeed shown promising results (Brunner et al, 2003; Foon et al, 2004). More recently, the influence of the cellular micromilieu has started to be investigated in the context of uncontrolled proliferation, invasion of tumors, and modification of the cellular radiation response (Park et al,
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Lisiak and Cordes: Integrins and the cellular radiation response cytoskeleton, the property for which integrins are named. Via diverse cytoplasmic signaling molecules, including focal adhesion kinase (FAK), Src-family kinases and integrin-linked kinase (ILK), the stimuli generated through ligation of integrin receptors are transduced for regulating cellular events (Hannigan et al, 1996; Schaller, 2001).
B/AKT (AKT) and inhibition of the proapoptotic molecules caspase-9 and the Bcl-2 family member Bad (Datta et al, 1999). AKT can also activate NF-kĂ&#x;, which induces the expression of a set of survival factors such as osteoprotegerin and prevents release of cytochrome C from mitochondria (Hofbauer and Heufelder, 2001; Wang et al, 2001). To note, extensive investigations revealed strong synergistic cooperation between integrin- and growth factor receptor-mediated signaling, which triggered and optimized signal transduction (Moro et al, 2002; Yamada and Even-Ram, 2002) (Figure 1). Integrins were first studied in platelets and thrombocytes (Davignon et al, 1981; Hansen and Clemmensen, 1982). Nowadays e.g. endothelial cells are intensively under investigation to identify potential therapeutic targets for inhibition of angiogenesis in tumors (Wary, 2004). In fact, both Ă&#x;3 and Ă&#x;1 integrin subunits are expressed by endothelial cells and regulate critical adhesive interactions with a variety of ECM proteins including fibronectin, vitronectin, laminin, collagens and fibrinogen (Ingber, 2002). Cytokine- and growth factorstimulated
II. Integrin signaling in normal and cancer cells Normal cells such as fibroblasts, endothelial or epithelial cells need both adhesion to ECM proteins and stimulation of growth factors in order to survive and proliferate. Matrix protein binding by integrins initiates interactions of FAK with Src, which links FAK to survival and proliferation signaling via mitogen-activated protein kinase (MAPK) (Schlaepfer et al, 1997). Integrinmediated regulation of survival furthermore involves integrin-linked kinase (ILK) and growth factor receptorrelated phosphatidylinositol-3-kinase (PI3K) (Delcommenne et al, 1998). These events modulate apoptotic responses via phosphorylation of protein kinase
Figure 1. Schematic diagram of converging and mutually modifying integrin-RTK signaling in focal adhesions. The ligand binding, e.g. extracellular matrix proteins or growth factors, to their receptors activates downstream protein kinases. Stimulation and inhibition of the different signaling pathways as well as the crosstalk between integrin and growth factor receptor pathways optimize the regulation of cell survival, cell cycle progression, apoptosis, DNA repair, adhesion, migration, protein synthesis, and gene transcription. Further structural and signaling molecules of these membranous multiprotein signaling complexes (actin, gelsolin, talin, !-actinin) are not displayed. However, these components are substantial for the regulation of diverse cellular functions. Additionally, most of the delineated signaling events are not located at the cell membrane where they usually occur to avoid overcrowding. RTK, receptortyrosine-kinase; GF, growth factor.
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Cancer Therapy Vol 2, page 273 endothelial cells express a wide spectrum of cell adhesion receptors as compared to quiescent cells (Ingber, 1992). In particular, the function of !vß3 integrins was elucidated in survival and apoptotic processes with regard to the identification of a specific target molecule for innovative anti-tumor therapies (Kumar et al, 2001). This integrin is only minimally expressed on quiescent blood vessels, but highly upregulated on cells undergoing angiogenesis (Ingber, 1992). Recent studies provided promising evidence that !vß3 integrins may also serve as a useful diagnostic indicator. In animal studies, anti-!vß3 antibodies coupled to a gadolinium-containing liposome or iodine radioisotopes specifically targeted to tumorassociated blood vessels and, thus, proved useful in MRI and scintigraphy (Blankenberg et al, 2002). However, blockade of this integrin disturbed angiogenic processes in the chicken chorioallantoic membrane, mouse retina and rabbit cornea (Friedlander et al, 1995; Luna et al, 1996; Kumar et al, 2001). Moreover, !vß3 or ß1 integrin knockout mice developed serious vascular malformations (Hodivala-Dilke et al, 1999). These observations led to the conclusion that !vß3 integrin facilitates the survival of stimulated endothelial cells. In fact, systemic administration of !vß3 antagonists to animals with ongoing angiogenesis showed blood vessels containing high levels of apoptotic endothelial cells. Similar effects were observed in vitro by comparing endothelial cells of different origins. Testing of a first anti-angiogenic compound, which is a specific !vß3 integrin antibody, has recently started in clinical oncological trials (Eskens et al, 2003). In contrast to normal cells, cancer cells are often able to proliferate and survive in the absence of adhesion to ECM proteins (Schwartz, 1997). These characteristics are important for cancer progression and metastases formation. Intense studies performed in a wide spectrum of cancer cells including melanoma, breast, prostate or colon gave rise to the hypothesis that resistance of cancer cells to medical treatment is partly due to alteration or loss of cell-matrix interactions (Lewis et al, 2002). Because of difficulties in demonstrating a positive or negative correlation between tumor progression and integrin expression, which was due to the heterogeneity of tumors and to the fact that changes in the expression level of a single integrin subunit always had to be judged against the background of the expression levels of all other integrins, the results are still controversial. For example, !2ß1 integrin expression seems to promote invasion in pancreatic carcinomas but is downregulated in bladder and colon cancer (Orian-Rousseau et al, 1998; Arao et al, 2000). On the other hand, a strong correlation between the enhancement of !vß3 integrin expression and the metastatic potential of melanoma cells was detected, thus rating expression of !vß3 as prognostic factor (Hieken et al, 1996). Contrary, expression of ß8 integrin was only observed in normal airway epithelium but not in lung cancer cells (Fjellbirkeland et al, 2003). Tumor progression and ability to form distant metastases has been correlated with the elevation of integrin-dependent protein kinases activity such as FAK or ILK (Gabarra-Niecko et al, 2003; Persad and Dedhar, 2003). A major observation
in these studies was a strong converging communication between oncogenic Ras and Raf with components of integrin signaling cascades (Kinbara et al, 2003). Ras and Raf activate prosurvival pathways and are able to induce a significant degree of anchorage-independent growth. Considering the role of integrins in malignant disease, this family of cell adhesion molecules presents as attractive drug target but the wide range of functions in different cell types is daunting. Understanding of integrinmediated signaling, function and cross-talk with growth factor receptor-regulated pathways is crucial for the optimization and development of innovative chemo- and radiotherapeutic approaches. Nevertheless, a handful of therapeutic approaches have been initiated that implicate engineered anti-integrin antibodies or peptidomimetics with clinical success (Mousa, 2002). Most of the compounds tested with high degree of specificity were anti-!vß3 or anti-RGD peptides for blocking the RGD receptor subset of integrins (Tucker, 2002). While !5ß1 integrins are predominantly involved in survival signalling and anti-RGD peptides possess anti-migratory potential, !vß3-integrins mainly promote angiogenic events (Tucker, 2003). Interestingly, neither systemic increase in bleeding nor impairment of wound healing repair have yet been observed. These findings provide first successful results that can serve as basis for further detailed preclinical and clinical trials.
III. Cell-extracellular interactions modulate the behavior after exposure to radiation
matrix cellular ionizing
Experimental data demonstrated that cancer cells are often more resistant against cell killing by conventional antimitotic drugs in the presence of ECM proteins compared to standard in vitro culture conditions. Cell adhesion-mediated drug resistance (CAM-DR) is thought to be partially caused by integrin signaling (Damiano et al, 1999; Cordes et al, 2004). Sethi et al, (1999) showed that adhesion of small-cell lung cancer cells to ECM protects from the chemotherapeutic agents doxorubicin, etoposide, cyclophosphamide, and cisplatin. This effect is mediated by ß1 integrins, which activate intracellular protein kinases in response to therapy-induced DNA-damage. PI3K was uncovered to participate in drug resistance of breast cancer cells (Clark et al, 2002) and increased drug resistance could also be correlated with overexpressed !4ß1 integrin in human myeloma cells (Damiano et al, 1999). Similar findings were seen in cell adhesion-mediated radioresistance (CAM-RR) studies and the specific impact of integrin-mediated signaling on decreased cellular radiosensitivity will be outlined in detail below.
A. ECM-dependent mediated cell survival exposure
and after
integrinradiation
The cytoprotective effects of physiological substrata were detected both in irradiated normal cells like skin and lung fibroblasts, endothelial cells, and keratinocytes as 273
Lisiak and Cordes: Integrins and the cellular radiation response well as in transformed cells of different origin (lung, pancreas, skin, brain, breast, melanoma, colon) (Figure 2; Rose et al, 1999; Cordes and Meineke, 2003; Cordes and van Beuningen, 2003; Cordes and van Beuningen, 2004). Clonogenic survival experiments provided evidence that the interaction of cells with the physiological integrin ligands fibronectin, laminin, collagen-III and vitronectin before and at the time of irradiation essentially promotes survival compared to cells irradiated on culture plastic. Interestingly, this effect demonstrated only marginal variation between the different matrix proteins, complex matrix compositions like Matrigel (matrix extract from the Englebreth-Holm-Swarm mouse sarkoma) or matrix produced by bovine and human endothelial cells. Except from a variety of human glioblastoma cell lines (Cordes et al, 2003), our studies clearly show that the transformed cell lines used are all susceptible to matrix signals and that these signals strongly impact on the cellular resistance to IR. In agreement with our studies on different ILK overexpressing mutants (Cordes, 2004), Lewis et al, (2002) and Truong et al, (2003) observed adhesion to matrix as one of the major determinants for the ability to execute apoptosis after induction of genotoxic stress.
cellular phenomena have been intensively investigated in radiobiology and are regulated by cyclins, cyclindependent kinases (CDK) and their inhibitors (Bernhard et al, 1999) (Figure 3). Unfortunately, only few studies have considered an impact of cell-matrix interactions on the regulation of cell cycling in irradiated cells (Gadbois et al, 1997; Dimitrijevic-Bussod et al, 1999). Recently, we showed that adhesion of normal lung fibroblasts to fibronectin enabled pronounced radiation-induced accumulation of cells in the G2/M phase of the cell cycle relative to culture plastic (Cordes and van Beuningen, 2004). Whether improved ECM-dependent survival is caused by optimization of DNA repair mechanisms at fibronectin presence is currently not known but studies to clarify this aspect are ongoing in our laboratory. Confirmatory observations were reported by Gadbois et al. (1997) and Dimitrijevic-Bussod et al. (1999) who showed that fibronectin and collagen-IV significantly reduced the portion of cells arrested in G1 and S and that radiationinduced cell cycle arrest of human normal fibroblasts is substratum-dependent.
C. Radiation-dependent modification of integrin cell surface presentation, cell motility and invasion
B. Adhesion of cells to ECM proteins modulates radiation-induced cell cycle arrest
In vitro experiments using primary and permanent cell cultures of diverse origin demonstrated that IR is able to induce significant up-regulation of cell surface Ă&#x;1-, Ă&#x;3-,
Cell survival and cell cycling are closely linked cell functions. After irradiation, cells are blocked in the G1- or G2-phase of the cell cycle, which is hypothesized to provide time for DNA-repair (O`Connor, 1997). These
Figure 2. Fibronectin-mediated radiation resistance. In contrast to polystyrene (Poly), growth of cells on the matrix protein fibronectin (FN) improves survival of irradiated human normal and transformed cells. Radiation doses " 2 Gy for normal skin (HSF1) and lung (CCD32) fibroblasts and for radiation doses " 4 Gy for A172 (glioblastoma) and A549 (lung carcinoma) cells grown on FN resulted in a p < 0.05 compared to cells grown on polystyrene. * p < 0.05.
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Figure 3. Schematic diagram delineating the regulation of the G2 checkpoint response after genotoxic stress (e.g. ionizing radiation) in vitro. During G2/M-phase transition, the activity of various cell cycle proteins is tightly regulated by phosphorylation, dephosphorylation and protein interactions, mechanisms that are modulated by cell-matrix interactions. Chk1 and Chk2 phosphorylate Cdc25C, which is proposed to be critical for G2 checkpoint regulation. After binding of 14-3-3, the 14-3-3-Cdc25C complex is degraded in the cytoplasm thus preventing the activation of the Cdc25C substrate and G2-phase driving protein kinase Cdk1 (p34cdc2) by dephosphorylation. Furthermore, Chk1 and Chk2 are able to phosphorylate p53 on serine-20, a site implicated in p53 stabilization through p53-MDM2 interactions. While subsequent induction of CDK-inhibitor p21 prevents cleavage of the pRb-E2F-complex via inhibition of cyclin D1/CDK4/CDK6 activation responsible for G1-phase arrest, p53 is also required to sustain but not to initiate a G2 arrest.
and !5-integrin expression within 48 to 96 h (Meineke et al, 2002; Cordes et al, 2002). This effect presented always dose- and frequently matrix-dependent. Interestingly, in most cell lines there was a transient decrease in ß1 or ß3 integrin cell surface presentation detectable during the first 6 h following IR. Evaluation of the adhesive functionality of the newly expressed integrin receptors revealed a strong dose-dependent elevation of cell adhesion to various matrix proteins. To assess the importance of single integrin subunits in this process, blocking experiments were carried out and identified both ß1- and ß3-integrins to play an essential role in adhesion of irradiated cells. Similar observations were reported by other investigators in endothelial cells (Hallahan et al, 2003). One elegant therapeutic approach used enhancement of !vß3 integrin
expression on sublethally irradiated endothelial cells undergoing angiogenesis to deliver drugs or immunoradioisotopes directly into the irradiated endothelium of a malign tumor (Hallahan et al, 2003). This might be a reasonable strategy for tumor-specific integrin trageting in selected tumor entities. Besides adhesion, integrins serve in invasion and migration events of normal but especially cancer cells. The impact of irradiation on these two cell functions is still controversial. Wild-Bode et al, (2001) showed enhanced migration and invasiveness of glioma cells, which correlated with elevated expression of !vß3 integrins and activity of matrix-metalloproteinases (MMPs). In contrast, we showed differential invasion-impairing potential of irradiation in glioma cells (Cordes et al, 2003). Radiation275
Lisiak and Cordes: Integrins and the cellular radiation response Burridge K, Fath K, Kelly T, Nuckolls G, and Turner C (1988) Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton. Annu Rev Cell Biol 4, 487-525. Clark AS, West K, Streicher S, and Dennis PA (2002) Constitutive and inducible Akt activity promotes resistance to chemotherapy, trastuzumab, or tamoxifen in breast cancer cells. Mol Cancer Ther 1, 707-717. Cordes N (2004) Overexpression of hyperactive integrin-linked kinase leads to increased cellular radiosensitivity. Cancer Res, in press Cordes N, and Beinke C (2004) Fibronectin alters A549 human lung cancer cell survival after irradiation. Cancer Biol Ther 3, 47-53. Cordes N, and Meineke V (2003) Cell adhesion-mediated radioresistance (CAM-RR): Extracellular matrix-dependent improvement of cell survival in human tumor and normal cells in vitro. Strahlenther Onkol 179, 337-344. Cordes N, and van Beuningen D (2003) Cell adhesion to the extracellular matrix protein fibronectin modulates radiationdependent G2 phase arrest involving integrin-linked kinase (ILK) and glycogen synthase kinase-3# (GSK-3#) in vitro. Brit J Cancer 88, 1470-1479. Cordes N, and van Beuningen D (2004) Arrest of human lung fibroblasts in G2 phase after irradiation is regulated by converging phosphatidylinositol-3 kinase and beta1-integrin signaling in vitro. Int J Radiat Oncol Biol Phys 58, 453462. Cordes N, Beinke C, Plasswilm L, and van Beuningen D (2004) Irradiation and various cytotoxic drugs enhance tyrosine phosphorylation and #1-integrin clustering in human A549 lung cancer cells in a substratum-dependent manner in vitro. Strahlenther Onkol 180, 157-164. Cordes N, Blaese MA, Meineke V, and van Beuningen D (2002) Ionizing radiation induces up-regulation of functional #1integrin in lung tumour cell lines in vitro. Int J Radiat Biol 78, 347-357. Cordes N, Hansmeier B, Beinke C, Meineke V, and van Beuningen D (2003) Irradiation differentially affects substratum-dependent survival, adhesion, and invasion of glioblastoma cell lines. Brit J Cancer 89, 2122-2132. Damiano JS, Cress AE, Hazlehurst LA, Shtil AA, and Dalton WS (1999) Cell adhesion mediated drug resistance (CAMDR): role of integrins and resistance to apoptosis in human myeloma cell lines. Blood 93, 1658-1667. Datta SR, Brunet A, and Greenberg ME (1999) Cellular survival: a play in three Akts. Genes Dev 13, 2905-2927. Davignon D, Martz E, Reynolds T, Kurzinger K, and Springer TA (1981) Lymphocyte function-associated antigen 1 (LFA1): a surface antigen distinct from Lyt-2,3 that participates in T lymphocyte-mediated killing. Proc Natl Acad Sci USA 78, 4535-4539. Delcommenne M, Tan C, Gray V, Ruel L, Woodgett J, and Dedhar S (1998) Phosphoinositide-3-OH kinase-dependent regulation of glycogen synthase kinase 3 and protein kinase B/Akt by the integrin-linked kinase. Proc Natl Acad Sci USA 95, 11211-11216. Dimitrijevic-Bussod M, Balzaretti-Maggi VS, and Gadbois DM (1999) Extracellular matrix and radiation G1 cell cycle arrest in human fibroblasts. Cancer Res 59, 4843-4847. Eskens FA, Dumez H, Hoekstra R, Perschl A, Brindley C, Bottcher S, Wynendaele W, Drevs J, Verweij J, and van Oosterom AT (2003) Phase I and pharmacokinetic study of continuous twice weekly intravenous administration of Cilengitide (EMD 121974), a novel inhibitor of the integrins alphavbeta3 and alphavbeta5 in patients with advanced solid tumours. Eur J Cancer 39, 917-926.
induced Ă&#x;1 and Ă&#x;3 integrin cell surface presentation in combination with elevated MMP-2 activity suggested that the invasion-inhibiting effect in irradiated cells is in part due to integrin-mediated local anchorage and disproportional MMP-2-mediated degradation of ECM proteins. Further controversy data on this issue indicated that exposure of endothelial cells to 6 Gy of X-rays promotes migration of endothelial cells in vitro (Sonveaux et al, 2003). Studies on the area vasculosa or the chorioallantoic membrane of the fertilized egg clearly showed that angiogenesis is inhibited after low doses of IR such as 2 Gy but is stimulated after higher doses like 10 Gy (Hatjikondi et al, 1996; Plasswilm et al, 1999).
IV. Discussion In many cases refractory tumor responses are clinically observable during both chemo- and radiotherapy. The resistance of tumors or tumor cell clones is still a major problem to be solved and integrin-mediated cell-ECM interactions are very likely to play an essential role in this context. It is widely accepted that a combination of molecularly targeted anticancer therapies may be more effective than conventional approaches. The presented data on the role of integrin receptors in CAMRR could provide a new, challenging, and effective basis for the development of innovative multi-targeted oncological strategies that are able to augment the cytotoxic powers against the primary tumor as well as its metastases. Further investigations on tumor-specific integrin-mediated signaling and cross-talk between integrins and growth factor receptor signaling may contribute to the identification of new molecular targets and the design of interacting substances for adjuvant chemo- and radiotherapy strategies.
References Arao S, Masumoto A, and Otsuki M (2000) Beta1 integrins play an essential role in adhesion and invasion of pancreatic carcinoma cells. Pancreas 20, 129-137. Assoian RK (1997) Anchorage-dependent cell cycle progression. J Cell Biol 136, 1-4. Barcellos-Hoff MH (2001) It takes a tissue to make a tumor: epigenetics, cancer and the microenvironment. J Mammary Gland Biol Neoplasia 6, 213-221. Beinke C, van Beuningen D, and Cordes N (2003) Ionizing radiation modulates expression and tyrosine phosphorylation of the focal adhesion-associated proteins focal adhesion kinase (FAK) and its substrates p130cas and paxillin in A549 human lung carcinoma cells in vitro. Int J Radiat Biol 79, 721-731. Bernhard EJ, McKenna WG, and Muschel RJ (1999) Radiosensitivity and the cell cycle. Cancer J Sci Am 5, 194204. Blankenberg FG, Mari C, and Strauss HW (2002) Development of radiocontrast agents for vascular imaging: progress to date. Am J Cardiovasc Drugs 2, 357-365. Brakebusch C, and Fässler R (2003) The integrin-actin connection, an eternal love affair. EMBO J 22, 2324-2333. Brunner TB, Hahn SM, Gupta AK, Muschel RJ, McKenna WG, and Bernhard EJ (2003) Farnesyltransferase inhibitors: an overview of the results of preclinical and clinical investigations. Cancer Res 63, 5656-5668.
276
Cancer Therapy Vol 2, page 277 Fjellbirkeland L, Cambier S, Broaddus VC, Hill A, Brunetta P, Dolganov G, Jablons D, and Nishimura SL (2003) Integrin alphavbeta8-mediated activation of transforming growth factor-beta inhibits human airway epithelial proliferation in intact bronchial tissue. Am J Pathol 163, 533-542. Foon KA, Yang XD, Weiner LM, Belldegrun AS, Figlin RA, Crawford J, Rowinsky EK, Dutcher JP, Vogelzang NJ, Gollub J, Thompson JA, Schwartz G, Bukowski RM, Roskos LK, and Schwab GM (2004) Preclinical and clinical evaluations of ABX-EGF, a fully human anti-epidermal growth factor receptor antibody. Int J Radiat Oncol Biol Phys 58, 984-990. Friedlander M, Brooks PC, Shaffer RW, Kincaid CM, Varner JA, and Cheresh DA (1995) Definition of two angiogenic pathways by distinct alpha v integrins. Science 270, 15001502. Gabarra-Niecko V, Schaller MD, and Dunty JM (2003) FAK regulates biological processes important for the pathogenesis of cancer. Cancer Metastasis Rev 22, 359-374. Gadbois DM, Bradbury EM, and Lehnert BE (1997) Control of radiation-induced G1 arrest by cell-substratum interactions. Cancer Res 57, 1151-1156. Garcia-Barros M, Paris F, Cordon-Cardo C, Lyden D, Rafii S, Haimovitz-Friedman A, Fuks Z, and Kolesnick R (2003) Tumor response to radiotherapy regulated by endothelial cell apoptosis. Science 300, 1155-1159. Giancotti FG, and Ruoslahti E (1999) Integrin signaling. Science 285,1028-1032. Hallahan D, Geng L, Qu S, Scarfone C, Giorgio T, Donnelly E, Gao X, and Clanton J (2003) Integrin-mediated targeting of drug delivery to irradiated tumor blood vessels. Cancer Cell 3, 63-74. Hanahan D, and Weinberg RA (2000) The hallmarks of cancer. Cell 100, 57-70. Hannigan GE, Leung-Hagesteijn C, Fitz-Gibbon L, Coppolino MG, Radeva G, Filmus J, Bell JC, and Dedhar S (1996) Regulation of cell adhesion and anchorage-dependent growth by a new #1-integrin-linked protein kinase. Nature 379, 9196. Hansen MS, and Clemmensen I (1982) A fibronectin-binding glycoprotein from human platelet membranes. Biochem J 201, 629-633. Hatjikondi O, Ravazoula P, Kardamakis D, Dimopoulos J, and Papaioannou S (1996) In vivo experimental evidence that the nitric oxide pathway is involved in the X-ray-induced antiangiogenicity. Br J Cancer 74, 1916-1923. Henning T, Kraus M, Brischwein M, Otto AM, and Wolf B (2004) Relevance of tumor microenvironment for progression, therapy and drug development. Anticancer Drugs 15, 7-14. Hieken TJ, Farolan M, Ronan SG, Shilkaitis A, Wild L, and Das Gupta TK (1996) Beta3 integrin expression in melanoma predicts subsequent metastasis. J Surg Res 63, 169-73. Hodivala-Dilke KM, McHugh KP, Tsakiris DA, Rayburn H, Crowley D, Ullman-Cullere M, Ross FP, Coller BS, Teitelbaum S, and Hynes RO (1999) Beta3-integrin-deficient mice are a model for Glanzmann thrombasthenia showing placental defects and reduced survival. J Clin Invest 103, 229-238. Hofbauer LC, and Heufelder AE (2001) Role of receptor activator of nuclear factor-kappaB ligand and osteoprotegerin in bone cell biology. J Mol Med 79, 243-253. Hynes RO (2002) Integrins: Bidirectional, allosteric signaling machines. Cell 110, 673-687. Ingber DE (1992) Extracellular matrix as a solid-state regulator in angiogenesis: identification of new targets for anti-cancer therapy. Semin Cancer Biol 3, 57-63.
Jacks T, and Weinberg RA (2002) Taking the study of cancer cell survival to a new dimension. Cell 111, 923-925. Kasahara T, Koguchi E, Funakoshi M, Aizu-Yokota E, and Sonoda Y (2002) Antiapoptotic action of focal adhesion kinase (FAK) against ionizing radiation. Antioxid Redox Signal 4, 491-499. Kinbara K, Goldfinger LE, Hansen M, Chou FL, and Ginsberg MH (2003) Ras GTPases: integrins' friends or foes? Nat Rev Mol Cell Biol 4, 767-776. Kumar CC, Malkowski M, Yin Z, Tanghetti E, Yaremko B, Nechuta T, Varner J, Liu M, Smith EM, Neustadt B, Presta M, and Armstrong L (2001) Inhibition of angiogenesis and tumor growth by SCH221153, a dual alpha(v)beta3 and alpha(v)beta5 integrin receptor antagonist. Cancer Res 61, 2232-2238. Lewis JM, Truong TN, and Schwartz MA (2002) Integrins regulate the apoptotic response to DNA damage through modulation of p53. Proc Natl Acad Sci USA 99, 3627-3632. Luna J, Tobe T, Mousa SA, Reilly TM, and Campochiaro PA (1996) Antagonists of integrin alpha v beta 3 inhibit retinal neovascularization in a murine model. Lab Invest 75, 56373. Meineke, Gilbertz KP, Schilperoort K, Cordes N, Sendler A, Moede T, and van Beuningen D (2002) Ionizing radiation modulates cell surface integrin expression and adhesion of COLO-320 cells to collagen and fibronectin in vitro. Strahlenther Onkol 178, 709-714. Moro L, Dolce L, Cabodi S, Bergatto E, Erba EB, Smeriglio M, Turco E, Retta SF, Giuffrida MG, Venturino M, GodovacZimmermann J, Conti A, Schaefer E, Beguinot L, Tacchetti C, Gaggini P, Silengo L, Tarone G, and Defilippi P (2002) Integrin-induced epidermal growth factor (EGF) receptor activation requires c-Src and p130Cas and leads to phosphorylation of specific EGF receptor tyrosines. J Biol Chem 277, 9405-9414. Mousa SA (2002) Anti-integrin as novel drug-discovery targets: Potential therapeutic ad diagnostic implications. Curr Opin Chem Biol 6, 534-541. O´Connor PM (1997) Mammalian G1 and G2 phase checkpoints. Cancer Surv 29, 151-182. Orian-Rousseau V, Aberdam D, Rousselle P, Messent A, Gavrilovic J, Meneguzzi G, Kedinger M, and SimonAssmann P (1998) Human colonic cancer cells synthesize and adhere to laminin-5. Their adhesion to laminin-5 involves multiple receptors among which is integrin alpha2beta1. J Cell Sci 111, 1993-2004. Park CC, Bissell MJ, and Barcellos-Hoff MH (2000) The influence of the microenvironment on the malignant phenotype. Mol Med Today 6, 324-9. Persad S, and Dedhar S (2003) The role of integrin-linked kinase (ILK) in cancer progression. Cancer Metastasis Rev 22, 375-384. Plasswilm L, Hoper J, Cordes N, and Tannapfel A (1999) Investigation of microvessel density after irradiation. Radiat Res 151, 454-460. Rose RW, Grant DS, Oâ&#x20AC;&#x2122;Hara MD, and Williamson SK (1999) The role of laminin-1 in the modulation of radiation damage in endothelial cells and differentiation. Radiat Res 152, 1428. Schaller MD (2001) Biochemical signals and biological responses elicited by the focal adhesion kinase. Biochem Biophys Acta 1540, 1-21. Schlaepfer DD, Broome MA, and Hunter T (1997) Fibronectinstimulated signaling from a focal adhesion kinase-c-Src complex: involvement of the Grb2, p130cas, and Nck adaptor proteins. Mol Cell Biol 17, 1702-1713. Schwartz MA (1997) Integrins, oncogenes, and anchorage independence. J Cell Biol 139, 575-578.
277
Lisiak and Cordes: Integrins and the cellular radiation response Schwartz MA (2001) Integrin signaling revisited. Trends Cell Biol 12, 466-470. Sethi T, Rintoul RC, Moore SM, MacKinnon AC, Salter D, Choo C, Chilvers ER, Dransfield I, Donnelly SC, Strieter R, and Haslett C (1999) Extracellular matrix proteins protect small cell lung cancer cells against apoptosis: a mechanism for small cell lung cancer growth and drug resistance in vivo. Nature Med 5, 662-668. Sonveaux P, Brouet A, Havaux X, Gregoire V, Dessy C, Balligand JL, and Feron O (2003) Irradiation-induced angiogenesis through the up-regulation of the nitric oxide pathway: implications for tumor radiotherapy. Cancer Res 63, 1012-1019. Truong T, Sun G, Doorly M, Wang JY, and Schwartz MA (2003) Modulation of DNA damage-induced apoptosis by cell adhesion is independently mediated by p53 and c-Abl. Proc Natl Acad Sci USA 100, 10281-10286. Tucker GC (2002) Inhibitors of integrins. Curr Opin Pharmacol 2, 394-402. Tucker GC (2003) Alpha v integrin inhibitors and cancer therapy. Curr Opin Investig Drugs 4, 722-731. Wang XQ, Sun P, and Paller AS (2001) Inhibition of integrinlinked kinase/protein kinase B/Akt signaling: mechanism for ganglioside-induced apoptosis. J Biol Chem 276, 4450444511. Wary KK (2004) Molecular targets for anti-angiogenic therapy. Curr Opin Mol Ther 6, 54-70. Watt FM (2002) Role of integrins in regulating epidermal adhesion, growth and differentiation. EMBO J 21, 19-26.
Wild-Bode C, Weller M, Rimner A, Dichgans J, and Wick W (2001) Sublethal irradiation promotes migration and invasiveness of glioma cells: implications for radiotherapy of human glioblastoma. Cancer Res 61, 2744-2750. Yamada KM, and Even-Ram S (2002) Integrin regulation of growth factor receptors. Nat Cell Biol 4, E75-E76. Zahir N, and Weaver VM (2004) Death in the third dimension: apoptosis regulation and tissue architecture. Curr Opin Genet Dev 14, 71-80.
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Cancer Therapy Vol 2, page 279 Cancer Therapy Vol 2, 279-290, 2004
Potential application of desmopressin as a perioperative adjuvant in cancer surgery. Biological effects, antitumor properties and clinical usefulness Review Article
Daniel E. Gomez*, Giselle V. Ripoll, Santiago Gir!n and Daniel F. Alonso Laboratory of Molecular Oncology, Department of Science and Technology, Quilmes National University, Bernal, Buenos Aires, Argentina
__________________________________________________________________________________ *Correspondence: Dr. Daniel E. Gomez, R. S!enz Peâ&#x20AC;˘a 180, Bernal B1876BXD Buenos Aires, Argentina; Phone: +54 11 4365-7100 extension 171; Fax: +54 11 4365-7132; e-mail: degomez@unq.edu.ar Key words: Desmopressin, coagulation, fibrinolysis, surgery, breast cancer, metastasis Abbreviations: Desmopressin (DDAVP); extracellular matrix (ECM); tissue-type plasminogen activator (tPA) Received: 15 July 2004; Accepted: 27 July 2004; electronically published: September 2004
Summary Desmopressin (1-deamino-8-D-arginine vasopressin), the synthetic derivative of the antidiuretic hormone, is a welltolerated and convenient haemostatic drug that can be used in a number of clinical conditions with bleeding diathesis. It has several effects on the haemostatic and fibrinolytic system, causing release of coagulation factor VIII, von Willebrand factor and tissue-type plasminogen activator, among others. In this work we review the biological effects exerted by desmopressin and analyze the clinical experience of its use, to show its clinical relevance and safety. Also, we present a growing body of preclinical evidence indicating that adjuvant desmopressin therapy may impair the spread of cancer cells. Perioperative treatment with desmopressin dramatically reduced lymph node involvement and lung metastasis in an animal model of tumor manipulation. Available experimental evidence indicates antitumor effects of desmopressin in breast cancer, and similar benefits in other aggressive solid tumors are expected. The potential dual role of desmopressin in surgical oncology, reducing blood loss and limiting tumor recurrence or metastasis, warrants further investigation. If similar findings are obtained in humans, pharmacologic modulation of hemostasis and fibrinolysis using desmopressin should become a priority in the management of cancer patients undergoing surgery.
dose of 2-20 Âľg, DDAVP has a plasma half-life between 50 and 160 min. When administered by intranasal route, the half-life is about of 90 min. Although DDAVP is absorbed orally, the doses needed to reach an antidiuretic effect are much higher than the ones needed using the endovenous route. Metabolization of DDAVP is carried out in liver and kidney but slower than vasopressin. Approximately 60% of the compound is released by the kidney without metabolization (Richardson and Robinson 1985).
I. Introduction Peptide hormones released at the neurohypophysis are derived from neurophysins, and display a wide spectrum of biological properties. Oxytocin induces milk ejection and contraction of the uterus, while vasopressin is mainly involved in water balance, causing antidiuresis and increase in blood pressure (North 1987). As shown in Figure 1, the peptide sequence of vasopressin includes 9 aminoacid residues, having a disulfide bridge between positions 1 and 6. Desmopressin (DDAVP, 1-deamino-8-D-arginine vasopressin) is a synthetic analog of vassopresin described for the first time during the sixties (Zaoral et al, 1967). With homocystein deamination in sequence position 1 the antidiuretic effect is prolonged and the substitution of Darginine for L-arginine in position 8 decreases the pressor effect of the molecule (Figure 1). After an endovenous
II. Biological effects of DDAVP A. Receptors In contrast to vasopressin, which binds to different cell membrane receptors (V1a, V1b, V2 and V3), DDAVP is a selective agonist for the V2 receptor. This vasopressin
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Gomez et al: Potential application of desmopressin in cancer surgery Figure 1. Chemical structure of the nonapeptide hormone vasopressin. The synthetic analog DDAVP differs from the natural hormone by deamination of homocystein at position 1 and Darginine substitution at position 8 (arrowheads).
2. Increase of plasma levels of coagulation factor VIII
receptor subtype is expressed in the kidney collecting duct and mediates the antidiuretic effect of the hormone (Kaufmann et al, 2003a). The V2 receptor is also expressed in endothelial cells (Kaufman et al, 2003b), mediating most of the non-renal effects of DDAVP. Interestingly, the presence of vasopressin receptors was reported in transformed epithelial cells, and also documented in several tumor variants, including breast and lung cancer (North 2000). In addition, neuropeptide receptor expression was detected in different human tumor cell lines (Petit et al 2001).
DDAVP induces an increase in plasma levels of coagulation factor VIII, a cofactor of activated coagulation factor IX, responsible for the activation of factor X of the intrinsic coagulation pathway, leading to the formation of a fibrin clot. The effect of DDAVP on circulating levels of coagulation factor VIII remains poorly understood. The plasma level of any substance results from the balance between production and removal. Thus, DDAVP could induce factor VIII release from its producing cells. Alternately, factor VIII could be protected from proteolytic degradation, by DDAVP-induced increase in plasma von Willebrand factor, as explained above.
B. Renal effects In the kidney collecting duct, DDAVP activates V2 receptors and causes water retention by inducing the translocation of the water channel aquaporin-2 from intracellular stores to the apical plasma membrane, an example of cAMP-mediated exocytosis (Topal et al, 2003).
3. Release activator (tPA)
of
tissue-type
plasminogen
The role of DDAVP in fibrinolysis was one of the first effects described. The profibrinolytic activity of DDAVP is due to an increase in tPA, a proteolytic enzyme that converts plasminogen to plasmin and thus initiates fibrin degradation. The vascular endothelium is thought to be the main source of plasma tPA. In cultured endothelial cells, tPA is expressed at low levels. Its synthesis is upregulated, usually at the transcriptional level, in response to fluid shear stress, thrombin, histamine, retinoic acid, vascular endothelial growth factor and sodium butyrate. In addition, there is both in vivo and in vitro evidence that tPA is acutely released from preformed stores. A rapid increase in plasma tPA levels is observed in response to DDAVP, as well as beta-adrenergic agents administered systemically (Wall et al, 1998). Co-localization of von Willebrand factor and tPA in the same compartment could account for the coordinate effect of DDAVP on the plasma level of the two proteins. The identification of a storage compartment for tPA, distinct from Weibel-Palade bodies, remains unexplained (Emeiss et al, 1997).
C. Other effects 1. Release of von Willebrand factor The von Willebrand factor is a large glycoprotein playing a role in primary haemostasis, by mediating adhesion of platelets to the subendothelium. It also functions as a carrier protein for coagulation factor VIII, protecting it from proteolytic degradation. The von Willebrand factor is synthesized as a precursor protein in endothelial cells and megakaryocytes. This precursor undergoes dimerization, glycosylation, proteolytic cleavage into von Willebrand factor, and propeptide assembly of the dimmers into large multimers (500-15,000 kDa). Multimerized von Willebrand factor, together with equimolar amounts of propeptide, is stored in specialized secretory granules called Weibel-Palade bodies (Kaufmann et al, 2003a). DDAVP-induced secretion of von Willebrand factor results from V2 receptor-mediated, cAMP-dependent exocytosis from Weibel-Palade bodies. Some patients treated with repetitive doses of DDAVP during short periods present a progressive decrease in the response of coagulation factor VIII and of von Willebrand factor. Probably, this fact is related with a negative feed-back in the receptors of endothelial cells. Another interesting fact is that taquifilaxia does not occur regarding the release of catecholamines, tPA or urokinase (Vicente et al, 1991).
4. Vasodilation DDAVP is known to have vasodilator properties, as shown by an increase in heart rate and a decrease in systolic and diastolic blood pressure, as well as facial flushing (Derkx et al, 1983). Perfusion studies have demonstrated that vasopressin and DDAVP exert a direct vasodilator effect after intraarterial administration by a mechanism dependent of nitric oxide (Hayoz et al, 1997). These observations suggest a direct activation of
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Cancer Therapy Vol 2, page 281 endothelial nitric oxide synthase in the skeletal muscle vasculature, in a V2 receptor-dependent, cAMP-mediated manner.
Registered thrombotic episodes are few. An interesting review analyzed the number of people treated between 1985 and 1988, estimated in approximately 433,000, and the number of published thrombotic episodes was 10. The author concludes that the prothrombotic risk was of 0.0001% (Rodheghiero et al, 1991).
5. Expression of P-selectin The adhesion molecule P-selectin is expressed in both endothelial cells and megakaryocytes/platelets, in Weibel-Palade bodies and "-granules, respectively (McEver et al, 1989). Kanwar et al demonstrated that DDAVP induced a significant but transient increase in Pselectin expression on human umbilical vein endothelial cells, as well as on rat and human platelets. Earlier studies have shown that endothelial cell expression of P-selectin is important for the very early leukocyte-endothelial cell interaction, known as leukocyte rolling, an absolute prerequisite for leukocyte adhesion and migration (Kanwar et al, 1995). As blood monocytes have been identified as a target for DDAVP, Pereira et al demonstrated that DDAVP enhanced the ability of blood monocytes to bind activated platelets, mainly by increasing the expression of P-selectin sialylated ligands on the monocyte surface (Pereira et al, 2003).
IV. Clinical use DDAVP was primarily used for its antidiuretic properties in the treatment of conditions such as central diabetes insipidus and enuresis. In 1977 DDAVP was used for the first time to treat patients with hemophilia A and von Villebrand disease, the most frequent congenital bleeding disorders. The clinical indications for DDAVP quickly expanded beyond these diseases. We will analyze its clinical utility, in order to provide evidence about the widespread use and good tolerance.
A. Diabetes insipidus Diabetes insipidus is an uncommon condition characterized by polyuria and polydipsia. The symptoms and biochemical changes of this condition result from either a lack of the antidiuretic hormone vasopressin, or renal insensitivity to its effect. Failure to produce or release the hormone may result from cranial pathology. The renal insensitivity to vasopressin that occurs in patients with nephrogenic diabetes insipidus may be caused by genetic factors, drugs (especially lithium) or specific disease processes. Patients may compensate for polyuria and nocturia by excessive water intake but show marked decreases in urine specific gravity and osmolality. Patients with severe and uncompensated symptoms develop dehydration, neurological symptoms and encephalopaty. Vasopressin “replacement” with DDAVP is the treatment of choice in patients with cranial diabetes insipidus, although extreme caution is required when treating babies or small children because of the danger of fluid overload. The treatment of nephrogenic diabetes insipidus is difficult and typically involves therapy with a diuretic such as chlorothiazide, as well as indomethacin. These agents enhance urine osmolality by their renal effect on solute and water handling (Cheetham et al, 2002).
6. Release of catecholamines Researchers have shown that intravenous DDAVP (0.3 µg/kg) increased 2-fold the plasma levels of norepinephrine (Grant et al, 1988). Concomitantly, other authors have demonstrated that central and peripheral administration of DDAVP increase locomotor activity in rats in doses that alter brain dopamine neurochemistry. By using different catecholamine manipulating agents, they reported that the central stimulatory action of DDAVP involves granula-mediated dopamine release and subsequent activation of dopamine receptors, and that alpha-adrenoceptors possibly also are involved (Di Michele et al, 1988).
III. Secondary effects The primary adverse reaction associated with DDAVP is hypotonic hyponatremia. Hyponatremia has been reported in adults treated with DDAVP for Von Willebrand#s disease and hemophilia, and in children and adults treated for enuresis (Shulman et al, 1990). Water intoxication is uncommon when the drug is used with proper precautions. The strong antidiuretic action of DDAVP has some potential problems that are negligible in adults and older children when water intake is restricted. In infants and small children under the age of 18 months, however, DDAVP should be used with caution in order to prevent water intoxication and electrolyte imbalance. Extreme caution should be exercised when the patients receive parenteral fluid substitution (Sutor, 1998). Other side effects observed in the treatment of bleeding disorders are mild and transient, including facial flushing, transient headache, increased pulse rate and drop in systolic blood pressure. They can be minimized when the dose is not exceeding 0.3 µg/kg body weight, and the infusion lasts at least 20 to 30 minutes.
B. Congenital hemostatic pathology Von Willebrand disease is an autosomal dominantly inherited hemorrhagic disease caused by a deficiency in von Willebrand factor. Most patients have a mild disease that may go undiagnosed until trauma or surgery. Symptomatic individuals manifest easy bruisability and mucosal surface bleeding. The goals of therapy consist of correcting the deficiencies in von Willebrand factor protein activity to above 50% of normal and coagulation factor VIII activity to levels appropriate for the clinical situation. DDAVP (0.3 µg/kg in endovenous saline infusion or 150 µg intranasally for adults) is the recommended treatment for type 1 von Willebrand disease, eliminating potential exposure to blood-borne pathogens that replacement therapies may contain. DDAVP administration should be avoided in most
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Gomez et al: Potential application of desmopressin in cancer surgery individuals with type 2B variant of the disease (Mannucci, 1997). Hemophilias are sex-linked recessive disorders. Hemophilia A is caused by a deficiency of coagulation factor VIII and hemophilia B is caused by a deficiency of factor IX. A deficiency of either of these two intrinsic coagulation pathway components result in inefficient and inadequate generation of thrombin, which cannot be circumvented or supplemented by a normal extrinsic pathway because of the strong modulatory effects of tissue factor pathway inhibitor. Severe cases are characterized by frequent spontaneous bleeding events in joints (hemarthrosis) and soft tissues, and by profuse hemorrhage with trauma or surgery. DDAVP is useful in patients with mild hemophilia A since an adecuate incremental rise in factor VIII activity can circumvent the use of clotting factor concentrates (De La Fuente et al, 1995). Regarding congenital trombocitopenias, the use of DDAVP varies according with the pathology. Storage pool disease is an autosomal dominant disorder, whereas platelet storage granules are decreased in number and/or content, presumably because of abnormal granule formation in megakaryocytes. The bleeding diathesis is mild and affects mostly women. With the deficiency in dense granules, platelets aggregate abnormally. Densegranule storage pool disease is also associated with several other congenital disorders, including oculocutaneous albinism in both the Hemansky-Pudlak and ChĂŠdiakHigashi syndromes. The Bernard-Soulier syndrome, is an autosomal recessive disorder caused by a deficiency of a platelet membrane glycoprotein complex. As a result, giant platelets appear in the peripheral blood smear. Physiologically, platelets fail to adhere normally to subendothelial connective tissue because of defective binding of Von Willebrand factor. Glanzmann#s thrombasthenia, is an autosomal recessive bleeding disorder characterized by a prolonged bleeding time and platelets that fail to aggregate normally when stimulated. Some patients with storage pool disease are responsive to DDAVP administration (Nieuwenhuis et al, 1988). Furthermore, in the Hermansky-Pudlak syndrome the use of ristocetin and collagen with DDAVP produce a shortening in the bleeding time and improve platelet aggregation (Wijermans et al, 1989). In the rest of the pathology analysed although use of DDAVP has improved bleeding time and aggregation, there are no parameters that allow to foresee whether a patient will be responsive or not to DDAVP infusion.
congenital disease, but responses are unpredictable (Tefferi et al, 1997).
D. Acquired inhibitors of factor VIII Autoantibody inhibitors occur spontaneously in individuals with previously normal hemostasis. Although approximately 50% of the cases have no obvious underlying etiology, the remainder is associated with autoimmune diseases, lymphoproliferative disorders, idiosyncratic drug associations or pregnancy. Patients typically have massive hemorrhagic disorders. Treatment usually includes replacement therapy, porcine factor VIII concentrate and immunosuppressive therapy with steroids and cytotoxic agents. There are some reports of patients with this pathology satisfactory treated with DDAVP (Cohen et al, 1996).
E. Renal failure Platelets function abnormally in patiens with renal failure. The uremic metabolites responsible for this disfunction are uncertain, but certain phenolic compounds that accumulate in uremia may inhibit platelet aggregation. Uremic bleeding is usually mucocutaneous and reflects abnormal platelet or vascular hemostatic functions. DDAVP is usually a good and safe alternative for profilaxis and treatment of hemorrhagic alterations associated with terminal uremia (Lens et al, 1988).
F. Hepatic failure Platelet function is sometimes abnormal in liver disease, but the mechanisms and the extent to which it contributes to bleeding are unclear. DDAVP has been reported to improve the bleeding time in these circumstances (Mannucci et al, 1986).
G. Drug-induced bleeding disorders DDAVP counteracts the effect on hemostasis of some antithrombotic drugs. It shortens the prolonged bleeding time of individuals taking widely used antiplatelet agents, such as aspirin and ticlopidine, and the prolonged bleeding time and activated partial thromboplastin time of patients receiving heparin. It also counteracts the antihemostatic effects of dextran, with no apparent impairment of the antithrombotic properties. Although, more clinical evidence is needed, DDAVP may provide an opportunity to control drug-induced bleeding without stopping treatment and perhaps avoiding recurrence or progression of thrombosis (Butler et al, 1993).
H. Blood saving agent in surgery
C. Acquired Von Willebrand disease
Several investigators have evaluated whether DDAVP was beneficial during surgical operations in which blood loss is large and for which multiple blood transfusions are needed. Open-heart surgery with extracorporeal circulation is the epitome of operations that warrant the adoption of blood-saving measures. Conflicting results using DDAVP in open-heart surgery were obtained and they might be due to the fact that most studies were of small size and had insufficient
Acquired Von Willebrand disease is a rare condition and usually occurs as a complication of autoimmune, myeloproliferative, or lymphoproliferative disorders. The acquired disease associated with neuroblastoma is secondary to proteolysis of Von Willebrand factor by tumor-secreted hyaluronidase. Abnormal multimeric composition of Von Willebrand factor is a hallmark of these syndromes. Treatment is similar to that for
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Cancer Therapy Vol 2, page 283 statistical power to detect true differences in blood loss. A meta-analysis of 17 randomized, double-bind, placebocontrolled trials, which included 1171 patients undergoing open heart surgery, has attempted to overcome this pitfall. Overall, DDAVP reduced postoperative blood loss by 9%. Although DDAVP had no blood-saving effect when the total blood loss was low, the compound seems to be beneficial in cardiac operations associated with blood loss larger than 1 liter (Cattaneo et al, 1998).
three sequential steps: adhesion of tumor cells to the basement membrane and extracellular matrix (ECM), disruption of the basement membrane by proteolytic digestion, and migration through the modified basement membrane (Liotta, 1986). A biological similitude between tumor invasion and neovascularization underlines a cooperative function of cancer cells and endothelial cells during tumor progression. Adhesion of tumor cells to the basement membrane involves specific anchoring glycoproteins of ECM, such as fibronectin, laminin and collagens, which bind to a variety of tumor cell surface receptors. To penetrate ECM, the invading cells must disrupt local segments in the organized structure of the basement membrane, a tightly regulated process involving proteolytic enzymes. Once the tumor cells enter the stroma, they can easily gain access to lymphatic and blood vessels for further dissemination. Four major classes of proteases are important in the invasion process: serine, aspartyl, cysteinyl, and metal iondependent proteases. Many subclasses of metalloproteases have been described, including interstitial collagenase, type IV collagenases, and stromelysin. There is evidence that tumor cells elaborate different types of proteases, which together with proteases expressed by surrounding host cells such as endothelial cells, fibroblasts and inflammatory cells, are capable of degrading the complex network of ECM barries (Thorgeirsson et al, 1994). Tumor invasion and metastasis require active cell motility, not only for the endothelial cells in the process of angiogenesis but also for the tumor cells. Migration is initiated by pseudopodia, followed by translocation of the entire cell. The locomotion involves assembly and disassembly of cross-linked actin filaments, govern by specific cell signals (Gomez et al, 1999). Once the tumor cells gain access to a blood vessel, it is ready to circulate into the blood and reach distant sites.
I. Enuresis and nocturia Nocturnal enuresis is a prevalent clinical problem in childhood and adolescence. It is a heterogeneous disorder with various underlying mechanisms, causing a mismatch between the nocturnal bladder capacity and the amount of urine produced during sleep at night, in association with a simultaneous failure of conscious arousal in response to the sensation of bladder fullness. Children with increase nocturnal urine production usually have a good response to DDAVP therapy (Eggert et al, 2001). Patients with a small bladder almost invariably have various types of occult bladder dysfunction, but otherwise have a completely normal circadian rhythm of urine production. These patients generally have a poor response to DDAVP treatment, but would benefit more from combination therapy with enuretic alarm, urotherapy and antimuscarinic agents, in addition to DDAVP. Nocturia is also common in elderly men and women. The circadian rhythm of arginine vasopressin present in younger individuals, is lost in the elderly. The efficacy of DDAVP treatment (0.1 mg oral at bedtime) in patients of 65 years-old and older with nocturia was investigated and found safe and effective (Kwo, 2003).
V. DDAVP as a potential antitumor agent Having described the current clinical use, we will describe the process of invasion and metastasis, and then analyze the antitumor properties of DDAVP in preclinical animal models. The known body of literature about the compound will be related with the new evidence showing DDAVP as a potential perioperative adjuvant for cancer surgery.
B. Rheologic metastatic cell
characteristics
of
the
It has been long-recognized that although a large number of cancer cells may be released each day from primary tumors, comparatively few metastases develop from these cells. This â&#x20AC;&#x153;metastatic inefficiencyâ&#x20AC;? has been well-documented by observations on humans, and several experiments in animal models. A precise estimation of the inefficiency of circulating cancer cells in forming tumors, is obtained from counts of pulmonary colonies in mice after receiving tail-vein injections of metastatic tumor cell suspensions (Weiss et al, 1982). Even with highly aggressive transplantable tumors, efficiencies of less than 0.1% are common. When combined with cancer cell loss and delay associated with intravasation, this constitutes a high degree of operational metastatic inefficiency. Kinetic studies in mice point to the massive destruction of cancer cells in the microcirculation. As a result of interactions with microvessel walls, it appears that some tumor cells are killed relatively slowly, over minutes or hours by various arms of the inflammatory and/or immunologic response, whereas others are killed rapidly over seconds by mechanical damage (Weiss et al, 1983).
A. Biology of tumor cell invasion To form secondary growths, cancer cells at the primary tumor must invade the surrounding tissue, penetrate vessels, and travel to other sites where they arrest and resume growth (Figure 2). Metastasis is the major cause of mortality in cancer patients (Fidler, 1991). Of all the tumor cells that enter into the circulation, only 0.01% will survive to produce secondary tumors. Metastatic capacity depends in part on angiogenesis, a process by which the tumor induces the formation of new blood vessels, beginning with capillary buds and progressing to a vascular network. The new blood vessels within and around the tumor mass provide nutrients for tumor growth and create access to circulation for metastasis (Thorgeirsson et al, 1994). The invasion process can be classically divided into
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Gomez et al: Potential application of desmopressin in cancer surgery
Figure 2. Critical steps in tumor invasion and metastasis. 1) Intravasation from a primary growth. 2) Circulation in blood, whereas cells can be destroyed or protected in a tumor emboli. 3) Extravasation. 4) Secondary growth and angiogenesis.
Following this early evidence, recently Topal et al have demonstrated that aggregated colon cancer cells have a higher metastatic efficiency in the liver compared with non-aggregated cells. Hepatic metastases were observed in 81% of the rats after intraportal injection of aggregates equivalent to 0.5 x 106 cancer cells. A significant lower metastatic efficiency (16%) was found after the injection of the same number of non-aggregated cancer cells (Topal et al, 2003). Similar results were obtained by other authors who found that in contrast with viable single or nonaggregated cells that often fail to form metastases, tumor cell clumps result in a high metastatic efficiency after injection via the portal vein (Panis et al, 1992). Aggregated cancer cells may remain in large clusters of viable cells, and trapped in venous or arterial branches where they get attached to the endothelial cells. Here they may be able to evade host defense mechanisms and form secondary tumors. On the contrary, non-aggregated cancer cells may be unable to form clusters of viable cells and be challenged with mechanical forces and immune defenses. Metastatic tumor cells entering into the blood stream interact with components of the haemostatic system. This interaction results in fibrin deposition around tumor cells, determining the formation of microthrombi that increase the efficiency of the metastatic cascade (Constantini et al, 1992). Fibrin deposition may determine an enhanced intravascular tumor cell aggregation and trapping in the target organ, and also protects tumor cells from destruction by host immunity (Gunji et al, 1998). In this regard, we have reported an enhancement of lung colonization by
mammary tumor cells administering a synthetic inhibitor of the profibrinolytic enzyme urokinase during the first stages of metastasis formation (Alonso et al, 1996).
C. Effect of tumor manipulation on metastasis Although the metastatic process is highly inefficient, any release of tumor cells into the circulation should be avoided. It has been suggested that surgical manipulation can provoke liberation of viable cancer cells. The presence of cancer cells in the peripheral blood has been confirmed by reverse transcription-polymerase chain reaction in patients undergoing breast cancer surgery (Brown et al, 1995). Similarly, conventional chemotherapy may cause a mobilizing effect on cancer cells. Other authors have reported the recruitment of tumor cells into the peripheral blood after the first courses of primary chemotherapy in patients with breast cancer enrolled in a prospective study. (Sabbatini et al, 2000). Other authors reported the histological findings in a series of axillary lymph node dissections taken approximately 2 weeks after breast biopsy (Carter et al, 2000). They described the presence of epithelial cells in the subcapsular sinus of draining lymph nodes that may be attributed to mechanical transport of tumor breast epithelium secondary to the previous needle or surgical manipulation. Recently, Moore et al investigated by immunohistochemical staining whether the pattern of sentinel lymph node metastasis in breast cancer is related to tumor manipulation. Interestingly, the data suggested 284
Cancer Therapy Vol 2, page 285 that the frequency of positive nodes is increased after instrumentation of the tumor site (Moore et al, 2004). Several experimental studies with animal models have confirmed that intrabdominal tumor manipulation was the main factor acting on metastatic dissemination using conventional laparotomy or laparoscopy (Mutter et al, 1999). It has been shown that port site tumor recurrence rates decreased with increased surgical experience in a mouse adenocarcinoma model of laparoscopic splenectomy, suggesting that a poor surgical technique was the main cause of recurrence (Lee et al, 2000). In the same line, interesting results were obtained in an experimental model of breast cancer. Syngeneic mice were inoculated into the mammary fat pad with TA3Ha adenocarcinoma cells and the resulting tumors were surgically excised with a curative intent. Under these conditions, perioperative chemotherapy with doxorubicin reduced local recurrence, axillary metastasis, and lung metastasis, and also improved disease-free survival (Murthy et al, 1996).
obtained with administration of DDAVP 24 h after tumor cell inoculation (Alonso et al, 1999). Interestingly, in vitro pretreatment of tumor cells with comparable concentrations of DDAVP followed by peptide washout did not reduce the incidence of lung colonies, ruling out the possibility that DDAVP was mediating its antimetastatic activity through a direct effect on tumor cells. Extrapulmonary tumor colonies were not found in any of the control mice or mice treated with DDAVP. Our experiments suggested for the first time that adjuvant DDAVP therapy can impair successful implantation of circulating cancer cells.
3. Effects on tumor manipulation and surgical excision Considering the antimetastatic effect of DDAVP in animal studies, as well as its well-known hemostatic and profibrinolytic properties, the compound is an excellent candidate for adjuvant therapy both during and immediately after tumor surgery. Therefore, we investigated the effect of DDAVP on lymph node and lung metastasis, using a preclinical mouse mammary carcinoma model of subcutaneous tumor manipulation and surgical excision. We developed an experimental instrument for the application of controlled pressures on subcutaneous tumors. It consists on a mobile platform that transmits pressure though an axis to a small surface of 6 cm2. The platform is loaded with the appropriate weight and the instrument discharges a stable and controlled pressure on the tumor mass. Tumor-bearing mice were anesthetized and subcutaneous tumors subjected to experimental manipulations using pressures of 0.5 kg/cm2 during 2 min. To examine the antimetastatic properties of DDAVP, subcutaneous tumors were subjected to 2 or 3 weekly experimental manipulations, followed by surgical excision. DDAVP was administered intravenously 30 min before and 24 h after each manipulation or surgery, at a dose of 2 Âľg/kg. At the end of the experiment, mice were sacrificed and necropsied (Giron et al, 2002). Tumor manipulation induced massive dissemination to axillary nodes and increased up to 6-fold the number of metastatic lung nodules. Perioperative treatment with DDAVP dramatically reduced regional metastasis. The incidence of lymph node involvement in manipulated animals was 12% with DDAVP therapy and 87% without treatment (Figure 3). Histopathological analysis of most axillary nodes from DDAVP-treated animals showed sinusal histiocytosis and no evidence of cancer cells. Histiocytic reaction of the regional lymph nodes is considered a strong indicator of antitumor resistance in patients with breast cancer (Loboda et al, 1982). In contrast, axillary nodes from control mice bearing manipulated mammary tumors and administered with the saline vehicle evidenced massive metastasis and lacked sinusal histiocytosis. As shown in Figure 3, metastatic lung nodules were also reduced about 65% in animals treated with DDAVP (Giron et al, 2002). Perioperative DDAVP apperead to be safe at the dosage employed, and antitumor resistance was obtained without overt toxic effects.
D. DDAVP effects on tumor spread in a breast cancer model 1. In vitro and ex vivo studies We have examined the effects of neuropeptide hormones on our mouse mammary carcinoma model F3II (Alonso et al, 1997). We reported that vasopressin and its synthetic derivative DDAVP can modulate tumor cell growth in vitro and the secretion of urokinase, a profibrinolytic enzyme involved in hematogenous metastasis. In this regard, enhancement of pericellular fibrinolysis may prevent coating of intravascular tumor emboli with fibrin, therefore decreasing the survival of tumor cells in the circulation (Alonso et al, 1996). The formation of multicellular aggregates of mammary tumor cells in the presence of plasma from control or DDAVP-treated mice was investigated in ex vivo assays. After a short time, control plasma induced a significant aggregation of the tumor cell suspension. Also, a clot was formed in tubes and tumor cell clumps were trapped in a fibrin gel matrix. In contrast, in the presence of plasma from DDAVP-treated mice, most of the mammary tumor cells remained as a single cell suspension (Alonso et al, 1999). DDAVP did not reduce cell viability of tumor cell suspensions at the doses employed. Similarly, semiconfluent monolayers were not affected after continue in vitro culture in the presence of DDAVP.
2. Inhibition of experimental colonization of mammary tumor cells
lung
We have examined the effects of DDAVP on experimental lung colonization of highly metastatic mammary tumor cells in syngeneic Balb/c mice. Coinjection of DDAVP (1-2 Âľg/kg body weight) at the time of endovenous inoculation of F3II carcinoma cells significantly inhibited the formation of experimental lung metastases. Similar results were obtained with the parental LM3 mammary adenocarcinoma cells. In both cases, the number of lung nodules was reduced up to 70% in DDAVP-treated mice. Inhibition of metastasis was also
285
Gomez et al: Potential application of desmopressin in cancer surgery Figure 3. Effect of perioperative DDAVP on lymph node and lung metastasis in mice bearing subcutaneous F3II mammary carcinoma subjected to repeated experimental manipulation. Each group represent the combined data of a minimum of 6 animals. *p<0.05 versus its respective control in manipulation plus saline group. Kruskal-Wallis test. **p<0.02 versus its respective control in manipulation plus saline group. Chi square test. *** p<0.01 versus its respective control in manipulation plus saline group. Chi square test.
In addition, DDAVP increases intravascular fibrinolysis, helping to dissolve the protective fibrin shield of circulating tumor cells and reducing tumor cell aggregation (Alonso et al, 1999). As mentioned above, fibrin deposition around cancer cells entering into the blood stream ameliorates cell survival and facilitates trapping in the target organ. In accordance, implantation of mammary tumor cells at sites of trauma in an experimental mouse model was inhibited by injection of profibrinolytic agents, such as streptokinase and recombinant tPA (Murthy et al, 1991). DDAVP effect is exerted in the early stages of metastasis, not only by inducing rapid encapsulation of residual tumor tissue and limiting the formation of intravascular tumor cell emboli, but also altering the interaction of cancer cells with endothelium (Table 1). For instance, DDAVP may modify tumor cell attachment at the target organ by altering P-selectin expression on endothelial cells (Kanwar et al, 1995) or platelets (Wun et al, 1995). DDAVP may also alter hemodynamics of blood flow or induce lysis of tumor cells through the production of nitric oxide from the microvasculature (Hirano, 1997). Furthermore, we cannot exclude direct biological effects of DDAVP on tumor cells during intravasation and formation of metastatic foci. It has been described that breast and small-cell lung cancer cells contain normal genes for all vasopressin receptors and express normal vasopressin V1a and V1b receptor proteins, plus both normal and abnormal forms of the V2 receptor (North, 2000).
4. Putative mechanisms of antitumor action The biological effects of DDAVP on both endothelial and tumor cells are complex, and further investigations will determine the precise mechanisms of antitumor action. Nevertheless, the hemostatic effect of DDAVP seems to be pivotal, since it improves and accelerates the postoperative healing process. In this context, local and distant recurrence of breast cancer may be because of the perioperative stimulation of residual cancer cells (Reid et al, 1997). The perioperative period is also characterized by immunosuppression that may predispose to tumor spread (Vallejo et al, 2003). Perioperative DDAVP may offer the opportunity to modulate the early wound environment and reduce locoregional cancer recurrence rates. Enhanced coagulation after tumor manipulation may contribute to a rapid encapsulation of residual tumor tissue, limiting intravasation of tumor cells. It is known that proangiogenic molecules are locally produced in response to wounding and cancer. Recently, very high local concentrations of angiogenic factors were detected in surgical wound fluid samples from breast cancer patients, suggesting that they may need to be antagonized using perioperative systemic or local therapy (Hormbrey et al, 2003). Other blood-saving agents have been used during cancer surgery. Administration of perioperative and postoperative tranexamic acid reduced the frequency of wound complications in women with breast cancer undergoing lumpectomy or mastectomy (Oertli et al, 1994). Similarly, intraoperative infusion of the hemostatic agent aprotinin, a nonspecific protease inhibitor, was associated with a significant survival benefit in patients underwent liver resection for colorectal cancer metastasis (Lentschener et al, 1999).
VI. Conclusions and perspectives DDAVP has been used in patients with diabetes insipidus and in a variety of bleeding disorders. DDAVP is a safe and effective hemostatic agent for use during surgery in patients with hemophilia or von Willebrand 286
Cancer Therapy Vol 2, page 287 mouse mammary tumor cells. Int J Oncol 10, 375-379. Alonso DF, Skilton G, Farina HG, De Lorenzo MS, Gomez DE (1999) Antimetastatic effect of desmopressin in a mouse mammary tumor model. Breast Cancer Res Treat 57, 271275. Brown DC, Purushotham AD, Birnie GD, George WD (1995) Deteccion of intraoperative tumor cell dissemination in patients with breast cancer using reverse transcription and polymerase chain reaction. Surgery 117, 95-101. Butler KD, Dolan SL, Talbot MD, Wallis RB (1993) Factor VIII and DDAVP reverse the effect of recombinant desulphatohirudin (CGP 39393) on bleeding in the rat. Blood Coagul Fibrinol 4, 459-464. Carter BA, Jensen RA, Simpson JF, Page DL (2000) Benign transport of breast epithelium into axillary lymph nodes after biopsy. Am J Clin Pathol 113, 259-265. Cattaneo M, Harris AS, Stromberg U, Mannucci PM (1995) The effect of desmopressin on reducing blood loss in cardiac surgery. A meta-analysis of double-blind, placebo-controlled trials. Thromb Haemost 74, 1064-1070. Cheetham T, Baylis PH (2002) Diabetes insipidus in children, patophysiology, diagnosis and management. Paediatr Drugs 4, 785-796. Cohen AJ, Kessler CM (1996) Acquired inhibitors. Baillieres Clin Haematol 9, 331-354. Constantini V, Zacharski LR (1992) The role of fibrin in tumor metastasis. Cancer Metastasis Rev 11, 283-290. De la Fuente B, Kasper CK, Rickles FR, Hoyer LW (1985) Response of patients with mild and moderate hemophilia A and von Willebrand disease to treatment with desmopressin. Ann Intern Med 103, 6-14. Derkx FH, Man in#t Veld AJ, Jones R, Reid JL, Schalekamp MA (1983) DDAVP (1-desamino-8-D-arginine vasopressin), an antagonist of the pressor action of endogenous vasopressin? J Hypertens Suppl 1, 58-61. Di Michele S M Ericson U Sillen JA Engel, Soderpalm B (1998) The role of catecholamines in desmopressin induced locomotor stimulation. J Neural Transm 105, 1103-1115. Eggert P, Chavez-Kattau K, Muller D (2001) Age-and weightrelated dosage of dDAVP in the treatment of primary noctural enuresis). Klin Padiatr 213 (6), 317-320. Emmeis JJ, van den Eijnden-Schrauwen Y, van den Hoogen CM, de Priester W, Westmuckett A, Lupu F (1997) An endothelial storage granule for tissue-type plasminogen activator. J Cell Biol 139, 245-56 Fidler IJ (1991) Cancer metastasis. Br Med Bull 47, 157-177. Giron S, Tejera AM, Ripoll GV, Gomez DE, Alonso DF (2002) Desmopressin inhibits lung and lymph node metastasis in a mouse mammary carcinoma model of surgical manipulation. J Surg Oncol 81, 38-44. Gomez DE, Skilton G, Alonso DF, Kazanietz MG (1999) The role of protein kinase C and novel phorbol ester receptors in tumor cell invasion and metastasis. Oncol Rep 6, 1363-1370. Grant MB, Guay C, Lottenberg R (1988) Desmopressin stimulates parallel norepinephrine and tissue plasminogen activator release in normal subjects and patients with diabetes mellitus. Thromb Haemost 59, 269-272. Gunji Y, Gorelik E (1998) Role of fibrin coagulation in protection of murine tumor cells from destruction by cytotoxic cells. Cancer Res 48, 5216-5221. Hayoz D, Weber R, Pechere A, Burnier M, Brunner HR (1997) Heterogeneous vascular response to vasopressin, radial artery versus forearm blood flow. J Hypertens 15, 35-41. Hirano S (1997) In vitro and in vivo cytotoxic effects of nitric oxide on metastatic cells. Cancer Lett 115, 57-62. Hormbrey E, Han C, Roberts A, McGrouther DA, Harris AL (2003) The relationship of human wound vascular endothelial growth factor (VEGF) after breast cancer surgery
disease. Antitumor properties of DDAVP in tumor models were obtained administering endovenous doses close to the ones previously used and proved enhanced antidiuretic or hemostatic effect (0.3-4 µg/kg). These doses have the advantage of being well characterized from a pharmacological point of view (Lethagen, 1994; Mannucci, 1997). Our preclinical observations strongly suggest the application of DDAVP as a perioperative adjuvant in cancer surgery. The potential dual role of DDAVP in surgical oncology, reducing blood loss and limiting tumor recurrence or metastasis, warrant further investigation. If similar findings are obtained in humans, pharmacologic modulation of hemostasis and fibrinolysis using DDAVP should become a priority in the management of cancer patients undergoing surgery. Surgical manipulation and tissue trauma enhance the growth and dispersement of many types of malignant cells. However, as wounds develop and healing is complete the surgical site becomes less favorable to tumor implantation (Murthy et al, 1989). Thus, local recurrence found in conjunction with widespread metastatic disease is likely to have been established by perioperative seeding rather than as a late phenomenon (Hormbrey et al, 2003). Available experimental evidence indicates antitumor effects of DDAVP in breast cancer, and similar benefits in other aggressive solid tumors are expected, such as prostate cancer, ovarian cancer, head and neck cancer, colorectal cancer, esophageal cancer, lung cancer, sarcomas, melanoma and central nervous system tumors. In this regard, a panel of synthetic peptide analogs has been developed in our laboratory in the search for improved efficacy in particular tumor variants. In the future, we will gain a better understanding of the complex biological events that occur during the perioperative period in cancer patients. Whichever the mechanisms of action involved, the hemostatic and profibrinolytic compound DDAVP appears as a new agent which could be able to act cooperatively with cancer surgery, as well as with other standard therapies, to reduce recurrences and improve survival of patients. It seems that perioperative treatment strategies will be a fruitful area for cancer research in the next years and deserve further clinical investigation.
Acknowledgements The authors want to thank Genesica for its useful advice and management of the intellectual property of the findings. This work was supported by the R&D Priority Grant Program from Quilmes National University (53A048) to D.E.G. and D.F.A. To Guillermo Skilton, in memoriam.
References Alonso DF, Bertolesi GE, Far£as EF, Gomez DE, Bal de Kier Joffé E (1996) Inhibition of fibrinolysis by a synthetic urokinase inhibitor enhances lung colonization of metastatic murime mammary tumor cells. Oncol Rep 3, 1055-1058. Alonso DF, Skilton G, Farias EF, Bal de Kier Joffe E, Gomez DE (1997) Modulation of growth and urokinase secretion by vasopressin and closely related nonapeptides in metastatic
287
Gomez et al: Potential application of desmopressin in cancer surgery to circulating VEGF and angiogenesis. Clin Cancer Res 9, 4332-4339. Kanwar S, Woodman RC, Poon MC, Murohara T, Lefer AM, Davenpeck KL, Kubes P (1995) Desmopressin induces endothelial P-selectin expression and leukocyte rolling in postcapillary venules. Blood 86, 2760-2766. Kauffmann JE, Iezzi M, Vischer UM (2003b) Desmopressin (DDAVP) induces NO production in human endothelial cells via V2 receptor- and cAMP-mediating signalling. J Thromb Haemos 1, 821-828. Kaufmann JE, Vischer UM (2003a) Cellular mechanisms of the hemostatic effects of desmopresin (DDAVP). J Thromb Haemos 1, 682-689. Kwo H-Ch (2002) Efficacy of desmopressin in treatment of refractory nocturia in patients older than 65 years. Urology 59, 485-489. Lee SW, Gleason NR, Bessler M, Whelan RL (2000) Port site recurrence rates in a murine model of laparoscopic splenectomy decreased with increased experience. Surg Endosc 14, 805-811. Lens XM, Caasals J, Oliva JA, Pascual R, Carri$ J, Mallafré JM (1988) Trastornos de la coagulaci$n en la insuficiencia renal, modificaciones por la desamino-8-D-arginina vasopresina. Med Clin (Barc) 90, 603-606. Lentschener C, Li H, Franco D (1999) Intraoperativelyadministered aprotinin and survival after elective liver resection for colorectal cancer patients. Fibrinol Proteol 13, 39-45. Lethagen S (1994) Desmopressin (DDAVP) and hemostasis. Ann Hematol 69, 173-180. Lethagen S (1997) Desmopressin-a haemostatic drug, state-ofthe-art review. Eur J Anaesthesiol 14 (Suppl 14), 1-9. Liotta LA (1986) Tumor invasion and metastasis-role of extracellular matrix. Cancer Res 46, 1-7. Loboda VI, Grinevich IA (1982) Sinus histiocytosis of the regional lymph nodes as an indicator of antitumor resistance in breast cancer. Arkh Patol 44, 45–49. Mannucci PM (1997) Desmopressin (DDAVP) in the treatment of bleeding disorders, the first 20 years. Blood 90, 25152521. Mannucci PM, Vicente V, Vianello L, Cattaneo M, Alberca I, Coccato MP, Faioni E Mari D (1986) Controled trial of desmopressin (DDAVP) in liver cirrosis and other conditions associated with prolonged bleeding time. Blood 67, 11481153. McEver RP, Beckstead JH, Moore KL, Marshall-Carlson L, Bainton DF (1989) GMP-140, a platelet alpha-granule membrane protein, is also synthesized by vascular endothelial cells and is localized in Weilbel-Palade bodies. J Clin Invest 84, 92-99. Moore KH, Thaler HT, Tan LK, Borgen PI, Cody HS (2004) Immunohistochemically detected tumor cells in the sentinel lymph nodes of patients with breast carcinoma, biologic metastasis or procedural artifact? Cancer 100, 929-934. Murthy MS, Goldschmidt RA, Rao LN, Ammirati M, Buchmann T, Scanlon EF (1989) The influence of surgical trauma on experimental metastasis. Cancer 64, 2035-2044. Murthy MS, Scanlon EF, Reid SE, Xang XF (1996) Pre-, peri-, and postoperative chemotherapy for breast cancer, Is one better than the other? J Surg Oncol 61, 273-277. Murthy MS, Summaria LJ, Miller RJ, Wyse TB, Goldschmith RA, Scanlon EF (1991) Inhibition of tumor implantation sites of trauma by plasminogen activators. Cancer 68, 17241730. Mutter D, Hajri A, Tassetti V, Solis-Caxaj C, Aprahamian M, Marescaux J (1999) Increased tumor growth and spread after laparoscopy vs laparotomy, Influence of tumor manipulation in a rat model. Surg Endosc 13, 365-370.
Nieuwenhuis HK, Sixma JJ (1988) 1-Desamino-8-d-arginine vasopressin (Desmopressin) shortens the bleeding time in storage pool deficiency. Ann Intern Med 198, 65-67. North WG (1987) Biosynthesis of vasopressin and neurophysins. In, Vasopressin, Principles and properties. D.M. Gash and G.J Boer (eds) Plenum Press, New York, pp175-209. North WG (2000) Gene regulation of vasopressin and vasopressin receptors in cancer. Exp Physiol 85, 27S-40S. Oertli D, Laffer U, Haberthuer F, Kreuter U, Harder F (1994) Perioperative and postoperative tranexamic acid reduces the local wound complication rate after surgery for breast cancer. Br J Surg 81, 856-859. Panis Y, Ribeiro J, Chrétien Y, Nordlinger B (1992) Dormant liver metastases, an experimental study. Br J Surg 79, 221223. Pereira A M Del Valle Honorato, Sanz C (2003) DDAVP enhances the ability of blood monocytes to form rosettes with activated platelets by increasing the expression of Pselectin sialylated ligands on the monocyte surface. Br J Haematol 120, 814-820. Petit T, Davidson KK, Lawrence RA, von Hoff DD, Izbicka E (2001) Neuropeptide receptor status in human tumor cell lines. Anticancer Drugs 12, 133-136. Reid SE, Kaufman MW, Murthy S, Scanlon EF (1997) Perioperative stimulation of residual cancer cells promotes local and distant recurrence of breast cancer. J Am Coll Surg 185, 290-306. Richardson DW, Robinson AG (1985) Desmopressin. Ann Intern Med 103, 228-239. Rodeghiero F, Castaman G, Manucci PM (1991) Clinical indications for desmopressin (DDAVP) in congenital and acquired von Willebrand disease. Blood Rev 5, 155-161. Sabbatini R, M Morselli F, Depenni M, Cagossi R, Luppi K, Torelli M, Silingardi G (2000) Detection of circulating tumor cells by reverse transcriptase polymerase chain reaction of maspin in patients with breast cancer undergoing conventional-dose chemotherapy. J Clin Oncol 18, 31963197. Shulman LH, Miller JL, Rose LI (1990) Desmopressin for diabetes insipidus, hemostatic disorders and enuresis. Am Fam Physician 42, 1051-1057. Sutor AH (1998) Desmopressin (DDAVP) in bleeding disorders of childhood. Semin Thromb Hemost 24, 555-566. Tefferi A, Nichols WL (1997) Acquired von Willebrand disease, concise review of occurrence, diagnosis, pathogenesis, and treatment. Am J Med 103, 536-540. Thorgeirsson UP, Lindsay CK, Cottam DW, Gomez DE (1994) Tumor invasion, proteolysis and angiogenesis. J Neuro Oncol 18, 89-103. Topal B, Roskams T, Fevery J, Penninckx F (2003) Aggregated colon cancer cells have a higher metastatic efficiency in the liver compared with non-aggregated cells, an experimental study. J Surg Res 112, 31-37. Vallejo R, Hord ED, Barna SA, Palma JS, Ahmed S (2003) Perioperative immunosuppression in cancer patients. J Environ Pathol Toxicol Oncol 22, 93-100. Vicente V, Laso J, Alberca I, Moraleda J, Estelles A, Aznar J (1991) Repeated infusions of DDAVP induce low response of VIII, C and vW, F but not of plasminogen activators (tPA and u-PA). Thromb Haemost 65, 977. Wall U, Jern S, Tengborn L, Jern C (1998) Evidence of a local mechanism for desmopressin-induced tissue-type plasminogen activator release in human forearm. Blood 91, 529-537. Weiss L, Mayhew E, G Rapp D, Holmes JC (1982) Metastatic inefficiency in mice bearing B16 melanomas. Br J Cancer 45, 55. Weiss L, Ward PM, Holmes JC (1983) Liver to lung traffic of
288
Cancer Therapy Vol 2, page 289 cancer cells. Int J Cancer 32, 79. Wijermans PW, Dorp DB (1989) Hermansky-Pudlak syndrome, Correction of bleeding time by 1-desamino-8D-argininve vasopressin. Am J Hematol 30, 154-157. Wun T, Paglieroni TG, Lachant NA (1995) Desmopressin stimulates the expression of P-selectin on human platelets in vitro. J Lab Clin Med 126, 401-409.
Zaoral M, Kole J, Sorm F (1967) Synthesis of 1-deamino-8-Damino-butyrine vasopressin, 1-deamino-8-D-lysine vasopressin and 1-deamino-8-D-arginine vaspressin. Collection Czechoslov Chem Commun 32, 1250-1257.
From left to right: Dr. Daniel E. Gomez, Dr. Giselle V. Ripoll, Dr. Santiago Gir$n, Dr. Daniel F. Alonso
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Cancer Therapy Vol 2, page 291 Cancer Therapy Vol 2, 291-296, 2004
A pilot clinical study of a combination of docetaxel and doxifluridine for the treatment of advanced/recurrent gastric cancer with prior chemotherapy Research Article
Hajime Kase*, Naoyasu Saito, Natsuki Tokura, Naohiro Washizawa, Makoto Kikuchi and Kazuo Kobayashi The First Department of Surgery, Toho University School of Medicine, 6-11-1 Omori-nishi, Ota-ku, Tokyo 143-8541, Japan
__________________________________________________________________________________ *Correspondence: Hajime Kase, The First Department of Surgery, Toho University School of Medicine, 6-11-1 Omori-nishi, Ota-ku, Tokyo 143-8541, Japan; Telephone: +81-3-3762-4151; Fax: +81-3-3298-4348; e-mail: hajimejjj@aol.com Key words: Docetaxel, doxifluridine, combination therapy, gastric cancer Abbreviations: best supportive care, (BSC); cisplatin, (CDDP); confidence interval, [CI]; docetaxel, (TXT); doxifluridine, (5’-DFUR); granulocyte colony stimulating factor, (G-CSF); Methotrexate, (MTX); National Cancer Institute common toxicity criteria version 2, (NCI-CTC ver.2); partial response, (PR); performance status, (PS); platelet-derived endothelial cell growth factor, (PD-ECGF); progression of disease, (PD); proximal margin, (PM); stabilization of the disease, (SD); thymidine phosphorylase, (dThdPase) Received: 23 July 2004; Accepted: 09 August 2004; electronically published: August 2004
Summary A pilot study was conducted to assess the activity and tolerability of a combination chemotherapy regimen of docetaxel (TXT) and doxifluridine (5’-DFUR) in patients with advanced/recurrent gastric cancer. Eligibility criteria for patients included the following: (1) histologically proven gastric cancer, (2) performance status of 2 or less, (3) age 75 years or younger, (4) one or more prior chemotherapy regimens, (5) adequate bone marrow, liver, renal, and cardiac functions, and (6) provision of written informed consent. The treatment consisted of TXT (60 mg/m2) on Day 1 and 5’-DFUR (600 mg/day) on Days 1 to 21, repeated every 4 weeks. Objective responses to chemotherapy in measurable or evaluable lesions were evaluated using standard World Health Organization criteria. Nine patients were enrolled in the study. The antitumor effect was evaluated in 8 of the 9 patients, excluding 1 patient who had no evaluable lesion. The overall response rate was 37.5% (3 of 8 patients, 95% confidence interval [CI], 8.5% to 75.5%) and the disease control rate (partial response and stabilization of disease) was 87.5% (7 of 8 patients, 95% CI, 47.4% to 99.7%). The median survival time was 531 days (19.0 months) for all patients. Grade 3 leukopenia was observed in 1 patient (11.1%), but grade 3 or 4 diarrhea, which is commonly encountered in patients undergoing chemotherapy, was not observed. Other adverse reactions were mild. No treatment-related deaths occurred. This combination chemotherapy regimen was active and well tolerated. The results suggest that this combination therapy is an appropriate regimen for future Phase 2 trials. who received the best supportive care (BSC). In most of the chemotherapy regimens for the treatment of advanced/recurrent gastric cancer in Japan, 5-FU is used as the base therapy and is combined with cisplatin (CDDP), with the response rate being reported to be 34.3 to 65.9% (Sasaki et al, 1997; Kusaba et al, 1999; Tsuji et al, 1999; Saji et al, 2002). However, the prognosis is poor in the case of gastric cancer for which the first-line chemotherapy is ineffective and no active second-line
I. Introduction In recent years, the survival rate for gastric cancer has been dramatically improved by early detection and curative surgery. However, the prognosis for patients with unresectable advanced/recurrent gastric cancer is still poor. Some randomized studies (Murad et al, 1993; Glimelius et al, 1994; Pyrhonen et al, 1995) have reported that the median survival time of patients with advanced/recurrent gastric cancer is 6 to 9 months in those who received chemotherapy and about 3 months in those
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Kase et al: Docetaxel and doxifluridine combination therapy for gastric cancer granulocyte colony stimulating factor (G-CSF) and antidiarrheal drugs.
therapy has been established. Therefore, the establishment of an effective second-line therapy is required. Docetaxel (TXT) is a taxoid that possesses an antitumor effect and is extracted from the needle leaves of the European yew tree (Taxus baccata). TXT exerts its antitumor activity by promoting the polymerization of microtubule proteins and inhibiting mitosis (Diaz and Andreu, 1993). In cases of gastric cancer, TXT has been shown to exhibit antitumor activity and has been used clinically. However, the efficacy of TXT as a monotherapy is insufficient, with the response rate being about 20% (Taguchi et al, 1998; Mai et al, 1999). Therefore, it is necessary to use TXT in combination with a drug with which a synergistic effect can be expected. However, in gastric cancer, there has been only one case report on a drug that showed efficacy in such a combination regimen (Sato et al, 2002), although a few clinical studies have been performed in the past. Therefore, the present study was conducted as a pilot study to confirm the activity and tolerability of the combination regimen of TXT and 5’DFUR in the treatment of gastric cancer.
C. Evaluation Objective responses to chemotherapy in measurable or evaluable lesions were evaluated using standard World Health Organization criteria. A survival curve was generated using the Kaplan-Meier method. For the evaluation of adverse reactions, the National Cancer Institute common toxicity criteria version 2 (NCI-CTC ver.2) was applied.
III. Results A. Patient characteristics Nine patients with gastric cancer were registered from June 2000 to June 2001 and evaluated for treatment response and adverse events. However, Patient No. 5, who was a resected but non-cured case judged positive for cancer infiltration at the resection margin [proximal margin (PM): +] after surgery, was not evaluated for treatment response because no evaluable lesion was detected on the CT image. Table 1 shows patient demographics. The age range of the patients at the start of treatment was 51 to 74 years (median: 66 years), and the number of males and females was 8 and 1, respectively. All of these patients were rated as 0 or 1 for PS. Histologically, poorly differentiated adenocarcinoma was observed in 4 patients (44.4%), tubular adenocarcinoma and mucinous adenocarcinoma in 2 patients (22.2%) each, and papillary adenocarcinoma in 1 patient (11.1%). The metastatic lesions in patients who had evaluable metastatic lesions were located in the liver (3 patients: 33.3%), lymph nodes (3 patients: 33.3%) and peritoneum (4 patients: 44.4%). The response of these patients to chemotherapy prior to registration in the present study was progression of the disease (PD) (Table 1). In addition, all patients underwent a cisplatin (CDDP) +5-fluorouracil (5-FU) regimen, and 5 underwent 2 regimens as prior therapy.
II. Materials and methods A. Eligibility criteria Patients registered for the present study were required to satisfy the following eligibility criteria: 1) presenting with resected but non-cured or recurrent histologically proven gastric cancer; 2) a performance status (PS) of 2 or less on the scale of the Eastern Clinical Oncology Group; 3) aged 75 years or younger: 4) one or more prior chemotherapy regimens which were completed at least 4 weeks before registration; 5) normal (adequate) bone marrow function (white blood cell count ! 4,000/µL, absolute neutrophil count ! 2,000/µL, platelet count ! 10,000/µL), liver function (serum bilirubin level " 2.0mg/dL, serum transaminase level " 3-fold the normal limit), renal function (serum creatinine level " 1.5mg/dL, blood urea nitrogen level " 25mg/dL, creatinine clearance ! 50ml/min); 6) normal cardiac function; 7) absence of any other medical condition; 8) absence of any other active tumor; and 9) provision of written informed consent before commencement of the study.
B. Response and survival Of 8 patients who had measurable or evaluable lesions, 3 patients were rated as exhibiting a partial response (PR), 4 patients as exhibiting stabilization of the disease (SD), and 1 patient as exhibiting progression of the disease (PD). Thus, the response rate was 37.5% (3 of 8 patients; 95% CI: 8.5% to 75.5%). The disease control rate (partial response and stabilization of disease) was 87.5% (7 of 8 patients; 95% CI: 47.4% to 99.7%). The response rates in patients who had metastatic lesions in the liver, lymph nodes and peritoneum were 33.3% (1 of 3 patients), 66.6% (2 of 3 patients) and 25% (1 of 4 patients), respectively (Table 2). The median survival time was 531 days for all patients, and the 1-year survival rate was 66.7% (Figure 1).
B. Treatment schedule On Day 1, TXT (60 mg/m2) was administered by intravenous injection over a period of more than 1 hour. On Day 1 to Day 21, doxifluridine (5’-DFUR) was administered orally at 600 mg/day. This therapy was repeated every 4 weeks as long as there was no progression of disease (PD), no refusal by a patient to continue the study or no unacceptable adverse reactions. In the event of the occurrence of any of the following in patients at the start of the subsequent treatment cycle, the administration of TXT and 5’-DFUR was to be postponed until the resolution of the adverse reaction: Leukopenia, neutropenia or thrombocytopenia of Grade 2 or higher; or diarrhea or infections of Grade 1 or higher. If the start of the next administration was delayed 1 week or more due to these adverse reactions, the therapy was to be discontinued. In the event of Grade 3 or 4 hematological adverse reactions or diarrhea, the administration of TXT and 5’-DFUR was to be suspended temporarily. After resolution of these reactions, the therapy was to be resumed with a reduction in the dose of TXT of 20%. Granisetron was routinely used before administration of TXT. No restriction was placed on the supportive care to be provided in the event of the occurrence of serious adverse events, including the use of
C. Adverse reactions The total number of treatment cycles was 107 (median: 10 cycles per patient; range: 4 to 18 cycles). Adverse reactions observed with this regimen are summarized in Table 3. Grade 3 leukopenia was observed in 1 patient (11.1%) and was resolved quickly by a single
Table 1. Patient demographics 292
Cancer Therapy Vol 2, page 293 Patient No.
Sex
Age
Site evaluated
Histological type
number of survival days course
1
m
64
Lymph node
por 1
18
2
f
62
Lymph node/peritoneum
tub 2
3
m
51
Lymph node
4
m
72
5
m
6
response
Previous tretment
840
SD
FP, MTX+5-FU
10
385
PR
FP
por 2
16
827
PR
FP (NAC), MTX+5-FU
Peritoneum
por 2
9
300
SD
FP
60
None
muc
16
757
NE
FP (NAC)
m
74
Peritoneum
por 2
4
124
PD
FP
7
m
66
Liver metastatic lesion/peritoneum
tub 2
8
294
SD
FP (NAC), 5-FU (HAI)
8
m
70
Liver metastatic lesion
muc
10
1011
PR
FP, MTX+5-FU, 5-FU (HAI)
9
m
71
Liver metastatic lesion
pap
16
531
SD
FP, 5-FU (HAI)
HAI: hepatic arterial infusion chemotherapy NAC: neoadjuvant chemotherapy FP: 5-FU plus cisplatin NE: not evaluated
Table 2. Response rate Response rates CR
PR SD 3 4 The degree classified by part for the response rate CR
PD 1
Total 8
PR
SD
Liver
1
2
3
Lymph nodes
2
1
3
Peritoneum
1
2
administration of G-CSF. Other hematological adverse reactions were mild. The most frequently observed non-hematological adverse reactions were alopecia (100%) and nail changes (88.9%). However, these reactions were rated as lower than Grade 3 and the patients were eligible to continue the study. Although diarrhea was observed in 4 patients (44.4%), it was rated as Grade 1 in all cases, and therefore there was no dose reduction or prolongation or cessation of treatment due to diarrhea. The diarrhea in these patients was resolved by the use of loperamide or other appropriate antidiarrheal drugs. Other non-hematological adverse reactions were mild. There was no cessation of treatment due to adverse reactions and no treatment-related deaths.
PD
1
Total
4
were able to receive the treatment on an outpatient basis as scheduled.
E. Case report The CT images (T3, N3, H1, P0, Cy0, Stage IV, and pap) of Patient No. 8 with multiple liver metastatic tumors, who was rated as exhibiting PD after prior therapy with CDDP+5-FU, Methotrexate (MTX)+5-FU and 5-FU (hepatic arterial infusion), are shown. After administration of TXT+5â&#x20AC;&#x2122;-DFUR, the liver metastatic lesion shrunk, and a PR was attained on the 4th treatment cycle (Figure 2).
IV. Discussion The present pilot study was conducted to assess the antitumor effect and tolerability of a combination chemotherapy regimen of docetaxel (TXT) and doxifluridine (5â&#x20AC;&#x2122;-DFUR) in patients with advanced/recurrent gastric cancer who failed to respond to
D. Dosage adjustments The patient who developed Grade 3 leukopenia was eligible to continue the treatment after reducing the dose of TXT by 20%. There was no case in which the treatment was delayed due to adverse reactions. Thus, all patients
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Kase et al: Docetaxel and doxifluridine combination therapy for gastric cancer Figure 1. The survival rate of all the patients (n=9)
Table 3. Adverse reactions* NCI-CTC Grade (no of patients) Adverse event
1
2
3
4
(%)
Grade3 and 4 (%)
Leukopenia
1
2
1
-
44.4
11.1
Neutropenia
-
3
-
-
33.3
-
Edema
1
2
-
-
22.2
-
Fervescence
2
-
-
-
22.2
-
Alopecia
4
5
-
-
100
-
Changes in nails
6
2
-
-
88.9
-
Skin
6
-
-
-
44.4
-
Anorexia
3
-
-
-
33.3
-
Diarrhea
4
-
-
-
44.4
-
Nausea/ vomiting
2
-
-
-
22.2
-
* NCI-CTC version 2
prior therapy. Patients with gastric cancer who fail to respond to the first-line chemotherapy regimen and for whom no second-line therapy has been established generally have a poor prognosis. In the present study, TXT was selected because it is an anti-cancer agent possessing a completely new mechanism of action, and because it is reported to have little cross-tolerance to currently used anti-cancer agents. In addition, 5’-DFUR was selected (Sawada et al, 1998) because it has been clinically demonstrated to produce a synergistic effect when used concomitantly with TXT in other cancers such as breast cancer.
The most likely mechanism by which the synergistic effect is produced is that TXT upregulates thymidine phosphorylase (dThdPase), an enzyme responsible for the metabolism of 5’-DFUR. dThdPase has been shown to exist more abundantly in tumor tissues than in normal tissues and has recently been found (Furukawa et al, 1992) to be identical to Platelet-derived endothelial cell growth factor (PD-ECGF), which is involved in neovascularization. 5’-DFUR is an intermediate metabolite of capecitabine, which is used globally for the treatment of large intestine cancer and breast cancer. 5’DFUR is a pro-drug that exerts its action following conversion to 5-FU by dThdPase, and its efficacy and
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Cancer Therapy Vol 2, page 295 report (Sato et al, 2002) on this combination regimen in gastric cancer. Therefore, the present study was necessary to confirm the activity and tolerability of the combination regimen of TXT and 5’-DFUR in gastric cancer. The overall response rate in the combination regimen was 37.5% (3 of 8 patients), and the disease control rate (partial response and stabilization of disease) was 87.5% (7 of 8 patients; 95% CI: 47.4% to 99.7%). In particular, 2 of 4 patients rated as exhibiting SD maintained this status for more than 6 months. In 3 patients with multiple liver metastatic tumors for whom hepatectomy was not applicable, the hepatic artery infusion therapy had to be discontinued because of catheter obstruction. Following treatment with the combination regimen, one of the 3 patients was rated as exhibiting a PR, and the remaining 2 patients were rated as exhibiting SD. Their survival times were also prolonged to 294 days, 1011 days and 531 days. This result suggests that the combination regimen is effective in patients for whom hepatic artery infusion therapy is not suitable. Two Japanese clinical studies (Taguchi et al, 1998; Mai et al, 1999) of TXT in the second-line management of advanced/recurrent gastric cancer reported the response rate of TXT monotherapy at 60 mg/m2 to be 20% and 22%, respectively. In a Phase II clinical study (Taguchi et al, 1985) of 5’-DFUR, the response rate was reported to be about 15.8%. Taking these data into consideration, the results from our study suggest that TXT and 5’-DFUR used in combination may produce a synergistic antitumor effect. In this study, the median survival time was 531 days. Thus, since the survival time was prolonged in these patients with advanced/recurrent gastric cancer who had undergone prior therapy, this combination regimen is considered to give a survival benefit. The adverse event with the highest incidence in this regimen was nail changes, such as perionychia, nail deformation and onychoptosis. In Phase II clinical studies (Taguchi et al, 1998; Mai et al, 1999) where TXT monotherapy at 60 mg/m2 was administered every 3 to 4 weeks to patients with gastric cancer, the subjective and objective adverse events observed included no adverse events related to nails. The incidence of adverse events related to nails was also low among the adverse events observed with 5’-DFUR. Therefore, the nail changes were considered to be adverse events specific to the concomitant use of TXT and 5’-DFUR. In the most serious case, a subungual abscess accompanied by pain was observed, followed by nail avulsion, but none of the changes resulted in cessation of treatment. For the resolution of these adverse events, it was necessary to use analgesics and antibiotics, and such treatment from the early stage following the onset of the adverse events was effective. The dose of 5’-DFUR was determined based on the Phase I study of Tominaga et al, (2001). However, in that study, one case of non-hematotoxic nausea/vomiting of Grade 2 and two cases of stomatitis of Grade 2 were observed, whose relation with the test drug was not completely ruled out. Therefore, the dose was set at 600 mg/day that is 25% lower than the usual dose of 800 mg/day, in order to reduce the incidence of adverse event
A: before treatment
B: after treatment Figure 2. The CT images of Patient No. 8 before and after the treatment with TXT+5'-DFUR. the solid vertical arrow indicates a stent in common bile duct, and the horizontal arrow indicates an obstructed infusion catherter. : Stent in common bile duct : Obstructed infusion catherter
safety in the treatment of solid cancers including gastric cancer have been recognized in Japan, Korea, and Italy, among other countries. Recent basic studies (Endo et al, 1999; Sato et al, 2002) have reported that several types of anticancer agent, including TXT, specifically upregulate dThdPase in tumor tissues, and that concomitant use of these agents with 5’DFUR/capecitabine can produce a synergistic effect. O’Shaughnessy et al, (2002) conducted a clinical study to compare TXT and TXT/capecitabine, and demonstrated the usefulness of the TXT/capecitabine combination regimen, with the response rate being 30% and 42%, and the MST being 11.5M and 14.5M, respectively. In Japan, Tominaga et al, (2001) conducted a Phase I study of the combination regimen of TXT and 5’-DFUR in advanced/recurrent breast cancer, and reported a synergistic effect with the combination regimen, with the response rate being 58.3%. However, although there are clinical reports on the combination regimen of TXT and 5’-DFUR in breast cancer, there is only one clinical case 295
Kase et al: Docetaxel and doxifluridine combination therapy for gastric cancer gastric cancer. Cancer 72, 37-41. O’shaughnessy J, Miles D, Vukelja S, Moiseyenko V, Ayoub JP, Cervantes G, Fumoleau P, Jones S, Lui WY, Mauriac L, Twelves C, Van Hazel G, Verma S, and Leonard R (2002) Superior survival with capecitabine plus docetaxel combination therapy in anthracycline-pretreated patients with advanced breast cancer, phase III trial results. J Clin Oncol 20, 2812-2823. Pyrhonen S, Kuritunen T, Nyandoto P and Kouri M (1995) Randomized comparison of fluorouracil, epidoxorubicin and methotrexate (FEMTX) plus supportive care with supportive care alone in patients with non-resectable gastric cancer. Br J Cancer 71, 587-91. Saji S, Toge T, Kurosu Y, Hirata K, Gochi A, Tominaga S, and Inokuchi (2002) Interim report of JFMC study no. 23--phase III randomized clinical trial on the effectiveness of low-dose cisplatin plus 5-FU as a postoperative adjuvant chemotherapy for advanced gastric cancer. Jpn J Cancer Chemother 29, 2499-2507. Sasaki K, Hirata K, Denno R, Oikawa I, Mukaidani M, Hiraike N (1997) Combination chemotherapy of continuous infusion 5fluorouracil and low-dose cisplatin in advanced gastrointestinal and lung adenocarcinoma. Jpn J Cancer Chemother 24, 959-964. Sato A, Shimada K, Nakamachi M, Ushio J, Yamamoto W, Kurihara M and Matsukawa M (2002) Effectiveness of doxifluridine (5'-DFUR)/doxitaxel against advanced/recurrent gastric cancer showing resistance to various anticancer drug regimens. Gastric Cancer 5, 233236. Sawada N, Ishikawa T, Fukase Y, Nishida M, Yoshikubo T, and Ishitsuka H (1998) Induction of thymidine phosphorylase activity and enhancement of capecitabine efficacy by taxol/taxotere in human cancer xenografts. Clin Cancer Res 4, 1013-1019. Taguchi T, Sakai K, Terasawa T, Irie K, Yamamoto M, Kawahara T, Satomi T, Tomita K, Yamaguchi A et al, (1985) Phase II study of 5'-DFUR (5'-deoxy-5-fluorouridine) by the Cooperative Study Group. Jpn J Cancer Chemother 12, 2179-2184. Taguchi T, Sakata Y, Kanamaru R, Kurihara M, Suminaga M, Ota J, and Hirabayashi N (1998) Late phase II clinical study of RP56976 (Docetaxel) in patients with advanced/recurrent gastric cancer, A Japanese cooperative study group trial (Group A). Jpn J Cancer Chemother 25, 1915-1924. Tominaga K, Nishimura R, Aoyama H, Iwase H, Mitsuyama S,. Asaga T, and Kimura M (2001) A phase I study of docetaxel (TXT) and doxifluridine (5'-DFUR) combination therapy in patients with advanced and recurrent breast cancer. Jpn J Cancer Chemother 28, 965-972. Tsuji A, Morita S, Horimi T, Takazaki M, Takahashi I, and Shirasaka T (1999) Chemotherapy with low-dose CDDP and continuous 5-FU for the treatment of advanced gastric cancers. Jpn J Cancer Chemother 26, 933-938.
such as diarrhea, since it was to be used in combination with TXT in gastric cancer treatment. Another consideration in setting the dose was that, by reducing the dose level used in the present study by 20 %, the chemotherapeutic dosage and administration could be performed on an outpatient basis. As a result, although the incidence of diarrhea is usually high among the adverse events observed with 5’-DFUR, diarrhea of Grade 1 was observed only in 4 patients in the present study. Therefore, the occurrence of diarrhea is not problematic, and the patients were able to continue the treatment. The incidence (11.1%) of leukopenia of Grade 3 or higher was relatively low and the condition was improved quickly following treatment with G-CSF. There was no case of infection. The leukopenia and neutropenia observed in the present study did not result in postponement or cessation of the subsequent treatment cycle with TXT and 5’-DFUR, and there were no treatment-related deaths. Thus, the adverse reactions observed in the present study were controllable, and all patients were able to receive the chemotherapy on an outpatient basis. In conclusion, because the efficacy of the combination regimen is high and adverse reactions to it are controllable, it was considered to be a useful therapy because it can be administered safely on an outpatient basis over a prolonged period of time to patients with advanced/recurrent gastric cancer who have undergone prior therapy. The combination regimen is considered to have a high potential for clinical use and merits investigation in further clinical studies.
References Diaz JF and Andreu JM (1993) Assembly of purified GOTtubulin into microtubules induced by taxol and taxotere, reversibility, ligand stoichiometry and competition. Biochemistry 32, 2747-2755. Endo M, Shinbori N, Fukase Y, Sawada N, Ishikawa T, Ishituka T, and Tanaka Y (1999) Induction of thymidine phosphorylase expression and enhancement of efficacy of capecitabine or 5'-deoxy-5-fluorouridine by cyclophosphamide in mammary tumor models. Int J Cancer 83, 127-134. Furukawa T, Yoshimura A, Sumizawa T, Haraguchi M, Akiyama S, Fukui K, Ishizawa M, and Yamada Y (1992) Angiogenic factor. Nature 356, 668. Glimelius B, Hoffman K, Haglund V, Nyren O and Sjoden PO (1994) Initial or delayed chemotherapy with best supportive care in advanced gastric cancer. Ann Oncol 5, 189-190. Kusaba H, Mitsugi K, Nakano S, and Saijou N (1999) Problems and prospects for combined chemotherapy with 5fluorouracil and low-dose cisplatin. Jpn J Cancer Chemother 26, 1575-1580. Mai M, Sakata Y, Kanamaru R, Kurihara M, Suminaga M, Ota J, Hirabayashi N, Taguchi T, and Furue H (1999) A late phase II clinical study of RP56976 (Docetaxel) in patients with advanced/recurrent gastric cancer, A Japanese cooperative study group trial (Group B). Jpn J Cancer Chemother 26, 487-496. Murad AM, Santiago FF, Petroiam A, Rocha PRS, Rodorigues MAG and Rausch M (1993) Modified therapy with 5fluorouracil, doxorubicin, and methotrexate in advanced
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Cancer Therapy Vol 2, page 297 Cancer Therapy Vol 2, 297-304, 2004
Promiscuous and specific anti-cancer drugs: combatting biological complexity with complex therapy Review Article
Maria C. Shoshan* and Stig Linder Cancer Center Karolinska, Department of Oncology-Pathology, Karolinska Institute and Hospital, S-171 76 Stockholm.
__________________________________________________________________________________ *Correspondence: Maria C. Shoshan, Cancer Center Karolinska, CCK R8:03, Karolinska Institute, S-171 76 Stockholm, Sweden; Tel.: 46-8-51775460; Fax: 46-8-339031; E-mail: mimmi.shoshan@onkpat.ki.se Key words: anti-cancer drugs, tumor cell complexity, drug screening Abbreviations: cyclin-dependent kinases, (CDKs); daunorubicin, (DNR); doxorubicin, (DXR); farnesylation transferase inhibitors, (FTIs); National Cancer Institute, (NCI); protein kinase C, (PKC); reactive oxygen species, (ROS) Received: 4 August 2004; Accepted: 31 August 2004; electronically published: September 2004
Summary Most anticancer drugs in clinical use have each been assigned a generally acknowledged mechanism of action, e.g., DNA damage or microtubule disruption. These mechanisms, however, are most likely not the unique cause of the antiproliferative effects of each drug. In fact, it may be induction of multiple and likely dose-dependent signals that explains the efficiency of many anticancer drugs, as illustrated by agents which have the same basic proposed mechanism of action, but not the same spectrum of clinical efficiency, nor the same side effects. By contrast, many novel, single-target drugs, e.g., inhibitors of specific growth factor receptors or other enzymes, show few side effects, but also limited efficiency, in particular as single agents. Does this purported inseparability of desired effects and side effects spell the end of developing cytotoxic drugs? Not necessarily, provided that the molecular signaling of each drug is properly mapped in order to identify optimal drug combinations, or that techniques for localized treatment are improved. However, this concept of drug efficiency does affect current specific-target approaches to drug development, and complicates the protocols for defining and evaluating drug resistance/sensitivity. We will here present the hypothesis that clinically efficient drugs are efficient by virtue of their “promiscuity” in the sense that they induce multiple targets/signals. Because this concept of drug efficiency is at variance with specific-target approaches to drug development, future perspectives will also be discussed. testicular cancer, even at advanced stages and is also rather effective in ovarian cancer. It is perhaps ironic that cisplatin is clinically widely effective, while a sophisticated VEGF inhibitor may be less so. One explanation might be the ability of cisplatin to rapidly induce pro-apoptotic reactive oxygen species (ROS) – an effect which has long been known to underlie its oto- and nephrotoxicity. There is no reason why similar “side effects” would not be involved in its antitumoral activities, and it has indeed been shown that scavengers of ROS will block cisplatin-induced apoptosis in cultured tumor cells. Again, it is also ironic that had the antiproliferative effect of cisplatin not been discovered in the sixties (Rosenberg et al, 1965), this drug would not have been considered for evaluation by current chemogenomics, due to its chemical reactivity and lack of drug-likeness (“druggability”) as
. Introduction Sophisticated molecular-level knowledge of cancer cell biology has proven difficult to translate into drugs that are clinically effective and the prognosis of patients with metastatic carcinoma is generally as poor today as 30 years ago. Although this is sometimes put down to preclinical researchers not understanding oncology and oncologists not understanding cell biology, a biological explanation appears more adequate, i.e., the insight that the lack of single "wonder drugs" is due to cancer being a complex set of diseases, and to each cancer cell phenotype being defined by combinations of severe functional disturbances. Some single drugs of course do work wonders. Despite its serendipitous discovery as a toxic compound formed at platinum electrodes during electrolysis of bacterial cultures, the widely used drug cisplatin does cure
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Shoshan and Linder: Anti-cancer drugs: combatting biological complexity with complex therapy generally defined by lipophilicity, H-bonding properties etc (Lipinski, 2000).
some of the most efficient and wide-spectrum anticancer drugs ever developed. Despite the classification, topoIImediated DNA lesions do not correlate well with DXRmediated tumor cell killing (Minotti et al, 2004). The toxic side effects, notably cardiotoxicity, of DXR have long been known to involve oxidative events. These were recently shown to be pro-apoptotic and to involve oxidization of DNA bases leading to a varied spectrum of DNA lesions (Minotti et al, 2004). Significant increases in at least nine different oxidized, cytotoxic DNA bases were identified in vivo in peripheral blood cells from patients undergoing DXR infusions (Doroshow et al, 2001). The complex chemistry of anthracyclines allows the intracellular formation of a range of free radicals and other reactive or toxic metabolites, and oxidative damage is indeed now considered important also for the proapoptotic and antitumoral effect of anthracyclines (Minotti et al, 2004). Daunorubicin (DNR) differs from DXR only in that the side chain of DXR ends with a primary alcohol, whereas in DNR it ends with a methyl. Nevertheless, DXR and DNR are used in different spectra of disease: DXR in breast cancer, childhood solid tumors, soft tissue sarcomas and in lymphomas, whereas DNR is used in acute lymphoblastic or myeloblastic leukemias (Minotti et al, 2004). The structural difference between DXR and DNR may alter lipophilicity and thence cellular uptake. However, the possibility that these drugs induce in part different signaling is supported by the fact that DNR and DXR appear in different mechanistic clusters in the response pattern map of growth inhibitory drugs created by the National Cancer Institute (see section V).
II. Standard chemotherapy drugs induce multiple effects Cisplatin and its various derivative forms are efficient against a wide range of solid tumors. All of them are classified as DNA-damaging agents which form bifunctional DNA adducts. However, it is only about 1% of the cellular uptake of cisplatin that actually forms DNA adducts (Eastman, 1991). The activated cisplatin molecule is electrophilic and highly reactive also towards, e.g., cysteine and methionine residues in polypeptide chains (Peleg-Shulman et al, 2002). Nephrotoxicity is a major dose-limiting side effect of cisplatin which depends on induction of antioxidant-sensitive lipid peroxidation in renal cortical cells (Hannemann and Baumann, 1988). Cisplatin-induced apoptosis as well as necrosis in renal cortical cells were shown to involve peroxidation and calcium release from intracellular stores (Kim et al, 1997; Baek et al, 2003). Ototoxicity is another serious side effect of cisplatin, and there are indeed numerous reports on the protective in vivo and in vitro effects of antioxidants on cisplatin-induced toxicity (Kim et al, 1997; Baliga et al, 1999; Kalkanis et al, 2004; Ekborn et al, 2003; Schaaf et al, 2002) and many more. The rapidity (minutes â&#x20AC;&#x201C; hours) of ROS induction, peroxidation and lethal cell injury in these - and other nonproliferating - systems suggests the existence of cisplatin targets other than DNA. There is no reason to believe that these targets would not be affected also in tumor cells. To test the possibility that cisplatin has other targets besides DNA, we treated tumor cell cytoplasts, i.e., cells from which nuclei have been removed, with cisplatin at clinically relevant concentrations and found that apoptosis was induced also in the absence of nuclear DNA, conclusively demonstrating a non-nuclear effect in the presence as well as absence of p53 (Mandic et al, 2003). This finding may furthermore help explain why p53 is not always a determinant of the anticancer action of cisplatin, despite induction of DNA damage. Indisputably, DNA damage and p53 are involved in the total efficiency of cisplatin, and are of particular importance under conditions of low intracellular cisplatin levels leading not to apoptosis, but to an irreversible damage-induced senescent state. The role of senescence as a major response determinant (Roninson, 2004) is of increasing interest and in agreement with the hypothesis of multiple, dose-dependent effects. In our hands, cisplatin at low doses (up to 2-5 ÂľM depending on cell line) does induce DNA damage, inhibition of proliferation by senescence, but not apoptosis, whereas more than five times higher doses (15-30 ÂľM) are required for induction of apoptosis (manuscript in preparation). The total antitumor effect of cisplatin is therefore the sum of nuclear and non-nuclear effects, or, differently put, the sum of two types of proliferation inhibition. Anthracyclines, such as doxorubicin (DXR), are classified as inhibitors of topoisomerase-II, and have, like cisplatin, been in widespread clinical use for decades as
Figure 1. General model of multiple drug effects Many standard drugs induce various growth inhibitory effects via different types of DNA damage. However, concentration-dependent drug effects on non-nuclear targets may also contribute to the net effect, either by directly inducing cell death and/or via lipid peroxidation which in turn can lead to secondary DNA damage. In non-proliferating non-tumor cells, effects on non-nuclear targets probably dominate, and subsequent apoptosis in sensitive organs leads to undesired side effects, e.g., neuro- and nephrotoxicity.
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Cancer Therapy Vol 2, page 299 Figure 1 illustrates that the total antitumoral effect of many drugs, such as cisplatin and DXR, is the sum of DNA damage plus additional multifunctional damage originating in non-nuclear compartments. The toxic side effects in differentiated, low-proliferative normal cells are primarily caused by these non-nuclear effects. The plant alkaloid ellipticine is a topoisomerase-II inhibitor, and is used in the treatment of, e.g., gliomas. Similar to cisplatin, it was found to induce DNA-damage independent apoptosis in cells lacking nuclei (Hägg et al, 2004). Etoposide, a topoisomerase-II inhibitor, classically induces DNA-damage and ensuing apoptosis but can at higher doses also induce a distinct effect on mitochondria (Robertson et al, 2000, 2002). In accordance with effects on mitochondria rather than primarily on DNA, etoposide treatment of HL60 cells induces ROS already after 15 min, protein carbonylation within one hour, and cell death at 4 h posttreatment is reduced by half by a ROS scavenger (England et al, 2004). Methotrexate is an antimetabolite which prevents DNA/RNA synthesis, but which also has antiinflammatory and immunosuppressive effects. Depending on cell type it can induce reactive oxygen within 4 h and massive apoptosis within 16 h, or growth arrest. Both responses were inhibitable by scavengers of ROS or by
GSH (Phillips et al, 2003). Paclitaxel exerts an antiproliferative effect via mitosis-inhibiting stabilization of microtubuli. When added to purified mitochondria, this drug induced loss of mitochondrial membrane potential and formation of reactive oxygen (Andre et al, 2000); paclitaxel-induced release of cytochrome c subsequent to these same events has also been reported (Varbiro et al, 2001). Added to intact cells at 10 µM, paclitaxel induces release of mitochondrial Ca2+ within seconds probably via an effect on the mitochondrial permeability transition pore (Kidd et al, 2002). Although 10 µM would appear an unphysiologically high concentration, similar levels have indeed been observed in plasma (Hajnoczky et al, 1994). Long-term incubation with paclitaxel at low concentrations is also known to lead to its accumulation to 40 µM in cells and tissues (Jordan et al, 1993). Together, these and other examples (Table 1) suggest to us that although the generally accepted classification of drugs according to basic mechanisms of action is correct, the very labels may prevent viewing the drugs in terms of other possibilities.
Table 1. Additional targets or effects of some standard chemotherapeutic drugs
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Shoshan and Linder: Anti-cancer drugs: combatting biological complexity with complex therapy full activity, farnesylation transferase inhibitors (FTIs) have been developed. For those which have been successful in clinical studies, it has been clear for some time, however, that this is not the key mode of action. For instance, FTIs have shown activity in leukemias and some solid tumors regardless of the Ras mutational status (Russo et al, 2004). The antioncogenic protein RhoB, which is structurally and functionally related to Ras, was early on believed to be a candidate alternative target of FTIs, and it was recently suggested that FTIs act by recruiting RhoB to interfere with pro-oncogenic signaling (Zeng et al, 2003). FTIs may also inhibit farnesylation of other proteins, e.g., prelamins A and B and centromere proteins (Russo et al, 2004). R115777 is one of the most promising FTIs in clinical trials and shows activity in, e.g., myelomas, breast, lung and gastrointestinal carcinomas. In a study on advanced myeloma it induced stable disease in more than 60% of the patients, although this effect did not correlate with inhibition of farnesyltransferase (Santucci et al, 2003). In an in vitro myeloma system, it induced apoptosis also when Ras remained farnesylated, and this apoptosis appeared to involve both the mitochondria and the ER (Beaupré et al, 2004).
III. Investigational and novel drugs Signal transduction pathways specific for tumor cells have long been principal targets for drug development. Because activation of protein kinase C (PKC) promotes tumor formation, inhibitors of PKC were early on investigated as potential new drugs. Several of these, e.g., UCN-01 (7-hydroxystaurosporine) and bryostatin-1, were indeed found to have antitumor activity in clinical trials, although it remains unclear which PKC isoforms are critical to the these effects. However, the major effect of UCN-01 is mediated by inhibition of cyclin-dependent kinases (CDKs), inhibition of AKT and PKC-independent induction of apoptosis (Dai and Grant, 2004). Similar to the tumor promoters phorbol ester and mezerein, bryostatin-1 can either activate or downregulate PKC, depending on the duration of exposure. Unlike the tumor promoters, however, bryostatin-1 presents a range of antiproliferative effects in tumor cell lines, e.g., proliferation inhibition, differentiation and apoptosis, suggesting a degree of promiscuity in its mode of action in vitro. Nevertheless, in clinical trials, the use of PKC inhibitors appears to lie more in combinations than as single agents (Swannie and Kaye, 2002; Kortmansky and Schwartz, 2003). Similar to PKC inhibitors, the antisense oligonucleotide ISIS3521 (LY900003) directed towards PKCalpha has modest effects in clinical trials. Since ISIS3521 by nature is more specific than the inhibitors, this suggests that at best PKCalpha is crucial only to certain types or subtypes of cancer cells. Several small molecule inhibitors of receptor and non-receptor tyrosine kinases have recently been developed for anticancer therapy (Smith et al, 2004). These drugs fall into three categories: inhibitors of the EGFR tyr kinase family (e.g., gefitinib (ZD1839; Iressa)), inhibitors of the split kinase domain tyrosine kinases (e.g., SU11248), and inhibitors of tyrosine kinases from multiple subgroups (e.g., STI571 (Gleevec/Glivec)) (Laird and Cherrington, 2003). Gefitinib was originally intended to target lung cancers due to their often high levels of EGFR. However, as a single agent it yielded few responders in clinical trials; neither was there any correlation between response and EGFR levels. This was later explained by the finding that gefitinib blocks only such EGFR that contain an activating mutation (Lynch et al, 2004; Paez et al, 2004), probably making gefitinib a highly specific drug both in terms of target and of disease. Other tyr kinase inhibitors may be fortuituously or purposely designed to be promiscuous, i.e., to inhibit a certain group of kinases rather than one specific. SU11248 is a multi-target tyrosine kinase inhibitor affecting FLT3 which is often activated in acute myeloid leukemia (O’Farrell et al, 2003). It also blocks c-Kit and PDGFR (Abrams et al, 2003). STI571 was originally found to inhibit the Bcr-Abl chimeric kinase in CML patients, but is now known to inhibit also the c-Kit tyrosine kinase. A perhaps unexpected effect of STI571 in a murine in vivo system was its tumor-specific enhancement of uptake of another drug, epothilone B (Pietras et al, 2003). Ras is the most frequently mutated protooncogene in human cancers. Because farnesylation of the Ras protein provides the plasma membrane association required for its
IV. Therapy complexity reflects tumor cell complexity The above examples suggest that cornerstone conventional drugs are promiscuous and therefore efficient, but have toxic side effects, whereas new drugs may be more specific and less toxic, but act primarily as sensitizers in combinations with conventional drugs, including various antibody therapies. Novel drugs have usually not been fully evaluated yet, but when potentially efficient as single agents, e.g., the FTIs, they appear to have several targets. This is entirely consistent with the biological insight that the numerous functional disturbances in cancer cells are determined by likewise numerous combinations of molecular signals, i.e., that no single oncogenic mutation can on its own lead to, or maintain, cancer. It is also in accordance with empirical as well as recent experimental experience showing that combination therapies of many kinds are generally more efficient. This concept of promiscuous drug efficiency is at some variance with “classical” medicinal chemistry, which, however, is mainly aimed at diseases with a biologically less complex etiology than cancer. Combinations of several different single-target treatments are illustrated, e.g., by a murine xenograft model where the complete, but not the partial, combination of antisense Bcl2/Bcl-xL, antisense protein kinase A and ZD1839 (Iressa) resulted in 50% growth inhibition and 50% tumor-free mice after 5 weeks (Tortora et al, 2003). Combinations of novel inhibitors of cyclindependent kinases with other single-target agents have also shown preclinical promise (Dai and Grant, 2004). It is reasonable to believe that future scenarios will involve rationally developed combinations of low-dose cytotoxic, promiscuous agents plus targeted sensitizers. This allows individual adjustment of treatment according to cancer type, and possibly also to each individual patient. 300
Cancer Therapy Vol 2, page 301 Alternatively, a great number of targeted drugs, one each for a large number of well-defined sets of signal transduction disturbances, might be envisaged to constitute a “building-block” set from which the individual treatment is to be rationally created. In either case, adequate diagnostic tools for identifying the specific combinations of dysregulated signal transduction pathways of each individual tumor is a prerequisite. These scenarios thus require detailed knowledge of signal transduction pathways and how they interact. This knowledge must be not only on a molecular and intracellular level, but should incorporate also microenvironmental, pharmacological and pharmacokinetic parameters. The order, timing and uptake of drugs in a certain combination are important, as are the drug effects on and influence of surrounding tissue. The type, concentration-dependence and kinetics of cell death are crucial, since these parameters affect the choice of therapy protocol as well as the induction of secondary effects and feedback loops which affect the net outcome. Some of these aspects are summarized in Figure 2. It should also be kept in mind that cell death type and kinetics are to a great extent determined also by factors such as the oxygenation and energy status of each tumor cell.
implicated in the phenotype. With this approach, parameters such as cell permeability, general propensity for macromolecule interactions (“stickiness”) and the tumor cell killing potential of the drugs are investigated only at later steps, making late attrition of impossible leads an expensive problem. Computational design of molecules to fit a specific target molecule structure is another approach to drug discovery which also requires that the actual cell kill of each candidate be assessed after its synthesis. Cell-based screening is therefore on the increase, its obvious advantage being that the desired effect – proliferation inhibition or cell death – is also the endpoint. Thus, all non-permeable compounds are immediately excluded, and, importantly, cell-based screening also directly confronts the complexity of tumor cell signaling. However, as indicated in Figure 2, any experimental protocol and interpretations should take into careful consideration the time-scale, the concentration range and the type of endpoint determination. Using the simple colorimetric MTT assay, the National Cancer Institute (NCI) has screened more than 100,000 compounds for growth inhibition over three days in 60 different human tumor cell lines representing leukemia, melanoma and cancers of the lung, colon, brain, ovary, breast, prostate and kidney. This has led to, for instance, the use of ellipticine in glioma treatment, and in identification of the kinase inhibitor UCN-01 as a lead molecule. This type of project also contributes to tumor biology knowledge, by revealing patterns of cellular responsiveness summarized in “maps” which can be
V. Cell based drug screening Many current drug discovery projects use vast drug libraries screened for molecules showing in vitro binding to and inhibition of a specific target, often a protein shown to be overexpressed in tumors or to be otherwise
Figure 2. General model of concentration- and time-dependent anti-proliferauve effects of many drugs on cancer cells. Low doses can induce states of arrest and senescence during which the cells remain viable as seen by, e.g., MTT assays. Over-lapping or higher concentrations may induce expression of various signaling factors such as c-Jun, NF!B, TRAIL and Fas ligand etc, in the entire cell population, or parts thereof. Such factors may over time have either anti-proliferative or protective effects, either intra-cellularly or as part of extracellular signaling loops. Apoptosis which develops over several days time can thus be a secondary effect of such signaling. Higher drug concentrations can lead to acute apoptosis within, e.g., 24 h. The apoptotic "concentration window" before induction of necrosis may vary depending on drug and cell type.
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Shoshan and Linder: Anti-cancer drugs: combatting biological complexity with complex therapy obtained at http://spheroid.ncifcrf.gov/scripts /mapviewer.cfm. In such a map, compounds cluster in distinct regions believed to reflect distinct mechanisms of action. Thus, cisplatin and DXR are found in a cluster of known DNA-damaging agents, whereas microtubuleactive compounds cluster elsewhere. The same map, however, also shows incongruities. We have mentioned the divergent localizations of DXR and DNR; also, many drugs occur in several different clusters which is likely explained by multiple mechanisms of action. Using a caspase-/apoptosis-specific assay, we have screened the mechanistic subset of the NCI library in order to find drugs showing apoptosis synergy within 24 h in combination with cisplatin, and identified ellipticine as one such drug (Hägg et al, 2004). This assay system was used also to assess the role of p53 in acute apoptosis induction by drugs in the same drug library. Most of the proapoptotic drugs induced apoptosis also in p53-null cells, and this apoptosis was markedly dependent on early permeabilization of the lysosomes (Erdal et al, submitted). Necrosis is another type of cell death. Since agents with unspecific toxic effects may induce necrosis and hence present a very small therapeutic window, assessment of necrosis is important for evaluating drug candidates. However, like apoptosis, necrosis can be a regulated process and is not necessarily to be avoided. For many drugs in vivo, the balance between apoptosis and necrosis likely depends not only on the signal transduction “wiring” of the tumor cell, but also on oxygenation and energy status. Necrosis can even be the expected outcome: it has been reported that cell death induced by alkylating agents such as nitrogen mustard is mainly necrotic due to the NAD-/ATP-depleting effect of PARP activity induced by DNA damage (Zong et al, 2004). Studies on drug responses in vivo should therefore include necrosis as well as apoptosis. We have developed a method to assess the relative contributions of apoptosis and necrosis in the same sample in vitro and in post-treatment serum samples from cancer patients, and found that induction of necrosis may be a quite common response to many drugs (Kramer et al, 2004; Erdal et al, submitted). As exemplified above in the section on cisplatin, treatment-induced senescence (which in some aspects differs from replicative senescence) is yet another possible growth inhibitory outcome (Roninson, 2004), but it will not be detected by apoptosis/necrosis assays and may confuse the interpretation of viability assays such as the MTT. Therefore, because they show a net antiproliferative result, clonal outgrowth assays may be appropriate for assessing total drug effects. However, the difficulties in adapting them to high-throughput systems are a major drawback; other disadvantages include cell culture conditions which may be even more nonphysiological than in shorter-term monolayer cultures (e.g., extremely low cell density and the subsequent alterations in gene expression, decay of nutrients during the long incubation; also, the unphysiological global stimulation when media are changed after a period of starvation can per se induce death). As the NCI screening projects and our own results show, screening systems are useful not only for drug
discovery, but also for investigating signal transduction. In this context, clonal outgrowth assays are unsuitable, precisely because they reflect the net influence of many pathways, feedback loops and types of cell death. In view of the roles of microenvironmental factors, cell-based screening systems for specific types of cell death must, however, be improved. A first step is to introduce threedimensional cell cultures in high-throughput screens; later steps involve development of tissue-like and monitorable reconstructions of in vivo situations. Systems reflecting the differing responses of normal and tumor cells, respectively, are also required. The future screening systems will thus be able to determine the net outcome of multiple drug effects in combination with biological conditions, i.e., they will resolve many questions in very few steps.
VI. Conclusion To use a drastic metaphor, the anticancer drugs that are promiscuous, but toxic, provide us with a variety of deadly bombs, whereas the single-target drugs constitute carefully aimed guns. While the bombs will efficiently remove several species of deadly prey but cause a great deal of collateral damage, the smaller guns are efficient only at close range, but will therefore leave other species of the forest unharmed. To continue this analogy, future improvements in cancer therapy require knowledge of forest ecology and the habits of the quarry, but also of weapons technology. Because tumor cell signaling complexity must be countered with complex combinations of medicinal weapons, future drugs will range from widespectrum multi-target compounds to specially designed molecules targetting tumor-specific, mutated forms of signaling proteins.
Acknowledgements We would like to acknowledge the support from Cancerföreningen in Stockholm, and from the Swedish Cancer Society.
References Abrams T, Lee L, Murray L, Pryer N and Cherrington J (2003) SU11248 inhibitis KIT and PDGFR-beta in preclinical models of human small cell lung cancer. Mol Cancer Ther 2, 471-478. Andre N, Braguer D, Brasseur G, Goncalves A, LemesleMeunier D, Guise S, Jordan M and Briand C (2000) Paclitaxel induces release of cytochrome c from mitochondria isolated from human neuroblastoma cells. Cancer Res 60, 5349-5353. Babiak RM, Campello AP, Carnieri EG and Oliveira MB (1998) Methotrexate: pentose cycle and oxidative stress. Cell Biochem Funct 16, 283-293 Baek S, Kwon C, Kim J, Woo J, Jung J and Kim Y (2003) Differential roles of hydrogen peroxide and hydroxyl radical in cisplatin-induced cell death in renal proximal tubular epithelial cells. J Lab Clin Med 142, 178-186. Baliga R, Ueda N, Walker P and Shah S (1999) Oxidant mechanisms in toxic acute renal failure. Drug Metab Rev 31, 971-997. Beaupré D, Cepero E, Obeng E, Boise L and Lichtenheld M
302
Cancer Therapy Vol 2, page 303 (2004) R115777 induces Ras-independent apoptosis of myeloma cells via multiple intrinsic pathways. Mol Cancer Ther 3, 179-186. Bordoni A, Biagi P and Hrelia S (1999) The impairment of essential fatty acid metabolism as a key factor in doxorubicin-induced damage in cultured rat cardiomyocytes. Biochim Biophys Acta 1440, 100-106 Buchdunger E, Cioffi CL, Law N, Stover D, Ohno-Jones S, Druker BJ and Lydon NB (2000) Abl protein-tyrosine kinase inhibitor STO571 inhibits in vitro signal transduction mediated by c-Kit and PDGF receptors. J Pharmacol Exp Ther 295, 139-145 Dai Y and Grant S (2004) Small molecule inhibitors targeting cyclin-dependent kinases as anticancer agents. Curr Oncol Rep 6, 123-130. Doroshow J, Synold T, Somlo G, Akman S and Gajewski E (2001) Oxidative DNA base modifications in peripheral blood mononuclear cells of patients treated with high-dose infusional doxorubicin. Blood 97, 2839-2845. Eastman A (1991) Mechanisms of resistance to cisplatin. Cancer Treat Res 57, 233-249. Ekborn A, Laurell G, Ehrsson H and Miller J (2003) Intracochlear administration of thiourea protects against cisplain-induced outer hair cell loss in the guinea pig. Hear Res 181, 109-115. England K, O’Driscoll C and Cotter T (2004) Carbonylation of glycolytic proteins is a key response to drug-induced oxidative stress and apoptosis. Cell Death Diff 11, 252-260. Erdal H, Berndtsson M, Mandic A, Castro J, Brunk U, Shoshan M and Linder S (2004) Lysosomal membrane permeabilization is a common mechanism for p53independent apoptosis. (submitted) Hägg M, Mandic A, Zhou R, Shoshan M and Linder S (2004) ER stress induced by ellipticine plant alkaloids. Mol Cancer Ther 3, 489-497. Hajnoczky G, Lin C and Thomas AP (1994) Luminal communication between intracellular calcium stores modulated by GTP and the cytoskeleton. J Biol Chem 269, 10280-10287 Hannemann J and Baumann K (1988) Cisplatin-induced lipid peroxidation and decrease of gluconeogenesis in rat kidney cortex: different effects of antioxidants and radical scavengers. Toxicology 51, 119-132. Hrelia S, Fiorentini D, Maraldi T, Angeloni C, Bordoni A, Biagi PL and Hakim G (2002) Doxorubicin induces early lipid peroxidation associated with changes in glucose transport in cultured cardiomyocytes. Biochim Biophys Acta 1567, 1506 Jin JS, Yao CW, Chin TY, Chueh SH, Lee WH and Chen A (2004) Adriamycin impairs the contraction of mesangial cells through the inhibition of protein kinase C and intracellular calcium. Am J Physiol Renal Physiol 287, F188-94 Jordan MA, Toso RJ, Thrower D and Wilson L (1993) Mechanism of mitotic block and inhibition of cell proliferation by Taxol at low concentrations. Proc Natl Acad Sci USA 90, 9552-9556 Kalkanis J, Whitworth C and Rybak L (2004) Vitamin E reduces cisplatin ototoxicity. Laryngoscope 114, 538-542. Kidd J, Pilkington M, Schell M, Fogarty K, Skepper J, Taylor C and Thom P (2002) Paclitaxel affects cytosolic calcium signals by opening the mitochondrial permeability transition pore. J Biol Chem 277, 6504-6510. Kim Y, Jung J, Lee S and Kim Y (1997) Effects of antioxidants and Ca2+ in cisplatin-induced cell injury in rabbit renal cortical slices. Toxicol Appl Pharmacol 146, 261-269. Kortmansky J and Schwartz G (2003) Bryostatin-1: a novel PKC inhibitor in clinical development. Cancer Invest 21, 924. Kramer G, Erdal H, Mertens H, Nap M, Mauermann J, Steiner G,
Marberger M, Bivén K, Shoshan M and Linder S (2004) Differentiation between cell death modes using measurements of different soluble forms of cytokeratin 18. Cancer Res 64, 1751-1756. Laird A and Cherrington J (2003) Small molecule tyrosine kinase inhibitors: clinical development of anticancer agents. Expert Opin Investig Drugs 12, 51-64. Lipinski C (2000) Drug-like properties and the causes of poor solubility and poor permeability. J Pharmacol Toxicol Methods 44, 235-249. Lynch T, Bell D, Sordella R, Gurubhagavatula S, Okimoto R, Brannigan B, et al (2004) Activating mutations in the EGFR underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 350, 2129-2139. Mandic A, Hansson J, Linder S and Shoshan M (2003) Cisplatin induces ER stress and nucleus-independent apoptotic signaling. J Biol Chem 278, 9100-9106. Minotti G, Menna P, Salvatorelli E, Cairo G and Gianni L (2004) Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacological Reviews 56, 185-229. O’Farrell A, Abrams T, Yuen H, Ngai T, Louie S, Yee K, Wong L, Hong W, Lee L, Town A, et al (2003) SU11248 is a novel FLT3 tyrosine kinase inhibitor with potent activity in vitro and in vivo. Blood 101, 3597-3605. Paez J, Janne P, Lee J, Tracy S, Greulich H, Gabriel S, et al (2004) EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 304, 14971500. Pattacini L, Mancini M, Mazzacurati L, Brusa G, Benvenuti M, Martinelli G, Baccarani M and Santucci MA (2004) Endoplasmic reticulum stress initiates apoptotic death induced by STI571 inhibition of p210 bcr-abl tyrosine kinase. Leuk Res 28, 191-202 Peleg-Shulman T, Najajreh Y and Gibson D (2002) Interactions of cisplatin and transplatin with proteins. J Inorg Biochem 91, 306-311. Peng Y, Li C, Chen L, Sebti S and Chen J (2003) Rescue of mutant p53 transcription function by ellipticine. Oncogene 22, 4478-87 Phillips D, Woollard K and Griffiths H (2003) The antiinflammatory actions of methotrexate are critically dependent upon the production of reactive oxygen species. Br J Pharm 138, 501-511. Pietras K, Stumm M, Hubert M, Buchdunger E, Rubin K, Heldin CH, McSheehy P, Wartmann M and Ostman A (2003) STI571 enhances the therapeutic index of epothilone B by a tumor-selective increase of drug uptake. Clin Cancer Res 9, 3779-3787. Robertson J, Enoksson M, Suomela M, Zhivotovsky B and Orrenius S (2002) Caspase-2 acts upstream of mitochondria to promote cytochrome c release during etoposide-induced apoptosis. J Biol Chem 277, 29803-29809. Robertson J, Gogvadze V, Zhivotovsky B and Orrenius S (2000) Distinct pathways for stimulation of cytochrome c release by etoposide. J Biol Chem 275, 32438-32443. Roninson I (2004) Tumor senescence in cancer treatment. Cancer Research 63, 2705-2715. Rosenberg B, Van Camp L and Krigas T (1965) Inhibition of cell division in Escherichia coli by electrolysis products from a platinum electrode. Nature 205, 698-9 Russo P, Loprevite M, Cesario A and Ardizzoni A (2004) Farnesylated proteins as anticancer drug targets: from laboratory to the clinic. Curr Med Chem Anti-Canc Agents 4, 123-138. Santucci R, Mackley P, Sebti S and Alsina M (2003) Farnesyltransferase inhibitors and their role in the treatment of multiple myeloma. Cancer Control 10, 384-387.
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Shoshan and Linder: Anti-cancer drugs: combatting biological complexity with complex therapy Schaaf G, Maas R, Groene E and Fink-Gemmels J (2002) Management of oxidative stress by heme oxygenase-1 in cisplatin-induced toxicity in renal tubular cells. Free Radic Res 36, 835-843. Smith J, Mamoon N and Duhe R (2004) Emerging roles of targeted small molecule protein-tyrosine kinase inhibitors in cancer therapy. Oncol Res 14, 175-225. Swannie H and Kaye S (2002) Protein kinase C inhibitors. Curr Oncol Rep 4, 37-46. Tortora G, Caputo R, Damiano V, Caputo R, Troiani T, Veneziani B, Placido S D, Bianco A, Zangemeister-Wittke U and Ciardello F (2003) Combined targeted inhibition of bcl2, bcl-xL, EGF receptor and protein kinase A type I causes potent antitumor, apoptotic and antiangiogenic activity. Clin Cancer Res 9, 866-871.
Tsang WP, Chau SP, Kong SK, Fung KP and Kwok TT (2003) Reactive oxygen species mediate doxorubicin-induced p53independent apoptosis. Life Sci 73, 2047-58 Varbiro G, Veres B, Gallyas F and Sumegi B (2001) Direct effect of Taxol on free radical formation and mitochondrial permeability transition. Free Radic Biol Med 31, 548-558. Zeng P, Rane N, Du W, Chintapalli J and Prendergast G (2003) Role for RhoB and PRK in the suppression of epithelial cell transformation by farnesyltransferase inhibitors. Oncogene 22, 1124-1134. Zong W, Ditsworth D, Bauer D, Wang Z and Thompson C (2004) Alkylating DNA damage stimulates a regulated form of necrotic cell death. Genes & Dev 18, 1272-1282.
From left to right Maria Shoshan and Stig Linder
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Cancer Therapy Vol 2, page 305 Cancer Therapy Vol 2, 305-316, 2004
The current management of primary ovarian cancer: a review Review Article
Anish Bali1*, Karina Reynolds2 1 2
Royal Marsden Hospital, London, UK St. Bartholomew’s Hospital, London, UK
__________________________________________________________________________________ *Correspondence: Anish Bali, Dept. Gynaecology Oncology, Royal Marsden Hospital, London, SW3 6JJ, UK; Tel: +44 7813904009; Fax: +44 2078082258; E-mail: anishbali@doctors.org.uk New address: 4 The willows, Old Park Road, Ballymena, N. Ireland, BT421QN Key words: primary ovarian cancer, pathology, prognostic markers, symptoms and signs, ultrasound, computed tomography (CT) and magnetic resonance imaging (MRI), serum tumour markers, surgery, chemotherapy, gene therapy Abbreviations: cancer services collaborative, (CSC); carcino-embryonic antigen, (CEA); Chemotherapy OR Upfront Surgery, (CHORUS); Computed tomography, (CT); Epithelial Ovarian Cancers, (EOC); European Organisation for Research and Treatment of Cancer, (EORTC); human milk factor globulins, (HMFG); intra-peritoneal, (i.p); intra-venous, (i.v); macrophage colony stimulating factor, (M-CSF); magnetic resonance imaging, (MRI); National Cancer Research Network, (NCRN); National Institute of Clinical Excellence, (NICE); negative predictive value, (NPV); placental alkaline phosphatase, (PLAP); positive predictive value, (PPV); positron emission tomography, (PET); Risk of Malignancy Index, (RMI); risk of ovarian cancer, (ROC); tumour suppressor genes, (TSG) Received: 03 August 2004; Accepted: 23 August 2004; electronically published: August 2004
Summary Traditionally ovarian cancer has been thought of as the ‘silent killer’. In developed countries it is the leading cause of cancer related mortality among women with gynaecological cancers. In the UK several approaches are being followed that encompass provision of cancer care within the remit of the National Health Service, screening trials such as UKCTOCS, and randomisation of patients on well constructed clinical trials to improve the outcome from this disease. Established surgical and adjuvant treatments are discussed as are the various on-going clinical and experimental trials in combating this disease. The remit of this review is to provide an overview of recent advances in the diagnosis and management of ovarian cancer. Epithelial cancer is the most common variant and is the primary focus of this review. The management of rare ovarian malignancies, such as germ cell tumours, hereditary ovarian cancer and recurrent disease is not described.
I. Introduction Ovarian cancer affects one in seventy women and is the 4th leading cause of cancer related mortality amongst women in developed countries. Typically this cancer has an insidious onset and over 70% of women present with disease that has spread beyond the ovary and in this population, the 5-year survival is approximately 30% despite optimal surgery and aggressive chemotherapy. Each year in the UK there are 6000 new cases of ovarian cancer and 4500 deaths. In the UK, with the development of national cancer networks providing a comprehensive care package, a multidisciplinary approach and recruitment of patients onto well-constructed clinical trials, significant inroads are being made to improve the overall and palliative outcome of patients with ovarian cancer. In addition, a better understanding of cancer aetiology is emerging with a flourish of activity in the fields of cancer genetics and molecular biology.
II. Organisation of care In 1995, the Calman/Hine report "A policy framework for commissioning cancer services" was published in response to concerns about variations in treatment polices across the UK (A report by the expert advisory group on cancer to the chief medical officers of England and Wales. 1995; www.doh.gov.uk). In order to provide high quality cancer care, it recommended that cancer services be delivered at three levels - primary care based in the community, cancer units based in district/local hospitals, and cancer centres mainly in larger teaching hospitals. A multidisciplinary approach was 305
Bali and Reynolds: The current management of primary ovarian cancer recommended in all settings. It envisaged the less common cancers and more advanced cancers be referred and treated at the cancer centres where affiliated services such as radiotherapy and advanced chemotherapy was readily available. In October 1999 delivery of this "hub and spoke" model of cancer care provision was identified as top priority by the government, so as to raise the standard and quality of cancer care across the country. To implement the necessary infrastructure, development of 34 cancer networks became mandatory, each serving a population of 1 to 2 million people. In 2001, the Cancer Services Collaborative (CSC) published its experience of working with 9 cancer networks in redesigning services necessary to achieve the required changes (Cancer Services Collaborative: Ovarian cancer-Service improvement guide. 2001; www.nhs/npat). In summary, the CSC were able to demonstrate a significant reduction in the waiting times for diagnosis and treatment of patients with suspected ovarian cancer, an improved and standarised management of patients by the formal introduction of multidisciplinary teams, and a much greater patient satisfaction with care. Although multidisciplinary care for challenging cases has always existed in the National Health Service (NHS), a formal specialised multidisciplinary team for each cancer type is being established in all cancer units and cancer centres. For gynaecological malignancies these teams in general consist of specialist surgeons, medical and clinical oncologists, dedicated radiologists and pathologists, clinical nurse specialists, dieticians and psychosexual/social counselors. Ovarian cancer is now largely managed within the remit of a cancer centre. It is recommended that all patients with pelvic masses suspicious of ovarian cancer be referred to the cancer centre for further surgical and possibly medical intervention. On completion of their initial treatment, continued follow up of patients is generally provided within the cancer units.
result in a decreased risk of developing this disease. Indeed multiparity, breast feeding, late menarche and early menopause, that all result in decreased ovulation, have all been shown to confer a protective effect. Use of the combined oral contraceptive pill is associated with up to a 70% reduction in developing EOC after 10 years or more of use compared with no-use, although this reduction appears to be vastly disproportionate to the decrease in number of ovulations inhibited (Gross and Schlesselman, 1994). Progestins have a potent apoptotic effect on ovarian epithelium as demonstrated in a study of primates receiving either combined oestrogen and progestin or progestin alone as opposed to oestrogen only therapy. A four to six-fold increase in the apoptotic activity of ovarian epithelium was noted in primates receiving a combined or single progestin regime (Rodriguez et al, 1998). Pathways regulating progestin induced apoptosis is complex, with evidence suggesting a role of differential regulation of p53 and TGF!11 in this process (Rodriguez et al, 2002). More recently, epidemiological data suggest an increase in the risk ratio of ovarian cancer amongst women taking oestrogen replacement in the menopause compared to those who are not (Noller, 2002). Studies have also demonstrated a small increased risk in those women taking sequential progestins along with oestrogen as compared to those taking a continuous combined form of HRT. One possible mechanism accounting for this increased risk may be the direct effect of oestrogen on epithelial ovarian cells. In cell line experiments, exposure to oestrogen results in a rapid increase in the proliferation of ovarian cancer cells (Chein et al, 1994). In addition, tamoxifen, an antioestrogen, has been reported to have beneficial effects in a few women with ovarian cancer. Hereditary ovarian cancer is uncommon. Germline mutations in the BRCA1 and BRCA2 breast/ovarian susceptibility genes overwhelmingly account for most hereditary subtypes. BRCA1 is located on chromosome 17q21 and encodes a large 220KDa protein expressed abundantly in testis, breast and ovarian tissue. BRCA1 is thought to function as a tumour suppressor gene and carriers of a germline mutation have a 30-80% life time risk of developing ovarian or breast cancer. Similarly ovarian cancer has been reported to occur in 10-35% of carriers with BRCA2 mutation located on chromosome 13 (Venktaraman, 2002). In hereditary nonpolyposis colorectal cancer, women with a mismatch repair gene mutation have been reported to have cumulative lifetime risk of 8-12% for ovarian cancer. The surveillance options and the preventative medical and surgical management of these high risk individuals are not discussed within this remit.
III. Pathology Ninety to ninety-five percent of ovarian cancer is sporadic in nature with the remainder being familial. Of the sporadic cases, 90% are defined as Epithelial Ovarian Cancers (EOC), denoting their origin from the surface epithelium of the ovary or its corresponding invaginations into the ovarian stroma. Histologically EOC's comprise of Serous, Endometrioid, Mucinous, Clear Cell and Brenner subtypes, with the first being the most common. In relation to surgical and medical management of ovarian cancer this review refers to sporadic epithelial ovarian neoplasia. Although the aetiology of sporadic ovarian cancer is unknown, epidemiological studies identify numerous factors that may influence the risk of developing it. The number of ovulatory cycles in a women's reproductive lifetime is the most significant of these associations giving rise to the "incessant ovulation" hypothesis (Fathalla, 1971). Theoretically, the repetitive disruption and repair of the surface epithelium of the ovary during ovulation may lead to spontaneous genetic mutations that in turn may confer an oncogenic genotype to the epithelial cells. Therefore, reducing the number of ovulatory cycles may
IV. Prognostic markers Numerous clinicopathological variables have been identified that impact on the overall and disease-free survival of patients with ovarian cancer. These include clinical stage; volume of post-operative residual disease; histological grade including mitotic index, DNA ploidy, and cell type; presence of ascites; pre-operative rupture of ovarian tumour; pre-treatment level of serum CA125 and age and performance status of the patient. Whilst most 306
Cancer Therapy Vol 2, page 307 factors in various settings have been shown to be independent predictors of survival, the most important remain clinical stage and residual disease. The FIGO staging for ovarian cancer is shown in Table 1. Many oncogenes and tumour suppressor genes (TSG) have been implicated in the pathogenesis of ovarian cancer. Several of these genes are important in cell cycle regulation, in particular G1 to S phase transition, and their aberrant expression is associated with poor prognosis. In various settings, the loss of cyclin dependent kinase inhibitors such as p21Waf1/Cip1, p27Kip1, inactivation of wild type p53 and overexpression of cyclin D1 have all been implicated
as markers of poor outcome in ovarian cancer. Other genetic mutations that present in ovarian cancer include overexpression/amplification of HER-2/neu, c-MYC, BAX and c-FMS. With the recent completion of the human genome and utilisation of complimentary techniques, such as oligonucleotide microarrays, the list of novel genes implicated in ovarian tumourogenesis is likely to grow. Table 2 summarises the clinically useful prognostic variables predictive of disease outcome in ovarian cancer at various time points during treatment (Eisenhauer et al, 1999).
Table 1. FIGO staging system for ovarian cancer. Stage I Growth limited to ovaries Ia Growth limited to one ovary, no malignant cells in ascitic fluid or peritoneal washings; no tumour on external surface; capsule intact Ib Growth limited to both ovaries, no malignant cells in ascitic fluid or peritoneal washings; no tumour on external surface; capsule intact Ic As with 1a or 1b but with tumour on the surface of one or both ovaries; or with malignant cells in ascitic fluid or peritoneal washings Stage II Growth involving one or both ovaries with pelvic extension IIa Extension and/or metastases to the tubes and/or uterus No malignant cells in ascitic fluid or peritoneal washings IIb Extension to other pelvic tissues No malignant cells in ascitic fluid or peritoneal washings IIc As with 2a or 2b but with tumour on the surface of one or both ovaries; or with malignant cells in ascitic fluid or peritoneal washings Stage III Tumour involving one or both ovaries with peritoneal implants outside the pelvis and/or positive retroperitoneal or inguinal lymph nodes Superficial liver metastases equals stage 3 IIIa Tumour grossly limited to true pelvis with negative nodes but histologically confirmed microscopic seeding of abdominal peritoneal surfaces IIIb Histologically confirmed tumour implants of abdominal peritoneal surfaces, none exceeding 2 cm in diameter Nodes negative IIIc Abdominal tumour implants exceeding 2 cm in greatest dimension and/or positive retroperitoneal or inguinal lymph nodes Stage IV Growth involving one or both ovaries with distant metastases Positive pleural effusions and/or parenchymal liver disease
Table 2. Prognostic variables predictive of disease outcome in ovarian cancer at indicated time points (modified table from Eisenhauer et al, 1999). Events Surgery Post surgery During Relapse Pre-chemotherapy chemotherapy Prognostic Specialty of Age Type of Time since last chemotherapy variables surgeon chemotherapy treatment Adequacy of Performance status CA125 decrease Disease bulk surgery Stage Number of disease sites Grade Performance status Histology - cell type Histology Residual disease Time since diagnosis Ascites Haemoglobin DNA ploidy Capsule rupture (stage 1 disease) CA125 level Albumin level
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Bali and Reynolds: The current management of primary ovarian cancer tumours have increased blood flow associated with neovascularity and a low resistivity index due to a lack of smooth muscle support. However, combining Doppler colour flow or Doppler arterial resistance with morphology does not appear to significantly improve the sensitivity or specificity of ultrasound in determining malignant from benign ovarian masses. Combining these techniques requires additional expertise, is expensive, and vastly more time consuming. In addition, the reproducibility of Doppler arterial resistance measurements in ovarian masses has not been verified (Kinkel et al, 2000). The use of ultrasound in determining the morphology of ovarian masses has been studied extensively. Taken in isolation, and depending on the scoring system used, studies have reported a sensitivity of 60 – 100%, specificity of 70 – 95%, positive predictive value (PPV) of 30 – 88% and negative predictive value (NPV) of 80 – 100% in determining the probability of malignant ovarian pathology. Table 3 gives one example of a scoring system which was developed by Sassone et al, (1991) for a study population of 143 patients. This scoring system achieved a sensitivity of 100% and specificity of 83%, although the PPV remained low at 37%. High scores for many benign masses such as teratomas, endometriomas and ovarian fibromas contributed to the low PPV. This scoring system was later modified by Lerner (by removing wall thickness and adding shadowing defined as loss of acoustic echo), but with disappointing results (Lehner et al, 1998). To overcome this problem, research has focused on combining demographic, clinical, biochemical and radiological features to distinguish benign from malignant pathology. An example is an algorithm developed by Jacobs et al, (1991). The algorithm, termed Risk of Malignancy Index (RMI) is the product of serum CA125 level, the ultrasound score (expressed as 0,1 or 3 - Table 4) and the menopausal status. A sensitivity of 85% and specificity of 97% was reported with an RMI score of 200 or more in predicting ovarian malignancy. With a RMI cut-off at 50 the sensitivity improved to 95% but the specificity decreased to 76%. The RMI in this study was derived from the same population that was used to evaluate it. In 1993, these results were validated in a new study population that reported 87% sensitivity, 89% specificity and 75% PPV with a RMI cut-off level of 200 (Davies et al, 1993)
V. Diagnosis A. Symptoms and signs As previously mentioned, 70% of patients with ovarian cancer are diagnosed when the disease has spread beyond the ovary. Symptoms are generally vague, often resulting in a lengthy delay in diagnosis. A survey of 1700 patients carried out in the USA and Canada showed that 22% of women ignored their symptoms for a significant length of time prior to presentation to their local health authority. Subsequently, a further delay in diagnosis of 2 months occurred in 55% of women, 3-6 months in 19%, 712 months in 15% and more than 12 months in 11%. Patients frequently present with a history of indigestion (30%), non-specific abdominal discomfort (40%), abdominal distension (60%), lethargy (50%), urinary frequency (30%) and, more rarely, with weight loss, abnormal menses and post menopausal bleeding (Griffith RW, The not-so-silent killer. www.healthandage.com /Phome/gid2=1056). Abdominal examination may reveal a mass arising from the pelvis, although the presence of a smaller pelvic mass can only be revealed by combined vaginal and rectal examination. However, vaginal examination lacks the sensitivity and specificity, even when carried out by experienced clinicians to differentiate between benign adnexal masses and malignant ones. The presence of ascites is highly suggestive of malignancy and with late stage disease there may be a pleural effusion, palpable disease in the recto-vaginal septum or supra-clavicular or inguinal lymphadenopathy.
B. Ultrasound Ultrasound remains the most important form of radiological imaging in the initial evaluation of adnexal masses. Transvaginal ultrasound provides higher resolution in delineating the characteristics and site of smaller adnexal masses, whereas transabdominal ultrasound remains important for assessing larger cystic masses, the upper abdomen and other intra-abdominal organs. Architectural features consistent with malignant ovarian pathology include the size of the mass, multiloculated cysts with septae measuring > 3mm, solid components, papillary excrescences and the presence of ascites. When used in conjunction with Doppler colour flow, pelvic ultrasound is useful in determining the blood flow within ovarian tumours. Characteristically, malignant Table 3 Value
Inner wall structure
Wall thickness (mm)
Septa (mm)
Echogenicity
1 2 3
Smooth Irregularities ! 3mm Papillarities > 3mm
Thin ! 3mm Thick > 3mm Not applicable, mostly solid
No septae Thin ! 3mm Thick > 3mm
4
Not applicable, solid
Sonolucent Low echogenicity Low echogenicity with echogenic core Mixed echogenicity
3
3
5 Maximum
4
mostly
Reproduced from Sassone et al, 1991 with kind permission from Obstetrics & Gynecology
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High echogenicity 5
Cancer Therapy Vol 2, page 309 Table 4 Ultrasound (1 point for each characteristic) Multilocular cyst Evidence of solid areas Evidence of metastases Presence of ascites Bilateral lesions
Menopausal status Premenopausal Postmenopausal RMI = U x M x serum CA125 level
Ultrasound score (U) 0 for 0 points 1 for 1 point 3 for 2-5 points
Menopausal status score (M) 1 3
Reproduced from Jacobs and Oram, 1990 with kind permission by Charman & Hall
More recently use of artificial neural networks and logistic regression models have been introduced, although their value remains to be proven. The rationale of developing an ideal scoring system would be that appropriate referral to a specialist oncology centre could be triggered quickly and appropriately, ensuring optimal treatment for that individual.
overall accuracy for the diagnosis of malignancy was reported as 91%. One particular subset of patients that prove difficult, and a diagnostic dilemma, are premenopausal women in whom there are multiple reasons for an elevated CA125 and an adnexal mass. Subgroup analyses were not performed in above-mentioned studies and the potential use of MRI in the premenopausal patient remains unanswered. Several small studies have addressed pre-operative staging of ovarian cancer using cross-sectional imaging with reported accuracy of 70 â&#x20AC;&#x201C; 85%. One limitation is the unreliable detection of lesions less than 1cm located on the bowel surface, mesentery or peritoneum, and of lymph node involvement in early stage disease. The use of positron emission tomography (PET), spiral scanning CT and fat-suppressed, gadolinium-enhanced studies and phased array multicoils with MRI may improve the detection rate but have yet to be tested in clinical trials. Surgical intervention carries a degree of morbidity, and in those patients who have been sub-optimally or minimally debulked (the â&#x20AC;&#x153;open-closeâ&#x20AC;? laparotomy), this morbidity significantly delays the initiation of adjuvant chemotherapy. Forstner et al. reported high positive and negative predictive values for CT and MRI in predicting tumour resectability (Forstner et al, 1995). The criteria used for non-resectability had been derived from an earlier study by Nelson et al, (1993). These included tumour deposits > 2 cm at the root of the mesentery, in the porta hepatis, the lesser sac of the omentum, gastro-splenic ligament, diaphragm, dome of liver and lymph nodes > 1cm at or superior to the celiac axis (Nelson et al, 1993). With current trials examining the role of neoadjuvant chemotherapy for patients with advanced ovarian cancer, the issue of operability needs to be formalised.
C. Computed tomography (CT) and magnetic resonance imaging (MRI) The use of cross-sectional imaging in the preoperative assessment of primary ovarian cancer remains unclear. Important issues that need to be addressed are whether these modalities can: 1) Better distinguish between benign and malignant adnexal pathology than ultrasound. 2) Accurately stage the disease and improve the preoperative predictability of achieving optimal and gross tumour resectability. 3) Accurately evaluate retroperitoneal lymph node status, thereby allowing conservative surgery in early stage disease without compromising optimal surgical staging and subsequent treatment. To date there are no prospective trials comparing the use of gray-scale ultrasound to CT or contrast-enhanced MRI in distinguishing benign from malignant adnexal masses. Morphological features of malignant change on non-enhanced MRI and contrast enhanced CT are similar to those used in ultrasound, and have roughly equivalent ability in predicting disease, but the cost and time differential vastly favours the use of ultrasound. Recent advances in MRI such as the use of paramagnetic contrast agents (gadolinium) and multicoils have dramatically improved its specificity, and this examination may have a role to play in the second-line evaluation of patients suspected of ovarian malignancy on the basis of ultrasound alone or in combination with other parameters. Using gadolinium enhanced MRI in a study population of 128 patients with 187 adnexal masses, Hricak et al, (2000) reported 93% and 95% accuracy in defining benign and malignant ovarian masses respectively. Inter and Intra observer agreement was extremely high, and the lesions that were not detected were generally less than 2cm in maximum dimension. Similar findings have also been published by Sohaib et al, (2003) in 104 patients. The
D. Serum tumour markers Bast et al, (1983), first described the use of CA125 for ovarian cancer. In a population of 101 patients, 82% were found to have elevated levels of CA125 >35u/ml as compared with 6% of patients with non-malignant disease (Bast 1983). This high molecular weight glycoprotein is secreted by endothelial cells of most pelvic organs including normal ovary, and by mesothelial cells of peritoneum, pericardium and pleura. Levels are elevated in 1% of the normal population, 6% of patients with benign disease such as endometriosis and pelvic infections and 309
Bali and Reynolds: The current management of primary ovarian cancer 28% of patients with non-gynaecological intra-abdominal malignancies. In EOC, levels are raised in 90% of stage II, III and IV tumours but in only 50% of stage I tumours. Therefore, the specificity of using CA125 as a solitary diagnostic test in detecting ovarian malignancy is poor. To improve this specificity, the addition of one or more tumour markers to CA125 has been examined. These include placental alkaline phosphatase (PLAP), macrophage colony stimulating factor (M-CSF), OVX1 and human milk factor globulins (HMFG). However, only a modest improvement in specificity is achieved and the addition of other tumour markers is not routine. In the UK, a prospective randomised controlled trial involving 200,000 women is presently underway examining the roles of CA125 measurements and ultrasound in screening for ovarian cancer. This multimodel screening strategy has been optimised by use of a sophisticated way to interpret CA125 levels. The risk of ovarian cancer (ROC) is calculated using an algorithm based on Bayes theorem, which compare each individualâ&#x20AC;&#x2122;s serial CA125 levels to the pattern in cases and controls. The UKCTOCS (UK Collaborative Trial of Ovarian Cancer Screening) trial is still underway but depending upon results may alter the manner in which patients present with ovarian cancer. Presently in all patients with suspected ovarian malignancy, serum "HCG and #FP should be taken to diagnose germ cell tumours and carcino-embryonic antigen (CEA) to exclude other primaries, in particular from the gastro-intestinal tract. However CEA may also be raised in ovarian mucinous cystadenocarcinomas.
likelihood of side effects and development of chemoresistant disease. In addition, surgery offers the immediate relief of pressure symptoms, although the longterm quality of life of patients that have been successfully debulked compared to those that have not has not been adequately addressed. Adequate intra-abdominal exposure is achieved through a vertical incision. A sample of ascites or peritoneal washings using saline is taken and sent for cytological evaluation. This step is unnecessary when widespread intra-abdominal disease is visualized. Following this, a systematic exploration of the intraabdominal contents is performed as described above. Cytoreductive surgery typically involves a total hysterectomy, bilateral salpingoophorectomy, infra-colic omentectomy and resection of any bulky disease from the peritoneal surfaces or from the intestines. For stage Ia and Ib disease where no other indications for chemotherapy exist (such as adverse histopathological cell type or grade of tumour) and bulky nodal disease, retroperitoneal lymph node dissection should be performed, the value of which is discussed later. If there is no evidence of macroscopic metastatic disease, biopsies or scrapings of the peritoneal surfaces including the area under the diaphragm should be performed. All intraabdominal adhesions should also be biopsied. Ultraradical surgery involving peritoneal stripping, splenectomy and en-bloc resection of the sigmoid colon with the reproductive organs to attain optimal debulking is questionable. Morbidity from these procedures is high and there is no good evidence of a survival benefit for patients (Bristow, 2000). Conservative surgery can be considered for young women wishing to preserve their fertility. These patients need to as thoroughly staged as possible and unilateral oophorectomy only considered for presumed stage I, grade I tumours. Careful follow up is essential, arguably in the form of transvaginal ultrasound surveillance and CA125 monitoring, as recurrence rates of up to 9% in the remaining ovary have been reported. Consideration should be given to removal of the uterus and remaining ovary once child-bearing has been completed (Zanetta et al, 1997). The use of minimally invasive surgery in gynaecological practice has grown rapidly in the past decade. Possible benefits include shorter hospitalisation and time to recovery, less patient discomfort and analgesic requirements and, importantly, decreased cost. Laparoscopic surgery for a presumed benign mass has become routine. Data suggests that 10% of presumed 'benign' masses will appear suspicious on laparoscopy and of these 50% will be malignant (Manolitsas and Fowler, 2001). Therefore the unexpected malignant mass encountered during laparoscopy is not uncommon, and to date there are no clear recommendations for the subsequent management of such patients. What is not in doubt is that full surgical staging should be performed, as up to 25% of apparent stage I ovarian cancers are actually stage III disease on account of retroperitoneal lymph node involvement. In a retrospective study of 192 patients, Kindermann et al. reported that a delay in staging laparotomy of greater than 8 days after laparoscopy was
VI. Treatment A. Surgery The role of primary surgery for ovarian cancer can be broadly classified into 3 categories: 1) Diagnosis - to establish a definitive diagnosis of ovarian cancer. Although the presence of a pelvic mass with ascites is highly suggestive of ovarian malignancy, surgical resection is required to confirm this diagnosis. Consequently the histological cell type and grade of tumour can be confirmed by pathological analysis. 2) Staging - accurate surgical staging requires a systematic examination of the pelvic organs, the peritoneal surfaces of the abdominal cavity including the paracolic gutters and subdiaphragmatic area, the surfaces of the small and large bowel, liver and omentum, and palpation with resection of any suspicious pelvic and/or para-aortic lymph nodes. 3) Therapy - cytoreductive surgery with the aim of leaving < 1 cm of macroscopic disease. There are now several published series confirming an advantage in disease-free interval and overall survival for patients that have been optimal debulked and indeed even further benefit of leaving no macroscopically visible disease. There are also theoretical benefits for successful delivery of chemotherapeutic agents to small volume disease. Small well perfused tumour beds, with high cell growth fraction, may favor increased cell kill to cytotoxic agents, as might the removal of large, poorly vasularised tissue which may not respond to drug manipulation. The possible use of fewer cycles of chemotherapy would reduce the 310
Cancer Therapy Vol 2, page 311 associated with significant adverse effects (Kindermann et al, 1996). Port site metastases occurred in 56% of apparent stage IC-II cancers and in 47% of stage III cancers. In addition, there also appeared a rapid progression to stage III disease in 39% of presumed stage I cancers. However, interpretation of this data is difficult. The majority of cases were associated with capsule rupture as techniques such as aspiration, biopsy and morcellation were used, with only 7% of masses being removed intact in an endobag. Research also suggests that carbon dioxide, used routinely to create a pneumoperitoneum, may have a growth stimulating effect on tumour cells and intra-peritoneal dissemination (Volz et al, 1999). There are no well constructed prospective trials examining the short term morbidity and long term prognosis of patients with ovarian malignancy managed by laparoscopy as compared to those managed by laparotomy. With advancing technology and growing expertise in pelvic and paraaortic lymph node dissection, adequate staging for apparent stage I ovarian cancer by laparoscopy is possible (Pomel et al, 1995). The adequacy of lymph node yields by laparoscopic surgery has been addressed in several studies. These studies are limited to cases of cervical cancer, and there are only anecdotal reports of laparoscopic restaging of ovarian malignancies (i.e. for cases referred for definitive management when malignancy had been diagnosed unexpectedly elsewhere). Nevertheless, in laparoscopic lymphadenectomy for cervical cancer the average node count appears to be only marginally less than that removed by an open approach (Childers et al, 1992). As more post menopausal women are now entering ovarian cancer screening trials, laparoscopic oophorectomy has become the norm for those who fall into the moderate or high risk categories. Hence, the finding of an incidental malignant mass at laparoscopy is likely to increase, and there is need to examine the role of minimal access surgery in the complete management of patients with stage I disease. The interval to completion staging also needs to be addressed as does the disease-free interval and overall survival of such patients.
failed to demonstrate a survival advantage, although initial clinical response was much greater with platinum therapy, and the quality of life for patients was improved. A significant survival benefit for platinum was finally demonstrated by results of a meta-analysis in 1998, which showed a 5% improvement in survival of patients at 2 years (45%-50%) and 5 years (25%-30%) (Aabo et al, 1998). The two platinum drugs currently in use are cisplatin and carboplatin. Carboplatin developed in the 1980â&#x20AC;&#x2122;s, has significantly fewer side effects than cisplatin with almost no renal or neurotoxicicty and a decreased frequency of nausea and vomiting. Trials, such as ICON2, comparing the use of these two drugs in over 2000 patients have shown no difference in efficacy between them, and given that carboplatin is significantly better tolerated than cisplatin, carboplatin alone or in combination have become adopted for first-line therapy for ovarian cancer (The ICON collaborators, 1998).
2. Taxane drugs Paclitaxel is derived from the bark of the pacific yew tree and was first introduced in the treatment for ovarian cancer in the 1990â&#x20AC;&#x2122;s. Side effects of paclitaxel include total hair loss, hypersensitivity reactions, myelosuppression and sensory neuropathy. Nausea and vomiting are generally mild. The first trial examining the efficacy of paclitaxel was the GOG111 trail (McGuire et al, 1996). Four hundred and ten women with suboptimally debulked stage III/IV disease were recruited from 1990 to 1992 and were randomised to receive, cisplatin plus cyclophospomide or cisplatin plus paclitaxel. An improvement of 5 months in the progression free interval (13 vs. 18 months) and 14 months in overall survival (24 vs. 38 months) was observed in those receiving cisplatin plus paclitaxel. A further randomised trial (OV10) with a similar design to GOG111 supported these finding in a group of 680 women (Piccart et al, 2000). An absolute improvement of 4 and 10 months was observed in progression free survival and overall survival respectively in patients receiving a paclitaxel regime. In contrast to these findings, the results of GOG132 and ICON3 show no survival advantage for patients receiving paclitaxel. In the GOG132 trial, a large number of the 645 participants in all arms crossed to alternative treatments before progression, thus confounding the results and making interpretations difficult (Muggia et al, 2000). For the ICON3 trial, 2074 patients were recruited between 1995 and 1998 (The ICON collaborators, 2002). Entry criteria included patients with all stages of ovarian cancer requiring chemotherapy. Patients were randomised to receive paclitaxel plus carboplatin or a control (either carboplatin alone or cyclophosphamide, doxorubicin and cisplatin). Meaningful comparison of this trial to the previously published positive data on paclitaxel is immediately difficult due to the broad entry criteria in ICON3. Although clinical significance is not reached, subgroup analysis of non-optimally debulked stage III/IV cancer patients in ICON3, does demonstrate a trend towards improved survival with paclitaxel. These are the only group of patients that can be compared with those
B. Chemotherapy Chemotherapy plays a central role in the management of EOC and adjuvant combination therapy is usually given after the surgical treatment and accurate staging of this disease (Harries and Gore, 2002). As a result of numerous prospective randomised clinical trials, the combination of a platinum based agent (Cisplatin / Carboplatin) and paclitaxel has become the standardised therapy for ovarian cancer in developed countries. The role of neo-adjuvant chemotherapy for advanced disease and adjuvant chemotherapy for early stage disease is controversial and is discussed later.
1. Platinum drugs Cisplatin was first introduced in the treatment for ovarian cancer in the 1970â&#x20AC;&#x2122;s for patients who had failed to respond to alkylating agents such as melphalan and chlorambucil. Subsequently clinical trials examining the role of cisplatin as first-line treatment, either in combination with alkylating agents or as a single agent, 311
Bali and Reynolds: The current management of primary ovarian cancer participants in GOG111 and OV10 trials. Further confounding factors may include a combination of paclitaxel with carboplatin rather than cisplatin, and the differing time and dose regimes of the various chemotherapeutic agents. Despite the conflicting results, paclitaxel may have a role in the management of ovarian cancer although its use may have to be tailored to those with poor risk advanced disease. The National Institute of Clinical Excellence (NICE) in the UK offers guidance on the use of taxanes for ovarian cancer. Paclitaxel is licensed for use in the UK and is recommended for treatment in combination with cisplatin in patients with advanced disease or residual disease after initial surgical management. Docetaxel, a more recent family member of the taxane group with fewer side effects, is not licensed for use, though there is a trial underway comparing the use of docetaxel/platinum versus paclitaxel/platinum.
chemotherapy. The trial is currently open and recruitment will hopefully be complete in 18 months. A similar trial in 800 patients organised by the European Organisation for Research and Treatment of Cancer (EORTC) is also currently underway. Once again the questions being addressed include: the quality of life of patients receiving either treatment, whether optimal debulking is more frequent in those treated with neoadjuvant chemotherapy and if this translates to a prolonged disease-free interval and overall survival of patients.
4. Adjuvant chemotherapy for early stage ovarian cancer Stages I â&#x20AC;&#x201C; IIa ovarian cancers account for 30% of all EOC. Over the past decade there has been limited and controversial data regarding the impact of adjuvant chemotherapy in such cases on the disease-free interval and overall survival of patients. The ACTION and ICON1 trials were initiated by the EORTC and MRC respectively to address these questions and were closed in January 2000. The individual and combined results of these trials were published in January 2003 (The ICON collaborators, 2003a, 2003b; Trimbos et al, 2003). In brief, patients were randomised to platinum based chemotherapy or no treatment following surgery. Both trials allowed considerable flexibility about the chemotherapy regimes used as long as a platinum drug was included. The recruitment criteria for patients entered in the ACTION trial were strict. Optimal staging surgery was required and only those with stages Ia and Ib, grade 2 and 3, Ic and IIa, all grades were entered. ICON1 recruitment was more liberal, not requiring adequate staging and randomising all cases of early stage ovarian cancer on the basis of the physicianâ&#x20AC;&#x2122;s uncertainty about the need for chemotherapy. In the combined analysis of the 925 patients entered (477 in ICON1 and 448 in ACTION), overall survival at 5 years was 82% in the chemotherapy arm and 74% in the observation arm. Recurrence free survival at 5 years also followed a similar and significant trend (76% in the chemotherapy arm and 65% in the observation arm). However, these results need to be interpreted with caution. In ICON1, with its liberal entry criteria, many patients with good prognostic factors were included, as were poor prognosis patients with possible occult stage 3 disease. Indeed, ICON1 reported an increase of 7% (82% vs. 75%) in overall 5 year survival for patients receiving chemotherapy. In contrast, the ACTION trial with its strict entry criteria showed no overall survival advantage and only a marginal benefit in recurrence free survival with adjuvant chemotherapy. However this benefit appeared to be limited to patients with non-optimal staging. In this trial four staging categories were defined ranging from optimal to inadequate, and on closure, only one-third of patients were deemed to have been optimally staged. Certainly lymph node involvement is present in 14% - 20% of apparent stage I disease, which under current FIGO staging would upgrade these tumours to stage III. Retroperitoneal pelvic and paraaortic lymph node sampling or indeed full systematic lymphadenectomy requires considerable expertise and increases patient morbidity. This needs to be balanced against a possibly
3. Neoadjuvant chemotherapy The role of neoadjuvant chemotherapy in the treatment of ovarian cancer is yet to be established. There is evidence to suggest that interval debulking may be technically easier, with less intra-operative and postoperative morbidity, in patients with advanced FIGO stage ovarian cancer. In addition, this modality of treatment may also offer a better quality of life for patients without significantly compromising their disease-free interval and overall survival. In a retrospective study by Schwartz et al, (1999) 59 women were identified having received neoadjuvant chemotherapy on the basis of clinical and radiological evidence of advanced ovarian cancer and in whom optimal debulking surgery would be unlikely. The outcome of this cohort was compared to that of 206 patients over a similar time frame receiving conventional treatment (i.e. cytoreductive surgery followed by adjuvant chemotherapy). The three surgical parameters measured were estimated blood loss, intensive care days and postoperative hospital days. All parameters favoured the neoadjuvant chemotherapy group without altering the overall and disease-free survival of patients. In contrast, a smaller but more recent prospective trial demonstrated a significantly higher median survival time for patients with advanced disease treated with neoadjuvant chemotherapy compared to those treated conventionally (42 months vs. 23 months). Time spent in surgery, the requirement for blood transfusion and the intra-operative morbidity and mortality were not significantly different between the two groups (Kuhu et al, 2001). In the UK, the overall benefits of neoadjuvant chemotherapy are currently being studied in a randomised feasibility trial of 150 patients organised and funded by the clinical trials unit of the medical research council. This pilot study termed CHORUS (Chemotherapy OR Upfront Surgery) will investigate the impact of timing of surgery and chemotherapy in newly diagnosed patients with advanced ovarian, peritoneal or fallopian tube cancer, in terms of overall and progression free survival, and quality of life. Eligible patients will receive either up front surgery or interval debulking after 3 cycles of carboplatin based
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Cancer Therapy Vol 2, page 313 unnecessary use of chemotherapy in cases where the disease is actually confined to the ovary. Prior to the publication of the above data and despite the increased morbidity, there has already been a shift to performing optimal surgical staging for apparent early stage disease thereby permitting a logical argument for the use of adjuvant chemotherapy. This is likely to continue. Subsequent studies need to focus on molecular markers and microarray profiles that may further distinguish between good and bad prognosis in early stage ovarian cancer thereby allowing even more tailored treatments.
5. New chemotherapy agents Numerous drugs have shown activity in recurrent ovarian cancer. Some of these are now being evaluated as first-line agents in treating patients with ovarian cancer. A large five-arm phase III randomized trial is currently underway comparing 4 experimental drug regimes to the standard treatment with placitaxel and carboplatinum. This international collaborative trial funded by the MRC in the UK and the NIH in the USA (Protocol identification: GOG0182, ICON5, SWOG-GO182) will better define the role of these agents. The recruitment target is estimated at 5000 patients and is some years away to completion. The various arms of this trial are summarised below. Doses and intervals can be obtained from the MRC.
ARM I
Carboplatin + Paclitaxel
8 cycles (every 3 weeks)
ARM II
Carboplatin + Paclitaxel + Gemcitabine
8 cycles
ARM III
Carboplatin + Paclitaxel + Doxorubicin
8 cycles
ARM IV
Carboplatin + Topotecan
4 cycles
(Paclitaxel IV over 3 hours and Carboplatin IV over 30 minutes â&#x20AC;&#x201C; Day 1) (Chemotherapy as in arm 1. Gemcitabine IV over 30 minutes days 1 and 8) (Chemotherapy as in arm 1. Doxorubicin IV over 1 hour cycles 1,3,5 and 7) (Topotecan IV over 30 minutes on days 1-3. Carboplatin IV over 30 minutes on day 3)
Followed by Carboplatin + Paclitaxel ARM IV Carboplatin + Gemcitabine
4 cycles 4 cycles
(Chemotherapy as in arm 1) (Gemcitabine IV over 30 minutes on days 1 and 8, Carboplatin IV over 30 minutes day 8)
Followed by Carboplatin + Paclitaxel
4 cycles
(Chemotherapy as in arm 1)
The sequential delivery of these drugs either as a single agent or in pairs is aimed at reducing the toxic side effects associated with triple therapy. Research examining the combination of other new regimes is also continuing. The Scottish Randomised Trials in Ovarian Cancer (SCOTROC) has recently reported on the use of docetaxel in combination with carboplatin in advanced diseased compared to standard paclitaxel and carboplatin (Vasey, 2002). Docetaxel is a semi-synthetic taxane with a different toxicity profile to paclitaxel, but with at least similar efficacy. Although survival data are not yet available, the disease-free interval is not significantly different. The use of Docetaxel appears to be associated with less neurotoxicity but greater myelosuppression. The use of epirubicin in combination with carboplatin and paclitaxel as first-line treatment has also been disappointing with a significant increase in toxicity, and has largely been abandoned.
cancer tends to remain confined to the abdominal cavity and spread superficially. A direct effect on the cancer cells would possibly limit systemic side-effects as compared to those treated with high dose intra-venous (i.v) therapy. In addition there may be a pharmacokinetic advantage of IP delivery of some agents such as paclitaxel where IP delivery results in > 1000 fold higher than achieved with IV delivery of the same drug. However, practical difficulties in administrating IP chemotherapy remain, including catheter insertion, intra-peritoneal infections and acceptability to patients. In 1996, a combined SWOG and GOG trial reported a significant improvement in the median survival and in the reported and measured side effects of women receiving 6 cycles of intra-peritoneal cisplatin compared to those receiving it via the IV route (Markman et al, 2001). Patients in both arms of this trail received concomitant IV cyclophosphamide. Of the 546 patients eligible for evaluation, median survival was significantly longer in the IP group versus the IV group (49 months vs. 42 months). In addition, those in the IP arm suffered fewer and milder neurotoxic events relating to tinitus, hearing loss and neuromuscular effects. A further GOG study (GOG 172) has recently been reported (Armstrong et al, 2002). Four hundred and seventeen patients were randomised to receive 6 cycles of either IV paclitaxel and cisplatin or IV
6. Intra-peritoneal therapy Few studies have evaluated the use of intraperitoneal (i.p) therapy in ovarian cancer. Due to the limited penetration of drugs used in this manner, only patients optimally debulked have been assessed in clinical trials. The rationale for this route of therapy is that ovarian
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Bali and Reynolds: The current management of primary ovarian cancer paclitaxel followed by IP cisplatin and IP paclitaxel on day 8. Although the data reported are very immature, there appears to be an improvement in disease-free survival in the IP group (24.3 months vs. 19.3 months) and a trend towards improved overall survival. Interestingly, however, there does appear to be more neurological, gastrointestinal, renal and haematological side effects in the IP treated group. The use of IP chemotherapy remains experimental but given the results of these recent trials further investigation is warranted.
large prospective trials examining the role of new chemotherapeutic agents in treating advanced malignancy and the value of complete surgical staging in early disease, the next few years are likely to change the shape of current management in ovarian cancer. Listed below are some of the recent and anticipated advances in the management of patients presenting with ovarian cancer. All patients suspected to have ovarian cancer should be referred to and managed in cancer centres. This would allow rapid and appropriate recourse to all cancer services that include dedicated radiology, pathology, nursing, oncology and social support. 1) All patients should be considered for recruitment onto clinical trials whether they are locally or nationally run. The National Cancer Research Network (NCRN) in England was established April 2001 to provide the NHS with an infrastructure to support prospective trials of cancer treatments and well designed studies. By improving the speed, quality and integration of research undertaken on a national basis the overall aim is to provide better patient care. 2) The routine use of cross-sectional imaging in ovarian cancer requires further assessment, and given cost and time implications its use should be tailored to individual cases. 3) In light of recent data, selected cases with presumed stage 1 ovarian cancer should have appropriate surgical staging that includes pelvic and para-aortic lymphadenectomy. The use of new chemotherapeutic agents (in addition to standard Paclitaxel and Platinum drugs) should only be considered in the setting of clinical trials. An example of such a trial is the five-arm randomised trial funded by the MRC and NIH, examining the efficacy and side effects of new chemotherapy treatments. There are also trials examining the impact of neoadjuvant chemotherapy although selected patients are offered this outside the setting of a trial (for example those deemed unfit for appropriate surgery or those presenting with stage IV disease).
C. Gene therapy Cancer development and progression is thought to arise from an accumulation of mutations in multiple genes that are important for normal cellular function resulting in uncontrolled proliferation and tumour growth. Therefore the rationale for using gene therapy to treat cancer is the potential targeting of cells at a molecular level. Current gene therapy approaches involve introduction of TSG, pro-apoptotic genes, or factors blocking oncogenic effect with a wide range of viral factors. Different vectors provide different benefits / problems, such as integration of the desired gene into the host genome, limitation of gene size for transfer, induction of host immune reponse and so on. Gene mutations and/or overexpression of mutated p53 (a TSG) have been reported in 30% - 80% of EOCâ&#x20AC;&#x2122;s. Accumulation of this aberrant protein is associated with reduced disease-free interval and overall survival of patients with ovarian cancer. In addition, a few studies also report a significant relationship between loss of functional wild-type p53 and chemoresistant disease. Long term follow up of a phase I/II trial using recombinant wild-type p53 gene replacement with an adenoviral vector has been recently reported (Buller et al, 2002). Patients with recurrent disease in a heavily pretreated population were given either a single dose or multiple doses of IP p53 gene replacement with concurrent Placitaxel and Carboplatin. Median survival was significantly longer in those receiving multiple doses, and almost double that of those treated with palliative radiotherapy or following paclitaxel failure (Buller, 2002). Other genes being targeted include Her-2/neu and BRCA1. Although expertise in this field is expanding, numerous obstacles remain which include the identification of an ideal vector, efficient gene transfer, effective transgene expression and reduction in the host immune response (Russell, 2002).
Acknowledgments We would like to thank Sanjeev Bali and Kopal Tandon for their advice and help in editing this manuscript.
References Aabo K, Adams M, Adnitt P et al, (1998) Chemotherapy in advanced ovarian cancer: Four systematic meta-analyses of individual patient data from 37 randomized trials. Br J Cancer 78, 1479-1487. A report by the expert advisory group on cancer to the chief medical officers of England and Wales. 1995; www.doh.gov.uk Armstrong DK, Bundy BN, Baergen R et al, (2002) Randomized phase II study of intravenous (IV) paclitaxel and cisplatin versus IV paclitaxel, intraperitoneal (IP) cisplatin and IP paclitaxel in optimal stage III epithelial ovarian cancer (OC): A Gynecologic Oncology Group trial (GOG 172). Proc Am Soc Clin Oncol (Abstr 803).
VII. Conclusion Ovarian cancer is the 4th most common cancer amongst women in the UK, and is the leading cause of gynaecological cancer related death in this country. Many women are still managed outside the setting of a cancer centre without appropriate multidisciplinary support and treatment, although various steps to rectify this are underway. The majority of patients with early and advanced disease are not recruited for prospective clinical trials, which may reflect the poor co-ordination of trials at a national level in the UK. With several of these issues now being addressed and anticipated completion of some
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Cancer Therapy Vol 2, page 315 Bast RC, Klug TL, St John E, Jenison E et al, (1983) A radioimmunoassay using a monoclonal antibody to monitor the course of epithelial ovarian cancer. N Engl J Med 309, 883-887. Bristow RE (2000) Surgical standards in the management of ovarian cancer. Curr opin Oncol 12, 474-80. Buller RE, Shahin MS, Horowitz JA et al, (2002) Long term follow-up of patients with recurrent ovarian cancer after Ad p53 gene replacement with SCH 58500. Cancer Gene Ther 9, 567-572. Cancer Services Collaborative: Ovarian cancer-Service improvement guide. 2001; www.nhs/npat Chein CH, Wang FF, Hamilton TC (1994) Transcriptional activation of c-myc proto-oncogene by estrogen in human ovarian cancer cell lines. Mol Cell Endocrinol 99, 11-19. Childers J, Hatch K, Surwit E (1992) The role of laparoscopic lymphadenectomy in the management of cervical carcinoma. Gynecol Oncol 47, 38-43. Davies AP, Jacobs I, Woolas R, Fish A, Oram D (1993) The adnexal mass: benign or malignant? Evaluation of a risk of malignancy index. Br J Obstet Gynaecol 100, 927-931. Eisenhauer EA, Gore M, Neijt JP (1999) Ovarian cancer: should we be managing patients with good and bad prognostic factors in the same manner? Ann Oncol 10, 9-15. Fathalla MF (1971) Incessant ovulation-a factor in ovarian neoplasia? Lancet 17, 163. Forstner R, Hricak H, Occhipinti KA et al, (1995) Ovarian cancer: Staging with CT and MR imaging. Radiology 197, 619-626. Griffith RW. The not-so-silent killer. www.healthandage.com/Phome/gid2=1056 Gross TP, Schlesselman JJ (1994) The estimated risk of oral contraceptive use on the cummulative risk of epithelial ovarian cancer. Obstet Gynecol 83, 419-424. Harries M, Gore M (2002) Part I: Chemotherapy for epithelial ovarian cancer-treatment at first diagnosis. Lancet Oncol 3, 529-536. Hricak H, Chen M, Coakley FV et al, (2000) Complex adnexal masses: Detection and characterization with MR Imaging â&#x20AC;&#x201C; multivariate analysis. Radiology 214, 39-46. Jacobs IJ, Oram DH (1990) Potential screening tests for ovarian cancer. Sharp F, Mason WP, Leake RE (eds). Ovarian cancer. Charman & Hall: 197-205. Kindermann G, Massen V, Kuhn W et al, (1996) Laparoscopic management of ovarian tumours subsequently diagnosed as malignant. J Pelvic Surg 2, 245-251. Kinkel K, Hricak H, Lu Y et al, (2000) US characterization of ovarian masses: a meta-analysis. Radiology 217, 803-811. Kuhu W, Rutke S, Spathe K et al, (2001) Neoadjuvant chemotherapy followed by tumour debulking prolongs survival for patients with poor prognosis in international federation of gynecology and obstetrics stage IIIc ovarian carcinoma. Cancer 92, 2585-2591. Lehner R, Wenzl R, Heinzl H et al, (1998) Influence of delayed staging laparotomy after laparoscopic removal of ovarian masses later found malignant. Obstet Gynecol 92, 967-971. Manolitsas TP, Fowler JM (2001) Role of laparoscopy in the management of the adnexal mass and staging of gynecologic cancers. Clin Obstet Gynecol 44, 495-520. Markman M, Bundy BN, Alberts DS et al, (2001) Phase III trial of standard-dose intravenous cisplatin plus paclitaxel versus moderately high-dose carboplatin followed by intravenous paclitaxel and intraperitoneal cisplatin in small-volume stage III ovarian carcinoma: An intergroup study of the Gynecologic Oncology Group, Sowthwestern Oncology Group, and Eastern Cooperative Oncology Group. J Clin Oncol 19, 1001-1007.
McGuire WP, Hoskins WJ, Brady MF et al, (1996) Cyclophosphamide and cisplatin compared with paclitaxel and cisplatin in patients with stage III and stage IV ovarian cancer. N Engl J Med 334, 1-6. Muggia FM, Braly PS, Brady MF et al, (2000) Phase III randomised study of cisplatin versus paclitaxel versus cisplatin and paclitaxel in patients with suboptimal stage III or stage IV ovarian cancer: a Gynecologic oncology group study. J Clin Oncol 18, 106-115. Nelson BE, Rosenfield AT, Schwartz PE (1993) Preoperative abdominopelvic computed tomographic prediction of optimal cytoreduction in epithelial carcinoma. J Clin Oncol 11, 166172. Noller KL (2002) Estrogen replacement therapy and risk of ovarian cancer. JAMA 288, 368-377. Piccart MJ, Bertelsen K, James K et al, (2000) Randomised intergroup trial of cisplatin-paclitaxel versus cisplatincyclophosphamide in women with advanced ovarian cancer: Three-year result. J Natl Cancer I 92, 699-708. Pomel C, Provencher D, Dauplat J et al, (1995) Laparoscopic staging of early ovarian cancer. Gynecol Oncol 58, 301-306. Rodriguez GC, Nagarsheth NP, Lee KL et al, (2002) Progestininduced apoptosis in the macaque ovarian epithelium: Differential regulation of transforming growth factor-b. J Natl Cancer I 94, 50-60. Rodriguez GC, Walmer DK, Cline M et al, (1998) Effect of progestin on the ovarian epithelium of macaques: cancer prevention through apoptosis? J Soc Gynecol Investig 5, 271-276. Russell W (2002) Adenovirus gene therapy for ovarian cancer. J Natl Cancer I 94, 706-707. Sassone AM, Timor-Tritsch IE, Artner A et al, (1991) Transvaginal sonographic characterization of ovarian disease. Ostet Gynecol 78, 70-76. Schwartz PE, Rutherford MD, Chambers JT et al, (1999) Neoadjuvant chemotherapy for advanced ovarian cancer: Long-term survival. Gynecol Oncol 72, 93-99. Sohaib SA, Sahdev A, Van Trappen P, Jacobs IJ, Reznek RH (2003) Characterization of adnexal mass lesions on MR imaging. AJR Am J Roentgenol 180, 1297-304. The ICON collaborators (1998) ICON2: Randomised trial of single agent carboplatin against three-drug combination of CAP (cyclophosphamide, doxorubicin and cisplatin) in women with ovarian cancer. Lancet 352, 1571-1576. The ICON collaborators (2002) ICON3: Paclitaxel plus carboplatin versus standard chemotherapy with either singleagent carboplatin or cyclophosphamide, doxorubicin and cisplatin in women with ovarian cancer: the ICON3 randomised trial. Lancet 36, 505-515. The ICON collaborators (2003a) International collaborative ovarian neoplasm trial 1 and adjuvant chemotherapy in ovarian neoplasm trial: Two parallel randomized phase III trials of adjuvant chemotherapy in patients with early-stage ovarian carcinoma. J Natl Cancer I 95, 105-112 The ICON collaborators (2003b) International collaborative ovarian neoplasm trial 1: A randomised trial of adjuvant chemotherapy in women with early-stage ovarian cancer. J Natl Cancer I 95, 125-132. Trimbos JB, Vergote I, Bolis G et al. (2003) Impact of adjuvant chemotherapy and surgical staging in early ovarian carcinoma: European organisation for research and treatment of cancer-Adjuvant chemotherapy in ovarian neoplasm trial. J Natl Cancer I 95, 113-125. Vasey PA (2002) Survival and longer-term toxicity results of the SCOTROC study: docetaxel-carboplatin (DC) vs. placitaxelcarboplatin (PC) in epithelial ovarian cancer (EOC). Proc Am Soc Clin Oncol (Abstr 804).
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Bali and Reynolds: The current management of primary ovarian cancer Venktaraman AR (2002) Cancer susceptibility and the functions of BRCA1 and BRCA2. Cell 108, 171-182. Volz J, Koster S, Spacek Z et al, (1999) The influence of pnemoperitoneum used in laparoscopic surgery on an intraabdominal tumour growth. Cancer 86, 770-774. Zanetta G, Chiari S, Rota S et al, (1997) Conservative surgery for stage 1 ovarian cancer in women of childbearing age. Br J Obstet Gynaecol 104, 1030-1035.
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Cancer Therapy Vol 2, page 317 Cancer Therapy Vol 2, 317-328, 2004
Receptor tyrosine kinases as targets for cancer therapy Review Article
Francesca De Bacco, Michela Fassetta and Andrea Rasola* Division of Molecular Oncology, University of Torino Medical School, Candiolo, Italy
__________________________________________________________________________________ *Correspondence: Andrea Rasola, Division of Molecular Oncology, IRCC, Institute for Cancer Research, University of Torino Medical School, Strada Provinciale 142, Km 3.95, 10060 Candiolo (TO), Italy; Phone: +390119933210; Fax: +390119933225; Email: andrea.rasola@ircc.it Key words: Receptor tyrosine kinases, Cancer therapy, Signal transduction Abbreviations: dermatofibrosarcoma protuberans, (DFSP); Epidermal growth factor receptors, (EGFRs); Fibroblast growth factor receptors, (FGFRs); gastrointestinal stromal tumors, (GIST); Hepatocyte growth factor receptor, (HGFR); human epidermal receptors, (HERs); Insulin-like growth factor I receptor, (IGF-IR); monoclonal antibodies, (mAbs); myeloproliferative disorders, (MPDs); nonsmall-cell lung cancer, (NSCLC); oligonucleotides, (ODNs); phosphotyrosine binding, (PTB); Platelet-derived growth factor receptors, (PDGFRs); Receptor tyrosine kinases, (RTKs); small interfering RNAs, (siRNAs); Src homology 2, (SH2); Vascular endothelial growth factor receptors, (VEGFRs) Received: 15 July 2004; Accepted: 7 September 2004; electronically published: September 2004
Summary Receptor tyrosine kinases (RTKs) transduce into the cell a complex network of environmental signals, orchestrating in a tightly regulated fashion mandatory processes for the development of multicellular organisms, such as cell growth, differentiation, migration and survival. Perturbation of RTK signaling caused by genetic or functional alterations results in development and progression of cancer. Therefore, strategies aimed at targeting RTKs are under intensive investigation in order to achieve highly selective anti-tumor devices. In this review we summarize the different approaches directed towards the interception of aberrant RTK activation in cancer therapy.
is frequently deregulated in cancer cells. When mutated, overexpressed or structurally altered, RTKs can become potent oncoproteins, and actually they are aberrantly activated in most tumor types. Hence, RTKs establish a promising class of molecular targets for the design of tailored therapies aimed at the selective eradication of tumor cells.
I. Introduction Tumorigenesis is an extremely complex multistep process, which leads to the pathological expansion of a tissue resulting in morbidity (Green and Evan, 2002). Tumor cells gain an unlimited replicative potential as a result of imbalance among the finely tuned proliferative, growth-inhibitory and apoptotic signals. In parallel, dynamic changes in the host microenvironment are required to convey signals that favor the expansion of the tumor mass. The orchestrated execution of the different strategies operated during tumorigenesis can eventually lead to cell invasion and settling in secondary body sites, i.e. to the acquisition of a malignant phenotype. Cells need to breach several defense barriers in order to acquire the cancer phenotype. Disruption of the tightly regulated network that controls tissue homeostasis occurs as a result of perturbed signal transduction, whereby certain transduction pathways, namely those affecting proliferation, survival, cell migration, invasiveness and angiogenesis, are preferentially targeted by oncogenic changes. Ligand-mediated activation of receptor tyrosine kinases (RTKs) plays a pivotal role in these processes and
A. Structure and function of RTKs RTKs are a class of approximately 60 molecules divided into 20 subfamilies, whose enzymatic activity is the transfer of !-phosphate groups from ATP to the hydroxil group of tyrosine residues on signal transduction molecules. They are transmembrane receptors that share a similar structure (Figure 1): an extracellular domain, usually glycosilated, is connected to the cytoplasmic region by a single transmembrane helix. The extracellular portion contains the binding site for polypeptide growth factors, whereas in the cytoplasmic domain are located the conserved protein tyrosine kinase core and several regulatory sequences (Pawson, 2002). RTKs transduce
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Figure 1. Schematic structure of the main human RTK families. Abbreviations are: TK, tyrosine kinase domain; CRD, cystein-rich domain; LD, leucine domain; FNIII, fibronectin type III-like domain; AB, acidic box; CadhD; cadherin-like domain; LRD, leucine-rich domain; IgD, immunoglubulin-like domain.
signals from the extracellular environment to the cytoplasm, ultimately modulating gene transcription within the nucleus. When strictly controlled, their activity regulates several fundamental cell processes, as metabolism and growth, survival, proliferation, differentiation and migration (Schlessinger, 2000). RTK enzyme activity is stimulated by the selective binding of cognate growth factors. Ligand binding elicits or stabilizes receptor oligomerization, causing the reciprocal transphosphorylation of receptor protomers on tyrosine residues in the activation loop of the catalytic region and in regulatory domains. Thus, receptor autophosphorylation enables full kinase activation, but also provides docking sites for molecular recruitment. In fact, phosphorylated tyrosines are recognized by SH2 (Src homology 2)- and PTB (phosphotyrosine binding)-domain containing proteins, which in turn harbor other interaction motifs and promote the assembly of molecular platforms based upon modular interactions between definite peptide domains (Pawson and Nash, 2000). Signaling molecules in these complexes include adaptor proteins, scaffold and docking molecules and enzymes. Hence, in response to RTK stimulation the formation of molecular piers facilitates the activation of specific effectors, induces the amplification of single transduction pathways and promotes the bridging among different cascades, thus eliciting a network of receptor-dependent effects. Among the best characterized intracellular signaling pathways downstream to RTK activation there are: the MAP kinase cascades, composed by three protein kinases organized in a modular
amplification sequence; the nuclear translocation of the latent cytoplasmic transcription factors STATs; and the stimulation of phosphoinositol metabolism, which generates multiple second messengers (Schlessinger, 2000). The final output of these signaling events is the phosphorylation and activation of specific transcription factors, whose targets are involved in the control of a plethora of metabolic processes, including cell cycle, cell migration, proliferation, differentiation, survival and cell shape. It remains an open question how a given biological response is obtained by the stimulation of relatively common repertoires of signal transducers (Comoglio, 2001). Nonetheless, pathway specificity can be determined through different strategies. These include: the combinatorial recruitment of specific molecular subsets at each step of the pathway; the insulation through scaffolding and subcellular compartimentalization of molecular assemblies; differences in signal threshold caused by variations in input duration and amplitude; differences in the molecular complement of specific signal transducers expressed in a given cell in a certain moment; and signal redundancy due to the cross-talks with other metabolic pathways (Schlessinger, 2000).
B. Deregulation of RTKs in human cancer Deregulated activation of such carefully orchestrated signaling networks can lead to the transformation of RTKs into potent oncoproteins, and more than half of the known
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Cancer Therapy Vol 2, page 319 RTKs are associated with human malignancies (BlumeJensen and Hunter, 2001). Multiple brakes operate as safeguard autoinhibitory mechanisms to ensure a correct RTK activation. The acquisition of oncogenic properties results from relief or perturbation of these control devices, leading to RTKs with constitutive or enhanced signaling capacity. The resulting stimulation of signaling pathways enables cells to survive independently of their environment (Porter and Vaillancourt, 1998) and can make them resistant to genotoxic therapies (Navolanic et al, 2003). This aberrant activity of RTKs can be originated by receptor overexpression or mutation, or by formation of ligand-receptor autocrine loops (Robertson et al, 2000; Trusolino and Comoglio, 2002; Zwick et al, 2002). RTK overexpression can occur following gene amplification (Zwick et al, 2001) or reduced degradation (Petrelli et al, 2002), raising the response of cancer cells to growth factor levels and favoring receptor oligomerization and activation even in the absence of the specific ligand (Zwick et al, 2002). Somatic and germline mutations include chromosomal translocations, which result in fusion protein formed by the RTK catalytic domain and peptides providing constitutive dimerization capabilities (Braun and Shannon, 2004), and gain-of-function point mutations or small deletions either within the extracellular domain or in the catalytic region (Robertson et al, 2000). In addition, mutations in the transmembrane motif can lead to ligandindependent kinase activation (Zwick et al, 2001). The establishment of autocrine-paracrine circuitries requires that a RTK is aberrantly expressed in the presence of its cognate ligand, or vice versa, thus relieving cells from dependence on a paracrine supply of growth factors (Hunter, 2000, Schlessinger, 2000, Trusolino and Comoglio, 2002, Zwick et al, 2002).
antisense oligonucleotides (Figure 2) (Al-Obeidi and Lam, 2000). The success of these approaches implies a dominant role of the oncogenic RTK in subverting cellular signaling networks (oncogene addiction), so that targeted therapies could be specifically detrimental to tumor cells (Hingorani and Tuveson, 2003).
1. Monoclonal immunoconjugates
antibodies
and
Mechanistically, monoclonal antibodies (mAbs) directed against the extracellular domain of RTKs might specifically interfere with ligand binding, hence neutralizing the growth factor-driven signaling, and/or induce receptor downregulation or internalization. It is now evident that mAbs can also alter the intracellular signaling pathway in target cells, leading to growth inhibition or apoptosis (Cragg et al, 1999; Zwick et al, 2001). In addition, mAbs can bind epitopes on cancer cells, eliciting immune responses such as complementmediated cell lysis or antibody-dependent cytotoxicity by macrophages or natural killer cells (Zwick et al, 2002). Furthermore, the use of mAbs in combination therapy sensitizes neoplastic cells to a range of conventional therapeutic regimens such as DNA-damaging chemotherapy or irradiation. Improved response to the traditional chemotherapeutics is generally obtained without additional toxicity. However, conventional therapeutic approaches are in principle highly mutagenic, and should therefore be used with the utmost attention in order not to generate unwanted resistance to targeted agents (Hingorani and Tuveson, 2003). The applicability of mAbs in cancer therapy is reduced by immunogenicity, suboptimal biodistribution and severe side effects. With the aim to circumvent these problems, in the last few years several recombinant technologies were developed. Recombinant molecules include: (I) chimeric or humanized antibodies, composed by fused human and animal portions; (II) bivalent or multivalent antibody derivatives, which simultaneously bind antigens on target tumor cells and on immune effector cells; (III) intracellular antibodies (intrabodies); (IV) single-chain Fv, i.e. antibody sequences that lack constant regions and Fc domains; (V) antibodies linked to toxins (immunotoxins) by chemical coupling or recombinant technology; (VI) immune cells genetically modified for exposing antibodybased receptors on the surface or for secreting therapeutic antibodies. Several of these compounds are undergoing clinical trials (Sanz et al, 2004, Wels et al, 2004).
C. Intervention strategies Since RTKs are major players in the evolution of a variety of cancer types, efforts are underway to develop treatment strategies that target these molecules or their downstream effectors. This approach has great potential as it relies on blocking specific transduction pathways by taking advantage of the molecular differences between normal and tumor cells (Vlahovic and Crawford, 2003). The final goal is to obtain a higher therapeutic index and safety with respect to traditional chemotherapeutics, which induce a generic cytotoxic effect that hardly distinguishes malignant from non-malignant cells (Johnstone et al, 2002). The recent development of a breed of agents selectively directed against RTKs, and their ability to bridle the proliferation of tumor cells expressing the target RTK in vivo, show that inhibition of a deregulated, dominant oncogenic receptor is often enough to slow tumor progression. Strategies towards the prevention or interception of aberrant RTK enzymatic activity include the development of modified protein ligands, as inhibitory monoclonal antibodies or new generation immunoconjugates (Sanz et al, 2004; Wels et al, 2004), and of small inhibitor molecules, as kinase inhibitors or
2. Small-molecule RTK inhibitors A promising approach to inhibit aberrant RTK signaling is the development of small-molecule drugs that selectively interfere with their intrinsic tyrosine kinase activity and block receptor autophosphorylation and activation. The three-dimensional structure of the kinase domain and its modes of regulation have been rather well elucidated by NMR, crystallographic and biochemical approaches for several RTKs (Mohammadi et al, 1996;
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Figure 2. Strategies to selectively prevent RTK signaling. Monoclonal antibodies (mAbs) can be used alone, associated to a toxin (immunotoxin) or to another antibody recognized by effector cells (Bispecific Abs). Engineered receptors (decoy soluble receptors or mini-receptors) or decoy ligands (e.g. NK4) act as dominant negative molecules and abrogate signal transduction pathways. Small tyrosine kinase inhibitors (TKI) compete with ATP in binding the activation domain. Antisense oligonucleotides DNA (ODN)/RNA, small RNA interfering (siRNAs) and ribozymes inhibit RTK transcription and/or translation.
Till et al, 2001; Ekman et al, 2002; Bohmer et al, 2003; Chiara et al, 2003, 2004; Jorissen et al, 2003; Mol et al, 2003; Schiering et al, 2003; Manley et al, 2004). Hence, the rational design of kinase inhibitors can now be pursued by the integration of high-throughput screens, as the use of peptide chips of kinase substrates, with combinatorial chemistry (Schlessinger, 2002, Heldin, 2001, Noble et al, 2004). These small-molecule drugs can be substrate competitive, ATP competitive, i.e. they target the Mg-ATP complex pocket in the enzyme catalytic domain, or bisubstrate competitive (competitive against both the substrate and ATP). Despite the high sequence homology among the ATP binding sites of RTKs, these inhibitors are frequently endowed with an elevated degree of specificity towards their targets (Levitzki, 2002). The first classes of these molecules were natural products, such as quercetin, staurosporine, herbimycin A and genistein. However, their selectivity and efficacy was low, and they have mainly served as starting points for the development of many classes of tyrosine kinase inhibitors. These include: (I) compounds that compete at the ATP binding site of RTKs, as quinazolines, pyridopyrymidines or phenylaminopyrimidines; (II) molecules that act in a non-competitive fashion against ATP or peptide substrates, as indoles or
oxindoles; and (III) tyrphostins, which are competitive inhibitors of RTKs at either or both ATP and substrate binding sites (Al-Obeidi and Lam, 2000). Many of these compounds are moving to clinical trials and they are displaying an enormous clinical potential, either as single agent therapeutics or, more frequently, when supplied in association with more traditional therapeutic approaches.
3. Targeting RTK mRNA The use of antisense oligonucleotides (ODNs), antisense RNA, triple helix or small interfering RNAs (siRNAs) is aimed at the inhibition of RTK transcription and/or translation (Surmacz, 2003). ODNs are short fragments of synthetic DNA or RNA that are designed to interact with the mRNA to block the transcription and the expression of specific target proteins by the production of heteroduplex, recognized and cleaved by RNase H (Marcusson et al, 1999). Even though ODNs can be degraded upon internalization by cellular nucleases, preclinical and clinical studies suggest that antisense therapy could be useful for the treatment of solid tumors (Pawlak et al, 2000). Antisense RNA vectors containing fragments of RTK cDNA cloned in 3"-5" orientation have given promising results in animal models, inducing 320
Cancer Therapy Vol 2, page 321 massive apoptosis of tumor cells. The triple helix approach utilizes an oligoribonucleotide capable of forming a triple helix in target DNA, inhibiting the passage of RNA polymerase and thus the transcription of specific genes (Surmacz, 2003). siRNAs take advantage of the cell RNA interference machinery. Specific enzymes recognize double-strand RNA and cleave it in 21-28 nucleotide fragments (siRNAs) that are incorporated in a protein complex, where they direct the degradation of complementary mRNA sequences (Dorsett and Tuschl, 2004). Recent works report a negative regulation of RTK expression when targeted by siRNA approaches (Bohula et al, 2003; Filleur et al, 2003; Reich et al, 2003). In the following sections we focus on therapeutic strategies targeting RTK families involved in the main tumor phenotypes.
inhibiting the downstream signaling cascades. Pertuzumab is currently in phase II clinical trials (Franklin et al, 2004; Badache and Hynes, 2004). These antibodies could act by inducing receptor down-regulation, antibody-dependent cell cytotoxicity or alteration of vessel development. A better comprehension of their mode of action is highly warranted in order to tailor adequate therapies for the consistent number of non-responsive patients (Menard et al, 2003). Cetuximab (C225, Erbitux) is a human-murine chimeric anti-EGFR antibody that mediates complement fixation and inhibits cell cycle progression and cellular proliferation by receptor down-modulation (Thomas and Grandis, 2004). Cetuximab is used either as a monotherapy or in combination with chemo- and radiotherapy, and is currently in phase II/III clinical trials in non-small cell lung and head-and-neck cancer patients (Janmaat and Giaccone, 2003; Caponigro, 2004; Ciardiello et al, 2004; Laskin and Sandler, 2004). ABX-EGF is a fully humanized monoclonal antibody that binds EGFR with high affinity. It eradicates xenograft tumors in animal models, probably by inactivating EGFR-mediated growth pathways. ABX-EGF displays a good anti-tumoral activity with minimal side effects in phase I trials, and in combination therapy it enhances the activity of chemotherapeutics (Foon et al, 2004). MDX-447 (Medarex) is a bispecific antibody that simultaneously binds to EGFR on target cells and to CD64 on monocytes, macrophages and neutrophils, thus increasing antibodydependent cell-mediated cytotoxicity (Laskin and Sandler, 2004). Among small-molecule EGFR kinase inhibitors, the quinazoline derivatives Gefitinib (Iressa, ZD1839) and Erlotinib (Tarceva, OSI-774) act by selectively preventing EGFR phosphorylation. These compounds inhibit proliferation, cell cycle progression and angiogenesis, and increase apoptosis. Gefitinib has a significant anti-tumor effect in cells that co-express EGFR and TGF# and overexpress HER2/neu (Blackledge and Averbuch, 2004). Gefitinib enhances tumor growth inhibition in combination therapies with paclitaxel or cisplatin (Janmaat and Giaccone, 2003), and it is undergoing phase II clinical trials for the treatment of several solid tumors (Laskin and Sandler, 2004). Erlotinib phase III studies are now ongoing in non-small-cell lung cancer (NSCLC) and pancreatic cancer patients (Herbst, 2003; Thomas and Grandis, 2004). Bispecific inhibitors targeting both EGFR and HER2 (GW572016 and CI-1033) are presently in early clinical trials (Laskin and Sandler, 2004). Antisense oligonucleotides decrease EGFR expression, inhibiting proliferation and inducing apoptosis of EGFR-expressing cells. Although these molecules have anticancer activity in xenograft models, they have not entered clinical testing. The only exception is TP-38 (Laskin and Sandler, 2004), an ODN conjugated to the TGF#/mutated Pseudomonas exotoxin presently in phase I clinical trial for the treatment of head and neck squamous cell (Thomas and Grandis, 2004).
II. Epidermal growth factor receptors (EGFRs) The EGFRs or human epidermal receptors (HERs) form a family of four related RTKs broadly expressed in epithelial and mesenchimal tissues: EGFR (HER1, erbB1), HER2 (neu, erbB2), HER3 (erbB3) and HER4 (erbB4) (Jorissen et al, 2003). They bind six known ligands, including EGF, TGF# and neuregulins (Harris et al, 2003). Ligand binding induces homo- or heterodimerization of HERs, thus eliciting the transduction of cell growth, survival and proliferation signals. HER2/neu lacks any specific ligand, but it is the preferred dimerization partner for the other HERs (Burgess et al, 2003; Ross et al, 2003). HERs are overexpressed and activated in two thirds of all solid tumors (Yano et al, 2003), where they play a pivotal role in angiogenesis, invasion and metastasis (Ritter and Arteaga, 2003). HER aberrant activation correlates with poor prognosis and can result from mutation, overexpression or stimulation through autocrine loops (Burgess et al, 2003). Hence, HERs are promising targets for the development of cancer therapeutics (Dancey and Freidlin, 2003; Laskin and Sandler, 2004), and a variety of anti-EGFR agents and strategies are currently used or are undergoing pre-clinical or clinical trials (Ciardiello and Tortora, 2004; Thomas and Grandis, 2004). The anti-HER2/neu humanized antibody trastuzumab (herceptin) was the first therapeutic agent applied on the basis of the genetic characteristics of a tumor (Zwick et al, 2002). Trastuzumab is now the standard therapy in women with HER2-overexpressing metastatic breast cancer, and in clinical studies it induces long-term stable remission (Ross et al, 2003). Recently, the combination of trastuzumab with chemotherapeutics such as taxanes, platinum salts and gemcitabine has proved to be active also against HER2-overexpressing early-stage breast cancers (Burstein et al, 2003; Spigel and Burstein, 2003; Montemurro et al, 2004). Trastuzumab is currently undergoing clinical trials in non-small cell lung cancer patients (Hirsch and Langer, 2004). Another antiHER2/neu antibody, pertuzumab, could have additional potentialities with respect to trastuzumab, as it blocks HER2/neu association with its partner receptors, thus
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De Bacco et al: Receptor tyrosine kinases as targets for cancer therapy in combination studies and phase III trials in metastatic colorectal cancer have now commenced. ZD6474 potently inhibits several RTKs and is undergoing phase III clinical trials for a variety of solid tumors (Manley et al, 2004; Ferrara et al, 2004; Sridhar and Shepherd, 2003). The development of second-generation kinase inhibitors is largely based upon molecular modeling aimed at targeting the active ATP-binding pocket. AAL993 is a molecule designed upon the PTK787 structure. It displays good biopharmaceutical properties and an excellent oral bioavailability in animal models. Clinical trials have not yet initiated (Manley et al, 2004). CP-547,632 acts specifically against VEGFR-2 tyrosine kinase activity. In vitro it inhibits endothelial cell proliferation and in xenografts it retards tumor growth. Phase I data suggest encouraging safety profile and pharmacokinetic parameters (Sridhar and Shepherd, 2003). Bay 43-9006 is a broad spectrum RTK inhibitor, now in phase III trials for metastatic renal-cell carcinoma (Ferrara et al, 2004). A different approach is constituted by angiozyme, a stabilized ribozyme directed against the pre-mRNA of the VEGFR-1 gene. Angiozyme is now in phase II studies on patients with metastatic colorectal cancer with promising results. The high cost of manufacturing and the necessity for parental application are limiting features of this drug (Manley et al, 2004).
factor receptors (VEGFRs) The formation of new blood vessels from preexisting ones, termed angiogenesis, requires the integration of highly complex and coordinated processes and is crucial for the expansion and progression to malignancy of solid tumors (Carmeliet, 2000). The signaling pathways triggered by binding of vascular endothelial growth factor isoforms (VEGFs) to their cognate receptors (VEGFRs) are mandatory for tumor neovascularization (McMahon, 2000). VEGFs bind two high-affinity RTKs, VEGFR-1 and VEGFR-2, on endothelial cells. VEGFR-1 expression is upregulated by hypoxia and controls the release of tissue-specific growth factors. VEGFR-2 is the major mediator of the mitogenic, angiogenic and permeability-enhancing effects of VEGF (Ferrara et al, 2003). Since formation of solid tumors is angiogenesis dependent, it is expected that blocking angiogenesis will be an efficient therapeutic approach against many tumor types (Ferrara, 2002). VEGFs and VEGFRs are expressed at high levels in many types of human solid tumors, including glioma, lung, breast, renal, ovarian and gastrointestinal tract carcinomas (Ferrara et al, 2004). Strategies for targeting the VEGF signaling utilize a vast array of molecules, and the combination of antiangiogenesis therapies with chemo- or radiotherapy results in greater antitumor effects. Bevacizumab (rhuMab VEGF; Avastin) is a recombinant humanized monoclonal antibody raised against VEGF that potently suppresses angiogenesis and growth of human tumor xenografts in nude mice as a single agent or synergistically with chemotherapy. Phase I clinical trials showed that bevacizumab was relatively non-toxic, and phase II trials displayed encouraging efficacy in metastatic prostate cancer, NSCLC and in colorectal cancer (Sridhar and Shepherd, 2003; Manley et al, 2004). Phase III trials are under way on NSCLC and renal-cell cancer. In metastatic colorectal cancer, Bevacizumab in combination with a 5fluorouracil/leucovorin/irinotecan-based chemotherapy increases response rates, time to disease progression and patient survival and has been recently approved as a firstline treatment (Ferrara et al, 2004). The VEGF-trap is a decoy-soluble receptor created by fusing two distinct extracellular domains of the receptor to an IgG constant region, with the aim of increasing the binding affinity to VEGFR. In vivo the VEGF-trap suppresses tumor growth and vascularization, and at present it is in phase I clinical trials for several different cancer types (Holash et al, 2002; Ferrara et al, 2004). Small-molecule inhibitors target the ATP-binding pocket of VEGFRs. The first generation of these compounds includes indoline derivatives, the phthalazine derivative PTK787 (Vatalanib), and ZD6474, that is based upon a quinazoline template. Despite in vitro inhibition of endothelial cell proliferation and in vivo anti-tumor activity in mice xenografts, indoline derivatives show an unacceptable toxicity profile. PTK787 and ZD6474 are instead well tolerated and have anti-tumor activity in animal models. PTK787 has been used as a single agent or
IV. Platelet-derived growth factor receptors (PDGFRs) PDGFRs are composed by one # and one $ chain that can variously homo- or hetero-dimerize upon binding of five different PDGF isoforms. Their activation induces cell proliferation, survival and migration, and is required for a plethora of biological processes. These include wound healing, regulation of the interstitial fluid pressure and villus formation in the gut. Moreover, PDGFRs are crucial for the development of several cell types, as mesangial cells in the kidney, smooth muscle cells in vessels and alveoli, hair follicle cells and oligodendrocytes (Betsholtz et al, 2001, Heldin et al, 2002). Tumorigenic activation of PDGFRs can occur by gene amplification or mutation in high-grade gliomas (Fleming et al, 1992, Clarke and Dirks, 2003) and in a subset of patients with gastrointestinal stromal tumors (GIST) (Heinrich et al, 2003), or by chromosomal translocation in myeloproliferative disorders (MPDs) (Cortes and Kantarjian, 2004, Magnusson et al, 2001) and in dermatofibrosarcoma protuberans (DFSP) (Shimizu et al, 1999). PDGFR signaling plays important roles in the autocrine stimulation of cancer cells, in the paracrine stimulation of stromal fibroblasts and of perivascular cells, and in angiogenesis (Ostman and Heldin, 2001). The most important inhibitor of PDGFRs is STI571 (Gleevec, Glivec, Imatinib), which is also used to inhibit the oncogenic activity of Kit and Abl in GIST and chronic myelogenous leukemia, respectively (Capdeville et al, 2002). In seminal studies on MPD patients with PDGFRactivating translocations, STI571 causes a persistent disappearance of translocation-positive cells and a rapid normalization of blood counts (Apperley et al, 2002; 322
Cancer Therapy Vol 2, page 323 Magnusson et al, 2002) (Cortes and Kantarjian, 2004). Case reports of DFSP patients display marked tumor reduction, even though not all patients are responsive (Maki et al, 2002) (Rubin et al, 2002). Furthermore, in neuroblastoma and pancreatic adenocarcinoma xenografts, STI571-mediated tumor regression is associated with suppression of PDGFR activity (Hwang et al, 2003; Beppu et al, 2004). An antiangiogenic activity of STI571 has been reported in prostate cancer bone metastases, possibly due to the inhibition of PDGFR-mediated pericyte recruitment (Uehara et al, 2003), and in human ovarian cancers grafted in nude mice, due to apoptosis of tumorassociated endothelial cells (Apte et al, 2004). These results pave the way for the combination of PDGFR inhibitors with anti-endothelial compounds (Erber et al, 2004). As PDGFRs are expressed in the stroma of the majority of tumors, STI571 has been used in animal models that express PDGFRs in stromal and perivascular cells. When supplied as a single agent, STI571 is ineffective on tumor growth. Instead, a striking response is obtained by using STI571 in association with chemotherapeutics. Remarkably, the increased efficacy of chemotherapy is not due to enhanced tumor cell sensitivity or to reduced angiogenesis, but to an augmented drug uptake by tumor cells, possibly related to a PDGFR antagonist-mediated reduction in tumor interstitial fluid pressure (Pietras et al, 2001, 2002, 2003). Recently, other small-molecule PDGFR inhibitors have been developed. PKC412 is effective for treatment of STI571-resistant MPD in animal models (Cools et al, 2003). SU11248 and SU6668 are broad spectrum RTK inhibitors that potently enhance the regression of different PDGFR-expressing tumor types in preclinical models, particularly when supplied in combination with chemo- or radiotherapy (Abrams et al, 2003; Garofalo et al, 2003; Mendel et al, 2003; Schueneman et al, 2003; Marzola et al, 2004). Synthetic agents that bind to PDGFs can antagonize the activation of PDGFRs. One such molecule, GFB-111, binds PDGF with a high degree of selectivity over other growth factors. GFB-111 inhibits PDGFR autophosphorylation and activation of downstream effectors, and abolishes tumor growth in nude mice grafted with different tumor cell lines, possibly interfering with angiogenesis (Sebti and Hamilton, 2000).
V. Insulin-like receptor (IGF-IR)
growth
factor
involved in the development of several malignancies, as carcinomas of breast, prostate, liver, colon, pancreas and lung, and many compounds fully accomplish their tumorigenic potential only in the presence of IGF-IR (Surmacz, 2003). Several strategies to inactivate IGF-IR are being developed. Blocking antibodies or single-chain humanized scFv-Fc immunoconjugates raised against the # subunit of the receptor downregulate IGF-IR, inhibit the survival of cancer cells and cause regression of pancreatic tumor xenografts in mice (Hailey et al, 2002; Maloney et al, 2003; Sachdev et al, 2003). The low-molecular weight kinase inhibitor NVP-ADW742 abrogates the growth of small cell lung cancer cells (Warshamana-Greene et al, 2004), whereas NVP-AEW541 reduces the growth of IGFIR-driven fibrosarcomas in mouse xenografts (GarciaEcheverria et al, 2004). Mutant IGF-IRs delivered to target cells act as dominant-negative receptors. In particular, truncated IGF-IR mutants lacking the $ subunit cause massive apoptosis of cancer cells in animals, reducing tumor growth and metastatization and also displaying bystander effects on neighboring cells (Hongo et al, 2003; Min et al, 2003; Sachdev et al, 2004). Furthermore, since the C terminal tail inhibits the anti-apoptotic activity of the receptor, the expression of this portion of IGF-IR linked to a membrane-anchoring signal (mini-receptor) abrogates tumorigenesis in nude mice by inducing apoptosis of tumor cells (Surmacz, 2003). Alternative strategies to target IGF-IR or IGFs are still in early discovery phases, and include: (I) increase of receptor internalization by molecule that act on chaperones; (II) use of ligand mimetic peptides, whose efficacy in vivo remains to be determined; (III) antisense oligodeoxynucleotides, antisense RNAs, ribozymes or siRNAs, which are currently under intensive investigation in animal models (Bohula et al, 2003a, 2003b; Surmacz, 2003; Grzmil et al, 2004; Nielsen et al, 2004).
VI. Fibroblast growth factor receptors (FGFRs) FGFRs make up a family of four receptor types that bind to 23 different ligands, the fibroblasts growth factors (FGFs), whose prototypic members are acidic FGF (aFGF or FGF-1) and basic FGF (bFGF or FGF-2). FGFs can be secreted from cells or localized on the cell surface, and they also interact with heparin or heparan sulphate proteoglycans (McKeehan et al, 1998). FGFR activation triggers a number of transduction events in epithelial and mesenchimal cells (Groth and Lardelli, 2002). Biological responses include proliferation, differentiation, migration and survival of cells, and also organogenesis, inflammation, hematopoiesis and wound healing (Ornitz and Itoh, 2001; Cronauer et al, 2003). In addition, the activation of FGFRs plays a major role in angiogenesis and in the degradation of extracellular matrix, thus facilitating metastatization (Christofori, 2003; Manetti and Botta, 2003). FGFs and/or FGFRs are aberrantly expressed in several malignancies, and a high FGF serum level correlates with poor prognosis and resistance to chemotherapeutics (Cronauer et al, 2003). An aberrant
I
IGF-IR is an ubiquitously expressed RTK composed of two extracellular # subunits, which bind the IGF-I and IGF-II ligands, and of two cytoplasmic $ subunits that contain the kinase domain. The signaling cascades triggered by the binding of IGFs to the receptor are mainly involved in cell survival and proliferation, but they also affect migration, invasion, and cell-substrate adhesion. Tumorigenesis by IGF-IR requires the activation of antiapoptotic pathways following constitutive overexpression and/or hyperactivation of the receptor, which may also result from autocrine or paracrine stimulation. IGF-IR is 323
De Bacco et al: Receptor tyrosine kinases as targets for cancer therapy tyrosine kinase activity of FGFR1 is generated by chromosomal translocations that generate fusion proteins responsible for myeloproliferative disorders (Braun and Shannon, 2004). The solving of the crystallographic structures of the FGFR TK domain has provided the basis for the rational design of small-molecule kinase inhibitors, some of which are undergoing preclinical trials (Manetti and Botta, 2003). The development of pyrido-pyrimidine derivatives led to the development of an array of agents (PD166285, PD173074, PD161570, PD166866) that selectively inhibit FGFR autophosphorylation and signaling in various cell models (Bansal et al, 2003, Skaper et al, 2000). The association of a broad-spectrum (PD166285) and of a selective (PD173074) kinase inhibitor of this family in combination with photodynamic therapy causes tumor regression in animal models (Manetti and Botta, 2003). The quinazoline derivatives ZD4190 and ZD6474 abrogate the proliferation of endothelial cells, and are currently under preclinical evaluation (Manetti and Botta, 2003). The oxindole derivative compounds SU6668, SU5416 and SU5402 inhibit vascularization and growth of tumor xenografts of diverse origins. They also induce regression of established tumors and abrogation of metastasis formation, with a massive apoptosis of tumor and endothelial cells. Currently SU6668, which is also used in combination with chemotherapeutics, is undergoing phase II clinical trials (Laird et al, 2000; Shaheen et al, 2001; Garofalo et al, 2003; Manetti and Botta, 2003; Udayakamur et al, 2003).
VII. Hepatocyte receptor (HGFR)
growth
The furanosylated indolocarbazole K252a and the pyrrole-indolinone PHA-665752 competitively inhibit the binding of ATP to the Met catalytic domain. Both compounds abrogate the activation of the Met pathway and prevent the in vitro oncogenic properties driven by cMet. Unfortunately, K252a is inactive in vivo (Morotti et al, 2002), whereas the K252a analogues CEP-751 and CEP-701 have to be tested on Met-driven tumors. Instead, PHA-665752 causes growth inhibition or regression of tumors at well-tolerated doses in mouse models (Christensen et al, 2003, Wang et al, 2003). The indoline derivative SU11274 is a highly selective Met inhibitor that induces G1 cell cycle arrest and apoptosis by targeting key regulators of the PI3K pathway (Sattler et al, 2003). Tumorigenesis induced by mutant forms of Met is strictly dependent on ligand stimulation and can therefore be inhibited by HGF antagonists (Comoglio and Trusolino, 2002). An alternatively spliced form of HGF, NK2, antagonizes the growth of Met-expressing melanoma xenografts in NK2-HGF bitransgenic mice, but proved not to be useful for therapeutic development as it facilitates metastasis (Otsuka et al, 2000). Another HGF fragment, NK4, binds but does not activate the receptor, acting as an antagonist of the biological activity of HGF, and also as an angiogenesis inhibitor (Kuba et al, 2000). In animal models, NK4 suppresses tumor growth and inhibits metastasis even in cases of advanced diseases (Maemondo et al, 2002; Matsumoto and Nakamura, 2003). A similar result has been recently obtained with a soluble decoy Met receptor (Michieli et al, 2004). Alternatively, peptides directed against the cytosolic tail of Met can impair the catalytic and biological properties of the receptor, thus providing another selective targeting strategy (Bardelli et al, 1999; Maulik et al, 2002). Furthermore, the comprehension of the machinery that recycles the receptor on plasma membrane (Petrelli et al, 2002) will allow its selective down-modulation. Geldanamycins are antibiotics that interfere with the chaperone function of Hsp90, thus reducing the expression on plasma membrane of several RTKs by not completely understood mechanisms (Neckers et al, 1999). These drugs down-regulate Met protein expression and inhibit HGF/SF-mediated cell motility and invasion in NIH3T3 cells transformed by mutant forms of Met (Webb et al, 2000). The use of receptor as a possible apoptosis enhancer constitutes an attractive therapeutic possibility. We have recently observed that, in ovarian cancer cells overexpressing Met, the apoptotic effect of chemotherapeutics is increased by stimulation with HGF (Rasola et al, 2004). HGF constructs devoid of proinvasive properties could improve the feasibility of this approach (Michieli et al, 2002). Targeting either HGF or Met with recombinant ribozymes carrying antisense sequences (anti-HGF or antiMet U1snRNA/ribozyme) reduces HGF/Met expression and abolishes Met-driven biological responses. When delivered in mouse glioma xenografts, these ribozymes inhibit cancer growth by inducing tumor cell apoptosis and blocking tumor angiogenesis (Abounader et al, 2002).
factor
The HGFR, encoded by the proto-oncogene MET, is functionally deregulated in a variety of cancer types. Met overexpression strictly correlates with higher metastatic potential and poor prognosis in a plethora of aggressive tumors, including thyroid and colorectal carcinomas, whereas its tyrosine kinase activity is increased by germline and somatic mutations in papillary renal, gastric and hepatocellular carcinomas, and in lymph-node metastases of head and neck squamous-cell carcinomas. HGFdependent autocrine loops are found associated with osteosarcomas, rhabdomyosarcomas and breast carcinomas (Danilkovitch-Miagkova and Zbar, 2002; Trusolino and Comoglio, 2002). To date, Met is the only known proto-oncogene whose activation can elicit and orchestrate all the biological programs required for invasive growth, a complex process in which apparently independent events such as proliferation, migration, survival, polarization and matrix degradation, are integrated to confer cells the capability to invade secondary districts. Hence, selective Met inhibitors would be endowed with the unique feature of hampering cancer progression towards malignancy and metastasis spreading. Nevertheless, Met inhibitors are far from being tested in clinical trials, even though in recent years several prototype molecules have been developed in pre-clinical studies.
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Cancer Therapy Vol 2, page 325 CE, Dimitrijevic S, Mahon FX, Marin D, Nikolova Z, Olavarria E, Silberman S, Schultheis B, Cross NC and Goldman JM (2002) Response to imatinib mesylate in patients with chronic myeloproliferative diseases with rearrangements of the platelet-derived growth factor receptor beta. N Engl J Med 347, 481-487. Apte SM, Fan D, Killion JJ and Fidler IJ (2004) Targeting the platelet-derived growth factor receptor in antivascular therapy for human ovarian carcinoma. Clin Cancer Res 10, 897-908. Bansal R, Magge S and Winkler S (2003) Specific inhibitor of FGF receptor signaling: FGF-2-mediated effects on proliferation, differentiation and MAPK activation are inhibited by PD173074 in oligodendrocyte-lineage cells. J Neurosci Res 74, 486-493. Bardelli A, Basile ML, Audero E, Giordano S, Wennstrom S, Menard S, Comoglio PM and Ponzetto C (1999) Concomitant activation of pathways downstream of Grb2 and PI 3-kinase is required for MET-mediated metastasis. Oncogene 18, 1139-1146. Betsholtz C, Karlsson L and Lindahl P (2001) Developmental roles of platelet-derived growth factors. Bioessays 23, 494507. Blackledge G and Averbuch S (2004) Gefitinib ('Iressa', ZD1839) and new epidermal growth factor receptor inhibitors. Br J Cancer 90, 566-572. Blume-Jensen P and Hunter T (2001) Oncogenic kinase signalling. Nature 411, 355-365. Bohmer FD, Karagyozov L, Uecker A, Serve H, Botzki A, Mahboobi S and Dove S (2003) A single amino acid exchange inverts susceptibility of related receptor tyrosine kinases for the ATP site inhibitor STI-571. J Biol Chem 278, 5148-5155. Bohula EA, Salisbury AJ, Sohail M, Playford MP, Riedemann J, Southern EM and Macaulay VM (2003) The efficacy of small interfering RNAs targeted to the type 1 insulin-like growth factor receptor (IGF1R) is influenced by secondary structure in the IGF1R transcript. J Biol Chem 278, 1599115997. Braun BS and Shannon K (2004) The sum is greater than the FGFR1 partner. Cancer Cell 5, 203-204. Burgess AW, Cho HS, Eigenbrot C, Ferguson KM, Garrett TP, Leahy DJ, Lemmon MA, Sliwkowski MX, Ward CW and Yokoyama S (2003) An open-and-shut case? Recent insights into the activation of EGF/ErbB receptors. Mol Cell 12, 541552. Capdeville R, Buchdunger E, Zimmermann J and Matter A (2002) Glivec (STI571, imatinib), a rationally developed, targeted anticancer drug. Nat Rev Drug Discov 1, 493-502. Carmeliet P (2000) Mechanisms of angiogenesis and arteriogenesis. Nat Med 6, 389-395. Chiara F, Bishayee S, Heldin CH and Demoulin JB (2004) Autoinhibition of the Platelet-derived Growth Factor {beta}Receptor Tyrosine Kinase by Its C-terminal Tail. J Biol Chem 279, 19732-19738. Chiara F, Michieli P, Pugliese L and Comoglio PM (2003) Mutations in the met oncogene unveil a "dual switch" mechanism controlling tyrosine kinase activity. J Biol Chem 278, 29352-29358. Christensen JG, Schreck R, Burrows J, Kuruganti P, Chan E, Le P, Chen J, Wang X, Ruslim L, Blake R, Lipson KE, Ramphal J, Do S, Cui JJ, Cherrington JM and Mendel DB (2003) A selective small molecule inhibitor of c-Met kinase inhibits cMet-dependent phenotypes in vitro and exhibits cytoreductive antitumor activity in vivo. Cancer Res 63, 7345-7355. Comoglio PM (2001) Pathway specificity for Met signalling. Nat Cell Biol 3, E161-162.
VIII. Conclusions The development of targeted RTK inhibitors represents a major breakthrough both in the understanding of the molecular mechanisms of cancer and in the rational design of therapeutic tools. Novel-generation compounds will be designed following the integration of combinatorial chemistry, high-throughput screening, genomic and/or proteomic profiling, comprehension of the threedimension molecular structure, together with a better understanding of signal transduction mechanisms. Several important issues need to be addressed in order to maximize the enormous potential of receptor targeting strategies. The efficacy of this approach depends upon the differential magnitude of receptor expression by the tumor vs normal cells, as well as by accessibility of tumor cells to drug administration. Receptor levels might not simply correlate with response, and the selection of potentially responsive patients must be accurately achieved by molecular profiling and by improved methods to find activated receptors. New inhibitors must be carefully designed in order to mitigate toxicity and to increase selectivity, but non-selective compounds could be required in some cases for overcoming redundancies in deregulated signal cascades. The abrogation of signaling cascades at the cell membrane is generally required for a complete inhibition of downstream transduction networks, but in certain situations retaining a subset of signals could be advantageous. In this frame, the development of animal models that better reflect the pathogenesis of human malignancies is highly warranted. Moreover, potential applications for RTK inhibitors in chemoprevention and in the chronic or long-term treatment settings are being investigated. Finally, it remains to be fully understood why does targeted therapy works, as it is not specific for receptor mutations. This possibly depends on the particular sensitivity of tumor cells to the disruption of dominant oncogenic pathways (oncogene addiction). Therefore, tumor resistance could appear following selection of cells no longer addicted to the targeted oncogene. The targeting of cancer cell microenvironment, an accurate molecular characterization of the tumor and the improvement of crossing approaches with more conventional anticancer treatments will help to tailor more adequate and efficient therapies.
Acknowledgements We are indebted with Prof. Paolo M. Comoglio for continuous support.
References Abounader R, Lal B, Luddy C, Koe G, Davidson B, Rosen EM and Laterra J (2002) In vivo targeting of SF/HGF and c-met expression via U1snRNA/ribozymes inhibits glioma growth and angiogenesis and promotes apoptosis. Faseb J 16, 108110. Al-Obeidi FA and Lam KS (2000) Development of inhibitors for protein tyrosine kinases. Oncogene 19, 5690-5701. Apperley JF, Gardembas M, Melo JV, Russell-Jones R, Bain BJ, Baxter EJ, Chase A, Chessells JM, Colombat M, Dearden
325
De Bacco et al: Receptor tyrosine kinases as targets for cancer therapy Comoglio PM and Trusolino L (2002) Invasive growth: from development to metastasis. J Clin Invest 109, 857-862. Cools J, Stover EH, Boulton CL, Gotlib J, Legare RD, Amaral SM, Curley DP, Duclos N, Rowan R, Kutok JL, Lee BH, Williams IR, Coutre SE, Stone RM, DeAngelo DJ, Marynen P, Manley PW, Meyer T, Fabbro D, Neuberg D, Weisberg E, Griffin JD and Gilliland DG (2003) PKC412 overcomes resistance to imatinib in a murine model of FIP1L1PDGFRalpha-induced myeloproliferative disease. Cancer Cell 3, 459-469. Cortes J and Kantarjian H (2004) Beyond chronic myelogenous leukemia: potential role for imatinib in Philadelphia-negative myeloproliferative disorders. Cancer 100, 2064-2078. Cronauer MV, Schulz WA, Seifert HH, Ackermann R and Burchardt M (2003) Fibroblast growth factors and their receptors in urological cancers: basic research and clinical implications. Eur Urol 43, 309-319. Dancey JE and Freidlin B (2003) Targeting epidermal growth factor receptor--are we missing the mark? Lancet 362, 6264. Dorsett Y and Tuschl T (2004) siRNAs: applications in functional genomics and potential as therapeutics. Nat Rev Drug Discov 3, 318-329. Ekman S, Kallin A, Engstrom U, Heldin CH and Ronnstrand L (2002) SHP-2 is involved in heterodimer specific loss of phosphorylation of Tyr771 in the PDGF beta-receptor. Oncogene 21, 1870-1875. Erber R, Thurnher A, Katsen AD, Groth G, Kerger H, Hammes HP, Menger MD, Ullrich A and Vajkoczy P (2004) Combined inhibition of VEGF and PDGF signaling enforces tumor vessel regression by interfering with pericyte-mediated endothelial cell survival mechanisms. Faseb J 18, 338-340. Ferrara N (2002) VEGF and the quest for tumour angiogenesis factors. Nat Rev Cancer 2, 795-803. Ferrara N, Gerber HP and LeCouter J (2003) The biology of VEGF and its receptors. Nat Med 9, 669-676. Ferrara N, Hillan KJ, Gerber HP and Novotny W (2004) Case history: Discovery and development of bevacizumab, an antiVEGF antibody for treating cancer. Nat Rev Drug Discov 3, 391-400. Filleur S, Courtin A, Ait-Si-Ali S, Guglielmi J, Merle C, HarelBellan A, Clezardin P and Cabon F (2003) SiRNA-mediated inhibition of vascular endothelial growth factor severely limits tumor resistance to antiangiogenic thrombospondin-1 and slows tumor vascularization and growth. Cancer Res 63, 3919-3922. Foon KA, Yang XD, Weiner LM, Belldegrun AS, Figlin RA, Crawford J, Rowinsky EK, Dutcher JP, Vogelzang NJ, Gollub J, Thompson JA, Schwartz G, Bukowski RM, Roskos LK and Schwab GM (2004) Preclinical and clinical evaluations of ABX-EGF, a fully human anti-epidermal growth factor receptor antibody. Int J Radiat Oncol Biol Phys 58, 984-990. Garcia-Echeverria C, Pearson MA, Marti A, Meyer T, Mestan J, Zimmermann J, Gao J, Brueggen J, Capraro HG, Cozens R, Evans DB, Fabbro D, Furet P, Porta DG, Liebetanz J, Martiny-Baron G, Ruetz S and Hofmann F (2004) In vivo antitumor activity of NVP-AEW541-A novel, potent and selective inhibitor of the IGF-IR kinase. Cancer Cell 5, 231239. Green DR and Evan GI (2002) A matter of life and death. Cancer Cell 1, 19-30. Groth C and Lardelli M (2002) The structure and function of vertebrate fibroblast growth factor receptor 1. Int J Dev Biol 46, 393-400. Harris RC, Chung E and Coffey RJ (2003) EGF receptor ligands. Exp Cell Res 284, 2-13.
Heinrich MC, Corless CL, Duensing A, McGreevey L, Chen CJ, Joseph N, Singer S, Griffith DJ, Haley A, Town A, Demetri GD, Fletcher CD and Fletcher JA (2003) PDGFRA activating mutations in gastrointestinal stromal tumors. Science 299, 708-710. Heldin CH, Eriksson U and Ostman A (2002) New members of the platelet-derived growth factor family of mitogens. Arch Biochem Biophys 398, 284-290. Hingorani SR and Tuveson DA (2003) Targeting oncogene dependence and resistance. Cancer Cell 3, 414-417. Hirsch FR and Langer CJ (2004) The role of HER2/neu expression and trastuzumab in non-small cell lung cancer. Semin Oncol 31, 75-82. Hunter T (2000) Signaling--2000 and beyond. Cell 100, 113-127. Janmaat ML and Giaccone G (2003) The epidermal growth factor receptor pathway and its inhibition as anticancer therapy. Drugs Today (Barc) 39 Suppl C, 61-80. Johnstone RW, Ruefli AA and Lowe SW (2002) Apoptosis: a link between cancer genetics and chemotherapy. Cell 108, 153-164. Jorissen RN, Walker F, Pouliot N, Garrett TP, Ward CW and Burgess AW (2003) Epidermal growth factor receptor: mechanisms of activation and signalling. Exp Cell Res 284, 31-53. Kuba K, Matsumoto K, Date K, Shimura H, Tanaka M and Nakamura T (2000) HGF/NK4, a four-kringle antagonist of hepatocyte growth factor, is an angiogenesis inhibitor that suppresses tumor growth and metastasis in mice. Cancer Res 60, 6737-6743. Laskin JJ and Sandler AB (2004) Epidermal growth factor receptor: a promising target in solid tumours. Cancer Treat Rev 30, 1-17. Levitzki A (2002) Tyrosine kinases as targets for cancer therapy. Eur J Cancer 38 Suppl 5, S11-18. Magnusson MK, Meade KE, Brown KE, Arthur DC, Krueger LA, Barrett AJ and Dunbar CE (2001) Rabaptin-5 is a novel fusion partner to platelet-derived growth factor beta receptor in chronic myelomonocytic leukemia. Blood 98, 2518-2525. Magnusson MK, Meade KE, Nakamura R, Barrett J and Dunbar CE (2002) Activity of STI571 in chronic myelomonocytic leukemia with a platelet-derived growth factor beta receptor fusion oncogene. Blood 100, 1088-1091. Maki RG, Awan RA, Dixon RH, Jhanwar S and Antonescu CR (2002) Differential sensitivity to imatinib of 2 patients with metastatic sarcoma arising from dermatofibrosarcoma protuberans. Int J Cancer 100, 623-626. Manetti F and Botta M (2003) Small-molecule inhibitors of fibroblast growth factor receptor (FGFR) tyrosine kinases (TK). Curr Pharm Des 9, 567-581. Manley PW, Bold G, Bruggen J, Fendrich G, Furet P, Mestan J, Schnell C, Stolz B, Meyer T, Meyhack B, Stark W, Strauss A and Wood J (2004) Advances in the structural biology, design and clinical development of VEGF-R kinase inhibitors for the treatment of angiogenesis. Biochim Biophys Acta 1697, 17-27. Marcusson EG, Yacyshyn BR, Shanahan WR, Jr. and Dean NM (1999) Preclinical and clinical pharmacology of antisense oligonucleotides. Mol Biotechnol 12, 1-11. Maulik G, Shrikhande A, Kijima T, Ma PC, Morrison PT and Salgia R (2002) Role of the hepatocyte growth factor receptor, c-Met, in oncogenesis and potential for therapeutic inhibition. Cytokine Growth Factor Rev 13, 41-59. McKeehan WL, Wang F and Kan M (1998) The heparan sulfatefibroblast growth factor family: diversity of structure and function. Prog Nucleic Acid Res Mol Biol 59, 135-176. McMahon G (2000) VEGF receptor signaling in tumor angiogenesis. Oncologist 5 Suppl 1, 3-10.
326
Cancer Therapy Vol 2, page 327 Menard S, Pupa SM, Campiglio M and Tagliabue E (2003) Biologic and therapeutic role of HER2 in cancer. Oncogene 22, 6570-6578. Michieli P, Cavassa S, Basilico C, Luca AD, Mazzone M, Asti C, Chiusaroli R, Guglielmi M, Bossu P, Colotta F, Caselli G and Comoglio PM (2002) An HGF MSP chimera disassociates the trophic properties of scatter factors from their pro-invasive activity. Nat Biotechnol 20, 488-495. Michieli P, Mazzone M, Basilico C, Cavassa S, Sottile A, Naldini L and Comoglio PM (2004) Targeting the tumor and its microenvironment by a dual-function decoy Met receptor. Cancer Cell 6, 1-13. Mohammadi M, Schlessinger J and Hubbard SR (1996) Structure of the FGF receptor tyrosine kinase domain reveals a novel autoinhibitory mechanism. Cell 86, 577-587. Mol CD, Lim KB, Sridhar V, Zou H, Chien EY, Sang BC, Nowakowski J, Kassel DB, Cronin CN and McRee DE (2003) Structure of a c-kit product complex reveals the basis for kinase transactivation. J Biol Chem 278, 31461-31464. Morotti A, Mila S, Accornero P, Tagliabue E and Ponzetto C (2002) K252a inhibits the oncogenic properties of Met, the HGF receptor. Oncogene 21, 4885-4893. Navolanic PM, Steelman LS and McCubrey JA (2003) EGFR family signaling and its association with breast cancer development and resistance to chemotherapy (Review). Int J Oncol 22, 237-252. Neckers L, Schulte TW and Mimnaugh E (1999) Geldanamycin as a potential anti-cancer agent: its molecular target and biochemical activity. Invest New Drugs 17, 361-373. Ostman A and Heldin CH (2001) Involvement of platelet-derived growth factor in disease: development of specific antagonists. Adv Cancer Res 80, 1-38. Otsuka T, Jakubczak J, Vieira W, Bottaro DP, Breckenridge D, Larochelle WJ and Merlino G (2000) Disassociation of metmediated biological responses in vivo: the natural hepatocyte growth factor/scatter factor splice variant NK2 antagonizes growth but facilitates metastasis. Mol Cell Biol 20, 20552065. Pawlak W, Zolnierek J, Sarosiek T and Szczylik C (2000) Antisense therapy in cancer. Cancer Treat Rev 26, 333-350. Pawson T (2002) Regulation and targets of receptor tyrosine kinases. Eur J Cancer 38 Suppl 5, S3-10. Pawson T and Nash P (2000) Protein-protein interactions define specificity in signal transduction. Genes Dev 14, 1027-1047. Petrelli A, Gilestro GF, Lanzardo S, Comoglio PM, Migone N and Giordano S (2002) The endophilin-CIN85-Cbl complex mediates ligand-dependent downregulation of c-Met. Nature 416, 187-190. Porter AC and Vaillancourt RR (1998) Tyrosine kinase receptoractivated signal transduction pathways which lead to oncogenesis. Oncogene 17, 1343-1352. Rasola A, Anguissola S, Ferrero N, Gramaglia D, Maffe A, Maggiora P, Comoglio PM and Di Renzo MF (2004) Hepatocyte growth factor sensitizes human ovarian carcinoma cell lines to paclitaxel and cisplatin. Cancer Res 64, 1744-1750. Reich SJ, Fosnot J, Kuroki A, Tang W, Yang X, Maguire AM, Bennett J and Tolentino MJ (2003) Small interfering RNA (siRNA) targeting VEGF effectively inhibits ocular neovascularization in a mouse model. Mol Vis 9, 210-216. Ritter CA and Arteaga CL (2003) The epidermal growth factor receptor-tyrosine kinase: a promising therapeutic target in solid tumors. Semin Oncol 30, 3-11. Robertson SC, Tynan JA and Donoghue DJ (2000) RTK mutations and human syndromes. When good receptors turn bad. Trends Genet 16, 265-271. Ross JS, Fletcher JA, Linette GP, Stec J, Clark E, Ayers M, Symmans WF, Pusztai L and Bloom KJ (2003) The Her-
2/neu gene and protein in breast cancer 2003: biomarker and target of therapy. Oncologist 8, 307-325. Rubin BP, Schuetze SM, Eary JF, Norwood TH, Mirza S, Conrad EU and Bruckner JD (2002) Molecular targeting of platelet-derived growth factor B by imatinib mesylate in a patient with metastatic dermatofibrosarcoma protuberans. J Clin Oncol 20, 3586-3591. Sattler M, Pride YB, Ma P, Gramlich JL, Chu SC, Quinnan LA, Shirazian S, Liang C, Podar K, Christensen JG and Salgia R (2003) A novel small molecule met inhibitor induces apoptosis in cells transformed by the oncogenic TPR-MET tyrosine kinase. Cancer Res 63, 5462-5469. Schiering N, Knapp S, Marconi M, Flocco MM, Cui J, Perego R, Rusconi L and Cristiani C (2003) Crystal structure of the tyrosine kinase domain of the hepatocyte growth factor receptor c-Met and its complex with the microbial alkaloid K-252a. Proc Natl Acad Sci U S A 100, 12654-12659. Schlessinger J (2000) Cell signaling by receptor tyrosine kinases. Cell 103, 211-225. Sebti SM and Hamilton AD (2000) Design of growth factor antagonists with antiangiogenic and antitumor properties. Oncogene 19, 6566-6573. Shimizu A, O'Brien KP, Sjoblom T, Pietras K, Buchdunger E, Collins VP, Heldin CH, Dumanski JP and Ostman A (1999) The dermatofibrosarcoma protuberans-associated collagen type Ialpha1/platelet-derived growth factor (PDGF) B-chain fusion gene generates a transforming protein that is processed to functional PDGF-BB. Cancer Res 59, 37193723. Skaper SD, Kee WJ, Facci L, Macdonald G, Doherty P and Walsh FS (2000) The FGFR1 inhibitor PD 173074 selectively and potently antagonizes FGF-2 neurotrophic and neurotropic effects. J Neurochem 75, 1520-1527. Sridhar SS and Shepherd FA (2003) Targeting angiogenesis: a review of angiogenesis inhibitors in the treatment of lung cancer. Lung Cancer 42 Suppl 1, S81-91. Surmacz E (2003) Growth factor receptors as therapeutic targets: strategies to inhibit the insulin-like growth factor I receptor. Oncogene 22, 6589-6597. Thomas SM and Grandis JR (2004) Pharmacokinetic and pharmacodynamic properties of EGFR inhibitors under clinical investigation. Cancer Treat Rev 30, 255-268. Till JH, Ablooglu AJ, Frankel M, Bishop SM, Kohanski RA and Hubbard SR (2001) Crystallographic and solution studies of an activation loop mutant of the insulin receptor tyrosine kinase: insights into kinase mechanism. J Biol Chem 276, 10049-10055. Trusolino L and Comoglio PM (2002) Scatter-factor and semaphorin receptors: cell signalling for invasive growth. Nat Rev Cancer 2, 289-300. Uehara H, Kim SJ, Karashima T, Shepherd DL, Fan D, Tsan R, Killion JJ, Logothetis C, Mathew P and Fidler IJ (2003) Effects of blocking platelet-derived growth factor-receptor signaling in a mouse model of experimental prostate cancer bone metastases. J Natl Cancer Inst 95, 458-470. Vlahovic G and Crawford J (2003) Activation of tyrosine kinases in cancer. Oncologist 8, 531-538. Wang X, Le P, Liang C, Chan J, Kiewlich D, Miller T, Harris D, Sun L, Rice A, Vasile S, Blake RA, Howlett AR, Patel N, McMahon G and Lipson KE (2003) Potent and selective inhibitors of the Met [hepatocyte growth factor/scatter factor (HGF/SF) receptor] tyrosine kinase block HGF/SF-induced tumor cell growth and invasion. Mol Cancer Ther 2, 10851092. Warshamana-Greene GS, Litz J, Buchdunger E, Hofmann F, Garcia-Echeverria C and Krystal GW (2004) The insulin-like growth factor-I (IGF-I) receptor kinase inhibitor NVPADW742, in combination with STI571, delineates a
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De Bacco et al: Receptor tyrosine kinases as targets for cancer therapy spectrum of dependence of small cell lung cancer on IGF-I and stem cell factor signaling. Mol Cancer Ther 3, 527-535. Webb CP, Hose CD, Koochekpour S, Jeffers M, Oskarsson M, Sausville E, Monks A and Vande Woude GF (2000) The geldanamycins are potent inhibitors of the hepatocyte growth factor/scatter factor-met-urokinase plasminogen activatorplasmin proteolytic network. Cancer Res 60, 342-349. Yano S, Kondo K, Yamaguchi M, Richmond G, Hutchison M, Wakeling A, Averbuch S and Wadsworth P (2003) Distribution and function of EGFR in human tissue and the effect of EGFR tyrosine kinase inhibition. Anticancer Res 23, 3639-3650. Zwick E, Bange J and Ullrich A (2001) Receptor tyrosine kinase signalling as a target for cancer intervention strategies. Endocr Relat Cancer 8, 161-173. Zwick E, Bange J and Ullrich A (2002) Receptor tyrosine kinases as targets for anticancer drugs. Trends Mol Med 8, 17-23.
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Cancer Therapy Vol 2, page 329 Cancer Therapy Vol 2, 329-344, 2004
Imaging of pancreatic cancer: a promise for early diagnosis through targeted strategies Research Article
Zdravka Medarova, Anna Moore* Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, Charlestown, USA
__________________________________________________________________________________ *Correspondence: Anna Moore, Ph.D., MGH/MIT/HMS Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, Bldg. 149, 13th St, Rm. 2301, Charlestown, MA 02129; tel: (617)724-0540; fax: (617)7267422; E-mail:amoore@helix.mgh.harvard.edu Key words: Clinical imaging of pancreatic cancer, Molecular imaging of pancreatic cancer, Abbreviations: Abdominal ultrasonography, (US); charged coupled device, (CCD); Computerized tomography, (CT); dextran coated iron oxide, (CLIO); Endoscopic retrograde cholangiopancreatography, (ERCP); Endoscopic ultrasonography guided fine needle aspiration, (EUS-FNA); fluorescence mediated tomography, (FMT); Magnetic resonance cholangio pancreatography, (MRCP); Magnetic resonance imaging, (MRI); matrix metalloproteinases, (MMPs); multidetector CT, (MDCT); Pancreatic intraepithelial neoplasia, (PanIN); Positron emission tomography, (PET); single photon emission computed tomography, (SPECT) Received: 24 August 2004; Accepted: 08 September 2004; electronically published: September 2004
Summary Pancreatic cancer is a disease characterized by high invasiveness, acute resistance to chemo- and radiotherapy, and consequently represents one of the most difficult malignancies to detect and treat. As a result, the diagnosis of pancreatic cancer is essentially a death sentence. Current strategies for pancreatic cancer detection using noninvasive imaging techniques rely on non-targeted morphological approaches. Combining the present knowledge about the molecular nature of the disease on the one hand, and the capacity of imaging to provide a real-time, global view of processes within a living organism, on the other, molecular imaging has the potential to greatly advance our ability for early detection of pancreatic cancer. This review will discuss a targeted imaging approach to explore the specific molecular events during carcinogenesis.
Despite significant efforts to improve the prognosis associated with this disease, progress towards the management of pancreatic cancer over the past 50 years has been limited and still close to 100% of patients diagnosed with pancreatic cancer will develop metastases and die. This outlook, however, can be improved dramatically by the availability of early diagnostics. An essential element of diagnosis is represented by noninvasive imaging. Imaging techniques are in active development both in the laboratory and the clinic, since the conception of novel approaches ultimately leading to therapy for pancreatic cancer is an urgent priority.
I. Introduction st
At the beginning of the 21 century, pancreatic cancer remains one of the most devastating human cancers, responsible for 30,000 deaths per year in the USA (Jemal et al, 2004). The lack of specific symptoms until late in the pathology, as well as the high proliferative and metastatic potential of the disease hamper early diagnosis and consequently, effective treatment. As a result, disease prognosis is poor, with a 5-year survival rate of only around 4% (Jemal et al, 2004). The only potentially curative treatment remains surgical resection. At the time of presentation, approximately 15-20% of patients have resectable disease. Nevertheless, despite the possibility for radical intervention, due to the aggressive nature of the disease, only 20% of these patients survive to 5 years. Adjuvant chemotherapy and radiation therapy only provide marginal palliative benefits due to the low chemosensitivity of the disease with response rates to conventional agents of less than 10% (Kollmannsberger et al, 1998).
II. course
Pancreatic
cancer:
molecular
The progression to pancreatic cancer appears to follow the Vogelstein model described for colorectal cancers (Vogelstein et al, 1988). Namely, tumorigenesis is the result of the violation of multiple checkpoints
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Medarova and Moore: Imaging of pancreatic cancer as an early diagnosis promise controlling cell proliferation, differentiation, and death. This process, characterized by serial perturbations in a variety of cell signaling pathways, ultimately leads to the acquisition of a survival and growth advantage by select cells which escape the normal safeguards to uncontrollable proliferation and invasiveness, and consequently results in carcinogenesis (Cowgill and Muscarella, 2003). Pancreatic cancer, in particular, is believed to originate from mutations in pancreatic duct cells. Pancreatic intraepithelial neoplasia (PanIN) is defined as the increased incidence of abnormal ductal structures seen in patients with pancreatic carcinoma. The similarity in spatial distribution between PanIN lesions and malignant tumors led to the hypothesis that these lesions in fact represent incipient pancreatic adenocarcinomas. Morphologically, PanIN lesions are quite heterogeneous and can be classified based on graded stages of increasingly dysplastic growth (Bardeesy and DePinho, 2002). Both tumor suppressor genes, responsible for arresting cell growth at critical points in the cellâ&#x20AC;&#x2122;s life, and oncogenes, promoting cell division, have been identified as possible culprits for the development of pancreatic cancer. Some of these have been reviewed in more detail (Cowgill and Muscarella, 2003). One of the most frequently mutated genes in pancreatic cancer is the k-ras oncogene, altered in nearly 90% of pancreatic malignancies (Almoguera et al, 1988). k-ras is a key element in cell-growth signaling cascades, linking growth factor and hormone receptors to downstream mediators of cell proliferation and differentiation. In pancreatic carcinomas, k-ras mutations are generally the first detected alterations in the progression series. In fact, k-ras mutations are seen in about 30% of early pancreatic lesions and increase in frequency with disease progression (Bardeesy and DePinho, 2002). However, k-ras, is also altered in some benign conditions, which makes it a poor candidate for a diagnostic marker. The tumor suppressor gene, p16, which is part of the well-characterized Rb tumor-suppressive pathway, responsible for global cell-cycle regulation, is also commonly mutated in pancreatic malignancy. p16 inactivation has been identified in 27% to 82% of primary tumors (Caldas et al, 1994). Furthermore, p16 inactivation has been linked to prognosis, altered more frequently in short term survivors (85% of the cases) compared to longterm survivors (50% of the cases) (Gerdes et al, 2002). Another tumor suppressor, p53, is inactivated in between 40% and 75% of pancreatic cancers (Pellegata et al, 1994). p53 is a mediator of DNA-damage induced cellcycle arrest and programmed cell death. Interestingly, in pancreatic carcinomas, p53 is often found mutated in association with k-ras mutations, suggesting cooperativity between the two genes in pancreatic cancer tumorigenesis (Scarpa et al, 1993). Still, p53 is a later marker of tumorigenesis, mutated in PanINs which demonstrate a high degree of dysplasia. DPC4, a component of the TGF-beta signaling pathway, is deleted in approximately 50% of pancreatic adenocarcinomas (Hahn et al, 1996). It has been suggested
that mutations in DPC4 correlate with invasiveness (Takaku et al, 1998). Still, the precise role of this tumor suppressor in the development of pancreatic cancer is poorly understood. In addition to classical oncogenes and tumorsuppressor genes implicated in the development of pancreatic cancer, many growth factors and growth factor receptors have been found to be overexpressed in carcinomas of the pancreas. Among them are: the epidermal growth factor (Korc, 1998), vascular endothelial growth factor (Shi et al, 2001), fibroblast growth factor (Yamanaka et al, 1993), as well as multiple cytokines, including transforming growth factor beta (Kleeff et al, 1999), interleukin 1 (Blanchard et al, 2000), interleukin 6 (Saito et al, 1998), tumor necrosis factor alpha (Watanabe et al, 1997), and interleukin 8 (Shi et al, 1999) reviewed in (Li et al, 2004). For some of these molecules, a direct link has been established between their upregulated expression and pancreatic cancer. Vascular endothelial growth factor for instance is a key angiogenic factor regulated by hypoxia, common to most solid tumors, among which is pancreatic cancer (Li et al, 2004). Angiogenesis is one mechanism conducive to local and systemic expansion of the tumor mass and as a result is implicated in primary tumor growth and indirectly, metastasis. Pancreatic cancer is characterized by its high invasivenes and metastatic potential. The molecules involved in the metastatic process fall into two groups: cell adhesion molecules and extracellular proteases. One cell adhesion molecule mutated in pancreatic cancers is Ecadherin (Karayiannakis et al, 2001). E-cadherin couples adjacent cells via E-cadherin bridges and in that way is directly implicated in the transmission of antigrowth signals in response to cell-cell contacts. Loss of Ecadherin function is thus one mechanism leading to cancer cell invasion and metastasis. Adhesion molecules overexpressed in pancreatic cancer include ICAM-1, VCAM-1 (Tempia-Caliera et al, 2002), and integrins (Keleg et al, 2003). Overexpression of these adhesion molecules imparts to cancer cells the ability to migrate and home in to distant sites where they form metastatic lesions. Another class of molecules also implicated in cancer invasiveness and metastasis are extracellular proteases. Extracellular proteases facilitate the invasion of cancer cells into the adjacent stroma, across blood vessels and to a metastatic site by mediating the degradation of the extracellular matrix. Studies on pancreatic cancer have clearly established the tendency for upregulation of proteases, downregulation of protease inhibitors and conversion of inactive zymogens into active enzymes (Coussens and Werb, 1996). Evidence exists for induction of urokinase expression in stromal cells (Cantero et al, 1997) and overexpression of a variety of matrix metalloproteinases (MMPs) in pancreatic cancer (Gress et al, 1995) reviewed in (Keleg et al, 2003). Understanding of the genetic alterations contributing to the progression to pancreatic cancer is an essential starting point for future research, which would hopefully lead to the construction of a detailed map of the intricate molecular interactions characterizing the disease. It is
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Cancer Therapy Vol 2, page 331 clear now that the aggressive nature of the disease necessitates early intervention as the most promising treatment strategy. Commonly used clinical tests for pancreatic cancer tumor markers are serum-based immunoassays for tumor-specific antigens, such as CA199, which is a mucin-associated marker (Ringel and Lohr, 2003). This test, however, is not specific to pancreatic cancer, since elevated marker levels are also present in pancreatitis and other cancers. Therefore, timely diagnosis and characterization of the malignancy through noninvasive imaging of the pancreas is an essential first step towards disease management. Targeted imaging which utilizes the molecular alterations specific to pancreatic cancer, has the potential to advance our capacity for identification and characterization of the malignancy in its initial stages.
some of the drawbacks of abdominal ultrasonography based on the endoscopic positioning of the ultrasound probe adjacent to the pancreas. This allows the detailed assessment of local anatomy and permits the identification of small lesions normally not detected by abdominal ultrasound. The reported sensitivity, specificity, and accuracy of endoscopic ultrasonography are significantly higher than conventional CT, with an accuracy of 85-100%, compared to 64-66% for CT and 61-64% for abdominal ultrasound (Rosch et al, 1992; Palazzo et al, 1993; Yasuda et al, 1993). The superiority of endoscopic ultrasonography over CT is particularly evident for lesions smaller than 3 cm in size. Furthermore, endoscopic ultrasound is a good staging tool for pancreatic cancer. Generally, staging is based on a TNM classification. T stage refers to tumor characteristics including vascular involvement. N stage reflects regional lymph node involvement, and M stage assesses metastatic spread. Endoscopic ultrasound is reliable for the identification of certain types of vascular involvement of the portal and splenic veins into the tumor. However, despite its sensitivity in terms of T and N staging, endoscopic ultrasound is not suitable for assessing M stage due to its limited tissue penetration (Santo, 2004).
III. Imaging of pancreatic cancer A. Clinical imaging of pancreatic cancer Currently pancreatic cancer imaging in the clinic relies on nontargeted morphologically-based modalities. Ultrasonographic methods, such as abdominal ultrasonography, endoscopic ultrasonography, as well as helical CT and MRI have evolved as the major tools for pancreatic cancer detection and staging (Rosewicz and Wiedenmann, 1997; Hosten et al, 2000). More recently, these techniques have been complemented by the biochemically-based detection procedure of FDG-PET.
3. Endoscopic ultrasonography guided fine needle aspiration (EUS-FNA) EUS-FNA combines the ability to visualize primary tumors, lymph nodes, and the liver with the capacity to obtain a tissue sample during the diagnostic procedure. Its sensitivity ranges from 45% to 100%. Its specificity approaches 100% (Pinto et al, 1988). Still the diagnostic benefits of the procedure are limited by the ability to visualize the lesion in the first place, since endoscopic ultrasound cannot differentiate between inflamed and metastasis-bearing lymph nodes as well as focal pancreatitis from a tumor (Hawes et al, 2000). The major advantage of this procedure lies in its relative noninvasiveness. Traditionally, the high mortality and morbidity associated with pancreatidoduodenectomy has discouraged the progress to surgery without tissue diagnosis. The morbidity associated with percutaneous FNA has been minimal, despite fears that percutaneous biopsy would lead to peritoneal seeding of tumor cells along the needle tract (Clarke et al, 2003). Obtaining a tissue sample would allow one to distinguish pancreatitis from malignancy, and in cases where adjuvant chemo- or radiotherapy is implemented, biopsy of a suspected lesion is necessary and beneficial.
1. Abdominal ultrasonography (US) Ultrasonography is the most widely used clinical imaging modality because of its low cost, availability, and safety. Ultrasound images are obtained when highfrequency (>20-kHz) sound waves are emitted from a transducer placed against the skin and the ultrasound is reflected back from the internal organs under examination. Contrast in the images obtained depends on the imaging algorithm used, backscatter, attenuation of the sound, and sound speed. Some of the drawbacks of ultrasound imaging, however, extend from the presence of bone and air artefacts, due to the tendency of air and bone to not transmit sound waves. Consequently ultrasound is characterized by limited depth penetration. Abdominal ultrasonography represents a good initial screening tool. However, US has a fairly low sensitivity and specificity of 67% and 40%, respectively for pancreatic cancer (Rosch, 1995). Furthermore, US is operator-dependent and affected by local artefacts. Nevertheless, US is suitable for detection of tumors over 2 cm in diameter, dilated biliary and pancreatic ducts, and extrapancreatic spread (Furukawa, 2002).
4. Computerized tomography (CT) CT is the most commonly used modality for the initial diagnosis, staging, and evaluation of response to therapy of pancreatic cancer. The reported accuracy of CT in determining that a tumor is unresectable approaches 100%. However, about a third of the cases considered resectable based on CT, in fact are unresectable (Freeny et al, 1988; Freeny et al, 1993; Bluemke et al, 1995). Signal in computed tomography (CT) results from differential absorption of X-rays by component tissues and
2. Endoscopic ultrasonography Endoscopic ultrasonography is a sensitive and reliable modality for the detection, staging, and surgical evaluation of pancreatic cancer. It uses a high-frequency sonographic transducer that is introduced into the gastrointestinal tract by a side-viewing endoscope. This setup permits the operator to obtain real-time crosssectional images of the gastrointestinal wall and soft-tissue (Tamm et al, 2003). Endoscopic ultrasound overcomes 331
Medarova and Moore: Imaging of pancreatic cancer as an early diagnosis promise media, namely, bone, air, fat, and water. Volumetric data are collected as an X-ray source and a detector rotate around the subject. Limiting factors affecting the level of resolution of this imaging modality include the pixel sampling size, the size of the X-ray source, and blurring in the phosphor screen which constitutes an element in the signal detector system (Massoud and Gambhir, 2003). A major drawback of CT is the poor soft tissue contrast, which necessitates the administration of iodinated contrast agents which pass through different tissues at different rates. CT has a relatively high spatial resolution (50µm) and is characterized by fast acquisition times (Weissleder, 2002). CT is a commonly applied clinical imaging modality and is traditionally used as a cancer diagnostic tool. More recently modifications of the CT procedure have shown improved sensitivity and diagnostic accuracy. Helical CT provides thin-section, motion-free images. It permits imaging of the entire pancreas and tissues adjacent to it in different circulatory phases. The different phases are defined by variability in scan delay. The pancreatic phase (scan delay of 40 s) has an enhanced capacity to differentiate between pancreatic parenchyma and blood vessels, compared to portal-vein phase imaging (scan delay 60-70 s) (Bluemke et al, 1995). On the other hand, imaging during the portal-vein phase is best for imaging of liver metastases. Therefore, the assessment of tumor stage and metastasis is derived from a “dual-phase” technique. However, circulation times vary between patients, which is a source of error in the use of this application. The reported accuracy of helical CT for T staging is 77%, for N staging 58%, and for M staging 79% (Zeman et al, 1997). Thin section helical CT reduces the obscuring impact of volume averaging on the detection of small lesions. Typically slices ranging between 3 and 5 mm in thickness are obtained using this procedure (Tamm et al, 2003). In general, helical CT can reveal the presence of the tumor and its location in relation to surrounding structures, such as the superior mesenteric artery and vein, the portal vein, and the coeliac axis. The evaluation of resectability is therefore dependent on the capacity of CT to determine whether the tumor is invading the superior mesenteric artery and the coeliac axis, as well as to detect liver and distant metastases, which is an indicator of unresectability (Li et al, 2004). The latest advance in CT imaging of the pancreas combines volume rendering of CT data with a threedimensional display and is referred to as multidetector CT (MDCT). The advantages of this technique lie in the ability of the operator to optimize the visualization of structures, which allows key elements of the anatomy to be enhanced. Still, the accuracy and reliability of this procedure remain to be determined. Furthermore, its demand for extensive computer memory and relative expense make it less popular (Clarke et al, 2003).
5. Endoscopic retrograde pancreatography (ERCP)
pancreatic duct stenosis. It is recommended for patients who present with symptoms of obstructive jaundice, i.e., renal failure or cholangitis, and is a way of alleviating biliary obstruction. ERCP has several drawbacks as a diagnostic tool. Due to the indirect determination of parenchymal abnormalities, a normal pancreatogram does not exclude the possibility for the presence of a tumor. Chronic pancreatitis and pancreatic cancer cannot be differentiated predictably. Lesions in certain areas of the pancreas are less likely to be detected by this method (Clarke et al, 2003). Improvements in ERCP utilize the capacity of the procedure to obtain tissue specimens from the location of interest. ERCP with secretin stimulation and brush biopsy have been utilized in order to collect material for further analysis (Davis et al, 1975; Warshaw and Fernandez-del Castillo, 1992). However, the use of EUS-FNA as a diagnostic/sampling tool will likely replace ERCP due to its limited invasiveness and enhanced specificity.
6. Magnetic resonance imaging (MRI) The principle behind MRI is founded upon the tendency of unpaired nuclear spins (dipoles), e.g. hydrogen atoms in water and organic molecules, to align themselves along an externally applied magnetic field. This external field is produced by a strong magnet surrounding the subject. Following the magnetic pulse delivered by the magnet, the dipoles return to their baseline orientation. That event is detected as a change in electromagnetic flux and is characterized by a differential rate of magnetic relaxation depending on the local environment. For example, fat and hydrocarbon-rich environments have short relaxation times, whereas aqueous environments have relatively long relaxation times. The measurement of dipole relaxation is translated into an MR signal with contrast provided by the differential nature of relaxation rate (Massoud and Gambhir, 2003).The most commonly used timing parameters are known as T1 and T2 and reflect the differential relaxation of the dipoles in the longitudinal and transverse directions, respectively. Whereas the resolution of MRI is high (10-100 µm), its sensitivity is quite low (10-3-10-5 moles/L). Nevertheless, the capacity to derive both anatomical and molecular/physiologic information simultaneously through MRI make it one of the most promising imaging modalities. Its application in the clinic is expanding despite its relatively high cost. Furthermore, as a research tool, MRI has been used to image specific molecular interactions by the use of chemical agents capable of altering MR signal intensity. Paramagnetic metal cations, such as gadolinium or superaparamagnetic iron oxide nanoparticles have been used as targeted MRI probes (Moore et al, 1997, 2000). Still, the low sensitivity of MRI makes it necessary to deliver very high concentrations of probe at the target site in order to achieve sufficient contrast for reliable imaging. Nevertheless, magnetic resonance imaging has been used for multiple applications included but not limited to cell trafficking (Josephson et
cholangio
ERCP with stent placement is a relatively invasive procedure which identifies visual symptoms of biliary and
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Cancer Therapy Vol 2, page 333 al, 1999; Moore et al, 2002), and imaging of gene expression (Weissleder et al, 2000; Moore et al, 2001). Originally, conventional MRI had a limited diagnostic value for pancreatic cancer due to motion artifacts (respiratory, vascular, peristaltic). Recently, however, the use of more advanced MRI techniques, such as dynamic contrast enhanced MRI, has led to considerable sensitivity levels, surpassing even those reported for dual-phase helical CT. A comparative study found that MRI had an accuracy of 96% for predicting resectability vs. 81% for helical CT (Sheridan et al, 1999). Compared to endoscopic ultrasound, MRI was found to have a positive predictive value of 77% vs. 69% for EUS. In determining resectability, the negative predictive value (defining unresectability) was 76% (Ahmad et al, 2000). Furthermore, MR imaging is reported to have higher sensitivity for small liver metastases compared to CT (Santo, 2004). Additional advantages of MRI over CT derive from the fact that it offers better soft-tissue contrast, prior to the administration of iodinated contrast agents and that images can be acquired in multiple planes. However, CT reportedly provides higher spatial resolution (Tamm et al, 2003). The imaging protocols for detection of pancreatic malignancy involve both T1 and T2-weighted sequences, or dynamically-enhanced T1-weighted sequences. On T1weighted images, the normal pancreas has higher signal intensity than any other abdominal organ. Its short T1 relaxation time has been attributed to the abundant protein and rough endoplasmic reticulum contained within it. Fatsaturated T1-weighted sequences are useful for distinguishing normal from abnormal pancreatic parenchyma (Winston et al, 1995). Also, T1-weighted sequences have been shown to be more efficient at reducing motion artifacts than T2-weighted sequences (Steiner et al, 1989). T2-weighted sequences are typically used to differentiate between benign and malignant liver lesions. Dynamic contrast-enhanced MRI estimates blood flow using a computational algorithm. Its capability to identify pancreatic malignancy rests on the fact that pancreatic cancers are hypovascular relative to normal pancreas (Siegelman et al, 1995). With its high sensitivity and tissue contrast, specifically in applications involving contrast agents, e.g. gadolinium as a T1 contrast agent, and its capability to simultaneously provide anatomical and functional information, as well as by the diversity of its applications, i.e. pancreatography by means of MRCP and angiography by means of dynamic contrast-enhanced MRI, the magnetic resonance imaging modality is likely to replace other imaging tools, such as helical CT for instance, as the method of choice in the diagnosis of pancreatic cancer.
7. Magnetic resonance pancreatography (MRCP)
pancreatic juice and bile and the hypointense signal produced by blood and solid organs (Furukawa, 2002). The sensitivity of MRCP for diagnosis of pancreatic and biliary duct abnormalities is 93-100% (Soto et al, 1995). As a result, MRCP is suitable for the assessment of obstructive jaundice. Still, its limited diagnostic potential necessitates the complementary use of alternative imaging modalities.
8. Positron emission tomography (PET) One of the most sensitive imaging modalities is positron emission tomography (PET). The sensitivity of PET ranges between 10-11 and 10-12 mole/L and is independent of the location depth of the contrastproducing probe. This makes PET a very attractive modality for metabolic/physiological characterization of the tumor microenvironment. The principle of PET relies on the labeling of biological molecules with a positronemitting isotope, such as 15O, 13N, 11C, 18F, 14O, 64Cu, 124I, 76 Br, 82Rb, and 68Ga. This positron-emitting isotope is capable of generating two !-rays by releasing a positron from its nucleus. The released positron subsequently annihilates with an electron in its vicinity which results in the production of two ! rays located at 180째 apart. These emitted rays are detected using scintigraphic equipment, which converts the energy of the ! rays into visible light (Massoud and Gambhir, 2003). Alternatively ! emitting isotopes, such as 99mTc, 111In, 123 I, and 131I, can be used for imaging but require different equipment, namely gamma cameras, which can generate tomographic information by rotating around the subject. This modality is known as single photon emission computed tomography (SPECT). SPECT is at least a log less sensitive than PET, less quantitative compared to PET but allows the simultaneous detection of multiple molecular events since it is capable of detecting several isotopes with different energy ! rays (Massoud and Gambhir, 2003). Unlike the imaging modalities listed above, which largely rely on morphological parameters to detect and assess, PET delivers biochemical/metabolic information about tumor biology. For detection of pancreatic cancer, PET traditionally uses FDG, a glucose analogue, labeled with the radioisotope 18F. The principle behind the preferential uptake of this contrast agent by cancers is the enhanced metabolic activity associated with malignancy. FDG enters cells in the same manner as glucose, and is trapped there after being phosphorylated by endogenous kinases to a form, which cannot be further metabolized. On PET, pancreatic cancer appears as an intense region of radiotracer uptake. The reported values for PET sensitivity and specificity vary greatly and range between 64% and 100% for specificity and 71% to 100% for sensitivity (Zimny and Schumpelick, 2001). The main advantage of PET over other imaging modalities is its enhanced capacity to identify metastatic disease and clarify uncertain CT findings in the liver (Mertz et al, 2000). PET can detect lesions less than 2 cm in diameter (Clarke et al, 2003). One major drawback of PET imaging is its poor spatial resolution and anatomic accuracy compared to MRI
cholangio
MRCP is a noninvasive procedure, which is replacing ERCP for diagnosis of the biliary and pancreatic ducts. It is based on magnetic resonance imaging and utilizes T2 weighted imaging with long echo times to deliver optimal contrast between the hyperintense signal of
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Medarova and Moore: Imaging of pancreatic cancer as an early diagnosis promise and CT. Novel combined PET-CT scanners, however, can overcome that weakness. A case study in which FDG-PET results were superimposed on CT-generated scans permitted the identification of a region of atrophy visible on CT as a tumor by virtue of its increased FDG uptake (Hosten et al, 2000). The application of PET to pancreatic cancer imaging is an exciting and very promising new strategy, particularly in view of the recent progress in developing multimodal PET-CT technology for image collection and analysis. The sensitivity of PET combined with the good spatial resolution of CT could become one of the leading strategies for diagnosis and staging of this malignancy.
1. Nuclear imaging of pancreatic cancer The majority of studies investigating the potential of nuclear imaging for pancreatic cancer targeting have focused on the use of radiolabeled tumor-specific antibodies. A recent investigation uses PAM4 which is an antibody targeting the tumor-associated MUC-1 antigen (Cardillo et al, 2004). The basis for the study is the success of initial clinical trials utilizing 131I- and 99m Tc-lebeled PAM4 whole IgG (Mariani et al, 1995; Gold et al, 2001). Cardillo et al. used a bispecific chimeric antibody consisting of PAM4 Fabâ&#x20AC;&#x2122; and murine anti-indiumdiethylenetriaminepentaacetic acid Fabâ&#x20AC;&#x2122; fragments to target subcutaneously implanted pancreatic cancer employing a pretargeting enhancement strategy. The pretargeting strategy exploits the use of a primary targeting monoclonal antibody carrying a secondary recognition moiety, which is targeted later with a radiolabeled hapten. This system achieves high tumor:nontumor signal ratios after injection of the hapten, and rapid tumor penetration and clearance from the circulation (Figure 1). In addition to the traditional methods for pancreatic cancer imaging by PET involving a radiolabeled glucose analogue, different groups have reported the use of radiolabeled nucleotide or amino-acid tracers for PET or SPECT imaging exploiting the increased metabolic rate of tumors (Seitz et al, 2001; Samnick et al, 2004). These investigations show good tumor localization and kinetics. Furthermore, tumor uptake is directly related to proliferative rate, unlike imaging with radiolabeled glucose analogues. 111 bsRIT-Pretargeted In-pepetide 111 In-pepetide Alone
B. Molecular imaging of pancreatic cancer Molecular imaging is a novel field which attempts to combine the global anatomical/physiologic scale of in vivo imaging with the detailed molecular/cellular scale of biochemistry and cell and molecular biology in order to obtain a visual representation and characterization of biological processes at the cellular/sub-cellular level in living subjects (Massoud and Gambhir, 2003). This approach would allow the unraveling of complex disease pathways, the diagnosis of disease at the earliest causative stages characterized by the first signs of metabolic or molecular disturbance, and the noninvasive real-time monitoring of disease progression as well as response to therapy in authentic physiologic environments. There are very few described attempts at imaging pancreatic cancer using targeted molecular approaches. All of these studies have been conducted in murine models and will be discussed below. 111 bsPAM4-Pretargeted In-cPAM4 IgG 111 In-pepetide Direct-Labeled
Figure 1. Immunoscintigraphy of pancreatic tumor xenografts. Athymic nude mice bearing CaPan1 tumor xenografts were injected with bispecific PAM4 (bsPAM4; 1.5 x 10-10 mol) followed by administration of 111In-peptide (35 ÂľCi; 1.5 x 10-11 mol) 40 h later. A comparison of the images taken at 48 h after injection of radiolabeled peptide was made between these mice and mice that were injected with 111In-cPAM4 whole IgG, or pretargeted with control bsRIT, or given radiolabeled peptide alone. Reproduced from Cardillo et al, 2004 with kind permission from Clinical Cancer Research.
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Cancer Therapy Vol 2, page 335 900nm). Tissue scattering at that wavelength range is minimal and tissue penetrance is highest, thus partially overcoming the difficulties associated with other fluorescence imaging molecules, such as GFP. Targets located deeper into the tissue can be imaged using fluorescence mediated tomography (FMT), which delivers tomographic reconstruction of the image by mathematical modeling of diffusion and scattering (Ntziachristos et al, 2002). This method has achieved resolution of 1-2mm and nanomolar sensitivity (Weissleder, 2002). A study published by the group of Robert Hoffman (Bouvet et al, 2002), generated orthotopic pancreatic tumor models in immunocompromised mice using GFPtransformed human pancreatic cancer cell lines. The contribution of this type of model for the study of pancreatic tumor progression and metastasis is significant in that it allows the real-time tracking of tumor growth and dissemination in a living animal. After orthotopic implantation, tumor development was followed by whole-body optical imaging. Furthermore, consecutive whole body simultaneous images of the primary tumor as well as spleen, bowel, and omentum metastases were obtained and quantitated for up to 64 days (Figure 2). The described model is particularly useful for the assessment of therapeutic progress and the study of tumor progression. However, it has no direct clinical applications due to its reliance on artificial, transgenic strategies for tumor cell labeling.
2. Optical molecular imaging of pancreatic cancer Optical imaging is still an experimental modality for small animal imaging. Optical imaging draws its charm from the fact that it is easy, relatively cheap to perform, and image acquisition times are short. Optical imaging is also relatively sensitive, ranging between 10-9 and 10-17 mole/L, depending on the precise strategy used. Nevertheless, the depth penetration of optical imaging is only 1-2cm into the tissue due to the low efficiency of light transmission through an opaque object and the significant tissue scattering effects on image acquisition. As a general rule, muscle and skin have a high transmission index, whereas highly vascularized tissues have low transmission. Furthermore, the images obtained by optical imaging lack tomographic resolution which is another drawback of this imaging modality. Optical imaging is defined by two main imaging strategies, fluorescence imaging and bioluminescence imaging. Bioluminescence imaging detects photons released from cells genetically engineered to express luciferases- photoroteins which upon contact with their substrate (luciferin or coelentrazine), induce the release of a photon which can then be detected using a charged coupled device (CCD) (Weissleder, 2002). In fluorescence imaging, the contrast agent is illuminated with light of a certain wavelength which results in a shifted-wavelength emission of light from the contrast agent. The most promising strategy to date involves imaging in the near-infrared spectrum (700-
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Figure 2. A, consecutive external whole-body images of internally-growing BxPC-3-GFP tumors. A series of external fluorescence images of the BxPC-3-GFP pancreatic tumor in a single animal was obtained from days 46 to 64 after SOI of BxPC-3-GFP in a nude mouse. B, growth curves for primary pancreatic tumor (P), splenic metastasis (S), omental metastases (O), and bowel metastasis (B) as determined by whole-body imaging. Reproduced from Bouvet et al, 2002 with kind permission from Cancer Research.
design multi-modal imaging probes that would combine the advantages of both methods. In order to target the uMUC-1 antigen on tumor cells in vivo we synthesized an imaging probe that consists of CLIO nanoparticles, modified with Cy5.5 fluorochrome and carrying EPPT peptides (Hussain et al, 1996), specifically recognizing uMUC-1, attached to its dextran coat (Figure 3). The resultant target specific probe designated CLIOEPPT was tested in mice, bilaterally implanted with a uMUC-1-positive and a uMUC-1-negative tumor. The CLIO-EPPT probe demonstrated selective and specific accumulation in the uMUC-1 positive tumors and produced signal on MR and optical images. Quantitation of MR imaging-derived signal intensities indicated a reduction in T2 following injection of the contrast agent (as a measure of probe accumulation) of 53% for pancreatic tumors compared to 13-18% for MUC-1 negative control tumors (Moore et al, 2004). Following these encouraging results, we attempted to apply CLIO-EPPT to the imaging of orthotopicallyimplanted pancreatic cancer. MR imaging of the mouse pancreas, however, represents a challenge not found in the clinical imaging of human pancreas since it is not a whole solid organ but rather a thin, membrane-like tissue spread under the liver, and extending downward and outward under the stomach and intestines. In order to identify the pancreas on an MR image, one has to first localize it within the abdomen and optimize imaging parameters. In a study done by our group, we delineated the mouse pancreas in a live animal within the abdomen and identified crucial “landmarks” which allowed
3. Multimodal NIRF/MR molecular imaging of pancreatic cancer As mentioned earlier, each of the currently available imaging modalities suffers specific drawbacks. Whereas MRI has unlimited depth penetration and high spatial resolution (25-100 µm), its sensitivity is low (10-3–10-5 M). Optical imaging, on the other hand has a high sensitivity (10-9 -10-12 M), but limited depth penetration (<1 cm) and low resolution (2-3 mm) (Massoud and Gambhir, 2003). Therefore, it would be highly beneficial to synthesize imaging probes that would combine these two modalities and take advantage of their best features. Studies by our group utilized a novel multimodal optical/MR approach for detection of subcutaneous and orthotopically implanted human pancreatic tumors in the mouse model. We developed a multimodal imaging probe, specific for various epithelial adenocarcinomas, including pancreatic cancer. The molecular target for our probe is epithelial cell mucin, the product of the MUC-1 gene, which becomes overexpressed and underglycosylated in a variety of malignancies. As a result of these properties, epitopes on underglycosylated MUC-1 (uMUC-1), which are cryptic in the nontransformed state, become exposed and available for targeting by imaging or therapeutic probes. With the recent development of new crosslinked superparamagnetic dextran coated iron oxide nanoparticles (CLIO) for MR imaging (Josephson et al, 1991; Moore et al, 1997) and near-infrared probes (Cy5.5 dye) for optical imaging (Petrovsky et al, 2003), it became possible to
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Figure 3. A, the core protein of the MUC-1 tumor antigen. The immunodominant region of the tandem repeat is recognized by the EPPT1 peptide derived from an ASM2 monoclonal antibody (25). B, synthesis (left) and scheme of the probe (right). C, the absorption spectrum of CLIO-EPPT showed the presence of three peaks corresponding to FITC, Cy5.5, and iron oxide nanoparticles. Reproduced from Moore et al, 2004 with kind permission from Cancer Research.
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Figure 4. (a) Anatomic positioning of the pancreas (P, black frame) within the mouse abdomen. The sacrificed animal was opened with a median incision and the pancreas was exposed carefully. D, duodenum; L, liver; V, stomach; S, spleen. (b-f) Coronal MRI of the pancreas on T1-weighted spin echo sequence without (b and c) and with (d) Gd-DTPA and on T2-weighted sequence (e and f). The pancreas is framed red, delineating the splenic (c and f) and the duodenal (b) parts of the pancreas. Reproduced from Grimm et al, 2003 with kind permission from International Journal of Cancer
differentiating the pancreatic tissue from adjacent gut structures (Grimm et al, 2003) (Figure 4). The tail of the pancreas is closely adjacent to the spleen and the stomach.
The spleen appears as a triangular shaped structure on coronal slices; the stomach presents as an oval with a low (or mixed) signal lumen, continuing to the right into the
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Cancer Therapy Vol 2, page 339 duodenum adjacent to the right lobe of the liver. The pancreatic tail is situated distal of the stomach, partly overlaying the spleen and the upper pole of the left kidney, filling a triangular-shaped space between the stomach, the kidney and the spleen. Following the tail of the pancreas to the right allows easy delineation of the body of the pancreas, located immediately adjacent to the duodenum. Having resolved some of the challenges involved in localization of the structure of the pancreas by MR imaging, we utilized our probe as a tumor targeting tool. For that purpose, we utilized an orthotopic model of pancreatic cancer that we had previously established (Grimm et al, 2003) by delivering CAPAN-2 human pancreatic adenocarcinoma cells directly into the tail of the pancreas in nude mice. Twenty one days following tumor implantation, mice were injected i.v. with CLIOEPPT (10mg Fe/kg) and subjected to MR and NIRF imaging pre- and 24 h post-injection As evidenced in Figure 5A, both on coronal and transverse T2-weighted MR images, there is a clearly
defined area of signal reduction medial to the spleen and superior to the kidney, a region corresponding to the coordinates of the mouse pancreas. Based on the observed MR contrast effects of CLIO-EPPT accumulation in ectopic tumors, the highly localized nature of the observed signal, and the good spatial correlation between coronal and transverse views of the lesion, we hypothesize that this discrete T2 shortening is due to specific accumulation of the probe. Further support for our hypothesis came from the detection of a high-intensity NIRF signal associated with the area of the pancreas in tumor-implanted animals (Figure 5B). The ability to detect orthotopic tumors opens the possibility not only for recognition of lesions growing in a natural physiologic environment but also for monitoring of tumor progression and the collection of time-course data defining tumor response to therapy. To that end, we performed time course imaging of mice orthotopically implanted with human pancreatic adenocarcinoma.
Figure 5. A. Coronal and transverse T2 weighted MR images of a CAPAN-2 pancreatic adenocarcinoma bearing mouse 24h after administration of CLIO-EPPT. Arrowheads point to regions of T2 shortening associated with accumulation of the probe. B. Coronal and saggital light (left), NIRF (middle), and color-coded NIRF (right) images of the same mouse. The visible signal enhancement in the region of the pancreas is representative of probe accumulation.
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Medarova and Moore: Imaging of pancreatic cancer as an early diagnosis promise MR and NIRF imaging utilizing CLIO-EPPT as a tumor contrast agent were performed on 19th and 26th days after tumor implantation. MR imaging was performed before and 24 hours after injection of the CLIO-EPPT probe on both days. As shown in Figure 6A, the implanted growing tumors were identified on MR images
as discrete areas of T2 reduction at both time points in the transverse and coronal planes. NIRF imaging confirmed the presence of CLIO-EPPT-mediated signal associated with the area of the pancreas suggesting the consistent localization of uMUC-1-expressing tumor cells in that region throughout the study (Figure 6B).
Figure 6. A. T2 weighted coronal and transverse MR images of a CAPAN-2 pancreatic adenocarcinoma bearing mouse 24h after CLIOEPPT administration. MR imaging of the same mouse was performed on days 19 and 26 after tumor implantation. Arrowheads point to areas of T2 shortening associated with probe accumulation. B. Light (left), NIRF (middle), and color-coded NIRF (right) images of the same mouse. The signal intensity in the region of the pancreas is evident on both day 19 and day 26 after tumor implantation.
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Cancer Therapy Vol 2, page 341 pancreatic adenocarcinoma: spiral CT assessment with surgical and pathologic correlation. Radiology 197, 381-385. Bouvet M, Wang J, Nardin SR, Nassirpour R, Yang M, Baranov E, Jiang P, Moossa AR, and Hoffman RM (2002) Real-time optical imaging of primary tumor growth and multiple metastatic events in a pancreatic cancer orthotopic model. Cancer Res 62, 1534-1540. Caldas C, Hahn SA, da Costa LT, Redston MS, Schutte M, Seymour AB, Weinstein CL, Hruban RH, Yeo CJ, and Kern SE (1994) Frequent somatic mutations and homozygous deletions of the p16 (MTS1) gene in pancreatic adenocarcinoma. Nat Genet 8, 27-32. Cantero D, Friess H, Deflorin J, Zimmermann A, Brundler MA, Riesle E, Korc M, and Buchler MW (1997) Enhanced expression of urokinase plasminogen activator and its receptor in pancreatic carcinoma. Br J Cancer 75, 388-395. Cardillo TM, Karacay H, Goldenberg DM, Yeldell D, Chang CH, Modrak DE, Sharkey RM, and Gold DV (2004) Improved targeting of pancreatic cancer: experimental studies of a new bispecific antibody, pretargeting enhancement system for immunoscintigraphy. Clin Cancer Res 10, 3552-3561. Clarke DL, Thomson SR, Madiba TE, and Sanyika C (2003) Preoperative imaging of pancreatic cancer: a managementoriented approach. J Am Coll Surg 196, 119-129. Coussens LM, and Werb Z (1996) Matrix metalloproteinases and the development of cancer. Chem Biol 3, 895-904. Cowgill SM, and Muscarella P (2003) The genetics of pancreatic cancer. Am J Surg 186, 279-286. Davis JL, Milligan FD, and Cameron JL (1975) Septic complications following endoscopic retrograde cholangiopancreatography. Surg Gynecol Obstet 140, 365367. Freeny PC, Marks WM, Ryan JA, and Traverso LW (1988) Pancreatic ductal adenocarcinoma: diagnosis and staging with dynamic CT. Radiology 166, 125-133. Freeny PC, Traverso LW, and Ryan JA (1993) Diagnosis and staging of pancreatic adenocarcinoma with dynamic computed tomography. Am J Surg 165, 600-606. Furukawa H (2002) Diagnostic clues for early pancreatic cancer. Jpn J Clin Oncol 32, 391-392. Gerdes B, Ramaswamy A, Ziegler A, Lang SA, Kersting M, Baumann R, Wild A, Moll R, Rothmund M, and Bartsch DK (2002) p16INK4a is a prognostic marker in resected ductal pancreatic cancer: an analysis of p16INK4a, p53, MDM2, an Rb. Ann Surg 235, 51-59. Gold DV, Cardillo T, Goldenberg DM, and Sharkey RM (2001) Localization of pancreatic cancer with radiolabeled monoclonal antibody PAM4. Crit Rev Oncol Hematol 39, 147-154. Gress TM, Muller-Pillasch F, Lerch MM, Friess H, Buchler M, and Adler G (1995) Expression and in-situ localization of genes coding for extracellular matrix proteins and extracellular matrix degrading proteases in pancreatic cancer. Int J Cancer 62, 407-413. Grimm J, Potthast A, Wunder A, and Moore A (2003) Magnetic resonance imaging of the pancreas and pancreatic tumors in a mouse orthotopic model of human cancer. Int J Cancer 106, 806-811. Hahn SA, Schutte M, Hoque AT, Moskaluk CA, da Costa LT, Rozenblum E, Weinstein CL, Fischer A, Yeo CJ, Hruban RH, and Kern SE (1996) DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science 271, 350-353. Hawes RH, Xiong Q, Waxman I, Chang KJ, Evans DB, and Abbruzzese JL (2000) A multispecialty approach to the diagnosis and management of pancreatic cancer. Am J Gastroenterol 95, 17-31.
Currently, we are undertaking studies to evaluate the CLIO-EPPT probe as a diagnostic tool for monitoring the response of orthotopically-implanted pancreatic tumors to various forms of chemotherapy. Our progress on multimodal imaging of pancreatic cancer combines the sensitivity of optical imaging with the high resolution of MR imaging and allows not only the detection of primary tumor and metastases (MUC-1 is overexpressed and underglycosylated on metastatic lesions) but also, potentially, the tracking of tumor progression and response to therapy non-invasively. The development of new therapeutics for pancreatic cancer can be very costly. Therefore, there has been an increased demand for accurate and non-invasive assessment of their effectiveness that can be achieved using our approach.
IV. Future outlook Carcinogenesis of pancreatic cancer is a multistep signal transduction process, in which the most likely early events involve activation of oncogenes, such as k-ras (Kawesha et al, 2000), overexpression of growth signal receptors, the EGFR (Korc, 1998) in particular, and mutations in various tumor suppressor genes, including DPC4 (Takaku et al, 1998), p53 (Rozenblum et al, 1997), and p16 (Gerdes et al, 2002). Based on the currently available information regarding tumor origin and progression, there is an abundance of potential strategies for targeting malignancy, whether for the purpose of imaging or therapy. Monoclonal antibodies to the EGFR, probes/inhibitors of tumor-upregulated proteases, apoptosis-related molecules, such as caspase-3 and tumor necrosis factor family members, all constitute prospective targets and are actively being investigated. Detailed knowledge about these targets and their interactions represents the basis upon which one can design various diagnostic approaches by combining the specificity and selectivity of molecular interrogation of malignancy with the capacity of the presently available imaging modalities to provide a real-time global view of anatomical characteristics and physiologic processes in a living subject. The potential implications of these methodologies for the management of pancreatic cancer become obvious in view of the aggressiveness of the disease and its high mortality rate.
References Ahmad NA, Lewis JD, Siegelman ES, Rosato EF, Ginsberg GG, and Kochman ML (2000) Role of endoscopic ultrasound and magnetic resonance imaging in the preoperative staging of pancreatic adenocarcinoma. Am J Gastroenterol 95, 19261931. Almoguera C, Shibata D, Forrester K, Martin J, Arnheim N, and Perucho M (1988) Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell 53, 549-554. Bardeesy N, and DePinho RA (2002) Pancreatic cancer biology and genetics. Nat Rev Cancer 2, 897-909. Blanchard JA, 2nd, Barve S, Joshi-Barve S, Talwalker R, and Gates LK, Jr. (2000) Cytokine production by CAPAN-1 and CAPAN-2 cell lines. Dig Dis Sci 45, 927-932. Bluemke DA, Cameron JL, Hruban RH, Pitt HA, Siegelman SS, Soyer P, and Fishman EK (1995) Potentially resectable
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Medarova and Moore: Imaging of pancreatic cancer as an early diagnosis promise Hosten N, Lemke AJ, Wiedenmann B, Bohmig M, and Rosewicz S (2000) Combined imaging techniques for pancreatic cancer. Lancet 356, 909-910. Hussain R, Courtenay-Luck NS, and Siligardi G (1996) Structure-function correlation and biostability of antibody CDR-derived peptides as tumour imaging agents. Biomed Pept Proteins Nucleic Acids 2, 67-70. Jemal A, Tiwari RC, Murray T, Ghafoor A, Samuels A, Ward E, Feuer EJ, and Thun MJ (2004) Cancer statistics, 2004. CA Cancer J Clin 54, 8-29. Josephson L, Groman E, and Weissleder R (1991) Contrast agents for magnetic resonance imaging of the liver. Targeted Diagn Ther 4, 163-187. Josephson L, Tung CH, Moore A, and Weissleder R (1999) High-efficiency intracellular magnetic labeling with novel superparamagnetic-Tat peptide conjugates. Bioconjug Chem 10, 186-191. Karayiannakis AJ, Syrigos KN, Polychronidis A, and Simopoulos C (2001) Expression patterns of alpha-, betaand gamma-catenin in pancreatic cancer: correlation with Ecadherin expression, pathological features and prognosis. Anticancer Res 21, 4127-4134. Kawesha A, Ghaneh P, Andren-Sandberg A, Ograed D, Skar R, Dawiskiba S, Evans JD, Campbell F, Lemoine N, and Neoptolemos JP (2000) K-ras oncogene subtype mutations are associated with survival but not expression of p53, p16(INK4A), p21(WAF-1), cyclin D1, erbB-2 and erbB-3 in resected pancreatic ductal adenocarcinoma. Int J Cancer 89, 469-474. Keleg S, Buchler P, Ludwig R, Buchler MW, and Friess H (2003) Invasion and metastasis in pancreatic cancer. Mol Cancer 2, 14. Kleeff J, Ishiwata T, Maruyama H, Friess H, Truong P, Buchler MW, Falb D, and Korc M (1999) The TGF-beta signaling inhibitor Smad7 enhances tumorigenicity in pancreatic cancer. Oncogene 18, 5363-5372. Kollmannsberger C, Peters HD, and Fink U (1998) Chemotherapy in advanced pancreatic adenocarcinoma. Cancer Treat Rev 24, 133-156. Korc M (1998) Role of growth factors in pancreatic cancer. Surg Oncol Clin N Am 7, 25-41. Li D, Xie K, Wolff R, and Abbruzzese JL (2004) Pancreatic cancer. Lancet 363, 1049-1057. Mariani G, Molea N, Bacciardi D, Boggi U, Fornaciari G, Campani D, Salvadori PA, Giulianotti PC, Mosca F, Gold DV, and et al. (1995) Initial tumor targeting, biodistribution, and pharmacokinetic evaluation of the monoclonal antibody PAM4 in patients with pancreatic cancer. Cancer Res 55, 5911s-5915s. Massoud TF, and Gambhir SS (2003) Molecular imaging in living subjects: seeing fundamental biological processes in a new light. Genes Dev 17, 545-580. Mertz HR, Sechopoulos P, Delbeke D, and Leach SD (2000) EUS, PET, and CT scanning for evaluation of pancreatic adenocarcinoma. Gastrointest Endosc 52, 367-371. Moore A, Josephson L, Bhorade RM, Basilion JP, and Weissleder R (2001) Human transferrin receptor gene as a marker gene for MR imaging. Radiology 221, 244-250. Moore A, Marecos E, Bogdanov A, Jr., and Weissleder R (2000) Tumoral distribution of long-circulating dextran-coated iron oxide nanoparticles in a rodent model. Radiology 214, 568574. Moore A, Medarova Z, Potthast A, and Dai G (2004) In vivo targeting of underglycosylated MUC-1 tumor antigen using a multimodal imaging probe. Cancer Res 64, 1821-1827. Moore A, Sun PZ, Cory D, Hogemann D, Weissleder R, and Lipes MA (2002) MRI of insulitis in autoimmune diabetes. Magn Reson Med 47, 751-758.
Moore A, Weissleder R, and Bogdanov A, Jr. (1997) Uptake of dextran-coated monocrystalline iron oxides in tumor cells and macrophages. J Magn Reson Imaging 7, 1140-1145. Ntziachristos V, Tung CH, Bremer C, and Weissleder R (2002) Fluorescence molecular tomography resolves protease activity in vivo. Nat Med 8, 757-760. Palazzo L, Roseau G, Gayet B, Vilgrain V, Belghiti J, Fekete F, and Paolaggi JA (1993) Endoscopic ultrasonography in the diagnosis and staging of pancreatic adenocarcinoma. Results of a prospective study with comparison to ultrasonography and CT scan. Endoscopy 25, 143-150. Pellegata NS, Sessa F, Renault B, Bonato M, Leone BE, Solcia E, and Ranzani GN (1994) K-ras and p53 gene mutations in pancreatic cancer: ductal and nonductal tumors progress through different genetic lesions. Cancer Res 54, 15561560. Petrovsky A, Schellenberger E, Josephson L, Weissleder R, and Bogdanov A, Jr. (2003) Near-infrared fluorescent imaging of tumor apoptosis. Cancer Res 63, 1936-1942. Pinto MM, Avila NA, and Criscuolo EM (1988) Fine needle aspiration of the pancreas. A five-year experience. Acta Cytol 32, 39-42. Ringel J, and Lohr M (2003) The MUC gene family: their role in diagnosis and early detection of pancreatic cancer. Mol Cancer 2, 9. Rosch T (1995) Staging of pancreatic cancer. Analysis of literature results. Gastrointest Endosc Clin N Am 5, 735739. Rosch T, Lorenz R, Braig C, and Classen M (1992) Endoscopic ultrasonography in diagnosis and staging of pancreatic and biliary tumors. Endoscopy 24 Suppl 1, 304-308. Rosewicz S, and Wiedenmann B (1997) Pancreatic carcinoma. Lancet 349, 485-489. Rozenblum E, Schutte M, Goggins M, Hahn SA, Panzer S, Zahurak M, Goodman SN, Sohn TA, Hruban RH, Yeo CJ, and Kern SE (1997) Tumor-suppressive pathways in pancreatic carcinoma. Cancer Res 57, 1731-1734. Saito K, Ishikura H, Kishimoto T, Kawarada Y, Yano T, Takahashi T, Kato H, and Yoshiki T (1998) Interleukin-6 produced by pancreatic carcinoma cells enhances humoral immune responses against tumor cells: a possible event in tumor regression. Int J Cancer 75, 284-289. Samnick S, Romeike BF, Kubuschok B, Hellwig D, Amon M, Feiden W, Menger MD, and Kirsch CM (2004) p-[123I]iodoL-phenylalanine for detection of pancreatic cancer: basic investigations of the uptake characteristics in primary human pancreatic tumour cells and evaluation in in vivo models of human pancreatic adenocarcinoma. Eur J Nucl Med Mol Imaging 31, 532-541. Santo E (2004) Pancreatic cancer imaging: which method? Jop 5, 253-257. Scarpa A, Capelli P, Mukai K, Zamboni G, Oda T, Iacono C, and Hirohashi S (1993) Pancreatic adenocarcinomas frequently show p53 gene mutations. Am J Pathol 142, 1534-1543. Seitz U, Wagner M, Vogg AT, Glatting G, Neumaier B, Greten FR, Schmid RM, and Reske SN (2001) In vivo evaluation of 5-[(18)F]fluoro-2'-deoxyuridine as tracer for positron emission tomography in a murine pancreatic cancer model. Cancer Res 61, 3853-3857. Sheridan MB, Ward J, Guthrie JA, Spencer JA, Craven CM, Wilson D, Guillou PJ, and Robinson PJ (1999) Dynamic contrast-enhanced MR imaging and dual-phase helical CT in the preoperative assessment of suspected pancreatic cancer: a comparative study with receiver operating characteristic analysis. AJR Am J Roentgenol 173, 583-590. Shi Q, Abbruzzese JL, Huang S, Fidler IJ, Xiong Q, and Xie K (1999) Constitutive and inducible interleukin 8 expression by hypoxia and acidosis renders human pancreatic cancer cells
342
Cancer Therapy Vol 2, page 343 more tumorigenic and metastatic. Clin Cancer Res 5, 37113721. Shi Q, Le X, Abbruzzese JL, Peng Z, Qian CN, Tang H, Xiong Q, Wang B, Li XC, and Xie K (2001) Constitutive Sp1 activity is essential for differential constitutive expression of vascular endothelial growth factor in human pancreatic adenocarcinoma. Cancer Res 61, 4143-4154. Siegelman ES, Outwater EK, Vinitski S, and Mitchell DG (1995) Fat suppression by saturation/opposed-phase hybrid technique: spin echo versus gradient echo imaging. Magn Reson Imaging 13, 545-548. Soto JA, Barish MA, Yucel EK, Clarke P, Siegenberg D, Chuttani R, and Ferrucci JT (1995) Pancreatic duct: MR cholangiopancreatography with a three-dimensional fast spin-echo technique. Radiology 196, 459-464. Steiner E, Stark DD, Hahn PF, Saini S, Simeone JF, Mueller PR, Wittenberg J, and Ferrucci JT (1989) Imaging of pancreatic neoplasms: comparison of MR and CT. AJR Am J Roentgenol 152, 487-491. Takaku K, Oshima M, Miyoshi H, Matsui M, Seldin MF, and Taketo MM (1998) Intestinal tumorigenesis in compound mutant mice of both Dpc4 (Smad4) and Apc genes. Cell 92, 645-656. Tamm EP, Silverman PM, Charnsangavej C, and Evans DB (2003) Diagnosis, staging, and surveillance of pancreatic cancer. AJR Am J Roentgenol 180, 1311-1323. Tempia-Caliera AA, Horvath LZ, Zimmermann A, Tihanyi TT, Korc M, Friess H, and Buchler MW (2002) Adhesion molecules in human pancreatic cancer. J Surg Oncol 79, 93100. Vogelstein B, Fearon ER, Hamilton SR, Kern SE, Preisinger AC, Leppert M, Nakamura Y, White R, Smits AM, and Bos JL (1988) Genetic alterations during colorectal-tumor development. N Engl J Med 319, 525-532. Warshaw AL, and Fernandez-del Castillo C (1992) Pancreatic carcinoma. N Engl J Med 326, 455-465.
Watanabe N, Tsuji N, Kobayashi D, Yamauchi N, Akiyama S, Sasaki H, Sato T, Okamoto T, and Niitsu Y (1997) Endogenous tumor necrosis factor functions as a resistant factor against hyperthermic cytotoxicity in pancreatic carcinoma cells via enhancement of the heart shock elementbinding activity of heart shock factor 1. Chemotherapy 43, 406-414. Weissleder R (2002) Scaling down imaging: molecular mapping of cancer in mice. Nat Rev Cancer 2, 11-18. Weissleder R, Moore A, Mahmood U, Bhorade R, Benveniste H, Chiocca EA, and Basilion JP (2000) In vivo magnetic resonance imaging of transgene expression. Nat Med 6, 351355. Winston CB, Mitchell DG, Outwater EK, and Ehrlich SM (1995) Pancreatic signal intensity on T1-weighted fat saturation MR images: clinical correlation. J Magn Reson Imaging 5, 267271. Yamanaka Y, Friess H, Buchler M, Beger HG, Uchida E, Onda M, Kobrin MS, and Korc M (1993) Overexpression of acidic and basic fibroblast growth factors in human pancreatic cancer correlates with advanced tumor stage. Cancer Res 53, 5289-5296. Yasuda K, Mukai H, Nakajima M, and Kawai K (1993) Staging of pancreatic carcinoma by endoscopic ultrasonography. Endoscopy 25, 151-155. Zeman RK, Cooper C, Zeiberg AS, Kladakis A, Silverman PM, Marshall JL, Evans SR, Stahl T, Buras R, Nauta RJ, Sitzmann JV, and al-Kawas F (1997) TNM staging of pancreatic carcinoma using helical CT. AJR Am J Roentgenol 169, 459-464. Zimny M, and Schumpelick V (2001) [Fluorodeoxyglucose positron emission tomography (FDG-PET) in the differential diagnosis of pancreatic lesions]. Chirurg 72, 989-994.
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Cancer Therapy Vol 2, page 345 Cancer Therapy Vol 2, 345-352, 2004
The peripheral benzodiazepine receptor: A target for innovative diagnostic and therapeutic approaches in gastrointestinal oncology Review Article
Kerstin Maaser, Andreas P. Sutter, and Hans Scherübl* Medical Clinic I, Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin, 12200 Berlin, Germany
__________________________________________________________________________________ *Correspondence: Hans Scherübl, Medical Clinic I, Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany, phone: +493084453534, fax: +493084454481, e-mail: hans.scherubl@charite.de Key words: gastrointestinal oncology, peripheral benzodiazepine receptor (PBR) Abbreviations: diazepam binding inhibitor, (DBI); extracellular signal-regulated kinase, (ERK); growth arrest and DNA damage protein, (gadd); mitogen-activated protein kinase, (MAPKinase); peripheral benzodiazepine receptor, (PBR); permeability transition pore, (PTP); positron emission tomography, (PET) Received: 27 August 2004; Accepted: 13 September 2004; electronically published: September 2004
Summary The peripheral benzodiazepine receptor (PBR) plays a role in the regulation of cellular proliferation, immunomodulation, porphyrin transport and heme biosynthesis, regulation of steroidogenesis and apoptosis. The PBR has been implicated in the growth control of various cancers. The PBR displays antiapoptotic and proliferative functions. PBR was found to be overexpressed in several cancers including colorectal, breast, or brain cancers. In colorectal cancer, high PBR overexpression correlates with poor prognosis. PBR-specific ligands were shown to have antiproliferative effects in cancer cells. This review focuses on the functional role of PBR in gastrointestinal cancers. It describes the PBR expression in normal and neoplastic gastrointestinal tissues, presents recent reports on possible diagnostic and prognostic applications of PBR and its ligands, and discusses PBR- and PBR ligandbased therapeutic approaches. Thomas et al, 2000; Casellas et al, 2002; Papadopoulos, 2003). Different endogenous compounds have been identified as ligands of the PBR. The diazepam binding inhibitor (DBI) is a polypeptide which binds to PBR with intermediate affinity (Bovolin et al, 1990). Other PBR binding molecules include the porphyrins protoporphyrin IX, mesoporphyrin, and hemin (Verma et al, 1987). Moreover, PBR binds cholesterol and is involved in its transport to the inner mitochondrial membrane which is the rate-limiting step of steroid biosynthesis (Papadopoulos et al, 1997). Specific synthetic ligands have been widely used to pharmacologically characterize the PBR: the benzodiazepine 4-chlorodiazepam (Ro5-4864), the isoquinoline carboxamide PK 11195, and the indoleacetamide FGIN-1-27, all of which bind with high affinity to the PBR but not to the central benzodiazepine binding site, the GABAA receptor (Le Fur et al, 1983; Kozikowski et al, 1993). The PBR has been implicated in growth control of various cancers for two reasons: First, PBR has been shown to be overexpressed in a variety of tumors, and
I. Introduction The peripheral benzodiazepine receptor (PBR) was first described as a high affinity binding site for diazepam in rat kidney (Braestrup and Squires, 1977). In fact, the receptor is ubiquitously expressed in peripheral tissues as well as in the brain but to very different extents. Its expression levels range from very high in steroidproducing tissues to relatively low in the brain, breast or gut mucosa. The PBR is an evolutionary conserved 18 kD protein which contains five transmembrane domains. It is mainly localized in the outer mitochondrial membrane (Anholt et al, 1986), but has also been detected in the plasma membrane (Garnier et al, 1993), and in the nucleus (Hardwick et al, 1999). In the mitochondrial membrane, PBR is associated with the voltage dependent anion channel (VDAC) and the adenine nucleotid translocator (ANT). VDAC and ANT are known to be part of the permeability transition pore (PTP) which is involved in the initiation and regulation of apoptosis. PBR has been implicated in various cellular processes, including steroidogenesis, immune response, apoptosis, and proliferation (reviewed in Beurdeley345
Maaser et al: PBR as a target for diagnosis and therapy of gastrointestinal cancer second, specific PBR ligands inhibit cancer cell proliferation. Proliferative and antiapoptotic properties have been ascribed to PBR. Direct evidence for the antiapoptotic action of PBR was provided by the findings that transfection and subsequent overexpression of PBR protected cells against UV- or hydrogen peroxide-induced apoptosis (Carayon et al, 1996; Stoebner et al, 2001). Moreover, PBR knockout Leydig cells grew much slower than their wild-type counterparts (Amri et al, 1999). In breast cancer cell lines, the level of PBR expression correlated inversely with the doubling time (Beinlich et al, 2000) and positively with the ability of the tumors to grow in SCID mice (Hardwick et al, 2001). Gastrointestinal cancers comprise biologically different entities. The aim of this report is to give an overview about the occurrence and functional role of PBR in colorectal adenocarcinoma, esophageal adenocarcinoma and squamous cell cancer, and hepatocellular carcinoma. Innovative PBR-based diagnostic, prognostic, and therapeutic approaches will be described.
II. PBR expression in gastrointestinal cancers The association of PBR with cancer development was first suggested when the binding capacities for PBRspecific ligands were found to be increased in cancers in comparison to benign tissues. An increase in PBR binding was detected in a variety of cancers including cancers of the colon (Katz et al, 1990b), liver (Venturini et al, 1998), ovary (Katz et al, 1990a), brain (Cornu et al, 1992), and breast (Beinlich et al, 1999). In the gastrointestinal tract, PBR expression is low in the mucosa of the esophagus (Sutter et al, 2002) and the colorectum (Maaser et al, 2002; Han et al, 2003; Bribes et al, 2004) (Figure 1). In the small intestine, PBR was found to be heterogeneously expressed and a strong expression was detected in the mucosa of the stomach (Bribes et al, 2004; Ostuni et al, 2004). In tissues with a low basal expression, PBR was found to be overexpressed upon neoplastic transformation.
Figure 1. Immunohistochemical detection of PBR overexpression. Immunohistochemical assessment of PBR expression in gastrointestinal cancers in comparison to normal tissues of the same patient. Sections of normal colorectal mucosa (A) and colorectal carcinoma (B), normal esophageal squamous mucosa (C) and esophageal squamous cell carcinoma (D), and liver tissue (E) with nonneoplastic components (no) and hepatocellular carcinoma (HCC) were incubated with monoclonal anti-PBR antibody 8D7. Specific antibody-binding was detected using the APAAP “fast-red system”. Bar = 20 µM (B, D), bar = 50 µM (E).
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Cancer Therapy Vol 2, page 347 The frequency of PBR overexpression is tissue specific. In esophageal squamous cell carcinoma only one third of the patients showed an increase in PBR expression in cancer in comparison to the normal squamous epithelium (Sutter et al, 2002). In patients with colorectal carcinoma 88% of the cancers expressed more PBR than the normal colorectal mucosa of the same patient (Maaser et al, 2002). Preliminary data of our group suggest that PBR upregulation is an early event in colorectal carcinogenesis. PBR overexpression was observed already in early adenomas and persisted even in metastases of colorectal cancers. The results concerning PBR expression in liver tissues are variable. Different groups detected no or weak PBR expression in normal liver tissue but increased in hepatocellular carcinoma (HCC) (Figure 1) (Venturini et al, 1998; Venturini et al, 1999; Sutter et al, 2004b; Bribes et al, 2004). In contrast, Han et al. (2003) detected a heterogenous, partly strong PBR expression in normal liver tissues and no further increase in hepatocellular carcinomas. The increase of PBR expression in cancers has been associated with development and aggressiveness of cancers: PBR was shown to be a negative prognostic marker in stage III colorectal cancer (Maaser et al, 2002). In astrocytoma, PBR expression correlated with the grade of malignancy (Miettinen et al, 1995). The aggressiveness of breast cancer cells correlated not only with the extent of PBR expression but also with its nuclear or perinuclear localization (Hardwick et al, 1999). The fact that PBR was found to be overexpressed in a variety of cancers, not only within the gastrointestinal tract but also in other tumors including breast, brain, ovary, prostate, and lung cancer (Katz et al, 1990a, 1990b; Cornu et al, 1992; Miettinen et al, 1995; Beinlich et al, 1999; Hardwick et al, 1999; Maaser et al, 2002; Sutter et al, 2002; Han et al, 2003), indicates that PBR upregulation is a common feature during malignant transformation. Nevertheless it is noteworthy that in tissues with a relatively high basal PBR expression such as normal adrenal and normal testis, PBR expression tends to be decreased in the respective cancers (Han et al, 2003). PBR was shown to be highly expressed in gastric mucosa (Bribes et al, 2004; Ostuni et al, 2004). Recent data demonstrate that in gastric mucosa PBR expression was functionally coupled to Ca2+-dependent but H+independent chloride secretion possibly involved in gastric mucosa protection (Ostuni et al, 2004). The molecular mechanisms of PBR overexpression have rarely been investigated in neoplasms yet. An important factor may be gene amplification, since it was shown that the PBR gene was amplified in a highly PBR expressing, aggressive breast cancer cell line but not so in a non-aggressive cell line that contained low levels of PBR (Hardwick et al, 2002). Moreover, differences in the expression of transcription factors and usage of promoters have recently been shown for steroidogenic and nonsteroidogenic cell lines expressing different levels of PBR (Giatzakis and Papadopoulos, 2004). However, it is not known yet if these mechanisms are involved in PBR overexpression by gastrointestinal cancers.
Whether PBR overexpression is the cause or the consequence of malignant transformation has yet to be elucidated. However, its overexpression in various cancers points to the growth regulating properties which have been attributed to PBR. PBR has been associated with apoptotic and mitotic processes. It was shown that transfectioninduced PBR overexpression protected lymphocytes against UV-induced apoptosis (Stoebner et al, 2001). The ability of breast cancer cells to grow in SCID mice correlated with PBR expression (Hardwick et al, 2001). In different glioma cell lines it was recently shown that PBR density correlated positively with the proliferation rate but inversely with spontaneous apoptosis (Veenman et al, 2004). PBR!s proliferative and/or apoptosis-protective effects probably contribute to the malignant transformation during carcinogenesis. The abundant PBR overexpression in a variety of cancers qualifies PBR as a target for diagnostic approaches, as a prognostic marker, and as a promising novel therapeutic target.
III. Diagnostic and prognostic value of PBR PBR overexpression proved to be of prognostic relevance in colorectal and breast cancer. In UICC III colorectal cancers a high PBR overexpression correlated with a poor survival of the patients (Maaser et al, 2002). Thus, it will be interesting to investigate the prognostic relevance of PBR overexpression in other gastrointestinal cancers whose tumor biology differs from colorectal carcinoma. The prognostic value of PBR overexpression is not limited to colorectal cancers. Recently, it was shown for breast cancer that a high PBR expression level was significantly correlated with a shorter disease-free survival in lymph node-negative patients (Galiegue et al, 2004). In astrocytoma, the extent of PBR expression correlated with the grade of malignancy and therefore with the survival of the patients (Miettinen et al, 1995). The suitability and efficacy of PBR overexpression to detect gastrointestinal malignancies has not been investigated yet. However, PBR-based imaging by positron emission tomography (PET) has been widely studied for neuroinflammatory and neurodegenerative diseases and the use of radio-labeled PBR-specific ligands has been well established (Starosta-Rubinstein et al, 1987; Junck et al, 1989; Banati et al, 2000; Henkel et al, 2004). Both, in colorectal carcinogenesis and tumor spread PBR is frequently up-regulated (Maaser et al, 2002 and unpublished data). The surrounding tissues of colorectal primary carcinomas and of most metastases express PBR at much lower levels than colorectal cancer cells. This indicates that PBR-based imaging might well identify residual cancer tissues or micrometastases. Therefore, it is promising to apply radio-labeled PBR ligands to detect residual cancer cells or micrometastases. Another diagnostic approach does not rely on the PBR itself, but involves its endogenous ligand diazepam binding inhibitor (DBI). Venturini et al, (1998) showed that the DBI blood level was increased in patients with
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Maaser et al: PBR as a target for diagnosis and therapy of gastrointestinal cancer HCC and recommended the blood DBI level as a marker of hepatocellular carcinogenesis. Taken together, these data suggest that PBR qualifies as a target for both diagnostic and /or prognostic purposes in clinical gastrointestinal oncology.
IV. PBR-based approaches
lonidamine, gemtuzumab ozogamicin, etoposide, doxorubicin, or arsenite in different tumor entities (Ravagnan et al, 1999; Decaudin et al, 2002; Walter et al, 2004). The PBR specificity of these exogenous PBR ligands was shown using structural analogues with no affinity to PBR, which did not affect proliferation, apoptosis, or cell cycle regulation (Maaser et al, 2001; Sutter et al, 2002, 2004b). Nevertheless, the specificity of PBR ligands is discussed controversially since their antiproliferative effects are induced at micromolar ligand concentrations only thereby far exceeding the nanomolar binding affinities. However, several factors like the cellular uptake of the ligands (Sutter et al, 2002), ligand absorption to the serum of the culture medium (Lockhart et al, 2003), or different states of PBR may be responsible for the discrepancy. Kletsas et al recently showed that the antiproliferative effects of PBR ligands were independent of the extent of PBR expression in fibroblast and fibrosarcoma cells suggesting that in these cells PBR ligands target other structures than PBR (Kletsas et al, 2004). As lipophilic agents, synthetic PBR ligands are mainly metabolized in the liver. Diazepam, which also binds to PBR with intermediate affinity, is known to have hepatotoxic side effects. Therefore possible hepatotoxic effects of synthetic PBR ligands have to be investigated. PBR ligands such as Ro5-4864 and PK 11195 were shown to inhibit protoporphyrin IX uptake, suggesting that ligand binding to PBR antagonizes its function in tetrapyrrole transport, possibly leading to cytotoxicity in long-term treatments (Wendler et al, 2003). However, PBR ligands have been safely administered in vivo without short-term toxicity and being well tolerated (Decaudin et al, 2002; Walter et al, 2004). In line with these results, no acute toxicity was observed in rat hepatocytes (Fischer et al, 2001). Nevertheless, long-term effects of PBR ligands should be addressed in future studies. Thus, the studies presented suggest that the model of using PBR ligands is a promising approach in gastrointestinal oncology. Yet it is necessary to understand the exact mechanisms and signaling pathways of PBR ligand action to build a rational base for the use of PBR ligands. Future approaches may include the use of existing or new PBR ligands alone or in combination with other anti-neoplastic drugs.
therapeutic
The abundant PBR overexpression in colorectal and other cancers qualifies PBR as a target for tumor-specific therapies. There are different PBR-based therapeutic approaches that warrant testing. Many photosensitizing agents used for photodynamic therapy are structurally related to protoporphyrin IX, an endogenous ligand of PBR. Sensitivities of tumor cell lines to photodynamic therapy with porphyrins correlate positively with their densities of PBR, suggesting that porphyrin-based photodynamic therapy is mediated by PBR (Verma et al, 1998). Photodynamic therapy has already been successfully applied in the treatment of Barrett!s mucosa as well as for dysplasia and early cancer of the esophagus (Prosst et al, 2003). A direct approach to PBR-based therapy might involve the modulation of PBR expression. Following the hypothesis that PBR displays antiapoptotic and proliferative properties, a depletion of PBR expression might lead to apoptosis or abrogation of cell division. Papadopoulos and co-workers, (2000) showed that PBR expression can be reduced by ginkgolide B, a component of a Ginkgo biloba leaf extract. The decrease of PBR expression was associated with reduced cell proliferation and reduced tumor weight of xenografts implanted in nude mice. Moreover, the high PBR expression in cancer could be used to direct chemotherapeutics to neoplastic tissues and thereby to increase the tumor specificity of chemotherapeutics. Enhanced cytotoxicity and increased tumor selectivity were shown for melphalan or gemcitabine in brain tumors, when these agents were coupled to synthetic PBR ligands (Kupczyk-Subotkowska et al, 1997; Guo et al, 2001). In addition to the use of PBR ligands as a vehicle for chemotherapeutics, the ligands were shown to have antineoplastic potential themselves. The synthetic PBR ligands were shown to directly inhibit proliferation or to sensitize cancer cells to cytostatics. Synthetic PBR ligands such as PK 11195, FGIN-1-27, and Ro5-4864 induced apoptosis and cell cycle arrest in colorectal and esophageal cancer cells (Maaser et al, 2001; Sutter et al, 2002), in hepatocellular carcinoma cells (Sutter et al, 2004b), as well as in several other tumor entities including breast, melanoma, testis, and astrocytoma (Garnier et al, 1993; Neary et al, 1995; Landau et al, 1998; Beinlich et al, 1999; Carmel et al, 1999). Several studies showed indirect antineoplastic effects of PBR-specific ligands demonstrating that PBR ligands increased apoptosis induced by other chemotherapeutics. In hepatocellular carcinoma PK 11195 and FGIN-1-27 enhanced the chemosensitivity to paclitaxel, docetaxel, doxorubicin, and the Bcl-2 inhibitor HA14-1 (Sutter et al, 2004b). Similar sensitizing effects were observed by the use of anti-CD95,
V. Signaling pathways modulated by PBR ligands A crucial step in the apoptotic process is the opening of the permeability transition pore (PTP) in the mitochondrial membrane. The PBR is located in the outer mitochondrial membrane being associated with the PTP. This localization suggests that PBR ligands induce apoptosis by affecting the PTP. In fact, it was shown that PBR ligands induce swelling of mitochondria and a decrease of the mitochondrial membrane potential (Maaser et al, 2001; Sutter et al, 2002). The functional relevance of PTP opening in PBR ligand-induced apoptosis was shown by using cyclosporin A. Cyclosporin A is known to inhibit
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Cancer Therapy Vol 2, page 349 the opening of the PTP and was shown to prevent PBR ligand-induced decrease of the mitochondrial membrane potential and subsequent apoptosis (Sutter et al, 2002). However, it still remains to be investigated whether PBR ligands directly affect the PTP, e.g. by changing the conformation of the PBR, or by homogeneous or heterogeneous protein interactions, or if additional signaling pathways are required. Moreover, the PTP is under the control of proteins of the Bcl-2 family. A functional interaction of PBR with the anti-apoptotic protein Bcl-2 has been suggested (Marchetti et al, 1996). In line with this, PBR ligand-induced apoptosis was shown to be associated with a decrease of Bcl-2 expression in hepatocellular carcinoma cells (Sutter et al, 2004b) and hepatic stellate cells (Fischer et al, 2001). However, in other tissues, such as pancreatic islet cells, Bcl-2 expression was not affected by PBR ligand treatment (Marselli et al, 2004). Other pathways induced by PBR ligands involve the p38 mitogen-activated protein kinase (p38MAPKinase) pathway. PBR ligands were shown to activate the p38MAPKinase, partly via caspase-3 activation, leading to overexpression of growth arrest and DNA damage-inducible protein (gadd) 45 and gadd153, apoptosis and cell cycle arrest (Sutter et al, 2003). In addition, PBR ligands were shown to modulate the extracellular signal-regulated kinase (ERK) 1/2, though the mode and time course of ERK1/2 modulation seems to be cell-type specific. In esophageal cancer cells, PBR ligands transiently activated ERK1/2 (Sutter et al, 2004a), whereas in colorectal cancer cells ERK1/2 was inactivated (Maaser et al, 2004). However, blocking the ERK1/2 pathway enhanced the PBR ligand-induced antiproliferative effects (Sutter et al, 2004a; Maaser et al, 2004), independently of the direct effect of PBR ligands on ERK1/2 activation. The antiproliferative effects of PBR ligands involve the induction of apoptosis as well as cell cycle arrest. In esophageal and colorectal cancer cells, PBR ligands induced an arrest in the G1/G0 phase of the cell cycle which occurred due to an increased expression of the cell cycle inhibitors p21Cip1 and p27Kip1, and a decrease of the cyclins D1 and B (Maaser et al, 2001, 2004; Sutter et al, 2002). Many molecules and pathways involved in PBR ligand-induced apoptosis and cell cycle regulation have already been identified, but other important interactions and signaling pathways have not yet been addressed in this context. Moreover, future studies will have to clarify which of the pathways are universal and which are celltype specific. This knowledge will help to establish if, how, and which PBR ligands are of therapeutic value, either alone or in combination with established chemotherapeutics.
gastrointestinal cancers.
Acknowledgements We thank the Deutsche Forschungsgemeinschaft, Deutsche Krebshilfe, Berliner Krebshilfe, and WilhelmSander-Stiftung.
References Amri H, Li H, Culty M, Gaillard JL, Teper G, and Papadopoulos V (1999) The peripheral-type benzodiazepine receptor and adrenal steroidogenesis. Curr Opin Endocrin Diab 6, 179186. Anholt RR, Pedersen PL, De Souza EB, and Snyder SH (1986) The peripheral-type benzodiazepine receptor. Localization to the mitochondrial outer membrane. J Biol Chem 261, 576583. Banati RB, Newcombe J, Gunn RN, Cagnin A, Turkheimer F, Heppner F, Price G, Wegner F, Giovannoni G, Miller DH, Perkin GD, Smith T, Hewson AK, Bydder G, Kreutzberg GW, Jones T, Cuzner ML, and Myers R (2000) The peripheral benzodiazepine binding site in the brain in multiple sclerosis: quantitative in vivo imaging of microglia as a measure of disease activity. Brain 123, 2321-2337. Beinlich A, Strohmeier R, Kaufmann M, and Kuhl H (1999) Specific binding of benzodiazepines to human breast cancer cell lines. Life Sci 65, 2099-2108. Beinlich A, Strohmeier R, Kaufmann M, and Kuhl H (2000) Relation of cell proliferation to expression of peripheral benzodiazepine receptors in human breast cancer cell lines. Biochem Pharmacol 60, 397-402. Beurdeley-Thomas A, Miccoli L, Oudard S, Dutrillaux B, and Poupon MF (2000) The peripheral benzodiazepine receptors: a review. J Neurooncol 46, 45-56. Bovolin P, Schlichting J, Miyata M, Ferrarese C, Guidotti A, and Alho H (1990) Distribution and characterization of diazepam binding inhibitor (DBI) in peripheral tissues of rat. Regul Pept 29, 267-281. Braestrup C and Squires RF (1977) Specific benzodiazepine receptors in rat brain characterized by high- affinity (3H)diazepam binding. Proc Natl Acad Sci U S A 74, 38053809. Bribes E, Carriere D, Goubet C, Galiegue S, Casellas P, and Simony-Lafontaine J (2004) Immunohistochemical assessment of the peripheral benzodiazepine receptor in human tissues. J Histochem Cytochem 52, 19-28. Carayon P, Portier M, Dussossoy D, Bord A, Petitpretre G, Canat X, Le Fur G, and Casellas P (1996) Involvement of peripheral benzodiazepine receptors in the protection of hematopoietic cells against oxygen radical damage. Blood 87, 3170-3178. Carmel I, Fares FA, Leschiner S, Scher端bl H, Weisinger G, and Gavish M (1999) Peripheral-type benzodiazepine receptors in the regulation of proliferation of MCF-7 human breast carcinoma cell line. Biochem Pharmacol 58, 273-278. Casellas P, Galiegue S, and Basile AS (2002) Peripheral benzodiazepine receptors and mitochondrial function. Neurochem Int 40, 475-486. Cornu P, Benavides J, Scatton B, Hauw JJ, and Philippon J (1992) Increase in omega 3 (peripheral-type benzodiazepine) binding site densities in different types of human brain tumours. A quantitative autoradiography study. Acta Neurochir 119, 146-152. Decaudin D, Castedo M, Nemati F, Beurdeley-Thomas A, De Pinieux G, Caron A, Pouillart P, Wijdenes J, Rouillard D, Kroemer G, and Poupon MF (2002) Peripheral
VI. Concluding remarks PBR is an attractive target for diagnostic, prognostic, and therapeutic approaches in gastrointestinal cancers. Clinical studies showed its prognostic value in colorectal carcinoma, whereas most of the therapeutic approaches have not been studied in patients yet. Therefore a series of clinical trials is required to evaluate PBR-targeted strategies in the diagnosis, prognosis, and therapy of 349
Maaser et al: PBR as a target for diagnosis and therapy of gastrointestinal cancer benzodiazepine receptor ligands reverse apoptosis resistance of cancer cells in vitro and in vivo. Cancer Res 62, 13881393. Fischer R, Schmitt M, Bode JG, and Haussinger D (2001) Expression of the peripheral-type benzodiazepine receptor and apoptosis induction in hepatic stellate cells. Gastroenterology 120, 1212-1226. Galiegue S, Casellas P, Kramar A, Tinel N, and SimonyLafontaine J (2004) Immunohistochemical assessment of the peripheral benzodiazepine receptor in breast cancer and its relationship with survival. Clin Cancer Res 10, 2058-2064. Garnier M, Boujrad N, Oke BO, Brown AS, Riond J, Ferrara P, Shoyab M, Suarez-Quian CA, and Papadopoulos V (1993) Diazepam binding inhibitor is a paracrine/autocrine regulator of Leydig cell proliferation and steroidogenesis: action via peripheral-type benzodiazepine receptor and independent mechanisms. Endocrinology 132, 444-458. Giatzakis C and Papadopoulos V (2004) Differential utilization of the promoter of peripheral-type benzodiazepine receptor by steroidogenic versus nonsteroidogenic cell lines and the role of Sp1 and Sp3 in the regulation of basal activity. Endocrinology 145, 1113-1123. Guo P, Ma J, Li S, Guo Z, Adams AL, and Gallo JM (2001) Targeted delivery of a peripheral benzodiazepine receptor ligand-gemcitabine conjugate to brain tumors in a xenograft model. Cancer Chemother Pharmacol 48, 169-176. Han Z, Slack RS, Li W, and Papadopoulos V (2003 ) Expression of peripheral benzodiazepine receptor (PBR) in human tumors: relationship to breast, colorectal, and prostate tumor progression. J Recept Signal Transduct Res 23, 225-238. Hardwick M, Cavalli LR, Barlow KD, Haddad BR, and Papadopoulos V (2002) Peripheral-type benzodiazepine receptor (PBR) gene amplification in MDA-MB-231 aggressive breast cancer cells. Cancer Genet Cytogenet 139, 48-51. Hardwick M, Fertikh D, Culty M, Li H, Vidic B, and Papadopoulos V (1999) Peripheral-type benzodiazepine receptor (PBR) in human breast cancer: correlation of breast cancer cell aggressive phenotype with PBR expression, nuclear localization, and PBR-mediated cell proliferation and nuclear transport of cholesterol. Cancer Res 59, 831-842. Hardwick M, Rone J, Han Z, Haddad B, and Papadopoulos V (2001) Peripheral-type benzodiazepine receptor levels correlate with the ability of human breast cancer MDA-MB231 cell line to grow in SCID mice. Int J Cancer 94, 322327. Henkel K, Karitzky J, Schmid M, Mader I, Glatting G, Unger JW, Neumaier B, Ludolph AC, Reske SN, and Landwehrmeyer GB (2004) Imaging of activated microglia with PET and [(11)C]PK 11195 in corticobasal degeneration. Mov Disord 19, 817-821. Junck L, Olson JM, Ciliax BJ, Koeppe RA, Watkins GL, Jewett DM, McKeever PE, Wieland DM, Kilbourn MR, and Starosta-Rubinstein S (1989) PET imaging of human gliomas with ligands for the peripheral benzodiazepine binding site. Ann Neurol 26, 752-758. Katz Y, Ben-Baruch G, Kloog Y, Menczer J, and Gavish M (1990a) Increased density of peripheral benzodiazepinebinding sites in ovarian carcinomas as compared with benign ovarian tumours and normal ovaries. Clin Sci 78, 155-158. Katz Y, Eitan A, and Gavish M (1990b) Increase in peripheral benzodiazepine binding sites in colonic adenocarcinoma. Oncology 47, 139-142. Kletsas D, Li W, Han Z, and Papadopoulos V (2004) Peripheraltype benzodiazepine receptor (PBR) and PBR drug ligands in fibroblast and fibrosarcoma cell proliferation: role of ERK, c-Jun and ligand-activated PBR-independent pathways. Biochem Pharmacol 67, 1927-1932.
Kozikowski AP, Ma D, Brewer J, Sun S, Costa E, Romeo E, and Guidotti A (1993) Chemistry, binding affinities, and behavioral properties of a new class of "antineophobic" mitochondrial DBI receptor complex (mDRC) ligands. J Med Chem 36, 2908-2920. Kupczyk-Subotkowska L, Siahaan TJ, Basile AS, Friedman HS, Higgins PE, Song D, and Gallo JM (1997) Modulation of melphalan resistance in glioma cells with a peripheral benzodiazepine receptor ligand-melphalan conjugate. J Med Chem 40, 1726-1730. Landau M, Weizman A, Zoref-Shani E, Beery E, Wasseman L, Landau O, Gavish M, Brenner S, and Nordenberg J (1998) Antiproliferative and differentiating effects of benzodiazepine receptor ligands on B16 melanoma cells. Biochem Pharmacol 56, 1029-1034. Le Fur G, Perrier ML, Vaucher N, Imbault F, Flamier A, Benavides J, Uzan A, Renault C, Dubroeucq MC, and Gueremy C (1983) Peripheral benzodiazepine binding sites: effect of PK 11195, 1-(2-chlorophenyl)-N-methyl-N-(1methylpropyl)-3-isoquinolinecarboxamide. I. In vitro studies. Life Sci 32, 1839-1847. Lockhart A, Davis B, Matthews JC, Rahmoune H, Hong G, Gee A, Earnshaw D, and Brown J (2003) The peripheral benzodiazepine receptor ligand PK11195 binds with high affinity to the acute phase reactant alpha1-acid glycoprotein: implications for the use of the ligand as a CNS inflammatory marker. Nucl Med Biol 30, 199-206. Maaser K, Grabowski P, Sutter AP, Höpfner M, Foss HD, Stein H, Berger G, Gavish M, Zeitz M, and Scherübl H (2002) Overexpression of the peripheral benzodiazepine receptor is a relevant prognostic factor in stage III colorectal cancer. Clin Cancer Res 8, 3205-3209. Maaser K, Höpfner M, Jansen A, Weisinger G, Gavish M, Kozikowski AP, Weizman A, Carayon P, Riecken EO, Zeitz M, and Scherübl H (2001) Specific ligands of the peripheral benzodiazepine receptor induce apoptosis and cell cycle arrest in human colorectal cancer cells. Br J Cancer 85, 1771-1780. Maaser K, Sutter AP, Krahn A, and Scherübl H (2004) Cell cycle-related signaling pathways modulated by peripheral benzodiazepine receptor ligands in colorectal cancer cells. BBRC, In Press. Marchetti P, Hirsch T, Zamzami N, Castedo M, Decaudin D, Susin SA, Masse B, and Kroemer G (1996) Mitochondrial permeability transition triggers lymphocyte apoptosis. J Immunol 157, 4830-4836. Marselli L, Trincavelli L, Santangelo C, Lupi R, Del Guerra S, Boggi U, Falleni A, Gremigni V, Mosca F, Martini C, Dotta F, Di Mario U, Del Prato S, and Marchetti P (2004) The role of peripheral benzodiazepine receptors on the function and survival of isolated human pancreatic islets. Eur J Endocrinol 151, 207-214. Miettinen H, Kononen J, Haapasalo H, Helen P, Sallinen P, Harjuntausta T, Helin H, and Alho H (1995) Expression of peripheral-type benzodiazepine receptor and diazepam binding inhibitor in human astrocytomas: relationship to cell proliferation. Cancer Res 55, 2691-2695. Neary JT, Jorgensen SL, Oracion AM, Bruce JH, and Norenberg MD (1995) Inhibition of growth factor-induced DNA synthesis in astrocytes by ligands of peripheral-type benzodiazepine receptors. Brain Res 675, 27-30. Ostuni MA, Marazova K, Peranzi G, Vidic B, Papadopoulos V, Ducroc R, and Lacapere JJ (2004) Functional characterization and expression of PBR in rat gastric mucosa: stimulation of chloride secretion by PBR ligands. Am J Physiol Gastrointest Liver Physiol 286, G1069-G1080.
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Cancer Therapy Vol 2, page 351 Papadopoulos V (2003) Peripheral benzodiazepine receptor: structure and function in health and disease. Ann Pharm Fr 61, 30-50. Papadopoulos V, Amri H, Boujrad N, Cascio C, Culty M, Garnier M, Hardwick M, Li H, Vidic B, Brown AS, Reversa JL, Bernassau JM, and Drieu K (1997) Peripheral benzodiazepine receptor in cholesterol transport and steroidogenesis. Steroids 62, 21-28. Papadopoulos V, Kapsis A, Li H, Amri H, Hardwick M, Culty M, Kasprzyk PG, Carlson M, Moreau JP, and Drieu K (2000) Drug-induced inhibition of the peripheral-type benzodiazepine receptor expression and cell proliferation in human breast cancer cells. Anticancer Res 20, 2835-2847. Prosst RL, Wolfsen HC, and Gahlen J (2003) Photodynamic therapy for esophageal diseases: a clinical update. Endoscopy 35, 1059-1068. Ravagnan L, Marzo I, Costantini P, Susin SA, Zamzami N, Petit PX, Hirsch F, Goulbern M, Poupon MF, Miccoli L, Xie Z, Reed JC, and Kroemer G (1999) Lonidamine triggers apoptosis via a direct, Bcl-2-inhibited effect on the mitochondrial permeability transition pore. Oncogene 18, 2537-2546. Starosta-Rubinstein S, Ciliax BJ, Penney JB, McKeever P, and Young AB (1987) Imaging of a glioma using peripheral benzodiazepine receptor ligands. Proc Natl Acad Sci U S A 84, 891-895. Stoebner PE, Carayon P, Casellas P, Portier M, Lavabre-Bertrand T, Cuq P, Cano JP, Meynadier J, and Meunier L (2001) Transient protection by peripheral benzodiazepine receptors during the early events of ultraviolet light-induced apoptosis. Cell Death Differ 8, 747-753. Sutter AP, Maaser K, Barthel B, and Scherübl H (2003) Ligands of the peripheral benzodiazepine receptor induce apoptosis and cell cycle arrest in oesophageal cancer cells: involvement of the p38MAPK signalling pathway. Br J Cancer 89, 564572. Sutter AP, Maaser K, Gerst B, Krahn A, Zeitz M, and Scherübl H (2004a) Enhancement of peripheral benzodiazepine receptor ligand-induced apoptosis and cell cycle arrest of esophageal cancer cells by simultaneous inhibition of MAPK/ERK kinase. Biochem Pharmacol 67, 1701-1710. Sutter AP, Maaser K, Grabowski P, Bradacs G, Vormbrock K, Höpfner M, Krahn A, Heine B, Stein H, Somasundaram R, Schuppan D, Zeitz M, and Scherübl H (2004b) Peripheral
benzodiazepine receptor ligands induce apoptosis and cell cycle arrest in human hepatocellular carcinoma cells and enhance chemosensitivity to paclitaxel, docetaxel, doxorubicin and the Bcl-2 inhibitor HA14-1. J Hepatol . Sutter AP, Maaser K, Höpfner M, Barthel B, Grabowski P, Faiss S, Carayon P, Zeitz M, and Scherübl H (2002) Specific ligands of the peripheral benzodiazepine receptor induce apoptosis and cell cycle arrest in human esophageal cancer cells. Int J Cancer 102, 318-327. Veenman L, Levin E, Weisinger G, Leschiner S, Spanier I, Snyder SH, Weizman A, and Gavish M (2004) Peripheraltype benzodiazepine receptor density and in vitro tumorigenicity of glioma cell lines. Biochem Pharmacol 68, 689-698. Venturini I, Alho H, Podkletnova I, Corsi L, Rybnikova E, Pellicci R, Baraldi M, Pelto-Huikko M, Helen P, and Zeneroli ML (1999 ) Increased expression of peripheral benzodiazepine receptors and diazepam binding inhibitor in human tumors sited in the liver. Life Sci 65, 2223-2231. Venturini I, Zeneroli ML, Corsi L, Avallone R, Farina F, Alho H, Baraldi C, Ferrarese C, Pecora N, Frigo M, Ardizzone G, Arrigo A, Pellicci R, and Baraldi M (1998) Up-regulation of peripheral benzodiazepine receptor system in hepatocellular carcinoma. Life Sci 63, 1269-1280. Verma A, Facchina SL, Hirsch DJ, Song SY, Dillahey LF, Williams JR, and Snyder SH (1998) Photodynamic tumor therapy: mitochondrial benzodiazepine receptors as a therapeutic target. Mol Med 4, 40-45. Verma A, Nye JS, and Snyder SH (1987) Porphyrins are endogenous ligands for the mitochondrial (peripheral-type) benzodiazepine receptor. Proc Natl Acad Sci U S A 84, 2256-2260. Walter RB, Raden BW, Cronk MR, Bernstein ID, Appelbaum FR, and Banker DE (2004) The peripheral benzodiazepine receptor ligand PK11195 overcomes different resistance mechanisms to sensitize AML cells to gemtuzumab ozogamicin. Blood 103, 4276-4284. Wendler G, Lindemann P, Lacapere JJ, and Papadopoulos V (2003) Protoporphyrin IX binding and transport by recombinant mouse PBR. Biochem Biophys Res Commun 311, 847-852.
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Cancer Therapy Vol 2, page 353 Cancer Therapy Vol 2, 353-364, 2004
A single monoclonal antinuclear autoantibody with nucleosome-restricted specificity inhibits growth of diverse human tumors in nude mice Research Article
Ananthsrinivas R. Chakilam1, Santhosh Pabba1, Dmitry Mongayt, Leonid Z. Iakoubov2, Vladimir P. Torchilin* Department of Pharmaceutical Sciences, Bouve College of Health Sciences, Northeastern University, 360 Huntington Avenue, Boston MA 02115, USA 1 ARC and SP participated equally in this study 2 Present address: Chronix Biomedical, 1735 N First Street, Suite 309, San Jose CA, 95112, USA
__________________________________________________________________________________ *Correspondence: Vladimir Torchilin, Ph.D., D.Sc., Department of Pharmaceutical Sciences, Northeastern University, Mugar Building, Room 312, 360 Huntington Avenue, Boston, MA 02115, USA; Tel: 617-373-3206; Fax: 617-373-8886; e-mail: v.torchilin@neu.edu Key words: cancer immunotherapy; human tumors; nude mice; monoclonal antibody; anti-nuclear autoantibody; nucleosome-specific antibody Abbreviations: antibody-dependent cellular cytotoxicity, (ADCC); antinuclear autoantibodies, (ANAs); counts per minute, (CPM); Diethylene triamine pentaacetic acid anhydride, (DTPA); Hank’s balanced salt solution, (HBSS); nucleosome, (NS)
VPT and LZI hold a US patent on antitumor antibodies described in this paper. The patent is assigned to Procyon Biopharma, Inc. (Dorval, Canada) for commercial development. Received: 22 September 2004; Accepted: 27 September 2004; electronically published: September 2004
Summary Advances in cancer immunotherapy with monoclonal antibodies reemphasized the role of humoral immunity in neoplasia control. Still, most antibodies used in cancer diagnosis and treatment, induced either by active immunization or preexisting as natural antibodies, possess restricted tumor-type specificity. The development of a “universal” antibody targeting diverse cancers remains an unresolved issue. Earlier, we showed that certain natural antinuclear autoantibodies recognize the surface of numerous tumor cells but not normal cells, possess nucleosomerestricted specificity, mediate antibody-dependent cellular cytotoxicity of tumor cells in vitro, and inhibit the development of murine tumor in syngeneic mice. Here, we have shown that a single monoclonal antinuclear nucleosome-specific autoantibody, mAb 2C5, specifically recognizes multiple unrelated human tumor cell lines in flow cytometry experiments and accumulates at high tumor-to-normal ratio (between 2.7 and 13.4) in various human tumors in nude mice. Immunotherapy with mAb 2C5 in two different models – prophylactic therapy and established tumor therapy – results in significant suppression of the growth of several human tumors (including a multidrug resistant tumor) in nude mice. This is the first experimental evidence that a single monoclonal anticancer antibody can inhibit the growth of various unrelated human tumors in nude mice.
suppressive activity (David et al, 1996) support their possible role in tumor immunosurveillance. Such antibodies usually belong to the IgM isotype (Brandlein et al, 2003), which might limit their infiltration into solid tumors (compared to smaller IgGs) and restrict their antitumor effector function since no antibody-dependent cellular cytotoxicity (ADCC) is known for IgMs. In addition, the applicability of natural anticancer antibodies may be limited by their restricted specificity against only
I. Introduction Recent success with mAbs as cancer therapeutics (Bell, 2002; Boye et al, 2003; Mendelsohn, 2003; Zelenetz, 2003) has renewed the attention to the anticancer role of humoral immunity, including natural humoral immunity. Natural anti-tumor antibodies are common for healthy individuals (Colnaghi et al, 1982), and experimental in vivo data on their strong tumor-
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Chakilam et al: A single monoclonal antinuclear autoantibody inhibits growth of diverse human tumors certain tumor types (Cote et al, 1983). We have identified a subset of natural IgG class antibodies capable of binding to the surface of a broad spectrum of various cancer cells but not normal cells (Iakoubov, Torchilin, 1997). These antibodies are natural antinuclear autoantibodies (ANAs) that are present in a proportion of healthy rodents and humans, especially in the aged (Xavier et al, 1995). Our hypothesis that these ANAs participate in tumor immunosurveillance (Torchilin et al, 2001) was strongly supported by the results of pre-clinical in vivo experiments with mAb 2C5, a monoclonal tumor cell surface-reactive nucleosome (NS)-specific ANA of IgG2a isotype obtained from a non-immunized healthy aged Balb/c mouse. Within syngeneic mouse models, mAb 2C5 suppressed the growth of EL4 T lymphoma and led to a prolonged survival time in B16 melanoma-bearing mice (Iakoubov and Torchilin, 1997). Existing data on the tumor reactivity and anti-tumor properties of certain ANAs related to autoimmune pathology or induced by immunization also favor this hypothesis (Brinkman et al, 1993; Johnson, Shin, 1983; Sorace, Johnson, 1990). Numerous additional data supporting the hypothesis on ANAsâ&#x20AC;&#x2122; role in tumor immunosurveillance have been reviewed recently (Torchilin et al, 2003). ADCC was considered as a most probable in vivo mechanism for mAb 2C5 anti-tumor activity because of significant ADCC effect of this antibody in vitro (Iakoubov and Torchilin, 1997). Though in vitro experiments did reveal neither complement-mediated cytotoxicity, nor direct inhibition of tumor cell proliferation, some additional mechanisms might be involved in vivo. By forming immune complexes with free NSs in the circulation, mAb 2C5 and similar antibodies might induce the enhanced release of inflammatory cytokines and proteolytic enzymes from certain immune cells (Lucisana and Mantovani, 1984), as well as toll receptor-involving events (Leadbetter et al, 2002), and/or empowerment of dendritic cell (Schuurhuis et al, 2002). The ability to recognize tumor but not normal cells was observed for mAb 2C5 and another similarly obtained ANA, mAb 1G3. Both mAbs demonstrated NS-restricted specificity. Thus, tumor cell surface-bound NSs (supramolecular constituents of nuclear material consisting of well-characterized individual monoNS composed of DNA and four pairs of histones arranged in a characteristic pattern) were proposed to be their target on tumor cells (Iakoubov, Torchilin, 1997). The binding of extracellular NSs to tumor cell surface might be mediated by specific NS receptors that have been repeatedly reported by several investigators to be present on the surface of tumor cells (Jacob et al, 1989; Koutouzov et al, 1996; Seddiki et al, 2001). As for the origin of tumor cell-bound NSs, extracellular NSs were found in tumor cell cultures (Bell, Morrison, 1991) and in patients with tumors (Le LannTerrisse et al, 1994), where they arise from apoptotic tumor cells present in every in vivo developing tumor (Wyllie, 1993). In cell culture experiments, NSs released from apoptotic S49 T lymphoma cells after sub-optimal dexamethasone treatment were able to attach to the surface of surviving cells. This was accompanied by a 50-fold increase in monoclonal ANA 2C5 binding to these cells
(Iakoubov and Torchilin, 1998). Elevated free extracellular nucleochromatin has also been observed under other, non-cancerous conditions accompanied by massive apoptotic death, such as lupus erythematosis and AIDS (Licht et al, 2001). It is especially important, that NSs are exposed on the cell surface of apoptotic cells (Radic et al, 2004). Functionally, extracellular chromatin fragments have been shown to inhibit the tumor cell killing by NK cells in vitro (Le Lann-Terisse et al, 1994, 1997). Such data allow considering the NS release by dying tumor cells as a tumor self-defense mechanism that protects the surviving tumor cells from host immune attack. In this case, the increased production of NS-specific cytotoxic autoantibodies by a tumor-bearing organism may be contemplated a response that acts to overcome the tumor self-defense. The data demonstrating a prolonged time to disease progression and an increased survival rate in cancer patients with elevated serum ANAs (Blaes et al, 2000; Syrigos et al, 2000), and a higher remission rate in chronic myelogenous leukemia patients who develop autoimmune phenomena (known to be accompanied with a rise in ANAs) as a result of alphainterferon therapy vs those who do not (Sacchi et al, 1995) support a possibility of anti-tumor activity of ANAs. Here, we present the first experimental data on strong anti-tumor activity of mAb 2C5 against diverse human tumors in in vivo models. In addition, the specificity of mAb 2C5 and similar antibodies against a broad variety of tumors is a prerequisite for their broad applicability as vehicles for targeting different therapeutic and diagnostic agents to various tumors (Torchilin et al, 2003). Supporting data on mAb 2C5â&#x20AC;&#x2122;s ability for intratumoral accumulation are also presented.
II. Materials and methods A. Materials All cell culture media, heat-inactivated fetal bovine serum, and other cell culture reagents were obtained from Cellgro (Herndon, VA). Isotype-matching control antibody UPC10 and fluorescein-conjugated goat anti-mouse antibody were purchased from ICN Pharmaceuticals, Inc. (Cosa Mesa, CA). Diethylene triamine pentaacetic acid anhydride (DTPA) for radiolabeling mAb 2C5 with 111In and all other chemicals and buffer solution components were obtained from Sigma (St. Louis, MO) and were of analytical grade. 111In with specific radioactivity of 395 Ci/mg was from Perkin-Elmer (Boston, MA).
B. Monoclonal antinuclear autoantibody 2C5 (mAb 2C5) mAb 2C5 [IgG2a] (Iakoubov, Torchilin, 1997) was purified from the supernatant of 2C5 hybridoma cells grown in the CM10 culture medium by using a standard saturated ammonium sulfate precipitation and affinity chromatography on a protein G column (Exalpha Biologicals, Inc., Watertown, MA). Non-reducing SDS-PAGE of the purified antibody (Laemmli, 1970), and ELISA with purified monoNSs (Li et al, 1995) were performed to test mAb 2C5 purity and immunoreactivity. The purified antibody was stored at -80°C as a 5 mg/ml solution. To label the mAb 2C5 with 111In, it was first modified with chelating residues DTPA and loaded with 111In by transchelation from the 111In-citrate complex (Samokhin et al, 2001).
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Cancer Therapy Vol 2, page 355 following protocol #011022 approved by the Institutional Animal Care and Use Committee in accordance with Principles of Laboratory Animal Care (NIH publication #85-23, revised in 1985). The animals were allowed free access to food and water. Approximately 8x106 of H69(AR) cells, 10-12x106 of COLO2O5, PC3, C32TG, and NCI-H82 cells, or 40x106 of BT20 cells in 0.25 ml of RPMI, were inoculated s.c. into the left flanks of the mice. Mice were examined every 2-3 days until palpable tumors were detected.
C. Determination of the dissociation constant KD of monoNSs-mAb 2C5 complexes in solution by ELISA (Friguet et al, 1985) The referenced protocol includes the incubation of the antigen [monoNSs purified as in (Li et al, 1995), in this particular case] at various concentrations with mAb 2C5 at constant concentration until equilibrium is reached and subsequent determination of the free antibody concentration by an indirect ELISA. Samples of monoNSs at various concentrations (0.016 - 16 µg/ml) were mixed with the constant amount of antibody (1 µg/ml) in TBS, 2mM EDTA, pH 7.5, supplemented with 10 mg/ml BSA. After a 15 h incubation at 20°C, 100µl of each mixture was transferred and incubated for 30 min at 20°C into the wells of a microtiter plate coated with monoNSs [100 µl per well, at 10 µg/ml in TBST-Cas (TBS containing 0.05% w/v Tween-20 and 2 mg/ml casein), pH 7.5, for 1 h at 20°C]. After washing with TBS supplemented with 0.05% Tween 20, the wells were incubated for 1 h at room temperature with 100 µl of 1:5000 diluted goat anti-mouse IgG peroxidase conjugate (ICN Biomedicals Inc., OH) in TBST-Cas. After incubation, the wells were washed three times with 200 µl of TBST and each well was incubated with 100µl of enhanced Kblue® TMB peroxidase substrate (Neogen Corporation, KY) for 15 min. Finally, the plate was read at a dual wavelength of 620 nm with the reference filter at 492 nm using a Labsystems Multiskan MCC/340 microplate reader installed with GENESISLITE windows based microplate software.
G. Tumor accumulation of mAb 2C5 Mice with tumors 1-2 cm in diameter were injected via the tail vein with 111In-labeled mAb 2C5. After 48 hrs, the mice were anaesthetized and body distribution and tumor accumulation of mAb 2C5-associated 111In radioactivity was visualized using an Ohio Nuclear 400 "camera equipped with high-energy collimator and Technicare 560 dedicated computer with whole body pictures taken by digital camera. Tumor and adjacent normal tissues were then collected, and 111In radioactivity was quantified (as counts per minute, CPM) using a Beckman 5500B "counter (Fullerton, CA). Then tumor-to-normal tissue ratios of the %injected dose/g of tissue were calculated.
H. Immunotherapy with mAb 2C5 Immunotherapy was performed in nude mice with COLO2O5, PC3, NCI-H82, and H69(AR) tumors. Since our previous and current preliminary experiments have revealed no anti-tumor activity of control injections of PBS and isotypematched antibody UPC10, in our full scale experiments, in order to minimize the number of animals, we have alternatively used as controls PBS or isotype-matched UPC10 antibody, but normally not both within the same model. In the model of prophylactic treatment, nude mice were injected with mAb 2C5 and control PBS, pH 7.4 (in PC3 and NCI-H82 tumor models) or isotype-matched antibody UPC-10 (in COLO2O5 tumor model) prior to and after the inoculation with tumor cells. 150 µg of mAb 2C5 in 100 µl of PBS or the same volume/quantity of control injections per mouse were given via tail vein on days –1, 6, 13 and 19 in COLO2O5- and NCIH82-bearing mice, and on days –1, 6 and 13 in PC3-bearing mice. In the established tumor treatment model, mAb 2C5 and control injections were initiated after the formation of a visible tumor 7-14 days after the inoculation of tumor cells. Tumorbearing mice were injected with mAb 2C5 and control PBS [in PC3, NCI-H82, and H69(AR) tumor models] or UPC-10 (in COLO2O5 tumor model). 150 µg of mAb 2C5 in 100 µl of PBS or the same volume/quantity of control injections per mouse were given via tail vein on days 13 and 20 post inoculation in COLO2O5-bearing mice. In case of PC3-, NCI-H82-, and H69(AR)-bearing mice, injections were given on days 7, 11 & 15; 7, 14 & 21; and 9, 14, 20, and 27, respectively. Anticancer activity of mAb 2C5 was assessed by following the tumor volumes during the treatment and average tumor weights at the completion of treatment in the control and mAb 2C5-treated groups. The apparent tumor volume was calculated on different days during the treatment using the formula (Geran et al, 1972), tumor volume (mm3) = (Length X Width2)/2, length being greater than width. At the end of treatment, the mice were sacrificed and tumors were removed and weighed.
D. Human tumor cell lines NCI-H82 (small cell lung carcinoma), COLO2O5 (colorectal adenocarcinoma), PC-3 (prostate adenocarcinoma), H9 (lymphoma), C32TG (amelanotic melanoma), BT-20 (breast adenocarcinoma), MCF7 (estrogen receptor-positive breast adenocarcinoma), and H69(AR) (adriamicin-resistant small cell lung carcinoma) cell lines were obtained from the American Type Culture Collection (Rockville, MD). NCI-H82, COLO2O5, PC3, H9, and H69(AR) cells were grown at 37!C in the RPMI 1640 medium with 2mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/l glucose, 10 mM HEPES, 1.0 mM sodium pyruvate, and 10% fetal bovine serum. C32TG, BT-20, and MCF7 cells were propagated at 37!C using the minimum essential medium (Eagle) with 2 mM L-glutamine and Earl's BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, and 10% fetal bovine serum.
E. FACS analysis of surface binding of mAb 2C5 to human tumor cell lines (Iakoubov and Torchilin, 1997) Approximately 10,000 cells of different cell lines in each sample were used to check for mAb 2C5 binding using the isotype-matching non-specific UPC10 antibody as a negative control. Cells were washed with Hank’s balanced salt solution (HBSS) and then with 1% BSA in PBS, pH 7.4, to prevent a nonspecific binding. Cells were then incubated for 30 min at room temperature with mAb 2C5 or UPC10 and then stained with fluorescein-conjugated goat anti-mouse IgG. Finally, the cells were fixed with 4% paraformaldehyde solution in PBS, livegated using forward vs. side scatter to exclude debris and dead cells, and analyzed for surface fluorescence using FACScan (Beckton Dickinson, Franklin Lakes, NJ).
I. Statistics Differences in apparent tumor volumes during the treatment and post-mortem tumor weights were compared using the non-parametric two-tailed Wilcoxon rank sum test for two independent samples. For easy representation of the difference in tumor volumes during the treatment, the average tumor volume (mm3) of the group vs. days after tumor cell inoculation was
F. Tumors in nude mice All experiments were performed in 6-8 week old female nu/nu mice (Charles River Laboratories, Wilmington, MA)
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(Figure 1, A,B). We have also confirmed antibody immunoreactivity against NSs purified from the rat liver (Li et al, 1995) using a standard ELISA protocol (Figure 1, C). The determination of mAb 2C5 KD with monoNS is presented on Figure 2 (here, the KD value characterizes avidity or relative affinity because of bivalent nature of an antibody).
III. Results A. Properties of mAb 2C5 We confirmed the purity of mAb 2C5 by the presence of the single peak in the protein G column antibody elution profile and the single band corresponding to an IgG protein after polyacrylamide gel electrophoresis
Figure 1. Purification and characterization of mAb 2C5. (A) The elution profile of mAb 2C5 from a protein G column; (B) SDS-PAGE showing purified mAb 2C5 (lane 1) and Bio-Rad high-range MW standards (lane 2, numbers on the right show corresponding MW values); and (C) The immunoreactivity of purified mAb 2C5 with nucleosome as a substrate in ELISA.
Figure 2. Determination of KD for mAb 2C5 interaction with NSs. (A) Calibration curve for the binding of mAb 2C5 to monoNSs in ELISA; (B) The Scatchard plot for the binding of mAb 2C5 to monoNSs. v is the fraction of the bound antibody and a the concentration of free antigen at equilibrium. The total concentration of the antibody (as NS binding sites) was 6.6 x 10-9 M.
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Cancer Therapy Vol 2, page 357 and similar antibodies as universal vehicles for in vivo intra-tumor delivery of drugs and imaging agents (Gao et al, 2003; Torchilin et al, 2003).
According to the protocol used for determination of the dissociation constant, we worked in the linear binding concentration range of mAb 2C5 to monoNSs. One hundred per cent saturation was achieved, thus we used only the linear concentration range as shown in the standard curve to determine the dissociation constant. As shown in Figure 2A, the dependence of the absorbance (resulting from the enzymatic activity of the immunoconjugate retained in the wells) on the initial concentration of the mAb 2C5 remains linear in the whole range of concentrations used. This linear dependence allows for the determination of the free antibody concentration at equilibrium, provided that the total antibody concentration is known. Figure 2B shows the results, plotted according to the Scatchard equation for the binding of the pure mAb 2C5 to monoNSs. From the linear regression, the apparent KD value for the mAb 2C5 is 2.36Âą0.64 x 10-10 M (actually, it might be somewhat lower because of the bivalency of the whole antibody and possible presence of more than one antibody-binding site on the monoNS) that shows high specificity of antibody binding to NSs.
D. Anti-cancer activity of mAb 2C5 in different models in vivo Most important, however, is the fact that, similar to shown earlier with two syngeneic murine tumors (Iakoubov and Torchilin, 1997), mAb 2C5 significantly inhibited the growth of several unrelated human tumors in athymic mice in both a prophylactic model (antibody administration was started prior to the tumor inoculation) and a therapeutic model (antibody administration began after the formation of visible tumors). The data presented in Figures 5 and 6 clearly indicate that in all models used [COLO205, PC3, NCI-H82, H69(AR)] tumor growth was strongly inhibited by the administration of mAb 2C5, and tumor weight at the end of the experiment was significantly lower in mAb 2C5-treated mice. Naturally, the data on the tumor volume are less precise because of the nature of the measurements. Interestingly, mAb 2C5 appeared to inhibit the growth of the adriamicin-resistant tumor, H69(AR) (Figure 6). We have intentionally chosen the more difficult-to-treat â&#x20AC;&#x153;established tumorâ&#x20AC;? model in case of this particular tumor.
B. Recognition of diverse human cancer cells by mAb 2C5 in vitro The results of FACS analysis (Figure 3) demonstrate an effective recognition by the mAb 2C5 of all human cancer cell lines tested. For our studies, we have selected COLO205, PC3, NCI-H82, BT-20, H9, MCF-7, C32TG, and adriamicin-resistant H69(AR) cell lines to represent a broad variety of unrelated tumors. These results are in good agreement with the previous data and confirm the broad anti-tumor specificity of this ANA with NSrestricted specificity (Iakoubov and Torchilin, 1997). We used several of these cell lines in further experiments on mAb 2C5 intra-tumor accumulation and anti-tumor activity.
IV. Discussion Our findings, although rather unusual, are supported by the whole set of circumstantial evidence found in the literature. Many publications provide direct and indirect evidence that ANAs may carry certain anti-tumor properties. Thus, the presence of ANAs is a characteristic feature of systemic autoimmunity (von Muhlen and Tan, 1995). At the same time, the mortality rate from cancer in autoimmune patients was long ago found to be significantly less than that in the healthy population (Palo et al, 1977). Recently, it was found that lupus patients are better protected from cancer (Huges, 2001). On the other hand, the experimental suppression of autoimmune manifestations (resulting in a decrease of autoantibody, including ANAs, production) in spontaneously autoimmune mice was found to sharply increase the incidence of spontaneous tumors (Hahn et al, 1975; Walker et al, 1978). In addition, as was already mentioned, circulating ANAs in patients with lung cancer (Blaes et al, 2000) and colorectal carcinoma (Syrigos et al, 2000) were associated with a prolonged survival without disease progression. Actually, the intentional induction of
C. Tumor accumulation of radiolabeled mAb 2C5 in vivo The investigation of tumor accumulation of the antibody-bound 111In radioactivity in several selected human tumors in mice clearly showed an enhanced accumulation of mAb 2C5 in all tested tumors, although the extent of this enhancement depended on the tumor type. Table 1 presents the data on an enhanced tumor accumulation of the antibody-bound 111In radioactivity in several selected human tumors in mice with the tumor-tonormal tissue accumulation ratio varying between 2.7 and 13.4. We have additionally confirmed the efficient tumor accumulation of mAb 2C5 by direct gammaimmunoimaging of tumor-bearing mice showing an increased accumulation of the antibody-associated radioactivity in the tumor area (typical images are presented in Figure 4 for an NCI-H82 tumor-bearing mouse). At the same time, non-specific antibodies were repeatedly shown to have very little (if any) accumulation in various tumor models in mice (De Santes et al, 1992; Camera et al, 1993; Xu et al, 1997; Palm et al, 2003). These data strongly support the application of mAb 2C5
Table 1. Accumulation of mAb 2C5-Bound 111In radioactivity in various human tumors in nude mice (expressed as the tumor-to-normal tissue radioactivity ratio) Tumor type Tumor-to-normal tissue ratio NCI-H82 5.99 PC3 3.17 BT-20 13.40 COLO2O5 2.72 C32TG 3.89
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Figure 3. Surface binding of mAb 2C5 to different human tumor cell lines as shown by flow cytometry. _____ cells only; _____ fluorescein-conjugated goat anti-mouse IgG; ____ isotype-matching control antibody UPC10; ____ mAb 2C5.
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Figure 4. Typical gamma-immunoscintigraphic images obtained at different time points post-injection of 111In-radiolabeled mAb 2C5 in a NCI-H82 tumor model.
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Figure 5. Prophylactic tumor treatment model in COLO2O5, PC3, and NCI-H82 human tumors in athymic mice. Within each model, the tumor growth in mAb 2C5-treated group and control group is illustrated by calculated tumor volumes during the treatment period (left columns) and by post-mortem tumor weights (right columns). Tumor volumes are plotted as average tumor volume (mm3) vs. days after tumor cell inoculation with arrows indicating days of injections. Post-mortem tumor weights are shown as box plots showing the group median, quartiles, and extreme values.
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Figure 6. Established tumor treatment model in COLO2O5, PC3, NCI-H82 and multi-drug resistant H69 (AR) human tumors in athymic mice. See details in the legend to Figure 5.
autoimmunity was seriously considered as an antineoplastic therapeutic strategy (Pardoll, 1999). Though the exact autoimmune components with antitumor function are not known, ANAs seem to be a good candidate. Two major mechanisms of action are presently being considered to explain anti-tumor activity of therapeutic monoclonal antibodies (Houghton and Scheinberg, 2000). The first is based on the antibody-mediated killing of tumor cells by various immune effectors such as complement or cytotoxic immune cells. The second involves antibody bioactivity un-related to immunologic function, such as blocking the access of growth factors to tumor cells or tumor vasculature. A convincing case for the in vivo involvement of immune effectors in monoclonal antibody-based tumor suppression was obtained recently in immunocompetent as well as athymic mice (Clynes et al, 2000). However, for each particular antibody, the conclusion on the involvement of immune killing is traditionally based on the indirect evidence, where the ability of the antibody to mediate complementor cytotoxic cell-dependent killing is demonstrated in vitro (Herlyn et al 1980). In addition, according to this in vitro criteria, effector cells from nude mice are known to mediate levels of ADCC of equal or even greater magnitude than cells from normal mice (Kohl et al, 1984). Our in vitro data on the ability of mAb 2C5 to mediate ADCC serve as indirect evidence for this mechanism of tumor suppression in both syngeneic tumor models in normal mice (Iakoubov and Torchilin, 1997) and xenogeneic models in nude mice. We can also reasonably expect at least one additional activity for the mAb 2C5. Since most tumors are expected to bear NSs on the surfaces of apoptotic cells (Radic et al,
2004) and result in elevated blood NSs (Holdenrieder et al, 2001), external anti-NS antibodies should lead to elevated immune complexes known to activate the immune system (Lucisano, Mantovani, 1984). This activation could be of a significant importance in opposing the tumor-mediated immunosuppression characteristic of cancer patients (Pollock, Roth, 1989). Thus, the efficiency of mAb 2C5 and similar antibodies might be in part based on their ability to turn on some additional immune mechanisms not characteristic for conventional immunization-induced monoclonal antibodies. In addition to their probable ability to block to a certain extent the earlier mentioned NKinhibiting action of extracellular NSs (chromatin) (Le Lann-Terisse et al, 1994, 1997), these antibodies can also interfere with the recently reported neoangiogenic activity of NSs (Tanner, 2003). The competition between NSspecific antibodies and C-reactive protein (Robey et al, 1985) for the binding with chromatin may exist in the blood, however the results of our experiments clearly demonstrate that in vivo the activity of mAb 2C5 is not blocked by this hypothetical competition. Earlier, monoclonal antibodies belonging to IgG2a and IgG3 isotypes have been described demonstrating ADCC against tumor cells over-expressing corresponding antigens, such as anti-ganglioside antibodies (Lin et al, 2001; Nakamura et al, 2001; Parajuli et al, 2001). However, these antibodies have been obtained by immunization and, to the best of our knowledge, none of them was ever shown to demonstrate anti-tumor activity against a variety of unrelated human tumors. As potential anti-tumor agents, natural ANAs may have a number of advantages compared to conventional anti-tumor antibodies. ANAs appear to be effective against a broad spectrum of various tumor types. Their anti-tumor 361
Chakilam et al: A single monoclonal antinuclear autoantibody inhibits growth of diverse human tumors response in mice as shown by monoclonal antibodies. J Immunol 128, 2757-2762. Cote RJ, Morrissey DM, Houghton AN, Beattie EJ Jr, Oettgen HF, Old LJ (1983) Generation of human monoclonal antibodies reactive with cellular antigens. Proc Natl Acad Sci USA 80, 2026-2030. David K, Ollert MW, Juhl H, Vollmert C, Erttman R, Vogel CW, Bredehorst R (1996) Growth arrest of solid human neuroblastoma xenografts in nude rats by natural IgM from healthy humans. Nat Med 2, 686-689. De Santes K, Slamon D, Anderson SK, Shepard M, Fendly B, Maneval D, Press O (1992) Radiolabeled antibody targeting of the HER-2/neu oncoprotein. Cancer Res 52, 1916-1923. Friguet B, Chaffotte AF, Djavadi-Ohaniance L, Goldberg ME (1985) Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay. J Immunol Meth 77, 305-319. Gao Z, Lukyanov AN, Chakilam AR, Torchilin VP (2003) PEGPE/phosphatidylcholine mixed immunomicelles specifically deliver encapsulated taxol to tumor cells of different origin and promote their efficient killing. J Drug Target 11, 87-92. Geran RI, Greenberg NH, MacDonald MM, Schumacher AM, Abbot BJ (1972) Protocols for screening chemical agents and natural products against animal tumors and other biological systems. Cancer Chemother Rep 3, 1-103. Hahn BH, Knotts L, Ng M, Hamilton TR (1975) Influence of cyclophosphamide and other immunosuppressive drugs on immune disorders and neoplasia in NZB/NZW mice. Arthritis Rheum 18, 145-152. Herlyn DM, Steplewski Z, Herlyn MF, and Koprowski H (1980) Inhibition of growth of colorectal carcinoma in nude mice by monoclonal antibody. Cancer Res 40, 717-721. Holdenrieder S, Stieber P, Bodenmuller H, Fertig G, Furst H, Schmeller N, Untch M, Seidel D (2001) Nucleosomes in serum as a marker for cell death. Clin Chem Lab Med 39, 596-605. Houghton AN, Scheinberg DA (2000) Monoclonal antibody therapies-a 'constant' threat to cancer. Nat Med 6, 373-374. Huges GR (2001) Lupus patients are protected from cancer. Lupus 10, 833-834. Iakoubov LZ, Torchilin VP (1997) A novel class of antitumor antibodies: nucleosome-restricted antinuclear autoantibodies (ANA) from healthy aged nonautoimmune mice. Oncol Res 9, 439-446. Iakoubov LZ, Torchilin VP (1998) Nucleosome-releasing treatment makes surviving tumor cells better targets for nucleosome-specific anticancer antibodies. Cancer Detect Prev 22, 470-475. Jacob L, Viard JP, Allenet B, Anin MF, Slama FB, Vandekerckhove J, Primo J, Markovits J, Jacob F, Bach JF (1989) A monoclonal anti-double-stranded DNA autoantibody binds to a 94-kDa cell-surface protein on various cell types via nucleosomes or a DNA-histone complex. Proc Natl Acad Sci USA 86, 4669-4673. Johnson RJ, Shin HS (1983) Monoclonal antibody against a differentiation antigen on human leukemia cells: crossreactivity with rat leukemia and suppression of rat leukemia in vivo. J Immunol 130, 2930-2936. Kohl S, Tang JP, Loo LS (1984) Antibody-dependent cellular cytotoxicity and natural killer cytotoxicity of peritoneal cells from nude mice to herpes simplex virus-infected cells. Microbiol Immunol 28, 439-449. Koutouzov S, Cabrespines A, Amoura Z, Chabre H, Lotton C, Bach JF (1996) Binding of nucleosomes to a cell surface receptor: redistribution and endocytosis in the presence of lupus antibodies. Eur J Immunol 26, 472-486.
mechanisms are probably multimodal and hence more efficient. Their side-effects should be minimal, since their natural presence in the blood does not seem to be harmful to the host. In addition, assuming that the ADCC and other immune effectors possibly activated by circulating mAb 2C5/NS immunocomplexes are independent of MDR mechanisms, such as the p-glycoprotein pump, mAb 2C5 and similar antibodies might serve as a treatment of choice against MDR tumors. This assumption is well supported by our results with the H69(AR) tumor. One can also speculate that such antibodies may be used together with apoptosis-inducing agents required to convert the tumor cell chromatin into NSs and subsequently provoke the release of these NSs and their binding to the surface of living tumor cells to make them better targets for the antibodies. The question if the mAb 2C5 (and similar antibodies) only inhibit tumor growth and have to be used as a component of the combination therapy, or, if optimized administration protocols are established, they can completely eliminate tumors, as well as the exact nature of NS-binding sites on the surface of tumor cells and major mechanisms of anti-tumor activity of NS-restricted ANAs are currently under investigation in our laboratory.
Acknowledgements This study was funded in part by the NIH grant R01 EB02995 to VPT. The authors would like also to acknowledge the researcher-friendly policy of partial overhead return at Northeastern University.
References Bell DA. and Morrison, B (1991) The spontaneous apoptotic cell death of normal human lymphocytes in vitro: the release of, and immunoproliferative response to, nucleosomes in vitro. Clin Immunol Immunopathol 60, 13-26. Bell R (2002) What can we learn from Herceptin trials in metastatic breast cancer? Oncology 63 Suppl 1, 39-46. Blaes F, Klotz M, Huwer H, Straub U, Kalweit G, Schimrigk K, Schafers HJ (2000) Antineural and antinuclear autoantibodies are of prognostic relevance in non-small cell lung cancer. Ann Thorac Surg 69, 254-258. Boye J, Elter T, and Engert A (2003) An overview of the current clinical use of the anti-CD20 monoclonal antibody rituximab. Ann Oncol 14, 520-535. Brandlein S, Pohle T, Ruoff N, Wozniak E, Muller-Hermelink HK, and Vollmers HP (2003) Natural IgM antibodies and immunosurveillance mechanisms against epithelial cancer cells in humans. Cancer Res 63, 7995-8005. Brinkman K, Termaat R, Berden JH, and Smeenk RJ (1990) Anti-DNA antibodies and lupus nephritis: the complexity of crossreactivity. Immunol Today 11, 232-234. Camera L, Kinuya S, Pai LH, Garmestani K, Brechbiel MW, Gansow OA, Paik CH, Pastan I, Carrasquillo JA (1993) A. Preclinical evaluation of 111In-labeled B3 monoclonal antibody: biodistribution and imaging studies in nude mice bearing human epidermoid carcinoma xenografts. Cancer Res 53, 2834-2839. Clynes RA, Towers TL, Presta LG, and Ravetch JV (2000) Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets. Nat Med 6, 443-446. Colnaghi MI, Menard S, Tagliabue E, and Torre GD (1982) Heterogeneity of the natural humoral anti-tumor immune
362
Cancer Therapy Vol 2, page 363 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685. Le Lann AD, Fournie GJ, Boissier L, Toutain PL, Benoist H (1994) In vitro inhibition of natural-killer-mediated lysis by chromatin fragments. Cancer Immunol Immunother 39, 185-192. Le Lann-Terrisse AD, Fournie GJ, Benoist H (1997) Nucleosome-dependent escape of tumor cells from naturalkiller-mediated lysis: nucleosomes are taken up by target cells and act at a postconjugation level. Cancer Immunol Immunother 43, 337-344. Leadbetter EA, Rifkin IR, Hohlbaum AM, Beaudette BC, Shlomchik MJ, Marshak-Rothstein A (2002) Chromatin-IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors. Nature 416, 603-607. LI Q, Bjork U, Wrange O (1995) Assays for interaction of transcription factor with nucleosome. Meth Enzymol 304, 314-317. Licht R, van Bruggen MC, Oppers-Walgreen B, Rijke TP, Berden JH (2001) Plasma levels of nucleosomes and nucleosome-autoantibody complexes in murine lupus: effects of disease progression and lipopolyssacharide administration. Arthritis Rheum 44, 1320-1330. Lin CC, Shen YC, Chuang CK, Liao SK (2001) Analysis of a murine anti-ganglioside GD2 monoclonal antibody expressing both IgG2a and IgG3 isotypes: monoclonality, apoptosis triggering, and activation of cellular cytotoxicity on human melanoma cells. Adv Exp Med Biol 491, 419429. Lucisano YM, Mantovani B (1984) Lysosomal enzyme release from polymorphonuclear leukocytes induced by immune complexes of IgM and of IgG. J Immunol 132, 2015-2020. Mendelsohn J (2003) Antibody-mediated EGF receptor blockade as an anticancer therapy: from the laboratory to the clinic. Cancer Immunol Immunother 52, 342-346. Nakamura K, Tanaka Y, Shitara K, Hanai N (2001) Construction of humanized anti-ganglioside monoclonal antibodies with potent immune effector functions. Cancer Immunol Immunother 50, 275-284. Palm S, Enmon RM Jr, Matei C, Kolbert KS, Xu S, Zanzonico PB, Finn RL, Koutcher JA, Larson SM, Sgouros G (2003) Pharmacokinetics and Biodistribution of (86)Y-Trastuzumab for (90)Y dosimetry in an ovarian carcinoma model: correlative MicroPET and MRI. J Nucl Med 44, 1148-1155. Palo J, Duchesne J, Wikstrom J (1977) Malignant diseases among patients with multiple sclerosis. J Neurol 216, 217222. Parajuli P, Yanagawa H, Hanibuchi M, Takeuchi E, Miki T, Yano S, Sone S (2001) Humanized anti-ganglioside GM2 antibody is effective to induce antibody-dependent cellmediated cytotoxicity in mononuclear cells from lung cancer patients. Cancer Lett 165, 179-184. Pardoll DM (1999) Inducing autoimmune disease to treat cancer. Proc Natl Acad Sci USA 96, 5340-5342. Pollock RE, Roth JA (1989) Cancer-induced immunosuppression: implications for therapy? Semin Surg Oncol 5, 414-419. Radic M, Marion T, Monestier M (2004) Nucleosomes are exposed at the cell surface in apoptosis. J Immunol 172, 6692-6700. Robey AF, Jones AD, Steinberg AD (1985) C-reactive protein mediates the solubilization of nuclear DNA by complement in vitro. J Exp Med 161, 1344-1356. Sacchi S, Kantarjian H, O'Brien S, Cohen PR, Pierce S, Talpaz M (1995) Immune-mediated and unusual complications during interferon alfa therapy in chronic myelogenous leukemia. J Clin Oncol 13, 2401-2407.
Samokhin GP, Mongayt DA, Iakoubov, LZ, Levchenko TS, Torchilin VP (2001) Negatively charged polymers protect antinuclear antibody against inactivation by acylating agents. Anal Biochem 292, 245-249. Schuurhuis DH, Ioan-Facsinay A, Nagelkerken B, van Schip JJ, Sedlik C, Melief CJ, Verbbek JS, Ossendorp F (2002) Antigen-antibody immune complexes empower dendritic cells to efficiently prime specific CD8+ CTL responses in vivo. J Immunol 168, 2240-2246. Seddiki N, Nato F, Lafaye P, Amoura Z, Piette JC, Mazie JC (2001) Calreticulin, a potential cell surface receptor involved in cell penetration of anti-DNA antibodies. J Immunol 166, 6423-6429. Sorace JM, Johnson RJ (1990) A cytotoxic monoclonal antileukemia antibody binds to histone H1. Hybridoma 9, 419427. Syrigos KN, Charalambopoulos A, Pliarchopoulou K, Varsamidakis N, Machairas A, Mandrekas D (2000) The prognostic significance of autoantibodies against dsDNA in patients with colorectal adenocarcinoma. Anticancer Res 20, 4351-4353. Tanner JE (2003) Apoptotic tumor cells promote angiogenesis through the release of free nucleosomes or nucleosomes associated with apoptotic bodies. Proceedings of the AACR, Volume 44, 2nd ed. #2058. Torchilin VP, Iakoubov LZ, Estrov Z (2001) Antinuclear autoantibodies as potential antineoplastic agents. Trends Immunol 22, 424-427. Torchilin VP, Iakoubov LZ, Estrov Z (2003) Therepeutic potential of antinuclear autoantibodies in cancer. Cancer Therapy 1,179-190. Torchilin VP, Lukyanov AN, Gao Z, Papahadjopoulos-Sternberg B (2003) Immunomicelles: targeted pharmaceutical carriers for poorly soluble drugs. Proc Natl Acad Sci USA 100, 6039-6044. von Muhlen CA, Tan EM (1995) Autoantibodies in the diagnosis of systemic rheumatic diseases. Semin Arthritis Rheum 24, 323-358. Walker SE, Anver MR, Schechter SL, Bole GG (1978) Prolonged lifespan and high incidence of neoplasms in NZB/NZW mice treated with hydrocortisone sodium succinate. Kidney Int 14, 151-157. Wyllie AH (1993) Apoptosis (the 1992 Frank Rose Memorial Lecture). Br J Cancer 67, 205-208. Xavier RM, Yamauchi Y, Nakamura M, Tanigawa Y, Ishikura H, Tsunematsu T, Kobayashi S (1995) Antinuclear antibodies in healthy aging people: a prospective study. Mech Ageing Dev 78, 145-154. Xu, FJ, Yu YH, Bae DS, Zhao XG, Slade SK, Boyer CM, Bast RC Jr, Zalutsky MR (1997) Radioiodinated antibody targeting of the HER-2/neu oncoprotein. Nucl Med Biol 24, 451-459. Zelenetz AD (2003) A clinical and scientific overview of tositumomab and iodine I 131 tositumomab. Semin Oncol 30, 22-30.
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The multidisciplinary management of pain and palliative care in cancer patients: a review Review Article
Frank E. Mott1*, Carl Chakmakjian2 and Joel Marcus3 1
Hematology/Oncology, Lung Cancer Clinic and Pain & Palliative Care Clinic, Medical Oncology, 3 Depts of Psychiatry and Internal Medicine, Division of Hematology/Oncology, Psycho-Oncology, Scott & White Clinic, Texas A&M University HSC, Temple, TX, USA 2
__________________________________________________________________________________ *Correspondence: Frank E. Mott, MD FACP, Assistant Professor of Medicine, Hematology/Oncology, Director, Medical Oncology Fellowship Program, Director, Lung Cancer Clinic, Attending, Pain & Palliative Care Clinic, Scott & White Clinic, Texas A&M University HSC, Temple, TX; Tel: 254-724-7048; Fax: 254-724-4904; e-mail: fmott@swmail.sw.org Key words: cancer pain, assessment, treatment, hypnosis Abbreviations: Brief Pain Inventory, (BPI); nonsteroidal anti-inflammatory drugs, (NSAIDS); Psychosocial Collaborative Oncology Group, (PSYCHOG); World Health Organization, (WHO); Received: 14 September 2004; revised: 12 October 2004 Accepted: 15 October 2004; electronically published: October 2004
Summary Pain afflicts many cancer patients and it can create significant distress for the patient and family members. Appropriate assessment of the pain and patient/family as a whole is imperative for proper management. Opioid analgesics are the backbone of treatment; but other adjunctive agents, as well as non-pharmacologic methods to manage the pain and its associated symptoms are an important part of the care of the cancer patient with pain. Multi-disciplinary pain and palliative care clinics can facilitate a collegial approach and coordinate the various modalities necessary for the global care of these patients. these symptoms are important. This is the underlying reason for the emergence of palliative care and supportive oncology. The term palliative is derived from the Latin pallium: to cloak or cover. Many definitions of palliative care exist, but the common theme is its goal of delivering the utmost quality of life to patients and their families when dealing with an incurable illness. The definition of palliative care put forth by the Canadian Palliative Care Association in 1995 takes into account this broad scope (Canadian Palliative Care Association, 1995): Palliative care, as a philosophy of care, is the combination of active and compassionate therapies intended to comfort and support individuals and families who are living with a life-threatening illness. During periods of illness and bereavement, palliative care strives to meet physical, psychological, social, and spiritual expectations and needs, while remaining sensitive to personal, cultural, and religious values, beliefs, and practices. Palliative care may be combined with therapies aimed at reducing or curing the illness, or it may be the total focus of care. It is quite obvious from this that patients with advanced cancer who also suffer from pain do not do so in
I. Introduction Pain is a common symptom in an Oncology practice. Surveys have indicated that pain is experienced by 3060% of cancer patients during active therapy and by more than two-thirds of those with advanced disease (Bonica et al, 1990). In addition to pain, patients with advanced cancer also report a high prevalence of fatigue, generalized weakness, dyspnea, delirium, nausea, and vomiting (Reuben et al, 1988; Coyle et al, 1990; Curtis et al, 1991; Donnelly and Walsh, 1995). Psychiatric disturbances occur in over 60% of this group, with adjustment disorders, depression, anxiety, and delirium being the most common (Massie et al, 1983; Bukberg et al, 1984; Beitbart and Passik, 1993). Regardless of the technological advances that medicine may have achieved or expects to see in coming years, the relief of suffering has been and always will be at the very core of its endeavors and should be the guiding principle in anything a physician or other health care provider does. In Oncology, where over 50% of patients will die of their disease (Walker and Bruera, 2002), debilitating symptoms are common and programs that focus on the appropriate assessment and management of
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Mott et al: Pain and palliative care in cancer patients a void, free of other symptoms. Pain leads to emotional distress and sleep disturbance, which in turn create fatigue. Pain medications can cause nausea and/or constipation. Appetite is affected and dysphoria can result from the central nervous system disturbances. Thus, managing cancer-related pain is a complex, inter-related program that incorporates a number of disciplines. There is more to pain management than simply writing a prescription for an opiate analgesic. As such, the litany of physical and psychological influences that affect or result from the pain need to be addressed as well. Most cancer centers now provide pain management as part of their program, often in the format of a separate, multidisciplinary team that addresses the global needs of the patient with cancer-related pain and its associated symptoms. These teams comprise personnel with expertise in the requisite disciplines that are vital to attain successful pain management with a balance of quality of life. These include medical and radiation oncologists, often with special interest and focused expertise in pain management and palliative care. There are now accredited societies in Hospice and Palliative Care with one-year fellowships leading to board certification in this subspecialty. Pharmacists are critical and provide proper dosing recommendations, help prevent serious drug interactions in a population of patients who are often taking multiple medications, and offer helpful counseling to patients and, more importantly, family members who are often primary care-givers and monitor the administration of medications at home. Nursing personnel, social workers, physical therapists, and chaplains are also important members of the team. Psychologists, often with focus in the area of Psycho-oncology, can provide significant benefit by helping patients cope with anxiety, depression, and fear of death. The gradual and successful transition to more palliative care includes close interaction with hospice personnel. Ancillary services, such as pain block clinics, interventional radiology, complementary and alternative medicine experts, acupuncturists, hypnosis, etc. all have a potential role in the overall care of patients with cancerrelated pain.
allow a patient to better discriminate subtle changes in their pain character. It also helps the health care provider better quantify the effect of their intervention.
III. Associated pain factors In addition to the level of pain, several associated factors are important in the overall assessment. These include the quality of the pain, its timing and relation to other activities or events, the location and distribution of the painful area(s), and the effect of any interventions on either relieving or, perhaps producing the pain. The patient’s description of the pain can be very helpful in determining its cause, as well as what diagnostic and/or therapeutic interventions are indicated. Somatic pain is usually focal, sharp, aching, throbbing, or pressurelike. Isolated bone metastases can produce this type of pain. Visceral pain is more diffuse and described as crampy or gnawing and is seen with soft tissue involvement such as diffuse liver metastases or bowel obstruction. Neuropathic pain is burning, tingling, and often radiating or lancinating. This type of pain is common when nerve plexuses are involved or spinal cord lesions press on nerve roots. It can also be seen as a consequence of some chemotherapy agents, which damage peripheral nerve endings. When the pain occurs, either during the day or night, can be helpful for making recommendations to modify behavior so as to reduce the pain syndrome. Most pain is divided into acute and chronic. Acute pain is characterized by a recent onset, usually well defined by the patient or family, often associated with a temporal event such as a fall or other injury, and usually transient in nature. Chronic pain has been defined as usually more than three months in duration, constant or even progressive, and often associated with a chronic pathologic process, such as cancer (Bonica et al, 1990). Chronic cancer-related pain can have acute exacerbations, which may indicate progression of the underlying disease. Pain location is helpful in discerning its cause. Most cancer patients will experience pain in more than one site (Portenoy et al, 1992). Focal pain usually is due to an underlying, localized lesion; however, focal pain may also be referred from another site. For example, shoulder pain that is not due to pathology involving the shoulder itself requires investigation of the apical lung area to exclude tumors invading the underlying brachial plexus, and the region immediately above and below the ipsilateral diaphragm, which can produce referred pain to the shoulder. Asking the patient to point to the area(s) of pain is one method, but a more useful approach is to use a drawing of the front and back of the body. The patient can mark the involved area. This method is simple and provides an easy means for tracking changes in pain locations. It also provides a way to show if the pain is focal or diffuse. Patients may have already tried their own remedies to treat the pain and it is important to ascertain their effectiveness. Over-the-counter analgesics are the most common agents employed, but it is not unusual for a wellintentioned friend or family member to provide the patient
II. Pain: the “Fifth vital sign” Pain, if not reported by the patient and not asked about by the physician or nurse, will be overlooked and under-treated. The prevention of and relief from pain is vital to optimal health status. A number of palliative care programs have endorsed the concept of pain as the “fifth vital sign”, as integral to the care of a patient as their temperature, pulse, respirations, and blood pressure (Abrahm and Snyder, 2001; Lynch, 2001). The National League of Nursing has promoted it and in many centers, it is a common part of the nursing assessment of not only the cancer patient, but also any patient with chronic pain. The development of an appropriate, reproducible pain scale is critical to the assessment of pain. Asking the patient to quantify the severity of their pain on a scale of one to five or one to ten, with the higher number representing more severe pain, is a useful method of determining how much pain a patient has. We find that one to ten is more reliable since the increased gradations 366
Cancer Therapy Vol 2, page 367 with one of their unused prescription analgesics. Nonpharmacologic approaches such as topical ice packs or heating pads, range of motion exercises or immobilization, etc. are all likely to have been tried in one fashion or another. The use of complementary and alternative therapies is popularly accepted by many patients and they may have resorted to some of these interventions. If the health care provider does not ask, the patient may not mention them. Even when asked, patients may be reluctant to accurately describe pain, or even report it at all. Reasons for this vary and probably range from denial to stoicism. The following list includes some of the reasons why patients may be reluctant to report pain (Ward et al, 1993). • Do not want to be perceived as complainers or “whiners” • May think reporting pain will draw attention away from the disease itself • Acknowledging increased pain will indicate a worsening of their condition • May view pain as an “inevitable” part of cancer • Concern about the treatment of pain, e.g. opiates, radiation, etc • Potential side effects of pain medications • Fear that taking opiates too early will prevent adequate future pain control • Concerned about the costs of pain medications • May not want to distress or burden family members In addition, cultural backgrounds may affect a patient’s attitude about pain and its treatment. There may be a fear of “addiction” to opiates or that once an opiate is started that means the “end is near” or the patient is “giving up”. Patients need to be reassured often and encouraged to report pain.
with family and friends, sleep, and quality of life. In so doing, the management of the pain and its associated factors will be more easily facilitated.
VI. Medical management of pain It can often be a difficult decision when attempting to determine the most appropriate medical regimen to alleviate a patient’s suffering. A basis by which pain management may be approached was created by the World Health Organization (WHO), (1996) in Geneva, Switzerland. This logical approach to pain management has been endorsed by the Royal College of Physicians, the European Association for Palliative Care, and the Education for Physicians on End-of-Life Care (Emanuel et al, 1999; Medicine Committee of the Royal College of Physicians, 2000; Hanks et al, 2001; Thomas and von Gunten, 2003). In this summary a “three step ladder” is described. Step 1 is characterized by mild pain. Mild pain would be classified as a one to three on a ten point scale by the patient. Step 2 is moderate pain, which would be a four to six on a ten point patient verbal scale. Finally, step 3 is severe pain which would be described as a seven to ten on a ten point scale (World Health Organization, 1996; Thomas and von Gunten, 2003). Each step of the ladder is associated with specific medications which should be utilized. In addition, adjuvant pain medications may be used alone or more commonly in combination with the recommended medications at any step to achieve better pain control. It is important to note that the ladder is not a concrete demarcation between pain levels, but more a transition. It provides a broad approach to pain management, but by no means does it imply a rigid recipe.
A. Step 1 The first step of the WHO ladder primarily recommends use of non-opioid analgesic medications. Non-opioid analgesics include nonsteroidal antiinflammatory drugs (NSAIDS) and acetaminophen. Nonopioid analgesics are especially useful (alone or in combination) for painful bony metastases or pain secondary to infiltration of muscle or soft tissue. NSAIDS and acetaminophen are subject to a “ceiling effect.” In other words, increasing the dose above recommended levels will not provide further analgesic effect but may only increase the likelihood of experiencing side effects (World Health Organization, 1996; Thomas and von Gunten, 2003). NSAIDS mechanism of action is inhibition of the cyclo-oxygenase enzyme. This decreases the production of pro-inflammatory cytokines. The main side effects associated with NSAIDS include renal insufficiency, platelet inhibition and gastrointestinal upset. It is believed that the severity of the latter two side effects may be decreased with the use of newer generation NSAIDS that are selective in the inhibition of the cyclooxygenase two enzyme (Thomas and von Gunten, 2003). The WHO dosing guidelines references ibuprofen (a commonly used NSAID) at a dose of 400 mg every 4-6 hours with a maximum cumulative dose of 3 grams in a twenty-four hour period (World Health Organization, 1996).
V. Pain impact It is important to assess the person with pain and not just the pain itself (Turk et al, 2002). It has been established that pain rated at four or greater on an 11-point scale can significantly reduce the ability of patients to function (Serlin et al, 1995). Pain, or the anticipation of it, can create anxiety, which, in turn, can lead to a number of physiologic disturbances such as fatigue, sleep disturbance, anorexia, nausea, and additional sensation of pain. In an effort to address the impact of pain on the patient’s functional ability, several scales have been developed. The McGill Pain Questionnaire (Melzack, 1975) addresses pain quality with no less than 78 adjectival descriptors. A more reasonable modification of this that uses just 15 descriptors was described by Melzack (1987). The Brief Pain Inventory (BPI) is a 16-question, comprehensive self-reporting survey that includes numeric ratings of pain severity at its worst, least, currently, and average; a figure drawing to locate pain, and pain impact (Daunt et al, 1983). By incorporating these and/or similar scales into the initial and subsequent assessments of cancer-related pain, the clinician can gain valuable information about the impact of pain on the patient’s mood, work, interactions 367
Mott et al: Pain and palliative care in cancer patients
codeine and have one fifth the potency of morphine (World Health Organization, 1996). Usual doses are 50100 mg every four to six hours. Appropriate dose adjustments should be made in elderly patients and those with renal or hepatic impairments (impairment (Micromedex Helthcare Series, 2004). In our experience, tramadol is a relatively weak opioid analgesic. Hydrocodone is supplied in 5-10 mg increments in combination with acetaminophen or ibuprofen. This can be a very effective medication for patients with moderate pain. Dosing is usually limited by the acetaminophen or ibuprofen component of the medication.
Acetaminophen is metabolized in the liver. Maximum dosing should be up to 4 grams in a twenty-four period. In patients with liver disease, 2 grams should not be exceeded in a twenty-four hour period.
B. Step 2 The second step of the WHO ladder recommends the use of opioid analgesics with or without concurrent use of non-opioid analgesics or adjuvant medications for the treatment of moderate pain. The medications primarily included in the second rung of the WHO ladder are codeine, hydrocodone and tramadol (Thomas and von Gunten, 2003). These three drugs are classified as analgesic opioids. Common side effects in this class include drowsiness, constipation and nausea (Micromedex Helthcare Series, 2004). Codeine sulfate is supplied in 15 mg, 30 mg and 60 mg tablets (Micromedex Helthcare Series, 2004). It may be administered orally in doses up to 120 mg every 4 hours. Above this dose, side effects begin to outweigh the benefits of analgesic relief (World Health Organization, 1996). Appropriate dose adjustments should be made in patients with renal impairment (Micromedex Helthcare Series, 2004). In addition, codeine is metabolized to morphine and, in some patients, this ability is impaired or inhibited by other drugs, such as fluoxetine. Oxycodone, when used alone, is a potent opiate and a schedule II narcotic. However, in combination with acetaminophen (Percocet), it may be a useful agent in this stage of pain management. Tramadol is supplied in a 50mg tablet. It has both opioid and nonopioid properties allowing it to cause less constipation in addition to reducing the other opioid side effects as well. It is estimated to be twice as potent as
C. Step 3 The final step of the WHO ladder endorses the use of potent analgesic opioids with or without concurrent use of non-opioid analgesics or adjuvant medications in the treatment of severe pain. Morphine, oxycodone, fentanyl, hydromorphone and methadone provide the pharmaceutical foundation for the treatment of severe pain. Side effects of these medications include nausea, emesis, constipation, pruritis, sedation, urinary retention, dry mouth, and respiratory depression (Micromedex Helthcare Series, 2004). Morphine sulfate should really be considered the â&#x20AC;&#x153;backboneâ&#x20AC;? of opioid analgesics. It may be administered via multiple routes to include oral, sublingual, rectal, subcutaneous, intravenous, intramuscular or epidural/intrathecal. Oral preparations come in immediate release (pill or elixir) and extended realease formulations. Dose adjustments should be made in renal impairment and cirrhotic patients (Micromedex Helthcare Series, 2004). The dose of the morphine sulfate should be titrated upward until pain control is achieved. The oral to intranvenous conversion factor is three to one. 368
Cancer Therapy Vol 2, page 369 Oxycodone is structurally similar to codeine. It has good oral bioavailability, but can be administered rectally as well. It also is manufactured as a long acting or immediate release preparation. Potency is similar to morphine sulfate (World Health Organization, 1996). Dose adjustments should be made accordingly for the following populations: liver disease, renal impairment, geriatric patients, and patients requiring the use of other central nervous system depressants. Fentanyl is yet another opioid analgesic. It is unique because it is supplied as a transdermal patch. This gives it a special niche for patients without intravenous access who are unable to swallow a pill. It is also supplied as a “lollypop” which is absorbed across the oral mucosa and an intravenous formulation that provides effective analgesia with a short half-life. Hydromorphone is a particularly potent opiate. When administered orally, the potency is about eight-fold greater than morphine. The intravenous preparation is approximately six-fold greater than intravenous morphine (World Health Organization, 1996). Hydromorphone has metabolites that are cleared renally and, in the face of renal insufficiency, can accumulate and lead to neurotoxicity. Methadone may often be overlooked by physicians because it is also approved for narcotic detoxification and treatment. However, it has been very effective for use in patients with pain that is requiring large amounts of other opiates like morphine or hydromorphone; or in patients developing analgesic tolerance to increasing doses of other opiates. It is a synthetic opioid analgesic. The plasma halflife is variable (World Health Organization, 1996). In our practice, it is typically prescribed every eight hours. Methadone offers the following advantages: no neuroactive metabolites, low cost, good oral bioavailability (~80%) (Walker and Bruera, 2002). Methadone does have a unique pharmacodynamic profile which affects its equianalgesic conversion from morphine as the morphine doses increase, (see opioid conversion table). Drugs that should be avoided in the management of chronic cancer pain include meperidine, pentazocine, butorphanol, and propoxyphene. Meperidine has a very short half-life and its metabolite, nor-meperidine, can accumulate, especially in the face of renal insufficiency, and lead to seizures. Both pentazocine and butorphanol have agonist and antagonist narcotic properties, which diminish their effectivenss and can lead to acute withdrawal symptoms. Propoxyphene is an extremely weak opioid. The analgesic benefit of propoxyphene products usually is derived more from the acetaminophen that is included in the formulation.
which have activity via NMDA receptors, which modulate neuroapathic pain stimuli. Antiepileptic drugs, antidepressants and corticosteroids are the main classes of medications utilized either alone or in combination with opioids or nonsteroidal anti-inflammatories. Antiepileptic drugs have primarily been studied in the treatment of nonmalignant forms of neuropathic pain (Walker and Bruera, 2002). The believed mechanism of action lies in their effect on neuronal discharge (Thomas and von Gunten, 2003). Antiepileptic drugs used include gabapentin, carbamazepine, phenytoin and clonazepam. Gabapentin is the most commonly used drug in this class. It is well tolerated with the most troubling side effect being lethargy (Thomas and von Gunten, 2003). It should be started at a low dose of one hundred to three hundred milligrams per day and titrated upward as tolerated (Walker and Bruera, 2002). Tricyclic antidepressants are the most frequently utilized class of anidepressants with regard to neuropathic pain. The most troubling side effects of tricyclic antidepressants are the anticholinergic properties that include dry mouth, fatigue, constipation, and urinary retention (Walker and Bruera, 2002, Thomas and von Gunten, 2003). Although amitriptyline has been the most frequently studied drug in this class, it is also known to have the most anticholinergic properties. For this reason, other tricyclics such as desipramine and nortriptyline have been effectively administered for control of neuropathic pain (Max et al, 1992; Watson et al, 1998; Thomas and von Gunten, 2003). Corticosteroids play an important role in adjuvant analgesia in oncologic patients. They are potent antiinflammatory agents that may be helpful for neuropathic or nociceptive pain (Thomas and von Gunten, 2003). Corticosteroids are indicated for use in pain control in the following situations: nerve or spinal cord compression, headache secondary to increased intracranial pressure, bone pain, pain secondary to capsular distension or duct obstruction (World Health Organization, 1996; Walker and Bruera, 2002). Dexamethasone has minimal mineralocorticoid properties compared to other steroids, making it an ideal option in terminal patients (Swartz and Dluhy, 1978; Demoly and Chung, 1998; Thomas and von Gunten, 2003).
E. Opioid conversions To effectively manage a patient’s pain, it is imperative to understand the concept of opioid conversions. Many references provide conversion tables to assist clinicians in the care of their patients. Table 1 shows the conversion ratios employed at our institution. Occasional patients that develop intractable, uncontrolled pain may require hospitalization to achieve adequate pain control in a timely fashion. In these instances, we may use a PCA (patient controlled analgesia) pump to determine the patient’s opioid need over a twenty-four hour period. Equianalgesic conversions are subsequently calculated to the oral dose equivalency and administered. The pump dose is reduced by fifty
D. Adjuvant analgesics As described above, adjuvant analgesics may be given in conjunction with Step 1, Step 2, or Step 3 medications to optimize pain control. Alternative causes of pain such as neuropathic pain are not very responsive to opioid therapy (Walker and Bruera, 2002), with the exception perhaps of levorphanol and methadone, both of
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Mott et al: Pain and palliative care in cancer patients Table 1. Drug Name Codeine Fentanyl* Hydrocodone Hydromorphone Methadone Morphine (IR)! Morphine (ER) ! Oxycodone (IR) Oxycodone (ER)
Approximate Intravenous 130 0.1 1.5-2 10
Equianalgesic Oral 200 30 6 Variable** 30 30 20 20
Dose Rectal
6 20
Duration (hours) 4-6 1-2 4-6 2-4 6-12 3-4 12 4-6 12
*The approximate ratio for a fentanyl patch is as follows: 50 microgram/hour patch: 90 mg per day of oral morphine. **As noted above, the conversion factor from morphine to methadone is variable. For patients requiring 30-90 mg of oral morphine per day, the conversion factor is 4:1 (morphine to methadone). For those who require 90-300 mg of oral morphine per day, the conversion factor is 8:1 (morphine to methadone). Finally, those requiring greater than 300 mg of morphine per day, the conversion factor is 12:1 (morphine to methadone). ! extended-release (ER), immediate-release (IR)
become disabling, such as depression, anxiety, panic, social isolation, and existential and spiritual crisis Twenty to forty percent of cancer patients will demonstrate significant distress (Roth et al, 1998; Zabora et al, 2001). The levels of distress correlate with the cancer site and type, age, and other variables. Under-treated cancer pain negatively affects sleep, energy and normal activity. It can lead to anxiety, depression, and an adverse quality of life that further exacerbates the patientâ&#x20AC;&#x2122;s distress (Montour and Chapman, 1991). Non-pharmacologic pain relief methods can be integrated within cancer pain treatment programs (Clinical Practice Guidelines, 1994). This is consistent with the consensus statement from the National Cancer Institute Workshop on cancer pain (National Cancer Institute, 1990): Under treatment of pain and other symptoms of cancer is a serious and neglected public health problem and ...every patient with cancer should have the expectation of pain control as an integral aspect of his/her care throughout the course of the disease The use of non-pharmacologic methods to reduce distress and help patients cope with their cancer and its related symptoms is an important part of the palliative care team. As such, clinical psychologists play a significant role in the care of cancer patients. At our institution, we have incorporated such personnel into the program under the global auspices of â&#x20AC;&#x153;Psycho-Oncologyâ&#x20AC;?. A number of methodologies are utilized, including psycho-social assessment, bio-feedback, relaxation exercises, and hypnosis. We have found that, for many patients, hypnosis can play an important role in developing helpful coping strategies.
percent six hours after the administration of the first oral dose and discontinued twelve hours after the first oral dose. Opiate side effects such as nausea and sedation are usually transient and can be managed by carefully introducing the opiate and titrating gradually as needed. Patients need to be counseled and reassured regarding the self-limiting nature of these side effects. In some cases, concurrent use of anti-emetics for the first few days can reduce the nausea associated with a new opiate or a dosage increase. Sedation can be off-set by timing the dosage administration; in some cases brief use of a stimulant such as methylphenidate may be helpful. Constipation is a common side effect of opiates that does not usually dissipate with time and, therefore, requires continuous and concurrent use of laxatives like senna. In cases of severe obstipation, orally administered naloxone may improve bowel function, again without systemic withdrawal. Respiratory depression with opiates is rare, if dosing guidelines are followed and dosage increments are done gradually. Severe respiratory depression can be reversed by inhaled naloxone without precipitating a systemic withdrawal.
VII. Psychosocial aspects of pain management Cancer pain can cause suffering that is both physically and psychologically devastating. Distress is the term used to characterize the adverse psychological components of cancer care. While distress is an umbrella term, it can help the patient to define their subjective level of discomfort surrounding the disease and its treatment. Holland described distress as follows (Holland, 1999): Distress is a multifactorial unpleasant emotional experience of a psychological (cognitive, behavioral, emotional), social, and/or spiritual nature that may interfere with the ability to cope effectively with cancer, its physical symptoms and its treatment. Distress extends along a continuum, ranging from common normal feelings of vulnerability, sadness, and fears to problems that can
VIII. Assessment considerations and mind-body interventions with cancer patients Working with cancer patients requires the clinician to see the patient and their symptoms on a multitude of levels. To conceptualize the patient, their disease, distress and pain the clinician must see the patient in their totality. 370
Cancer Therapy Vol 2, page 371 A cognitive behavioral assessment will lend itself to the development of specific interventions that will address the entirety of the patient. Using the DSM III criteria (American Psychiatric Association, 1980), the Psychosocial Collaborative Oncology Group (PSYCHOG) studied the psychiatric disorders in cancer patients in three cancer centers (Derogatis et al, 1983). Of the 215 randomly studied patients, 47% met the criteria for a psychiatric diagnosis. Ninety percent of those were in response or manifestation to the cancer diagnosis or treatment. Thirty-nine percent of the cancer patients diagnosed with a psychiatric disorder were also experiencing significant pain. Anxiety may be assumed to be present whenever the patient presents for therapy with the diagnosis of a possibly life-threatening disease. While it may never reach the threshold of diagnostic credence, that does not mitigate its existence nor its impact on the patient. On occasion, overt symptoms of anxiety may not be evident. Further probing may reveal a more typical constellation of symptoms of chronic anxiety such as sweating, sleeplessness, muscle tension, tachycardia, and so on. Constant repeated exposure of the body to these anxiety symptoms will produce a stress reaction within the patient that can further debilitate their physical condition, frequently manifesting itself in greater fatigue. This, in turn, further aggravates the anxiety, leading to more stress. These symptoms are all very amenable to hypnotic intervention.
reported a reduction in anxiety and depression (Kraft, 1990). Our experience has indicated that hypnotherapy is well accepted by cancer patients and is a powerful adjunct to the usual standard of oncology care (Marcus et al, 2003 a, b, c, d; 2004 a, b, c). Pain should be considered in its totality of impact. Pain must also be considered in its temporal existence. Every patient will be able remember a time prior to the advent of the cancer and its attendant pain. Pain exists in the moment, and that is generally the patient’s primary concern. The clinician needs to keep in mind that the pain should be treated in a prophylactic manner. When pain is present, a certain amount of anxiety must be considered to be in evidence. The anxiety may be overtly visible or it may be covertly in evidence by its conspicuous absence. Anxiety may manifest itself in the family. Understanding and awareness of the patient’s anxiety about impending pain and the clinician’s role in preventive management needs to be conveyed to the patient to allay this anxiety. Interventions such as hypnosis can increase the patient’s feeling of self-efficacy and mastery of their internal and external environments. As the patient becomes less anxious and increasingly competent in their use of self-hypnosis to manage their pain, their attendant anxiety frequently is diminished. This may have a similar effect on the family system as family members see their loved ones coping better with the pain.
X. Conclusions Cancer pain management requires a directed history to localize, quantify, and qualify the pain. The assessment should include all ancillary symptoms as well as effects on family members and immediate caregivers. The patient’s co-morbidities must be considered. Psychological symptoms like depression, anxiety, remorse, guilt, and other components of “distress” need to be addressed as part of the global management of cancer pain. A multidisciplinary team incorporating medical, nursing, psychology, and social services can best facilitate this protocol. The World Health Organization Analgesic Ladder can provide a simple approach to the initial medical regimen that is selected. Opiates play a vital role in the medical management of pain, but the use of adjunctive agents provides valuable integration in the relief of pain and ancillary symptoms. By assessing the patient and their pain in a holistic fashion, appropriate palliation can be achieved more effectively. Adequate analgesic relief improves one’s quality of life. Integrating mind-body interventions can assist the patient in controlling pain and help develop a sense of mastery and self-efficacy that can improve the treatment process.
IX. Hypnotic management of pain and distress Hypnotic relaxation is the most frequently cited form of non-pharmacologic cognitive pain control. Hypnotic relaxation may be defined as a deeply relaxed state involving mental imagery (Woody et al, 1992; Hammond and Elkins, 1994; Elkins, 1997). Hypnotic relaxation in the treatment of cancer patients involves the use of relaxation and mental imagery to induce relaxation, reduce anxiety and distress, and help patients detach themselves from obsessional thoughts (Araoz, 1983). Hypnotic relaxation has been found to be of significant benefit in reducing anxiety (Wadden and Anderton, 1982; Elkins, 1986). Furthermore, patients who develop anxiety disorders may be more hypnotizable than others (Frankel, 1974). In the use of hypnotic relaxation for pain management, the focus is on instructing the patient in relaxation and mental imagery. The patient learns a cognitive method of pain management which is utilized at the discretion of the patient and within the patient's own control. The successful effect is to introduce a non-pharmacologic method of pain control that may decrease unnecessary dependency on analgesics for pain. Hypnotic relaxation is a safe method, which, when properly used, has no harmful side effects. Cancer patients frequently experience anxiety due to anticipation about the illness, anticipation of potential treatment-related side effects such as nausea and vomiting, or anticipation of entering the final stages of life (Roberts et al, 1997). Kraft studied hypnotic relaxation in the management of 12 terminally ill cancer patients and
References Abrahm JL, Snyder L (2001) Pain assessment and management. Prim Care 28, 269-97. American Psychiatric Association. Diagnostic and statistical manual of mental disorders, 3rd ed. Washington, DC, 1980.
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Mott et al: Pain and palliative care in cancer patients Araoz DL (1983) Use of hypnotic techniques with oncology patients. J Psycho Soc Oncol 1, 47-54. Beitbart W, Passik SD (1993) Psychiatric aspects of palliative care. In Doyle D, Hanks GW, MacDonald N (eds), Oxford Textbook of Palliative Medicine. Oxford, Oxford University Press, pp 609-626. Bonica JJ, Ventafridda V, Twycross RG (1990) Cancer Pain. In, Bonica JJ, ed. The management of pain, 2nd ed. Philadelphia, Lea & Febiger, pp400-460. Bukberg J, Penman D, Holland JC (1984) Depression in hospitalized cancer patients. Psychosom Med 46, 199-212. Canadian Palliative Care Association. Palliative care (1995) Towards a consensus in standardized principles and practice. Ottowa, Ontario, Canadian Palliative Care Association. Clinical Practice Guidelines, Number 9, Management of Cancer Pain (1994) U.S. Department of Health and Human Services, Public Health Service, Agency for Health Care Policy and Research. Coyle N, Adelhardt J, Foley KM, et al (1990) Character of terminal illness in the advanced cancer patient, Pain and other symptoms during last four weeks of life. J Pain Symptom Manage 5, 83-89. Curtis EG, Krech R, Walsh TD (1991) Common symptoms in patients with advanced cancer. J Palliative Care 7, 25-29. Daunt RL, Cleeland CS, Flanery RC (1983) Development of the Wisconsin Brief Pain Questionnaire to assess pain in cancer and other diseases. Pain 17, 197-210. Demoly P, Chung KF (1998) Pharmocology of corticosteroid. Respir Med Mar 92, 385-4. Derogatis L, Morrow G, Fetting J, et al (1983) The prevalence of psychiatric disorders among cancer patients. JAMA 249, 751-7. Donnelly S, Walsh D (1995) The symptoms of advanced cancer. Semin Oncol 22 (suppl 2), 67-72. Elkins GR (1986) Hypnotic Treatment of Anxiety. In Wester, W.C (Ed.) Clinical Hypnosis, A Case Management Approach. Cincinnati, C.J. Krebbiel Co. Elkins GR (1997) My Doctor Does Hypnosis. Chicago, ASCH Press. Emanuel LL, von Gunten CF, Ferris FF, editors. Module 4, pain management. In, EPEC participants handbook. Chicago (IL), EPEC Project, Robert Wood Johnson Foundation, 1999, M41-35. Frankel FH (1974) Trance capacity and the genesis of phobic behavior. Arch Gen Psychiat 31, 261-263. Hammond DC and Elkins GR (1994) Standards of Training in Clinical Hypnosis, Chicago, ASCH Press. Hanks GW, Conno F, Cherny N, et al (2001) Morphine and alternative opioids in cancer pain, the EAPC recommendations. Br J Cancer 84, 587-93. Holland J (1999) NCCN practice guidelines for the management of psychosocial distress. National Comprehensive Cancer Network. Oncology, 13, 113-147. Kraft T (1990) Use of hypnotherapy in anxiety management in the terminally ill, A preliminary study. Brit J Exp Clin Hypn 7, 27-33. Lynch M (2001) Pain as the fifth vital sign. J Intraven Nurs 24, 85-94.. Marcus J, Elkins G, Mott FE (2003a) A model of hypnotic intervention for palliative care (Abstract/poster presentation) Cancer Care Conference. Washington, DC. April. Marcus J, Elkins G, Mott FE (2003b) A model of hypnotic intervention for palliative care. Adv Mind Body Med 19, 24-7 Marcus J, Elkins G, Mott FE (2003c) The integration of hypnosis into a model of palliative care. Integr Cancer Ther 2, 36570. Marcus J, Elkins G, Mott FE (2004a) Solution focused therapy,
Creating goals and meaning for the palliative care patient. Psychooncology 13, S55, Abstract P16-5. Marcus J, Elkins G, Mott FE, Palamara L, Herrington J (2004b) Development of a triage system for referrals to a Pain & Palliative Care Clinic (PPCC). Psychooncology 13, S28, Abstract P3-2. Marcus J, Elkins G, Mott FE, Wong L (2004c) Hypnosis as an integral component in comprehensive cancer care. Psychooncology 13, S51, Abstract P14-2 Marcus J, Elkins G, Young R, Mott FE, Cheung A, Rajab H, Palamara L, Herrington J (2003d) Translating clinical phenomenon into researchable questions and clinical practice, A multidisciplinary quest. Cancer Care Conference. Washington, DC. April. Massie MJ, Holland JC, Glass E (1983) Delirium in terminally ill cancer patients. Am J Psychiatry 140, 1048-1050. Max, MB, Lynch SA, Muir J, et al (1992) Effects of desipramine, amitriptyline, and fluoxetine on pain in diabetic neuropathy. N Eng J Med 326, 1250-6. Melzack R (1975) The McGill Pain Questionnaire, major properties and scoring methods. Pain 1, 277-99. Melzack R (1987) The short-form McGill Pain questionnaire. Pain 30, 191-7. Montour C.M, Chapman C.R (1991) Pain management and quality of life in cancer patients. In, Lehmann R.K.A, Zech D, editors. Transdermal fentanyl, a new approach to prolonged pain control. Berlin, Springer-Verlag; p.42-63. National Cancer Institute. NCI Workshop on Cancer Pain. September 14-15, 1990, Bethesda, M.D. Portenoy RK, Miransky J, Thaler HT, et al (1992) Pain in ambulatory patients with lung or colon cancer , prevalence, characteristics, and impact. Cancer 70, 1616-1624. Principles of pain control in palliatuve care for adults, guidance prepared by a working group of the Ethical Issues in Medicine Committee of the Royal College of Physicians (2000) J R Coll Physicians Lond 34, 350-352. Reuben DB, Mor V, Hiris J (1988) Clinical symptoms and length of survival in patients with terminal cancer. Arch Intern Med 148, 1586-1591. Roberts CS, Piper L, Denny J and Cuddeback G (1997) A support group intervention to facilitate young adults' adjustment to cancer. Health Soc Work, 22, 133-41. Roth, A.J, Kornblith, A, Batel-Copel, L (1998) Rapid screening for psychologic distress in men with prostrate carcinoma, a pilot study. Cancer 82, 1904-1908. Serlin RC, Mendoza TR, Nakamura Y, et al (1995) When is cancer pain mild, moderate, or severe ? Grading pain severity by its interference with function. Pain 61, 277-84. Swartz SL, Dluhy RG (1978) Corticosteroids, clinical pharmacology and therapeutic use. Drugs 16, 238-55. Thomas, J R, von Gunten, C F (2003) Pain in Terminally Ill Patients, Guidelines for Pharmacological Management. CNS Drugs, 17, 621-631. Thomson (2004), Micromedex Helthcare Series, Vol. 121, 1974-. Turk DC, Monarch ES, Williams AD (2002) Cancer patients in pain, considerations for assessing the whole person. Hematol Oncol Clin N Am 16, 511-525. Wadden TA and Anderton CH (1982) The clinical use of hypnosis. Psychol Bull, 91,215-243. Walker PW, Bruera ED (2002) Hematology/Oncology Clinics of North America-Palliative Care Medicine 530-1, 545. Walker PW, Bruera ED (2002) Palliative Care Medicine, Preface. Hem-Onc Clin N Amer 16; xiii-xiv. Ward SE, Goldberg N, Miller-McCauley V, et al (1993) Patient related barriers to management of cancer pain. Pain 52, 31924. Watson CP, Vernich L, Chipman M, et al (1998) Nortriptyline versus amitriptyline in postherpetic neuralgia, a randomized
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Cancer Therapy Vol 2, page 373 trial. Neurology 51, 1166-71. Woody EZ, Bowers KS and Oakman JM (1992) A conceptual analysis of hypnotic responsiveness, Experience individual differences and context. In Contemporary Hypnosis Research (Fromm, E. and Nash, M.R. Eds.), New York, Guilford Press. World Health Organization (1996) Cancer Pain Relief. Second
Frank E. Mott
Edition. Geneva, World Health Organization. Zabora, J, BrintzenhofeSzoc, K, Curbow, B, et al (2001) The prevalence of psychological distress by cancer site. Psychooncology, 10, 19-28.
Carl Chakmakjian
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Joel Marcus
Mott et al: Pain and palliative care in cancer patients
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Cancer Therapy Vol 2, page 375 Cancer Therapy Vol 2, 375-388, 2004
Reversal of immune suppression following vaccination with recombinant vaccinia virus expressing IL-2 in an orthotopic murine model of head and neck squamous cell carcinoma** Research Article
Santanu Dasgupta1, Malaya Bhattacharya-Chatterjee1, Bert W. Oâ&#x20AC;&#x2122;Malley, Jr.2, Sunil K. Chatterjee1* 1
Department of Internal Medicine and the Barrett Cancer Center, University of Cincinnati, Cincinnati, Ohio 45267. Department of Otolaryngology Head and Neck Surgery, University of Pennsylvania Health System, Philadelphia, Pennsylvania 2
__________________________________________________________________________________ *Correspondence: Sunil K. Chatterjee, Department of Internal Medicine and the Barrett Cancer Center, University of Cincinnati, Cincinnati, Ohio 45267; Telephone: (513) 558-0424; Fax: (513) 558-0505; E-mail: sunil.chatterjee@uc.edu Key words: head and neck cancer, vaccinia virus, interleukin-2, immune suppression Abbreviations: Dendritic cells, (DC); Head and neck squamous cell carcinoma, (HNSCC); Immunomodulatory molecules, (IMM); Inducible nitric oxide synthase (iNOS); Phosphate buffered saline (PBS); plaque forming units (pfu); Proliferating Cell nuclear antigen (PCNA); Recombinant vaccinia virus, (rvv); Tumor draining lymph nodes, (TDLN); Vascular Endothelial Growth Factor Receptor (VEGF-R) **This work was supported in part by the grant RO1-CA 89748 from the National Cancer Institute. Received: 20 September 2004; Revised: 20 October 2004 Accepted: 22 October 2004; electronically published: October 2004
Summary Immune cells from mice bearing SCC VII/SF tumors cells in the oral cavity did not respond to mitogenic stimulation. The immunosuppression was mediated by a number of immunomodulatory molecules (IMM), such as GM-CSF, IL-10, TGF-!1, iNOS and VEGF. IMM had been shown to be immunosuppressive in a significant number of cancer patients, including patients with HNSCC. A single subcutaneous injection of irradiated SCC VII/SF tumor cells, infected with a recombinant vaccinia virus expressing IL-2 (rvv-IL-2) reversed the expression of the IMM, both at the mRNA and protein levels, at least partially and restored the anti-tumor cellular immunity. Assay of cytokines from the immune cells on stimulation by SCC VII/SF tumor cells suggested the induction of Th1 type helper activities by the subcutaneous vaccination. Mature dendritic cells (DC) were recruited in the tumor sites and large numbers of both CD4+ and CD8+ T cells were detected near DC following the subcutaneous vaccination. The numbers of PCNA positive cells were reduced as a result of vaccination, suggesting inhibition of tumor cell proliferation. IMM inhibited the expression of MHC class I molecules, which were restored by the subcutaneous vaccination. The numbers of VEGF receptors (VEGF-R) were also reduced by subcutaneous vaccination. Subcutaneous immunization with irradiated SCC VII/SF cells, mixed with recombinant IL-2 induced Th1 helper and cytotoxic T cell activities, although these activities were lower compared to vaccination with rvv-IL-2 infected irradiated SCC VII/SF tumor cells. We concluded that a single subcutaneous injection with irradiated tumor cells infected with rvv-IL-2 or mixed with rIL-2 can reverse the immunosuppression mediated by IMM and restore tumor specific immunity. Squamous cell carcinoma of the head and neck (HNSCC) is the most common malignancy of the upper aero digestive tract, accounting for more than 90% malignancies in this area (Chen et al, 2003). The annual incidence of the disease is alarmingly high with 500,000 new cases worldwide (Kim et al, 2002). It is the fifth leading cause of cancer incidence and sixth leading cause
of cancer related death (Kim et al, 2002). Despite significant improvement in surgery, chemotherapy and radiation therapy, the overall survival for HNSCC patients has not improved in the last 30 years (Kim et al, 2002; Bray et al, 2003). It appears that with the currently applied methods for loco-regional treatment, small undetectable tumor deposits remain in the primary tumor site, resulting
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Dasgupta et al: Reversal of immune suppression in head and neck cancer in local and distant relapse within a short period of time after primary therapy (van Dongen et al, 1996). As a result the overall 5-year survival rate remained at 50%, with only a 30% 2-year survival rates for cancer stages III and IV (Bray et al, 2003). Thus, there is an urgent need for an effective adjuvant therapy for the eradication of residual disease after initial conventional therapy for HNSCC patients. Immuno/gene therapy represents a promising treatment strategy for these patients with minimal residual disease. HNSCC are ideal for immunological study as they are visible, easily biopsed and often removed with their regional lymph nodes (Hadden, 1997). Immunotherapy for cancer has been shown to be effective for only a small population of patients with melanoma and renal-cell carcinoma (Rosenberg et al, 1989). Melanoma and renal-cell carcinoma seem to be among the most immunogenic of human cancers, as evidenced by their high rate of spontaneous regression (Mastrangelo et al, 1975). Immunotherapy of HNSCC is a greater challenge, as patients with HNSCC are highly immunosuppressed (Vokes et al, 1993; Hadden et al, 1994; Gooding et al, 1995) in spite of the fact that these tumors are generally infiltrated with T lymphocytes (Heo et al, 1987; Whiteside et al, 1987). T cells from these patients show only partial response to mitogenic stimulation (Wanebo et al, 1975; Avradopoulos et al, 1997), suggesting that these cells are functionally defective. It has been proposed that the administration of T cell growth factor, such as IL-2 or INF-" may restore the T cell defect (Hamasaki and Vokes, 1995). Although systemic administration of rIL-2 in HNSCC patients caused little or no response, administration of natural or rIL-2 at the tumor site or around TDLN showed initial response. Unfortunately, all patients had recurrent tumor after an interval of 3-5 months and were refractory to further IL-2 treatment (De Stefani et al, 2002). Local administration of IL-2 probably was less effective for therapy of HNSCC patients due to the short half-life of this cytokine (Konrad et al, 1990). Continuous generation of IL-2 at the tumor site may be more effective. One approach to continuous delivery of IL-2 would be the use of recombinant replication-competent viruses expressing the desired cytokine. In addition, lytic viruses, such as vaccinia, may release potential tumor-associated antigens at the tumor sites and make the antigen available to the antigen presenting cells. Vaccinia virus is a strong immune adjuvant and has been shown to invoke humoral and cellular immunity against the transgene in numerous studies in animal and patients (Moss and Flexner, 1987). An additional advantage of using a recombinant vaccinia virus (rvv) for this purpose is that replication-competent virus can be injected directly into tumors. Moreover, a number of studies demonstrated that vaccinia virus is capable of infecting and replicating in tumor lesions despite the presence of systemic neutralizing antibodies (Tsang et al, 1995; Robinson et al, 1998; Mastrangelo et al, 1999; Chen et al, 2001). We demonstrated the antitumor efficacy of rvv expressing the cytokines GM-CSF and IL-2 in a number of animal models (Qin and Chatterjee, 1996a,b,c; Chatterjee et al, 1999; Qin et al, 2001; Dasgupta et al, 2003). The safety of vaccinia virus
in healthy population has been well established by the smallpox eradication program. Rvv has been also used safely in cancer patients (Tsang et al, 1995; Mastrangelo et al, 1999). In spite of that since HNSCC patients are severely immunocompromised, we are exploring the potential of this vaccine in an orthotopic model of head and neck cancer (SCC VII/SF) before translation to HNSCC patients. In order to translate results obtained in animal models to patients it is necessary that the model closely resemble the human disease. SCC VII/SF tumor model has the confirmed tumor histological characteristics of squamous cell carcinoma, the most common tumor in the head and neck region. The actual anatomical site and the initial loco-regional aggressiveness of this tumor, with early direct extension into the neck and later cervical lymph node and pulmonary metastasis, resemble the biological behavior of the tumor progression seen in HNSCC (O'Malley et al, 1997). Moreover, like human HNSCC immune cells isolated from the tumor bearing mice do not respond to mitogenic stimulation (Qin et al, 2001). Thus, the results with this model are likely to be translated for development of therapy for HNSCC. In earlier studies using this model we demonstrated that a combined subcutaneous vaccination with irradiated tumor cells, infected with rvv expressing IL-2 (rvv-IL-2) plus intratumoral rvv-IL-2 injection resulted in enhanced survival and tumor regression compared to intratumoral rvv-IL-2 injection alone (Dasgupta et al, 2003). Irradiated whole tumor cells for subcutaneous vaccination were used to provide tumor-associated antigens at the injection site. While intratumoral injection was performed for the expansion of T cells in the tumor bed by IL-2 from rvv-IL2 (Dasgupta et al, 2003). The subcutaneous immunization induced helper as well as cytotoxic T cell activities. However, complete eradication of tumors was not achieved by this treatment (Dasgupta et al, 2003) probably due to the presence of immune suppression. In this study we investigated the mechanism of reversal of immune suppression and resulting enhanced anti-tumor immunity induced by the subcutaneous immunization so that vaccination strategy can be improved and used for complete eradication of tumor.
II. Materials and methods A. Animals and reagents We obtained 6-8 week old female syngeneic C3H/HeJ mice form the Jackson Laboratory (Bar Harbor, ME, USA). We purchased tissue culture media and reagents from Life Technologies, Inc. (Gaithersburg, MD, USA). We bought recombinant human IL-2, purified rabbit anti-mouse anti-iNOS and anti-rabbit IgG antibodies from Chemicon International Inc. (Temecula, CA, USA) and murine GM-CSF from BD Pharmingen (San Diego, CA, USA). We also obtained the following anti-mouse monoclonal antibodies from BD Pharmingen: purified anti-CD4 (clone RM4-5), anti-CD8 (clone 53-6.7), CD11c (clone HL3), PCNA, Flk-1 (clone Avas 12#1), biotin-anti-H-2Kk (clone 36-7-5), biotin-anti-IgG. We bought cytokine ELISA kits for mouse TGF-!1, GM-CSF, IL-10, IL-2, IFN-", IL-4 and IL-5 from R&D Systems Inc. (Minneapolis, MN, USA). For the lysis of erythrocytes, RBC lysis buffer (Tris ammonium Chloride, pH 7.5) was purchased from Sigma
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Cancer Therapy Vol 2, page 377 Chemical Co. (St. Louis, MO, USA). All PCR primers were procured from Integrated DNA Technologies (Coralville, IA, USA).
F. Real-time RT-PCR analysis immunomodulatory molecules
the
For quantitative analysis, total RNA extracted was subjected to real-time RT-PCR using Cepheid Smart Cycler system (Sunnyvale, CA) in triplicate. Same primer pairs as above were used for these experiments. Before real-time analysis of the target genes, expression level of the internal control !-actin in all samples was normalized with respect to the threshold cycle number (Ct). Relative fold number of the target gene was then expressed with respect to the normalized internal control !-actin in each case. PCR reaction was carried out in 20µl reaction volume using SYBR Green QuantiTect RT-PCR kit (Qiagen, Valencia, CA, USA). We set the reaction condition according to the manufacturer’s instructions. Briefly, reverse transcription was carried out at 500C for 20-30 min. Inactivation of Reverse Transcriptase, HotStarTaq DNA polymerase activation and template cDNA denaturation were carried out at 95°C for 15 min. The final cDNA amplification step comprised of 40 cycles at 94°C for 30 s, 52°C for 30 s and 72°C for 1 min. Data analysis was done using 2-&& Ct method (Pfaffl, 2001; Miyazawa et al, 2004). Relative fold number of the target gene expression was determined by subtracting the Ct value of the reference gene from the Ct value of target gene in all cases.
B. Recombinant vaccinia virus We used two rvv, rvv-IL-2 expressing human IL-2 and rvvlacZ expressing Escherichia coli !-galactosidase for the preparation of the vaccines. The procedures for the preparation of rvv-IL-2 and rvv-lacZ have been described previously (Qin and Chatterjee, 1996c). For mock vaccination, we used 100µl of PBS and injected it into the flank or at the tumor site of the mice.
C. Cell line and tumor development We cultured murine SCCVII/SF, AG104A and C15 tumor cells in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum, 100 units/ml penicillin, 100µg/ml amphotericin B, and 2 mM L-glutamate. For the development of oral tumor, we anesthetized syngeneic C3H/HeJ mice with 3% Isoflurane (Abbott Laboratories, Chicago, IL) and then injected live SCCVII/SF cells (1 x 105) slowly into the floor of mouth with a 23-gauge needle at the depth of mylohyoid muscle (Qin et al, 2001; Dasgupta et al, 2003). This resulted in tumor development (~40-50 mg) in the floor of the mouth in 5-7 days (Dasgupta et al, 2003). All procedures were performed in accordance with the University of Cincinnati Institutional guidelines for the care and use of laboratory animals.
D. Treatment of mice by immunization with tumor cells
of
G. Preparation of tumor homogenate We excised the tumors and removed the necrotic parts. Tumor (20 mg) from each vaccinated mice were excised into small pieces and washed thoroughly to remove as much as possible infiltrating immune cells. Single cell suspension of tumors were made by mechanical dispersion and incubated for 45 h at 37°C to remove the remaining non-adherent immune cells. Adherent tumor cells were gently scrapped and homogenized by using a hand-held homogenizer. Following a brief centrifugation at 500g for 5 min, we harvested the clear supernatant for assay of the IMM by ELISA.
subcutaneous
We irradiated monolayer of SCC VII/SF cells with a total of 10,000 rad of "-radiation (Cesium-167). Irradiated cells (1X104) were mixed with 125 units of rIL-2 or GM-CSF in a total volume of 100µl of phosphate buffered saline (PBS) for subcutaneous injection into the left flank of the mice. When rvv were used for vaccine preparation, the cells were infected with 1X105 plaque forming units (pfu)/ml of rvv. After 24 h of infection, we injected 1X104 cells in 100µl of PBS as described above. Control vaccines were prepared using rvv-lacZ. For mock vaccination we used 100µl of PBS. We prepared and transplanted the tumor cells as above on day 0. Vaccination was given on day 7 when tumor weight reached $ 40- 50 mg. Tumors and immune cells from spleen and TDLN was isolated on day 9.
H. ELISA of immunomodulatory molecules from tumor homogenate and cytokines from immune cells following vaccination Levels of TGF-!1, GM-CSF and IL-10 in the tumor tissue homogenate of the vaccinated mice were determined using ELISA kits. We isolated splenocytes and immune cells from TDLN from mice as described earlier (Dasgupta et al, 2003) on day 9 after one time vaccination as described above. Briefly, we crushed the tissues and then passed the materials through 70 µm nylon mesh and collected the cells in culture medium (RPMI1640). Following two washes in the culture medium, we incubated the cells for 10 min in RBC lysis buffer. Levels of cytokines secreted by the immune cells following stimulation with SCC VII/SF cells were determined using ELISA kits. All experiments were performed in triplicate. The lower limit for the cytokine detection was 5pg/ml.
E. RT-PCR analysis of immunomodulatory molecules Total RNA prepared from tumor tissues of vaccinated mice was subjected to RT-PCR as described earlier (Qin et al, 2001). The reaction was carried out in a 50µl volume using Qiagen one step RT-PCR kit (Qiagen, Valencia, CA). The sequence of the primers are: GM-CSF: forward 5’CCCGCTCACCCATCACTG-3%, reverse 5%GGACTGGTTTTTTGCATTCAAAGG-3%, IL-10: forward 5%GTACAGCCGGGAAGACAA-3% reverse 5%TTTGATCATCATGTATGCTTC-3%, TGF-!1: forward 5%GAGGTACCGCCCGGCCCG-3%, reverse 5%GGTTCAGCCACTGCCGTA-3%, iNOS: forward 5%GGCCCCTGGTAGACCTCAGCT-3%, reverse 5’CCCACCGGTGAGGATGCTCAA-3% and VEGF: forward 5%GCCCTGGAGTGCGTGCCCACGTCAGAGAGCA-3%, reverse 5%-TGGCGATTTAGCAGCAGATA-3%. !–Actin was used as internal control. Amplified DNA fragments were analyzed by electrophoresis in 1.5% agarose gels.
I. Immunohistrochemistry Fresh frozen tumor sections (5µ) from vaccinated mice were fixed in 1% para-formaldehyde for 10 min. After washing 3 times in PBS, slides were incubated in 0.3% H2O2 followed by blocking with rabbit or mouse serum for 15 min. For staining of VEGF-R and iNOS, we incubated for 1 h tumor sections with purified anti-mouse Flk-1 and anti-iNOS antibodies respectively, followed by extensive washing with PBS. Biotin labeled goat anti-rat IgG and anti-rabbit IgG were used as secondary antibodies for Flk-1 and iNOS respectively. After extensive washing with PBS and 30 min incubation in pre-diluted streptavindin-HRP, we stained the sections with freshly prepared DAB solution for 5-10 min and rinsed with water. Sections were
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Dasgupta et al: Reversal of immune suppression in head and neck cancer counterstained in Harry’s hematoxyline. Matched isotypic controls were used in each case. We repeated each experiment at least 3 times. For staining of anti-C3H/HeJ mouse MHC class I molecules, we incubated sections in biotin labeled anti-mouse H2Kk antibody and stained and counterstained as described above. For counting of the positively stained cells or intensity measurement we used Metamorph software (Universal Imaging, Downington, PA). At least 10 fields were chosen at random for counting and data are expressed as mean±SE. For intensity measurement lowest value ('50.00) was represented by a single + sign and each fold increase was represented by additional + sign.
III. Results A. Expression in tumor microenvironment of mRNA encoding the immunomodulatory molecules and reversal by subcutaneous vaccination with irradiated, rvv-IL-2 infected tumor cells We demonstrated previously that immune cells from mice bearing SCC VII/SF tumors did not respond to mitogenic stimulation (Qin et al, 2001). Tumor induced immune suppression often is caused by the secretion of various immunomodulatory molecules (IMM) in tumor microenvironment (Kehrl et al, 1986; Mills, 1991; Pisa et al, 1992; Young et al, 1996a, b; Hsieh et al, 2000). In order to investigate whether tumor microenvironment in this model contains similar IMM, we compared the levels of expression of a number of such molecules in cultured SCC VII/SF cells and tumor lesions by RT-PCR. Levels of IL-10, GM-CSF, TGF-!1 and iNOS were significantly higher in tumor lesions compared to the cultured cells (Qin et al, 2001). Since a single subcutaneous injection of irradiated, rvv-IL-2 infected SCC VII/SF cells restored T helper as well as cytotoxic activities (Dasgupta et al, 2003), we examined whether this immunization also inhibited the expression of the IMM. Levels of the expression of mRNAs encoding GMCSF, IL-10, TGF-!1, VEGF and iNOS in tumor homogenates from vaccinated mice were compared by semi-quantitative RT-PCR. Mice were vaccinated once with PBS or irradiated, rvv (rvv-lacZ or rvv-IL-2) infected SCC VII/SF tumor cells. RT-PCR products were separated by agarose gel electrophoresis and the results are shown in Figure 1A. The levels of mRNAs encoding GM-CSF, iNOS and VEGF in tumors of mice treated with rvv-IL-2 (lane 3) were lower compared the rvv-lacZ (lane 2) or PBS treated mice (lane 1). Band for IL-10 was undetectable in the tumors of mice treated with rvv-IL-2 (lane 3). For quantitative determination of the levels of mRNAs encoding IMM, we performed Real-Time RTPCR and the results are shown in Figure 1B. Level of expression of GM-CSF mRNA was 1/4 in tumor from mice treated with rvv-IL-2 compared to rvv-lacZ and 1/6 compared to the PBS treated mice. Expression of IL-10 was negligible, almost undetectable in rvv-IL-2 treated mice. Levels of VEGF and iNOS were also reduced by vaccination with rvv-IL-2 (1.6-fold), although not as much as GM-CSF. These results suggested that subcutaneous immunization with irradiated, rvv-IL-2 infected SCC VII/SF cells caused significant reversal of expression of GM-CSF and IL-10 and partial (~40%) reversal of VEGF and iNOS in the tumor microenvironment. The expression of mRNA encoding TGF-!1, however, remained almost unaltered (<20% inhibition).
J. T cell proliferation assay Immune cells were isolated as described above. We placed 2 x 105 cells in each well of a 96-well flat bottom tissue culture plate in a volume of 100 µl. We added irradiated SCC VII/SF or control tumor cells (1x104/50 µl) as stimulators to each well. Irradiated splenic macrophages at a concentration of 3x104/50 µl was used as antigen presenting cells. We mixed the contents gently and incubated the plates for 5 days in a tissue culture incubator. We pulsed the cells with 1 µCi of [3H] thymidine 18 h before harvesting. We performed each assay in triplicate and calculated the mean from samples with SD of less than 10%. We determined the stimulation indices by dividing the mean counts per min of each sample by the counts in the medium without the stimulant. For positive control we used Concavalin A at a concentration of 200 ng/well. Other controls were irradiated fibrosarcoma cell line AG104A (H-2Kk) and colorectal carcinoma cell C15 (K-2b).
K. Assay of cytotoxic T cell activity We prepared the splenocytes and immune cells from TDLN as described above. Following washes in CTL medium (AMV VI, Invitrogen) we placed 4x106 cells/well contained in 1 ml culture medium in the wells of a 24-well culture plate. We added as stimulator 1x105 irradiated SCC VII/SF or other relevant target cells in 1 ml medium per well. We added human rIL-2 (1ng/well), mixed the contents gently and incubated the plates for 7 days in a tissue culture incubator. For the preparation of the target cells we labeled 1x106 cells with 200 µCi of [51Cr] and incubated at 37°C for 1 h. We washed the cells 4-5 times with cold medium and adjusted the volume to have a target cell concentration of 4x104/ml. We placed 100 µl of labeled target cells in a well of 96-well round bottom plate. We added immune cells (effector) to obtain an effector/target ratio 50:1 and 100:1 in a final volume of 200 µl. Target cells were SCC VII/SF (H-2Kk), AG104A fibrosarcoma (H-2Kk) and colorectal carcinoma cells, C15 (K-2b). For the calculation of spontaneous release, we added 100 µl of medium instead of effector cells per well and for the calculation of maximum release we added 5% Triton-X-100. Spontaneous release from all cell lines was less than 15%. For the lysis of the tumor cells, we incubated the plates for 6 h in a tissue culture incubator. We counted the radioactivity released in the aliquots of the incubation mixture by using a " counter. To calculate the percentage of specific lysis we used the formula [(sample counts-spontaneous release/maximum releasespontaneous release)]x 100. We performed each assay in triplicate and repeated the experiment at least two times.
B. Reduced synthesis of immunomodulatory proteins in tumor homogenate from mice following subcutaneous vaccination with irradiated, rvv-IL-2 infected tumor cells
L. Statistical analysis We used Student’s t test for normally distributed variables. When the data did not fulfill the criteria of being normally distributed, we used non-parametric statistics (Mann-Whitney rank sum test). We performed all statistical evaluation using SigmaStat software (Jandel, San Rafael, CA) and considered P< .05 to indicate statistical significance.
Due to differential degradation of mRNA in tissues, 378
Cancer Therapy Vol 2, page 379 the levels of mRNA encoding a protein may not reflect the levels of the encoded protein. We therefore determined the levels of immunomodulatory proteins in the tumor microenvironment. Levels of TGF-!1, GM-CSF, IL-10 and VEGF were determined in tumor homogenate by ELISA and the results are summarized in Table 1. Although the levels of mRNA encoding TGF-!1 were similar in 3 groups of treated mice (Figure 1) TGF-!1 protein was reduced by more than 3-fold in the tumor homogenate from mice treated with subcutaneous injection with irradiated, rvv-IL-2 infected SCC VII/SF tumor cells compared to PBS treated mice. Levels of GMCSF were also reduced by 4-fold when the vaccine was
prepared by rvv-IL-2. Levels of IL-10 in this group of mice were negligible at the detection level of 8 pg/ml. Levels of VEGF was reduced by a factor of about 8 by treatment with the rvv-IL-2 vaccine. Except IL-10 and VEGF, which was reduced by about 30% and 50% respectively, the levels of other IMM proteins remained unchanged in mice treated with the control vaccine prepared with rvv-lacZ. Since no antibody suitable for ELISA of iNOS was available, the expression of iNOS was evaluated by immunohistochemistry of sections of tumors following vaccination. Results shown in Figure 2 demonstrate significantly reduced levels of iNOS in
Figure 1. Expression of immunomodulatory molecules by the oral tumors of vaccinated mice. Total RNA was isolated from oral tumors on day 9 and subjected to conventional (A) and Real-Time RT-PCR analysis (B). !-Actin was used as internal control in the PCR reaction. In the conventional PCR assay (A), expression of GM-CSF, iNOS and VEGF was low in the rvv-IL-2 vaccinated mice (lane 3) compared to the rvv-lacZ (lane 2) or PBS (lane 1) group. Expression of IL-10 was undetectable in the rvv-IL-2 vaccinated mice after 40 cycles of PCR reaction. No marked difference in the expression of TGF-!1 was noted between the groups. By Real-Time RT-PCR analysis, relative fold expression of these genes with respect to control !-actin was determined and values were expressed as mean±SE of triplicate reaction. Expression fold level of GM-CSF, iNOS and VEGF was significantly lower and the level of expression of IL-10 was undetectable in the rvv-IL-2 vaccinated mice compared to the control rvv-lacZ or PBS group (P< .005). No significant fold difference in the expression of TGF-!1 was noted among the vaccinated groups.
Table 1. Assay of immunomodulatory proteins in the tumor homogenate following vaccination IMM proteinsa _______________________________________________________________________ Vaccine groups GM-CSF IL-10 TGF-! VEGF _______________________________________________________________________ PBS
124±13
87±7
2915±133
571±27
Rvv-lacZ
137±19
59±4
3097±142
256±22
Rvv-IL-2 29±8 8±2 937±89 75±7 ________________________________________________________________________ a
Amounts of immunomodulatory molecules (pg/ml±SE) in the tumor tissue homogenate were determined by ELISA. Homogenates were prepared from fresh tumor explants (20 mg) and equal amounts of cell free supernatant were used for ELISA. Details are described in Materials and Methods.
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Figure 2. Expression of iNOS in the vaccinated mice. Five-µm fresh frozen serial tumor sections on day 9 were stained with anti-mouse iNOS antibody and staining intensity of the positive cells was measured using Metamorph software from 10 randomly selected fields. Data were converted to mean±SE value. Lowest intensity value corresponds to single + sign and each fold increase was denoted by additional + sign. Expression of iNOS was significantly lower (+) in the rvv-IL-2 vaccinated mice compared to the control rvv-lacZ or PBS treated group (+++).
tumor microenvironment following one vaccination with irradiated tumor cells, infected with rvv-IL-2. Vaccination using the control rvv-lacZ resulted in minor reduction of protein expression in the tumor microenvironment. From these results we conclude that by a single subcutaneous vaccination with irradiated, rvv-IL-2 infected SCC VII/SF cells, suppression of tumor-induced immunity can be reversed.
of some Th1 type cells by vaccination with rvv-lacZ probably also took place.
D. Mechanism of action of IMM in immune suppression Chronic GM-CSF production by tumors suppressed antigen specific CD8+ T cell responses in mice as well as cancer patients. GM-CSF induced suppression of immunity is most common in HNSCC (Young et al, 1996a, b; Bronte et al, 1999). The immunosuppression by GM-CSF is due to the accumulation of immature myeloid cells (Young et al, 1996a, b; Kusmartsev and Gabrilovich, 2002). IL-10 (De Smedt et al, 1997; Girolomoni and Ricciardi-Castagnoli, 1997), TGF-!1 (Albini et al, 1987; Young et al, 1996b; Doran et al, 1997), iNOS (Young et al, 1996b) and VEGF (Oyama et al, 1998; Saito et al, 1998) all suppress the maturation of dendritic cells and suppress anti-tumor immunity. Immune suppression by secreted IMM could be mediated by the effect of these factors on DC. To investigate this possibility we immunostained serial sections of tumors by antibodies specific for mature DC (anti-CD11c) and anti CD4 and CD8 antibodies. Results presented in Figure 3 demonstrate significantly larger number of DC in to the tumor beds of mice vaccinated with rvv-IL-2 (88±6, range 59-114, P< .0001) compared to rvv-lacZ (11±2, range 317) or PBS vaccinated group (7±2, range 0-14). Number of CD4+ T cells adjacent to DC in tumor sections from mice treated with the rvv-IL-2 vaccine was also significantly higher (142±12, range 98-211, P< .0001) compared to rvv-lacZ (24±4, range 9-41) or PBS vaccinated group (10±2, range 9-17). This situation is likely to facilitate proper antigen presentation and antitumor activities. Similar to the CD4+ T cells, infiltrating CD8+ T cells were also significantly higher (79±8, range 34-129, P < .0001) into the tumor beds of rvv-IL-2 vaccinated group compared to the control rvv-lacZ (17±2, range 4-34) or PBS group (11±2, range 2-27). To determine anti-tumor activities of the T cells, we stained serial sections of tumors with anti-bodies specific for proliferating cell nuclear antigen (PCNA). PCNA is a marker reflecting the activity of cell proliferation, which is closely related to invasion and metastasis of malignant cells (Bantis et al, 2004; Kimos et al, 2004). Numbers of PCNA positive tumor cells in the tumor sections were
C. Secretion of cytokines in vitro by immune cells from vaccinated mice on stimulation with irradiated SCC VII/SF tumor cells We reported previously that subcutaneous injection of irradiated, rvv-IL-2 infected cells induced tumor specific helper and cytotoxic T cells (Dasgupta et al, 2003). For anti-tumor immunity, induction of Th1 subtype of helper cells are necessary. To investigate whether Th1 cells were induced by these vaccines, we determined the types and levels of signature cytokines secreted in the culture media by the immune cells following stimulation with irradiated SCC VII/SF tumor cells. Immune cells were isolated from spleen and TDLN cells of the vaccinated mice and the amounts were determined by ELISA. The results are summarized in Table 2. The amounts of Th1 cytokines, IL-2, INF-" secreted by splenocytes or immune cells from TDLN were significantly higher when isolated from mice treated with subcutaneous injection of irradiated, rvv-IL-2 infected SCC VII/SF cells compared to those secreted by immune cells from PBS treated mice (P <. 001). Th2 cytokines, IL4, IL-5 and IL-10, on the other hand, were significantly lower (P<.03) in these mice compared to PBS treated mice. Th1 cytokines secreted by immune cells from mice treated with control rvv-lacZ vaccine were also higher compared to those from PBS group however the differences between these two groups were lower. Except IL-10 from splenocytes and TDLN cells and IL-4 from splenocytes, levels of Th2 cytokines secreted by the immune cells from mice treated with rvv-lacZ vaccine were higher. From these results we concluded that a single subcutaneous immunization of mice with irradiated, rvvIL-2 infected SCC VII/SF cells resulted in induction of mostly tumor specific Th1 type of helper cells. Induction 380
Cancer Therapy Vol 2, page 381 drastically reduced by immunization of mice with the rvvIL-2 vaccine (10±2, range 0-18, P< .0001) compared to rvv-lacZ (39±5, range 19-61) or PBS (46±4, range 30-61) group. These data suggested that rvv-IL-2 vaccination resulted in the recruitment and maturation of DC leading to inhibition of tumor cell proliferation. IMM such as IL-10 may also protect tumors from cytotoxic T cells by down regulation of MHC class I molecules (Salazar-Onfray et al, 1997; Yue et al, 1997; Zeidler et al, 1997). Cultured SCC VII/SF cells, however,
express MHC class I molecules (Qin et al, 2001). To investigate whether loss of MHC class I molecule is a consequence of IL-10 secretion in tumor lesions in this model we evaluated the expression MHC class I molecule in fresh frozen tumor sections by immunohistochemistry. Results presented in Figure 4 demonstrate significantly lower number of MHC class I positive cells in the PBS (36±6, range 13-80) or rvv-lacZ (45±8, range 12-89) treated mice compared to the rvv-IL-2 (179±11, range 103-233 P < .0001) vaccinated mice. Notably, numerous
Figure 3. Distribution of DC, CD4+/CD8+ T cells and PCNA into the tumor beds of vaccinated mice. Five-µm fresh frozen serial tumor sections on day 9 were stained with anti-mouse CD11c, CD4, CD8 and PCNA antibodies. Counting of positive cells was carried out using Metamorph software from 10 randomly selected field and data were converted to mean±SE. Number of CD11c, CD4 and CD8 positive cells was always significantly higher into the tumor beds of rvv-IL-2 vaccinated mice compared to the control rvv-lacZ or PBS vaccinated mice (P < .0001). PCNA positive cells were significantly low in number in the rvv-IL-2 vaccinated mice compared to the controls (P < .0001). ( indicated positively stained cells.
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Table 2. Secretion of cytokines by immune cells following subcutaneous injection of rvv infected tumor cells.
__________________________________________________________________________________ Cytokinesa __________________________________________________________________________________________________ Source of IL-2 IFN-" IL-4 IL-5 IL-10 Immune Groups Cells __________________________________________________________________________________________________ Spleen PBS 49±7 125±46 100±21 48±15 122±11 Spleen rvv-lacZ 60±3 576±39 52±19 68±9 58±3 Spleen rvv-IL-2 177±7 1567±57 34±3 19±2 29±7 TDLN PBS 36±3 13±1 35±7 45±6 412±20 TDLN rvv-lacZ 2±9 44±13 60±9 131±10 50±11 TDLN rvv-IL-2 120±10 30±1 17±2 45±3 23±9 __________________________________________________________________________________________________ a
Amounts of cytokines (pg/ml±SE) secreted by the immune cells after stimulation with irradiated SCCVII/SF cells were determined using ELISA kit. Immune cells were isolated from spleen and TDLN as described in Materials and Methods. Mice were vaccinated on day 7 with PBS and irradiated SCCVII cells infected with rvv-lacZ or rvv-IL-2.
Figure 4. Expressions of MHC class I (H-2Kk) and VEGF-R in the vaccinated mice. Five-µm fresh frozen serial tumor sections on day 9 were stained with anti-mouse MHC class I (H-2Kk) and anti-VEGF-R (flk-1) antibodies. Counting of positive cells or measurement of staining intensity was carried out using Metamorph software from 10 randomly selected fields. In case of positively stained cells, data were converted to mean±SE and for intensity measurement, lowest intensity was represented as single + sign and each fold increase was denoted by additional + sign. Number of positive cells as well as intensity of H-2Kk was significantly higher in the rvv-IL-2 vaccinated group (179±11, +++, P< .0001)) compared to the control rvv-lacZ (45±8, +) or PBS group (36±6, +). Expression of VEGF-R was significantly lower in the rvv-IL-2 vaccinated group (+) compared to the control rvv-lacZ (+++) or PBS (+++) group. Thin arrow (() indicated positive staining where as thick arrow (!) indicated negatively stained cells in case of H-2Kk expression.
cells in the control rvv-lacZ or PBS group did not express MHC class I protein (thick arrows). In addition, the expression of MHC class I protein was also low in the rvvlacZ or PBS vaccinated group (+) compared to the rvv-IL2 treated group (+++). It appeared that treatment with rvvlacZ vaccine restored MHC class I to some extent, however, treatment with rvv-IL-2 vaccine significantly restored class I expression. VEGF stimulates the proliferation of endothelial cells through its specific receptor, such as flk-1 or flt-1 (Strawn et al, 1996) and induces formation of new blood vessels or angiogenesis. Results in Table 1 demonstrated
that tumor homogenates from PBS treated mice secreted VEGF, which is inhibited by treatment with rvv-IL-2 vaccine. To determine whether number of VEGF receptors (VEGF-R) is also increased in tumor tissues, we performed immunohistochemistry using anti-flk-1 antibody. Results presented in Figure 4 show that the tumor associated endothelial cells adjacent to the vessels stained strongly (+++) for flk-1 protein in the PBS and rvv-lacZ treated groups. The extent of staining of the tumor sections by flk-1 antibody from mice treated with rvv-IL-2 was considerably reduced (+). The number of positive foci was also higher in the control vaccinated 382
Cancer Therapy Vol 2, page 383 groups compared to the rvv-IL-2 vaccinated group. These results suggested that microenvironment of SCC VII/SF tumors favors angiogenesis. Treatment with subcutaneous injection of irradiated, rvv-IL-2 infected SCCV II/SF tumor cells resulted in inhibition of angiogenesis.
E. Role vaccination
of
rvv
in
by the splenocytes and TDLN cells from vaccinated mice and the results are presented in Table 3. Levels of Th1 cytokines secreted by splenocytes are similar in groups of mice vaccinated with rvv-IL-2 infected tumor cells and the groups vaccinated with tumor cells mixed with rhIL-2. Amounts of Th1 cytokines secreted by TDLN cells were, however, slightly lower in groups of mice vaccinated with tumor cells mixed with rhIL-2 compared to those vaccinated with tumor cells infected with rvv-IL-2. The levels of Th2 cytokines were, however, higher in rhIL-2 groups both in Spleen and TDLN cells, similar to levels with irradiated SCC VII/SF cells alone. Th1 cytokines secreted by immune cells from mice vaccinated with tumor cells mixed with rGM-CSF were lower than the levels obtained with mice treated with rvv-IL-2 infected tumor cells. Levels of Th2 cytokines were similar in groups of mice treated with irradiated SCC VII/SF cells and those treated with rvv-IL-2 infected cells. Cytotoxic T cell activities of immune cells from mice vaccinated with tumor cells infected with rvv-IL-2 and those vaccinated with tumor cells mixed with either rhIL-2 or rGM-CSF were also compared by Chromium release assays. Results in Figure 6 demonstrate that significant cytolytic activities of immune cells were found when mice were vaccinated with rhIL-2 mixed with tumor cells compared to irradiated tumor cells alone (P<. 02). However, these activities were lower compared to immune cells from mice vaccinated with rvv-IL-2 infected tumor cells (P<. 01). No differences in CTL activities of immune cells from mice vaccinated with irradiated SCC VII/SF cells mixed with rGM-CSF and immune cells from those vaccinated with irradiated tumor cells alone were found.
subcutaneous
Although the intratumoral growth of rvv was not affected by repeated intratumoral injections, subcutaneous injection of rvv may induce anti-vaccinia immunity (Schlom et al, 2003) and eventually neutralize the rvv vaccines. To overcome this potential problem we tested whether we could avoid the use of rvv for subcutaneous immunization. We used a combination of irradiated SCC VII/SF cells mixed with either 125 units of rhIL-2 or recombinant murine GM-CSF for the subcutaneous injection without infecting the cells with rvv. Helper T cell activities by Tcell proliferation assays of immune cells from mice vaccinated once with these vaccines were compared with those with irradiated, rvv-infected SCC VII/SF cells. Results of these experiments are summarized in Figure 5. Helper activities of immune cells from mice vaccinated with subcutaneous injection with irradiated SCC VII/SF cells mixed with rhIL-2 were significantly higher in splenocytes as well as TDLN cells compared to irradiated tumor cells alone (P< .02). However, these activities were lower compared to those with rvv-IL-2 vaccines (P< .004). More accurate tumor specific helper activities are reflected by the amounts and nature of cytokines secreted by the immune cells after stimulation with the tumor cells. We determined the levels of Th1 and Th2 cytokines secreted
Figure 5. Tumor specific helper T cell activities induced by single subcutaneous vaccination. T cell proliferation assay was performed and stimulation index (S.I.) of bulk splenocytes (A) and TDLN cells (B) was determined as described in Materials and Methods. Data represent MeanÂąSE value of triplicate well. Vaccination was administered on day 7 after tumor implantation. Bulk splenocytes or TDLN cells from 4 mice of each vaccinated group was harvested on day 9, pooled and stimulated for 5 days in a tissue culture incubator using irradiated SCCVII/SF or C15 tumor cells as stimulants. Concavalin A was used as positive control stimulant whereas culture medium served as the background stimulant. Stimulation indices of bulk splenocytes (A) or TDLN cells (B) was significantly higher in the group vaccinated with irradiated SCC cells plus rhIL-2 compared to the groups vaccinated with irradiated SCC cells alone, irradiated SCC cells plus GM-CSF, rvv-lacZ or PBS (P< .02). Higher stimulation index was achieved by rvv-IL-2 vaccination in spleen (A) as well as TDLN (B) compared to all the other groups (P< .004). Stimulation in the presence of antigen negative C15 cells was low compared to the SCCVII/SF cells.
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Dasgupta et al: Reversal of immune suppression in head and neck cancer Table 3. Secretion of cytokines by immune cells following subcutaneous injection with irradiated tumor cells plus recombinant IL-2 or GM-CSF
__________________________________________________________________________________ Cytokinesa __________________________________________________________________________________________________ Source of IL-2 IFN-" IL-4 IL-5 IL-10 Immune Groups Cells __________________________________________________________________________________________________ Spleen rvv-IL-2 177±7 1567±57 34±3 19±2 29±7 Spleen SCC 86±5 130±51 99±30 39±3 129±15 Spleen IL-2 122±12 1762±52 70±8 36±2 110±17 Spleen GM-CSF 107±18 774±54 89±8 32±3 104±15 TDLN rvv-IL-2 120±10 30±1 17±2 45±3 23±9 TDLN SCC 69±8 11±9 41±13 56±6 72±5 TDLN IL-2 105±7 19±1 42±11 53±12 56±9 TDLN GM-CSF 73±4 19±.5 45±12 46±5 53±6 __________________________________________________________________________________________________ a
Amounts of cytokines (pg/ml±SE) secreted by the immune cells were determined using ELISA kit. Immune cells were isolated from spleen and TDLN as described in Materials and Methods. Mice were vaccinated on day 7 with irradiated SCCVII/SF cells (SCC), irradiated SCCVII/SF cells infected with rvv-IL-2 or mixed with 125 units of either IL-2 or GM-CSF as described in Materials and methods. Figure 6. Induction of cytolytic activities of immune cells from spleen and TDLN by single subcutaneous vaccination. Mice were vaccinated on day 7 after tumor implantation. Cytolytic activities were assayed on splenocytes (A) and TDLN cells (B) of vaccinated mice on day 9. The immune cells were harvested on day 9 from 4 mice of each group, pooled and cultured in the presence of irradiated SCCVII cells and rhIL-2 for 7 days as described in Materials and Methods. [51Cr] labeled SCCVII/SF cells were used as target in the cytolytic assay. Cytolytic activities of bulk splenocytes (A) or TDLN cells (B) was significantly higher in the group vaccinated with irradiated SCC cells plus rhIL-2 compared to the groups vaccinated with irradiated SCC cells alone, irradiated SCC cells plus GM-CSF, SCC plus rvvlacZ or PBS (P< .02). Cytolytic activities were higher in the rvvIL-2 vaccinated group in spleen (A) as well as TDLN (B) compared to all the other groups (P< .01). Cytolytic activities were low and negligible in the groups vaccinated with irradiated SCC cells alone, SCC cells plus GM-CSF, SCC plus rvv-lacZ or PBS.
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Cancer Therapy Vol 2, page 385 CSF protein was marginally stimulated by this treatment (Table 1). IL-10 is a potent inhibitory factor for dendritic cells cell functions. Addition of IL-10 to splenic DC impairs their capacity to produce IL-12 and induce a Th1 response in vivo (De Smedt et al, 1997). In human, IL-10 has been shown to inhibit the ability of DC to stimulate T cells by preventing the induction of co-stimulatory molecules CD86 (Vicari et al, 2002). Ovarian cancer biopsies express higher levels of IL-10 mRNA compared to normal ovaries (Pisa et al, 1992). By subcutaneous injection of irradiated tumor cell, infected with rvv-IL-2 levels of IL-10 and the levels of mRNA encoding IL-10 were reduced by over 90%. Reduction of IL-10 by vaccination with the control rvv-lacZ vaccine was about 30% both at the mRNA and protein levels (Figure 1, Table 1). TGF-!1 also has been shown to suppress the maturation and functions of DC (Albini et al, 1987; Young et al, 1996b; Doran et al, 1997). Reduction of TGF-!1 by rvv-IL-2 vaccination was less than 20% at the mRNA level, although subcutaneous vaccination with rvv-IL-2 infected tumor cells resulted in about 70% reduction of the levels of TGF-!1 protein in the tumor homogenate (Figure 1, Table 1). iNOS also suppresses DC maturation (Young et al, 1996b). Significant reduction of iNOS resulted by the rvv-IL-2 vaccination both at the mRNA (Figure 1) and protein level (Figure 2). VEGF also suppresses the maturation and functions of DC (Oyama et al, 1998; Saito et al, 1998). Vaccination with rvv-IL-2 resulted in the reduction of the expression of mRNA encoding VEGF by about 40% and significantly inhibited the expression of VEGF protein by 90% (Figure 1, Table 1). Reduction of the levels of IMM in the tumor microenvironment of mice treated with subcutaneous injection of tumor cells infected with rvv-IL-2 resulted in the induction of both helper and cytotoxic T cells (Table 2, Table 3, Figure 5 and Figure 6). Helper T cells play a critical role in CD8+ T cell mediated anti-tumor immunity (Keene and Forman, 1982). Helper T cells have two subtypes, Th1 and Th2, which can be identified from the profile of cytokine secreted by the helper cells in response to the stimulation by the antigen. Th1 cells secrete IL-2 and IFN-", while Th2 cells secrete IL-4, IL5 and IL-10 (Swain, 1995; Abbas et al, 1996). Th1 cells are associated with induction of CTL responses, whereas Th2 cells are involved in promoting antibody responses. Cytokines produced by each cell subtype are antagonistic for the proliferation of the other subtype. As a result Th2 cells could negatively impact CTL generation, which is likely to reduce the efficacy of cancer immunotherapy. Results presented in Table 2 demonstrated that helper T cells generated by rvv-IL-2 vaccination are Th1 type. However, subtype of T helper cells generated by the control vaccine, rvv-lacZ is unbiased. For the activation of T cells to acquire anti-tumor immunity, tumor antigens need to be presented by antigen presenting cells (APC) to na誰ve helper and cytotoxic T cells. SCC VII/SF tumor cells cannot act as APC since these cells do not express MHC class II or B7 costimulatory molecules (Qin et al, 2001). Therefore,
IV. Discussion Intratumoral vaccination of mice bearing SCC VII/SF tumor cells in the floor of the mouth with rvv-IL-2 inhibited growth of tumors and enhanced survival, although tumor regression was not observed (Qin et al., 2001). We also found that the immune cells from tumor bearing mice only partially responded to mitogens. Comparison of the expression of a number of IMM molecules between the cultured tumor cells and tumor homogenate showed higher expression of IMM in tumor homogenate. We hypothesized that the failure to induce tumor regression was due to the synthesis of tumorinduced IMM in the tumor microenvironment (Qin et al, 2001). Removal of the IMM by specific antibodies or inhibition of their synthesis by anti-sense oligonucleotides could make the vaccine more effective. However, the vaccination protocol for patients will become extremely complicated. Subsequently we found that immunization with irradiated, rvv-IL-2 infected tumor cells combined with intratumoral injection of rvv-IL-2 induced tumor regression and induction of cellular and helper T cell activities (Dasgupta et al, 2003), suggesting the reversal of immune suppression. In this study we compared the levels of a number of IMM in tumor homogenate from 3 groups of mice vaccinated with (1) PBS, (2) by a single subcutaneous injection of irradiated SCC VII/SF cells, infected with rvvIL-2 as well as (3) the control vaccine rvv-lacZ. The results demonstrated that the vaccination with rvv-IL-2 reversed the expression of IMM in the tumor microenvironment. We also investigated the possible mechanism of IMM action in this murine model. GM-CSF has been found to be most potent in generating systemic anti-tumor responses (Danna et al, 2004). GM-CSF has multiple functions in immune regulation. Among these functions, GM-CSF has been shown to augment antigen presentation in a variety of cells (Morrissey et al, 1987), enhance major histocompatibility complex class II antigen expression on monocytes (Blanchard and Djeu, 1991), increase the expression of adhesion molecules on granulocytes and monocytes (Arnaout et al, 1986) and participate in the amplification of T-cell proliferation (Santoli et al, 1988). GM-CSF also induces the differentiation and maturation of hematopoietic cells (Metcalf, 1985). Tumor derived GMCSF, on the other hand, induces the proliferation of a population of CD34+ cells, which suppress the functions of intratumoral T cells. Patients whose HNSCC contain a higher number of CD34+ cells have increased incidence of subsequent recurrence and reduced survival (Young et al, 1996a; Young et al, 1997). Murine studies demonstrated that expression of GM-CSF by progressive tumors is associated with increased metastasis (Takeda et al, 1991; Young et al, 1996b). Tumor homogenates from PBS treated mice expressed significant amounts of GM-CSF, both at the mRNA and protein levels. Following subcutaneous injection with irradiated, rvv-IL-2 infected tumor cells expression of GM-CSF was reduced by about 80% both at the mRNA (Figure 1) and protein (Table 1) levels. Injection of the control rvv-lacZ vaccine only reduced the GM-CSF mRNA by 34% (Figure 1) and GM385
Dasgupta et al: Reversal of immune suppression in head and neck cancer induction of tumor specific T cell activation by the rvv-IL2 vaccine likely involved other types of APC, such as DC. Immunohistochemistry of tumor sections from mice treated with rvv-IL-2 and control vaccines showed only a few DC in the tumor bed from mice vaccinated with the control vaccines and numerous mature DC in the tumor beds from mice vaccinated with rvv-IL-2. Numbers of both CD4+ and CD8+ T cells were also high in parallel sections from rvv-IL-2 treated mice (Figure 3). Proximity of DC and the T cells in the tumor beds is likely to facilitate antigen presentation and induction of tumor specific immunity. Decreased number of PCNA positive tumor cells in the tumor beds of rvv-IL-2 mice suggests inhibition of tumor cell proliferation induced by the vaccine. Cytotoxic T cells recognize antigenic peptides from tumor antigens, which are bound in the cleft of MHC class I molecules. If tumor antigens are not presented bound to MHC class I molecule, tumor cells will not be recognized by the activated T cells. Thus, total or partial loss of MHC class I molecule is a mechanism of tumor immune escape (Garrido et al, 1993). MHC class I loss has been reported to occur frequently in human malignancies, including HNSCC (Grandis et al, 1995). Cultured SCC VII/SF cells express MHC class I molecules. However, fresh tumors from tumor bearing mice show low expression of MHC class I molecule (Figure 4). IL-10 has been reported to down regulate MHC class I molecule in tumor cells (Salazar-Onfray et al, 1997; Yue et al, 1997; Zeidler et al, 1997). Expression of IL-10 and the reversal by rvv-IL-2 vaccination also resulted in restoration of MHC class I molecule (Figure 4). Thus, the enhanced CTL activities by rvv-IL-2 vaccination may be also due to restoration of MHC class I expression. Although rvv-IL-2 vaccination appeared to result in the restoration MHC class I expression by the tumor cells in vivo, the exact mechanism is yet not clear. One possibility is the secretion of INF-" by the immune cells generated by the rvv-IL-2 vaccine. VEGF stimulates the proliferation of endothelial cells through its specific receptor, such as flk-1 or flt-1 (Strawn et al, 1996) and induces formation of new blood vessels or angiogenesis. Angiogenesis is required for solid tumor growth (Folkman, 1971, 1990) and facilitates tumor progression and metastasis (Weidner et al, 1991). Results in Table 1 demonstrated that tumor homogenates from PBS treated mice secreted VEGF, which is inhibited by treatment with rvv-IL-2 vaccine. To determine whether number of VEGF receptor was also increased in tumor tissues, we performed immunohistochemistry using antiflk-1 antibody. Results presented in Figure 4 showed that the tumor associated endothelial cells adjacent to the vessels stained strongly for flk-1 protein. The extent of staining of the tumor sections by flk-1 antibody from mice treated with rvv-IL-2 was considerably reduced, as was the number of positive foci. These results suggested that microenvironment of SCC VII/SF tumors favors angiogenesis, which may lead to tumor progression and metastasis. Treatment with subcutaneous injection of irradiated, rvv-IL-2 infected SCCVII/SF tumor cells resulted in inhibition of angiogenesis, thereby preventing tumor progression and metastasis.
For the subcutaneous vaccine to be effective the tumor cells were infected with rvv-IL-2 and rvv-lacZ had marginal effect. These suggested that the reversal of immune suppression was not the result of vaccinia virus. Most likely, the reversal of immune suppression was the result of the generation of IL-2 from rvv-IL-2. Although the growth of rvv in the tumors was not affected by repeated intratumoral injections of rvv-IL-2, subcutaneous injection of rvv may induce anti-vaccinia immunity (Schlom et al, 2003). To overcome this potential problem we tested whether we could avoid the use of rvv for subcutaneous immunization. We used rhIL-2 and murine rGM-CSF mixed with irradiated SCC VII/SF cells for subcutaneous vaccination. IL-2 has the potential to expand tumor specific T cells, while GM-CSF has been reported to enhance local recruitment of DC to the vaccine site and thereby increase antigen presentation. Results presented in Figure 5, Figure 6 and Table 3 demonstrate that while rGM-CSF had no effect, rhIL-2 stimulated both the helper and CTL activities, although rhIL-2 was less effective compared to rvv-IL-2. Helper T cell activities induced by rhIL-2 determined by T cell proliferation were higher compared to those of irradiated SCC VII/SF cells (Figure 5), although the differences were not statistically significant. However, the results were similar when the experiments were repeated one more time. Moreover, amounts of Th1 cytokine secretion from immune cells of mice vaccinated with rhIL-2 were similar to levels from mice treated with rvv-IL-2 (Table 3). In these experiments, we used only one concentration of cytokine, 125 Units/injection. Since the effects of these cytokines are strictly concentration dependent (Serafini et al, 2004), we need to explore various concentrations of these cytokines for vaccine preparations. One problem with the use of irradiated tumor cells in the subcutaneous vaccination is that the vaccine becomes more complicated and may become patient dependent. We have identified a number of proteins, which were highly expressed in tumor tissues and had low expression in normal organs. These proteins can be considered as tumorassociated antigens (unpublished data). In future studies we will explore the use these proteins for subcutaneous immunization, rather than the whole tumor cells.
References Abbas AK, Murphy KM, and Sher A (1996) Functional diversity of helper T lymphocytes. Nature 383, 787-93. Albini A, Iwamoto Y, Kleinman HK, Martin GR, Aaronson SA, Kozlowski JM, and Mcewan RN (1987) A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res 47, 3239-45. Arnaout MA, Wang EA, Clark SC, and Sieff CA (1986) Human recombinant granulocyte-macrophage colony-stimulating factor increases cell-to-cell adhesion and surface expression of adhesion-promoting surface glycoproteins on mature granulocytes. J Clin Invest 78, 597-601. Avradopoulos K, Mehta S, Blackinton D, and Wanebo HJ (1997) Interleukin-10 as a possible mediator of immunosuppressive effect in patients with squamous cell carcinoma of the head and neck. Ann Surg Oncol 4, 184-90. Bantis A, Giannopoulos A, Gonidi M, Liossi A, Aggelonidou E, Petrakakou E, Athanassiades P, and Athanassiadou P (2004)
386
Cancer Therapy Vol 2, page 387 Expression of p120, Ki-67 and PCNA as proliferation biomarkers in imprint smears of prostate carcinoma and their prognostic value. Cytopathology 15, 25-31. Blanchard DK, and Djeu JY (1991) Differential modulation of surface antigens on human macrophages by IFN-" and GMCSF: effect on susceptibility to LAK lysis. J Leukoc Biol 50, 28-34. Bray D, YU SZ, Koprowski H 2nd, Rhee J, Kumar S, Pericle F, Suntharalingam M, Van Echo DA, LI D, and O'Malley BW JR (2003) Combination nonviral interleukin 2 gene therapy and external-beam radiation therapy for head and neck cancer. Arch Otolaryngol Head Neck Surg 129, 618-22. Bronte V, Chappell DB, Apolloni E, Cabrelle A, Wang M, Hwu P, and Restifo NP (1999) Unopposed production of granulocyte-macrophage colony-stimulating factor by tumors inhibits CD8+ T cell responses by dysregulating antigenpresenting cell maturation. J Immunol 162, 5728-37. Chatterjee SK, Qin H, Manna S, and Tripathi PK (1999) Recombinant vaccinia virus expressing cytokine GM-CSF as tumor vaccine. Anticancer Res 19, 2869-73. Chen B, Timiryasova TM, Haghighat P, Andres ML, Kajioka EH, Dutta-Roy R, Gridley DS, and Fodor I. (2001) Low-dose vaccinia virus-mediated cytokine gene therapy of glioma. J Immunother 24, 46-57. Chen B, Van Den Brekel MW, Buschers W, Balm AJ, and Vna Velthuysen ML (2003) Validation of tissue array technology in head and neck squamous cell carcinoma. Head Neck 25, 922-30. Danna EA, Sinha P, Gilbert M, Clements VK, Pulaski BA, and Ostrand-Rosenberg S (2004) Surgical removal of primary tumor reverses tumor-induced immunosuppression despite the presence of metastatic disease. Cancer Res 64, 2205-11. Dasgupta S, Tripathi PK, Bhattacharya-Chatterjee M, O'Malley B JR, and Chatterjee SK (2003) Recombinant vaccinia virus expressing IL-2 generates effective anti-tumor responses in an orthotopic murine model of head and neck carcinoma. Mol Ther 8, 238-48. De Smedt T, Van Mechelen M, De Becker G, Urbain J, LEO O, and Moser M (1997) Effect of interleukin-10 on dendritic cell maturation and function. Eur J Immunol 27, 1229-35. De Stefani A, Forni G, Ragona R, Cavallo G, Bussi M, Usai A, Badellino F, and Cortesina G (2002) Improved survival with perilymphatic interleukin 2 in patients with resectable squamous cell carcinoma of the oral cavity and oropharynx. Cancer 95, 90-7. Doran T, Stuhlmiller H, Kim JA, Martin EW JR, and Triozzi PL (1997) Oncogene and cytokine expression of human colorectal tumors responding to immunotherapy. J Immunother 20, 372-6. Folkman J (1971) Tumor angiogenesis: therapeutic implications. N Engl J Med 285, 1182-6. Folkman J (1990) What is the evidence that tumors are angiogenesis dependent? J Natl Cancer Inst 82, 4-6. Garrido F, Cabrera T, Cohnca A, Glew S, Ruiz-Cabello F, and Stern PL (1993) Natural history of HLA expression during tumour development. Immunol Today 14, 491-9 Girolomoni G, and Ricciardi-Castagnoli P (1997) Dendritic cells hold promise for immunotherapy. Immunol Today 18, 1024. Gooding R, Riches P, Dadian G, Moore J, and Gore M (1995) Increased soluble interleukin-2 receptor concentration in plasma predicts a decreased cellular response to IL-2. Br J Cancer 72, 452-5. Grandis JR, Chang MJ, Yu WD, and Johnson CS (1995) Antitumor activity of interleukin-1# and cisplatin in a murine model system. Arch Otolaryngol Head Neck Surg 121, 197-200.
Hadden JW (1997) The immunopharmacology of head and neck cancer: an update. Int J Immunopharmacol 19, 629-44. Hadden JW, Endicott J, Baekey P, Skipper P, and Hadden EM (1994) Interleukins and contrasuppression induce immune regression of head and neck cancer. Arch Otolaryngol Head Neck Surg 120, 395-403. Hamasaki VK, and Vokes EE (1995) Interferons and other cytokines in head and neck cancer. Med Oncol 12, 23-33. Heo DS, Whiteside TL, Johnson JT, Chen KN, Barnes EL, and Herberman RB (1987) Long-term interleukin 2-dependent growth and cytotoxic activity of tumor-infiltrating lymphocytes from human squamous cell carcinomas of the head and neck. Cancer Res 47, 6353-62. Hsieh CL, Chen DS, and Hwang LH (2000) Tumor-induced immunosuppression: a barrier to immunotherapy of large tumors by cytokine-secreting tumor vaccine. Hum Gene Ther 11, 681-92. Keene JA, and Forman J (1982) Helper activity is required for the in vivo generation of cytotoxic T lymphocytes. J Exp Med 155, 768-82. Kehrl JH, Roberts AB, Wakefield LM, Jakowlew S, Sporn MB, and Fauci AS (1986) Transforming growth factor ! is an important immunomodulatory protein for human B lymphocytes. J Immunol 137, 3855-60. Kim ES, Kies M, and Herbst RS (2002) Novel therapeutics for head and neck cancer. Curr Opin Oncol 14, 334-42. Kimos MC, Wang S, Borkowski A, Yang GY, Yang CS, Perry K, Olaru A, Deacu E, Sterian A, Cottrell J, Papadimitriou J, Sisodia L, Selaru FM, Mori Y, Xu Y, Yin J, Abraham JM, and Meltzer SJ (2004) Esophagin and proliferating cell nuclear antigen (PCNA) are biomarkers of human esophageal neoplastic progression. Int J Cancer 111, 415-7. Konrad MW, Hemstreet G, Hersh EM, Mansell PW, Mertelsmann R, Kolitz JE, and Bradley EC (1990) Phermacokinetics of recombinant interleukin 2 in humans. Cancer Res 50, 2009-17. Kusmartsev S, and Gabrilovich DI (2002) Immature myeloid cells and cancer-associated immune suppression. Cancer Immunol Immunother 51, 293-8. Mastrangelo MJ, Bellet RE, Berkelhammer J, and Clark WH JR (1975) Regression of pulmonary metastatic disease associated with intralesional BCG therapy of intracutaneous melanoma metastases. Cancer 36, 1305-8. Mastrangelo MJ, Maguire HC JR, Eisenlohr LC, Laughlin CE, Monken CE, Mccue PA, Kovatich AJ, and Lattime EC (1999) Intratumoral recombinant GM-CSF-encoding virus as gene therapy in patients with cutaneous melanoma. Cancer Gene Ther 6, 409-22. Metcalf D (1985) The granulocyte-macrophage colonystimulating factors. Science 229, 16-22 Mills CD (1991) Molecular basis of "suppressor" macrophages. Arginine metabolism via the nitric oxide synthetase pathway. J Immunol 146, 2719-23 Miyazawa J, Mitoro A, Kawashiri S, Chada KK, and Imai K (2004) Expression of mesenchyme-specific gene HMGA2 in squamous cell carcinomas of the oral cavity. Cancer Res 64, 2024-9 Morrissey PJ, Bressler L, Park LS, Alpert A, and Gillis S (1987) Granulocyte-macrophage colony-stimulating factor augments the primary antibody response by enhancing the function of antigen-presenting cells. J Immunol 139, 1113-9. Moss B, and Flexner C (1987) Vaccinia virus expression vectors. Annu Rev Immunol 5, 305-25. O'Malley BW JR, Cope KA, Johnson CS and Schwartz MR (1997) A new immunocompetent murine model for oral cancer. Arch Otolaryngol Head Neck Surg 123, 20-4. Oyama T, Ran S, Ishida T, Nadaf S, Kerr L, Carbone DP, and Gabrilovich DI (1998) Vascular endothelial growth factor
387
Dasgupta et al: Reversal of immune suppression in head and neck cancer affects dendritic cell maturation through the inhibition of nuclear factor-)B activation in hemopoietic progenitor cells. J Immunol 160, 1224-32. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29, 2002â&#x20AC;&#x201C;2007 Pisa EK, Gerdin E, Hising C, Bucht A, Gerdin B, and Kiessling R (1992) Selective expression of interleukin 10, interferon ", and granulocyte-macrophage colony-stimulating factor in ovarian cancer biopsies. Proc Natl Acad Sci U S A 89, 7708-12. Qin H, and Chatterjee SK (1996a) Cancer gene therapy using tumor cells infected with recombinant vaccinia virus expressing GM-CSF. Hum Gene Ther 7, 1853-60. Qin H, and Chatterjee SK (1996b) Recombinant vaccinia expressing interleukin-2 for cancer gene therapy. Cancer Gene Ther 3, 163-7. Qin H, Valentino J, Manna S, Tripathi PK, BhattacharyaChatterjee M, Foon KA, O'Malley BW JR, and Chatterjee SK (2001) Gene therapy for head and neck cancer using vaccinia virus expressing IL-2 in a murine model, with evidence of immune suppression. Mol Ther 4, 551-8. Qin HX, and Chatterjee SK (1996c) Construction of recombinant vaccinia virus expressing GM-CSF and its use as tumor vaccine. Gene Ther 3, 59-66. Robinson BW, Mukherjee SA, Davidson A, Morey S, Musk AW, Ramshaw I, Smith D, Lake R, Haenel T, Garlepp M, Marley J, Leong C, Caminschi I, and Scott B (1998) Cytokine gene therapy or infusion as treatment for solid human cancer. J Immunother 21, 211-7. Rosenberg SA, Lotze MT, Yang JC, Aebersold PM, Linehan WM, Seipp CA, and White DE (1989) Experience with the use of high-dose interleukin-2 in the treatment of 652 cancer patients. Ann Surg 210, 474; discussion 484-5. Saito H, Tsujitani S, Ikeguchi M, Maeta M, and Kaibara N (1998) Relationship between the expression of vascular endothelial growth factor and the density of dendritic cells in gastric adenocarcinoma tissue. Br J Cancer 78, 1573-7. Salazar-Onfray F, Charo J, Petersson M, Freland S, Noffz G, Qin Z, Blankenstein T, Ljunggren HG, and Kiessling R (1997) Down-regulation of the expression and function of the transporter associated with antigen processing in murine tumor cell lines expressing IL-10. J Immunol 159, 3195202. Santoli D, Clark SC, Kreider BL, Maslin PA, and Rovera G (1988) Amplification of IL-2-driven T cell proliferation by recombinant human IL-3 and granulocyte-macrophage colony-stimulating factor. J Immunol 141, 519-26. Schlom J, Sabzevari H, Grosenbach DW, and Hodge JW (2003) A triad of costimulatory molecules synergize to amplify Tcell activation in both vector-based and vector-infected dendritic cell vaccines. Artif Cells Blood Substit Immobil Biotechnol 31, 193-228. Serafini P, Carbley R, Noonan KA, Tan G, Bronte V, and Borrello I (2004) High-dose granulocyte-macrophage colony-stimulating factor-producing vaccines impairs the immune response through the recruitment of myeloid suppressor cells. Cancer Res 64, 6337-43. Strawn LM, Mcmahon G, App H, Schreck R, Kuchler WR, Longhi MP, Hui TH, Tang C, Levitzki A, Gazit A, Chen I, Keri G, Orfi L, Risau W, Flamme I, Ullrich A, Hirth KP, and Svehawr LK (1996) Flk-1 as a target for tumor growth inhibition. Cancer Res 56, 3540-5. Swain SL (1995) T-cell subsets. Who does the polarizing? Curr Biol 5, 849-51.
Takeda K, Hatakeyama K, Tsuchiya Y, Rikiishi H, and Kumagai K (1991) A correlation between GM-CSF gene expression and metastases in murine tumors. Int J Cancer 47, 413-20. Tsang KY, Zaremba S, Nieroda CA, Zhu MZ, Hamilton JM, and Schlom J (1995) Generation of human cytotoxic T cells specific for human carcinoembryonic antigen epitopes from patients immunized with recombinant vaccinia-CEA vaccine. J Natl Cancer Inst 87, 982-90. van Dongen GA, Brakenhoff RM, Ten Brink CT, Van Gog FB, DE Bree R, Quak JJ and Snow GB (1996) Squamous cell carcinoma-associated antigens used in novel strategies for the detection and treatment of minimal residual head and neck cancer. Anticancer Res 16, 2409-13. Vicari AP, Caux C, and Trinchieri G (2002) Tumour escape from immune surveillance through dendritic cell inactivation. Semin Cancer Biol 12, 33-42. Vokes EE, Weichselbaum RR, Lippman SM, and Hong WK (1993) Head and neck cancer. N Engl J Med 328, 184-94. Wanebo HJ, Jun MY, Strong EW, and Oettgen H (1975) T-cell deficiency in patients with squamous cell cancer of the head and neck. Am J Surg 130, 445-51. Weidner N, Semple JP, Welch WR, and Folkman J (1991) Tumor angiogenesis and metastasis-correlation in invasive breast carcinoma. N Engl J Med 324, 1-8. Whiteside TL, Heo DS, Chen K, Adler A, Johnson JT, and Herberman RB (1987) Expansion of tumor-infiltrating lymphocytes from human solid tumors in interleukin-2. Prog Clin Biol Res 244, 213-22. Young MR, Wright MA, Lozano Y, Matthews JP, Benefield J, and Prechel MM (1996a) Mechanisms of immune suppression in patients with head and neck cancer: influence on the immune infiltrate of the cancer. Int J Cancer 67, 3338. Young MR, Wright MA, Lozano Y, Prechel MM, Benefield J, Leonetti JP, Collins SL, and Petruzzelli GJ (1997) Increased recurrence and metastasis in patients whose primary head and neck squamous cell carcinomas secreted granulocytemacrophage colony-stimulating factor and contained CD34+ natural suppressor cells. Int J Cancer 74, 69-74. Young MR, Wright MA, Matthews JP, Malik I, and Prechel M (1996b) Suppression of T cell proliferation by tumor-induced granulocyte-macrophage progenitor cells producing transforming growth factor-! and nitric oxide. J Immunol 156, 1916-22.. Yue FY, Dummer R, Geertsen R, Hofbauer G, Laine E, Manolio S, and Burg G (1997) Interleukin-10 is a growth factor for human melanoma cells and down-regulates HLA class-I, HLA class-II and ICAM-1 molecules. Int J Cancer 71, 6307. Zeidler R, Eissner G, Meissner P, Uebel S, Tampe R, Lazis S, and Hammerschmidt W (1997) Downregulation of TAP1 in B lymphocytes by cellular and Epstein-Barr virus-encoded interleukin-10. Blood 90, 2390-7.
Sunil K. Chatterjee
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Cancer Therapy Vol 2, page 389 Cancer Therapy Vol 2, 389-402, 2004
Development of a tetracycline-inducible system for expression of the Ca2+ permeable TRPL channel and the killing of prostate cancer cells Research Article
Prasit Ruengrairatanaroje, Michael Hahn, Helen M. Brereton, Lei Zhang, Mario Froscio, Tadija Petronijevic and Gregory J. Barritt* Department of Medical Biochemistry, School of Medicine, Faculty of Health Sciences, Flinders University, GPO Box 2100, Adelaide South Australia 5001, Australia.
__________________________________________________________________________________ *Correspondence: Professor GJ Barritt, Department of Medical Biochemistry, School of Medicine, Faculty of Health Sciences, Flinders University, GPO Box 2100, Adelaide South Australia 5001, Australia; Telephone: (+61 8) 8204 4260; Fax: (+61 8) 8374 0139; E-mail: Greg.Barritt@flinders.edu.au Key words: TRPL, Ca2+ channels, prostate cancer cells, Tet-On promoter, doxycycline Abbreviations: 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide, (MTT); cytoplasmic Ca2+ concentration, ([Ca2+]cyt); phosphate-buffered saline, (PBS); transient receptor potential, (TRP); transient receptor potential-like, (TRPL) Received: 11 August 2004; Accepted: 18 October 2004; electronically published: October 2004
Summary The artificial induction of high intracellular Ca2+ offers a strategy for killing rapidly-proliferating cells in diseased states, since sustained high concentrations of Ca2+ are toxic to all types of animal cells. We have previously shown that expression of the Drosophila melanogaster TRPL (transient receptor potential-like) Ca2+-permeable channel in a prostate cancer cell line leads to Ca2+-mediated cell death (Zhang L, Brereton HM, Hahn M, Froscio M, Tilley WD, Brown MP, Barritt GJ (2003) Cancer Gene Therapy 10, 611-625). The aim of the present experiments was to develop an inducible system for expression of the TRPL Ca2+-permeable channel. Androgen-insensitive PC-3 prostate cancer cells were stably-transfected with a plasmid encoding the inducible doxycycline-sensitive Tet-On promoter system to give clones of Tet-On PC-3 cells. When these Tet-On cells were transiently-transfected with a Tet-On-responsive plasmid harbouring TRPL cDNA, they exhibited doxycycline-induced expression of the TRPL protein, decreased cell viability and increased chromatin fragmentation and condensation. Obtaining clones of TetOn PC-3 cells stably-transfected with the Tet-On-responsive plasmid harbouring TRPL cDNA proved difficult, possibly due to some â&#x20AC;&#x153;leakinessâ&#x20AC;? of the Tet-On system and a low level of TRPL expression in the absence of doxycycline. However, several clones of Tet-On PC-3 cells stably-transfected with the Tet-On-responsive plasmid harbouring TRPL cDNA were obtained. These exhibited doxycycline-induced increases in: TRPL protein expression (although these were less than the increase observed in transiently-transfected Tet-On PC-3 cells), the basal cytoplasmic Ca2+ concentration, and in the proportion of large cells, cells exhibiting polymorphism, and of cells exhibiting chromatin condensation and fragmentation. Stably-transfected cell lines expressing TRPL also exhibited a decrease in the number of viable cells. It is concluded that the tetracycline expression system can be effectively used to control the expression of TRPL in PC-3 cells. The results illustrate the principle that inducibly regulated TRPL expression can provide controllable killing of prostate cancer cells. the concentration of androgen initiates apoptotic cell death in normal prostate epithelial cells and in prostate cancer cells. In early stage prostate cancer, androgen ablation leads to regression of the disease. However, androgenresistant clones of prostate epithelial cells often arise. These cells do not undergo apoptosis so that androgenindependent tumour cells proliferate and metastasise (Isaacs et al, 1992; Colombel et al, 1996; Denmeade and Isaacs, 1996; Gschwend, 1996). There is presently no
I. Introduction The development, growth and function of prostate epithelial cells are androgen dependent. Androgens elicit these effects via the androgen receptor (Isaacs et al, 1992; Colombel et al, 1996; Denmeade and Isaacs, 1996; Gschwend, 1996). Tumorigenic prostate epithelial cells exhibit uncontrolled cell growth which, in the early stages of the disease, is dependent on the presence of androgen. Any manipulation (e.g. androgen ablation) which reduces 389
Ruengrairatanaroje et al: Tetracycline-inducible system for prostate cancer effective way of killing androgen-independent tumour cells in prostate cancer patients (Isaacs et al, 1992; Colombel et al, 1996; Denmeade and Isaacs, 1996; Gschwend, 1996). The molecular mechanisms by which androgen ablation induces apoptosis in androgen-sensitive prostate cancer cells are not yet fully defined (Isaacs et al, 1992; Colombel et al, 1996; Denmeade and Isaacs, 1996; Gschwend, 1996). However, several studies suggest that apoptosis caused by androgen ablation is associated with a sustained increase in the cytoplasmic Ca2+ concentration ([Ca2+]cyt) (Martikainen et al, 1991; Steinsapir et al, 1991; Isaacs et al, 1992; Tombal et al, 1995; Denmeade and Isaacs, 1996; Gutierrez et al, 1999). Sustained high [Ca2+]cyt leads to cell toxicity and death through a variety of mechanisms in which the accumulation of Ca2+ in mitochondria and subsequent release of mitochondrial metabolites and proteins play major roles (McConkey and Orrenius, 1997; Porn-Ares et al, 1998; Kass and Orrenius, 1999; Jambrina et al, 2003). Thapsigargin (an inhibitor of the endoplasmic reticulum (Ca2+ + Mg2+)ATPase) has been shown to induce apoptosis in both androgen-sensitive and androgen-insensitive prostate cancer cell lines (Furuya et al, 1994). The apoptotic effects of thapsigargin are associated with a decrease in the concentration of the Ca2+ in the endoplasmic reticulum and, as a consequence of this, sustained Ca2+ inflow through store-operated Ca2+ channels in the plasma membrane (Furuya et al, 1994; Skryma et al, 2000; Putney et al, 2001). While the mechanism by which thapsigargin induces cell death is complex (it may involve a decrease in concentration of Ca2+ in the endoplasmic reticulum and other signalling pathways as well as a sustained high [Ca2+]cyt), the increase in [Ca2+]cyt appears to play a key role in the induction of apoptosis (Furuya et al, 1994; McConkey and Orrenius, 1997; Porn-Ares et al, 1998; Kass and Orrenius, 1999; Skryma et al, 2000). A sustained high [Ca2+]cyt can also be induced in cells by the heterologous expression of constitutively active Ca2+-permeable channels in the plasma membrane. For example, it has recently been shown that Ca2+ inflow through TRPM7 channels induces neuronal cell death in oxygen-glucose deprivation (Aarts et al, 2003) and through TRPV1 channels, the death of Jurkat cells transiently-transfected with TRPV1 (Jambrina et al, 2003). We have recently shown that heterologous expression of the Drosophila melanogaster TRPL (transient receptor potential like) non-selective cation channel (Phillips et al, 1992) in the androgen-sensitive LNCaP cell line induces Ca2+ inflow and cell death (Zhang et al, 2003). In these experiments, expression of the TRPL protein was placed under the control of the constitutive CMV promoter. However, in developing this system for the potential treatment of prostate cancer, it would be highly desirable to be able to control the amount and timing of expression of the TRPL protein. Therefore, the aim of the present experiments was to develop an inducible system for the regulated expression of TRPL and hence for the controlled killing of prostate cancer cells by a sustained increase in [Ca2+]cyt. The experiments have been conducted with the PC-3 androgen-insensitive prostate carcinoma cell line
(derived from metastatic tumour tissue obtained from the lumbar vertebra of a 62 year old patient) (Webber et al, 1997). The Tet-On (tetracycline-controlled transcription activation system activated by the tetracycline derivative, doxycycline (Gossen et al, 1993, 1995; Gossen and Bujard, 1995)) was used as the prototype inducible expression system. This has been successfully employed in the controlled expression of a number of different proteins in a variety of mammalian cell lines (Gossen et al, 1993, 1995; Gossen and Bujard, 1995). The results reported here show that the Tet-On tetracycline expression system can be effectively used to control the expression of TRPL in PC-3 cells. The consequences of increased TRPL expression include enhanced Ca2+ inflow and increased [Ca2+]cyt and cell death by apoptosis. These results provide an “in principle” demonstration that inducibly-controlled TRPL expression can provide controlled killing of prostate cancer cells.
II. Materials and methods A. Materials PC-3 prostate cancer cells and pBluescript SK(+), containing the Drosophila TRPL cDNA insert, were kindly provided by Professor Wayne Tilley, Hanson Research Institute, Adelaide and Dr. Leonard E. Kelly, Department of Genetics, University of Melbourne, Melbourne, respectively. Opti-MEM! and LipofectAMINETM2000 were obtained from GibcoBRL (Life Technologies, Melbourne, Australia); EDTA, doxycycline, 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), fura-2 acetoxymethyl ester (Fura-2/AM) and pluronic F127 from Molecular Probes (Eugene, OR, USA); FuGENETM 6 transfection reagent from Roche Applied Science (Castle Hill, NSW, Australia); plasmids for the Tet-On gene expression system (pTet-On, pTRE-Luc, pTK-Hyg, pBI-L and pTet-tTs) from Clontech (Palo Alto, Ca, USA); luciferase assay kit from Promega (NSW, Australia); PCR primers and pUC DNA markers from Geneworks Pty Ltd (South Australia).
B. Cell culture PC-3 cells were routinely grown in RPMI-1640 medium supplemented with 5% (v/v) foetal bovine serum (FBS), 100 U/ml penicillin and 100 µg streptomycin (complete RPMI-1640 medium) (Tilley et al, 1995). PT-20-1 (Tet-On PC-3) cells were routinely grown in complete RPMI-1640 medium supplemented with 100 µg/ml G418. MP25 and MP40 cells (Tet-On PC-3 cells harbouring the TRPL and luciferase genes) were routinely grown in complete RPMI-1640 medium supplemented with 100 µg/ml G418 and 25 µg/ml hygromycin.
C. Plasmids The plasmids pBluescript-TRPL, pTet-On, pTRE-Luc, pTK-Hyg, pBI-L, pBI-L-TRPL and pTet-tTS were transformed into competent bacterial cells (E. Coli XL1-Blue) by electroporation (Bio-Rad Gene Pulser) at 1.8 kV, 25 µF, and propagated using standard procedures. TRPL cDNA was subcloned from pBluescript SK(+) (Stratagene) into the pBI-L response plasmid using the Not I and Hind III restriction enzymes sites. PCR amplification was carried out to ensure that the full length of TRPL cDNA was successfully ligated into the pBI-L-TRPL plasmid and that the 5’-ends of the TRPL cDNA was located downstream of the minimal CMV promoter.
D. Transfection protocols Electroporation of PC-3 cells (Bio-Rad Gene Pulser) was performed at 500 volts and 25 µF. For transfection of PC-3 cells
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Cancer Therapy Vol 2, page 391 using FuGENETM6, cells (5 x 105) were plated in 2 ml RPMI1640-5% (v/v) FBS in a 35 mm culture dish and incubated until the cell density was at 50-80% confluency (48 h). FuGENE6/plasmid DNA complexes were prepared by adding a mixture of FuGENE6 (3 µl) and RPMI-1640 (serum free) (97 µl) into diluted plasmid DNA (10-1000 ng) and incubating at room temperature. After 15 min, the FuGENE6/plasmid DNA mixture was added into each 35 mm culture dish dropwise, and the cells incubated at 37oC in 5% CO2/95% O2 (v/v). For transienttransfection of PC-3 cells using LipofectAMINETM 2000, cells (5 x 105) were plated in 2 ml RPMI-5% (v/v) FBS in a 35 mm culture dish and incubated until the cell density was at 90-95% confluency (72 h), according to the manufacturer’s instructions. DNA-LF2000 was prepared by combining the diluted plasmid DNA (1 µg in 200 µl Opti-MEM!) with lipofectamine (3 µl in 200 µl Opti-MEM!) and incubating at room temperature for 20 min. The DNA-LF2000 reagent complex was then added directly to each culture dish, mixed by rocking, and the cells incubated at 37°C. After 4 h, the medium was replaced with fresh RPMI-5% (v/v) FBS.
Development of the inducible Tet-On system for expression of the TRPL gene in PC-3 cells required twoconsecutive stable transfections (Gossen et al, 1995). The first is to introduce the Tet-On plasmid, which contains the regulatory gene encoding the transcription activator rtetR/VP16 under the control of a CMV promoter. The second is to introduce the TRPL gene under the control of the TRE-element and a minimal CMV promoter. The TRE-element is activated when it binds to rtetR/VP16 in a doxycycline dependent manner (Gossen et al, 1995). This would be expected to promote expression of the TRPL gene. To generate double-stable PC-3 cell lines inducibly expressing both TRPL and luciferase, PT-20-1 Tet-On PC-3 cells (PC-3 cells stably-transfected with the Tet-On plasmid) were cotransfected with a given amount (100, 200, 600, 1000 or 2000 ng) of the pBI-L-TRPL plasmid (1000 ng) (which contains cDNA encoding the luciferase and TRPL proteins under the control of a bi-directional promoter) plus the pTK-Hyg (400 ng) plasmid, and grown in the presence of hygromycin (50 µg/ml) and G418 (100 µg/ml). All the individual hygromycin-resistant cell lines obtained from the transfections using different amounts of pBI-L-TRPL grew slowly, and many cells exhibited polymorphic morphology. A large number of dead cells was observed in each transfected cell line. (PT-20-1 cells transfected with the pBI-L plasmid (controls) grew at the same rate as untransfected PT-20-1 cells and exhibited normal morphology.) Since the cells which survived the co-transfection with pBI-L-TRPL and pTK-Hyg were unstable and polymorphic, and did not exhibit doxycycline-inducible luciferase, a second subcloning of those cells which survived from the 1000 ng pBIL-TRPL transfection was carried out in an attempt to obtain a stable and morphologically-homogeneous cell line expressing TRPL under control of the Tet-On promoter. Surviving cells were cultured in the presence of a higher concentration of hygromycin (150 µg/ml) (in order to increase the selection pressure) while maintaining the concentration of G418 at 100 µg/ml. After 8 weeks, a number of cell-colonies were isolated. Twenty of these individual isolated clonal cell lines were grown in the presence of 150 µg/ml hygromycin and G418 (100 µg/ml) for a further two passages, then maintained in the presence of a low concentration of hygromycin (25 µg/ml) plus G418 (100 µg/ml). These cells were tested for TRPL gene incorporation by PCR amplification using the FBTRPL2 / RBTRPL2 primers. A PCR band of 336 bp was obtained from each of 9 clones, indicating that the 5’-end of the TRPL gene had been incorporated into each clone. PCR amplification using the C20A/pBI-A1 primers, which amplify the 3’-end of the TRPL gene, was performed to further confirm incorporation of the TRPL gene in these cell lines. A predicted 1.5 kb fragment was obtained for all 9 clones, confirming that the TRPL gene had been stably incorporated into each of these cell lines. To test for incorporation of the luciferase gene, PCR amplification was carried out using the FLUC/RLUC primers. A predicted 280 bp fragment was obtained from all 9 clones, indicating that the luciferase gene had also been incorporated into each cell line. Two (MP25 and MP40) of the 9 clones described above showed the highest doxycycline induction of luciferase and were chosen for further study. When grown in early passages, MP25 and MP40 showed a high luciferase activity in the absence of doxycycline and a 3- and 2.5-fold induction, respectively, of luciferase following incubation with doxycycline (1 µg/ml for 48 h) (c.f. the amount of luciferase induction observed in other cell types (Isaacs et al, 1992; Colombel et al, 1996; Denmeade and Isaacs, 1996; Gschwend, 1996). (However in later passages, no luciferase induction was observed, even when a range of doxycycline concentrations was employed.) In order to confirm that the synthesis and action of the rTetR/VP16 transcription factor were effective in MP25 and MP40 cells, PC-20-1, MP25
E. Protocol for the isolation and storage of stably-transfected cell lines The isolation of adherent cell clones from cells grown in 35 mm culture dishes was conducted using 3-5 mm2 filter paper disks (Whatman Number 3) (Domann and Martinez, 1995). The cells on the filter paper were placed into a single well of a 24 well-plate containing 1 ml of complete RPMI-1640 medium supplemented with G418 or G418 plus hygromycin at the concentrations indicated in the text and legends to figures. The 24 well-plates were incubated in CO2 (5% in O2) at 37°C until the cells in each well (derived from a single isolated cell) had reached confluency. The cells were harvested by trypsinization, subcultured into 25 cm2 culture flasks containing complete RPMI-1640 medium supplemented with G418 and/or G418 plus hygromycin at the concentrations indicated, and the cells grown to near confluency. A sample of each clone was frozen in liquid N2.
F. Generation of stably-transfected clones of PC-3 cells harbouring the Tet-On plasmid PC-3 cells were transfected with the pTet-On plasmid by electroporation. The transfected PC-3 cells (3 x 104 cells/ml, 50 mm culture dish) were grown in the presence of 200 µg/ml G418 to allow for the selection of cells containing pTet-On. After 5 weeks in selective culture medium, 30 healthy and fast-growing G418-resistant cell colonies were isolated. To determine which of these expressed the rTetR/VP16 transcription activator (regulates gene expression from the pTRE-Luc plasmid), each isolated G418-resistant cell line was then transiently-transfected with 10 ng pTRE-Luc and grown at 1.5 x 104 cells/ml (35 mm culture dishes). After 48 h incubation in the absence, or presence, of 1 µg/ml doxycycline, cell extracts were prepared and analysed for luciferase activity. Out of the 30 G418-resistant clones isolated, three exhibited the highest degree of doxycyclineinduced luciferase reporter activities (24-, 12-, and 19-fold). One (PT-20-1), which exhibited the highest induction of luciferase in response to doxycycline and the lowest background luciferase activity in the absence of doxycycline, was chosen as the Tet-On PC-3 cell line to be used for subsequent transient expression of TRPL and for the generation of double-stable Tet-On PC-3 cells containing TRPL cDNA.
G. Generation of stable clones of Tet-On PC3 cells harbouring cDNA encoding the TRPL and luciferase genes
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Ruengrairatanaroje et al: Tetracycline-inducible system for prostate cancer and MP40 cells were each transiently-transfected with the pTRELuc plasmid In each case a 10- to 15-fold induction of luciferase by doxycycline was observed, thus indicating that the rTetR/VP16 transcription factor was active in these cells.
I. Western immunofluorescence
blot
analysis
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A polyclonal rabbit anti-TRPL antibody was raised (Zhang et al, 2003) against a synthetic peptide (fused with diphtheria toxin) corresponding to the 14 carboxy terminal amino acids of the Drosophila TRPL protein. The peptide sequence, which included a cysteine residue at the amino terminus to facilitate coupling to the carrier diphtheria toxoid, was CDSNFDIHVVDLDEK (Niemeyer et al, 1996). Serum from the fifth boost of peptide injection was used to precipitate IgG (using cold half-saturated ammonium sulfate). The precipitated protein was sedimented by centrifugation at 15,000 rpm at 4°C for 30 min and resuspended in PBS, dialysed twice at 4°C in PBS, then purified by affinity chromatography on immunizing peptide bound to epoxy-activated sepharose 6B (Pharmacia). The adsorbed antibody was eluted with 4.9 M MgCl2. The specificity of the resulting anti-TRPL antibody was assessed by Western blot analysis using a Drosophila head-extract as a source of the TRPL protein. Whole cell lysates were prepared by adding 100 µl of lysis buffer (1% (v/v) Triton-X100, 0.1% (w/v) SDS, 10 mM leupeptin, 5 mM pepstatin, 10 mM PMSF and 0.001% (v/v) "mercaptoethanol in PBS, pH 7.4) to the cell pellet (1 x 106 cells), mixing the suspension by pipetting, maintaining the suspension for 30 min at 4°C, centrifuging (1,000xg for 5 min at 4°C) and collecting the supernatant. Crude membrane extracts were prepared by adding 500 µl of 5 mM Tris-HCl, (pH 7.5), containing 5 mM EDTA, 10 mM leupeptin, 5 mM pepstatin, 10 mM PMSF and 0.0002% (v/v) "mercaptoethanol to the cell pellet (1 x 106 cells), lysing the cells at 4°C (by injecting the cell suspension through a 25-guage syringe needle 5-10 times), centrifuging (1,000xg for 5 min at 4°C), transferring the supernatant to a new tube, and centrifuging at 16,000xg for 30 min 4°C, and collecting the pellet. The pellet was then resuspended in 50 µl of 5 mM Tris-buffer and the solution stored at -20°C. Protein concentrations were measured using the Bradford method (Bradford and Bradford, 1976) with bovine serum albumin (Sigma) as a standard. Western blot analysis was performed as described previously (Zhang et al, 2003), using rabbit polyclonal antiTRPL antibody (1:100 dilution at 4°C overnight) and horseradish peroxidase-conjugated anti-rabbit IgG (1:100 dilution at room temperature for 2 h). Protein bands were visualised using enhanced chemiluminescence (ECL) (Amersham). For immunofluorescence (Zhang et al, 2003), cells were grown on glass coverslips in 35 mm culture dishes, fixed for 20 min in 4% (v/v) formaldehyde and permeabilized by incubation for 5 min in 0.5% (v/v) Triton-X100 in PBS on ice. Subsequently, the cells were washed three times (10 min each) with PBS and incubated for 1 h at room temperature with a 1:100 dilution of rabbit polyclonal anti-TRPL antibody in PBS containing 5 µg/ml BSA as the blocking agent. After incubation for 1 h, cells were washed three times (10 min each) with PBS, then incubated with 1:500 goat anti-rabbit-IgG labelled with Cy3 in PBS at room temperature for 1 h. The cells were washed three times (10 min each) with PBS, and the coverslips mounted on a microscope slide in glycerol:PBS (80:20). Cy3 fluorescence was examined using an Olympus BX50 fluorescence microscope and 515-550 nM (excitation) and 575-615 (emission) filters. A video camera was used for image acquisition and digital images were saved as TIFF files.
H. PCR and luciferase assay To amplify TRPL cDNA in the original or reversed orientation in the pBluescript-TRPL plasmid, the primer pairs C20A/RSP or C20A/USP were employed. RSP (5’GGAAACAGCTATGACCATG-3’) (antisense) and USP (5’GTAAAACGACGGCCAGT-3’) (sense) are two primers that flank the multiple cloning site of pBluescript. The C20A sense primer (5’-AGTGGAAGTTTGCCCGAACC-3’) corresponds to a short sequence within TRPL (2195-2214) (Phillips et al, 1992). The predicted size of the amplified product fragments for each pair C20A/RSP and C20A/USP is approximately 1.5 kb. To amplify TRPL cDNA in the pBI-L-TRPL plasmid and in the PC-3 cells incorporating the TRPL gene, three PCR primer pairs were designed. These were FETRPL1 (5’AACACTCGTGCCTCAGATGG-3’) (sense) and RETRPL1 (5’TCCCCAAACTCACCCTGAAG-3’) (antisense), which amplify the 3’-end of the TRPL DNA sequence (Zimmer et al, 2000) to yield a product of 354 bp; C20A (sense) and pBI-L A1 (5’AGAGATATCGTCGACAAG-3’) (antisense), which amplify the 3’ half of the TRPL DNA sequence (Zimmer et al, 2000) to yield a product of approximately 1.5 kb; and FBTRPL2 (5’CCATCCACGGTCTTTTGACC-3’) (sense) and RBTRPL2 (5’CGTCGGCAGCTTCTTTTTGC-3’) (antisense) which amplify the 5’-end of the TRPL DNA sequence and the PCMV-1 promoter in the pBI-L-TRPL plasmid, to yield a product of approximately 336 bp. To amplify a region of the luciferase gene in the pBI-LTRPL plasmid and in the luciferase gene incorporated in PC-3 cells, primers were designed to amplify a sequence of the PCMV-2 promoter and luciferase cDNA. The primers were FLUC (5’TAGCTTCTGCCAACCGAACG-3’) (sense) and RLUC (5’CCATCCACGCTGTTTTGACC-3’) (antisense), which yield a product of 280 bp. For PCR amplification of TRPL and luciferase genes in plasmids, plasmid DNA was purified using the QIAGEN QIA filter Plasmid Midi kit according to the manufacturers instructions. Bulk PCR reaction mixtures contained 2 mM MgCl2, 100 µM of each dNTP, 0.4 µM of each primer (sense, antisense), and 0.4 units Taq polymerase in 1x Taq PCR buffer. Each reaction mixture (23 µl) was overlaid with mineral oil then 2 µl of plasmid template DNA (diluted in sterile H2O) added. The thermal profile for PCR amplification (Hybaid Omn-E PCR machine) consisted of 1 cycle of 94°C for 5 min, 40 cycles at 94°C for 1 min (denaturation), at 55°C for 1 min (annealing), at 72°C for 1 min (extension), 1 cycle at 72°C for 5 min, and 1 cycle at 15°C for 1 min (completion). Amplified products were visualized (ethidium bromide) after separation on 1% agarose. For PCR amplification of regions of the TRPL and luciferase genes incorporated in PC-3 cells, the cells were harvested using 0.1% (w/v) trypsin/EDTA, and washed once with 1 ml PBS. 50 µl of cell lysis buffer (50 mM KCl, 10 mM Tris-HCl (pH 8.3), 0.1 mg/ml gelatin, 0.45% (v/v) Nonidet P40, 0.45% (v/v) Tween-20 and proteinase-K (6 µl of 10 mg/ml added just before use) was added to each pellet, and the mixtures incubated at 60°C for 1 h, then at 95°C for 15 min to denature enzyme activity. The resulting cell-lysate was used for PCR amplification. Luciferase activity was measured using a Promega Luciferase Assay kit, according to the manufacturer’s instructions.
J. Determination of cell viability chromatin condensation and fragmentation
and
For determination of the number of dead and living cells using the Trypan blue exclusion test, cells were grown in 35 mm culture dishes, the culture medium removed, and 1 ml of 0.1%
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Cancer Therapy Vol 2, page 393 (w/v) Trypan blue in PBS buffer added. The cells were incubated for 15-20 min at room temperature, the adhering cells viewed using a phase-contrast microscope (Nikon) equipped with an ocular square grid micrometer, and the number of Trypan bluestained cells and the total number of cells per square millimeter determined for 5 different regions of the culture dish. For estimation of the number of viable cells using 3-[4.5dimethylthiazol-2-yl]-diphenyltetrazolium bromide (MTT) (Park et al, 1987), cells were grown at 37°C in a 96 well-plate in 200 µl of culture medium per well. MTT (20 µl of 5 mg/ml water) was added to each well, incubation was continued for a further 4 h, then 100 µl of 20% (w/v) SDS in 0.02 M HCl was added, the plates covered with aluminium foil, and incubated at room temperature overnight. The optical density of each well was measured at 570 nm (MTT) and 630 nm (background) using a microplate reader (Bio-Rad). Optical density readings were converted to readings of cell numbers using a calibration curve. Chromatin DNA condensation and fragmentation in adherent and detached cells were assessed using Hoechst 33258 and by examination of nuclear morphology (Ferguson and Anderson, 1981; Yanagihara and Tsumuraya, 1992). Cells were grown in 35 mm culture dishes under the conditions described in the legends to figures. Detached cells from each dish were harvested by centrifugation (3,200xg at room temperature for 1 min), suspended in 0.5 ml of culture medium, mixed with 5 µl Hoechst 33258 (1 mg/ml in double-distilled water) and incubated for 10 min. A sample (15 µl) was transferred onto a glass slide. For analysis of adherent cells, the medium in the culture dishes containing the adherent cells was replaced with 1 ml culture medium, then 10 µl of Hoechst 33258 (1 mg/ml in doubledistilled water) was added and the cells were incubated at room temperature. After 10 min, the cells were examined by fluorescence microscopy and the number of cells exhibiting condensed or fragmented nuclei as well as the total number of cells were determined.
unpaired samples. Values of P < 0.05 were considered to be significant.
III. Results A. Effects of transient expression of the TRPL protein in Tet-On PC-3 cells on cell viability and nuclear fragmentation The TRPL protein and the luciferase reporter protein were transiently-expressed in PC-3 cells under the control of the Tet-On promoter by transfecting PT-20-1 cells (PC3 cells stably-transfected with the Tet-On regulator) with the pBI-L-TRPL plasmid (which contains cDNA encoding the luciferase and TRPL proteins under the control of a bidirectional promoter). PT-20-1 cells transfected with the pBI-L plasmid (which does not contain cDNA encoding TRPL) were used as controls. Western blot analysis indicated that PT-20-1 cells transiently-transfected with pBI-L-TRPL exhibited a very low level of TRPL expression in the absence of doxycycline, and a doxycycline-inducible expression of the TRPL protein (Figure 1). Densitometric analysis indicated a 12-fold increase in TRPL protein expression (mean of two determinations) in cells grown in the presence of doxycycline. The doxycycline-induced increase in luciferase, which is a measure of transfection efficiency (Storz et al, 1999), was 6.6 ± 1.1 (n = 3) fold. PT-20-1 cells transiently-transfected with pBI-LTRPL exhibited a substantial doxycycline-induced increase in the number of dead cells, assessed using Trypan blue (Figure 2A), and a substantial increase in the number of cells exhibiting chromosome damage as assessed using Hoechst 33258 (Figure 2B). No doxycycline-induced changes in these parameters were observed in PT-20-1 cells transfected with the pBI-L or in untransfected PT-20-1 cells (Figure 2). In these experiments, the induction of luciferase by doxycycline was 7.2 ± 0.6 and 15.5 ± 1.9 (means ± SEM, n = 4) for cells transfected with pBI-L-TRPL and pBI-L, respectively. The nuclear morphology of PT-20-1 cells transientlytransfected with the pBI-L or pBI-L-TRPL plasmids and stained with Hoechst 33258 (a fluorescent chromatinbinding dye (Ferguson and Anderson, 1981)) is shown in Figure 3. Both adherent and detached pBI-L-TRPLtransfected cells incubated in the absence of doxycycline exhibited some nuclear condensation (Figure 3E and 3G). The proportion of adherent and detached cells exhibiting nuclear condensation was increased when the pBI-LTRPL-transfected cells were incubated in the presence of doxycycline (Figure 3F c.f. 3E and Figure 3H c.f. 3G). Cells transfected with pBI-L exhibited little nuclear condensation, and no increase in nuclear condensation when incubated in the presence of doxycycline (Figure 3A-3D). No doxycycline-induced increase in nuclear condensation was observed in PT-20-1 (non-transfected) cells (results not shown). The photomicrographs at higher magnification (Figure 3I-3L) demonstrate a variety of
K. Cytoplasmic Ca2+ concentration and initial and sustained rates of Ca2+ inflow Cells were grown on glass coverslips in 35 mm culture dishes were washed once with 1 ml complete RPMI-1640 medium at 37°C then incubated in 1 ml of Fura-2/AM loading solution, which was composed of 1 µl Fura-2/AM (3 µM in DMSO), 6.6 µl pluronic acid F-127 (10% w/v in DMSO (final concentration 0.02% v/v)) and 3.3 ml RPMI-5% FBS, for 30 min (at 37°C in 5% CO2:95% O2). After 30 min, the cells were washed three times with RPMI-5% FBS (37°C), then the coverslip placed in a coverslip holder and washed three times at 37°C with Ca2+-free modified Hank’s solution. Modified Hank’s solution (300 µl) was added and the coverslip holder placed in the microscope incubation chamber. The measurement of fura-2 fluorescence and conversion of fluorescence ratios to Ca2+ concentrations were performed as described previously (Gregory and Barritt, 2003). The initial rate of Ca2+ inflow was determined by calculating the slope of the plot of fluorescence ratio as function of time for the period 0-2 min after extracellular Ca2+ (2 mM) addition. The sustained rate of Ca2+ inflow was determined by calculating the slope of the plot of fluorescence ratio as function of time at 5, 10, 15 and 20 min after added Ca2+.
L. Expression of results Unless otherwise indicated, results are expressed as mean ± S.E.M. with the number of experiments indicated in parentheses. Degree of significance were determined using Student’s t-test for
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Figure 1. Western blot analysis of the TRPL protein in extracts of Tet-On PC-3 cells transiently expressing TRPL. PT-20-1 (TetOn PC-3) cells were transiently-transfected with pBI-L-TRPL using FuGENE6 and incubated in the absence of doxycycline for 24 h, then for a further 48 h in the absence or presence of doxycycline (1 µg/ml). Cells were harvested, crude membrane preparations prepared, and Western blot analysis conducted as described in Materials and Methods. TRPL protein present in Drosophila head extract was employed as a positive control. The arrow (128 kDa) indicates the position of the TRPL protein. The results shown are those obtained in one of two experiments which each gave similar results.
apoptotic nuclear morphologies in adherent PT-20-1 cells transiently-transfected with pBI-L-TRPL. There were many degraded nuclear forms (apoptotic nuclei) such as chromatin condensation with leakage into the cytoplasmic space (Figure 3I and 3J), DNA droplets of different size scattered throughout the cytoplasmic space (Figure 3L), localised chromatin condensation (Figure 3K), and general chromatin condensation (Figure 3J). The numbers of condensed or fragmented nuclei in both adherent and detached cells were combined to estimate the total number of apoptotic nuclei. This was expressed as a percentage of all nuclei. For PT-20-1 cells transiently-transfected with pBI-L-TRPL grown in the presence of doxycycline the proportion of cells exhibiting condensed or fragmented nuclei was 19.5 ± 6.0 compared with 7.3 ± 3.0 for cells grown in the absence of doxycycline (mean ± SEM, n = 4), (P ! 0.05). There were no significant differences for either untransfected PT-20-1 cells or PT-20-1 cells transfected with pBI-L grown in the presence and absence of doxycycline (results not shown).
Tests were made for the presence of the TRPL and luciferase genes in the cells grown over a number of passages. The resulting PCR products indicated that both the TRPL and luciferase genes were permanently incorporated into the cells (results not shown). When compared with the parent PT-20-1 Tet-On PC-3 cells, which were more spindle-shaped, cells of the MP40 and MP25 clones grown in the absence of doxycycline exhibited a homogenous rounded morphology. When grown in the presence of doxycycline, larger cells with a rounder shape were observed. These features of the cell morphology were observed in MP25 and MP40 cells grown over a number of passages. When subjected to Western blot analysis, extracts of MP25 and MP40 cells grown in the absence of doxycycline exhibited a band at 128 kDa which corresponded in apparent size to the band of TRPL protein observed in Drosophila head extracts. When the cells were grown in the presence of doxycycline, the intensity of the 128 kDa band increased 1.3- and 1.4-fold (mean of two determinations) for MP25 and MP40 cells, respectively (results not shown). No band of 128 kDa was detected in extracts of PT-20-1 cells (Tet-On PC-3 cells which do not express TRPL) (results not shown). Immunofluorescence staining of the TRPL protein was carried out to further investigate TRPL expression and its intracellular localisation. There was no difference in immunofluorescence in PT-20-1 cells grown in the absence or presence of doxycycline (Figure 4B c.f. 4A). By contrast, in the presence of doxycycline, MP40 cells exhibited increased fluorescence (Figure 4D c.f. 4C). Examination of the immunostained cells also confirmed the polymorphism and presence of a number of larger cells in MP40 cells grown in doxycycline (Figure 4D c.f. 4C).Similar results were obtained for MP25 cells (not
B. Effects of stable expression of the TRPL protein on cell viability, intracellular Ca2+ and nuclear fragmentation The results described in Figures 1-3 show that transient expression of the TRPL protein in PC-3 cells can be controlled by the Tet-On promoter system. The next experiments were performed to see if the same control of TRPL protein expression could be achieved in PC-3 cells stably expressing TRPL. PT-20-1 Tet-On PC-3 cells were stably-transfected with the pBI-L-TRPL plasmid as described in Materials and Methods. Two clones, MP25 and MP40, which showed the highest induction of the luciferase reporter gene were chosen for further study.
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was observed around the perinuclear zone (Figure 4E-4H) indicating the presence of considerable amounts of TRPL protein in this region.
Figure 2. Transient expression of TRPL in Tet-On PC-3 cells leads to a doxycycline-inducible increase in cell death (assessed using Trypan blue) (A) and an increase in the number of cells exhibiting chromosome damage, chromatin condensation, and chromatin fragmentation (assessed using Hoechst 33258) (B). PT-20-1 (Tet-On PC-3) cells were transiently-transfected with the pBIL (control) or pBI-L-TRPL plasmids (1 µg) using Lipofectamine. After 4 h, the medium was changed and the cells grown in the absence and presence of doxycycline (1 µg/ml) for 48 h. The number of cells excluding Trypan blue and the number exhibiting chromatin condensation or fragmentation were determined as described in Materials and Methods. In each case, the results are expressed as a percentage of the total number of cells present under the given condition tested. The results are the means ± S.E.M. (n = 4)
Figure 3. Patterns of nuclear chromatin condensation and fragmentation in Tet-On PC-3 cells transiently expressing TRPL. PT20-1 (Tet-On PC-3) cells were transiently-transfected with pBI-L (control) (A-D) or pBI-L-TRPL (E-H and I-L) and incubated in the absence or presence of doxycycline (1 µg/ml) for 48 h, then stained with Hoechst 33258. Magnification: x200 (A-H) and x400 (I-L). Cell transfection and culture, the harvesting of detached cells, the staining of adherent and detached cells with Hoechst 33258, and fluorescence microscopy were performed as described in Materials and Methods. The images shown are those obtained from one of 5 or more experiments which each gave similar results.
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Figure 4. Immunofluorescence images of the TRPL protein expressed in two stable Tet-On PC–3 clones (MP25 and MP40) expressing TRPL. A-D. PT-20-1 (Tet-On PC-3) cells (A,B) and MP40 cells (Tet-On PC-3 cells stably expressing TRPL) (C,D) cultured for 48 h in the absence (A,C) or presence (B,D) of doxycyline (4 µg/ml). Magnification: x200. E-H. MP25 (Tet-On PC-3 cells expressing TRPL) (E,F) and MP40 (G,H) cells cultured for 48 h in the presence of doxycycline (4 µg/ml). Magnification: x400. Cell culture and the detection of TRPL by immunofluorescence were performed as described in Materials and Methods. The results shown are representative of those obtained for more than 20 fields of cells which showed similar morphology.
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Cancer Therapy Vol 2, page 397 Basal values of [Ca2+]cyt were measured in cells loaded with fura-2 and incubated in the absence of added extracellular Ca2+. Initial and sustained rates of Ca2+ inflow were measured following the addition of 2 mM extracellular Ca2+. When grown in either the presence or absence of doxycycline, PC-20-1 cells (control, no TRPL expression) exhibited no difference in basal [Ca2+]cyt or in the initial and sustained rates of Ca2+ inflow (Figure 5A and 5D-5F).
When compared with cells grown in the absence of doxycycline, MP25 and MP40 cells (expressing TRPL) grown in the presence of doxycycline exhibited an increased value of basal [Ca2+]cyt and greater initial and sustained rates of Ca2+ inflow (Figure 5B-5F). The effect of TRPL expression on cell viability was assessed using the MTT test. As described above, when grown in the presence of doxycycline, MP40 cell cultures exhibited numerous swollen large cells (Figure 6A).
Figure 5. Stable expression of TRPL in Tet-On PC-3 cells leads to a doxycycline-inducible enhancement of the sustained rate of Ca2+ inflow. A-C. Plots of fluorescence as a function of time for PT-20-1 (Tet-On PC-3) cells (A) and MP25 (B) and MP40 cells (C) (Tet-On cells stably expressing TRPL). Cells were grown for 48 h in the presence or absence of doxycycline (4 µg/ml), loaded with fura2, and fluorescence measured as a function of time, as described in Materials and Methods. Cells were initially incubated in Ca2+-free Hank’s medium in the absence of added extracellular Ca2+. At the time indicated by the arrows, CaCl2 (2 mM) was added to the incubation medium. The traces shown are representative of those obtained in three experiments which each gave similar results. D-F. Basal values of [Ca2+]cyt (D) and initial (E) and sustained (F) rates of Ca2+ inflow for PT-20-1, MP25 and MP40 cells. Values of [Ca2+]cyt, and initial and sustained rates of Ca2+ inflow were determined as described in Materials and Methods. The results are the means ± S.E.M. (n = 3).
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Figure 6. Tet-On PC-3 cells stably expressing TRPL exhibit a doxycycline-inducible decrease in cell proliferation (assessed using the MTT assay) when grown in culture. A. A photomicrograph of MP40 cells grown for 48 h in the presence of doxycycline (4 µg/ml). Magnification: x200. B-D. The number of viable cells plotted as a function of time for PT-20-1 (Tet-On PC-3) cells (B), MP25 (C) and MP40 (D) cells (Tet-On PC-3 cells stably expressing TRPL) grown in the presence or absence of doxycycline (4 µg/ml). PT-201 cells (B), and MP25 (C) and MP40 (D) cells were plated in 96 well-plates for 24 h, then grown in the absence or presence of doxycycline (4 µg/ml) and the number of viable cells determined using the MTT assay, as described in Materials and Methods. The results are the means ± S.E.M. (n = 4). The degree of significance (*), determined using Student’s t-test for unpaired samples, is P ! 0.05.
These large cells disintegrated easily, and detached from the surface of the culture dish. For PT-20-1 cells (control, no TRPL expression), culture in the presence of doxycycline did not cause any difference in the number of viable cells when assessed using the MTT test (Figure 6B). By contrast, growth of MP25 and MP40 cells in the presence of doxycycline reduced the number of viable cells (Figure 6C and 6D). The number of apoptotic nuclei was assessed by determining the level of chromatin condensation and nuclear fragmentation using Hoechst 33258. Representative photomicrographs of PT-20-1 and MP40 cell cultures stained with Hoechst 33258 are shown in Figure 7. For PT-20-1 cells grown in either the absence or presence of doxycycline, the number of adherent cells exhibiting chromatin condensation and fragmentation was small (Figure 7B c.f. 7A, and Figure 7H). Moreover, no detached (“floating”) cells were observed. For MP25 and MP40 cultures grown in the absence of doxycycline, the number of adherent cells exhibiting chromatin condensation and fragmentation at a given time was slightly greater compared to the number observed for PT20-1 cultures grown in the absence of doxycycline (Figure 7C and Figure 7H). In the presence of doxycycline, the
number of adherent cells exhibiting chromatin condensation and fragmentation at a given time after doxycycline addition was increased (Figure 7D and Figure 7H). Chromatin condensation was observed more frequently than fragmented nuclei. Examples are shown in Figure 7E-7G. The variability in the percentage of adherent cells exhibiting chromatin condensation and nuclear fragmentation (Figure 7H) is likely to be due to the disintegration of swollen cells (c.f. the cells shown in the photomicrography in Figure 6A).
IV. Discussion A. Tet-On provides an effective inducible system for expression of the Ca2+-permeable TRPL channel and inducible Ca2+- and Na+mediated cell toxicity The key observations reported here are that (i) expression of the TRPL protein in PC-3 prostate cancer cells can be controlled by the tetracycline-responsive TetOn promoter system, (ii) the Tet-On system is effective when TRPL (under the control of the TRE element) is expressed either transiently or stably, and (iii) tetracycline398
Cancer Therapy Vol 2, page 399 Figure 7. Tet-On PC-3 cells stably expressing TRPL exhibit a doxycycline-inducible increase in the number of cells with nuclear chromatin condensation and fragmentation (assessed using Hoechst 33258) when grown in culture. A-D. PT-20-1 (Tet-On PC3) cells (A,B) and MP40 cells (TetOn PC-3 cells stably expressing TRPL) (C,D) grown for 48 h in the presence (B,D) and absence (A,C) of doxycycline (4 µg/ml) then stained with Hoechst 33258. Magnification: x200. E-J. Adherent (E-G) MP40 cells grown for 48 h in the presence of doxycycline (4 µg/ml) then stained with Hoechst 33258. Magnification: x400. Cell culture, the harvesting of detached cells, staining with Hoechst 33258, and fluorescence microscopy were performed as described in Materials and Methods. The images shown are those obtained from one of 10 or more experiments which each gave similar results. H. Cells grown as described above for 48, 72 or 96 h. At each time point, the cells adherent to the culture dish were stained with Hoechst 33258, examined by fluorescence microscopy, and the number of cells exhibiting fragmented or condensed nuclei was determined and expressed as a percentage of the total number of cells present at that time point. The results are the means ± S.E.M (n = 3).
B. Comparison of the transient and stable transfection of PC-3 cells with TRPL DNA under control of the TRE element
induced expression of TRPL is associated with cell toxicity and death. The results provide an “in principle” demonstration that controlled expression of: the TRPL protein can kill prostate cancer cells. This inducible system could be further refined and developed by optimising TRPL expression and targetting expression of the TRPL protein and the inducible promoter to prostate cancer cells. The inducible TRPL Ca2+ channel expression system offers a potential therapeutic strategy for the killing of androgen-independent prostate cancer cells. It could also be applied to any other cancer cell type in which the TRPL protein can be over-expressed.
The transient transfection of Tet-On PC-3 cells with TRPL DNA under control of the TRE element provided a clear and significant doxycycline induction of expression of the TRPL (and luciferase reporter gene) proteins, decreased cell viability and increased apoptosis. The following were also clearly evident in Tet-On PC3 cells stably transfected with TRPL DNA under control of the TRE element. Doxycycline-induced expression of the TRPL and the luciferase proteins, increased basal [Ca2+]cyt, changes in cell morphology, and increased cell death and
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Ruengrairatanaroje et al: Tetracycline-inducible system for prostate cancer apoptosis. However, the degree of induction of both the TRPL and luciferase proteins was lower in stablytransfected cells than that observed in transientlytransfected cells. Also, it proved quite difficult to isolate stable Tet-On clones expressing TRPL. This may have been due to the presence in PC-3 cells of proteins other than the Tet-On transcription factor which can activate the TRE element in the absence of doxycycline. This, in turn, would induce TRPL expression in the absence of doxycycline and cause subsequent Ca2+ and Na+ inflow and some cell death. While TRPL was successfully expressed in PC-3 cells under the control of the Tet-On inducible promoter system, there was only a small response to doxycycline in the two stable double-transfected clones (MP25 and MP40) which were studied in detail. Moreover, the stable double-transfected clones exhibited a relatively high basal luciferase expression and only a modest doxycycline induction of luciferase which was lost in later passages. By contrast, when either PT-20-1 cells (Tet-On PC-3 cells expressing the Tet-On regulator (rTetR/VP16)) or stable double-transfected cells were transiently-transfected with the pTRE-Luc plasmid (harbouring DNA encoding the TRE-response element and luciferase), doxycycline induced a substantial increase in luciferase expression. This demonstrates the effectiveness of the Tet-On regulatory system in PC-3 cells and indicates that the induction machinery (expression of rTetR/VP16 and formation of the rTetRVP16-doxycycline complex) is intact. The degree of doxycycline-induced luciferase expression in cells transiently expressing pTRE-Luc was comparable with that observed by others for the Tet-On system in stable cell lines (Gschwend et al, 1997). While the induction by doxycycline of luciferase and TRPL in double-stable transfected PC-3 cells was small, it is within the wide range reported for the expression of proteins under the control of the TRE promoter element in other cell types (Nakamura et al, 2000; Gossen and Bujard, 1992). In contrast to the results reported here for luciferase expression in PC-3 cells, others have reported the successful stable expression of luciferase in PC-3 cells under the control of the CMV promoter (Rubio et al, 1998, 2000) and the inducible TRE promoter element in the Tetoff PC-3 cell line (Gschwend et al, 1997). However, the plasmids employed in these other studies (Gschwend et al, 1997; Rubio et al, 1998, 2000) differ from those employed here. High background levels (leakage) of proteins expressed under control of the TRE promoter element have also been reported for the Tet-Off system in PC-3 cells (Gschwend et al, 1997) and for the TRE system in other cell types (Park and RajBhandary, 1998; Keyvani et al, 1999). The high basal levels of luciferase and TRPL expression observed in the absence of doxycycline in double-stable transfected PC-3 clones (MP25 and MP40) may be due to the presence of endogenous proteins which activate the TRE promoter element and/or the minimal CMV promoters (Gschwend et al, 1997; Park and RajBhandary, 1998; Keyvani et al, 1999), a high basal activity of the minimal CMV promoter in the PC-3 cell environment (Gossen and Bujard, 1992, 1995; Gossen et
al, 1993), incomplete integration of the pBI-L-TRPL plasmid (Gossen and Bujard, 1992), and/or high plasmid copy number (Gill and Ptashne, 1988). However, it is noted that such high basal levels of expression were not observed in transiently-transfected PT-20-1 Tet-On PC-3 cells. Reasons for the lack of substantial induction by doxycycline of luciferase and TRPL expression may also include the possibility that expressed luciferase accumulates in peroxysomes and cannot be readily solubilized under the conditions of the luciferase assay (Keller et al, 1987), toxic effects of the expressed TRPL protein which affect expression of both luciferase and TRPL (Garcia-Gallo et al, 1999; Liu et al, 1999), and location of the minimal CMV promoter of the pBI-LTRPL plasmid in a region where it cannot be activated by the doxycycline-rTetR-VP16 complex (Gossen and Bujard, 1995; Freundlieb et al, 1999).
C. Possible mechanisms of TRPL-induced cell death The process by which TRPL expression kills PC-3 cells is most likely initiated by enhanced Ca2+ and Na+ inflow across the plasma membrane. The present results show that the toxic effects of expression of TRPL are associated with increased Ca2+ inflow and elevated [Ca2+]cyt. Since TRPL is a non-selective Ca2+ channel which admits Na+ as well as Ca2+ (Kunze et al, 1997), it is likely that an increased intracellular Na+ concentration accompanies the observed increase in [Ca2+]cyt. The idea that Na+ plays a role in cell toxicity is consistent with the observation that cell swelling followed by cell disintegration and chromosome degradation are not typical of cells undergoing apoptosis or necrosis (Kroemer et al, 1998; Hacker, 2000). This suggests that, at the very least, Na+ as well as Ca2+ is involved in the pathway of cell toxicity. The morphology of PC-3 cells stably expressing TRPL (large, polymorphic, and swollen cells) is similar to that of HepG2 and MKN42-P cells heterologously expressing the MDEG channel which induced a massive Na+ inflow (Horimoto et al, 2000). The TRPL-mediated increase in [Ca2+]cyt may cause other changes, including disruption of mitochondria (Beaver and Waring, 1994; McConkey and Orrenius, 1997; Berridge et al, 1998; Crompton, 1999; Jambrina et al, 2003), activation of Ca2+/Mg2+-dependent endonucleases (Cohen and Duke, 1984; Ribeiro and Carson, 1993) and K+ efflux (Speake et al, 1998) which, in part, lead to apoptosis. It is interesting to note that expression of Bcl-2 and Bcl-xL has been reported to decrease [Ca2+]cyt (McDonnell et al, 1992; Porn-Ares et al, 1998; Lebedeva et al, 2000; Scorrano et al, 2003). The presence of these proteins in PC-3 cells may counteract the effects of increased [Ca2+]cyt due to TRPL expression. Another possibility is that cation flow across intracellular membranes and/or the physical disruption of these membranes by large amounts of intracellular TRPL are partly responsible for some of the toxic effects of TRPL. A considerable amount of the stably-expressed TRPL protein appeared to be located at intracellular sites, especially around the nucleus. These sites may be the Golgi and endoplasmic reticulum. Such an intracellular 400
Cancer Therapy Vol 2, page 401 Domann R, Martinez J (1995) Alternative to cloning cylinders for isolation of adherent cell clones. Biotechniques. 18, 594595 Ferguson DJ, Anderson TJ (1981) Ultrastructural observations on cell death by apoptosis in the “resting” human breast. Virchows Arch A Pathol Anat Histol. 393, 193-203 Freundlieb S, Schirra-Muller C, Bujard H (1999) A tetracycline controlled activation/repression system with increased potential for gene transfer into mammalian cells. J Gene Med. 1, 4-12 Furuya Y, Lundmo P, Short AD, Gill DL, Isaacs TJ (1994) The role of calcium, pH, and cell proliferation in the programmed (apoptotic) death of androgen-independent prostatic cells induced by thapsigargin. Cancer Res. 54, 6167-6175 Garcia-Gallo M, Behrens MM, Renart J, Diaz-Guerra M (1999) Expression of N-methyl-D-asparate receptors using vaccinia virus causes exocytotic death in human kidney cells. J Cell Biochem. 72, 135-144 Gill G, Ptashne M (1988) Negative effect of the transcriptional activator GAL4. Nature. 334, 721-724 Gossen M, Bonin AL, Bujard H (1993) Control of gene activity in higher eukaryotic cells by prokaryotic regulatory elements. Trends Biochem Sci. 18, 471-475 Gossen M, Bujard H (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA. 89, 5547-5551 Gossen M, Bujard H (1995) Efficacy of tetracycline-controlled gene expression is influenced by cell type: commentary. Biotechniques. 19, 213-217 Gossen M, Freundlieb S, Bender G, Muller G, Hillen W, Bujard H (1995) Transcriptional activation by tetracyclines in mammalian cells. Science. 268, 1766-1769 Gregory RB, Barritt GJ (2003) Evidence that Ca2+-releaseactivated channels in rat hepatocytes are required for the maintenance of hormone-induced Ca2+ oscillations. Biochem J. 370, 695-702 Gschwend JE (1996) Apoptosis: principles and importance of programmed cell death for prostatic carcinoma. Urologe A. 35, 390-399 Gschwend JE, Fair WR, Powell CT (1997) Evaluation of the tetracycline-repressible transactivator system for inducible gene expression in human prostate cancel cell lines. Prostate. 33, 166-176 Gutierrez AA, Arias JM, Garcia L, Mas-Oliva J, GuerreroHernandez A (1999) Activation of a Ca2+-permeable cation channel by two different inducers of apoptosis in a human prostatic cancer cell line. J Physiol. 517, 95-107 Hacker G (2000) The morphology of apoptosis. Cell Tissue Res. 301, 5-17 Hofmann T, Schaefer M, Schultz G, Gudermann T (2002) Subunit composition of mammalian transient receptor potential channels in living cells. Proc Natl Acad Sci USA. 99, 7461-7466 Horimoto M, Sasaki Y, Ugawa S, Wada S, Toyama T, Iyoda K, Yakushijin K, Minami Y, Ito T, Hijioka T, Eguchi A, Nakanishi M, Shimada S, Tohyama M, Hayashi N, Hori M (2000) A novel strategy for cancer therapy by mutated mammalian degenerin gene transfer. Cancer Gene Therapy. 7, 1341-1347 Isaacs JT, Lundmo PI, Berges R, Martikainen P, Kyprianou N, English HF (1992) Androgen regulation of programmed death of normal and malignant prostatic cells. J Androl. 13, 457-464 Jambrina E, Alonso R, Alcalde M, del Carmen Rodr£guez M, Serrano A, Mart£nez-A C, Garc£a-Sancho J, Izquierdo M (2003) Calcium influx through receptor-operated channels induces mitochondria-triggered paraptotic cell death. J Biol Chem. 278, 14134-14145.
location of several heterologously-expressed TRP proteins has previously been observed by others (Wang et al, 1999; Schaefer et al, 2000; Hofmann et al, 2002). The location of TRPL at intracellular sites may represent the accumulation of large amounts of TRPL in vesicles which are part of the trafficking pathway to the plasma membrane (Wang et al, 1999; Schaefer et al, 2000; Hofmann et al, 2002) or may be part of a normal mechanism for the activation of members of the TRP channel family by exocytosis (e.g. Bezzerides et al, 2004). In conclusion, the results of the present study demonstrate, in principle, that TRPL expression in prostate cancer cells can be controlled by an inducible promoter system. The mechanism by which the expressed TRPL protein induces cell death is likely to involve increased intracellular Ca2+ and Na+, changes in the morphology and biochemistry of intracellular organelles, and the apoptotic pathway. This system offers a potential strategy for the controlled killing of prostate and other cancer cells.
Acknowledgements The authors gratefully acknowledge Dr. Len Kelly (Department of Genetics, University of Melbourne) for provision of TRPL cDNA, the advice of Dr. Tony Edwards, School of Medicine, Flinders University, and Ms. Courtney Bryans and Mrs. Diana Kassos for preparing the typescript. Support by a grant from the Cancer Council South Australia and the Thai Government for a postgraduate research scholarship are gratefully acknowledged.
References Aarts M, Iihara K, Wei WL, Xiong ZG, Arundine M. Cerwinski W, MacDonald JF, Tymianski M (2003) A key role for TRPM7 channels in anoxic neuronal death. Cell 115, 863877 Beaver JP, Waring P (1994) Lack of correlation between early intracellular calcium ion rises and the onset of apoptosis. Immunol Cell Biol. 72, 489-499 Berridge MJ, Bootman MD, Lipp P (1998) Calcium – a life and death signal. Nature. 395, 645-648 Bezzerides VJ, Ramsey IS, Kotecha S, Greka A, Clapham DE (2004) Rapid vesicular translocation and insertion of TRP channels. Nature Cell Biology 6, 709-720 Bradford HF, Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Adv Exp Med Biol. 69, 395-404 Cohen JJ, Duke RC (1984) Glucocortcoid activation of a calcium-dependent endonuclease in thymocyte nuclei leads to cell death. J Immunol. 132, 38-42 Colombel MG, Gil Diez S, Radvanyi F, Buttyan R, Thiery JP, Chopin D (1996) Apoptosis in prostate cancer. Molecular basis of study hormone refractory mechanisms. In: Castagnetta INL, Leon Bradlow H (eds). Basis for Cancer Management. The New York Academy of Sciences, New York, New York Crompton M (1999) The mitochondrial permeability transition pore and its role in cell death. Biochem J. 341, 233-249 Denmeade SR, Isaacs JT (1996) Activation of programmed (apoptotic) cell death for the treatment of prostate cancer. Adv Pharmacol. 35, 281-306
401
Ruengrairatanaroje et al: Tetracycline-inducible system for prostate cancer Kass GE, Orrenius S (1999) Calcium signalling and cytotoxicity. Environ Health Perspect. 107 Suppl 1, 25-35 Keller GA, Gould S, Deluca M, Subramani S (1987) Firefly luciferase is targeted to peroxisomes in mammalian cells. Proc Natl Acad Sci USA. 84, 3264-3268 Keyvani K, Baur I, Paulus W (1999) Tetracycline-controlled expression but not toxicity of an attenuated dipthelia toxin mutant. Life Sci. 64, 1719-1724 Kroemer G, Dallaporta B, Resche-Rigon M (1998) The mitochondrial death/life regulator in apoptosis and necrosis. Annu Rev Physiol. 60, 619-642 Kunze DL, Sinkins WG, Vaca L, Schilling WP (1997) Properties of single Drosophila Trpl channels expressed in Sf9 insect cells. Am J Physiol. 272, C27-C34 Lebedeva I, Rando R, Ojwang J, Cossum P, Stein CA (2000) Bcl-xL in prostate cancer cells: effects of overexpression and down-regulation on chemosensitivity. Cancer Res. 60, 60526060 Liu HS, Jan MS, Chou CK, Chen PH, Ke NJ (1999) Is green fluorescent protein toxic to the living cells? Biochem Biophys Res Commun. 260, 712-717 Martikainen P, Kyprianou N, Tucker RW, Isaacs JT (1991) Programmed death of nonproliferating androgen-independent prostatic cancer cells. Cancer Res. 51, 4693-4700 McConkey DJ, Orrenius S (1997) The role of calcium in the regulation of apoptosis. Biochem Biophys Res Commun. 239, 357-366 McDonnell TJ, Troncoso P, Brisbay SM, Logothetis C, Chung LW, Hsieh JT, Tu SM, Campbell ML (1992) Expression of the protooncogene bcl-2 in the prostate and its association with emergence of androgen-independent prostate cancer. Cancer Res. 52, 6940-6944 Nakamura K, Bossy-Wetzel E, Burns K, Fadel MP, Lozyk M, Goping IS, Opas M, Bleackley RC, Green DR, Michalak M (2000) Changes in endoplasmic reticulum luminal environment affect cell sensitivity to apoptosis. J Cell Biol. 150, 731-740 Niemeyer BA, Suzuki E, Scott K, Jalink K, Zuker CS (1996) The Drosophila light-activated conductance is composed of the two channels TRP and TRPL. Cell. 85, 651-659 Park H-J, RajBhandary UL (1998) Tetracycline-regulated suppression of amber codons in mammalian cells. Mol Cell Biol. 18, 4418-4425 Park JG, Kramer BS, Steinberg SM, Carmichael J, Collins JM, Minna JD, Gazdar AF (1987) Chemosensitivity testing of human colorectal carcinoma cell lines using a tetrazoliumbased colorimetric assay. Cancer Res. 47, 5875-5879 Phillips AM, Bull A, Kelly LE (1992) Identification of a Drosophila gene encoding a calmodulin-binding protein with homology to the trp phototransduction gene. Neuron. 8, 631642 Porn-Ares MI, Ares MPS, Orrenius S (1998) Calcium signalling and the regulation of apoptosis. Toxicol In Vitro. 12, 539543 Putney Jr JW, Broad LM, Braun FJ, Lievremont JP, Bird GS (2001) Mechanisms of capacitative calcium entry. J Cell Sci. 114, 2223-2229 Ribeiro JM, Carson DA (1993) Ca2+/Mg2+-dependent endonuclease from human spleen: purification, properties, and role in apoptosis. Biochem. 32, 9129-9136 Rubio N, Villacampa MM, Blanco J (1998) Traffic to lymph nodes of PC-3 prostate tumor cells in nude mice visualised using the luciferase gene as a tumor cell marker. Lab Invest. 78, 1315-1325 Rubio N, Villacampa MM, El Hilali N, Blanco J (2000) Metastatic burden in nude mice organs measured using
prostate tumor PC-3 cells expressing the luciferase gene as a quantifiable tumor cell marker. Prostate 44, 133-143 Schaefer M, Plant TD, Obukhov AG, Hofmann T, Gudermann T, Schultz G (2000) Receptor-mediated regulation of the nonselective cation channels TRPC4 and TRPC5. J Biol Chem. 275, 17517-17526 Scorrano L, Oakes SA, Opferman JT, Cheng EH, Sorcinelli MD, Pozzan T, Korsmeyer SJ (2003) BAX and BAK regulation of endoplasmic reticulum Ca2+: a control point for apoptosis. Science. 300, 135-139 Skryma R, Mariot P, Bourhis XL, Coppenolle FV, Shuba Y, Vanden Abeele F, Legrand G, Humez S, Boilly B, Prevarskaya N (2000) Store depletion and store-operated Ca2+ current in human prostate cancer LNCaP cells: involvement in apoptosis. J Physiol. 527, 71-83 Speake T, Douglas IJ, Brown PD (1998) The role of calcium in the volume regulation of rat lacrimal acinar cells. J Membr Biol. 164, 283-291 Steinsapir J, Socci R, Reinach P (1991) Effects of androgen on intracellular calcium of LNCaP cells. Biochem Biophys Res Commun. 179, 90-96 Storz P, Doppler H, Horn-Muller J, Groner B, Pfizenmaier K, Muller G (1999) A cellular reported assay to monitor insulin receptor kinase activity based on STAT 5-dependent luciferase gene expression. Anal Biochem. 276, 97-104 Tilley WD, Bentel JM, Aspinall JO, Hall RE, Horsfall DJ (1995) Evidence for a novel mechanism of androgen resistance in the human prostate cancer cell line, PC-3. Steroids. 60, 180186 Tombal B, Gailly P, Van Cangh PJ, Gillis JM (1995) Role of intracellular calcium in the programmed cell death of prostatic cancer cells. Acta Urol Belg. 63, 1-5 Wang W, Oâ&#x20AC;&#x2122;Connell B, Dykeman R, Sakai T, Delporte C, Swaim W, Zhu X, Birnbaumer L, Ambudkar IS (1999) Cloning of Trp1Ă&#x; isoform from rat brain: immunodetection and localization of the endogenous TRP1 protein. Am J Physiol. 276, C969-C979 Webber MM, Bello D, Quader S (1997) Immortalised and tumorigenic adult human prostatic epithelial cell lines: characteristics and applications Part 2. Tumorigenic cell lines. Prostate 30, 58-64 Yanagihara K, Tsumuraya M (1992) Transforming growth factor beta 1 induces apoptotic cell death in cultured human gastric carcinoma cells. Cancer Res. 52, 4042-4045 Zhang L, Brereton HM, Hahn M, Froscio M, Tilley WD, Brown MP, Barritt GJ (2003) Expression of the Drosophila Ca2+ permeable transient receptor potential-like (TRPL) channel protein in a prostate cancer cell line decreases cell survival. Cancer Gene Therapy. 10, 611-625 Zimmer S, Trost C, Wissenbach U, Philipp S, Freichel M, Flockerzi V, Cavalie A (2000) Modulation of recombinant transient-receptor-potential-like (TRPL) channels by cytosolic Ca2+. Pflugers Arch. 440, 409-417.
Gregory J. Barritt
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Cancer Therapy Vol 2, page 403 Cancer Therapy Vol 2, 403-414, 2004
Cellular drug resistance in lung cancer Review Article
J端rgen Mattern* Clinical Cooperation Unit Nuclear Medicine, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany
__________________________________________________________________________________ *Correspondence: J端rgen Mattern, M.D., Clinical Cooperation Unit Nuclear Medicine, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany, Tel: +49-6221-423221, Fax: +49-6221-423225, e-mail: j.mattern@dkfz.de Key words: Drug resistance, lung cancer, resistance-related proteins, protooncogenes, DNA microarray technology Abbreviations: antioxidant enzymes, (AOE); 50% inhibitory concentration, (IC50); called multidrug resistance, (MDR); cyclindependent kinases (cdks); glutathione peroxidase, (GPX); glutathione S-transferase, (GST); glutathione, (GSH); hypoxia-inducible factor-1, (HIF-1); lung resistance protein, (LRP); Metallothioneins, (MTs); non-small cell lung cancer, (NSCLC); O6-alkylguanine-DNA alkyltransferase, (ATase); of multidrug resistance-associated protein, (MRP); P-glycoprotein, (P-gp); small cell lung cancer, (SCLC); surface enhanced laser desorption/ionisation, (SELDI); Thymidylate synthase, (TS); topoisomerase II, (topo II); World Health Organization, (WHO) Received: 29 September 2004; Revised: 21 October 2004 Accepted: 25 October 2004; electronically published: November 2004
Summary Drug resistance is an important problem in the treatment of patients with lung cancer. Tumors become resistant not only to the drugs used initially, but also to those to which they have not yet been exposed. Data obtained from various sources indicate that multiple mechanisms contribute to drug resistance. Many of them are inter-related or independent of each other, but may exist simultaneously in cancer cells or subpopulation of cells, producing an overall drug-resistant phenotype. The resistance-related proteins P-glycoprotein, glutathione-dependent enzymes, topoisomerases, metallothioneins, thymidylate synthase and O6-alkyl-DNA alkyltransferase have been found in various human lung tumors, but these alone cannot explain the drug-resistant phenotype. Cell-cycle-related proteins, angiogenic factors, protooncogenes, and tumor suppressor genes also play a role in the resistant phenotype. In future, a key challenge will be to determine the relative quantitative contributions of each of these mechanisms to overall resistance. The use of DNA microarray technology in drug resistance in lung cancer will yield insight into the mechanisms of drug resistance and the rational design of more effective strategies to circumvent resistance. relapses and their tumors will be largely refractory to further treatment. NSCLC are inherent resistant and are generally nonresponsive to initial chemotherapy.
I. Introduction Lung cancer is a global problem. It is the most frequent cancer in the world, and the epidemic of lung cancer is still continuing. The global incidence of lung cancer is increasing at 0.5% per year. Consequently, lung cancer will remain a major cause of world wide cancer death in the 21th century (Haugen, 2000). The prognosis of lung cancer is very poor: nearly 80% of patients die within 1 year of diagnosis. Despite major advances in patient management, chemotherapy and radiotherapy made over the past decades, long-term survival is obtained in only 5-10% of the patients (Richardson and Johnson, 1993; Mattern et al, 2002). The major problem in lung cancer chemotherapy is the emergence of inherent and acquired drug resistance of the cancer cells. Resistance to anticancer agents is observed frequently in small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Most patients with SCLC have an initial response to chemotherapy, but the majority
II. Definition of drug resistance in vivo and in vitro Clinical drug resistance criteria are defined on the basis of tumor response criteria documented by the World Health Organization (WHO) or other such organizations. When a patient does not show more than a partial response after chemotherapy treatment, the tumor is considered to be resistant to the anticancer drugs used. Criteria for in vitro drug resistance are not clear. In most studies, the 50% inhibitory concentration (IC50) values are used. Various in vitro test procedures have been used to test the sensitivity or resistance of lung tumor cells to cytotoxic drugs prior to therapy. The soft agar clonogenic assay (Hamburger and Salmon, 1977), the most widely
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Mattern: Cellular drug resistance in lung cancer (P-glycoprotein) which is encoded by the mdr1 gene (Gros et al, 1986). Based on the homology of the mdr1 gene to a variety of genes encoding membrane transport proteins and on the ability of P-glycoprotein (P-gp) to bind drugs, it is believed that the multidrug resistance results from the function of P-gp as an energy-dependent drug efflux pump (Bradley et al, 1988). Relatively high levels of P-gp/mdr1 expression have been shown in many intrinsically drug-resistant tumors derived from tissues which normally express P-gp, including adrenal gland, kidney, liver, colon and rectum (Fojo et al, 1987). Generally low, but detectable levels of mdr1 gene expression on performance of RNA slot blot analysis or RT-PCR have been found in lung cancer as well as in normal lung tissue despite the intrinsic chemoresistance of lung cancer (Lai et al, 1989; Abe et al, 1994). Other studies using similar techniques also demonstrated that lung tumors contain low or undetectable levels of mdr1 mRNA (Goldstein et al, 1989; Shin et al, 1992). In contrast, using various monoclonal antibodies reactive with P-gp, some studies have revealed a relatively high expression of this protein in lung cancer (Radosevich et al, 1989). The inconsistency with the very low mdr1 levels found in lung tumors may be attributed to the varying sensitivity of the different methods to detect mdr1/P-gp expression (Herzog et al, 1992). Several SCLC and NSCLC cell lines made resistant to some MDRrelated drugs or derived from tumors relapsing after chemotherapy have been shown to express mdr1 mRNA and P-gp (Lai et al, 1989; Twentyman et al, 1986). In NSCLC, there appears to be a relationship between P-gp expression and drug resistance in vitro (Volm et al, 1991). The significance of mdr1/P-gp expression as a predictor for response to therapy in lung cancer is not yet clear. Studies including larger numbers of patients are necessary to answer this question. In conclusion, there is no evidence to suggest a major role of P-gp in the MDR of lung tumors. The overexpression of mdr1/P-gp alone does not completely explain intrinsic MDR, and thus other drug resistance mechanisms are thought to exist in lung cancers.
used test system for in vitro drug selection, has proven to be of limited practical use in lung cancer since drug testing can be performed in only a minority of cases because of technical problems. Renewed interest in in vitro testing has been generated by the use of dye exclusion assays which are simple colorimetric tests for cell proliferation and survival (Weisenthal et al, 1983). Our own research group described a simple test in which the uptake of radioactive nucleic acid precursors is determined in the cells after the addition of drugs (Volm et al, 1979). The use of xenografts for testing drugs in vivo has also been used (Mattern et al, 1984; 1987; 1988). In most instances good agreement was found between the in vitro and the in vivo results. However, it is common to all test systems used that tumors which demonstrate resistance during testing will not, with a high degree of probability, respond to therapy in the clinic (Mattern et al, 1982). Thus, lung cancers exhibit a drug resistance profile in vitro paralleling that observed in clinical practice. Cell lines established from untreated SCLC are often sensitive in vitro to cytotoxic drugs whereas cell lines from NSCLC or relapsed SCLC are resistant to multiple agents (Carmichael et al, 1985; Ruckdeschel at al., 1987). In an effort to understand better the mechanisms of drug resistance, many in vitro selected lines have been generated by continuous or pulsed exposure to drugs. Such induced drug resistance has been associated very often with changes in expression of certain intracellular and plasma proteins which are partly characteristic of resistance to certain drugs. Some examples of the types of proteins altered in drug-resistant cell lines are listed in Table 1.
III. Specific mechanisms of drug resistance A. Multidrug resistance One mechanism of resistance which has now been well characterized is the so-called multidrug resistance (MDR) which describes the broad pattern of crossresistance that is seen following the development of resistance to certain drugs. MDR cells consistently overexpress a 170 kDa membrane-associated glycoprotein
Table 1: Resistance-related proteins and influenced anticancer agents Resistance-related proteins P-glycoprotein Topoisomerase II Glutathione-S transferase-! Metallthionein 06-alkylguanine DNA alkyltransferase Thymidylate synthase Cell cycle-related proteins
Increased or decreased Increased Decreased Increased Increased Increased
Resistance to drugs
Increased Decreased
5-FU, MTX, DOX, DDP DOX, MTX, VCR, ARA-C, HU, 5-FU, CTX
DOX, ACT, VCR, VP16, VM26, COL, VDS, VBL, MTC DOX, ACT, VP16, VM26, MITOX, m-AMSA DOX, ACT, VP16, VM26, L-PAM, DDP, CTX, CHL, BCNU DOX, DDP, L-PAM, CHL BCNU, CCNU, ACNU, PC, CTX
Abbreviations: ACT, actinomycin D; ARA-C, cytosine arabinoside; m-AMSA, amsacrine, BCNU, carmustine; CCNU, lomustine; CHL, chlorambucil; COL, colchicine; CTX, cyclophosphamide; DDP, cisplatin; DOX, doxorubicin; 5-FU, 5-fluorouracil; HU, hydroxyurea; MITOX, mitoxantrone; MTC, mitomycin C; MTX, methotrexate; L-PAM, melphalan; PC, procarbacin; VBL, vinblastine; VCR, vincristine; VDS, vindesine; VM26, tenoposide; VP16, etoposide
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Cancer Therapy Vol 2, page 405 may be involved in drug resistance has come from the generation of drug-resistant cell lines in vitro and from transfection studies with GST cDNAs (Nakagawa et al, 1990). Changes in GST expression are most marked in cell lines made resistant to nitrogen mustard compounds and nitrosoureas and redox cycling drugs such as doxorubicin (Whelan et al, 1989). GST-! isoenzyme is also overexpressed in lung tumors of smokers compared to nonsmokers (Volm et al, 1991). It is suggested that GST overexpression may be a part of an adaptive response to enviromental stress to protect against toxic injury. Frequently, a coexpression of P-gp and GST-! is found (Volm et al, 1991; Linsenmeyer et al, 1992). But also a coordinated induction of MRP1 and GSH-related enzymes is reported in malignant cells after exposure to cytostatic agents (Van der Kolk et al, 1999). These observations led to the suggestions that these genes share common regulatory mechanisms, and that perhaps a single transcription factor or regulating protein may be involved in their regulation. However, it is unlikely that alterations in the GST are causally related to the development of drug resistance in lung tumors, but rather that co-modification along with other resistancerelated enzymes could mediate drug resistance. Besides these detoxifying enzymes, normal lung is also efficiently protected against exogenous free radicals by antioxidant enzymes (AOEs). Major human AOE include superoxide dismutases, catalase and enzymes associated with GSH metabolism, all of which are expressed in human lung. In addition, human lung also expresses several thiol-containing proteins including the families of thioredoxins, thioredoxin reductases and peroxiredoxins. Their expression in human lung is located mainly to alveolar macrophages, bronchial epithelial cells and alveolar epithelium, critical areas in the oxidant protection of human lung (Kinnula et al, 2004). These proteins not only have effects on cell proliferation and cell death, but also protect both non-malignant and malignant cells against radiation and chemotherapy. The redoxregulating proteins are highly expressed in lung tumors (Soini et al, 2001) and are associated with lymph node status and prognosis in NSCLC (Kakolyris et al, 2001). Peroxiredoxins also have effects on the progression and prognosis of lung cancer (Lehtonen et al, 2004).
Within the past few years a variety of further MDRassociated genes was identified: mainly the discovery of the MDR- associated protein MRP1 going along with its family members MRP2 to MRP6 of this transporter family (Cole et al, 1992; Borst et al, 1999) and the lung resistance-related protein (LRP) acting as the major vault protein in the nucleocytoplasmic transport (Scheper et al, 1993). They confer the MDR phenotype that is distinct in pattern from mdr1-related resistance, but includes many of the same drugs. MRP belongs to the ABC transporter gene superfamily and operates as an ATP-dependent primary active transporter for substrates conjugated with glucuronide or glutathione (Ishikawa et al, 2000). Overexpression of MRP and LRP is frequently observed in primary NSCLC, especially in the well-differentiated squamous cell carcinomas (Nooter et al, 1996) and expression is significantly higher in NSCLC samples when compared to SCLC samples (Dingemanns et al, 1996). There is also evidence that expression of MRP1 and LRP can be upregulated by sublethal exposure of lung cancer cells to some MDR-related drugs (Berger et al, 2000; Yoshida et al, 2001). Ota et al, (1995) examined the levels of expression of MRP in 104 NSCLC and found that MRP overexpression was associated with a worse prognosis in patients that received postoperative chemotherapy with MRP-related anticancer drugs (vindesine/etoposide). However, the concomitant operation of several resistance mechanisms may be often necessary to cause the MDR phenotype. Experimental data indicate that the genes coding for P-gp, MRP and LRP are differentially regulated by extracellular stimuli.
B. Glutathione-dependent enzymes Another important arsenal that cells utilize to detoxify the cytotoxic effects of anticancer drugs are glutathione (GSH) and the GSH-related enzymes glutathione S-transferase (GST) and glutathione peroxidase (GPX). GST catalyzes the conjugation of electrophilic metabolites to GSH to facilitate their excretion. GPX utilizes GSH to remove reactive oxygen intermediates. GSH and its related enzymes are ubiquitously distributed in many normal tissues as well as tumors and are involved in resistance to a wide variety of drugs such as alkylating agents, anthracyclines and vinca alkaloids (Tew, 1994). There is increasing evidence that these enzymes are a determinant factor in the sensitivity of lung tumors to anticancer drugs (Carmichael et al, 1988; Sharma et al, 1993). GST isoenzymes are found in significant amounts in bronchioles and alveoli of normal lung (Awasthi et al, 1987) and most intensely in the bronchial epithelium (Anttila et al, 1993). A number of studies have shown that the amount of GST isoenzymes is even higher in tumors of the lung relative to the surrounding normal tissue (Di Ilio et al, 1988; Clapper et al, 1991). In particular, high levels of glutathionedependent enzymes have been detected in cell lines derived from NSCLC compared to SCLC cell lines (Dâ&#x20AC;&#x2122;Arpa and Liu, 1989). It is speculated that these alterations could account for the differences in drug sensitivity between these tumor types. Evidence that GST
C. Topoisomerases In addition to P-gp and non-P-gp-mediated MDR, other mechanisms for resistance to multiple drugs have been described including frequent alterations of topoisomerase II (topo II) activity (Eijdems et al, 1985; Cole et al, 1991). Topo II is an ubiquous nuclear enzyme that is essential for many aspects of DNA function, including replication, recombination and transcription. There is evidence that this enzyme is the target of many clinically important antineoplastic drugs such as anthracyclines, ellipticines, amsacrines and epipodophyllotoxins (Dâ&#x20AC;&#x2122;Arpa and Liu, 1989; Zijlstra et al, 1990). These drugs stabilize the cleavable complex formed between topo II and DNA, resulting in increased DNA excision, detectable as DNA single-strand or double-strand
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Mattern: Cellular drug resistance in lung cancer breaks, and DNA-protein cross-links. Drug-induced cell destruction is proportional to the level of topo II, the more enzyme the greater the toxicity. This explains how a reduction in topo II could be a major mechanism of resistance to many antineoplastic drugs. Topo II has also been reported to play a role in cell proliferation. High levels of this enzyme are found in proliferating cells, and very low levels in quiescent cells (Zijlstra et al, 1990). In contrast, there are some reports that increased topo II is associated with resistance to certain DNA-damaging agents (Dingemans et al, 1999). It is also speculated that the increased affinity of topo II for cross-linked DNA in alkylating agents-resistant cells contribute to alkylator resistance by changing DNA topology, thereby facilitating DNA repair (Eder et al, 1995; Pu and Bezwoda, 1999). In surgical tissue samples of primary untreated lung tumors, significant intra- and intertumor variation in topo II expression has been observed. Topo II activity is higher in NSCLC as compared to breast cancer (McLeod et al, 1994). On the other hand, topo II activities of SCLC cell lines have been reported to be 2-fold higher than those for NSCLC cell lines, corresponding to their sensitivities to doxorubicin and etoposide (Kasahara et al, 1992). A correlation between topo II gene expression and sensitivity to doxorubicin, etoposide and cisplatin was also found in lung cancer cell lines not selected in vitro for drug resistance (Giaccone et al, 1992). Most lung cancer cell lines selected for resistance to doxorubicin demonstrate decreased levels of topo II expression in addition to P-gp overexpression (Eijdems et al, 1985). Therefore, low levels of topo II expression may predict reduced sensitivity of human lung cancer to several drugs. However, as with mdr1/P-gp expression, this cannot solely explain the drug-resistant phenotype of NSCLC.
SCLC cell lines to cisplatin (Kasahara et al, 1991). A transfected cell line that overexpresses MT proved not only resistant to cisplatin but also resistant to chlorambucil, melphalan and doxorubicin (Kelley et al, 1988). However, cells of various origins selected for cisplatin resistance often, but not always show increased MT expression, suggesting that an increased MT expression alone may not be the sole mediator of cisplatin resistance. Matsumoto et al, (1997) found that the proportion of MT-positive tumors was significantly higher in treated NSCLC compared with untreated NSCLC and treated SCLC, whereas Joseph et al, (2001) demonstrated that MT overexpression was predictive of short-term survival in patients with SCLC undergoing chemotherapy. In a study with human NSCLC, we found a significant relationship between MT expression and doxorubicin resistance in vitro (Mattern et al, 1992). Thus, a number of factors may be involved in the development of drug resistance in lung tumor cells and expression of MT may be one of them.
E.O6-alkylguanine-DNA alkyltransferase A number of DNA-damaging anticancer agents attack the O6 position on guanine, forming the potent cytotoxic DNA adduct. The DNA repair enzyme O6alkylguanine-DNA alkyltransferase (ATase), encoded by the gene MGMT, repairs alkylation at this site and is responsible for protecting tumor and normal cells from these agents. ATase activity varies widely among different organs, with lung tissue on average lower than others (Citron et al, 1991). However, ATase activity in normal peripheral lung tissue of smokers is significantly higher than that of nonsmokers (Drin et al, 1994; Mattern et al, 1998). Most human lung tumors contain amounts of ATase similar or greater than the tissue from which they originate (Kelley et al, 1988). In SCLC, ATase was found to be significantly lower than in NSCLC, but wide interindividual varaitions were observed (Oberli-Schrämmli et al, 1994). However, approximately 12% of human lung tumors are deficient in this enzyme (Citron et al, 1993). These ATase-deficient tumors are very sensitive to the cytotoxic effects of agents that alkylate the O6-position of guanine, such as nitrosoureas (Pegg, 1990). Thus, evidence suggesting a possible role for ATase in drug resistance of lung tumors comes from the following observations: (1) the level of ATase in tumor cells correlates well with the sensitivity to nitrosoureas (Brent et al, 1985), (2) transfection of the gene for alkyltransferase to ATase-deficient cells decreases the sensitivity to alkylating agents (Kaina et al, 1991), and (3) depletion of the activity of this enzyme by addition of O6benzylguanine significantly enhances toxicity (Dolan et al, 1993). Whether the ATase gene is a member of and coregulated with other stress-responsive genes and controlled by a common set of transcription factors remains to be elucidated.
D. Metallothioneins Metallothioneins (MTs) are intracellular proteins of low molecular weight (6-7 kDa) that are present in a wide variety of eukaryotes. MT are characterized by a high content of cysteine and the ability to bind heavy metal ions including zinc, copper, cadmium and platinum. The physiological function of MT is not well understood. Most mammalian tissues contain a basal level of MT, which may vary with the type of tissue. MT has also been demonstrated in a variety of malignancies including colorectal tumors (Ă&#x2013;fner et al, 1994), testicular germ cell tumors (Chin et al, 1993) and ovarian tumors (Murphy et al, 1991). MT in lung cancer tissue is significantly elevated when compared to nonmalignant lung tissue (Hart et al, 1993). The synthesis of MT is easily inducible in lung or other organs by certain hormones, cytokines, growth factors, tumor promotors and many other chemicals. Some stressful environmental conditions such as heat, cold and starvation also induce MT (Hamer, 1986). Recently, the synthesis of MT by tumor cells has been proposed as a possible mechanism for the intracellular inactivation of metal-containing chemotherapeutic agents such as cisplatin. MT content and MT mRNA levels correlated well with the sensitivity of 406
Cancer Therapy Vol 2, page 407 al, 1985; 1988; 1995). Thus, the determination of cell proliferation in clinical material provides a potentially useful marker to estimate sensitivity or resistance to anticancer drugs. Cell proliferation is regulated by both growthstimulatory and growth-inhibitory proteins (Sherr, 1993). Protein complexes that are composed of cyclins and cyclin-dependent kinases (cdks) are important factors for cellular proliferation. Cyclins are regulatory proteins for cdks and are differentially synthesized and degraded at specific points during the cell cycle (Cordon-Cardo, 1995). Five major classes of mammalian cyclins have been described (cyclin A-E). Cyclin C, D and E reach their peak of synthesis and activity during the G1 phase and regulate the transition from G1 to S-phase. Cyclins A and B achieve their maximum peaks during S- and G2-phases. We and others could demonstrate that cyclin A expression closely correlates with the proportion of S-phase cells measured by flow cytometry (Volm et al, 1997). Furthermore, patients with cyclin A-positive lung carcinomas had significantly shorter median survival times than patients with cyclin A-negative carcinomas. A significant correlation between expression of cyclin A and response of NSCLC to doxorubicin in vitro was also detected (Volm et al, 1997).
F. Thymidylate synthase Thymidylate synthase (TS) plays a central role in DNA biosynthesis and is the target of many chemotherapeutic agents, such as 5-fluorouracil, methotrexate and fluorodeoxyuridine (Washtien, 1982). Moreover, tumor cells resistant to cisplatin and doxorubicin display increased levels of this enzyme (Scanlon et al, 1988; Chu et al, 1991). Human NSCLC strongly express TS in a high percentage of cases (Volm and Mattern, 1992a; 1992b). This expression of TS is significantly related to doxorubicin resistance in vitro and associated with cross-resistance to 5-fluorouracil (Volm et al, 1979). Moreover, TS-positive lung tumors have been noted to be clinically progressive, the affected patients living a significantly shorter time than those with TSnegative tumors (Volm and Mattern, 1992a). In addition, evaluation of intratumoral TS activity accurately predicts responsiveness to 5-FU-based chemotherapy in NSCLC patients (Huang et al, 2000; Shintani et al, 2004). It has often been reported that the higher the TS level, the more resistant is the cell to antineoplastic drugs, in particular to 5-fluorouracil (Johnston et al, 1995). However, the biological relevance of TS relates not only to the importance of this enzyme as a chemotherapeutic target, but also as a DNA synthetic enzyme associated with cell division and proliferation (Stammler et al, 1995; Nakagawa et al, 2004). Nevertheless, high intrinsic levels of TS do not necessarily lead to higher proliferation rates than in cases with low levels of TS (Pestalozzi et al, 1995). A recent study has shown that TS protein binds to c-myc mRNA suggesting an involvement in the coordinate regulation of a number of other genes (Chu et al, 1994).
H. Hypoxia Because the rate of neovascularization frequently fails to keep pace with tumor growth, tumor vasculature is often inadequate for the tumor mass. Therefore, many solid tumors contain subpopulations of cells that are hypoxic and are relatively resistant to certain drugs (Teicher, 1994) and irradiation (HĂśckel et al, 1993). This is partly caused to poor vascularization that reduces the influx of cytostatic agents and lowers the levels of oxygen and nutrients. A growing body of evidence indicates that cells respond to hypoxic stress by altering the expression of specific genes or proteins (Wilson and Sutherland, 1989; Sutherland et al, 1996). Hypoxia is known to induce one or more transcription factors, the best characterized of which is hypoxia-inducible factor-1 (HIF-1), which in turn stimulates expression of several genes including those involved in drug resistance and endothelial cell growth. Hypoxia-induced resistance to doxorubicin and to methotrexate has been attributed to an amplification of the P-glycoprotein gene and the dihydrofolate reductase gene (Rice et al, 1986; 1987; Luk et et al, 1990; Kalra et al, 1993). Murphy et al, (1994) have recently shown that metallothionein IIA mRNA levels were significantly increased during hypoxia and during reoxygenation. Oâ&#x20AC;&#x2122;Dwyer et al, (1994) investigated the effects of hypoxia on the expression of a group of enzymes involved in drug metabolism. Exposing colon carcinoma cells to hypoxia resulted in a notably increased glutathione content. In a clinical study with NSCLC, it has been shown that poor vascularization, as measured by vessel density, correlates with an upregulation of glutathione Stransferase-!, metallothionein and thymidylate synthase (Koomägi et al, 1995). In another study involving rectal
G. Cell cycle-related proteins There exists general agreement that cancer chemotherapy is most successful when used on rapidly growing malignant cells (Valeriote and Van Putten, 1975). Experimental data obtained in a variety of systems ranging from mammalian cell cultures to transplanted rodent tumors show that proliferating cells are more sensitive to most cytotoxic agents than are resting cells (Drewinko et al, 1981). These experimental data are supported by the clinical observations that fast-growing tumors usually respond to treatment, whereas tumors with a low rate of proliferation very often show no response. To estimate the proliferative activity of cancer, various techniques including 3H-thymidine labeling (Alama et al, 1990) or flow-cytometric analysis (Volm et al, 1985; 1988) have been used. Several antibodies have also been produced that label preferentially the nuclei of proliferating (Volm et al, 1995a) and nonproliferating cells (Volm et al, 1995b). Although human lung tumors show a wide variation in proliferative activity and tumor doubling times, NSCLC have on average lower labeling indices and longer doubling times than SCLC, perhaps partly accounting for their resistance to cytotoxic drugs (Muggia, 1974; Arai et al, 1994). Moreover, patients whose lung tumors have a high proportion of cells in the S-phase generally die earlier than patients whose tumors have a low proportion of these cells (Alama et al, 1990; Volm et
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Mattern: Cellular drug resistance in lung cancer cancer, poor angiogenesis is also linked to an expression of glutathione S-transferase and metallothionein (Mattern et al, 1996). Moreover, lung tumors with low microvessel density and low VEGF expression were more frequently resistant to doxorubicin in vitro than tumors with high microvessel density and high expression of VEGF (Volm et al, 1996). These studies show that hypoxia or poor vascularization result in overexpression of certain detoxicating enzymes which provides an additional insight into cell resistance. Some studies have demonstrated the presence of drug resistance mechanisms in endothelial cells of normal and tumor tissue (Cordon-Cardo et al, 1990; Terrier et al, 1990). The MDR-associated P-glycoprotein and glutathione S-transferase have been localized in normal human endothelial cells and in the stroma of some tumors. Furthermore, Huang and Wright, (1994) found that some members of the fibroblast growth factor family, which are potent angiogenic peptides, may mediate resistance to some cytotoxic agents and modify gene amplification properties of tumor cells. Furthermore, there is experimental evidence that tumor cells and vascular endothelial cells within a solid tumor may stimulate each other by paracrine factors (Rak and Kerbel, 1996). On the basis of these studies, it seems reasonable to hypothesize that a highly vascularized tumor may produce elevated levels of angiogenic peptides that induce proliferation of chemoresistant endothelial cells which may confer tumor cell resistance to conventional anticancer therapy.
Sartorius and Krammer, 2002). However, several homologs of bcl-2 have been discovered, some of which function as inhibitors of cell death and others as promoters of apoptosis that oppose the actions of the bcl-2 protein. Thus, the role of bcl-2 as a clinically significant prognostic factor of drug resistance remained open (Martin et al, 2003). In a study with 85 human squamous cell lung carcinomas, we found a positive correlation between expression of bcl-2 and expression of the resistancerelated proteins P-gp and GST-!. Moreover, all bcl-2positive carcinomas were resistant to doxorubicin in an in vitro predictive test (Volm and Mattern, 1995). These results indicate that bcl-2 may contribute to drug resistance in NSCLC.
IV. Co-expression of resistance mechanisms and their putative regulation During the past few years it has become apparent that multiple mechanisms of resistance play a role in the clinical manifestaion of drug resistance. The study of drug resistance in lung cancer has not identified one single, specific mechanism as a major cause of the resistance observed in a clinical setting. There are now various reports that cell populations exist in human lung tumors which have several resistance mechanisms at once. The parallel assessment of drug resistance parameters in human tumors has shown that individual tumors exhibit different patterns: none, several or all of the monitored resistance markers are elevated. This indicates that each tumor has its own unique resistance factor profile. In lung tumors, Oberli-Schr채mmli et al, (1994) observed, in a majority of tumors, the concomitant overexpression of ATase and GSH-related parameters. In contrast, overexpression of ATase together with P-gp was never observed. There was no correlation between ATase and GSH or its enzymes in colorectal tumors (Redmond et al, 1991), however, ATase was frequently co-expressed with other drug resistance parameters in ovarain tumors (Joncourt et al, 1998). An increased expression of P-gp was detected not only concomitant to an overexpression of GST, but also accompanied by a coordinate overexpression of metallothionein and thymidylate synthase in human lung tumor (Volm and Mattern, 1992). A relationship exists between the extent of resistance measured in vitro and the number of detected resistance mechanisms. With an increasing extent of resistance, the number of resistance mechanisms increases (Volm et al, 1992). The reasons for the concomitant expression of different resistant mechanisms in human lung tumors are unknown. The increased expression of several resistance markers might be the result of induction of a cascade of resistance-related gene products triggered by chemotherapy or environmental factors. It was found from in vitro studies that NSCLC of smokers are more frequently resistant and express a higher degree of P-gp and GST-! than tumors of non-smokers (Volm et al, 1991). Thus, smoking may upregulate different detoxifying enzymes, depending on histopathological and clinicopathological variables, to protect the cells from
I. Programmed cell death The recent progress in the field of biology has indicated that programmed cell death (apoptosis) plays an important role in the chemotherapy-induced tumor cell killing. Since the different antineoplastic agents induce a similar pattern of cell death, it was suggested that a common pathway of apoptosis could exist in the druginduced apoptosis and the defect in the signaling pathway of apoptosis could cause a new form of multidrug resistance in tumor cells. Recent studies in human leukemia cells have demonstrated that chemosensitivity also depends on activation of caspases that are an integral part of the CD95 signaling pathway (Los et al, 1997). Inhibition of caspases not only retarded the apoptotic process but also provided protection from drug-induced death. In a study with NSCLC, caspase-3 expression correlated with a lower incidence of lymph node involvement and the median survival time was longer for patients with caspase-3-positive tumors than for those with caspase-3-negative tumors (Koom채gi and Volm, 2000). Thus, impairment in the protease effector phase of apoptosis may lead to chemoresistance against several anticancer drugs that is not due to other well-characterized resistance mechanisms such as overexpression of antiapoptotic bcl-2-related proteins or increased expression of P-gp (Friesen at al, 1997). Overproduction of bcl-2, a blocker of apoptosis, prevents cell death induced by nearly most anticancer drugs and radiation, thus contributing to treatment failures in patients with cancer (Miyashita and Reed, 1993;
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Cancer Therapy Vol 2, page 409 carcinogens but as a consequence render them resistant to drugs. The coordinate expression of different resistance mechanisms in the same tumor may explain why tumors are also resistant to drugs not involved in therapy and why a single marker, e.g. GST-!, may serve as a general marker for resistance and prognosis, irrespective of whether it is itself involved in the resistance mechanism (Mulder et al, 1995). Another explanation for the presence of different resistance mechanisms in human tumors is that tumors are mostly detected at a relatively late stage when they are already large and have metastasized. These tumors are for the most part hypoxic and the vascular network for supply of oxygen and nutrients is substantially lower (Mattern et al, 1996). In fact, it has been shown that various resistance parameters are upregulated in tumors with poor vascularization (Koom채gi et al, 1995) and that the reduced vascularization of tumors together with upregulated resistance-related proteins may represent an import contributing factor to the poor response to chemotherapy and irradiation. There are several hints that detoxifying systems may share common regulatory elements. One possibility is that the resistance factors present in human tumors belong to a set of genes that can be coordinately expressed to protect cells from injury and against different xenobiotics. Many oncogene products are implicated in the regulation of cellular proliferation and, because the growth rate of tumors is an important determinant for the response of tumors to chemotherapy, oncogenes might influence drug resistance by regulation of proliferative activity. It has been reported that c-fos is involved in growth control and cellular differentiation (Verma, 1986). The cfos protein is associated with the gene product of the proto-oncogene c-jun. The c-fos/c-jun protein complex binds specifically to a DNA sequence referred to as the AP-1 binding site and thereby affects the transcriptional expression of cellular genes (Sassone-Corsi et al, 1988). It has been also demonstrated that the promoter region of the Chinese hamster P-gp gene contains the AP-1 binding site and that this latter is essential for full promoter activity (Teeter et al, 1991). The promoter region of the genomic GST-! also contains an AP-1 motif, which suggests that this gene may be regulated by the cellular oncogenes c-fos and c-jun (Morrow et al, 1989). In a clinical study, surgical specimens of NSCLC of untreated patients were analyzed for expression of c-fos, c-jun and for resistance to doxorubicin. A significant association between drug resistance and expression of c-fos and c-jun proteins was found (Volm, 1993). With a c-fos-transfected cell line it was demonstrated that a ribozyme-mediated decrease in cfos expression was associated with reduced levels of thymidylate synthase, DNA polymerase " and metallothionein IIA (Scanlon et al, 1991). These results suggest that Fos may mediate DNA replication and repair processes through transcriptional activation of the aforementioned genes. A recent study has shown that thymidylate synthase protein binds to c-myc mRNA suggesting an involvement in the coordinate regulation of a number of other genes
(Chu et al, 1994). Whether the ATase gene is a member of and co-regulated with other stress-responsive genes and controlled by a common set of transcription factors remains to be elucidated.
V. Future directions The recent development of DNA microarray technology for large-scale analyses of gene expression has had a profound impact on biomedical research. Microarrays allow the simultaneous analysis of thousands of genes or proteins in a single experiment. Thus, it is not surprising that the old concept of prediction of drug response and individualized therapy is experiencing a revival. Staunton et al, (2001) determined whether the gene expression signatures of untreated cells are sufficient for the prediction of drug sensitivity. Using a panel of 60 human cancer cell lines, gene expression-based classifiers of sensitivity or resistance of 232 compounds were generated. They found that the accuracy of chemosensitivity prediction was considerably better than would be expected by chance. Kudoh et al, (2000) used the cDNA microarray to monitor the expression profiles of MCF-7 cells that are selected for resistance to doxorubicin. They found that a subset of genes was constitutively overexpressed in cells selected for resistance to doxorubicin. Ikehara et al, (2004) conducted a study with 47 human lung tumors (using cDNA microarray analysis) to determine whether expression levels of genes were correlated with survival after chemotherapy. They analyzed the expression levels of 1176 genes and found that three genes, G1/S-specific cyclin D2, type II cGMPdependent protein kinase and hepatocyte growth factorlike protein, were significantly correlated with survival. Wigle et al, (2002) performed expression profiling on tumor specimens from 39 NSCLC patients and could identify distinct profiles of gene expression correlating with disease-free survival. Significant technological advances in protein chemistry in the last decades have established mass spectrometry as a tool for protein study. The recently developed ProteinChip technology using surface enhanced laser desorption/ionisation (SELDI) mass spectrometry facilitate protein profiling of complex biological mixtures and could be used to discriminate e.g. normal vs. tumor tissues or treated vs. untreated cells. Preliminary results with this technology show that this method could be used to classify and predict histological subgroups as well as nodal involvement and survival in resected NSCLC (Yanagisawa et al, 2003; Zhukov et al, 2003). The promising aspect of all these new methods is the hope that it will be improve the ability to identify those patients who are at high risk of failing therapy.
VI. Conclusions The data obtained from multiple sources, including in vitro testing of lung tumors, determination of resistancerelated enzymes and patient response indicate that no single, specific currently known drug resistance mechanism can explain the drug resistance and poor
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Mattern: Cellular drug resistance in lung cancer cyclohexyl)-1-nitrosourea. Proc Natl Acad Sci 82, 29852989. Carmichael J, Mitchel JB, De Graff WG, Gamson J, Gazdar AF, Johnson BE, Glatstein E, and Minna JD (1985) Chemosensitivity testing of human lung cancer cell lines using the MTT assay. Br J Cancer 57, 540-547. Carmichael J, Mitchell JB, Friedman N, Gazdar AF, and Russo A (1988) Glutathione and related enzyme activity in human lung cancer cell lines. Br J Cancer 58, 437-440. Chin JL, Banerjee D, Kadhim SA, Kontozoglou TE, Chauvin PJ, and Cherian MG (1993) Metallothionein in testicular germ cell tumors and drug resistance. Cancer 72, 3029-3035. Chu E, Voeller DM, Jones KL, Takechi T, Maley GF, Maley F, Segal S, and Allegra CJ (1994) Identification of a thymidylate synthase ribonucleoprotein complex in human colon cancer cells. Mol Cell Biol 14, 207-213. Citron M, Decker R, Chen S, Schneider S, Graver M, Kleynerman L, Kahn LB, White A, Schoenhaus M, and Yarosh D (1991) O6-methylguanine-DNA methyltransferase in human normal and tumor tissue from brain, lung, and ovary. Cancer Res 51, 4131-4134. Citron M, Schoenhaus M, Graver M, Hoffman M, Lewis M, Wasserman P, Niederland M, Kahn L, White A, and Yarosh D (1993) O6-methylguanine-DNA methyltransferase in human normal and malignant lung tissue. Cancer Invest 11, 258-263. Clapper ML, Hoffman SJ, Carp N, Watts P, Seestaller LM, Weese JL, and Tew KD (1991) Contribution of patient history to the glutathione S-transferase activity of human lung, breast and colon tissue. Carcinogenesis 12, 1957-1961. Cole SP, Chanda ER, Dicke FP, Gerlach JH, and Mirski SEL (1991) Non-P-glycoprotein-mediated multidrug resistance in a small cell lung cancer cell line: Evidence for decreased susceptibility to drug-induced DNA damage and reduced levels of topoisomerase II. Cancer Res 51, 3345-3352. Cole SPC, Bhardwaj G, Gerlach JH, Mackie JE, Grant CE, Almquist KC, Stewart AJ, Kurz EU, Duncan AMV, and Deeley RG (1992) Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line. Science 258, 1650-1654. Cordon-Cardo C (1995) Mutation of cell cycle regulators. Biological and clinical implications for human neoplasia (review). Am J Pathol 147, 545-560. Cordon-Cardo C, Oâ&#x20AC;&#x2122;Brien JP, Boccia J, Casals D, Bertino JR, and Melaned MR (1990) Expression of the multidrug resistance gene product (P-glycoprotein) in human normal and tumor tissues. J Histochem Cytochem 38, 1277-1287. Dâ&#x20AC;&#x2122;Arpa P, and Liu LF (1989) Topoisomerase-targeting antitumor drugs. Biochim Biophys Acta 989, 163-177. Di Ilio C, Del Boccio G, Aceto A, Casaccia R, Mucilli F, and Federici G (1988) Elevation of glutathione transferase activity in human lung tumor. Carcinogenesis 9, 335-340. Dingemans ACM, Van Ark-Otte J, Van der Valk P, Apolinario RM, Scheper RJ, Postmus PE, and Giaccone G (1996) Expression of the human major vault protein LRP in human lung cancer samples and normal lung tissues. Ann Oncol 7, 625-630. Dingemans AM, Witlox MA, Stallaert RA, van der Valk P, Postmus PE, and Giaccone G (1999) Expression of DNA topoisomerase II alpha and topoisomerase II beta genes predicts survival and response to chemotherapy in patients with small cell lung cancer. Clin Cancer Res 5, 2048-2058. Dolan ME, Pegg AE, Moschel RC, and Grindey GB ( 1993) Effect of benzylguanine on the sensitivity of human colon tumor xenografts to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Biochem Pharmacol 46, 285-290. Drewinko B, Patchen M, Yang LY, and Barlogie B (1981) Differential killing efficacy of twenty antitumor drugs on
prognosis of patients with lung cancer. The mechanisms of resistance are numerous and depend on the detoxifying capacity of the cells, tissue-specific factors, repair capacity, drug delivery, cell proliferation and many others. Also mutations or amplification of specific genes involved in protective pathways or mutations of different oncogenes or suppressor genes may be responsible for the rsistance to chemotherapeutic drugs. Thus, the phenotype of resistance had to be understood as the net effect of a multifactorial process of a panel of resistance genes controlling an array of alternative resistance mechanisms (Stein, 2000). Consequently, clinical reversal of drug resistance may ultimately require intervention at several different sites in the tumor cell, ranging from blocking efflux pumps to inhibition of cytosolic detoxification enzymes and to inhibition of DNA repair. A key challenge for the future is to determine the relative quantitative contributions of each of these mechanisms to the drug-resistant phenotype. The recent development of DNA microarray technology for large-scale analysis of gene expression makes it possible to identify gene expression profiles in tumor cells which correlate with the treatment responsiveness such as drug resistance and clinical outcome of the disease. This could be of importance for better understanding of the biological behavior of the tumors and for better planning of treatment leading to a more rational, perhaps individualized choice of therapy.
References Abe Y, Nakamura M, Ota E, Ozeki Y, Tamai S, Inooue H, Ueyama Y, Ogata T, and Tamaoki N (1994) Expression of the multidrug resistance gene (mdr1) in non-small cell lung cancer. Jpn J Cancer Res 85, 536-541. Alama A, Constantini M, Repetto L, Conte PF, Serrano J, Nicolin A, Barbieri F, Ardizzoni A, and Bruzzi P (1990) Thymidin labelling index as prognostic factor in resected non-small cell lung cancer. Eur J Cancer 26, 622-625. Anttila S, Hirvonen A, Vainio H, Husgafvel-Pursiainen K, Hayes JD, and Ketterer B (1993) Immunohistochemical localization of glutathione S-transferases in human lung. Cancer Res 53, 5643-5648. Arai T, Kuroishi T, Saito Y, Kurita Y, Naruke T, Kaneko M, and Japanese Lung Cancer Screening Research Group (1994) Tumor doubling time and prognosis in lung cancer patients: Evaluation from chest films and clinical follow-up study. Jpn J Clin Oncol 24, 199-204. Awasthi YC, Singh SV, Ahmad H, and Moller PC (1987) Immunohistochemical evidence for the expression of GST1, GST2, and GST3 gene loci for glutathione S-transferase in human lung. Lung 165, 323-332. Berger W, Elbling L and Micksche M (2000) Expression of the major vault protein LRP in human non-small cell lung cancer cells: activation by short-term exposure to antineoplastic drugs. Int J Cancer 88, 293-300. Borst P, Evers R, Kool M, and Wijnholds J (1999) The multidrug resistance protein family. Biochim Biophys Acta 1461, 347357. Bradley G, Juranka PF, and Ling V (1988) Mechanisms of multidrug resistance. Biochim Biophys Acta 948, 87-128. Brent TP, Houghton PJ, and Houghton JA (1985) O6-alkyl DNA methyltransferase activity correlates with the therapeutic response of human rhabdomyosarcoma xenografts to 1-(2-chloroethyl)-3-(trans-4-methyl-
410
Cancer Therapy Vol 2, page 411 proliferating and nonproliferating human tumor cells. Cancer Res 41, 2328-2333. Drin I, Schoket B, Kostic S, and Vincze I (1994) Smokingrelated increase in O6-alkyl -DNA methyltransferase activity in human lung tissue. Carcinogenesis 15, 1535-1539. Eder JP, Chan VT-W, Ng S-W, Rizvi NA, Zacharoulis S, Teicher BA, and Schnipper LE (1995) DNA topoisomerase II # expression is associated with alkylating agent resistance. Cancer Res 55, 6109-6116. Eijdems EWHM, De Haas M, Timmermann AJ, Van der Schans GP, Kamst E, De Nooji J, Astaldi-Ricotti GCB, Borst P, and Baas F (1985) Reduced topoisomerase I activity in multidrug-resistant human non-small cell lung cancer cell lines. Br J Cancer 71, 40-47. Fojo AT, Ueda K, Slamon DJ, Poplack DG, Gottesman MM, and Pastan I (1987) Expression of multidrug-resistance gene in human tumors and tissues. Proc Natl Acad Sci 84, 265-269. Friesen C, Fulda S, and Debatin KM (1997) Deficient activation of the CD95 (APO-1/Fas) system in drug-resistant cells. Leukemia 11, 1833-1841. Giaccone G, Gazdar AF, Beck H, Zunino F, and Capranico G (1992) Multidrug sensitivity phenotype of human lung cancer cells associated with topoisomerase II expression. Cancer Res 52, 1666-1674. Goldstein LJ, Galski H, Fojo A, Willingham M, Lai SL, Gazdar A, Pirker R, Green A, Crist W, Brodeur GM, Lieber M, Cossman J, Gottesman MM, and Pastan I. J Natl Cancer Inst 81, 116-124. Gros P, Neriah YB, Croop JM, and Housman DE (1986) Isolation and expression of a complementary DNA that confers multidrug resistance. Nature 323, 728-731. Hamburger AW, and Salmon SE (1977) Primary bioassay of human tumor stem cells. Science 197, 461-463 Hamer DH (1986) Metallothionein. Annu Rev Biochem 55, 913-951. Hart BA, Voss GW, and Vacek PM (1993) Metallothionein in human lung carcinoma. Cancer Lett 75, 121-128. Haugen H (2000) Etiology of lung cancer. In: Hansen HH, ed. Textbook of lung cancer. London: Martin Dunitz, pp. 1-12. Herzog CE, Trepel JB, Mickley LA, Bates SE, and Fojo AT (1992) Various methods of analysis of mdr1/P-glycoprotein in human colon cancer cell lines. J Natl Cancer Inst 84, 711-716. Höckel M, Vorndran B, Schlenger K, Baussmann E, and Knapstein PG (1993) Tumor oxygenation: a new predictive parameter in locally advanced cancer of the uterine cervix. Gynecol Oncol 51, 141-149. Huang A, and Wright JA (1994) Fibroblast growth factor mediated alterations in drug resistance and evidence of gene amplification. Oncogene 9, 491-499. Huang CL, Yokomise H, Kobayashi S, Fukushima M, Hitomi S, and Wada H (2000) Intratumoral expression of thymidylate synthase and dihydropyrimidine dehydrogenase in non-small cell lung cancer patients treated with 5-FU-based chemotherapy. Int J Oncol 17, 47-54. Ikehara M, Oshita F, Sekiyama A, Hamanaka N, Saito H, Yamada K, Noda K, Kameda Y, and Miyagi Y (2004) Genome-wide cDNA microarray screening to correlate gene expression profile with survival in patients with advanced lung cancer. Oncol Rep 11, 1041-1044. Ishikawa T, Kuo MT, Furuta K and Suzuki M (2000) The human multidrug resistance-associated protein (MRP) gene family: from biological function to drug molecular design. Clin Chem Lab Med 38, 893-897. Joncourt F, Buser K, Altermatt HJ, Bacchi M, Oberli A, and Cerny T (1998) Multiple drug resistance parameter expression in ovarian cancer. Gynecol Oncol 70, 176-182.
Joseph MG, Banerjee D, Kocha W, Feld R, Stitt LW, and Cherian MG (2001) Metallothionein expression in patients with small cell carcinoma of the lung: correlation with other molecular markers and clinical outcome. Cancer 92, 836842. Kaina B, Fritz G, Mitra S, and Coquerelle T (1991) Transfection and expression of human O6-methylguanine-DNA methyltransferase (MGMT) cDNA in Chinese hamster cells: The role of MGMT in protection against the genotoxic effects of alkylating agents. Carcinogenesis 12, 1857-1867. Kakolyris S, Giatromanolaki A, Koukourakis M, Powis G, Souglakos J, Sivridis E, Georgoulias V, Gatter KC, and Harris AL (2001) Thioredoxin expression is associated with lymph node status and prognosis in early operable non-small cell lung cancer. Clin Cancer Res 7, 3087-3091. Kalra R, Jones AM, Kirk J, Adams GE, and Stratford IJ (1993) The effect of hypoxia on acquired drug resistance and response to epidermal growth factor in Chinese hamster lung fibroblasts and human breast-cancer cells in vitro. Int J Cancer 54, 650-655. Kasahara K, Fujiwara Y, Nishio K, Ohmori T, Sugimoto Y, Komiya K, Matsuda T, and Saijo N (1991) Metallothionein content correlates with the sensitivity of human small cell lung cancer cell lines to cisplatin. Cancer Res 51, 32373242. Kasahara K, Fujiwara Y, Sugimoto Y, Nishio K, Tamura T, Matsuda T, and Saijo N (1992) Determinants of response to the DNA topoisomerase II inhibitors doxorubicin and etoposide in human lung cancer cell lines. Cancer Res 84, 113-118. Kelley SL, Basu A, Teicher BA, Hacker MP, Hamer DH, and Lazo JS (1988) Overexpression of metallothionein confers resistance to anticancer drugs. Science 241, 1813-1815. Kinnula VL, Paakko P, and Soini Y (2004) Antioxidant enzymes and redox regulating thiol proteins in malignancies of human lung. FEBS Lett 569, 1-6. Koomägi R, and Volm M (2000) Relationship between the expression of caspase-3 and the clinical outcome of patients with non-small cell lung cancer. Anticancer Res 20, 493496. Koomägi R, Mattern J, and Volm M (1995) Up-regulation of resistance-related proteins in human lung tumors with poor vascularization. Carcinogenesis 16, 2129-2133. Kudoh K, Ramanna M, Ravatn R, Elkahloun AG, Bittner ML, Meltzer PS, Trent JM, Dalton WS, and Chin KV (2000) Monitoring the expression profiles of doxorubicin-induced and doxorubicin-resistant cancer cells by cDNA microarray. Cancer Res 60, 4161-4166. Lai SL, Goldstein LJ, Gottesman MM, Pastan I, Tsai CM, Johnson BE, Mulshine JL, Ihde DC, Kayser K, and Gazdar A (1989) MDR1 gene expression in lung cancer. J Natl Cancer Inst 81, 1144-1150. Lehtonen ST, Svensk AM, Soini Y, Paakko P, Hirvikoski P, Kang SW, Saily M, and Kinnula VL (2004) Peroxiredoxins, a novel protein family in lung cancer. Int J Cancer 111, 514-521. Linsenmeyer ME, Jefferson W, Wolf M, Matthews JP, Board PG, and Woodcock DM (1992) Levels of expression of the mdr1 gene and glutathione S-transferase genes 2 and 3 and response to chemotherapy in multiple myoloma. Br J Cancer 65, 471-475. Los M, Herr I, Friesen C, Fulda S, Schulze-Osthoff K, and Debatin KM (1997) Cross-resistance of CD95- and druginduced apoptosis as a consequence of deficient activation of caspases (ICE/Ced-3 proteases). Blood 90, 3118-3129. Luk CK, Veinot-Drebot L, Tjan E, and Tannock IF (1990) Effect of transient hypoxia on sensitivity to doxorubicin in human and murine cell lines. J Natl Cancer Inst 82, 684-692.
411
Mattern: Cellular drug resistance in lung cancer Martin B, Paesmans M, Berghmans T, Branle F, Ghisdal L, Mascaux C, Meert A-P, Steels E, Vallot F, Verdebout J-M, Lafitte J-J, and Sculier J-P (2003) Role of bcl-2 as a prognostic factor for survival in lung cancer: a systematic review of the literature with meta-analysis. Br J Cancer 89, 55-64. Matsumoto Y, Oka M, Sakamoto A, Narasaki F, Fukuda M, Takatani H, Terashi K, Ikeda K, Tsurutani J, Nagashima S, Soda H, and Kohno S (1997) Enhanced expression of metallothionein in human non-small cell lung carcinomas following chemotherapy. Anticancer Res 17, 3777-3780. Mattern J, and Volm M (1982) Clinical relevance of predictive tests for cancer chemotherapy. Cancer Treat Rev 9, 267298. Mattern J, and Volm M (1992) Increased resistance to doxorubicin in human non-small cell lung carcinomas with metallothionein expression. Int J Oncol 1, 687-689. Mattern J, Bak M, and Volm M (1987) Occurrence of a multidrug-resistant phenotype in human lung xenografts. Br J Cancer 56, 407-411. Mattern J, Bak M, Hahn EW, and Volm M (1988) Human tumor xenografts as model for drug testing. Cancer Metastasis Rev 7, 263-284. Mattern J, Kallinowski F, Herfarth C, and Volm M (1996) Association of resistance-related protein expression with poor vascularization and low levels of oxygen in human rectal cancer. Int J Cancer 67, 20-23. Mattern J, Wayss K, and Volm M (1984) Effect of five antineoplastic agents on tumor xenografts with different growth rates. J Natl Cancer Inst 72, 1335-1339. Mattern J, Koomägi R, and Volm M (1999) Smoking-related increase of O6-methylguanine-DNA methyltransferase expression in human lung carcinomas. Carcinogenesis 19, 1247-1250. Mattern J, Koomägi R, and Volm M (2002) Characteristics of long-term survivors of untreated lung cancer. Lung Cancer 36, 277-282. McLeod HL, Douglas F, Oates M, Symond RP, Prakash D, Van der Zee AGJ, Kaye SB, Brown R, and Keith WN (1994) Topoisomerase I and II activity in human breast, cervix, lung and colon cancer. Int J Cancer 59, 607-611. Morrow CS, Cowan KH, and Goldsmith ME (1989) Structure of the human genomic glutathione S-transferase ! gene. Gene 75, 3-11. Muggia FM (1974) Cell kinetic studies in patients with lung cancer. Oncology 30, 353-361. Mulder TPJ, Verspaget HW, Sier CFM, Roelof HMJ, Ganesh S, Griffioen G, and Peters WHM (1995) Glutathione Stransferase ! in colorectal tumors is predictive for overall survival. Cancer Res 55, 2696-2702. Murphy BJ, Laderoute KR, Chin RJ, and Sutherland RM (1994) Metallothionein IIA is up-regulated by hypoxia in human A431 squamous carcinoma cells. Cancer Res 54, 58085810. Murphy D, McGown AT, Crowther D, Mander A, and Fox BW (1991) Metallothionein levels in ovarian tumours before and after chemotherapy. Br J Cancer 63, 711-714. Miyashita T, and Reed JC (1993) Bcl-2 oncoprotein blocks chemotherapy-induced apoptosis in a human leukemia cell line. Blood 81, 151-157. Nakagawa K, Saijo N, Tsuchidas S, Sakai M, Tsunokawa Y, Yokota J, Muramatsu M, Sato K, Tereda M, and Tew KD (1990) Glutathione S-transferase-! as a determinant of drug resistance in transfectant cell lines. J Biol Chem 265, 42964301. Nakagawa T, Otake Y, Yanagihara K, Miyahara R, Ishikawa S, Fukushima M, Wada H, and Tanaka F (2004) Expression of thymidylate synthase is correlated with proliferative activity
in non-small cell lung cancer (NSCLC). Lung Cancer 43, 145-149. Nooter K, Bosman FT, Burger H, Van Wingerden KE, Flens MJ, Scheper RJ, Oostrum RG, Boersma AWM, Van der Gaast A, and Stoter G (1996) Expression of the multidrug resistance associated protein (MRP) gene in primary non-small cell lung cancer. Ann Oncol 7, 75-81. O’Dwyer PJ, Yao KS, Ford P, Godwin AK, and Clayton M (1994) Effects of hypoxia on detoxicating enzyme activity and expression in HT29 colon adenocarcinoma cells. Cancer Res 54, 3082-3087. Oberli-Schrämmli AE, Joncourt F, Stadler M, Altermatt HJ, Buser K, Ris HB, Schmid U, and Cerny T (1994) Parallel assessment of glutathione-based detoxifying enzymes, O6alkyl -DNA methyltransferase and P-glycoprotein as indicators of drug resistance in tumor and normal lung of patients with lung cancer. Int J Cancer 59, 629-636. Öfner D, Maier H, Riedmann B, Bammer T, Rumer A, Winde G, Böcker W, Jasan B, and Schmid KW (1994) Immunohistochemical metallothionein expression in colorectal adenocarcinoma: Correlation with tumour stage and patient survival. Virchows Arch 425, 491-497. Ota E, Abe Y, Oshika Y, Ozeki Y, Iwasaki M, Inoue H, Yamazaki H, Ueyama Y, Takagi K, and Ogata T (1995) Expression of the multidrug resistance-associated protein (MRP) gene in non-small cell lung cancer. Br J Cancer 72, 550-554. Pu QQ, and Bezwoda WR (1999) Induction of alkylator (melphalan) resistance in HL60 cells is accompanied by increased levels of topoisomerase II expression and function. Mol Pharmacol 56, 147-153. Radosevich JA, Robinson PG, Rittmann-Grauer LS, Wilson B, Leung JP, Maminta ML, Warren W, Rosen S, and Gould VE (1989) Immunohistochemical analysis of pulmonary and pleural tumors with the monoclonal antibody HYB-612 directed against the multidrug resistance (MDR-1) gene product, P-glycoprotein. Tumor Biol 10, 252-257. Rak JW, and Kerbel RS (1996) Reciprocal paracrine interactions between tumor cells and endothelial cells: The ‚angiogenesis progression‘ hypothesis. Eur J Cancer 32A, 2438-2450. Redmond SMS, Joncourt F, Buser K, Ziemiecki A, Altermatt HJ, Fey M, Margison G, and Cerny T (1991) Assessment of Pglycoprotein, glutathione-based detoxifying enzymes and O6-alkylguanine-DNA alkyltransferase as potential indicators of constitutive drug resistance in human colorectal tumors. Cancer Res 51, 2092-2097. Rice GC, Hoy C, and Schimke RT (1986) Transient hypoxia enhances the frequency of dihydrofolate reductase gene amplification in Chinese hamster ovary cells. Proc Natl Acad Sci 83, 5978-5982. Rice GC, Ling V, and Schimke RT (1987) Frequencies of independent and simultaneous selection of Chinese hamster cells for methotrexate and doxorubicin (adriamycin) resistance. Proc Natl Acad Sci 84, 9261-9264. Richardson GE, and Johnson BE (1993) The biology of lung cancer. Semin Oncol 20, 105-127. Ruckdeschel JC, Carney DN, Oie HK, Russel EK, and Gazdar AF (1987) In vitro chemosensitivity of human lung cancer cell lines. Cancer Treat Rep 71, 697-704. Sartorius UA, and Krammer PH (2002) Upregulation of bcl-2 is involved in the mediation of chemotherapy resistance in human small cell lung cancer cell lines. Int J Cancer 97, 584-592. Sassone-Corsi P, Lamph HW, Kamps M, and Verma IM (1988) Fos-associated cellular p39 is related to nuclear transcription factor AP-1. Cell 54, 553-563. Scanlon KJ, Jiao L, Wang W, Tone T, Rossi JJ, and KashaniSabet M (1991) Ribozyme-mediated cleavage of c-fos
412
Cancer Therapy Vol 2, page 413 mRNA reduces gene expression of DNA synthesis enzymes and metallothionein. Proc Natl Acad Sci 88, 10591-10595. Scheper RJ, Broxtermann HJ, Scheffer GL, Kaaijk P, Dalton WS, Van Heijningen THM, Van Kalken CK, Slovak ML, De Vries EGE, Van der Kalk P, Meijer CJLM, and Pinedo HM (1993) Overexpression of a Mr 110,000 vesicular protein in non-P-glycoprotein-mediated multidrug resistance. Cancer Res 53, 1475-1479. Sharma R, Singhal SS, Srivastava SK, Bajpai KK, Frenkel EP, and Awasthi S (1993) Glutathione and glutathione linked enzymes in human small cell lung cancer cell lines. Cancer Lett 75, 111-119. Sherr CJ (1993) Mammalian G1 cyclins. Cell 73, 1059-1065. Shin HJC, Lee JS, Hong WK, and Shin DM (1992) Study of multidrug resistance (mdr1) gene in non-small cell lung cancer. Anticancer Res 12, 367-370. Shintani Y, Ohta M, Hirabayashi H, Tanaka H, Iuchi K, Nakagawa K, Maeda H, Kido T, Miyoshi S, and Matsuda H (2004) Thymidylate synthase and dihydropyrimidine dehydrogenase mRNA levels in tumor tissues and the efficacy of 5-fluorouracil in patients with non-small cell lung cancer. Lung Cancer 45, 189-196. Soini Y, Kahlos K, Napankangas U, Kaarteenaho-Wiik R, Saily M, Koistinen P, Paakko P, Holmgren A, and Kinnula VL (2001) Widespread expression of thioredoxin and thioredoxin reductase in non-small cell lung carcinoma. Clin Cancer Res 7, 1750-1757. Staunton JE, Slonim DK, Coller HA, Tamayo P, Angelo MJ, Park J, Scherf U, Lee JK, Reinhold WO, Weinstein JN, Mesirov JP, Lander ES, and Golub TR (2001) Chemosensitivity prediction by transcriptional profiling. Proc Natl Acad Sci 98, 10787-10792. Stein US (2000) P-glycoprotein turned twenty: are we any closer to fight drug resistance in cancer? Onkologie 23, 316-317. Sutherland RM, Ausserer WA, Murphy BJ, and Laderoute KR (1996) Tumor hypoxia and heterogeneity: challenges and opportunities for the future. Sem Radiat Oncol 6, 59-70. Teeter LD, Eckersberg T, Tsai Y, and Kuo MT (1991) Analysis of the Chinese hamster P-glycoprotein/multidrug resistance gene pgp1 reveals that the AP-1 site is essential for full promoter activity. Cell Growth Differ 2, 429-437. Teicher BA (1994) Hypoxia and drug resistance. Cancer Metastasis Rev 13, 139-168. Terrier P, Townsend AJ, Coindre JM, Triche TJ, and Cowan KM (1990) An immuno-histochemical study of Pi class glutathione S-transferase expression in normal human tissues. Am J Pathol 137, 845-853. Tew KD (1994) Glutathione-associated enzymes in anticancer drug resistance. Cancer Res 54, 4313-4320. Twentyman PR, Fox NE, Wright KA, and Bleehen NM (1986) Derivation and preliminary characterisation of Adriamycin resistant cell lines of human lung cancer cells. Br J Cancer 53, 529-537. Valeriote F, and Van Putten L (1975) Proliferation-dependent cytotoxicity of anticancer agents: A review. Cancer Res 3, 2619-2630. Van der Kolk DM, Vellenga E, Muller M and de Vries EG (1999) Multidrug resistance protein MRP1, glutathione, and related enzymes. Their importance in acute myeloid leukemia. Adv Exp Med Biol 457, 187-198. Verma IM (1986) Protooncogene fos: a multifaceted gene. Trends Genet 2, 93-96. Volm M (1993) P-glycoprotein associated expression of c-fos and c-jun products in human lung carcinomas. Anticancer Res 13, 375-378. Volm M, and Mattern J (1992a) Elevated expression of thymidylate synthase in doxorubicin resistant non-small cell lung carcinomas. Anticancer Res 12, 2293-2296.
Volm M, and Mattern J (1992b) Expression of topoisomerase II, catalase, metallothionein and thymidylate synthase in human squamous cell lung carcinomas and their correlation with doxorubicin resistance and with patients‘ smoking habits. Carcinogenesis 13, 1947-1950. Volm M, and Mattern J (1995) Increased expression of bcl-2 in drug-resistant squamous cell lung carcinomas. Int J Oncol 7, 1333-1338. Volm M, Wayss K, Kaufmann M, and Mattern J (1979) Pretherapeutic detection of tumor resistance and the results of tumor chemotherapy. Eur J Cancer 15, 983-993. Volm M, Hahn EW, Mattern J, Müller T, Vogt-Schaden I, and Weber E (1988) Five-year follow-up study of independent clinical and flow cytometric prognostic factors for the survival of patients with non-small cell lung carcinoma. Cancer Res 48, 2923-2928. Volm M, Hecker S, Sauerbrey A, and Mattern J (1995a) Predictive value of statin, a Go-associated cell cycle protein, in childhood acute lymphoblastic leukemia. Int J Cancer (Pred Oncol) 64, 166-170. Volm M, Koomägi R, and Mattern J (1995b) Prognostic significance of proliferating cell nuclear antigen (PCNA) in adenocarcinoma of the lung. Int J Oncol 6, 359-362. Volm M, Koomägi R, and Mattern J (1996) Interrelationship between microvessel density, expression of VEGF and resistance to doxorubicin in non-small cell lung carcinoma. Anticancer Res 16, 213-218. Volm M, Koomägi R, Mattern J, and Stammler G (1997) Cyclin A is associated with an unfavourable outcome in patients with non-small cell lung carcinomas. Br J Cancer 75, 17741778. Volm M, Mattern J, and Samsel B (1991) Overexpression of Pglycoprotein and glutathione S-transferase-! in resistant nonsmall cell lung carcinomas of smokers. Br J Cancer 64, 700704. Volm M, Mattern J, Efferth T, and Pommerenke EW (1992) Expression of several resistance mechanisms in untreated human kidney and lung carcinomas. Anticancer Res 12, 1063-1068. Volm M, Mattern J, Sonka J, Vogt-Schaden M, and Wayss K (1985) DNA distribution in non-small cell lung carcinomas and ist relationship to clinical behavior. Cytometry 6, 348356. Weisenthal LM, Marsden JA, Dill PL, and Macaluso CK (1983) A novel dye exclusion method for testing in vitro chemosensitivity of human tumors. Cancer Res 43, 749-757 Whelan RDH, Hosking LK, Townsend AJ, Cowan KH, and Hill BT (1989) Differential increases in glutathione S-transferase activities in a range of multidrug-resistant human tumor cell lines. Cancer Commun 1, 359-365. Wigle DA, Jurisica I, Radulovich N, Pintilie M, Rossant J, Liu N, Lu C, Woodgett J, Seiden I, Johnston M, Keshavjee S, Darling G, Winton T, Breitkreutz BJ, Jorgenson P, Tyers M, Shepherd FA, and Tsao MS (2002) Molecular profiling of non-small cell lung cancer and correlation with disease-free survival. Cancer Res 62, 3005-3008. Wilson RE, and Sutherland RM (1989) Enhanced synthesis of specific proteins, RNA and DNA caused by hypoxia and reoxygenation. Int J Radiat Oncol Biol Phys 16, 957-961. Yanagisawa K, Shyr Y, Xu BJ, Massion PP, Larsen PH, White BC, Roberts JR, Edgerton M, Gonzalez A, Nadaf S, Moore JH, Caprioli RM, and Carbone DP (2003) Proteomic patterns of tumour subsets in non-small cell lung cancer. The Lancet 362, 433-439. Yoshida M, Suzuki T, Komiya T, Hatashita E, Nishio K, Kazuhiko N, and Fukuoka M (2001) Induction of MRP5 and SMRP mRNA by adriamycin exposure and its
413
Mattern: Cellular drug resistance in lung cancer overexpression in human lung cancer cells resistant to adriamycin. Int J Cancer 94, 432-437. Zijlstra JG, De Jongs S, De Vries EGE, and Mulder NH (1990) Topoisomerases, new targets in cancer chemotherapy. Med Oncol Tumor Pharmacother 7, 11-18. Zhukov TA, Johanson RA, Cantor AB, Clark RA, and Tockman MS (2003) Discovery of distinct protein profiles specific for lung tumors and pre-malignant lung lesions by SELDI mass spectrometry. Lung Cancer 40, 267-279.
Dr. J端rgen Mattern
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Cancer Therapy Vol 2, page 415 Cancer Therapy Vol 2, 415-422, 2004
Salted meat consumption and risk of squamous cell carcinoma of the oesophagus: a case-control study in Uruguay Research Article
Eduardo De Stefani1*, Paolo Boffetta2, Pelayo Correa3, Hugo Deneo-Pellegrini1, Mar£a Mendilaharsu1 and Alvaro L. Ronco4 1
Departamento de Patolog£a, Instituto Nacional de Oncolog£a, Montevideo, Uruguay. International Agency for Research on Cancer, Lyon, France. 3 Department of Pathology, Louisiana State University Health Sciences, New Orleans, Louisiana, USA. 4 Divisi!n de Epidemiolog£a, Instituto de Radiolog£a y Centro de Lucha contra el C"ncer, Hospital Pereira Rossell, Montevideo, Uruguay 2
__________________________________________________________________________________ *Correspondence: Dr. Eduardo De Stefani, Avenida Brasil 3080 dep. 402, Montevideo, Uruguay; Tel.: (598) 2 708 23 14; Fax: (598) 2 402 08 10; E-Mail: estefani@adinet.com.uy Key words: oesophageal cancer, salted meat, white meat, boiled meat, nitrosamines, salt
Supported by grant from International Agency for Research on Cancer, Lyon, France Received: 15 October 2004; Accepted: 22 October 2004; electronically published: November 2004
Summary A case-control study was conducted in Montevideo, Uruguay in the time period 1996-2003, in order to elucidate the role of meat consumption in oesophageal carcinogenesis. The study included 200 cases with squamous cell oesophageal carcinoma and 400 age- and sex-matched controls. Salted meat consumption was significantly associated with an increased risk for this malignancy (OR 2.34, 95 % CI 1.21-4.53). On the other hand, high intake of white meat was inversely associated with risk of oesophageal cancer (OR 0.55, 95 % CI 0.36-0.84). Also boiled meat, frequent component of stews, increased the risk of oesophageal cancer. The remaining types of meat were not related with the risk of squamous cell carcinoma of oesophagus. This study suffered of a low statistical power since it included only 82 cases (67 men and 15 women). Since then our database has been considerably enlarged. Also, our previous study compared the risks of different types of meat categorized in tertiles. This comparison was not entirely adequate, since different amounts of intake were used for it. In fact, for a reliable comparison it is necessary to calculate all OR using the same fixed amount of intake. We decided to conduct a new case-control study on the relationship between meat consumption and risk of squamous cell carcinoma of the oesophagus. In this study we will calculate the risk of several sets of meat categorized in quartiles. Furthermore, OR’s for the same subsets of meat will be calculated as continuous variables after adding a fixed amount of consumption (10 grams per day).
I. Introduction The role of meat consumption has been considered controversial in its relationship with squamous cell carcinoma of the oesophagus. Whereas several studies reported an inverse association with this malignancy (Pottern et al, 1981; Tuyns et al, 1987; Gao et al, 1994; Launoy et al, 1998), other reports suggested a positive association with oesophageal cancer (Victora et al, 1987; De Stefani et al, 1990; Cheng et al, 1992; Rol!n et al, 1995). Since meat industry is the main industry in Uruguay, this country is particularly well suited for studying the role of meat, more precisely red meat, in the aetiology of squamous cell oesophageal cancer. In fact, Uruguay is the leading country in the World regarding beef consumption (Matos and Brandani, 2002). A previous Uruguayan study reported that salted meat and meat from lamb were positively associated with the risk of oesophageal cancer (De Stefani et al, 1999).
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De Stefani et al: Meat consumption and oesophageal cancer. amount of 10 grams per day was added to each one. Each food item was calculated in grams per day by multiplying the frequency of consumption per the portion size for a middle age participant.
II. Material and methods A. Selection of cases In the period 1996-2003, 208 patients with newly diagnosed and microspically confirmed squamous cell oesophageal carcinoma were identified in the four major public hospitals located in Montevideo, Uruguay. Since hospitals located outside the capital city of Montevideo lacked facilities for diagnosis and treatment of cancer, almost all patients are admitted in hospitals of Montevideo. Three patients were diagnosed as adenocarcinoma of the oesophagus and five patients refused the interview. These eight patients were excluded from the study, leaving a final total of 200 cases with squamous cell carcinoma of the oesophagus (response rate 96.1 %). The case series included 162 males (81 %) and 38 females (19 %).
E. Statistical analysis Relative risks of squamous cell oesophageal cancer for foods, approximated by the odds ratios, were estimated by multiple unconditional logistic regression (Breslow and Day, 1980). The following variables were included in all models: age (categorical, 5 strata), sex, county of residence (dichotomized as Montevideo and other), birthplace (categorical, 3 strata), education (categorical, 3 strata), average number of cigarettes smoked per day (categorical, 5 strata), years since quit (categorical, 5 strata), smoking status (categorical, 3 strata), alcohol drinking (categorical, 5 strata), maté temperature (categorical, 4 strata), total energy intake (continuous) and total vegetables and fruits (categorical, 4 strata). Tests for trend were performed after entering categorical variables as ordinal in the same model. Departure from the multiplicative model was determined by assessing the likelihood ratio test statistic. An alpha of 0.05 was used as the indicator of statistical significance and, accordingly, 95 % CI s were reported. All p-values were derived from two-sided statistical tests. All the calculations were done with the STATA programme (1999).
B. Selection of controls In the same time period and in the same hospitals, 723 patients with diseases not related with tobacco smoking or alcohol drinking and without recent changes in their diets were considered eligible for the study. Twenty patients refused the interview leaving a final total of 703 potential controls (response rate 97.2 %). From this pool, 400 controls were frequency matched to the cases on age (in ten-years intervals) and sex. The patients presented the following diseases: abdominal hernia (103 patients, 25.7 %), eye disorders (88, 22.0 %), diseases of the skin (12.0 %), acute appendicitis (37, 9.2 %), urinary stones (29, 7.3 %), varicose veins (29, 7.3 %), injuries (26, 6.5 %), hydatid cyst (20, 5.0 %) and blood disorders (20, 5.0 %).
III. Results As expected for the frequency matched design, age and sex were identical (Table 1). There was a higher proportion of dwellers living outside Montevideo, of rural residents and of subjects born in the Northern part of the country among cases than among controls. Cases were significantly less educated than controls (OR 0.6, 95 % CI 0.4-0.9). Income (in US dollars) was rather similar in both series of participants. Cases were significantly leaner than controls (OR 0.5, 95 % CI 0.3-0.8). The consumption of total energy was slightly higher in cases than in controls. Heavy smoking was associated with an increased risk of 4.8 (95 % CI 2.6-9.1). There was no difference between smoking of black tobacco and of blond tobacco cigarettes (OR 1.2, 95 % CI 0.7-1.8). On the other hand, smoking of hand-rolled cigarettes displayed an OR of 1.7 (95 % CI 1.1-2.7) compared with commercial cigarettes. Heavy drinking of alcohol was associated with a four-fold increase in risk and drinking of very hot maté displayed an OR of 4.0 (95 % CI 1.5-10.3) compared with drinking of
C. Questionnaire All participants were submitted to a questionnaire shortly after admittance. The interviews were conducted by two trained social workers in the hospitals. No proxy interviews were accepted. The questionnaire included the following sections: sociodemographics, an occupational history based in the job titles and its duration, a family history of cancer among firstdegree relatives, a complete tobacco smoking history (including age at start, age at quit, average number of cigarettes smoked per day, type of tobacco, type of cigarette), a history of alcohol drinking (including number of drinks per day or week for beer, wine and hard liquor), a complete history of maté drinking (maté is the folk name of a local herbal tea which is drunk hot or very hot), menstrual and reproductive events and a food frequency questionnaire (FFQ) on 64 food items. This FFQ allowed the calculation of total energy intake and was tested for reproducibility with good results.
D. Definition of food groups All queries concerned the consumption of foods five years before the date of questionnaire. The following types of meat were included in the FFQ: fried beef, broiled beef, boiled beef, fried lamb, broiled lamb, boiled lamb, poultry, fish, bacon, sausage, blood pudding, liver, mortadella, salami, saucisson, hot dog, ham, salted meat (consumption in the adolescence and current intake). These items were selected on the basis of the consumption of the Uruguayan population. From these individual items, the following food groups were created: red meat (beef, lamb), white meat (poultry, fish), processed meat (bacon, sausage, blood pudding, mortadella, salami, saucisson, hot dog, ham, salted meat), fried meat (fried beef, fried lamb), broiled meat (broiled beef, broiled lamb), boiled meat (boiled beef, boiled lamb) and total meat (red meat, processed meat, white meat). In order to compare the odds ratios for these nested food groups, each variable was treated as continuous and the fixed
warm maté. Odds ratios of squamous cell esophageal carcinoma for different types of meat are shown in Table 2. Total meat, red meat and preserved meat were not associated with risk of esophageal carcinoma. Similarly, different types of red meat (beef and lamb) were not associated with this malignancy. On the other hand, white meat consumption (poultry plus fish) was associated with a significant reduction in risk (OR 0.46, 95 % CI 0.25-0.85, p-value for trend=0.005). When red meat was analyzed by the cooking method, neither fried nor barbecued meat were associated with risk. In fact, barbecued meat showed reductions in risk which were close to the significance level of 0.05. On the other hand, boiled meat was associated with positive gradient (OR for high consumption of boiled meat 1.90, 95 % CI 1.05-3.46, p-value for trend=0.01).
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Cancer Therapy Vol 2, page 417 Table 1. Distribution of controls and cases for sociodemographics and selected risk factors Cases Variable Age (years)
Sex Residence Urban/rural status Birthplace
Education (yrs)
Income (dollars)
Family history Body mass index
Total calories
Tobacco smoking Ex-smokers (years)
Current smokers (cigarettes/day)
Type of tobacco
Type of cigarette
Alcohol drinking (1)
Maté temperature
Controls Category 40-49 50-59 60-69 70-79 80-89 Males Females Montevideo Other counties Urban Rural Montevideo South North 0-2 3-5 6+ <=146 147+ Unknown No Yes <=23.1 23.2-25.3 25.4-27.3 27.4+ <=1851 1852-2260 2261-2644 2645+ Never smokers 20+ 10-19 1-9 1-9 10-19 20-29 30+ Blond Mixed Black Manufactured Mixed Hand-rolled Never drinkers 1-60 61-120 121-240 241+ Warm Hot Very hot
N° patients 1-In militers of ethanol per day
N° % 15 7.5 40 20.0 69 34.5 61 30.5 15 7.5 162 81.0 38 19.0 76 38.0 124 62.0 149 74.5 51 25.5 47 23.5 69 34.5 84 42.0 69 34.5 84 42.0 47 23.5 67 33.5 79 39.5 54 27.0 194 97.0 6 3.0 75 37.5 52 26.0 34 17.0 39 19.5 39 19.5 56 28.0 47 23.5 58 29.0 40 20.0 15 7.5 13 6.5 29 14.5 6 3.0
N° % 30 7.5 80 20.0 138 34.5 122 30.5 30 7.5 324 81.0 76 19.0 167 41.8 233 58.2 321 80.3 79 19.7 135 33.8 171 42.7 94 23.5 124 31.0 131 32.8 145 36.2 173 43.3 158 39.5 69 17.2 391 97.8 9 2.2 101 25.2 101 25.2 98 24.6 100 25.0 100 25.0 100 25.0 100 25.0 100 25.0 140 35.0 33 8.3 32 8.0 38 9.5 20 5.0
OR 95 % CI
21 36 40 77 26 57 41 21 98 62 37 41 34 26 11 144 35 200
61 15.2 47 11.8 29 7.2 119 45.8 65 25.0 76 29.2 94 36.2 37 14.2 129 49.6 195 48.8 85 21.2 61 15.2 39 9.8 20 5.0 25 7.4 295 86.8 20 5.8 400 100.0
1.2 2.7 4.8 1.0 0.6 1.2 1.0 1.3 1.7 1.0 1.4 2.1 2.7 4.1 1.0 1.1 4.0
10.5 18.0 20.0 48.1 16.3 35.6 25.6 13.1 61.3 31.0 18.5 20.5 17.0 13.0 5.8 75.8 18.4 100.0
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Not applicable Not applicable 1.0 1.2 0.8-1.6 1.0 1.4 0.9-2.1 1.0 1.1 0.7-1.8 2.6 1.6-4.0 1.0 1.1 0.8-1.7 0.6 0.4-0.9 1.0 1.3 0.9-1.9 - 1.0 1.3 0.5-3.8 1.0 0.7 0.4-1.1 0.5 0.3-0.8 0.5 0.3-0.8 1.0 1.4 0.9-2.3 1.2 0.7-2.0 1.5 0.9-2.4 1.0 1.6 0.8-3.2 1.4 0.7-2.9 2.7 1.4-4.9 1.1 0.4-2.8 0.6-2.2 1.5-4.7 2.6-9.1 0.4-1.1 0.7-1.8 0.7-2.5 1.1-2.7 0.8-2.2 1.3-3.5 1.6-4.8 2.1-8.0 0.5-2.5 1.5-10.3
De Stefani et al: Meat consumption and oesophageal cancer. Table 2. Odds ratios of squamous cell esophageal carcinoma for different types of meat, categorized in quartiles (1) Type of meat Total meat
Intake (grams/day) Cases/Controls OR 95 % CI <=160.5 49/100 1.0 160.6-203.0 40/100 0.48 0.26-0.89 203.1-251.9 44/100 0.56 0.31-1.01 252.0+ 67/100 0.96 0.54-1.70 p-value for trend 0.80 Red meat <=114.3 42/100 1.0 114.4-160.2 42/100 0.65 0.35-1.20 160.3-192.7 41/100 0.65 0.35-1.19 192.8+ 75/100 1.14 0.64-2.05 p-value for trend 0.49 Processed meat <=7.9 46/100 1.0 8.0-19.1 50/100 1.12 0.64-1.99 19.2-39.7 57/100 1.11 0.63-1.96 39.8+ 47/100 1.15 0.63-2.07 p-value for trend 0.68 White meat <=6.6 86/100 1.0 6.7-17.1 50/100 0.64 0.38-1.08 17.2-30.0 33/100 0.48 0.26-0.86 30.1+ 31/100 0.46 0.25-0.85 p-value for trend 0.005 Beef <=85.4 60/100 1.0 85.5-146.3 43/100 0.69 0.39-1.21 146.4-171.3 37/100 0.46 0.25-0.84 171.4+ 60/100 0.77 0.45-1.34 p-value for trend 0.25 Lamb 0 85/195 1.0 0.1-21.3 48/101 0.93 0.56-1.54 21.4+ 67/104 1.08 0.65-1.81 p-value for trend 0.80 Fried meat <=21.3 62/100 1.0 21.4-42.7 47/100 0.69 0.39-1.21 42.8-64.1 48/100 0.78 0.45-1.37 64.2+ 43/100 0.73 0.41-1.28 p-value for trend 0.35 Barbecued meat <=10.8 65/100 1.0 10.9-26.3 42/100 0.56 0.31-0.99 26.4-53.4 44/100 0.62 0.35-1.09 53.5+ 49/100 0.58 0.33-1.03 p-value for trend 0.09 Boiled meat <=32.0 33/100 1.0 32.1-74.7 37/100 1.02 0.54-1.94 74.8-96.1 49/100 1.38 0.75-2.55 96.2+ 81/100 1.90 1.05-3.46 p-value for trend 0.01 1-Adjusted for age, sex, residence, urban/rural status, birthplace, education, body mass index, smoking status, number of cigarettes smoked per day, years since quit, alcohol drinking, matĂŠ temperature and total energy intake. The analysis of types of meat as continuous variables after the addition of 10 g/day is shown in Table 3. Total meat consumption was not associated with risk of oesophageal cancer. When total meat was dissected in red meat, preserved meat and white meat, the last group was significantly and inversely associated with risk of squamous cell oesophageal cancer (OR 0.57, 95 % CI 0.38-0.86, p-value for trend=0.008). In model 3, red meat was partitioned in beef and lamb; preserved meat and white meat were retained in this model. Neither beef nor lamb meat were associated with risk of oesophageal
cancer. In model 4, red meat was dissected in fried, barbecued and boiled meat, according with the cooking method. Again preserved meat and white meat were retained in the model. Whereas fried and barbecued meat were not associated with risk, boiled meat displayed a modest increased risk which was non-significant (OR 1.29, 95 % CI 0.92-1.81, p-value for trend=0.14). In model 5, white meat was examined according to its nested components poultry and fish. Poultry intake was inversely associated with risk of oesophageal cancer (OR 0.65, 95 % CI 0.39-1.07, p-value for trend=0.09), whereas fish
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Cancer Therapy Vol 2, page 419
Table 3. Odds ratios of squamous cell esophageal carcinoma for types of meat included in the models as continuous variables after the addition of 10 g/day (1). Model OR 95 % CI p-value for linear trend 1 Total meat 1.26 0.66-2.40 0.40 2 Red meat 1.10 0.68-1.79 0.70 Processed meat 1.10 0.81-1.49 0.55 White meat 0.57 0.38-0.86 0.008 3 Beef 0.88 0.63-1.24 0.48 Lamb 1.01 0.77-1.32 0.94 Processed meat 1.08 0.79-1.47 0.63 White meat 0.57 0.38-0.86 0.007 4 Fried meat 0.91 0.68-1.21 0.51 Barbecued meat 0.78 0.58-1.05 0.11 Boiled meat 1.29 0.92-1.81 0.14 Processed meat 1.12 0.82-1.53 0.47 White meat 0.57 0.38-0.86 0.008 5 Red meat 1.12 0.69-1.81 0.66 Processed meat 1.11 0.81-1.50 0.51 Poultry 0.65 0.39-1.07 0.09 Fish 0.74 0.44-1.24 0.26 6 Red meat 1.16 0.70-1.91 0.57 Bacon 1.38 0.44-4.34 0.58 Sausage 0.68 0.37-1.26 0.32 Blood pudding 0.81 0.41-1.61 0.55 Liver 0.68 0.38-1.23 0.21 Mortadella 0.95 0.61-1.47 0.81 Salami 0.94 0.56-1.59 0.83 Saucisson 0.99 0.43-2.28 0.99 Hot dog 1.34 0.80-2.21 0.26 Ham 1.26 0.69-2.28 0.45 Salted meat 2.34 1.21-4.53 0.01 White meat 0.55 0.36-0.84 0.006 1-Adjusted for age, sex, residence, urban/rural status, birthplace, education, body mass index, smoking status, number of cigarettes smoked per day, years since quit, alcohol drinking, maté temperature, total energy intake and total vegetables and fruits. consumption displayed a modest reduction in risk. Finally, in model 6 preserved meat was dissected into its 10 components. Red meat and white meat were retained in this last model. Only salted meat (OR 2.34, 95 % CI 1.214.53, p-value for trend=0.01) and white meat (OR 0.55, 95 % CI 0.36-0.84, p-value for trend=0.006) were significantly associated with risk of squamous cell carcinoma of the oesophagus.
carcinogenesis. Also, previous studies conducted in Uruguay reported similar findings (De Stefani et al, 1999, 2003) for salted red meat. In fact before the advent of the refrigeration, red meat was preserved in rural areas of Uruguay by salting and air-drying (“charque”). These areas displayed very high incidence rates of oesophageal cancer, close to 60 cases per 100,000 inhabitants. The consumption of “charque” has declined in the last 30-40 years, paralelling the decline in the incidence and mortality of oesophageal carcinoma (Zheng et al, 1993; De Stefani et al, 1994). Salted meat is probably more active in the adolescence (Cheng et al, 1992) and is suspected as a rich source of nitrosamines and salt. Both agents (nitrosamines and salt) have been considered as important determinants in the process of oesophageal carcinogenesis (Craddock, 1992; Castellsagué et al, 2000). Other studies suggested that heterocyclic amines could be important aetiologic factors for oesophageal cancer (Ward et al, 1997). Barbecued and fried meat are precisely a rich source of HCA and in previous studies high consumption of barbecued meat was associated with a high risk of oesophageal carcinoma (De Stefani et al, 1990; Castelletto et al, 1994). In the present study, neither
IV. Discussion According to the results of our study boiled meat, salted meat and white meat were significantly associated with risk of oesophageal carcinoma. The remaining varieties of meat were not related with oesophageal carcinogenesis. Previous epidemiological studies reported similar results (Tavani et al, 1994; Bosetti et al, 2000; De Stefani et al, 2003). In particular, the role of salted meat in the development of oesophageal carcinoma has been considered a consisting finding (Cheng et al, 1992; De Stefani et al, 1999, 2003). According to Cheng et al, (1992), salted fish is an important factor in oesophageal
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De Stefani et al: Meat consumption and oesophageal cancer. fried nor barbecued meat were risk factors for this malignancy. Boiled meat, a main component of stew, was significantly associated with an increased risk of squamous cell oesophageal cancer in our study. The role of boiled meat was specially evident when this meat was analyzed as a categorical variable. When boiled meat was entered into the model as a continuous term, this type of meat was no longer significant. In is possible that boiled meat acted in the process of oesophageal carcinogenesis through thermal injury of oesophageal mucosa, since stews are ingested very hot by the Uruguayan population. This could induce chronic oesophagitis, considered as the first step in the development of oesophageal carcinoma (Mu•oz et al, 1987). White meat, that is poultry plus fish meat, was the only protective type of meat in our study. This type of meat is characterized by a lower amount of saturated fat when compared with red meat. Unfortunately, our questionnaire did not discriminate cooking methods for poultry and fish. It has been reported that, whereas low-fat types of white meat (skinless chicken and unfried fish) are protective, high-fat types of this meat are associated with risk of other malignancies (Ronco et al, 2003). Thus, it is possible that our participants consumed mainly low-fat types of white meat. It is not possible to discard the fact that white meat consumption could be a marker of a healthy diet. Previous studies reporting OR’s of oesophageal carcinoma for white meat consumption, consistently showed inverse associations (De Stefani et al, 2003). Further studies on mechanisms of white meat consumption in oesophageal carcinogenesis are needed. Residual confounding of our results from tobacco smoking, alcohol drinking, maté consumption and diet could result in severe distortions of the estimates. For these reasons we included terms for tobacco smoking (8 strata), alcohol drinking (5 strata), maté temperature (4 strata) and total vegetables and fruits (4 strata). As other hospital-based case-control studies, the present study has limitations. In first place, selection bias is almost impossible to exclude. We tried to minimize this bias by frequency matching cases and controls on age and sex. Also, residence and urban/rural status were rather similar. Recall bias, which is almost non-existent in prospective studies, is also a major drawback. We tried to minimize recall bias asking about the consumption five years before the date of the interview. Nevertheless, since the process of oesophageal carcinogenesis is of long duration, is possible that cases experimented difficulties in eating foods like meat, more difficult to swallow. This could led to differential misclassification. Since the role of diet in oesophageal carcinoma is mostly unknown in Uruguay, it is unlikely that both interviewers and participants of the study could be affected in its attitudes during the interviews. Probably, the major limitation of our study was the lack of validation of our FFQ. Aside from this drawback the FFQ was comprehensive and incred queries on 64 food items, representative of the usual diet of the Uruguayan population. On the other hand, our study has strengths. Perhaps the major strength is related with the high response rate observed in both series
of patients. Also, the exclusion of proxy interviews is another strength of the study. Finally, the restriction of the study to patients afflicted by squamous cell carcinoma of the oesophagus is a strength. In summary, our study replicates previous findings showing that salted meat is a major risk factor for squamous cell carcinoma of the oesophagus. On the other hand white meat intake was a protective factor, even after allowing for the effect of plant foods and other major confounders. Public health measures resulting in a lower intake of salt and in a substitution of red meat by white meat could prevent the risk of development of squamous cell oesophageal carcinoma.
References Bosetti C, La Vecchia C, Talamini R, Simonato L, Zambon P, Negri E, Trichopoulos D, Lagiou P, Bardini R and Franceschi S (2000) Food groups and risk of squamous cell esophageal cancer in Northern Italy. Int J Cancer 87, 289294. Breslow NE and Day NE (1980) Statistical methods in cancer research. Vol. I. The analysis of case-control studies. IARC scientific publications N° 32. IARC Lyon. Castelletto R, Castellsagué X, Mu•oz N, Iscovich J, Chopita N and Jmelnitsky A (1994) Alcohol tobacco diet mate drinking and esophageal cancer in Argentina. Cancer Epidemiol Biomarkers Prev 3, 557-564. Castellsagué X, Mu•oz N, De Stefani E, Victora CG, Castelletto R and Rol!n PA (2000) Influence of mate drinking hot beverages and diet on esophageal cancer risk in South America. Int J Cancer 88, 658-664. Cheng KK, Day NE, Duffy SW, Lam TH and Fox M (1992) Pickled vegetables in the aetiology of oesophageal cancer in the Hong King Chinese. Lancet 339, 1314-1318. Craddock VM (1992) Aetiology of oesophageal cancer some operative factors. Eur J Cancer Prev 1, 89-103. De Stefani E, Deneo-Pellegrini H, Ronco AL, Boffetta P, Brennan P, Mu•oz N, Castellsagué X, Correa P and Mendilaharsu M (2003) Food groups and risk of squamous cell carcinoma of the oesophagus a case-control study in Uruguay. Br J Cancer 89, 1209-1214. De Stefani E, Fierro L, Barrios L and Ronco A (1994) Cancer mortality trends in Uruguay 1953-1991. Int J Cancer 56, 634-639. De Stefani E, Mu•oz N, Estève J, Vassallo A, Victora C and Teuchmann S (1990) Mate drinking alcohol tobacco diet and esophageal cancer in Uruguay a case-control study. Cancer Res 50, 426-431. De Stefani E., Deneo-Pellegrini H, Boffetta P and Mendilaharsu M (1999) Meat intake and risk of squamous cell esophageal cancer a case-control study in Uruguay. Int J Cancer 82, 3337. Gao YT, McLaughlin JK, Gridley G, Blot WJ, Ji BT, Dai Q and Fraumeni JF Jr (1994) Risk factors for esophageal cancer in Shanghai China. II. Role of diet and nutrients. Int J Cancer 58, 197-202. Launoy G, Milan C, Day N.E, Pienkowski MC, Gignoux M and Faivre J (1998) Diet and squamous cell cancer of the esophagus a French multicentre case-control study. Int J Cancer 76, 7-12. Matos E and Brandani A (2002) Review on meat consumption and cancer in South America. Mutat Res 506-507, 243-249. Mu•oz N, Victora CG, Crespi M, Saul C, Braga NM and Correa P (1987) Hot maté drinking and precancerous lesions of the oesophagus an endoscopic survey in southern Brazil. Int J Cancer 39, 708-709.
420
Cancer Therapy Vol 2, page 421 Pottern LM, Morris LE, Blot WJ, Ziegler RG and Fraumeni JF Jr (1981) Esophageal cancer among black men in Washington DC. I. Alcohol tobacco and other risk factors. J Natl Cancer Inst 67, 777-783. Rol!n PA, Castellsagué X, Benz M and Mu•oz N (1995) Hot and cold mate drinking and esophageal cancer in Paraguay. Cancer Epidemiol Biomarkers Prev 4, 595-605. Ronco AL, De Stefani E and Fabra A (2003) White meat intake and the risk of breast cancer a case-control study in Montevideo Uruguay. Nutr Res 23, 151-162. STATA (1999) Stata reference guide. Release 6. College Station Texas. Tavani A, Negri E, Franceschi S. and La Vecchia C (1994) Risk factors for esophageal cancer in lifelong nonsmokers. Cancer Epidemiol Biomarkers Prev 3, 87-92. Tuyns AJ, Riboli E, Doornbos G and Péquignot G (1987) Diet and oesophageal cancer in Calvados (France). Nutr Cancer 9, 81-92.
Victora CG, Mu•oz N, Day NE, Barcelos LB, Peccin DA and Braga N (1987) Hot beverages and oesophageal cancer in southern Brazil a case-control study. Int J Cancer 39 710716. Ward MH, Sinha R, Heinman EF, Rothman N, Markin R, Weisenburger DD, Correa P and Hahm SH (1997) Risk of adenocarcinoma of the stomach and esophagus with meat cooking method and doneness preference. Int J Cancer 71, 14-19. Zheng W, Jin F, Devesa SS, Blot WJ, Fraumeni JF Jr, and Gao YT (1993) Declining incidence is greater for esophageal than gastric cancer in Shanghai People’s Republic of China. Br J Cancer 68, 978-982.
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De Stefani et al: Meat consumption and oesophageal cancer.
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Case report on combined radiation myelopathy and intramedullary metastases Case Report
Robbert JHA Tersteeg1, Sherif Y El Sharouni1*, Henk B Kal1, Gerard H Jansen2, Petra M De Jong 3, Jacobus S. Straver 4 1
Department of Radiation Oncology, University Medical Center, Utrecht, The Netherlands Department of Pathology, University Medical Center, Utrecht, The Netherlands 3 Department of Pulmonology, Hofpoort Ziekenhuis, Woerden, The Netherlands 4 Department of Neurology, Hofpoort Ziekenhuis, Woerden, The Netherlands 2
__________________________________________________________________________________ *Correspondence: SY El Sharouni, MD, University Medical Center Utrecht, Dept. of Radiation Oncology, Q 00.118, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands; Fax +31 30 2581226; e-mail: S.Y.ElSharouni@azu.nl Key words: radiation myelopathy, intramedullary metastases, MRI, neurological complications Abbreviations: biologically effective dose, (BED); cerebral spinal fluid, (CSF); computerized tomography, (CT); linear-quadratic, (LQ); Magnetic Resonance Imaging, (MRI) Received: 20 September 2004; Accepted: 27 September 2004; electronically published: November 2004
Summary Radiation myelopathy is one of the complications most feared in radiotherapy. The clinical picture consists of central motor neuron signs, sometimes without loss of sensibility. Radiation myelopathy is preceded by a latency period, during which the patient is asymptomatic. We present a patient with combined radiation myelopathy and intramedullary metastases. Our patient was a female, aged 47, who showed a large-cell squamous cell carcinoma of the lung. She received palliative irradiation to the lung. Nine months later the patient experienced paraesthesia in her left leg and difficulty in stair climbing especially with her left leg. Within a few weeks these complaints extended to her right leg and urine incontinence developed. MRI imaging of the thoracal spine showed intramedullary metastases at vertebrae Th5 / Th6. She was reirradiated; the irradiated part of the myelum was partially included in the prior treatment. Two months after the last radiation treatment she developed complaints of numbness of the right arm, without loss of strength. Further investigations confronted us with combined radiation myelopathy and intramedullary metastases. Diagnosis is in fact a diagnosis per exclusionem. The three criteria for myelopathy as a result of radiation treatment according to Pallis et al, (1961) are: 1. the spinal cord must have been included in the radiation field 2. the main neurological lesion must be within the segments of cord exposed to radiation and 3. metastases or other primary spinal cord lesions must be ruled out as the cause of the neurological disorder. Recommended for further diagnostics are lumbar puncture and myelography, as well as CT- (computerized tomography) and MRI-imaging (Magnetic Resonance Imaging) (Goldwein, 1987). Analysis of the cerebral spinal fluid (CSF) might reveal an elevated protein. The myelogram in some cases demonstrates mild cord atrophy. Myelography is only advised in case of contra-indications for MRI, because of the superior imaging quality of the latter. CT/MRI imaging is also important to rule out the presence of intramedullary tumour (Schiff and Oâ&#x20AC;&#x2122;Neill,
I. Introduction Radiation myelopathy is one of the complications most feared in radiotherapy. It was first described by Ahlbom (1941). The clinical picture consists of central motor neuron signs, sometimes without loss of sensibility (Goldwein, 1987). Diagnosis can be difficult with a paraneoplastic syndrome, herpes zoster or myelopathy as a result of chemotherapy and epidural / intramedullary of leptomeningeal metastases as differential diagnosis. Radiation myelopathy is preceded by a latency period, during which the patient is asymptomatic. Symptoms begin as paresthesia and/or inability to perceive pain and/or temperature. In literature the latency period has been reported to last from 1 month to 6 years. Typically, the median latency is 6 months. About three out of four patients develop myelopathy within 18 months after radiation treatment (Goldwein, 1987; Michikawa et al, 1991; Koehler et al, 1996).
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Tersteeg et al: Case report on combined radiation myelopathy and intramedullary metastases 1996). Other causes of myelopathy could be excluded by means of MRI. Pathological changes of the myelum are described in a number of studies. Both early and late changes can be distinguished (Schultheiss et al, 1988; Van Daal et al, 1989; Van der Kogel, 1997). A bimodal distribution of latencies has been described (Goldwein, 1987) with peaks occurring at 13 months and 26 months after irradiation. Patients with shorter intervals have, in general, been treated with higher radiation doses. This supports the hypothesis of two mechanisms of damage to the spinal cord: white matter necrosis at higher doses and vascular damage at lower doses. Patients undergoing a reirradiation showed a shorter latency period, as did paediatric patients. Other causes of progressive myelitis must be excluded, most notably extramedullary metastases, intramedullary tumour, necrotizing carcinomatous myelopathy and hypertrophy of the posterior vertebral facets of the laminae as a result of arthritis. We present a patient with combined radiation myelopathy and intramedullary metastases.
A chest X-ray showed a large density in the upper right lobe of the lung. A CT-scan of the thorax showed a large mass extending into the mediastinum and a second mass ventrally, with probable destruction of the rib, explaining the complaints of pain. A bronchoscopy showed a tumour, just in front of the ostium of the upper right lobe, causing a stricture to 60%, with submucosal growth. Pathological findings showed a large-cell squamous cell carcinoma. She received palliative irradiation to the lung, to a dose of 39 Gy, in fractions of 3 Gy, administered by two opposing AP/PA fields. The target area included the right upper lobe, the ipsilateral hilus and the mediastinum. As a consequence the thoracic vertebrae Th 3 / Th9 were also included (Figure 1). Nine months later the patient experienced paraesthesia in her left leg and difficulty in stair climbing especially with her left leg. Within a few weeks these complaints extended to her right leg and urine incontinence developed. On neurological examination hyperreflexia of both legs was found but at this time no sensory level at the trunk could be determined. MRI imaging of the thoracal spine showed intramedullary metastases at vertebrae Th5 / Th6. She was irradiated to a dose of 20 Gy in fractions of 4 Gy. The target area was Th4 / Th7, irradiated in one PA field, the dose was given at a depth of 6 cm. This was a reirradiation because the irradiated part of the myelum was partially included in the prior treatment (Figure 2). Despite reirradiation the symptoms got worse resulting in progressive paresis of both legs. Two months after the last radiation treatment she developed complaints of numbness of the right arm, without loss of strength.
II. Case report Our patient was a female, aged 47, without a medical history. In August 1996 she developed complaints of pain in the right shoulder. She also coughed, producing viscous sputum and there was some shortness of breath while exercising. Finally, she developed fever and night sweat. At the time of presentation she smoked two packs of cigarettes a day, no consumption of alcohol. No abnormalities were found at physical examination.
Figure 1. First irradiation field
Figure 2. Second irradiation field, re-irradiation
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Cancer Therapy Vol 2, page 425 Neurological investigation showed numbness in dermatome C8 with hyper-abduction of the right arm but no pareses of the arms. Loss of sensibility at the trunk (delineated at Th6 on both sides), hyperreflexia of the legs and finally a Babinski reflex on both sides were noted. Imaging of the cervico-thoracical spine by means of MRI showed abnormal signal in the myelum at C7 / Th5. In the cervical (not irradiated) part of the spine, ring-like enhancing lesions were seen, arousing suspicion of radiation myelopathy (Figure 3). In the thoracic (irradiated) part of the spine intramedullary metastases were suspected (Figure 4). The complaints were rapidly progressive; paresis in the legs became complete. There was total urine incontinence.
Figure 3. MRI cervical spine, showing suspected myelopathy
Finally she died within three months after reirradiation. Autopsy was performed. The autopsy showed some surprising findings. In the reirradiated area intramedullary metastases were found, histologically corresponding with a squamous cell carcinoma (Figure 5). These metastases were found at the level of Th7. At the levels C7 / Th1, corresponding with the neurological findings, changes were seen, but not based on tumour. The found abnormalities mainly consisted of vascular changes with subtotal necrosis and hyaline changes of the vessels (Figure 6). ). These findings match radiation myelopathy.
Figure 4. MRI thoracal spine, showing intramedullary metastases
Figure 5. Intramedullary metastases of squamous cell carcinoma in re-irradiated area
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Tersteeg et al: Case report on combined radiation myelopathy and intramedullary metastases
Figure 6. Vascular changes with subtotal necrosis and hyaline changes of the vessels at level C7 / Th1.
It is remarkable that both MRI and pathological investigation showed injury outside the irradiated area of both treatments. We were confronted with radiation myelopathy outside the irradiated volume. A contradiction in terms?
for myelopathy as a result of radiation treatment according to Pallis et al, (1961) are: A. The spinal cord must have been included in the radiation field; B. The main neurological lesion must be within the segments of cord exposed to radiation; C. Metastases or other primary spinal cord lesions must be ruled out as the cause of the neurological disorder. In our patient described, the spinal cord level of neurological disturbances was located inside as well as outside the irradiated area. For every fractionation scheme a Biologically Effective Dose (BED) can be calculated using the LinearQuadratic (LQ) model (Barendsen, 1982). The incidence of a biological effect according to the LQ model is I = n (!d+"d2), and the BED that can be derived from this linear-quadratic equation is nd(1+d/(!/")); n is the number of fractions, d is the fraction dose, and !/" is a tissue parameter with values of about 10 Gy for acute reacting tissues and !/" is about 2 Gy for late reacting tissues. A reasonable estimate of radiation myelopathy risk is 5% at 60 Gy in 30 fractions. This myelum tolerance dose results in a BEDtot value of 60(1+2/2) = 120 Gy (with !/" is 2 Gy). The retreatment tolerance dose that can be derived from reirradiated experimental animals and human patients is about 130% of the BEDtot (Stewart, 1994). A prerequisite is that the interval is at least 6 to 9 months and the first treatment dose has not exceeded about 65% of the BEDtot value. The total BED for reirradiation then is 156 Gy. With our patient the first treatment BED1 = 39(1+(3/2)) = 97.5 Gy (this is 81% of the BEDtot). The BED2 of the second treatment is 5*4(1+(4/2) = 60 Gy. This sums up to 157.5 Gy. This BED value of 157.5 Gy
III. Discussion Early changes in radiation myelopathy notably consist of focal areas with spongious demyelination, axonal swelling and the absence of observable vascular injury. Late changes are characterised by involvement of the entire myelum with a preference for the white matter, as well as vascular injury (Schultheiss et al, 1988; Ang and Stephens, 1994). Most studies report vascular injury as the primary pathophysiological cause of radiation myelopathy. This seems to be the explanation for the damage outside the irradiated volume as seen with this patient. Vascular changes as a result of the formation of hyaline membranes, necrosis and thrombosis can migrate into the course of a blood vessel. In our case, however, we observed a short interval between the second treatment and the complaints. Recent MRI studies in Japan have mentioned a number of characteristics that can almost be conclusive for myelopathy as a result of radiotherapy: 1) swelling of the spinal cord on T1-weighted images and an intramedullary high-intensity area on T2-weighted images (usually attributed to spinal cord oedema, since the cord size diminishes following steroid administration on subsequent MR images) and 2) a ring-like enhancing lesion with gadolinium on T1-weigthed images, resulting from bloodbrain barrier breakdown (Michikawa 1991; Yasui 1992). In our case swelling of the spinal cord as well as a ringlike enhancing lesion were indeed found. The three criteria
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Cancer Therapy Vol 2, page 427 Ang KK, Stephens LC (1994) Prevention and management of radiation myelopathy. Oncology 8, 71-76. Barendsen GW (1982) Dose fractionation, dose rate and isoeffect relationships for normal tissue responses. Int J Radiat Oncol Biol Phys 8, 1981-1997. Goldwein JW (1987) Radiation myelopathy: a review. Med Pediatr Oncol 15, 89-95. Koehler PJ, Verbiest H, Jager J, Vecht CJ (1996) Delayed radiation myelopathy: serial MR-imaging and pathology. Clin Neurol Neurosurg 98, 197-201. Michikawa M, Wada Y, Sano M, et al (1991) Radiation myelopathy: significance of gadolinium-DTPA enhancement in the diagnosis. Neuroradiology 33, 286-289. Pallis CA, Louis S, Morgan RL (1961) Radiation myelopathy. Brain 84, 460-479. Schiff D, O'Neill BP (1996) Intramedullary spinal cord metastases: Clinical features and treatment outcome. Neurology 47, 906-912. Schultheiss TE, Stephens LC, Maor MH (1988) Analysis of the histopathology of radiation myelopathy. Int J Radiat Oncol Biol Phys 14, 27-32. Stewart FA, Oussoren Y, Van Tinteren H, Bentzen SM (1994) Loss of reirradiation tolerance in the kidney with increasing time after single or fractionated partial tolerance doses. Int J Radiat Oncol Biol Phys 66, 169-179. Van Daal WA, Hoogenhout J, Keyser AJM (1989) Radiation myelopathy. Ned Tijdschr Geneeskd 133, 1655-1657. Van der Kogel J (1997) Radiation response and tolerance of normal tissues. In Steel GG (ed) Basic Clinical Radiobiology. London, Arnold 30-39. Yasui T, Yagura H, Komiyama M, Fu Y, Nagata Y, Tamura K, Khosla VK, Hakuba A (1992) Significance of gadoliniumenhanced magnetic resonance imaging in differentiating spinal cord radiation myelopathy from tumor. J Neurosurg 77, 628-631.
seems equivalent to the retreatment tolerance dose. However, the dose of the first treatment delivered to the myelum was 81% of the tolerance dose, and repair of induced damage was probably not complete between the first and second treatment. Experimental data with animals show that the tolerance dose after reirradiation is inversely proportional to the first dose. For instance, after an initial dose of 50% of the BEDtot and a sufficient time interval of at least 6 months, the retreatment tolerance dose would be approximately 80% of the BEDtot, while after an initial dose close to full tolerance (90% of the BEDtot), only a maximum of 40% of the BEDtot would be left for retreatment. Moreover there probably remained substantial radiation-induced damage after the first (high) dose in our patient. Thus the myelopathy observed in our patient could also well be radiation induced.
IV. Conclusion The diagnostic pitfall in this case is the erroneous assumption that the complaints of this patient can be attributed to the known intramedullary metastases at Th7, which was confirmed by autopsy. However, this level does not correspond with the eventual clinical findings, which point to a localisation at the cervico-thoracal part of the myelum. These findings, as we have shown by MRI and pathological investigations are most probably caused by radiation myelopathy. We recommend that in case of complaints after (re-) irradiation, accurate imaging is performed, preferably by means of MRI with gadolinium. One should always be aware of radiation myelopathy, even if the level is not corresponding with the irradiated part of the myelum. The retreatment dose of 20 Gy in 5 fractions of 4 Gy could better have been reduced, with respect to the relatively high first dose and short interval time, to 3 fractions, or to a scheme with a lower fraction dose, e.g. 5x3 Gy. The BED of this alternative scheme is about 37 Gy, significantly lower than the BED of 60 Gy. One of the three criteria of myelopathy as result of radiation according to Pallis et al: the main neurological lesion must be within the segments of cord exposed to radiation, is not obligatory. This criterion should be read as: â&#x20AC;&#x2122;the main neurological lesion must be in or near to the segments of the cord exposed to radiationâ&#x20AC;&#x2122;. To date we have not found a resembling case in literature.
References
Robbert JHA Tersteeg
Ahlbom HE (1941) The results of radiotherapy of hypopharyngeal cancer at the Radiumhemmet, Stockholm, 1930-1939. Acta Radiol Oncol 22, 155-171.
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Cancer Therapy Vol 2, page 429 Cancer Therapy Vol 2, 429-440, 2004
Genistein induces apoptosis via mitochondrial damage in acute lymphoblastic leukemia T-cell lines Research Article
Fayth K. Yoshimura1* 1
Department of Immunology and Microbiology, and the Karmanos Cancer Institute Wayne State University, Detroit, MI 48201
__________________________________________________________________________________ *Correspondence: Fayth K. Yoshimura, Department of Immunology and Microbiology, Wayne State University, 540 E. Canfield Ave., Detroit, MI 48201; Phone: (313) 577-1571; Fax: (313) 577-1155; e-mail: fyoshi@med.wayne.edu Key words: genistein, T-cell lymphoblastic leukemia, mitochondria, caspase Abbreviations: apoptosis activating factor-1, (Apaf-1); B-cell acute lymphoblastic leukemia, (B-ALL); bongkrekic acid, (BA); fetal bovine serum, (FBS); fluorescein isothiocyanate-conjugated annexin V, (annexin V-FITC); permeability transition pore, (PTP); phosphate buffered saline, (PBS); room temperature, (RT); sodium dodecylsulfate, (SDS); T-cell acute lymphoblastic leukemia, (TALL) Received: 21 October 2004; Accepted: 9 November 2004; electronically published: November 2004
Summary Genistein, a soy isoflavone, has anti-proliferative and apoptotic effects on different types of malignant cells. We examined its properties on human T-cell acute lymphoblastic leukemia (T-ALL) cell lines to assess the potential use of genistein for the treatment of this type of childhood leukemia. At concentrations of 15 ÂľM and greater, genistein was able to kill Jurkat and CCRF-CEM cells in a dosage- and time-dependent manner. Flow cytometric analysis of T-ALL cells stained with Annexin V-FITC and PI as well as assays for caspase-3 activation indicated that cell killing occurred via apoptosis. Cell-staining with the mitochondrial-specific dye JC-1 revealed that genistein produced mitochondrial damage as an early step in apoptotic signaling. The ability of bongkrekic acid to inhibit mitochondrial damage by genistein suggested that membrane depolarization occurred by sustained opening of the permeability transition pore. The activation of caspase-9 after mitochondrial damage was detected suggests that an early step in genistein-induced apoptosis involves the intrinsic pathway. Messina, 2003; Yamamoto et al, 2003). More specifically, consumption of genistein, which is the predominant soy isoflavone, strongly correlates with a reduced incidence of cancer in humans and other animals (Adlercreutz et al, 1993; Barnes, 1995; Lamartiniere et al, 1995a,b, 2002; Polkowski and Mazurek, 2000; Mentor-Marcel et al, 2001; Mizunuma et al, 2002; Wang et al, 2002). Because of these observations there has been a strong interest in the use of genistein for cancer prevention. Besides its potential as a preventive agent for certain cancers, there is considerable interest in the use of genistein for therapeutic purposes as well. This potential use of genistein is based on numerous in vitro and in vivo studies, which have demonstrated that genistein is able to kill malignant cells in contrast with normal cells (Traganos et al, 1992; Constantinou et al, 1998; Davis et al, 1998; Li et al, 1999b; Messina, 1999; Baxa and Yoshimura, 2003). Malignant cell killing by genistein has been detectable for cell lines derived from different types of cancer, including breast (Zava and Duwe, 1997), non-small-cell lung (Lian et al, 1999), head and neck squamous cell (Alhasan et al,
I. Introduction Epidemiological studies of different populations have indicated that there is a high correlation between a lower incidence of certain types of cancer and a high soy diet (Mills et al, 1989; Lee et al, 1991; Adlercreutz et al, 1995; Wu et al, 1996; Dai et al, 2001; Messina, 2003; Sarkar and Li, 2003; Yamamoto et al, 2003). It was found that Asian women who regularly consumed soy foods had a significantly lower risk of developing breast cancer (Lee et al, 1991; Wu et al, 1996; Dai et al, 2001; Yamamoto et al, 2003). These observations have been supported by additional studies of premenopausal women in different Asian populations, which showed a correlation between a reduced risk of breast cancer and a high intake of soy (Lee et al, 1992; Hirose et al, 1995). In addition, several studies have shown that consumption of soy products reduced the mortality rate from prostate cancer (Mills et al, 1989; Adlercreutz et al, 1993; Hebert et al, 1998; Messina, 2003). Isoflavones have been identified to be the major component of soy that is responsible for lowering the incidence of these cancers (Adlercreutz et al, 1995; 429
Yoshimura: Genistein induces apoptosis in T-ALL cells 1999), prostate (Geller et al, 1998; Li and Sarkar, 2002), and leukemia (Traganos et al, 1992; Spinozzi et al, 1994; Baxa and Yoshimura, 2003). It has furthermore been demonstrated for many of these cell types that cell killing occurs via apoptosis (Alhasan et al, 1999; Spinozzi et al, 1994; Davis et al, 1998; Li et al, 1999b;). Tumor cellkilling by genistein could be potentially exploited in the treatment of cancers that have become resistant to chemotherapeutic drugs and radiation therapy. The potential use of genistein as a treatment for cancer was initially recognized by the observation that this reagent is able to specifically inhibit tyrosine kinases (Akiyama et al, 1987), a class of proteins that are frequently involved in the regulation of cellular proliferation (Ullrich and Schlessinger, 1990). Besides this activity, additional studies have demonstrated that genistein can also inhibit DNA topoisomerase II, angiogenesis, metastasis, protein-histidine kinase, and 5!-reductase (Okura et al, 1988; Huang et al, 1992; Evans et al, 1995; Fotsis et al, 1995; Li et al, 1999a). Other activities of genistein include the downregulation of activated NF-"B and an inhibition of the Akt signaling pathway (Davis et al, 1999; Li and Sarkar, 2002; Baxa and Yoshimura, 2003; Gong et al, 2003). Which of these multiple activities of genistein is responsible for the induction of apoptosis most likely depends on the particular malignant cell type. Studies of the effect of genistein on malignant cells have been performed predominantly on solid tumors, such as breast and prostate (Kyle et al, 1997; Constantinou et al, 1998; Shao et al, 1998; Li et al, 1999b). Although fewer studies have been conducted on hematopoietic malignancies, it has been shown that genistein induces apoptosis in human T-cell lymphomas as well (Traganos et al, 1992; Spinozzi et al, 1994; Hayon et al, 2003). The mechanism of the induction of apoptosis in T-cells and other cell types is not well understood. To identify some of the early steps in the induction of apoptosis by genistein, we examined murine T-leukemia cell lines and demonstrated that an early step in apoptosis induction is damage to the mitochondrial membrane (Baxa and Yoshimura, 2004). Furthermore, we observed that mitochondrial damage resulted in more downstream apoptotic events, such as activation of caspase-9 and caspase-3, as well as DNA fragmentation. To determine whether this early effect of genistein in murine cells also occurs in human T-leukemia cells, in this study we examined two acute lymphoblastic leukemia (T-ALL) cell lines, Jurkat (Schneider et al, 1977) and CCRF-CEM (Foley et al, 1965). T-ALL accounts for 10 to 15% of newly diagnosed cases of childhood acute lymphoblastic leukemia, but it has a higher relapse rate than in patients with B-cell leukemias (Goldberg et al, 2003). Although with aggressive combinations of cytotoxic chemotherapeutic drugs and radiation, the 5-year survival rate has now reached 75% (Goldberg et al, 2003), there is still a need for more effective and less toxic treatments for these patients and those with refractory disease. Two well-characterized pathways for apoptosis induction are the extrinsic and intrinsic pathway. The extrinsic pathway involves cell surface signaling, such as
in the case of Fas/Fas L and the TNF-! receptor (Ashkenazi and Dixit, 1998), while the intrinsic pathway involves mitochondrial damage (Traganos et al, 1992; Spinozzi et al, 1994; Green and Reed, 1998; Desagher and Martinou, 2000). Activation of the intrinsic pathway is initiated by damage to the mitochondrial membrane, leading to the release of cytochrome c into the cytoplasm (Martinou et al, 2000). Released cytochrome c subsequently binds the apoptosis activating factor-1 (Apaf-1) and ATP, which results in the recruitment and activation of procaspase-9 (Pan et al, 1998). Caspase-9 activation subsequently results in other more downstream events of apoptosis, such as activation of caspase-3, which in turn is responsible for the execution of additional features of apoptosis (Thornberry and Lazebnik, 1998). A mechanism by which mitochondrial damage can occur involves the sustained opening of the mitochondrial permeability transition pore (PTP) (Kroemer et al, 1998; Bernardi et al, 1999; Desagher and Martinou, 2000). Opening of the PTP leads to mitochondrial depolarization and the influx of solutes, which result in outer membrane damage and the release of cytochrome c (Hirsch et al, 1998). Certain stress factors, such as an increase in intracellular calcium, generation of reactive oxygen species, and changes in cytosolic pH, induce mitochondrial damage via the PTP (Hirsch et al, 1998). In this study, we have made the novel observation that one of the early steps in the induction of apoptosis by genistein in human T-ALL cells involves mitochondrial damage via the permeability transition pore, thus implicating an early involvement of the intrinsic pathway.
II. Materials and methods A. Cell culture Jurkat (TIB-152) and CCRF-CEM (CCL-119) cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were maintained in RPMI 1640 supplemented with Lglutamine, 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 10 mM Hepes, and 20 units per ml penicillinstreptomycin at 37°C and 5% CO2. Cells were cultured at 5 x 105 cells per ml 24 hr prior to the addition of genistein (Toronto Research Chemicals, Toronto, Canada). Cells were washed once with RPMI 1640 and resuspended at 2 x 105 cells per ml at the time of genistein exposure. A genistein stock solution was prepared in DMSO at a concentration of 100 mM. The same volume of DMSO was added to control cells, resulting in a final concentration of DMSO of less than 0.05%. For bongkrekic acid studies, cells were treated with 150 µM bongkrekic acid (EMD Biosciences, Inc., La Jolla, CA) for 1 hr at 37!C prior to the addition of genistein.
B. Flow cytometric analysis of apoptotic cells and mitochondrial depolarization 1. Detection of apoptotic cells by Annexin V-FITC and PI cell-staining A quantitative analysis of viable, dead, and apoptotic cell populations was conducted by staining cells with propidium iodide (PI) (Sigma-Aldrich, St. Louis, MO) and fluorescein isothiocyanate-conjugated annexin V (annexin V-FITC) (PharMingen, BD Biosciences, San Diego, CA). 105 cells were washed twice with cold phosphate buffered saline (PBS) and stained with 0.5 µg per ml PI and 0.6 "g per ml annexin V-FITC in binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM
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Cancer Therapy Vol 2, page 431 CaCl2). Cells were incubated at room temperature (RT) in the dark for 15 min, after which time 400 µl of binding buffer was added. Cells were analyzed by flow cytometry using a Becton Dickinson FACScan flow cytometer (Wayne State University and Karmanos Cancer Institute Flow Cytometry Core Facility) within 1 hr of staining. Data were collected on 2 x 104 cells using the CELLQuest software (Becton Dickinson, BD Biosciences). Gating was established on single color controls.
early times after exposure, we undertook this study of human T-ALL cells to identify the early steps involved in cell death signaling. We chose this leukemic cell type to study because an understanding of the mechanism of cell killing by genistein should provide insights into its potential use for the treatment of this type of childhood Tcell leukemia, which normally has a poorer prognosis than childhood leukemia of B-cell origin (DeVita, 2001). For this study we chose the Jurkat and CCRF-CEM cell lines, which were derived from two different patients with childhood T-cell leukemia (Foley et al, 1965; Schneider et al, 1977). Similar to previous results from other laboratories, we observed that genistein induced cell killing in Jurkat and CCRF-CEM cells at concentrations of 15 µM and greater in a dosage- and time-dependent manner (Figure 1A and B). In addition, flow cytometric analysis of cells stained with Annexin V-FITC and PI confirmed that cell killing occurred via apoptosis (Figure 1C and D). It has been shown that flow cytometric analysis of cells stained with Annexin V and PI is an effective method to distinguish between live (PI-, annexin V-), apoptotic (PI-, annexin V+), and dead (PI+, annexin V+) cells (Koopman et al, 1994). Our detection of apoptosis for Jurkat cells confirmed the results of Spinozzi et al, 1994, who examined Jurkat cells at 48 hr after genistein treatment. Although the percentage of apoptotic cells peaked at 48 hr for both cell lines, our data showed that apoptosis occurred at an earlier time as well. As an independent assay for apoptosis, we examined caspase-3 activation, which is a hallmark of apoptosis that occurs via both the extrinsic and intrinsic pathways (Thornberry and Lazebnik, 1998). Activation of caspase-3 occurs by cleavage of the 35 kDa procaspase form to 17 kDa and 12 kDa protein products (Nicholson et al, 1995; Sun et al, 1999). Protein extracts were prepared from Jurkat and CCRF-CEM cells treated with 60 µM genistein for 24, 48, and 72 hr. The 35 kDa procaspase form and 17 kDa cleavage product were detectable by immunoblotting with a polyclonal antibody specific for caspase-3 (Cell Signaling Technology, Inc.) (Figure 2). An intermediate 19 kDa cleavage product was also detectable for our cell extracts. For both cell lines, we detected increasing amounts of the 19 kDa and 17 kDa cleavage products over time, which confirmed that genistein was inducing apoptosis in these cells. The largest amount of activated caspase-3 was detectable at 72 hr of genistein treatment for both cell lines (Figure 2A and B, lane 6). As a positive control for caspase-3 activation, we treated Jurkat and CCRF-CEM cells for 4 hr with 1 µM staurosporine, which is known to activate caspase-3 and induce apoptosis (Bijur, 2000) (Figure 2A and B, lane 7).
2. Detection of mitochondrial depolarization by JC-1 cell-staining 5,5’,6,6’-tetrachloro-1,1’,3,3’ tetraethylbenzimidazoly lcarbocyanine iodide (JC-1, Molecular Probes, Inc., Eugene, OR) was used as an indicator dye for mitochondrial depolarization. JC-1 was added to cells in culture to a final concentration of 5 µg per ml for 15 min at RT. Stained cells were pelleted, washed twice in cold PBS, and resuspended in 500 µl PBS. As a positive control, cells were treated with valinomycin (Sigma-Aldrich) at 4 µg per ml final concentration for 4 hr at 37!C. Cells were analyzed by two-color flow cytometry. Gating was established with untreated and valinomycin treated cells.
C. Immunoblot analysis of caspase-3 and caspase9 2 x 107 cells were collected, washed twice with PBS, and lysed in 200 µl 50 mM Tris-HCl, pH 7.5, containing 0.03% Nonidet P-40 and 1mM dithiothreitol. For positive controls for caspase cleavage, cells were treated with 1 µM staurosporine (Sigma-Aldrich) for 4 hr at 37#C. Cellular extracts were prepared as described (Pazirandeh et al, 2000). Protein amounts were measured using the bicinchoninic acid protein assay (Pierce, Rockford, IL). 50 µg protein was added to reducing buffer (62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 2% sodium dodecylsulfate (SDS), 0.01% bromophenol blue, 5% #-mercaptoethanol) and boiled for 4 min. Samples were electrophoresed in running buffer of 25 mM Tris, 192 mM glycine, 1% SDS, pH 8.3, through a 12% or 15% SDS-polyacrylamide gel at 120 volts for 1 hr and transferred to polyvinylidene difluoride membrane (BioRad, Hercules, CA) at 350 mAmp for 2 hr at 4#C. Membranes were probed with a rabbit antibody specific for either caspase-3, caspase-9 (Cell Signaling Technology, Beverly, MA), or #-actin (Sigma-Aldrich) overnight at 4#C. After washing, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit serum (Pierce) for 1 hr at RT. Protein bands were detected by enhanced chemiluminescence (ECL, Amersham Pharmacia, Piscataway, NJ) and visualized on Biomax MR film (Kodak, Rochester, NY).
III. Results A. Kinetic analysis of the effects of genistein on the growth of T-ALL cell lines Most of the previous studies of the effects of genistein on human T-cell leukemia lines have identified cellular changes that occurred after 24 hr or more of exposure (Traganos et al, 1992; Spinozzi et al, 1994; Markovits et al, 1995). Various studies have shown that after this exposure time, genistein has multiple effects on cells, including cell cycle arrest, inhibition of tyrosine kinases and DNA topoisomerase II, downregulation of NF-"B, and induction of apoptosis (Akiyama et al, 1987; Okura et al, 1988; Traganos et al, 1992; Spinozzi et al, 1994; Davis et al, 1999; Polkowski and Mazurek, 2000; Baxa and Yoshimura, 2003). Because there are few studies of some of the effects that genistein produces in cells at
B. Genistein induces mitochondrial depolarization via the permeability transition pore In a study of the effects of genistein on murine thymic lymphoma cells, we observed that mitochondrial damage occurred as an early step in the induction of apoptosis (Baxa and Yoshimura, 2004). To determine
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Yoshimura: Genistein induces apoptosis in T-ALL cells
Figure 1. Genistein kills T-ALL cells via apoptosis. Number of viable Jurkat (A) or CCRF-CEM (B) cells after treatment with different concentrations of genistein for various times. 4 x 105 cells for each cell line were treated with genistein at concentrations ranging from 0 to 60 µM. Cells were collected and stained with trypan blue at days 1 through 4 after genistein exposure. Percentage of apoptotic Jurkat (C) or CCRF-CEM (D) cells treated with 60 µM genistein for various times. Percentage of apoptotic cells was determined by flow cytometric analysis of cells stained with annexin V-FITC and PI. The results shown are the mean values and standard deviations calculated from duplicate samples from two independent experiments.
Figure 2. Caspase-3 activation by genistein. Jurkat (A) or CCRF-CEM (B) cells were treated with 60 µM genistein for 24, 48, and 72 hr. 50 µg cell extracts were analyzed by Western blotting with an antibody specific for caspase-3 (Cell Signaling Technology, Inc.). C, control cells; G, genistein-treated cells. STS, staurosporine-treated cells for caspase-3 cleavage control. Arrows indicate the 35 kDa procaspase-3 form, and 19 kDa and 17 kDa cleavage products. #-actin was detected for loading control.
whether this is also an early effect of genistein in T-ALL cells, we used the mitochondrion-specific dye JC-1 (Molecular Probes) to detect mitochondrial membrane damage. JC-1 was chosen for this analysis because it selectively enters normal mitochondria where it forms red fluorescent J-aggregates (Reers et al, 1991; Salvioli et al, 1997). Upon damage to the mitochondrial membrane, which results in a decrease in the transmembrane potential, JC-1 aggregates are disrupted, and monomers that emit a green fluorescence are produced. Thus, a shift of
fluorescence in cells from red to green is an indication of depolarization of the mitochondrial membrane. Jurkat and CCRF-CEM cells were treated with 60 µM genistein for 4 hr before JC-1 staining. Flow cytometric analysis of Jurkat cells showed that a total of 69.5% of Jurkat cells suffered mitochondrial damage as a result of genistein treatment (Figure 3B). Of these cells, 41.3% displayed an increase in green fluorescence, and 28.2% displayed both an increase in green fluorescence and decrease in red fluorescence. These populations
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Cancer Therapy Vol 2, page 433 represented only 1.4% of control cells (Figure 3A). As a positive control for JC-1 staining, we exposed cells to 4 µg per ml valinomycin, a potassium ionophore, that produces
mitochondrial depolarization (Inai et al, 1997). For valinomycin-treated cells, mitochondrial depolarization occurred in a total of 94.4% of cells (Figure 3C). Figure 3. Genistein causes mitochondrial depolarization in Jurkat cells. 106 cells were treated for 4 hr with either DMSO (A), 60 µM genistein (B), or 4 µg per ml valinomycin (C). JC-1 (5 µg per ml final concentration) was subsequently added to cells for 15 min, after which time two-color flow cytometric analysis was performed. Number in each quadrant indicates the percentage of total cells analyzed. Dot plots are representative data from three independent experiments.
Figure 4. CCRF-CEM cells undergo mitochondrial depolarization by genistein. 106 cells were treated for 4 hr with either DMSO (A), 60 µM genistein (B), or 4 µg per ml valinomycin (C). Cell analysis was performed and displayed as described for Figure 3.
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Yoshimura: Genistein induces apoptosis in T-ALL cells These results thus indicate that mitochondrial depolarization occurred as early as 4 hr after exposure to genistein in Jurkat cells. To determine whether mitochondrial depolarization is an early step that occurs in other T-ALL cells, we performed a similar analysis of CCRF-CEM cells. Flow cytometric analysis of cells treated with 60 µM genistein for 4 hr and subsequently stained with JC-1showed that a total of 66.8% of cells suffered mitochondrial damage (Figure 4B). This was in contrast to a total of only 0.5% of untreated cells (Figure 4A).
examine whether a similar mechanism is involved in the damage to mitochondria by genistein in T-ALL cells, we determined whether bongkrekic acid (BA), a specific inhibitor of the mitochondrial PTP (Zamzami et al, 1996), could reverse the mitochondrial depolarization induced by genistein. As we had seen previously, treatment of Jurkat cells with 60 µM genistein resulted in mitochondrial depolarization in a significant percentage of cells as detectable by JC-1 staining and flow cytometric analysis (Figure 5C). In this representative experiment, genistein produced mitochondrial depolarization in a total of 48.3% of Jurkat cells (Figure 5C), in contrast to control cells, which showed little mitochondrial damage (Figure 5A). On the other hand, Jurkat cells pretreated with 150 µM BA for 1 hr prior to a further 4 hr incubation with genistein (Figure 5D) resembled cells treated with 150 µM BA alone (Figure 5B).
C. Mitochondrial depolarization occurs via the permeability transition pore We and others have previously observed that genistein is able to induce mitochondrial depolarization in some cell types as a result of association with the membrane PTP (Yoon et al, 2000; Salvi et al, 2002). To
Figure 5. Mitochondrial depolarization occurs via the permeability transition pore in Jurkat cells. 106 Jurkat cells were either untreated (A), treated with 150 µM bongkrekic acid (BA) for 5 hr (B), treated with 60 µM genistein for 4 hr with no BA pretreatment (C), or pretreated with 150 µM BA for 1 hr before the addition of 60 µM genistein for an additional 4 hr (D). Cells were subsequently stained with JC-1 and analyzed by two-color flow cytometry as described for Figure 3. Number in each quadrant indicates the percentage of total cells analyzed. Dot plots are representative data from two independent experiments.
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Figure 6. Involvement of permeability transition pore in mitochondrial depolarization in CCRF-CEM cells. 106 CCRF-CEM cells were either untreated (A), treated with 150 µM bongkrekic acid (BA) for 5 hr (B), treated with 60 µM genistein for 4 hr with no BA pretreatment (C), or pretreated with 150 µM BA for 1 hr before the addition of 60 µM genistein for an additional 4 hr (D). Cell analysis was performed and displayed as described for Figure 5.
These data indicate that under these conditions BA nearly completely inhibited mitochondrial depolarization induced by genistein, suggesting that mitochondrial damage occurred via deregulation of the PTP. Analysis of CCRFCEM cells showed that the PTP is similarly involved in mitochondrial depolarization by genistein in these cells (Figure 6). Treatment with genistein alone induced mitochondrial depolarization in 77.5% of CCRF-CEM cells (Figure 6C). In cells pretreated with BA before the addition of genistein, only 4.8% of cells experienced mitochondrial depolarization (Figure 6D).
D. Genistein caspase-9
induces
activation
extracts from Jurkat and CCRF-CEM cells at various times after exposure to 60 µM genistein (Figure 7). Immunoblot analysis of Jurkat cell extracts with an antibody specific for caspase-9 (Cell Signaling Technology, Inc.) revealed an initial caspase-9 cleavage product of 37 kDa after 24 hr of genistein treatment (Figure 7A, lane 2). We detected an increase in this cleavage product with continuous exposure to genistein (lanes 4 and 6). At 48 hr of genistein exposure, we began to see a smaller 35 kDa cleavage protein, which corresponds to the active enzyme (lane 4) (Li et al, 1997; Sun et al, 1999). Both cleavage proteins began to appear in CCRF-CEM cells after 24 hr of genistein treatment (Figure 7B, lane 2). Both products increased with treatment time (lanes 4 and 6). It is not clear why procaspase-9 is processed differently in the two cell lines. As a positive control, cells were treated with 1 µM staurosporine, which induces apoptosis via the intrinsic pathway and produces caspase-9 cleavage (Figure 7A and B, lane 7) (Bijur et al, 2000; Scarlett et al, 2000).
of
Our observation that genistein produces mitochondrial damage as an early event in T-ALL cells suggested that apoptosis proceeds via the intrinsic pathway. To further examine this idea, we evaluated caspase-9 activation, which occurs as a result of cytochrome c release from damaged mitochondria (Li et al, 1997). To perform this analysis, we examined protein
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Yoshimura: Genistein induces apoptosis in T-ALL cells
Figure 7. Caspase-9 activation in T-ALL cells treated with genistein. Western blot detection of caspase-9 from Jurkat (A) or CCRFCEM (B) cells treated with 60 ÂľM genistein for 24, 48, and 72 hr. 50 Âľg cell extracts were analyzed with an antibody specific for caspase-9 (Cell Signaling Technology, Inc.). C, control cells; G, genistein-treated cells. STS, staurosporin-treated cells for caspase-9 cleavage control. Arrows indicate the 47 kDa procaspase-9 form, and 37 kDa and 35 kDa cleavage products. #-actin was detected for loading control.
Taken together, our results demonstrate that genistein is able to kill cultured human T-ALL cells in a time- and dosage-dependent manner. Furthermore, cell killing by genistein can be attributed to the induction of apoptosis, which involves mitochondrial membrane damage soon after exposure. Our data suggest that the intrinsic apoptotic pathway is activated by genistein as an early step in the killing of T-ALL cells.
analysis of cells treated with bongkrekic acid suggested that damage to mitochondria occurred via the mitochondrial membrane pore. Yoon et al, 2000 showed that this also occurred in genistein-treated RPEJ neuronal cells. A study of the effect of genistein on isolated rat liver mitochondria also supports the idea that genistein can target mitochondrial membrane pores to induce damage in cells (Salvi et al, 2002). The induction of apoptosis via mitochondrial damage has been well-studied and is known to be initiated by the release of cytochrome c from the mitochondrial membrane into the cytoplasm (Martinou et al, 2000). The subsequent binding of cytochrome c to Apaf-1results in the activation of caspase-9, which in turn, is involved in the execution of additional steps in the apoptosis cascade, such as caspase3 activation (Pan et al, 1998; Thornberry and Lazebnik, 1998). In our analysis of T-ALL cells, caspase-9 activation was detectable at 24 hr after the addition of genistein (Figure 7). Furthermore, our analysis showed that caspase-3 activation occurred after the activation of caspase-9 (Figure 2). This sequence of events suggests that the induction of apoptosis by genistein in T-ALL cells follows the steps commonly observed for the intrinsic pathway involving mitochondrial damage. Acute lymphoblastic leukemia, which is derived from either a precursor T or B cell, accounts for approximately 75% of childhood leukemias (DeVita, 2001). The prognosis for ALL of precursor T-cell origin is typically worse than for B-cell acute lymphoblastic leukemia (B-ALL) (DeVita, 2001). Even with aggressive chemotherapy, patients with T-ALL are less likely to enter remission and relapse more quickly than those with BALL (Goldberg et al, 2003). Our results from this study suggest that genistein may be effective in the treatment of children with T-ALL because of its ability to induce apoptosis via damage to the mitochondrial membrane.
IV. Discussion Previous studies have demonstrated that genistein is able to induce apoptosis in different types of tumor cells (Traganos et al, 1992; Spinozzi et al, 1994; Barnes, 1995; Zava and Duwe, 1997; Geller et al, 1998; Lian et al, 1998; Alhasan et al, 1999; Li et al, 1999b). Various cellular effects of genistein, such as cell cycle arrest, the inhibition of tyrosine kinases, downregulation of NF-"B, inhibition of Akt kinase, and mitochondrial damage (Akiyama et al, 1987; Traganos et al, 1992; Spinozzi et al, 1994; Davis et al, 1999; Polkowski and Mazurek, 2000; Yoon et al, 2000; Salvi et al, 2002; Baxa and Yoshimura, 2003; Gong et al, 2003) may potentially contribute to apoptosis induction. Another commonly observed effect of genistein, i.e., inhibition of topoisomerase II (Okura et al, 1988; Polkowski and Mazurek, 2000; Sarkar and Li, 2003), does not appear to be involved in apoptosis induction (Salti et al, 2000). Many of the cellular effects of genistein have been detected after exposure of cells to genistein for 24 hr or longer. In this study, our goal was to identify some of the earlier events that occur in T-ALL cells after exposure to genistein to better understand the mechanism of action involved in the induction of apoptosis. This information would be useful for the assessment of the potential use of genistein in the treatment of this type of early childhood leukemia. Our assays for cell killing and apoptosis confirmed observations by others that pharmacological concentrations of genistein are able to induce apoptosis in T-ALL cells in a dosage- and time-dependent manner. More importantly, examination of genistein-treated cells before apoptosis could be detected led to our novel observation that mitochondrial damage was an early step in apoptotic signaling. Furthermore, flow cytometric
Acknowledgements The author would like to thank Ruchi Rastogi for her technical assistance, and the members of the Flow Cytometry Core Facility, supported by Wayne State University and the Karmanos Cancer Institute, for their
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Cancer Therapy Vol 2, page 437 Fotsis T, Pepper M, Adlercreutz H, Hase T, Montesano R and Schweigerer L (1995) Genistein, a dietary ingested isoflavonoid, inhibits cell proliferation and in vitro angiogenesis. J Nutr 125, 790S-797S. Geller J, Sionit L, Partido C, Li L, Tan X, Youngkin T, Nachtsheim D and Hoffman RM (1998) Genistein inhibits the growth of human-patient BPH and prostate cancer in histoculture. Prostate 34, 75-79. Goldberg JM, Silverman LB, Levy DE, Dalton VK, Gelber RD, Lehmann L, Cohen HJ, Sallan SE and Asselin BL (2003) Childhood T-cell acute lymphoblastic leukemia: the DanaFarber Cancer Institute acute lymphoblastic leukemia consortium experience. J Clin Oncol 21, 3616-3622. Gong L, Li Y, Nedeljkovic-Kurepa A and Sarkar FH (2003) Inactivation of NF-"B by genistein is mediated via Akt signaling pathway in breast cancer cells. Oncogene 22, 4702-4709. Green DR and Reed JC (1998) Mitochondria and apoptosis. Science 281, 1309-1312. Hayon T, Atlas L, Levy E, Dvilansky A, Shpilberg O and Nathan I (2003) Multifactorial activities of nonsteroidal antiestrogens against leukemia. Cancer Detect Prev 27, 389-396. Hebert JR, Hurley TG, Olendzki BC, Teas J, Ma Y and Hampl JS (1998) Nutritional and socioeconomic factors in relation to prostate cancer mortality: a cross-national study. J Natl Cancer Inst 90, 1637-1647. Hirose K, Tajima K, Hamajima N, Inoue M, Takezaki T, Kuroishi T, Yoshida M and Tokudome S (1995) A largescale, hospital-based case-control study of risk factors of breast cancer according to menopausal status. Jpn J Cancer Res 86, 146-154. Hirsch T, Susin SA, Marzo I, Marchetti P, Zamzami N and Kroemer G (1998) Mitochondrial permeability transition in apoptosis and necrosis. Cell Biol Toxicol 14, 141-145. Huang J, Nasr M, Kim Y and Matthews HR (1992) Genistein inhibits protein histidine kinase. J Biol Chem 267, 1551115515. Inai Y, Yabuki M, Kanno T, Akiyama J, Yasuda T and Utsumi K (1997) Valinomycin induces apoptosis of ascites hepatoma cells (AH-130) in relation to mitochondrial membrane potential. Cell Struct Funct 22, 555-563. Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen R, Pals ST and van Oers MH (1994) Annexin V for flow cytometric detection of phosphatidylserine expression on B, cells undergoing apoptosis. Blood 84, 1415-1420. Kroemer G, Dallaporta B and Resche-Rigon M (1998) The mitochondrial death/life regulator in apoptosis and necrosis. Annu Rev Physiol 60, 619-642. Kyle E, Neckers L, Takimoto C, Curt G and Bergan R (1997) Genistein-induced apoptosis of prostate cancer cells is preceded by a specific decrease in focal adhesion kinase activity. Mol Pharmacol 51, 193-200. Lamartiniere CA, Moore J, Holland M and Barnes S (1995a) Neonatal genistein chemoprevents mammary cancer. Proc Soc Exp Biol Med 208, 120-123. Lamartiniere CA, Moore JB, Brown NM, Thompson R, Hardin MJ and Barnes S (1995b) Genistein suppresses mammary cancer in rats. Carcinogenesis 16, 2833-2840. Lamartiniere CA, Cotroneo MS, Fritz WA, Wang J, MentorMarcel R and Elgavish A (2002) Genistein chemoprevention: timing and mechanisms of action in murine mammary and prostate. J Nutr 132, 552S-558S. Lee HP, Gourley L, Duffy SW, Esteve J, Lee J and Day NE (1991) Dietary effects on breast-cancer risk in Singapore [see comments]. Lancet 337, 1197-1200. Lee HP, Gourley L, Duffy SW, Esteve J, Lee J and Day NE (1992) Risk factors for breast cancer by age and menopausal
help. This work was supported in part by the Childrenâ&#x20AC;&#x2122;s Research Center of Michigan, Detroit, MI.
References Adlercreutz CH, Goldin BR, Gorbach SL, Hockerstedt KA, Watanabe S, Hamalainen EK, Markkanen MH, Makela TH, Wahala KT and Adlercreutz T (1995) Soybean phytoestrogen intake and cancer risk. J Nutr 125, 757S770S. Adlercreutz H, Markkanen H and Watanabe S (1993) Plasma concentrations of phyto-oestrogens in Japanese men. Lancet 342, 1209-1210. Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe S, Itoh N, Shibuya M and Fukami Y (1987) Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem 262, 5592-5595. Alhasan SA, Pietrasczkiwicz H, Alonso MD, Ensley J and Sarkar FH (1999) Genistein-induced cell cycle arrest and apoptosis in a head and neck squamous cell carcinoma cell line. Nutr Cancer 34, 12-19. Ashkenazi A and Dixit VM (1998) Death receptors: signaling and modulation. Science 281, 1305-1308. Barnes S (1995) Effect of genistein on in vitro and in vivo models of cancer. J Nutr 125, 777S-783S. Baxa DM and Yoshimura FK (2003) Genistein reduces NF-"B in T lymphoma cells via a caspase-mediated cleavage of I"B!. Biochem Pharmacol 66, 1009-1018. Baxa DM and Yoshimura FK (2004) Genistein induces apoptosis in T lymphoma cells via mitochondrial damage. Nutr Cancer In Press. Bernardi P, Scorrano L, Colonna R, Petronilli V and Di Lisa F (1999) Mitochondria and cell death. Mechanistic aspects and methodological issues. Eur J Biochem 264, 687-701. Bijur GN, De Sarno P and Jope RS (2000) Glycogen synthase kinase-3# facilitates staurosporine- and heat shock-induced apoptosis. Protection by lithium. J Biol Chem 275, 75837590. Constantinou AI, Kamath N and Murley JS (1998) Genistein inactivates bcl-2, delays the G2/M phase of the cell cycle and induces apoptosis of human breast adenocarcinoma MCF-7 cells. Eur J Cancer 34, 1927-1934. Dai Q, Shu XO, Jin F, Potter JD, Kushi LH, Teas J, Gao YT and Zheng W (2001) Population-based case-control study of soyfood intake and breast cancerrisk in Shanghai. Br J Cancer 85, 372-378. Davis JN, Kucuk O and Sarkar FH (1999) Genistein inhibits NF"B, activation in prostate cancer cells. Nutr Cancer 35, 167174. Davis JN, Singh B, Bhuiyan M and Sarkar FH (1998) Genisteininduced upregulation of p21WAF1, downregulation of cyclin B and induction of apoptosis in prostate cancer cells. Nutr Cancer 32, 123-131. Desagher S and Martinou JC (2000) Mitochondria as the central control point of apoptosis. Trends Cell Biol 10, 369-377. DeVita Jr VT, Helms S and Rosenberg SA (2001) Cancer. Principles and practice of oncology. Lippincott-Raven Publishers, Philadelphia, PA. Evans BA, Griffiths K and Morton MS (1995) Inhibition of 5 alpha-reductase ingenital skin fibroblasts and prostate tissue by dietary lignans and isoflavonoids. J Endocrinol 147, 295302. Foley GE, Lazarus H, Farber S, Uzman BG, Boone BA and McCarthy RE (1965) Continuous culture of human lymphoblasts from peripheral blood of a child with acute leukemia. Cancer 18, 522-529.
437
Yoshimura: Genistein induces apoptosis in T-ALL cells status: a case-control study in Singapore. Cancer Causes Control 3, 313-322. Li D, Yee JA, McGuire MH, Murphy PA and Yan L (1999a) Soybean isoflavones reduce experimental metastasis in mice. J Nutr 129, 1075-1078. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES and Wang X (1997) Cytochrome C and dATPdependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91, 479-489. Li Y and Sarkar FH (2002) Inhibition of NF"B activation in PC3 cells by genistein is mediated via Akt signaling pathway. Clin Cancer Res 8, 2369-2377. Li Y, Upadhyay S, Bhuiyan M and Sarkar FH (1999b) Induction of apoptosis in breast cancer cells MDA-MB-231 by genistein. Oncogene 18, 3166-3172. Lian F, Bhuiyan M, Li YW, Wall N, Kraut M and Sarkar FH (1998) Genistein-induced G2-M arrest, p21WAF1 upregulation and apoptosis in a non-small-cell lung cancer cell line. Nutr Cancer 31, 184-191. Lian F, Li Y, Bhuiyan M and Sarkar FH (1999) p53-independent apoptosis induced by genistein in lung cancer cells. Nutr Cancer 33, 125-131. Markovits J, Junqua S, Goldwasser F, Venuat AM, Luccioni C, Beaumatin J, Saucier JM, Bernheim A and JacqueminSablon A (1995) Genistein resistance in human leukaemic CCRF-CEM cells: selection of a diploid cell line with reduced DNA topoisomerase II # isoform. Biochem Pharmacol 50, 177-186. Martinou JC, Desagher S and Antonsson B (2000) Cytochrome C, release from mitochondria: all or nothing. Nat Cell Biol 2, E41-43. Mentor-Marcel R, Lamartiniere CA, Eltoum IE, Greenberg NM, Elgavish A (2001) Genistein in the diet reduces the incidence of poorly differentiated prostatic adenocarcinoma in transgenic mice (TRAMP). Cancer Res 61, 6777-6782. Messina MJ (1999) Legumes and soybeans: overview of their nutritional profiles and health effects. Am J Clin Nutr 70, 439S-450S. Messina MJ (2003) Emerging evidence on the role of soy in reducing prostate cancer risk. Nutr Rev 61, 117-131. Mills PK, Beeson WL, Phillips RL and Fraser GE (1989) Cohort study of diet, lifestyle and prostate cancer in Adventist men. Cancer 64, 598-604. Mizunuma H, Kanazawa K, Ogura S, Otsuka S and Nagai H (2002) Anticarcinogenic effects of isoflavones may be mediated by genistein in mouse mammary tumor virusinduced breast cancer. Oncology 62, 78-84. Nicholson DW, Ali A, Thornberry NA, Vaillancourt JP, Ding CK, Gallant M, Gareau Y, Griffin PR, Labelle M, Lazebnik YA (1995) Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis [see comments]. Nature 376, 37-43. Okura A, Arakawa H, Oka H, Yoshinari T and Monden Y (1988) Effect of genistein on topoisomerase activity and on the growth of [Val 12]Ha-ras-transformed NIH 3T3 cells. Biochem Biophys Res Commun 157, 183-189. Pan G, O'Rourke K and Dixit VM (1998) Caspase-9, Bcl-XL and Apaf-1 form a ternary complex. J Biol Chem 273, 58415845. Pazirandeh A, Xue Y, Okret S and Jondal M (2000) Glucocorticoid resistance in thymocytes from mice expressing a T cell receptor transgene. Biochem Biophys Res Commun 276, 189-196. Polkowski K and Mazurek AP (2000) Biological properties of genistein. A review of in vitro and in vivo data. Acta Pol Pharm 57, 135-155.
Reers M, Smith TW and Chen LB (1991) J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry 30, 4480-4486. Salti GI, Grewal S, Mehta RR, Das Gupta TK, Boddie AW Jr and Constantinou AI (2000) Genistein induces apoptosis and topoisomerase II-mediated DNA breakage in colon cancer cells. Eur J Cancer 36, 796-802. Salvi M, Brunati AM, Clari G and Toninello A (2002) Interaction of genistein with the mitochondrial electron transport chain results in opening of the membrane transition pore. Biochim Biophys Acta 1556, 187-196. Salvioli S, Ardizzoni A, Franceschi C and Cossarizza A (1997) JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess $% changes in intact cells: implications for studies on mitochondrial functionality during apoptosis. FEBS Lett 411, 77-82. Sarkar FH and Li Y (2003) Soy isoflavones and cancer prevention. Cancer Invest 21, 744-757. Scarlett JL, Sheard PW, Hughes G, Ledgerwood EC, Ku HH and Murphy MP (2000) Changes in mitochondrial membrane potential during staurosporine-induced apoptosis in Jurkat cells. FEBS Lett 475, 267-272. Schneider U, Schwenk HU and Bornkamm G (1977) Characterization of EBV-genome negative "null" and "T" cell lines derived from children with acute lymphoblastic leukemia and leukemic transformed non-Hodgkin lymphoma. Int J Cancer 19, 621-626. Shao ZM, Wu J, Shen ZZ and Barsky SH (1998) Genistein exerts multiple suppressive effects on human breast carcinoma cells. Cancer Res 58, 4851-4857. Spinozzi F, Pagliacci MC, Migliorati G, Moraca R, Grignani F, Riccardi C and Nicoletti I (1994) The natural tyrosine kinase inhibitor genistein produces cell cycle arrest and apoptosis in Jurkat T-leukemia cells. Leuk Res 18, 431-439. Sun XM, MacFarlane M, Zhuang J, Wolf BB Green DR and Cohen GM (1999) Distinct caspase cascades are initiated in receptor-mediated and chemical-induced apoptosis. J Biol Chem 274, 5053-5060. Thornberry NA and Lazebnik Y (1998) Caspases: enemies within. Science 281, 1312-1316. Traganos F, Ardelt B, Halko N, Bruno S and Darzynkiewicz Z (1992) Effects of genistein on the growth and cell cycle progression of normal human lymphocytes and human leukemic MOLT-4 and HL-60 cells. Cancer Res 52, 62006208. Ullrich A and Schlessinger J (1990) Signal transduction by receptors with tyrosine kinase activity. Cell 61, 203-212. Wang J, Eltoum IE and Lamartiniere CA (2002) Dietary genistein suppresses chemically induced prostate cancer in Lobund-Wistar rats. Cancer Lett 186, 11-18. Wu AH, Ziegler RG, Horn-Ross PL, Nomura AM, West DW, Kolonel LN, Rosenthal JF, Hoover RN and Pike MC (1996) Tofu and risk of breast cancer in Asian-Americans. Cancer Epidemiol Biomarkers Prev 5, 901-906. Yamamoto S, Sobue T, Kobayashi M, Sasaki S and Tsugane S (2003) Soy, isoflavones and breast cancer risk in Japan. J Natl Cancer Inst 95, 906-913. Yoon HS, Moon SC, Kim ND, Park BS, Jeong MH and Yoo YH (2000) Genistein induces apoptosis of RPE-J cells by opening mitochondrial PTP. Biochem Biophys Res Commun 276, 151-156. Zamzami N, Marchetti P, Castedo M, Hirsch T, Susin SA, Masse B and Kroemer G (1996) Inhibitors of permeability transition interfere with the disruption of the mitochondrial transmembrane potential during apoptosis. FEBS Lett 384, 53-57.
438
Cancer Therapy Vol 2, page 439 Zava DT and Duwe G (1997) Estrogenic and antiproliferative properties of genistein and other flavonoids in human breast cancer cells in vitro. Nutr Cancer 27, 31-40.
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Cancer Therapy Vol 2, page 441 Cancer Therapy Vol 2, 441-454, 2004
How should a screening programme for prostate cancer be designed? A case of aiming at a moving target Review Article
Gabriel Sandblom1*, Eberhard Varenhorst2 1 2
Department of Surgery, Uppsala Academic Hospital, Sweden; Department of Urology and Surgery, Norrköping and Finsp¨ng Hospital, Sweden
__________________________________________________________________________________ *Correspondence: Gabriel Sandblom, MD, PhD, Department of Surgery, Uppsala Academic Hospital, 751 85 Uppsala, Sweden; Telephone: +46 18 611 46 07; Fax: +46 18 611 45 08; E-mail: gabriel.sandblom@surgsci.uu.se Key words: Prostate Cancer, Mass screening, PSA Abbreviations: !2-macroglobulin, (A2M); !1-antichyomotrypsin, (ACT); prostatic intra-epithelial neoplasia, (PIN); prostate specific antigen, (PSA); World Health Organisation, (WHO) Received: 23 August 2004; Revised: 12 November 2004 Accepted: 15 November 2004; electronically published: November 2004
Summary Despite the fact that prostate cancer is one of the most common forms of cancers amongst men in the Western world, and a major cause of cancer death, there is still no consensus about how prostate cancer mortality should be reduced. The outcomes of numerous studies involving several of the ten WHO screening criteria have provided gradually increasing support in favour of prostate cancer screening, but definite evidence is still lacking. The first results of two large multicentre studies from Europe and the United States are expected in a few years. Although these trials may eventually provide more reliable evidence regarding the benefit of screening, ultimate eradication of all uncertainties should not be expected. Despite the fact that a number of studies have shown that screening results in a stage shift towards identification of localised tumours and that treatment at this stage improves survival, implying that randomised controlled trials will eventually show improved survival with screening, many questions remain to be answered. These questions concern the methods of tumour detection, which age groups should be screened, screening interval and cost-effectiveness. As new techniques for early detection and methods of treatment evolve, the design of screening programmes must also be adapted. This causes problems since the slow progress of prostate cancer makes it impossible to evaluate an intervention before at least a decade has passed. Accordingly, most studies presenting data on survival often refer to principles of management that are not up to date. the United States (Gohagan, 1995) as well as in Europe (Schröder, 1999). Hopefully, these studies will provide definite results in 2005-2008 (de Koning, 2002). A population-based trial of screening has been performed in Quebec, showing a significant reduction in prostate cancer mortality in those men who fulfilled the screening (Labrie, 2004). Although this study has been criticised for the poor compliance in the group allocated to screening, the death rate from prostate cancer ten years after the study was initiated was reduced by 62% in screened men versus unscreened. This analysis was based on the 24% of the invited men who fulfilled the screening. Since no shift of men at greater risk of dying from prostate cancer from the screened group towards the non-compliers, the risk of bias in this study is probably small. Performing a study aimed at showing mortality reduction after the introduction of a screening programme
I. Introduction Prostate cancer is one of the most common forms of cancers amongst men in the Western world, and a major cause of cancer death. Despite this, there is still no consensus about how prostate cancer mortality should be reduced. The only primary prevention that has been prove to be effective so far is Finasteride (Thompson, 2003). Treatment with Finasteride, however, may adversely affect the sexual function. Furthermore, although it prevents or delays the appearence of prostate cancer, it may increase the risk of poorly differentiated cancers. Secondary prevention, i.e., screening, has been the subject of intense debate after the introduction of Prostate-Specific Antigen (PSA) in clinical practice in the end of the 1980’s. The benefit of screening for prostate cancer, however, still remains to be proved. Large-scale randomised controlled trials on prostate cancer screening have been initiated in 441
Sandblom and Varenhorst: How should a screening programme for prostate cancer be designed? requires extremely large resources. The Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial of the National Cancer Institute in the US was planned on the assumption that 74 000 men had to be recruited to achieve sufficient power to show a reduction in prostate cancer mortality after ten years (Gohagan, 1995). Furthermore, these studies must continue for a long period of time since prostate cancer progresses slowly. One way of testing the benefit of screening for reducing prostate cancer mortality is to compare two similar populations with different screening intensity, although such comparisons are confounded by differences in environmental factors, such as diet, lifestyle and regional differences in how health care is provided. In a study from the Federal State of Tyrol in Austria a significant shift towards detection lower stages of cancer was seen after the introduction of a screening programme (Bartsch, 2001). Mortality rates also declined more rapidly in the Tyrol than in other parts of the country where mass screening was not practised. However, the outcome of this study has been questioned since the decline in mortality was too rapid to be explained by early detection of prostate cancer alone. In a study with similar design, comparing cohorts from two different regions with different prostate cancer screening intensities, no difference in prostate cancer mortality was seen (Lu Yao, 2002). Targeted screening in men with a family history of prostate cancer is already well established (Bock, 2003) and less controversial, since the higher prevalence, earlier onset and more aggressive natural course of prostate cancer in this group gives a relatively better improvement in prostate cancer-specific survival with screening (Valeri 2002). Screening may also have a beneficial psychological effect for these men (Bratt, 2003). The conditions for targeted screening are thus somewhat different than for the whole population of men and therefore not further discussed here. While awaiting the outcome of the large-scale randomised controlled studies, we have to rely on studies on a smaller scale addressing surrogate endpoints to obtain temporary evidence regarding the effectiveness of prostate cancer screening. Ten criteria have been identified by WHO, which can be used as prerequisites for the introduction of mass screening programmes (Wilson, 1986). The ten criteria address the following issues: 1. Is the disease under study an important health problem? 2. There must be effective treatment for patients suffering from localised disease. 3. Facilities for further diagnosis and treatment must be available. 4. There must be an identifiable latent or early symptomatic stage of the disease. 5. The technique to be used for screening must be effective. 6. The tests must be acceptable to the screened population. 7. The natural history of the disease, including development of the latent phase, into clinical disease must be sufficiently known.
8. There must be a generally accepted strategy allowing determination of which patients should be treated and which should remain untreated. 9. The cost must be acceptable Management of the disease in it’s early stages must have a favourable impact on prognosis.
II. Is prostate cancer an important health problem? The impact of prostate cancer on general health in most Western countries leaves no doubt about its importance as a public health problem. It is the second most common cancer in the Western world (Ferlay, 2001), and causes more deaths than any other cancer in men aged 55-74 (World Health Organisation, 1996). An increase in incidence as well as mortality was seen in most industrialised countries during the 1980’s (Hsing, 2000), but the mortality rate became stable during the 1990’s (Oliver, 2001). Although quality of life is not profoundly affected for the majority of men with prostate cancer (Sandblom, 2001), it causes a considerable reduction when the cancer reaches an advanced stage, especially if associated with pain from skeletal metastases (Litwin, 2001; Melmed, 2002; Sandblom, 2004a).
III. Is there effective treatment for localised prostate cancer? With the introduction of nerve-sparing radical prostatectomy in the beginning of the 1980’s, a new method of radical treatment for localised prostate cancer without excessive blood loss and decreased risk for urinary incontinence and potency became available (Walsh, 1983). Radical prostatectomy can either be performed as retropubic prostatectomy, which is the more common approach today, or as perineal prostatectomy. Reports from specialised centres with high competence have shown very favourable results after radical prostatectomy (Han, 2001), but the same results have not been reproduced when the technique is spread to the hospital community at large (Lu Yao, 1996). An alternative to radical prostatectomy is radiotherapy with curative intent. Although no large-scale randomised studies comparing the outcome of radical prostatectomy with radiotherapy have been reported, it is often considered that radiotherapy is the treatment of choice for older men and men with tumours with suspected extracapsular growth (T3a (UICC, 1997)). A problem with radiotherapy is the side-effects, such as incontinence, erectile dysfunction and rectal bleeding. Although such side-effects are typically associated with radical prostatectomy, they may also appear after radiotherapy. With advances in radiotherapy over the last decade the dose delivered to the organs surrounding the prostate has been reduced by the introduction of conformal radiotherapy (Dearnaley, 1999). This has made it possible to escalate the dose, without intestinal side-effects. Brachytherapy has evolved as an alternative treatment to radical prostatectomy with less severe consequences for men with small tumours (Schellhammer, 2000). Radiotherapy may also be given as salvation therapy to 442
Cancer Therapy Vol 2, page 443 men with local recurrence after radical prostatectomy (Stephenson, 2004). For men with high-risk disease, the combination of brachytherapy and external radiotherapy is an alternative (Stone, 1999). The outcome after radiotherapy can also be improved by combining it with androgen ablative therapy (Bolla, 1997; Lawton, 2001; Pilepich, 2001; Ataman, 2004). The results from these studies are so convincing that it is now generally accepted that men who receive radiotherapy for locally advanced prostate cancer should always have at least short-term adjuvant hormone therapy. There are, however, still controversies regarding the duration of the hormone therapy (Hanks, 2000).
prostate cancer detection. As the first step in a screening programme it is used to select those men who should undergo further diagnostic procedures. It may be performed in combination with digital rectal examination and transrectal ultrasound, although this adds little to sensitivity or specificity and is often considered unnecessary (SchrĂśder, 1998). Regular screening programmes and uncontrolled opportunistic screening has lead to a dramatic increase in the detection of prostate cancer, although evidence supporting the benefits of screening are still lacking. In a retrospective review of men undergoing radical prostatectomy, it was found that widespread early detection programmes for prostate cancer resulted in a downward stage migration (Han, 2004), but the outcome from studies of this design are difficult to interpret and may be biased by other circumstances. Although PSA is one of the tumour markers with the highest predictive values available, it is still not optimal for screening purposes. In men with PSA below 4 ng/ml enrolled in the Prostate Cancer Prevention Trial in the United States, 15% were found to have prostate cancer (Thompson, 2004). Several strategies have therefore been suggested to increase the sensitivity and specificity of PSA. An improved specificity is desirable in order to avoid unnecessary anxiety associated with suspected cancer and the risk for complications from prostate biopsy (Crundwell, 1999). A high sensitvity reduces the number of tumours missed at screening, although the risk for overdiagnosis with too high a sensitivity must be taken into account (Etzioni, 2002).
IV. Are there facilities for further diagnosis and treatment of localised prostate cancer? The diagnosis of prostate cancer requires access to transrectal ultrasound with the possibility of core biopsy, as well as units where radical treatment as presented above can be provided. In most Western countries where screening with PSA is practised or considered, this is usually present. However, whereas a PSA test and digital rectal examination does not require large resources, a screening programme should not be initiated in a population where socioeconomic conditions do not allow follow-up of a positive test. In South Africa, only 19% of screened black patients with an elevated PSA undergo prostate biopsy, usually due to economic problems or because they did not have a postal address or telephone number (Heyns, 2003).
A. Free to total PSA One attempt to distinguish between the elevation of PSA caused by benign prostatic hyperplasia and that caused by prostate cancer is to estimate the free-to-total PSA. The majority of PSA is complexed with !1antichyomotrypsin (ACT) or !2-macroglobulin (A2M), while a smaller fraction remains unbound. A low quote of free PSA to the total amount of PSA is indicative of prostate cancer (Luderer, 1995; Catalona, 1998; Aus, 2004).
V. Is there an identifiable latent or early symptomatic stage of prostate cancer? It is generally accepted that prostate cancer can be treated radically as long as the primary tumour is confined to the prostate and there are no regional or distant metastases, and that the prognosis becomes much poorer if the tumour has spread beyond the capsule of the prostate (Epstein, 1993; Aus, 2003). Exploration of pelvic lymph nodes is usually done before treatment with curative intent is attempted, since local spread results in a poorer prognosis what ever treatment is given (Aus, 2003). All screening programmes are aimed at detecting prostate cancer whilst still confined to the prostate and has neither spread locally nor to the skeleton (T1-2, N0, M0 (UICC, 1997)) High-grade prostatic intra-epithelial neoplasia (PIN) is sometimes considered a pre-malignant lesion of the prostate (Häggman, 1997). However, there is not sufficient proof that PIN may develop into manifest prostate cancer to warrant screening directed at PIN detection.
B. PSA velocity Whereas men with benign prostatic hyperplasia may have a constantly elevated PSA, a rise in PSA over time is associated with the presence of a cancer. If PSA increases by 0.75 ng/ml per year there is a greater risk for prostate cancer than in men with a slower rise in PSA (Carter, 1992). PSA velocity is most effective for detecting tumours with a relatively low initial PSA level, preferably below 4.0 ng/ml.
C. Differentiated screening intervals Even if a tumour is not detected at biopsy or the PSA level does not exceed the indication threshold for biopsy, the risk for developing cancer before the next screening occasion is related to the PSA. By taking this into consideration when deciding on screening intervals, the specificity can be improved (Hugosson, 2003; Ito, 2004b). In a study based on a screening programme in Japan, it
VI. Are there effective techniques for prostate cancer screening? Since PSA evolved as a screening tool in the late 1980â&#x20AC;&#x2122;s, screening for prostate cancer has spread dramatically. PSA has become the major tool for early 443
Sandblom and Varenhorst: How should a screening programme for prostate cancer be designed? was suggested that the rescreening interval was set at 3-5 years with a PSA level below 1.0 ng/ml, 1-2 years with a PSA level of 1.1-2.0 ng/ml and 1 year with a PSA of 2.14.0 ng/m (Ito, 2004a). A screening programme with the intervals adapted to the baseline PSA levels may be designed as in Figure 1.
in PSA is considered sufficient to trigger further investigations. This can be avoided by confirming the result a few weeks later before proceeding with biopsy (Eastham, 2003). One way of doing this is shown in Figure 2.
G. Pro prostate specific antigen D. PSA density
Pro prostate-specific antigen is a precursor form of PSA having higher levels in tumour tissue compared to benign prostate tissue. It provides a more specific serum marker for prostate cancer than the combination of free and total PSA. It has been shown to give the best improvement in specificity in the PSA interval 2-4 ng/ml, but may be useful with PSA levels up to 10 ng/ml (Catalona, 2003). Pro prostate-specific antigen in combination with free prostate-specific antigen can be used for screening as shown in Figure 3.
On the assumption that the equivalent volume of a prostate tumour causes a greater elevation in PSA than benign prostatic hyperplasia, PSA density has been suggested as method of increasing the sensitivity (Benson, 1992). PSA density is defined as serum PSA divided by the prostate volume. This requires transrectal ultrasound, which limits the usefulness of PSA density.
E. Age-specific PSA A cut-off level of 3-4 ng/ml is commonly set as the level triggering further investigations, representing a balance between the risk for false negative and false positive results. If the natural increase in PSA with time is taken into consideration, the number of unnecessary biopsies may be reduced (Oesterling, 1993). Whereas for men below 50 years of age a PSA level of 2.5 ng/ml may be the optimal cut-off level, the cut-off level is gradually increased to 6.5 ng/ml for men older than 70 years in order to avoid false positive results. Although this also leads to an increasing number of false negative results, sensitivity and specificity is maintained at a reasonably stabile level through all age groups.
H. Kallikrein 2 Human kallikrein 2, like PSA, is secreted by the prostate epithelial cells with the function of converting the precursor form of PSA into active PSA. It is very similar to PSA and has been suggested as a measure used in combination with PSA to predict extraprostatic extension of the cancer (Haese, 2000). In men with PSA 3.0 ng/ml or greater with a negative prostate biopsy, measurement of human kallikrein 2 has been shown to be useful in combination with total and free PSA, giving a higher specificity than PSA alone (Becker, 2003). Human kallikrein could thus be used in follow-up screening to help select men who should undergo repeated biopsies if PSA continues to be elevated (Figure 4).
F. Repeated PSA tests There are natural fluctuations in PSA levels which may result in false positive results if an isolated elevation
Figure 1. Screening with differentiated intervals. Reproduced from Ito et al, 2004 with kind permission from International Journal of Cancer.
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Figure 2. Screening with repeated PSA measures. Reproduced from Eastham et al, 2003 with kind permission from The Journal of the American Medical Association.
Figure 3. Integrating Pro Prostate-Specific Antigen with PSA in Screening. Reproduced from Catalona et al, 2003 with kind permission from the Journal of Urology.
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Figure 4. Screening with total PSA, free PSA and Human Kallikrein 2. Reproduced from Becker et al, 2003 with kind permission from the Journal of Urology.
I. Biopsy technique
procedure to confirm the diagnosis in the case of a positive if PSA (Applewhite, 2001). Although core biopsies carry the risk of complications such as infections and bleeding, it is usually accepted by the population (Aus, 1993). However, a study from South Africa has shown poor compliance regarding further investigation after a positive PSA result (Heyns, 2003), possibly reflecting a reluctance to further investigations even when the suspicion of cancer has been raised. This emphasises the importance of adequate information before the first steps of screening are initiated in order to avoid interrupting the investigation when only halfway towards the diagnosis of a cancer.
Although the second step in the diagnostic process, i.e. transrectal ultrasound and core biopsy, has a limited sensitivity, especially for larger tumours (Rietbergen, 1998; Basillote, 2003; Ung, 2003), most efforts to improve screening effectiveness were previously focused on the first steps in the process. However, in recent years several reports on how biopsy technique can be improved have been published. Using computer-simulation models of the prostate, the sensitivity has been shown to increase from 66%, applying traditional sextant biopsies, to 91% (Kawata, 2003). Another way of increasing sensitivity is to take biopsy samples via through the transperineal approach and not the transrectal approach, which is the more common technique (Emiliozzi, 2003). With an extended biopsy scheme, including laterally directed sextant biopsies, the yield can be increased and the variance in prostate-specific and age-related cancer rates minimised (Presti Jr, 2003). The higher detection rate with laterally directed biopsies has also been confirmed in other studies (Fink, 2003; de la Taille, 2003; O'Connell, 2004). Furthermore, tumours detected in the lateral zone also seem to have more malignant (Kawata, 2003). Another way of increasing the sensitivity is to direct the biopsies towards the transitional zone (Damiano, 2003; de la Taille, 2003).
VIII. Is the natural history of prostate cancer sufficiently known? Autopsy series show a high prevalence of prostate cancer in men without symptoms. At the age of 50 years, approximately every third man has occult prostate cancer (Sakr, 1993). As the mortality in prostate cancer does not reach this level, it is thought that the majority of these tumours are indolent tumours that progress very slowly or remain stable for very long periods of time. On the other hand, there is a small number of men with highly malignant prostate cancer which progresses rapidly and eventually kills the host if not treated in time (Albertsen, 1998; Sandblom, 2000). So far, no studies have reliably identified which of the early stage tumours eventually develop into lethal cancer. The total population of men with prostate cancer seems to comprise a continuum from men with practically stationary tumours to those with rapidly progressing cancer. The natural history of a tumour can be divided into two phases: the interval from the point where the tumour becomes detectable until it presents with clinical
VII. Are the tests used to detect prostate cancer acceptable to the population concerned? Although PSA sampling itself is widely accepted in the screened population, the anxiety raised by a false positive result has to be considered. Transrectal ultrasound with directed biopsies of the prostate is a safe and simple 446
Cancer Therapy Vol 2, page 447 symptoms (sojourn time); and the interval from the point where the tumour presents with clinical symptoms until death. As with most malignant diseases, our knowledge of the two phases above is insufficient and partly dates from the time when the natural progress of the cancer was not suppressed by the methods of treatment used today. Androgen ablation has been practised since the 1950â&#x20AC;&#x2122;s (Huggins, 1941), which obscures our knowledge about the second phase. The purpose of screening is to detect a tumour as early as possible during the first phase. A major problem is that the natural course of the disease probably differs between different tumours. Whereas a well differentiated tumour with little malignant potential probably has a long sojourn time, it may also have a long interval between onset of clinical symptoms and reaching the stage when it may kill the host. This makes it easily detected in a screening programme, but reduces the relative benefit of early curative treatment. The opposite is the case for poorly differentiated, highly malignant tumours. This is reflected in the classic statement of Whitmore â&#x20AC;?Is cure possible in those for whom it is necessary, and is cure necessary in those for whom it is possibleâ&#x20AC;? (Whitmore Jr, 1990). However, although this may lead to a passive, fatalistic attitude to prostate cancer, it should not be ignored that it is always a potential threat to its host.
number of small indolent tumours, with a natural course that cannot be predicted. The time gained by detecting the tumour by screening as opposed to waiting until the tumour becomes symptomatic, i.e. the lead time, has also to be defined. The lead time depends on the methods used at screening as well as the screened population. The sojourn time can be divided into the interval from the inception of preclinical disease to the point at which it is detected at screening (delay time) and the interval between the point of detection at screening until clinical symptoms appear. Several attempts have been made to estimate the sojourn time and the lead time. In a study from Stockholm, the cumulative incidence of prostate cancer over a twelve year period in a population undergoing screening consisting of a combination of digital rectal examination, PSA and transrectal ultrasound was determined. By comparing the incidence with that of an age-matched population, the median lead time was estimated to be 4.5 years in men with serum levels >3.0 ng/ml (Tornblom, 2004). Based on survival data from the Surveillance, Epidemiology and End Results database, the mean lead time was estimated to be 9 years (Nicholson, 2002). Based on the European Randomised Study of Screening for Prostate Cancer, the mean lead time was estimated to be 12.3 years with a single screening test at the age of 55 years and 6.0 years if screening was performed at 75 years (Draisma, 2003). The diverging estimates in these studies may be explained by different age distributions and screening strategies.
A. The natural course of prostate cancer before the onset of clinical symptoms One of the most important problems to be resolved in screening for prostate cancer is the absence of reliable predictive factors which indicate those tumours detected at early stage which eventually may develop into highly malignant cancers. In a study of specimens from cystoprostatectomy for bladder cancer in men with a mean age of 60 years without clinical signs of prostate cancer, latent tumours were found in 44% of the step-sectioned glands (Hautmann, 2000). When systematic core biopsy and fine needle aspiration biopsy samples were taken from these specimens in the same fashion as in normal clinical practice, cancer was detected in about 5% of the cases. Although the tumours detected by biopsy in this way in general had larger volumes than those remaining undetected, there was no clear threshold volume at which cancer was found. Whether or not a latent tumour is found or remains undetected after systematic core biopsy or fine needle biopsy, seems thus to be a random process, with the probability of detecting cancer increasing with the tumour volume and diagnostic activity. Accordingly, the more you seek, the more you find, but the prognosis remains unpredictable. As a result of this, no conclusion regarding the progress and survival of tumours detected at an early stage can be made without taking into consideration how the sample was obtained. Although a study based on the Tyrol PSA screening project has indicated that early stage tumours exhibit potentially malignant features, including heterogeneity in differentiation, multifocality, ploidy and proliferation index (Horninger, 2004), the clinical significance of these findings remain to be established. Inevitably screening results in the detection of a large
B. Natural course of prostate cancer after the onset of clinical symptoms In the majority of cases prostate cancer has a protracted course and mostly affects older men. Competing mortality from intercurrent diseases is therefore high. Since essentially all men with advanced cancer receive hormonal treatment, at least if the cancer reaches the stage that it may be lethal, the true natural course of the disease can not be determined. When analysing the survival of men managed conservatively, the assumption is that they receive hormonal treatment at some stage in the disease before the cancer kills the host. A few studies on survival of men with localised prostate cancer managed conservatively have been published. By following men who would be potential candidates for treatment with curative intent, i.e. those with tumours confined to the prostate, the natural course of cancer in men who would otherwise be treated with radical prostatectomy or radiotherapy may be determined. In a population-based Swedish study, 223 men with localised prostate cancer were managed by watchful waiting, i.e. they were left untreated at the time of diagnosis and received hormonal treatment when the cancer showed signs of progress (Johansson, 1997). The patients in this group had a survival close to that of agematched men without prostate cancer in the same population. The high age at diagnosis of this cohort has raised the concern that a high competing mortality may have resulted in a relatively minor effect on the overall mortality from the cancer, and that an analysis of the 447
Sandblom and Varenhorst: How should a screening programme for prostate cancer be designed? prostate cancer specific mortality would have been less favourable. Even so, this study has caused debate about the benefit of curative treatment as well as early detection. In a recent publication based on the same cohort, it was shown that the probability of progression to a more aggressive and lethal phenotype increased after 15 years (Johansson, 2004). In another Swedish study a steadily increasing prostate cancer-specific mortality was likewise seen for men with prostate cancer when followed after ten years had elapsed since diagnosis (Hugosson, 1995). In the United State, the age at diagnosis is lower than that in Sweden. A study on men in Connecticut aged 55 to 74 years at diagnosis has shown a relatively small risk for death in prostate cancer in men with a Gleason score of 6 or less, whereas the prognosis was much poorer for those with Gleason scores of 7-10 (Albertsen, 1998). This pattern, with a relatively favourable prognosis for the majority of prostate cancer patients but a smaller group with rapidly progressing disease has been repeated in several other studies (Chodak, 1994; Lu Yao, 1997; Sandblom, 2000). The favourable natural course during the first 15 years shown for men with localised prostate cancer in these observational studies implies that there is not much room for improvement in survival by radical prostatectomy or radiotherapy unless the patient is very young and has a long expected survival. As a paradoxical contradiction to these longitudinal studies of men with localised prostate cancer, prostate cancer is still one of the leading causes of cancer death in Sweden as well as most other Western countries. One way of explaining this is that the majority of men dying of prostate cancer have rapidly progressing cancers that escape all efforts of early detection and treatment while they are still curable. Accordingly, screening results in the detection of less malignant tumours that do not benefit from curative treatment. A contrary explanation is that the cohorts of men with localised cancer studied in these observational studies do not have the same natural course as those who are detected in populations where screening is practised more actively (Walsh, 1997). Screening would in that case result in the detection of cancer in men who would have a greater number of years lost due their cancer than those followed in these studies, although this is difficult to assess due to the lead time bias of early tumour detection. The actual truth is probably somewhere between these two explanations.
indolent tumours has never been proven, uncritical aggressive treatment of small tumours inevitably results in unnecessary overtreatment of men without prolonging their life. Whereas Gleason grade (Egevad, 2002), PSA (Kattan, 2003) and the percentage of prostate biopsies involved by cancer (D'Amico, 2002; Grossfeld, 2002) are reliable predictive factors for tumours that have reached the stage that growth beyond the boundaries of the prostate is under question, these factors are not sufficient to predict prognosis for the smallest tumours. In recent years considerable efforts have been made to identify other markers able to predict prognosis at an early stage, such as bcl-2, E-cadherin, p53 (Wu 2003), p27kip1, Ki-67 (Vis 2000), CD44 (Ekici, 2002), chromosomal alterations (Alers, 2001) and DNA ploidy (Deliveliotis, 2003). Several of these markers have promising features, but they need further evaluation before they can be introduced into clinical practice. One way of avoiding the problem of uncertainty regarding prognosis at the very earliest stages of the disease is to postpone radical treatment and follow the patient with active surveillance. If the tumour eventually shows signs of progression after a period observation, radiotherapy or radical prostatectomy is offered.
X. Is the cost of prostate cancer screening acceptable? In a decision model of the cost of screening based on a randomised study in Sweden, including cost of administration, loss of patient time, diagnostic measures and management strategies, it was estimated that the incremental cost per extra detected localised cancer was 168 000 SEK (18.400 Euro) and per curatively treated cancer 356 000 SEK (40.000 Euro) (Sennfält, 2004). The cost is probably similar in other European countries, although price levels in general are quite high in Sweden. This cost should be compared with the life-time cost of palliative treatment of a man with advanced prostate cancer, which was estimated to be 198 400 SEK in the same population (Sennfält, 2003). If improved survival from early detection is eventually proved, a cost of 356 000 SEK may considered reasonable, although this has to be seen the context of other prioritisations made in the health-care budget.
XI. Does management of prostate cancer when it is still localised improve prognosis?
IX. Is there a generally accepted strategy that selects patients who should be treated and those who should remain untreated?
The survival of men with localised prostate cancer if left without curative management is one of the most crucial questions that must be resolved before mass screening for prostate cancer can be recommended. The favourable prognosis of men with localised cancer managed conservatively mentioned in Paragraph G has raised doubt about the potential benefit of radiotherapy and radical prostatectomy. Overdetection, i.e. the percentage of tumours diagnosed at screening that otherwise would not be detected within the patientâ&#x20AC;&#x2122;s lifetime, gives a minimum measure of how many patients
Management decisions for men with prostate cancer should ideally depend on accurate assessment of the biological potential of the tumour. As mentioned in paragraph G, indolent tumours that do not affect prognosis even without treatment have to be distinguished from those that progress and pose a risk to the health or life of the host. The sooner the tumour is detected, the more difficult this is to achieve. Although the presence of
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Cancer Therapy Vol 2, page 449 do not benefit from early detection and curative treatment. Based on the European Randomised Study of Screening for Prostate Cancer and the assumption of annual screening of men aged 55 to 67 years with PSA, overdetection was estimated to be 50% (Draisma, 2003). By performing a computer simulation on a hypothetical cohort aged 60-84 years in the United States, the rate of overdiagnosis was estimated to be 29% for whites and 44% for blacks (Etzioni, 2002). Another problem in interpreting survival of patients with screen-detected tumours is the risk for misclassification of the cause of death, which may cause a bias in favour of screening (Feuer, 1999). Despite the concern raised by the slow natural course of prostate cancer shown in several studies, the only randomised and sufficiently powered study comparing radical prostatectomy with conservative management published so far showed a significantly better prostate cancer-specific survival in the men undergoing radical treatment (Holmberg, 2002). No significant difference in overall survival was seen in this study, although a survival benefit may become apparent in the future as further years of follow-up pass by. However, this study was not based on a sample of patients with screen-detected tumours. As mentioned in Paragraph G, screen-detected tumours may have another natural course which could limit the benefit of aggressive treatment. No large studies comparing radiotherapy and conservative management have been published.
be used to reach optimal cost-effectiveness, sensitivity and specificity? Is a high sensitivity always desirable or does a too effective a screening programme result in unnecessary overdiagnosis of tumours that otherwise would have remained undetected throughout the manâ&#x20AC;&#x2122;s lifetime? Several reports in recent years have supported a screening interval longer than one year, typically 2-4 years (Ross, 2000; Yao, 2001; Draisma, 2003; Hugosson, 2003; van der Cruijsen, 2003; Postma, 2004; Sandblom, 2004b). Most screening programmes have included men in the range from 50-55 years to 70-75 years. The low prevalence of prostate cancer in men younger than 50 years makes it meaningless to screen earlier than this. Because of the slow natural course (see Paragraph G), radical treatment usually does not result in essential improvement of overall survival if the expected survival is not longer than 10 years. Men older than 75 years are therefore not included in most screening programmes. As new techniques of early detection and methods of treatment evolve, the design of screening programmes must also be adjusted. This causes problems, since the slow progress of prostate cancer makes it impossible to evaluate an intervention before at least a decade has passed. Accordingly, most studies presenting data on survival often refer to principles of management that are not up to date. When debate about screening for prostate cancer started at the end of the 1980â&#x20AC;&#x2122;s, digital rectal examination was the main method of detection. PSA appeared later as a complement to digital rectal examination. The first screening programmes therefore usually included digital rectal examination , PSA and sometimes transrectal ultrasound. Examples of such programmes are that in NorrkĂśping, Sweden (Figure 5), the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial in the United States (Figure 6) and the European Randomised Study of Screening for Prostate Cancer (Figure 7). Transrectal ultrasound has now been abandoned in the European Randomised Study of Screening for Prostate Cancer (de Koning, 2002). In recent years, screening has become increasingly dominated by PSA in combination with other serum markers. Several approaches have been used to optimise sensitivity, specificity, such as differentiated screening intervals (Figure 1), repeated PSA measures (Figure 2), using Pro Prostate-Specific Antigen (Figure 3), and free PSA or kallkrein-2 (Figure 4) as a complement to PSA. A common approach is to use an additional marker to decide on biopsy in intermediate PSA ranges. It may be that a combination of several of these approaches may prove useful in the future. One of the most important questions that remains to be answered is not how sensitivity should be improved, but rather which level of sensitivity is optimal to obtain a balance between overdiagnosis on the one hand and the risk for not detecting tumours that eventually kill the host (Zappa, 1998) on the other hand. Can a screening programme be designed to efficiently filter out the most malignant tumours and leaving the remaining pool of latent tumours undetected? The ideal for that purpose would be marker that is more sensitive for malignant cancers than latent tumours at the same cut-off level. It
XII. Conclusions Although many questions remain unanswered, several of the prerequisites for screening may be fulfilled. If the effectiveness in detecting tumours with screening is proved and the benefit of treatment while the tumour is still localised holds true, the randomised studies initiated in the United States may eventually confirm an improvement in survival with screening. The Prostate, Lung, Colorectal and Ovary trial in the United States and the European Randomised Screening for Prostate Cancer trial in Europe have the power to show definite results in 2005-2008 (de Koning, 2002). However, even if reliable evidence supporting screening is provided, several problems still remain to be resolved. More than 20 years after results from the first large-scale randomised studies on mammography were presented, clearing the way for mass screening programmes, screening for breast cancer still remains controversial and a the subject of intense debate (Miettinen, 2002; Jatoi, 2003; Green, 2003; Retsky, 2003). In a report from the Cochrane Collaborative, the validity of all randomised controlled studies on breast cancer screening were questioned on several points, in particular regarding differential exclusion of women with breast cancer and misclassification of cause of death (Olsen, 2001). Similar concerns may arise with the prostate cancer screening studies if careful steps are not taken in advance to avoid analogous problems. Even if future evidence of improved survival provides support for screening, the optimal screening schedule has still to be established. Which age groups should be included? Which methods of detection should 449
Sandblom and Varenhorst: How should a screening programme for prostate cancer be designed? may be that human kallikrein 2 could act in this way (Haese, 2003), although this remains to be proved in clinical practice. Even if the randomised studies in Europe and the United States will eventually show a significantly increased overall or prostate cancer-specific survival, reflecting the fact that death in prostate cancer was avoided for a part of those screened, there will inevitably
be a large group of men who have their tumours detected and treated early although the tumour would never have killed them, and another group of men who die of prostate cancer despite all efforts to avoid it. The natural course of prostate cancer will always tend to concentrate men with prostate cancer to the second and third groups.
Figure 5. The Screening programme in Norrkรถping, Sweden, from 1993. Reproduced from Sandblom et al, 2004b with kind permission from the European Urology.
Figure 6. The Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial. Reproduced from Gohagan et al, 1995 with kind permission from Cancer
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Figure 7. The Rotterdam branch of the European Randomised Study of Screening for Prostate Cancer. Reproduced from Postma et al, 2004 with kind permission from Cancer. introduction of prostate-specific antigen mass screening in the Federal State of Tyrol. Urology 58, 417-424 Basillote JB, Armenakas NA, Hochberg DA, Fracchia JA (2003) Influence of prostate volume in the detection of prostate cancer. Urology 61, 167-171 Becker C, Piironen T, Pettersson K, Hugosson J, Lilja H (2003) Testing in serume for human glandular kallikrein 2, and free and total prostate specific antigen in biannual screening for prostate cancer. J Urol 170, 1169-1174 Benson MC, Whang IS, Pantuck A, et al (1992) Prostate specific antigen density, a means of distiguishing benign prostatic hypertrophy and prostate cancer. J Urol 147, 815-816. Bock CH, Peyser PA, Gruber SB, Bonnell SE, Tedesco KL, Cooney KA (2003) Prostate cancer early detection practices among men with a family history of disease. Urology 62, 470-475 Bolla M, Gonzalez D, Warde P, Dubois JB, Mirimanoff RO, Storme G, Bernie J, Kuten A, Sternberg C, Gil T, Collette L, Pierart M (1997) Improved survival in patients with locally advanced prostate cancer treated with radiotherapy and Goserelin. Engl J Med 337, 295-300 Bratt O, Emanuelsson M, Gronberg H (2003) Psychological aspects of screening in families with hereditary prostate cancer. Scandinavian Journal of Urology & Nephrology 37, 5-9 Carter HB, Pearson JD, Metter J, Brant LJ, Chan DW, Andres R, Fozard JL, Walsh PC (1992) Longitudinal evaluation of prostate specific antigen levels with and without prostate disease. JAMA 267, 2215. Catalona WJ, Bartsch G, Rittenhouse HG, Evans CL, Linton HJ, Amirkhan A, Horninger W, Klocker H, Mikolajczyk SD (2003) Serum pro prostate specific antigen improves cancer detection compared to free and complexed prostate specific antigen in men with prostate specific antigen 2 to 4 ng/ml. J Urol 170, 2181-2185
References Albertsen PC, Hanley JA, Gleason DF, Barry MJ (1998) Competing risk analysis of men aged 55 to 74 years at diagnosis manageed conservatively for clinically localised prostate cancer. JAMA 280, 975-980 Alers J, Krijtenburg PJ, Vis AN, Hoedemaker RF, Wildhagen MF, Hop WCJ, van der Kwast TH, Schröder FH, Tanke HJ, van Dekken H (2001) Molecular cytogenetic analysis of prostatic adenocarcinomas from screening studies. Am J Pathol 158, 399-406 Applewhite JC, Matlaga BR, MacCullough DL, Hall MC (2001) Transrectal ultrasound an biopsy in the early diagnosis of prostate cancer. Cancer Control, Journal of the Moffitt Cancer Center 8, 141-150 Ataman F, Zurlo A, Artignan X, v Tienhoven G, Blank LE, Warde P, Dubois JB, Jeanneret W, Keuppens F, Bernier J, Kuten A, Collette L, Pierart M, Bolla M (2004) Late toxicity following conventional radiotherapy for prostate cancer, analysis of the EORTC trial 22863. Eur J Cancer 40, 16741681 Aus G, Becker C, Franzén S, Lilja H, Lodding P, Hugosson J (2004) Cumulative prostate cancer risk assessment with the aid of the free-to-total prostate specific antigen ratio. Eur Urol 45, 160-165 Aus G, Hermansson CG, Hugosson J, Pedersen K (1993) Transrectal ultrasound examination of the prostate, complications and acceptance by patients. Br J Urol 71, 457-459 Aus G, Nordenskjöld K, Robinson D, Rosell J, Varenhorst E (2003) Prognostic factors and survival in node-positive (N1) prostate cancer - A prospective study based on data from a Swedish population-based cohort. Eur Urol 43, 627-631 Bartsch G, Horninger W, Klocker H, Reissigl A, Oberaigner W, Schonitzer Deaft (2001) Prostate cancer mortality after
451
Sandblom and Varenhorst: How should a screening programme for prostate cancer be designed? Catalona WJ, Partin AW, Slawin KM, Brawer MK, Flanigan RC, Patel A, Richie JP, deKernion JB, Walsh PC, Scardino PT, Lange PH, Subong EN, Parson RE, Gasior GH, Loveland KG, Southwick PC (1998) Use of the percentage of free prostate-specific antigen to enhance differentiation of prostate cancer from benign prostatic disease, a prospective multicenter clinical trial. JAMA 279, 1542-1547 Chodak GW, Thisted RA, Gerber GS, Johansson JE, Adolfsson J, Jones GW, et al (1994) Results of conservative management of clinically localized prostate cancer. N Engl J Med 330, 242-248 Crundwell MC, Cooke PW, Wallace DM (1999) Patients' tolerance of transrectal ultrasound-guided prostatic biopsy, an audit of 104 cases. BJU International 83, 792-795 Damiano R, Autorino R, Perdona S, De Sio M, Oliva A, Esposito C, Cantiello F, Di Lorenzo G, Sacco R, D'Armiento M (2003) Are extended biopsies really necessary to improve prostate cancer detection? Prostate Cancer & Prostatic Diseases 6, 250-255 D'Amico AV, Kehaviah, Manola J, et al (2002) Clinical utility of the percentage of positive biopsies in pedicting prostate cancer-specific and overall survival after radiotherapy for patients with localised prostate cancer. Int J Radiat Oncol Biol Phys 53, 581-587 de Koning HJ, Auvinen A, Berenguer Sanchez A, Calais da Silva F, Ciatto S, Denis L, Gohagan JK, Hakama M, Hugosson J, Kranse R, Nelen V, Prorok PC, Schroder FH, European Randomized Screening for Prostate Cancer, International Prostate Cancer Screening Trials Evaluation G (2002) Largescale randomized prostate cancer screening trials, program performances in the European Randomized Screening for Prostate Cancer trial and the Prostate, Lung, Colorectal and Ovary cancer trial. Int J Cancer 97, 237-244 de la Taille A, Antiphon P, Salomon L, Cherfan M, Porcher R, Hoznek A, Saint F, Vordos D, Cicco A, Yiou R, Zafrani ES, Chopin D, Abbou CC (2003) Prospective evaluation of a 21sample needle biopsy procedure designed to improve the prostate cancer detection rate. Urology 61, 1181-1186 Dearnaley DP, Khoo VS, Norman AR, Meyer L, Nahum A, Tait D, Yarnold J, Horwich A (1999) Comparison of radiation side-effects of conformal and conventional radiotherapy in prostate cancer, a randomised trial. Lancet 353, 267-272 Deliveliotis C, Skolarikos A, Karayannis A, Tzelepis V, Trakas N, Alargof E, Protogerou V (2003) The prognostic value of p53 and DNA ploidy following radical prostaectomy. World J Urol 21, 171-176 Draisma G, Boer R, Otto SJ, van der Cruijsen IW, Damhuis RAM, SchrĂśder FH, de Koning HJ (2003) Lead times and overdetection due to prostate-specific antigen screening, estimates from the European Randomised Study of Screening for Prostate Cancer. J Natl Cancer Inst 95, 868-878 Eastham JA, Riedel E, Scardino PT, Shike M, Fleisher M, Schatzkin A, Lanza E, Latkany L, Begg CB, Polyp Prevention Trial Study G (2003) Variation of serum prostatespecific antigen levels, an evaluation of year-to-year fluctuations. JAMA 289, 2695-2700 Egevad L, Granfors T, Karlberg L, Bergh A, Stattin P (2002) Percent Gleason grade 4/5 as prognostic factor in prostate cancer diagnosed at transurethral resection. J Urol 168, 509513 Ekici S, Ayhan A, Kendi S, Ozen H (2002) Determination of prognosis in patients with prostate cancer treated with radical prostatectomy, prognostic value of CD44v6 score. J Urol 167, 2037-2041 Emiliozzi P, Corsetti A, Tassi B, Federico G, Martini M, Pansadoro V (2003) Best approach for prostate cancer detection, a prospective study on transperineal versus transrectal six-core prostate biopsy. Urology 61, 961-966
Epstein JI, Carmichael MJ, Pizov G, Walsh PC (1993) Influence of capsular penetration on progression following radical prostatectomy, a study of 196 cases with long-term followup. J Urol 150, 135-141 Etzioni R, Penson DF, Legler JM, di Tommasso D, Boer R, Gann P, Feuer EJ (2002) Overdiagnosis due to prostate-specific antigen screening, lessons from US prostate cancer incidence trends. J Natl Cancer Inst 94, 981-990 Ferlay J, Bray F, Pisani P, Parkin D (2001) Globocan 2000, Cancer incidence, mortality and prevalence worldwide, version 1.0. IARC Cancer Base, 5th ed Lyon, IARC Press. Anonymous Feuer EJ, Merrill RM, Hankey BJ (1999) Cancer surveillance series, interpreting treends in prostate cancer - part II, cause of death misclassification and the recent rise and fall in prostate cancer mortality. J Natl Cancer Inst 91, 1025-1032 Fink KG, Hutarew G, Pytel A, Esterbauer B, Jungwirth A, Dietze O, Schmeller NT (2003) One 10-core prostate biopsy is superior to two sets of sextant prostate biopsies. BJU International 92, 385-388 Gohagan J, Prorok P, Kramer B, Hayes R, Cornett J (1995) The prostate, lung, colorectal and ovarian cancer screening trial of the National Cancer Institute. Cancer 75, 1869-1873 Green BB, Taplin SH (2003) Breast cancer screening controversies. J Am Board Fam Pract 16, 233-241 Grossfeld GD, Latini DM, Lubeck DP, Broering JM, Li YP, Mehta SS, Carroll PR (2002) Predicting disease recurrence in intermediate and high-risk patients undergoing radical prostatectomy using percent positive biopsies, results from CaPSURE. Urology 59, 560-565 Haese A, Becker C, Noldus J, Graefen M, Huland E, Huland H, et al (2000) Human glandular kallikrein 2, a potential serum marker for predicting the organ confined versus non-organ confined growth of prostate cancer. J Urol 163, 1491-1497 Haese A, Graefen M, Steuber T, Becker C, Noldus J, Erbersdobler A, Huland E, Huland H, Lilja H (2003) Total and Gleason grade 4/5 cancer volumes are major contributors of human kallikrein 2, whereas free prostate specific antigen is largely contributed by benign gland volume in serum from patients with prostate cancer or benign prostatic biopsies. J Urol 170, 2269-2273 Häggman MJ, Macoska JA, Wojno KJ, Oesterling JE (1997) The relationship between prostatic intraepithelial neoplasia and prostate cancer, critical issues. J Urol 158, 12-22 Han M, Partin AW, Chan DY, Walsh PC (2004) An evaluation of the decreasing incidence of positive surgical margins in a large retropubic prostatectomy series. J Urol 171, 23-26 Han M, Partin AW, Pound CR, Epstein JI, Walsh PC (2001) Long-term biochemical disease-free and cancer specific survival following anatomic radical retropubic prostatectomy. The 15-year John Hopkins experience. Urol Clin North Am 28, 555-565 Hanks G, Lu J, Machtay M, Venkatesan V, Pinover W, Byhardt R, Rosenthal S (2000) Proc of Am Soc Ther Rad Oncol (ASTRO) , RTOG protocol 92-02, a phase III trial of the use of long term total androgen suppression following neoadjuvant hormonal cytoreduction and radiotherapy in locally advanced carcinoma of the prostate. Int J Radiat Oncol Biol Phys 48, 112 Hautmann SH, Conrad S, Henke RP, Erbersdobler A, Simon J, Strauss M, et al (2000) Detection rate of histologically insignificant prostate cancer with systematic sextant biopsies and fine needle aspiration cytology. J Urol 163, 1734-1738 Heyns CF, Mathee S, Isaacs A, Kharwa A, De Beer PM, Pretorius MA (2003) Problems with prostate specific antigen screening for prostate cancer in the primary healthcare setting in South Africa. Br J Urol 91, 785-788
452
Cancer Therapy Vol 2, page 453 Holmberg L, Bill-Axelsson A, Helgesen F, Jaakko OS, Folmerz P, Häggman P (2002) A randomized trial comparing radical prostatectomy with watchful waiting in early prostate cancer. N Engl J Med 347, 781-789 Horninger W, Berger AP, Rogatsch H, Gschwendtner A, Steiner H, Niescher M, Klocker H, Bartsch G (2004) Characteristics of prostate cancers detected at low PSA levels. Prostate 58, 232-237 Hsing AW, Tsao L, Devessa SS (2000) International trends and patterns of prostate cancer incidence and mortality. Int J Cancer 85, 60-67 Huggins C, Hodges CV (1941) Studies on prostate cancer, The effect of castration of estrogen and of androgen injection on serum phosphatases in metastatic carcinoma of the prostate. Cancer Res 1, 293. Hugosson J, Aus G, Bergdahl C, Bergdahl S (1995) Prostate cancer mortality in patients surviving more than 10 years after diagnosis. J Urol 154, 2115-2117 Hugosson J, Aus G, Lilja H, Lodding P, Pihl CG, Pileblad E (2003) Prostate specific antigen based biennial screening is sufficient to detect almost all prostate cancers while still curable. J Urol 169, 1720-1723 Ito K, Yamamoto T, Ohi M, Takechi H, Kurokawa K, Suzuki K, Yamanaka H (2004a) Cumulative probability of PSA increase above 4.0 ng/ml in population-based screening for prostate cancer. Int J Cancer 109, 455-460 Ito K, Yamamoto T, Suzuki K, Kurokawa K, Yamanaka H (2004b) The risk of rapid prostate specific antigen increase in men with baseline prostate specific antigen 2.0 ng/ml or less. J Urol 171, 656-660 Jatoi I, Miller AB (2003) Why is breast-cancer mortality declining? Lancet Oncology 4, 251-254 Johansson JE, Andrén O, Andersson SO, Dickman PW, Holmberg L, Magnusson A, Adami HO (2004) The natural history of early, localised prostate cancer. JAMA 291, 27132719 Johansson JE, Holmberg L, Johansson S, Bergström R, Adami HO (1997) Fifteen-year survival in prostate cancer. A prospective, population-based study in Sweden. JAMA 277, 467-471 Kattan MW, Eastham JA, Wheeler TM, Maru N, Scardino PT, Erbersdobler A, Graefen M, Huland H, Koh H, Shariat SF, Slawin KM, Ohori M (2003) Counseling men with prostate cancer, a nomogram for predicting the presence of small, moderately differentiated, confined tumors. J Urol 170, 1792-1797 Kawata N, Miller GJ, Crawford ED, Torkko KC, Stewart JS, Lucia MS, Miller HL, Hirano D, Werahera PN (2003) Laterally directed biopsies detect more clinically threatening prostate cancer, computer simulated results. Prostate 57, 118-128 Labrie F, Candas B, Cusan L, Gomez JL, Belanger A, Brousseau G, Chevrette E, Levesque J (2004) Screening decreases prostate cancer mortality, 11-year follow-up of the 1988 Quebec prospective randomized controlled trial. Prostate 59, 311-318 Lawton CA, Winter K, Murray K et al. (2001) Updated results of the phase III Radiation Therapy Oncology Group (RTOG) trial 85-31 evaluating the potential benefit of androgen suppression following standard radiation therapy for unfavorable prognosis carcinoma of the prostate. Int J Radiat Oncol Biol Phys 49, 937-946 Litwin MS, Lubeck DP, Stoddard ML, Pasta DJ, Flanders SC, Henning JM (2001) Quality of life before death for men with prostate cancer, results from the CaPSURE data base. J Urol 165, 871-875 Lu Yao G, Albertsen PC, Stanford JL, Stukel TA, WalkerCorkery ES, Barry MJ (2002) Natural exeriment examining
impact of aggressive screening and treatment on prostate cancer mortality in two fixed cohorts from Seattle area and Connecticut. BMJ 325, 740-745 Lu Yao G, Yao SL (1997) Population-based study of long-term survival in patients with clinically localised prostate cancer. Lancet 349, 906-910 Lu Yao GL, Potosky AL, Albertsen PC, Wasson JH, Barry MJ, Wennberg JE (1996) Follow-up prostate cancer treatments after radical prostatectomy, a population-based study. J Natl Cancer Inst 88, 166-173 Luderer AA, Chen YT, Soriano TF, Kramp WJ, Carlson G, Cuny C, Sharp T, Smith W, Petteway J, Brawer MK (1995) Measurement of the proportion of free to total prostatespecific antigen improves diagnostic performance of prostate-specific antigen in the diagnostic gray zone of total prostate-specific antigen. Urology 46, 187-194 Melmed GY, Kwan L, Reid K, Litwin MS (2002) Quality of life at the end of life, trends in patients with metastatic prostate cancer. Urology 59, 103-109 Miettinen OS, Henschke CI, Pasmantier MW, Smith JP, Libby DB, Yankelevitz DF (2002) Mammographic screening, no reliable supporting evidence? Lancet 359, 404-406 Nicholson PW, Harland SJ (2002) Survival prospects after screen-detection of prostate cancer. Br J Urol 90, 686-693 O'Connell MJ, Smith CS, Fitzpatrick PE, Keane CO, Fitzpatrick JM, Behan M, Fenlon HF, Murray JG (2004) Transrectal ultrasound-guided biopsy of the prostate gland, value of 12 versus 6 cores. Abdom Imaging 29, 132-136 Oesterling JE, Jacobsen SJ, Chute CG, Guess HA, Girman CJ, Panser LA, Lieber MM (1993) Serum prostate-specific antigen in a community-based population of healthy men. Establishment of age-specific reference ranges. JAMA 270, 860-864 Oliver SE, May MT, Gunnell D (2001) International trends in prostate-cancer mortality in the "PSA-era". Int J Cancer 92, 893-898 Olsen O, Gotzsche PC (2001) Screening for breast cancer with mammography. Cochrane Database of Systematic Reviews. (4) , CD001877. Pilepich MV, Winter K, John MJ et al (2001) Phase III Radiation Therapy Oncology Group (RTOG) trial 86-10 of androgen deprivation adjuvant to definite radiotherapy in locally advanced carcinoma of the prostate. Int J Radiat Oncol Biol Phys 50, 1243-1252 Postma R, Roobol M, Schroder FH, van der Kwast TH (2004) Potentially advanced malignancies detected by screening for prostate carcinoma after an interval of 4 years. Cancer 100, 968-975 Presti Jr JC, O'Dowd GJ, Miller MC, Mattu R, Veltri RW (2003) Extended peripheral zone biopsy schemes increase cancer detection rates and minimize variance in prostate specific antigen and age related cancer rates, results of a community multi-practice study. J Urol 169, 125-129 Retsky M, Demicheli R, Hrushesky W (2003) Breast cancer screening, controversies and future directions. Curr Opin Obstet Gynecol 15, 1-8 Rietbergen JBW, Boeken Kruger AE, Hoedemaker RF, Bangma CH, Kirkels WJ, Schröder FH (1998) Repeat screening for prostate cancer after 1-year followup in 984 biopsied men, clinical and pathological features of detected cancer. J Urol 160, 2121-2125 Ross KS, Carter HB, Pearson JD, Guess HA (2000) Comparative efficiency of prostate-specific antigen screening strategies for prostate cancer detection. JAMA 284, 1399-1405 Sakr WA, Haas GP, Cassin BF, Pontes JE, Crissman JD (1993) The frequency of carcinoma and intraepithelial neoplasia of the prostate in young male patients. J Urol 150, 379-385
453
Sandblom and Varenhorst: How should a screening programme for prostate cancer be designed? Sandblom G, Carlsson P, Sennfält K, Varenhorst E (2004a) A population-based study of pain and quality of life during the year before death in men with prostate cancer. Br J Cancer 90, 1163-1168 Sandblom G, Carlsson P, Sigsjo P, Varenhorst E (2001) Pain and health-related quality of life in a geographically defined population of men with prostate cancer. Br J Cancer 85, 497-503 Sandblom G, Dufmats M, Varenhorst E (2000) Long-term survival in a Swedish population-based cohort of men with prostate cancer. Urology 56, 442-447 Sandblom G, Varenhorst E, Löfman O, Rosell J, Carlsson P (2004b) Clinical consequences of screening for prostate cancer. 15 years follow-up of a randomised controlled trial. Eur Urol, in press Schellhammer PF, Moriarty R, Bostwick D, Kuban D (2000) Fifteen-year minimum follow-up of a prostate brachytherapy series, comparing the past with the present. Urology 56, 436439 Schröder FH, Kranse R, Riebergen J, Hoedemaeke R, Krikels W (1999) The European Randomized Study of Screening for Prostate Cancer (ERSPC) : an update. Members of the ERSPC, Section Rotterdam. Eur Urol 35, 539-543 Schröder FH, van der Maas P, Beemsterboer P, Kruger AB, Hoedemaker R, Rietbergen J, Kranse R (1998) Evaluation of the digital rectal examination as a screening test for prostate cancer. Rotterdam section of the European randomised study of screening for prostate cancer. J Natl Cancer Inst 90, 1817-1823 Sennfält K, Carlsson P, Thorfinn J, Frisk J, Henriksson M, Varenhorst E (2003) Technological changes in the management of prostate cancer result in increased healthcare costs. Scand J Urol Nephrol 37, 226-231 Sennfält K, Sandblom G, Carlsson P, Varenhorst E (2004) Costs and effects of prostate cancer screening in Sweden – a 15year follow-up of a randomised trial. Scand J Urol Nephrol 38, 291-298 Stephenson AJ, Shariat SF, Zelefsky MJ, Kattan MW, Butler EB, Teh BS, Klein EA, Kupelian PA, Roehrborn CG, Pistenmaa DA, Pacholke HD, Liauw SL, Katz MS, Leibel SA, Scardino PT, Slawin KM (2004) Salvage radiotherapy for recurrent prostate cancer after radical prostatectomy. JAMA 291, 1325-1332 Stone NN, Stock RG (1999) Prostate brachytherapy, treatment strategies. J Urol 162, 421-426 Thompson IM, Goodman PJ, Tangen CM, Lucia MS, Miller GJ, Ford LG, Lieber MM, Cespedes RD, Atkins JN, Lippman SM, Carlin SM, Ryan A, Szczepanek CM, Crowley JJ, Coltman Jr CA (2003) The influence of Finasteride on the development of prostate cancer. N Engl J Med 349,215-224 Thompson IM, Pauler DK, Goodman PJ, Tangen CM, Lucia MS, Parnes HL, Minasian LM, Ford LG, Lippman SM, Crawford ED, Crowley JJ, Coltman Jr CA (2004) Prevalence of prostate cancer among men with a prostate-specific antigen
level < or =4.0 ng per milliliter. N Engl J Med 350, 22392246 Tornblom M, Eriksson H, Franzen S, Gustafsson O, Lilja H, Norming U, Hugosson J (2004) Lead time associated with screening for prostate cancer. Int J Cancer 108, 122-129 UICC (1997) TNM Classification of Malignant Tumours (ed 5) . Geneva, Switzerland, International Union Against Cancer Ung JO, San Francisco IF, Regan MM, DeWolf WC, Olumi AF (2003) The relationship of prostate gland volume to extended needle biopsy on prostate cancer detection. J Urol 169, 130135 Valeri A, Cormier L, Moineau MP, Cancel-Tassin G, Azzouzi R, Doucet L, Baschet F, Cussenot I, L'Her J, Berthon P, Mangin P, Cussenot O, Morin JF, Fournier G (2002) Targeted screening for prostate cancer in high risk families, early onset is a significant risk factor for disease in first degree relatives. J Urol 168, 483-487 van der Cruijsen, van der Kwast TH, Schröder FH (2003) Interval carcinomas in the European Randomised Study of Screening for Prostate Cancer (ERSPC) Rotterdam. J Natl Cancer Inst 95, 1462-1466 Vis AN, Noordzij MA, Fitoz K, Wildhagen MF, Schroder FH, van der Kwast TH (2000) Prognostic value of cell cycle proteins p27(kip1) and MIB-1, and the cell adhesion protein CD44s in surgically treated patients with prostate cancer. J Urol 164, 2156-2161 Walsh PC, Brooks JD (1997) The Swedish prostate cancer paradox. JAMA 277, 497-498 Walsh PC, Lepor H, Eggleston JC (1983) Radical prostatectomy with preservation of sexual function, anatomical and pathological consideration. Prostate 4, 473-485 Whitmore Jr WF (1990) Natural history of low-stage prostate cancer and the impact of early detection. Urol Clin North Am 17, 689-697 Wilson J, Jungner G (1986) Principles and practice of screening for disease. Geneve WHO, Public Health Paper 1986, No 34. Anonymous World Health Organisation (1996) World health statistics annual 1995. Geneva, WHO Wu TT, Hsu YS, Wang JS, Lee YH, Huang JK (2003) The role of p53, bcl-2 and E-cadherin expression in predicting biochemical relapse for organ confined prostate cancer in Taiwan. J Urol 170, 78-81 Yao SL, Lu Yao G (2001) Interval after prostate specific antigen testing and subsequent risk of incurable prostate cancer. J Urol 166, 861-865 Zappa M, Ciatto S, Bonardi R, Mazzotta A (1998) Overdiagnosis of prostate carcinoma by screening, An estimate based on results of the Florence screening pilot study. Ann Oncol 9, 1297-1301
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Thermal ablation of liver tumors Review Article
Charles R. Scoggins*, John F. Gleason Jr, Robert C. G. Martin, Farid J. Kehdy, Julie R. Hutchinson, and Kelly M. McMasters Division of Surgical Oncology, Department of Surgery, University of Louisville, Louisville, KY
__________________________________________________________________________________ *Correspondence: Charles R. Scoggins, MD, Assistant Professor of Surgery, Division of Surgical Oncology, University of Louisville, Norton Healthcare Pavilion, 315 East Broadway, Suite 303, Louisville, KY 40202; Phone: (502). 629-3380; Fax: (502). 629-3393; email: charles.scoggins@nortonhealthcare.org Key words: Thermal ablation, liver tumors, Radiofrequency ablation, Laser-induced thermotherapy, Microwave ablation Abbreviations: computed tomography (CT); Hepatocellular carcinoma (HCC); laser-induced thermotherapy (LITT); magnetic resonance imaging (MRI); microwave ablation (MA); Percutaneous ethanol injection (PEI); radiofrequency ablation (RFA) Received: 16 November 2004; Accepted: 30 November 2004; electronically published: November 2004
Summary The liver is a common site of malignant disease, either primary liver cancer or metastatic disease. Resection remains the best treatment available, however for many patients resection is not an option. Newer techniques have evolved to allow for liver tumor destruction with acceptable rates of morbidity and mortality, and with promising short-term results.
available for treatment. Recently, progress in systemic chemotherapy for metastatic colorectal cancer has resulted in increased response rates and modest increases in median survival, but still there are few long-term survivors (Conti et al, 1996; Bajetta et al, 2004; Cunningham et al, 2004). For HCC or other hypervascular tumors, embolization or chemoembolization may provide hepatic disease control for patients that are neither resectable nor appropriate for transplantation (Llovet et al, 2002; Lo et al, 2002; O’Suilleabhain et al, 2003). There are several ablative therapies available for treatment of unresectable hepatic tumors. Percutaneous ethanol injection (PEI) can be effective for small HCC tumors, but usually requires multiple treatments (Lencioni et al, 1997). PEI has been shown to be an effective alternative to resection, but is most beneficial in patients with adequate hepatic function and small, wellcircumscribed tumors (Mazzanti et al, 2004). PEI has limited effects on colorectal metastases and non-vascular tumors. PEI may be combined with other cytotoxic agents to enhance tumor cell kill (Kurokohchi et al, 2004). Cryoablation has been used for a number of malignant liver tumors, however may be associated with a complication rate that is higher than some other ablative techniques (Sohn et al, 2003). The “heat sink” effect may limit the efficacy of cryoablation of tumor located near large hepatic blood vessels. The complication rate and the local recurrence rate have made cryoablation less
I. Introduction The American Cancer Society, (2004) estimates over 18,000 cases of primary liver cancer will be diagnosed United States in 2004, of which over one-half are hepatocellular carcinoma. Hepatocellular carcinoma (HCC) is one of the most prevalent solid cancers worldwide, especially in East Asia and South Africa, with over one million new cases annually (Qin and Tang, 2002). The liver also is a common site of metastasis, particularly from colorectal cancer (Weiss et al, 1986). Surgical resection is remains the treatment of choice for patients with hepatic malignancies, with contemporary series demonstrating 5-year survival rates of 25% to 40% for both colorectal malignancies (Sugihara et al, 1993; Rees et al, 1997; Fong et al, 1999) and HCC (Vauthey et al, 1995; Hanazaki et al, 2000; Poon et al, 2000; Ercolani et al, 2003). However, the majority of patients with liver tumors are not candidates for resection due to tumor size, location, multifocality, inadequate liver reserve, and other health problems (Nagorney et al, 1989). The treatment options for patients with unresectable hepatic tumors include systemic chemotherapy, chemoembolization, and locally ablative techniques (including ethanol ablation, cryoablation, and radiofrequency, laser or microwave ablation). Results for systemic chemotherapy have been abysmal for HCC, with short median survivals and low response rates (Friedman, 1983; Lin et al, 1997; Patt et al, 2003). Additionally, the presence of cirrhosis often limits the cytotoxic agents
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Scoggins et al: Thermal ablation of liver tumors attractive than other ablative therapies (Adam et al, 2002; Kerkar et al, 2004). Currently, the most widely used hepatic ablative strategies are the thermal ablative techniques of radiofrequency ablation (RFA), laser-induced thermotherapy (LITT), and microwave ablation (MA). These techniques share a common method for inducing tumor killing: they heat the tumor resulting in coagulative necrosis. Thermal ablative techniques result in permanent cellular damage and death by achieving temperatures above 50° to 60°C (McGahan et al, 1992; Patterson et al, 1998). These three techniques appear promising for the future treatment of liver malignancies because they may be used in a minimally invasive setting, are relatively inexpensive, versatile, and have acceptably low rates of morbidity and mortality.
ablations. Inflow occlusion during the ablation may help to mitigate this problem, and can result in larger areas of ablation, as well as aid in ablating tumors abutting large blood vessels. Occlusion of the hepatic inflow by the Pringle maneuver has been shown in several studies to increase the size and regularity of ablations and possibly to enhance tumor kill (Chinn et al, 2001; Chang et al, 2002; Yamasaki et al, 2002). In addition to its utility in the initial placement of the electrode, ultrasonography can be used to monitor the progression of the ablation during the procedure. Ablated areas become hyperechoic due to gas bubbles generated by the heated tissue. Gas bubbles generated by an ablation may interfere with accurate ultrasonography of tissues deep to the electrode, which can hinder repositioning of the electrode for overlapping ablation zones when treating larger tumors. Ablating the deepest portions of the tumor first and re-deploying the electrode serially as it is withdrawn may obviate this phenomenon. Imaging-related difficulties may be more pronounced with the percutaneous approach, as transcutaneous ultrasound may not be as accurate as intra-operative ultrasonography. Percutaneous RFA is the least invasive mode of delivery, and can be performed in an outpatient setting, making it an acceptable alternative for many patients. Laparoscopic RFA has the advantages of providing more accurate cancer staging using laparoscopy and laparoscopic ultrasound and minimizing the probability of injury to nearby organs, while still being minimally invasive. The advantages of an open approach to RFA include excellent cancer staging, accessibility to all sections of the liver, ease of intra-operative ultrasound examination, inflow occlusion (Pringle maneuver), minimal risk to peripheral organs, and the ability to perform a resection in conjunction with the ablation. The main disadvantage is its invasiveness, which leads to recovery time, and length of hospital stay (Machi et al, 2001). Whether RFA should be performed percutaneously, laparoscopically, or via an open approach should be individualized for each patient. Percutaneous RFA is well suited for patients who have a limited number of small tumors and who are not good candidates for more invasive procedures due to co-morbid conditions. Cirrhotic patients with a small hepatocellular carcinomas and patients with one or two intrahepatic recurrences following hepatectomy are good examples of patients who might be best served by the percutaneous approach. Laparoscopic RFA is a good option for patients with a small number of tumors for which percutaneous RFA would not be safe, such as peripherally situated tumors near adjacent organs such as the stomach or colon. Patients may recover more quickly from a laparoscopic RFA as opposed to an open RFA, yet substantial surgical judgment must be used in selecting patients for which the laparoscopic approach is best. Open surgical RFA is preferable for patients with large tumors, multiple tumors, and tumors near large blood vessels (because of heat sink effect). Hepatic inflow occlusion diminishes the heat sink effect from large intrahepatic vessels, and is easier with open surgery as opposed to laparoscopic surgery. Additionally, the open approach
II. Radiofrequency ablation RFA was first mentioned as a treatment for hepatic malignancies in 1990 by McGahan and Rossi. Since then, the popularity of RFA as a viable treatment for unresectable HCC and hepatic metastases has increased exponentially, mainly due to the technique’s versatility and effectiveness. RFA can be employed in a number of ways: through traditional open surgery, laparoscopically, or percutaneously under guidance of ultrasound, computed tomography (CT), or magnetic resonance imaging (MRI). RFA needle electrode probes release high-frequency (460 kHz) alternating current into the target tissue. The ions in the tissue are frictionally heated as they attempt to change direction with the alternating current. After frictional heating rises above 50° to 60°C, the tissue surrounding the probe forms an area of necrosis as the cells die. RFA systems include an electrical generator, grounding pads, and needle electrodes (ranging from 14gauge to 19-gauge). Three companies currently market commercially available RFA devices in the United States: Rita Medical Systems of Mountain View, CA (Mountain View, CA), Radiotherapeutics (Mountain View, CA) and Radionics (Burlington, MA) (McGhana, 2001; Wong et al, 2001). It is presently unclear whether any system is superior to another in terms of ablation size, reproducibility, or local tumor control (de Baere et al, 2001). Grounding pads are placed on the patient’s thighs prior to an RFA procedure. The needle electrode is advanced into the liver tumor either by an open, laparoscopic, or percutaneous approach, and ultrasoundguidance is used to position the electrode accurately. The RFA generator will heat the tissue and maintain the desired temperature by changing the power output over time. Once the ablation is complete, the electrode is generally removed slowly while the track is cauterized to prevent tumor seeding and minimize bleeding. Multiple overlapping ablations may be required for larger tumors. A successfully ablated lesion should have a region of complete necrosis encompassing the entire tumor and 1 cm of the surrounding normal hepatic parenchyma. The cooling effect of large vessels in close proximity to the electrode creates a heat sink, which limits the size of the ablation zone and can cause irregularly shaped 456
Cancer Therapy Vol 2, page 457 allows extremely accurate intra-operative ultrasound to be done, which facilitates ablation of large tumors near blood vessels (Figure 1). Peripherally situated tumors may be safely ablated by packing the adjacent organs away from the liver, thus providing a layer of protection difficult to achieve with the other approaches. Multiple studies about RFA of hepatic malignancies have been published since 1990; however, many of these studies are not well controlled in respect to patient selection, type of cancer, stage of disease, and treatment approach. The high variability of patients in each study make definitive conclusions difficult, but the results do point strongly toward RFA being an effective tool against hepatic malignancies. One concern for all of the ablative techniques has been local failure. An analysis of several of the larger RFA trials shows local recurrence per ablated tumor to range from 1.8% to 26.6% and overall recurrence (outside the ablation zone) to range from 18% to 73.3% (Curley et al, 1999, 2000; Rossi et al, 2000; Bowles et al, 2001; Wong et al, 2001; Kuvshinoff and Ota, 2002; Jiang et al, 2002; Kosari et al, 2002; Bleicher et al, 2003; Komorizono et al, 2003) (Table 1). In a large trial conducted at the M. D. Anderson Cancer Center, Abdalla
and colleagues (Abdalla et al, 2004) compared recurrence and survival between patients with colorectal cancer metastases treated with resection, resection and RFA, RFA alone, or chemotherapy alone (including some patients treated with hepatic infusional therapy). Overall and liveronly recurrences were more common following RFA compared to resection. Indeed, the liver-only failure rate was a full four-fold higher for ablated patients as opposed to resected patients (Abdalla et al, 2004). While this increased recurrence rate likely reflects the higher risk among unresectable patients because of a greater number of tumors and other factors, it supports the generally accepted notion that patients with resectable tumors should undergo resection, rather than ablation, whenever possible (Machi et al, 2001). Tumor size has been shown to be an important factor affecting the rate of local recurrence following RFA (Wood et al, 2000; Machi et al, 2001; Chan et al, 2002; Kosari et al, 2002; Kuvshinoff and Ota, 2002; Bleicher et al, 2003). Machi et al, (2001), Kuvshinoff and Ota, (2002) and Kosari et al, (2002) all noted significantly higher risk for local recurrence for tumors > 4 cm in greatest diameter. Interestingly, Machi (2001) also associated higher local recurrence rates with metastatic tumors than with primary hepatomas. Some studies have also shown that the approach chosen for delivery of RFA (open, laparoscopic, or percutaneous) may impact local recurrence. Kuvshinoff and Ota, (2002) and Scaife and Curley, (2003) showed lower rates of local recurrence for open and laparoscopic RFA versus a percutaneous approach. The increased access to the liver and use of intra-operative ultrasound are likely the main reasons for lower local recurrence rates for operative RFA. The complication rate associated with RFA is quite acceptable, ranging from 2.4% to 27% in several trials (Curley et al, 1999, 2000; Bowles et al, 2001; Wong et al, 2001; Jiang et al, 2002; Iannitti et al, 2002; Kosari et al, 2002; Kuvshinoff and Ota, 2002; Bleicher et al, 2003; Komorizono et al, 2003), (Table 1). In a large review by Scaife and Curley, (2003) reported an overall mortality rate of 0.5%, a major complication rate of 2%, and a minor complication rate of 6% by combining over 1300 patients from18 different studies. The complications following RFA include wound infections, intra-abdominal abscess, renal failure, hepatic abscess, biliary injury, pleural effusion, fever, pain, and minor hemorrhage. RFAassociated morbidity, mortality, and local recurrence are low enough to justify its use for unresectable liver tumors. In non-randomized studies, the available evidence suggests that RFA extends survival. As with other hepatic directed therapies, recurrence either in unablated areas of the liver or in other organs is common, even in the absence of local recurrence (Curley et al, 1999, 2000; Wood et al, 2000; Kosari et al, 2002). Kosari et al, (2002) reported distant failure in 51% of the patients following RFA and local failure in only 1.8% of the hepatic malignancies ablated, but new hepatic or systemic disease still developed in 27.6% of patients at a median follow-up of 15 months. These high figures for distant failure suggest that RFA may best improve long-term survival when
Figure 1. (A) CT demonstrating 2 hepatic metastases from an obstructing right colon adenocarcinoma. (B) Post-RFA CT demonstrating complete ablation of metastatic disease.
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Scoggins et al: Thermal ablation of liver tumors Table 1. Results for RFA of unresectable liver tumors Author, year Curley et al, 2000 Rossi et al, 2000 Wong et al, 2001
N 110 62 40
Bowles et al, 2001
76
Kuvshinoff and 2002 Jiang et al, 2002
Ota,
45 36
Kosari et al, 2002
45
Bleicher et al, 2003
153
Komorizono et al, 2003
56
Tumor type HCC 100% HCC 100% HCC 5% Mets 95% HCC 32.9% Mets 67.1% HCC 24.4% Mets 75.6% HCC 58.3% Mets 41.7% HCC 11% Mets 89% HCC 13.7% Mets 86.3% HCC 100%
Median F/U 19 12 NR
Recurrence Rate per tumor per patient 3.6% 45.5% 18% 30% 10% 27.5%
Morbidity 12.7% 12.9% 20%
15
9%
60%
17%
12
26.6%
73.3%
22%
10
16.7%
NR
11.1%
19.5
7.7%
73%
27%
11
20.9%
NR
11.7%
16
26%
18%
21.4%
N, number or patients; F/U, follow-up in months; HCC, hepatocellular carcinoma; Mets, metastases (including colorectal); NR, not reported.
combined with other systemic and intra-hepatic treatments. Some studies have now reported overall survival statistics after RFA with moderate follow-up time. Solbiati et al, (2001) reported overall survival to be 93%, 62%, and 41% at one, two, and three years, respectively. Iannitti et al, (2002) reported overall survival for patients with colorectal metastases treated with RFA to be 87%, 77%, and 50% at one, two, and three years, respectively. They also reported 92%, 75%, and 60% overall survival rates at one, two, and three years, respectively for patients with HCC treated with RFA (Iannitti et al, 2002). When compared to liver resection, however, RFA clearly benefits patients less, with four-year survival of 22% as compared to 65% for resection (Abdalla et al, 2004). Survival for unresectable patients with colorectal cancer metastases is better following RFA than with nonsurgical treatments (Abdalla et al, 2004).
RFA, can be performed by percutaneous, laparoscopic, or open surgical approaches. LITT application kits used for percutaneous procedures include a cannulation needle with a tetragonally sharpened tip, a guide wire, and a sheath system. The catheter aids in easy removal of the laser fibers and prevents direct contact between the fibers and the treated tissue. The applicator has magnetic markers on it that allow the use of MR-guidance for precise placement. The laser is transmitted via an optical fiber cable that is placed in the desired treatment area by advancing it through the applicator system (Vogl et al, 2001). One of the main advantages of LITT is its compatibility with MR-guidance. It does not interfere with MR-imaging like RFA and microwave ablation. Ultrasound or CT may be used for tumor targeting and placement of the laser fibers, followed by MR imaging of the ablation. Alternatively, intraoperative MRI can provide precise targeting and ablation in a single unit. MR imaging provides increased lesion visibility, three-dimensional laser navigation, and allows monitoring of temperature changes and tissue coagulation (Vogl et al, 1995; Dick et al 2003a, b). Real-time temperature monitoring is a notable advantage because it allows the clinician to assure temperatures are high enough in the tumor for a successful ablation and low enough in surrounding tissue to minimize collateral damage. However, present limitations of MRI thermal mapping have precluded this technique from widespread acceptance. There have been even fewer large-scale studies of LITT than of RFA (Heisterkamp et al, 1999). In one large European study, Mack et al, (2001) treated liver tumors with LITT and demonstrated impressive local control rates, survival data, and low morbidity. They used MRguided LITT and reported results for 1981 lesions in 705 patients, including 97.9% local tumor control at 6 months, 93% one-year survival, 74% two-year survival, 50% three-
III. Laser-induced thermotherapy Laser-induced thermotherapy (LITT) is another nonresectional therapy that has been used to treat liver malignancies. LITT, like other thermal ablative techniques, kills tumors by raising the temperature above 55째 to 60째 C. LITT uses photons from a low-intensity laser, which are absorbed by natural molecular chromophores in all human cells and converted into heat (Izzo, 203). Diode lasers (wavelength 800-980 nm) or NYAG lasers (wavelength 1064 nm) operating at low energy generate zones of ablation during LITT (Germer et al, 1998; Muralidharan and Christophi, 2001). Similar to RFA, LITT can be combined with blood flow occlusion and multiple overlapping laser applications to increase the volume of ablation (Heisterkamp et al, 1997; Sturesson et al, 1997). Placing multiple laser fibers into the target tissue may create overlapping zones of ablation (Ivarsson et al, 1998). LITT has the same indications as RFA: unresectable primary and secondary liver tumors, and like
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Cancer Therapy Vol 2, page 459 year survival, 30% five-year survival, and only a 7.5% rate of complications. In a study of 74 patients with single tumors < 4 cm or one to three tumors < 3 cm in greatest dimension, Pacella et al, (2001) reported a 6% local recurrence rate and overall survival rates of 99%, 95%, 68%, and 15% at one, two, three, and five years, respectively. An analysis of the complications associated with the LITT in 899 patients with 2520 lesions concluded that the procedure had an acceptably low morbidity (Vogl et al, 2002). It remains to be seen whether other centers can duplicate these intriguing results; however, the results of these authors are encouraging and warrant further study.
indicating this is a safe technique, even in patients with severe comorbid conditions (Dong et al, 2003).
V. Conclusion The past several years have seen rapid advances in the treatment of patients with liver tumors. Thermal ablative techniques have become a valid treatment option for patients with unresectable liver tumors and for patients whose underlying disease state or physical condition will not permit a hepatectomy. Resection remains the treatment of choice for liver malignancies; however, most patients have tumors that are not resectable. RFA, LITT, and MA appear to be safe, effective treatments for such patients. The available evidence from non-randomized studies suggests that, given the limited efficacy of systemic therapy, the ability to completely ablate liver tumors extends survival. The range of thermal ablation approaches also permits minimally invasive treatment of recurrence in the liver following resection or ablation. Further study is necessary to demonstrate conclusively the survival benefit of thermal ablation, to aid in patient selection for various approaches, and to determine the most effective ablation systems and techniques. With improvements in the technology, it also remains to be determined whether thermal ablation can replace resection in selected patients.
IV. Microwave ablation Microwave ablation developed out of technology first used for hemostasis and coagulation during hepatic resection (Tabuse and Katsumi, 1981; Tabuse et al, 1986). It has since been adapted and used to treat small HCC tumors in the liver and has been most extensively studied in Japan for the treatment of patients with cirrhotic HCC lesions. Microwaves create dielectric heat in tissue and cells by stimulating tissue water molecules, which leads to frictional heating and necrosis in a similar manner to RFA and LITT (Izzo, 2003). MA can be applied percutaneously, laparoscopically, or as an open surgical procedure. Indications for MA include unresectable hepatic malignancies and patients not suited for major hepatectomy. The needle electrode is advanced directly into the tumor under ultrasound guidance and microwave emission commences. As with the other thermal ablative techniques, multiple overlapping zones of ablation are created to ablate larger tumors. Newer antenna designs, including looping coils may allow for more precise geometric ablation zones to be created with shorter treatment times (Wright et al, 2003; Shock et al, 2004). Our initial experience in a cohort of 20 patients with a multiple antennae MA system (Vivant Medical, Mountain View, CA) has been very favorable; this system allows ablation of multiple tumors at the same time, large (5 to 7 cm) ablation zones, complete tumor ablation, rapid ablation times (5 to 10 minutes maximum), and does not require grounding pads that can potentially result in skin burns such as in RFA systems (Martin et al, unpublished data). There are few studies on MA with data on local recurrence and complications following treatment, and most of the studies that have been published have reported small numbers of patients. One study involving 19 patients with 31 tumors (mean tumor size 2.1 cm). reported 9.7% local recurrence per tumor (Sato et al, 1996). Microwave ablation has been shown to be a valid treatment option in cirrhotic patients with HCC. Local failure rates are similar to those seen with the other thermal ablative techniques (Xu et al, 2004). In a large study by Dong et al, (Dong et al, 2003) 234 patients with 339 HCC tumors were treated percutaneously with MA. Survival rates of 92% were seen at one year, and long-term survival at five years of 56.7% was reported (Dong et al, 2003). The majority (88.5%) of the patients were Childâ&#x20AC;&#x2122;s class B cirrhotics. There were no perioperative deaths and very few minor complications,
References Abdalla EK, Vauthey JN, Ellis LM, Ellis V, Pollock R, Broglio KR, Hess K, Curley SA (2004) Recurrence and outcomes following hepatic resection, radiofrequency ablation, and combined resection/ablation for colorectal liver metastases. Ann Surg 239, 818-825. Adam R, Hagopian EJ, Linhares M, Krissat J, Savier E, Azoulay D, Kunstlinger F, Castaing D, Bismuth H (2002) A comparison of percutaneous cryosurgery and percutaneous radiofrequency for unresectable hepatic malignancies. Arch Surg 137, 1332-1339. American Cancer Society.(2004) Cancer Facts and Figures. 158. 2004. Bajetta E, Beretta E, Di Bartolomeo M, Cortinovis D, Ferrario E, Dognini G, Toffolatti L, Buzzoni R (2004) Efficacy of treatment with irinotecan and oxaliplatin combination in FUresistant metastatic colorectal cancer patients. Oncology 66, 132-137. Bleicher RJ, Allegra DP, Nora DT, Wood TF, Foshag LJ, Bilchik AJ (2003) Radiofrequency ablation in 447 complex unresectable liver tumors, lessons learned. Ann Surg Oncol 10, 52-58. Bowles BJ, Machi J, Limm WM, Severino R, Oishi AJ, Furumoto NL, Wong LL, Oishi RH (2001) Safety and efficacy of radiofrequency thermal ablation in advanced liver tumors. Arch Surg 136, 864-869. Chan RP, Asch M, Kachura J, Ho CS, Greig P, Langer B, Sherman M, Wong F, Feld R, Gallinger S (2002) Radiofrequency ablation of malignant hepatic neoplasms. Can Assoc Radiol J 53, 272-278. Chang CK, Hendy MP, Smith JM, Recht MH, Welling RE (2002) Radiofrequency ablation of the porcine liver with complete hepatic vascular occlusion. Ann Surg Oncol 9, 594-598.
459
Scoggins et al: Thermal ablation of liver tumors Chinn SB, Lee FT, Jr., Kennedy GD, Chinn C, Johnson CD, Winter TC, III, Warner TF, Mahvi DM (2001) Effect of vascular occlusion on radiofrequency ablation of the liver, results in a porcine model. AJR Am J Roentgenol 176, 789795. Conti JA, Kemeny NE, Saltz LB, Huang Y, Tong WP, Chou TC, Sun M, Pulliam S, Gonzalez C (1996) Irinotecan is an active agent in untreated patients with metastatic colorectal cancer. J Clin Oncol 14, 709-715. Cunningham D, Humblet Y, Siena S, Khayat D, Bleiberg H, Santoro A, Bets D, Mueser M, Harstrick A, Verslype C, Chau I, Van Cutsem E (2004) Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer. N Engl J Med 351, 337-345. Curley SA, Izzo F, Delrio P, Ellis LM, Granchi J, Vallone P, Fiore F, Pignata S, Daniele B, Cremona F (1999) Radiofrequency ablation of unresectable primary and metastatic hepatic malignancies, results in 123 patients. Ann Surg 230, 1-8. Curley SA, Izzo F, Ellis LM, Nicolas Vauthey J, Vallone P (2000) Radiofrequency ablation of hepatocellular cancer in 110 patients with cirrhosis. Ann Surg 232, 381-91. de Baere T, Denys A, Wood BJ, Lassau N, Kardache M, Vilgrain V, Menu Y, Roche A.(2001) Radiofrequency liver ablation, experimental comparative study of water-cooled versus expandable systems. AJR Am J Roentgenol 176, 187-192. Dick EA, Joarder R, de Jode M, Taylor-Robinson SD, Thomas HC, Foster GR, Gedroyc WM (2003a) MR-guided laser thermal ablation of primary and secondary liver tumours. Clin Radiol 58, 112-120. Dick EA, Wragg P, Joarder R, de Jode M, Lamb G, Gould S, Gedroyc WM (2003b) Feasibility of abdomino-pelvic T1weighted real-time thermal mapping of laser ablation. J Magn Reson Imaging 17, 197-205. Dong B, Liang P, Yu X, Su L, Yu D, Cheng Z, Zhang J (2003) Percutaneous sonographically guided microwave coagulation therapy for hepatocellular carcinoma, results in 234 patients. AJR Am J Roentgenol 180, 1547-1555. Ercolani G, Grazi GL, Ravaioli M, Del Gaudio M, Gardini A, Cescon M, Varotti G, Cetta F, Cavallari A (2003) Liver resection for hepatocellular carcinoma on cirrhosis, univariate and multivariate analysis of risk factors for intrahepatic recurrence. Ann Surg 237, 536-543. Fong Y, Fortner J, Sun RL, Brennan MF, Blumgart LH (1999) Clinical score for predicting recurrence after hepatic resection for metastatic colorectal cancer, analysis of 1001 consecutive cases. Ann Surg 230, 309-18. Friedman MA (1983) Primary hepatocellular cancer--present results and future prospects. Int J Radiat Oncol Biol Phys 9, 1841-1850. Germer CT, Albrecht D, Roggan A, Buhr HJ (1998) Technology for in situ ablation by laparoscopic and image-guided interstitial laser hyperthermia. Semin Laparosc Surg 5, 195203. Hanazaki K, Kajikawa S, Shimozawa N, Mihara M, Shimada K, Hiraguri M, Koide N, Adachi W, Amano J (2000) Survival and recurrence after hepatic resection of 386 consecutive patients with hepatocellular carcinoma. J Am Coll Surg 191, 381-388. Heisterkamp J, van Hillegersberg R, Mulder PG, Sinofsky EL, IJzermans JN (1997) Importance of eliminating portal flow to produce large intrahepatic lesions with interstitial laser coagulation. Br J Surg 84, 1245-1248.
Heisterkamp J, van Hillegersberg R, IJzermans JN (1999) Interstitial laser coagulation for hepatic tumours. Br J Surg 86, 293-304. Iannitti DA, Dupuy DE, Mayo-Smith WW, Murphy B (2002) Hepatic radiofrequency ablation. Arch Surg 137, 422-426. Ivarsson K, Olsrud J, Sturesson C, Moller PH, Persson BR, Tranberg KG (1998) Feedback interstitial diode laser (805 nm) thermotherapy system, ex vivo evaluation and mathematical modeling with one and four-fibers. Lasers Surg Med 22, 86-96. Izzo F (2003) Other thermal ablation techniques, microwave and interstitial laser ablation of liver tumors. Ann Surg Oncol 10, 491-497. Jiang HC, Liu LX, Piao DX, Xu J, Zheng M, Zhu AL, Qi SY, Zhang WH, Wu LF (2002) Clinical short-term results of radiofrequency ablation in liver cancers. World J Gastroenterol 8, 624-630. Kerkar S, Carlin AM, Sohn RL, Steffes C, Tyburski J, Littrup P, Weaver D (2004) Long-term follow up and prognostic factors for cryotherapy of malignant liver tumors. Surgery 136, 770-779. Komorizono Y, Oketani M, Sako K, Yamasaki N, Shibatou T, Maeda M, Kohara K, Shigenobu S, Ishibashi K, Arima T (2003) Risk factors for local recurrence of small hepatocellular carcinoma tumors after a single session, single application of percutaneous radiofrequency ablation. Cancer 97, 1253-1262. Kosari K, Gomes M, Hunter D, Hess DJ, Greeno E, Sielaff TD (2002) Local, intrahepatic, and systemic recurrence patterns after radiofrequency ablation of hepatic malignancies. J Gastrointest Surg 6, 255-263. Kurokohchi K, Masaki T, Miyauchi Y, Funaki T, Yoneyama H, Miyoshi H, Yoshida S, Himoto T, Morishita A, Uchida N, Watanabe S, Kuriyama S (2004) Percutaneous ethanol and lipiodol injection therapy for hepatocellular carcinoma. Int J Oncol 24, 381-387. Kuvshinoff BW, Ota DM (2002) Radiofrequency ablation of liver tumors, influence of technique and tumor size. Surgery 132, 605-611. Lencioni R, Pinto F, Armillotta N, Bassi AM, Moretti M, Di Giulio M, Marchi S, Uliana M, Della CS, Lencioni M, Bartolozzi C (1997) Long-term results of percutaneous ethanol injection therapy for hepatocellular carcinoma in cirrhosis, a European experience. Eur Radiol 7, 514-519. Lin DY, Lin SM, Liaw YF (1997) Non-surgical treatment of hepatocellular carcinoma. J Gastroenterol Hepatol 12, S319-S328. Llovet JM, Real MI, Montana X, Planas R, Coll S, Aponte J, Ayuso C, Sala M, Muchart J, Sola R, Rodes J, Bruix J (2002) Arterial embolisation or chemoembolisation versus symptomatic treatment in patients with unresectable hepatocellular carcinoma, a randomised controlled trial. Lancet 359, 1734-1739. Lo CM, Ngan H, Tso WK, Liu CL, Lam CM, Poon RT, Fan ST, Wong J (2002) Randomized controlled trial of transarterial lipiodol chemoembolization for unresectable hepatocellular carcinoma. Hepatology 35, 1164-1171. Machi J, Uchida S, Sumida K, Limm WM, Hundahl SA, Oishi AJ, Furumoto NL, Oishi RH (2001) Ultrasound-guided radiofrequency thermal ablation of liver tumors, percutaneous, laparoscopic, and open surgical approaches. J Gastrointest Surg 5, 477-489. Mack MG, Straub R, Eichler K, Engelmann K, Zangos S, Roggan A, Woitaschek D, Bottger M, Vogl TJ (2001) Percutaneous MR imaging-guided laser-induced
460
Cancer Therapy Vol 2, page 461 thermotherapy of hepatic metastases. Abdom Imaging 26, 369-374. Mazzanti R, Arena U, Pantaleo P, Antonuzzo L, Cipriani G, Neri B, Giordano C, Lanini F, Marchetti S, Gentilini P (2004) Survival and prognostic factors in patients with hepatocellular carcinoma treated by percutaneous ethanol injection, A 10-year experience. Can J Gastroenterol 18, 611-618. McGahan JP, Browning PD, Brock JM, Tesluk H (1990) Hepatic ablation using radiofrequency electrocautery. Invest Radiol 25, 267-270. McGahan JP, Brock JM, Tesluk H, Gu WZ, Schneider P, Browning PD (1992) Hepatic ablation with use of radiofrequency electrocautery in the animal model. J Vasc Interv Radiol 3, 291-297. McGhana JP, Dodd GD, III (2001) Radiofrequency ablation of the liver, current status. AJR Am J Roentgenol 176, 3-16. Muralidharan V, Christophi C (2001) Interstitial laser thermotherapy in the treatment of colorectal liver metastases. J Surg Oncol 76, 73-81. Nagorney DM, Van Heerden JA, Ilstrup DM, Adson MA (1989) Primary hepatic malignancy, surgical management and determinants of survival. Surgery 106, 740-748. O'Suilleabhain CB, Poon RT, Yong JL, Ooi GC, Tso WK, Fan ST (2003) Factors predictive of 5-year survival after transarterial chemoembolization for inoperable hepatocellular carcinoma. Br J Surg 90, 325-331. Pacella CM, Bizzarri G, Magnolfi F, Cecconi P, Caspani B, Anelli V, Bianchini A, Valle D, Pacella S, Manenti G, Rossi Z (2001) Laser thermal ablation in the treatment of small hepatocellular carcinoma, results in 74 patients. Radiology 221, 712-720. Patt YZ, Hassan MM, Lozano RD, Brown TD, Vauthey JN, Curley SA, Ellis LM (2003) Phase II trial of systemic continuous fluorouracil and subcutaneous recombinant interferon Alfa-2b for treatment of hepatocellular carcinoma. J Clin Oncol 21, 421-7. Patterson EJ, Scudamore CH, Owen DA, Nagy AG, Buczkowski AK (1998) Radiofrequency ablation of porcine liver in vivo, effects of blood flow and treatment time on lesion size. Ann Surg 227, 559-565. Poon RT, Fan ST, Ng IO, Lo CM, Liu CL, Wong J (2000) Different risk factors and prognosis for early and late intrahepatic recurrence after resection of hepatocellular carcinoma. Cancer 89, 500-507. Qin LX, Tang ZY (2002) The prognostic significance of clinical and pathological features in hepatocellular carcinoma. World J Gastroenterol 8, 193-199. Rees M, Plant G, Bygrave S (1997) Late results justify resection for multiple hepatic metastases from colorectal cancer. Br J Surg 84, 1136-1140. Rossi S, Fornari F, Pathies C, Buscarini L (1990) Thermal lesions induced by 480 KHz localized current field in guinea pig and pig liver. Tumori 76, 54-57. Rossi S, Garbagnati F, Lencioni R, Allgaier HP, Marchiano A, Fornari F, Quaretti P, Tolla GD, Ambrosi C, Mazzaferro V, Blum HE, Bartolozzi C (2000) Percutaneous radio-frequency thermal ablation of nonresectable hepatocellular carcinoma after occlusion of tumor blood supply. Radiology 217, 119126. Sato M, Watanabe Y, Ueda S, Iseki S, Abe Y, Sato N, Kimura S, Okubo K, Onji M (1996) Microwave coagulation therapy for hepatocellular carcinoma. Gastroenterology 110, 15071514.
Scaife CL, Curley SA (2003) Complication, local recurrence, and survival rates after radiofrequency ablation for hepatic malignancies. Surg Oncol Clin N Am 12, 243-255. Shock SA, Meredith K, Warner TF, Sampson LA, Wright AS, Winter TC, III, Mahvi DM, Fine JP, Lee FT, Jr (2004) Microwave ablation with loop antenna, in vivo porcine liver model. Radiology 231, 143-149. Sohn RL, Carlin AM, Steffes C, Tyburski JG, Wilson RF, Littrup PJ, Weaver DW (2003) The extent of cryosurgery increases the complication rate after hepatic cryoablation. Am Surg 69, 317-322. Solbiati L, Livraghi T, Goldberg SN, Ierace T, Meloni F, Dellanoce M, Cova L, Halpern EF, Gazelle GS (2001) Percutaneous radio-frequency ablation of hepatic metastases from colorectal cancer, long-term results in 117 patients. Radiology 221, 159-166. Sturesson C, Liu DL, Stenram U, Andersson-Engels S (1997) Hepatic inflow occlusion increases the efficacy of interstitial laser-induced thermotherapy in rat. J Surg Res 71, 67-72. Sugihara K, Hojo K, Moriya Y, Yamasaki S, Kosuge T, Takayama T (1993) Pattern of recurrence after hepatic resection for colorectal metastases. Br J Surg 80, 10321035. Tabuse K, Katsumi M (1981) Application of a microwave tissue coagulator to hepatic surgery the hemostatic effects on spontaneous rupture of hepatoma and tumor necrosis. Nippon Geka Hokan 50, 571-579. Tabuse K, Katsumi M, Kobayashi Y, Noguchi H, Egawa H, Aoyama O, Kim H, Nagai Y, Yamaue H, Mori K (1985) Microwave surgery, hepatectomy using a microwave tissue coagulator. World J Surg 9, 136-143. Vauthey JN, Klimstra D, Franceschi D, Tao Y, Fortner J, Blumgart L, Brennan M (1995) Factors affecting long-term outcome after hepatic resection for hepatocellular carcinoma. Am J Surg 169, 28-34. Vogl TJ, Muller PK, Hammerstingl R, Weinhold N, Mack MG, Philipp C, Deimling M, Beuthan J, Pegios W, Riess H (1995) Malignant liver tumors treated with MR imaging-guided laser-induced thermotherapy, technique and prospective results. Radiology 196, 257-265. Vogl TJ, Eichler K, Straub R, Engelmann K, Zangos S, Woitaschek D, Bottger M, Mack MG (2001) Laser-induced thermotherapy of malignant liver tumors, general principals, equipment(s), procedure(s)--side effects, complications and results. Eur J Ultrasound 13, 117-127. Vogl TJ, Straub R, Eichler K, Woitaschek D, Mack MG (2002) Malignant liver tumors treated with MR imaging-guided laser-induced thermotherapy, experience with complications in 899 patients (2,520 lesions). Radiology 225, 367-377. Weiss L, Grundmann E, Torhorst J, Hartveit F, Moberg I, Eder M, Fenoglio-Preiser CM, Napier J, Horne CH, Lopez MJ (1986) Haematogenous metastatic patterns in colonic carcinoma, an analysis of 1541 necropsies. J Pathol 150, 195-203. Wong SL, Edwards MJ, Chao C, Simpson D, McMasters KM (2001) Radiofrequency ablation for unresectable hepatic tumors. Am J Surg 182, 552-557. Wood TF, Rose DM, Chung M, Allegra DP, Foshag LJ, Bilchik AJ (2000) Radiofrequency ablation of 231 unresectable hepatic tumors, indications, limitations, and complications. Ann Surg Oncol 7, 593-600. Wright AS, Lee FT, Jr., Mahvi DM (2003) Hepatic microwave ablation with multiple antennae results in synergistically larger zones of coagulation necrosis. Ann Surg Oncol 10, 275-283.
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Scoggins et al: Thermal ablation of liver tumors Xu HX, Xie XY, Lu MD, Chen JW, Yin XY, Xu ZF, Liu GJ (2004) Ultrasound-guided percutaneous thermal ablation of hepatocellular carcinoma using microwave and radiofrequency ablation. Clin Radiol 59, 53-61. Yamasaki T, Kurokawa F, Shirahashi H, Kusano N, Hironaka K, Okita K (2002) Percutaneous radiofrequency ablation therapy for patients with hepatocellular carcinoma during occlusion of hepatic blood flow. Comparison with standard percutaneous radiofrequency ablation therapy. Cancer 95, 2353-2360.
Charles R. Scoggins
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Cancer Therapy Vol 2, page 463 Cancer Therapy Vol 2, 463-468, 2004
Genetic variation in the P2X7 apoptosis purinoreceptor correlated with anti – nuclear and cytoskeleton autoantibodies induction in nasopharyngeal carcinoma Research Article
Majida Jalbout1, Noureddine Bouaouina1,2, and Lotfi Chouchane1* 1 2
Laboratoire d’Immuno-Oncologie Moléculaire, Faculté de Médecine de Monastir, Université du Centre, Tunisia, Department of Cancérologie Radiothérapie CHU Farhat Hached, Sousse, Tunisia
__________________________________________________________________________________ *Correspondence: Pr. Lotfi Chouchane, Laboratoire d’Immuno-Oncologie Moléculaire, Faculté de Médecine de Monastir, 5019 Monastir, Tunisie; Tel: 216-3-462 200; Fax: 216-3-460 737; E-mail: lotfi.chouchane@planet.tn Key words: Apoptosis, Nasopharyngeal carcinoma, Autoantibodies, Purinoreceptor P2X7. Abbreviations: antinuclear antibodies, (ANA); bovine serum albumin, (BSA); Epstein-Barr virus, (EBV); nasopharyngeal carcinoma, (NPC); nuclear antigens, (NA); odds ratio, (OR); single nucleotide polymorphism, (SNP); Statistical Package of Social Sciences, (SPSS); undifferentiated carcinoma of nasopharyngeal type, (UCNT)
This work was supported by le Ministère de la Recherche Scientifique et de Technologie, by le Ministère de l’Enseignement Supérieur, by le Ministère de la Santé Publique de la République Tunisienne. Received: 25 November 2004; Accepted: 29 November 2004; electronically published: December 2004
Summary Apoptosis is initiated by the stimulation of numerous cellular receptors. The P2X7 receptor is expressed on immune cells and induces their ATP-mediated apoptosis. The A1513C polymorphism in P2X7 gene causes a loss of function of the receptor, impairing the death of cells which express these mutated receptors. Recently, we showed high frequency of autoantibodies to cytoskeleton and nuclear proteins in sera of patients with Epstein-Barr virus (EBV)related nasopharyngeal carcinoma. We investigated the potential association between P2X7 genetic variation and anti – nuclear and cytoskeleton autoantibodies induction in patients with nasopharyngeal carcinoma.We developed an RFLP-PCR to analyze the A1513C polymorphism of the P2X7 gene, reflecting a deficit in apoptosis, in 88 patients with nasopharyngeal carcinoma.A highly significant association was found between lack of autoantibodies to cytoskeleton and nuclear proteins and the wild P2X7-A/A homozygote genotype (p = 0.0018). The heterozygote genotype A1513C (A/C) was significantly more frequent in patients presenting autoantibodies in their sera (p = 0.014). We suggest that patients with nasopharyngeal carcinoma carrying the homozygote genotype A/A of the P2X7 gene might be less susceptible to produce autoantibodies against the cytoskeleton and nuclear antigens. This genotype reflecting a fully functional receptor might inhibit or eliminate autoreactive B lymphocytes by an appropriate process of apoptosis. Conversely, the heterozygote P2X7 -A/C genotype might be associated to the immune response deregulation observed in nasopharyngeal carcinoma due to the potential deficiency in purging autoreactive B cells.
pathogenesis or severity of many diseases, including cancer and autoimmunity. Under pathological conditions such as stress or cell damages, large amount of ATP and other nucleotides can be rapidly released from different cellular sources. Particularly in the immune system, extracellular ATP triggers a variety of biological responses including suicide
I. Introduction Recent investigations estimated that over half of the major medical diseases can be attributed directly and indirectly to defective regulation of programmed cell death mechanisms (Reed et al, 2001). Defects in the regulation of apoptosis make important contributions to the
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Jalbout et al: P2X7 polymorphism in nasopharyngeal carcinoma of immune cells (Rassendren et al, 1997; Humphrey et al, 2000; Kusner et al, 2000). Apoptosis regulates B cell maturation and differentiation, as well as the development of memory B cells (Di Virgilio et al, 1995; Buell et al, 1996; Nihei et al, 2000). This effect of ATP is mediated through the activation of specific surface molecules called P2 purinoreceptors (Ralevic et al, 1998; Di Virgilio et al, 2001). The latest cloned member of the P2X family, the P2X7 receptor (previously termed P2Z), is unique in being constituvely highly expressed in immune cells, particularly B and T lymphocytes (Di Virgilio et al, 1995; Rassendren et al, 1997; Khakh et al, 1999). Depending on cell background, activation of the P2X7 receptors triggers diverse physiologic activities including cytokine secretion and induction of cell death (Chused et al, 1996; Surprenant et al, 1996). Ferrari et al, (1999) have shown that activation of the P2X7 receptor, following the exposure to ATP, rapidly induced apoptotic DNA fragmentation, accompanied by the proteolytic processing of multiple caspases and caspase substrates (Ferrari et al, 1999). The genomic structure of P2X7 consists of 13 exons with exon 12 and exon 13 encoding for the carboxyl terminal tail of this molecule. There is strong evidence that this long carboxyl terminus is necessary for the permeability properties of the P2X7 channel (Surprenant et al, 1996; Wiley et al, 1998). Recently, a single nucleotide polymorphism (SNP) has been identified in the exon 13 of the P2X7 gene. This polymorphism (A1513C) encodes for a glutamic acid to alanine substitution at amino acid 496 (Glu496Ala). Homozygosity for the rare P2X7 allele (C/C) produces non-functional P2X7 protein while the heterozygous state gives cells with half the function of cells with germline P2X7 protein (Gu et al, 2001). The Epstein-Barr virus (EBV) - associated nasopharyngeal carcinoma (NPC) has a striking geographic and ethnic distribution in different populations of the world. While it is rare in western countries, its incidence is high in certain regions such as Southeast Asia. In North Africa, the NPC has an intermediate incidence. In Asia, NPC mainly affects patients in the 4th or 5th decade of their life, whereas in North Africa an additional peak of incidence is found confined to young population. The association of NPC with widely distributed factors implies that exceptional circumstances combining some interactions between Epstein- Barr virus infection and a particular environment, lead to the development of a carcinoma of the nasopharynx, favored by a genetic disposition. Among NPCs, the histological type showing the most consistent worldwide association with EBV is the undifferentiated carcinoma of nasopharyngeal type (UCNT) (Fedder et al, 1985; Choi et al, 1993; Jeannel et al, 1999). EBV infects B lymphocytes with very high efficiency, both in vivo and in vitro. The virus attaches itself to B cells via interaction of the EBV gp350/220 envelope glycoprotein with the CD21 molecule (receptor CR2 of complement) on B cell and generates the activation, proliferation and immortalization of these cells.
The polyclonal activation of B lymphocytes is associated with the secretion of antibodies (Henderson et al, 1993). In a recent study, we showed that frequencies of autoantibodies produced against nuclear and cytoskeleton proteins were markedly higher in patients’ sera with nasopharyngeal carcinoma compared to those of control subjects (Jalbout et al, 2002). Given the importance of apoptosis in immune response regulation, aberrant apoptosis could conceivably associate with immune system deregulation. In this study, we hypothesize that the genetic variation in the P2X7, reflecting a loss of function of its product, might be among factors leading to the autoantibody induction in patients with NPC. In line with this hypothesis, we investigated the potential association of the P2X7 gene polymorphism in NPC patients with or without autoreactivity sera to cytoskeleton and nuclear proteins.
II. Materials and methods A. Patients Patients with UCNT were recruited from the Department of Radiation Oncology and Medical Oncology of Sousse Hospital, between 1991 and 2003. The patients with UCNT (67 males and 21 females) had a mean age of 40 ± 21.3 years. The clinical stages ranged from II to IV (TNM classification, 1987). The diagnosis of cancer was confirmed by histopathology analyses. The histology was undifferentiated carcinoma (type III, WHO classification) in all cases (Shanmugaratnam et al, 1978). Written informed consent was obtained from all subjects and, in the case of children, from the parents.
B. ELISA for autoantibodies Sera autoreactivity to the cytoskeleton and nuclear proteins was determined on serum samples obtained prior to cancer therapy. Each serum collected was tested individually. As negative control for the reaction, 82 sera from healthy individuals were pooled and used in each test. The autoantibodies detection was carried out as previously described (Jalbout et al, 2002).
C. Immunofluorescence analysis Identification of antinuclear antibodies (ANA) by indirect immunofluorescence was carried out using cryostat sections prepared from liver tissue of young rats. These liver sections were deposed on glass and incubated overnight at -80°C. Briefly, serum was diluted at 1/100 with PBS pH7.4 and deposited on liver sections. After 30 min of incubation and 3 washes with PBS, antibodies were revealed by anti-human Igs antibodies (IgG, IgA, IgM) labeled with fluorescein.
D. Genomic DNA extraction Genomic DNA was extracted from peripheral blood leukocytes as previously described (Olerup et al, 1992). Briefly, 10 ml of blood was mixed with Triton lysis buffer (1% Triton X, 5 mM MgCl2, H2O, 0.32 M sucrose, 10mM Tris-HCl, PH 7.5). Leukocytes were spun down and washed with H2O. The pellet was incubated with proteinase K at 56°C and subsequently salted out at 4°C using a saturated NaCl solution. Precipitated proteins were removed by centrifugation. The DNA in supernatant fluid was precipitated with ethanol. The DNA pellet was dissolved in 500 µl TE (Tris-EDTA).
E. P2X7 gene polymorphism analysis 464
Cancer Therapy Vol 2, page 465 A restriction map of the P2X7 gene was assessed using the infobiogen web site (http://www.infobiogen.org) in order to determine a couple of primers enclosing the 1513 SNP. This nucleotide variation lies in the recognition motif of AluI [AG!CT]. The substitution of the adenine to a cytosine causes a loss of the restriction site of AluI. A 91-pb DNA fragment containing 1513 SNP was amplified by PCR using the following primers: forward, 5’GCTGCCTCCCATCTCAACTCC-3’ and reverse, 5’CTCTGAGGTGGTGATGCAGGCC-3’. Primers cited above were used to amplify DNA fragments of 91 pb enclosing the 1513 SNP by PCR. Amplification reactions were done in a total volume of 30µl containing 100ng of genomic DNA, 1.5 mM MgCl2 , 200µM dNTPs, 50 pmol of each primer, and 0.5 unit of Taq (Amersham) in 1X Taq buffer. Cycling conditions were 94°C for 5 min, 30 cycles of 94°C for 30 s, 60°C for 60s, and 72°C for 60s, and a final 10 min extension at 72°C. DNA Fragment (91 pb) were revealed on a 3% agarose gel. Genotyping was done using restriction fragment-length polymorphism analysis. PCR products were digested at 37°C overnight with 4 unites of the restriction enzyme AluI (New England Biolabs) in a 20µl reaction mix containing 1 X restriction enzyme buffer 2 and bovine serum albumin (BSA). Juxtaposed heterozygote digested products of 46 and 45 pb were resolved on a 3% agarose gel.
We set up an RFLP-PCR to analyze the A1513C polymorphism in the P2X7 gene. The A to C nucleotide transition at the position 1513 causes a loss of AluI site. We used this restriction site to genotype 88 patients for the P2X7. After defining the serological profile of patients for autoreactivity against cytoskeleton and nuclear proteins, we stratified them into two groups according to their sera autoreactivity. A patient is considered as having autoreactive serum when he presents autoantibodies produced against at least one of the tested antigens. The cut-off value for reactivity was defined as twice the value of the average autoreactivity in our pool of negative controls. Table 1 shows genotype frequencies for the P2X7 gene in the two groups of patients with nasopharyngeal carcinoma. These distributions were in Hardy-Weinberg equilibrium for both groups. The frequency of the wild homozygote genotype P2X7 -A/A was significantly higher in patients with nonautoreactive sera (35/48, 72.9%) than in the group of patients having autoantibodies to nuclear and cytoskeleton antigens (16/40, 40%) (p = 0.0018). The heterozygote P2X7 –A/C genotype was significantly more frequent in patients presenting autoantibodies in their sera (52.5%) than those having no autoantibodies (27.1%) (p = 0.014). The homozygote C/C genotype was carried only by patients with autoreactive antibodies (3 versus 0 in patients with no autoreactivity).
F. Statistical analysis Genotypes frequencies were estimated by gene counting. A Chi-Square Test ("2) was used to compare observed number of each P2X7 genotype with those expected for a population in Hardy- Weinberg equilibrium. The chi-square test (or Fisher’s exact test when n <5) was used to determine whether significant differences (p value) in the autoantibodies induction to cytoskeleton and nuclear antigens were observed between carriers of different P2X7 genotypes. Statistical analyses were performed using the Statistical Package of Social Sciences for Windows (SPSS version 10.0). Differences between groups were tested for significance by the two-tailed test. A p value less than 0.05 was considered significant. The significance of the association was estimated by the odds ratio (OR).
B. P2X7 genetic variation autoreactivity to nuclear antigens
and
As previously shown (Jalbout et al, 2002), the most frequent sera autoreactivity, found in patient’s sera compared to controls, was towards nuclear antigens (NA) (22% in patients versus 1.2% in controls; p = 0.00003). A significant association was found between the presence of ANA and the P2X7 A/C heterozygote genotype (p = 0.030). The frequency of homozygote A/A genotype, corresponding to a complete function of the P2X7 receptor, was significantly higher in patients without ANA (p=0.016)
III. Results A. P2X7 polymorphism distribution and sera autoreactivity in patients with nasopharyngeal carcinoma
Table 1. P2X7 polymorphism distributions in patients with nasopharyngeal carcinoma with or without anti- nuclear and cytoskeleton autoantibodies Genotype Patients Patients OR Confidence Interval p With autoantibodies Without autoantibodies n= 40 n= 48 P2X7 n f n f A/A 16 0.40 35 0.29 0.25 [0.09 - 0.66] 0.0018 A/C 21 0.525 13 0.271 2.98 [1.12 - 8.01] C/C 3 0.075 0 0 NS The chi-square and the Fisher tests were used to determine whether significant differences (p value) were observed when patients’ having anti- nuclear and cytoskeleton autoantibodies group was compared with that of those without. f, frequency.
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Jalbout et al: P2X7 polymorphism in nasopharyngeal carcinoma the CD40 on B cells allows these cells to escape to CD95mediated apoptosis (Sbih-Lammali et al, 1999). Other proteins might be involved in the escapement of B cells to apoptosis via different alternatives including the P2X7 pathway. In conclusion, this study showed a significant association between genetic variation in the P2X7 receptor and anti- nuclear and cytoskeleton autoantibodies induction. Our data suggest that apoptosis dysfunction might be among mechanisms responsible for the immune deregulation observed in NPC.
IV. Discussion Regardless of patient origin, nasopharyngeal carcinoma is consistently associated with Epstein-Barr virus. The EBV genome is contained in malignant epithelial cells (Jeannel et al, 1999; Murray et al, 2001). This virus has a tropism for B lymphocytes and a propensity to oncogenicity. Infection of B lymphocytes with EBV is associated with their polyclonal activation and in vitro immortalization (Mannick et al, 1991; Hildesheim et al, 1993). Recently, we detected in sera of patients with NPC autoantibodies induced against the cytoskeleton and nuclear proteins (Jalbout et al, 2002). These antigens are involved in cell division and proliferation. In this study, we hypothesize that the deregulation in apoptosis of autreactive B cells might be among factors leading to the autoantibodies induction in patients with NPC. The P2X7 receptor is attracting increasing interest due to its restricted cells distribution such as macrophages, thymocytes and B cells, and its induction of these cells death (Chen et al, 1999; Ferrari et al, 2000; Gu et al, 2000). The C-terminal cytoplasmic tail of P2X7 receptor, which is 120 amino acids longer than in other P2X receptors, is an important modulator of the receptor functions. In this study, frequencies of the non-functional single nucleotide polymorphism of the C-terminal tail of P2X7 were assessed within patients with NPC stratified according to their sera autoreactivity to nuclear and cytoskeleton antigens. The frequency of the wild genotype (Glu496) was associated to the absence of significant level of autoantibodies in patients with nasopharyngeal carcinoma. The Glu496Ala genotype frequency is significantly higher in patients presenting anti- nuclear and cytoskeleton proteins antibodies compared to patients with no autoantibodies. Reflecting a deficiency in purging the autoreactive B cells, this heterozygote genotype might be a susceptibility marker for autoantibodies production in patients with NPC. This observation was highlighted by the distribution of the mutant homozygote genotype 496Ala which was found only in patients having antinuclear and cytoskeleton autoantibodies. The negative effect of this mutation on P2X7 function was evident in all leukocytes which express surface P2X7, namely B and T lymphocytes in patients with chronic lymphocytic leukemia (Gu et al, 2000). The transition of glutamic acid to an alanine found in P2X7 in these patients confers resistance to ATP-induced apoptosis of Blymphocytes (Gu et al, 2000). Moreover, Wilson and colleagues showed that the C-terminal domain of this receptor is implicated in the binding of epithelial membrane proteins, which regulates cell death (Wilson et al, 2002). Several lines of evidence support the idea that malignant cells are more prone to apoptosis in NPC than other head and neck cancers. It has been reported that NPC tumor-infiltrating lymphocytes, which often produce the anti-apoptotic CD40 ligand, may increase the survival of malignant cells, thereby enhancing tumor growth in patients. In fact, the interaction of the CD40 ligand with
Acknowledgements We gratefully acknowledge the technical assistance of S. Gabbouj. We thank the staff of the Departments of Radiation Oncology and medical oncology of CHU F. Hached, Sousse, for providing samples and clinical information.
References Reed JC (2001) Apoptosis-regulating proteins as targets for drug discovery. Trends Molec Med 7, 314-319. Kusner D and Admas J (2000) ATP-Induced killing of virulent Mycobacterium tuberculosis within human mcrophages requires phospholipase D. J Immunol 164, 379-388. Humphreys BD, Rice J, Kertesy SB, Dubyak GR (2000) Stressactivated protein kinase/JNK anctivation and apoptotic induction by the macrophage P2X7 nucleotide receptor. J Biol Chem 275, 26792-26798. Rassendren R, Buell G, Virginio C, North RA, Surprenant A (1997) The permeabilizing ATP receptor (P2X7), cloning and expression of a human cDNA. J Biol Chem 272, 5482-5486. Di Virgilio F (1995) The P2Z purinoceptor, An intriguing role in immunity, inflammation and cell death. Immunol Today 16, 524-528. Buell G, Gollo G, Rassendren F (1996) P2X receptors, an emerging channel family. Eur J Neurosci 8, 2221-2218. Nihei OK, Savino W, Alves LA (2000) Procedures to characterize and study P2Z/ P2X7 purinoceptor, Flow cytometry as a promising practical, reliable tool. Mem Inst Oswaldo Cruz, Rio de Janeiro 95, 415-428. Ralevic V and Burnstock G (1998) Receptors for purines and pyrimidines. Pharmacol Rev 50, 413-492. Di Virgilio F, Chiozzi Paola, Ferrari D, Falzoni S, Sanz JM, Morelli A, Torbolli M (2001) Nucleotide receptors, an emerging family of regulatory molecules in blood cells. Blood 97, 587-600. Khakh BS and Lester HA (1999) Dynamic selectivity filters in ion channels. Neuron 23, 653-658. Surprenant A, Rassendren F, Kawashima E, North RA, Buell G (1996) The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7 ). Science 272, 735-738. Chused TM, Sergey A, Sitkovsky M (1996) Murine T lymphocytes modulates activity of an ATP-activated P2Z-type purinoreceptor during differentiation. J Immunol 157, 13711380. Ferrari D, Los M, Bauer M, Vandenabeele P, Wesselborg S and Osthoff KS (1999) P2Z purinoreceptor ligation induces activation of caspases with distinct roles in apoptotic and necrotic alteration of cell death. FEBS Letters 447, 71-75. Wiley JS, Gargett E, Zhang W, Snook MB and Jamieson GP (1998) Partial agonists and antagonists reveal a second permeability state of human lymphocyte P2Z/ P2X7 channel. Am J Physiol, C1224-C1231.
466
Cancer Therapy Vol 2, page 467 Gu BJ, Zhang W, Worthington RA, Sluyter R, Dao-Ung P, Petrou S, Barden JA and Wiley JS (2001) A Glu-496 to Ala Polymorphism Leads to Loss of Function of the Human P2X 7 Receptor. J Biol Chem 276, 11135-11142. Fedder M, Gonzalez MF (1985) Nasopharyngeal carcinoma (Brief Review) Am J Med 79, 365-369. Choi P, Suen M, Huang D, Lo K.W and Lee J (1993) Nasopharyngeal carcinoma, genetic changes, Epstein-Barr virus infection, or both. A clinical and molecular study of 36 patients. Cancer 72, 2873-2878. Jeannel D, Bouvier G, Hubert A (1999) Nasopharyngeal carcinoma, an epidemiological approach to carcinogenesis. Cancer Surveys 33, 125-155. Henderson S, Huen D, Rowe M, Dawson C, Johnson G, Rickinson A (1993) Epstein-Barr virus-coded BHRF1 protein, a viral homologue of Bcl-2, protects human B cells from programmed cell death.. Proc Natl Acad Sci U S A. 90, 8479-8483. Jalbout M, Bel Hadj Jrad B, Bouaouina N, Gargouri J, Yacoub S, Zakhama A, Khlifa R and Chouchane L (2002) Autoantibodies to Tubulin are specifically associated with the young age onset of the nasopharyngeal carcinoma. Int J Cancer 101, 146-150. Shanmugaratnam K, Tye CY, Goh EH, Chia KB (1978) Etiological factors in nasopharyngeal carcinoma, a hospitalbased, retrospective, case-control, questionnaire study. IARC Sci Publ 20,199-212. Olerup O, Zetterquist H (1992) HLA-DR typing by PCR amplification with sequence specific primers (PCR-SSP) in 2 hours, An alternative to serological DR typing in clinical practice including donorrecipient matching in cadaveric transplantation. Tissue Antigens 39, 225-235. Murray PG, Young LS (2001) Epstein-Barr virus infection, basis of malignancy and potential for therapy. Expert Rev Mol Med 3,1-20.
Mannick JB, Cohen JI, Birkenbach M, Marchini A, Kieff E (1991) The Epstein-Barr virus nuclear protein encoded by the leader of the EBNA RNAs is important in B-lymphocyte transformation. J Virol 65, 6826-6837. Hildesheim A, Levine PH (1993) Etiology of nasopharyngeal carcinoma, a review. Epidemiol Rev 15, 466-485. Hartley SB, Cooke D.A, Fulcher AW, Harris S, Cory S, Basten A and Goodnow CC (1993) Elimination of self-reactive B lymphocytes proceeds in two stages, arrested development and cell death. Cell 72, 325-335. Chen W, Wang HG, Srinivasula S, Alnemri E and Cooper N (1999) B cell apoptosis triggered antigen receptor ligation proceeds via a novel caspases-dependent pathway. The Journal of Immunology 163, 2483-2491. Gu BJ, Zhang WY, Bendall LJ, Chessell IP, Buell GN, Wiley JS (2000) Expression of P2X7 purinoreceptors on human lymphocytes and monocytes, evidence for nonfunctional P2X7 receptors. Am J Physiol Cell Physiol 279, 1189-1197. Ferrari D, Los M, Bauer KA, Vandenabeele P, Wesselborg S, Shilze-Osthoff K (1999) P2Z purinoreceptor ligation induces activation of caspases with distinct roles in apoptotic and necrotic alterations of cell death. FEBS Letters 447, 71-75. Wilson HL, Wilson SA, Surprenant A, North RA (2002) Epithelial membrane proteins induce membrane blebbing and interact with the P2X7 receptor C terminus. J Biol Chem 277, 34017-34023. Sbih-Lammali F, Clausse B, Ardila-Osorio H, Guerry R, Talbot M, Havouis s, Ferradini L, Bosq J, Tursz T and Busson P (1999) control of apoptosis in Epstein-Barr virus-positive nasopharyngeal carcinoma cells, opposite effects of CD95 and CD40 stimulation. Cancer Res 59, 924-930.
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Jalbout et al: P2X7 polymorphism in nasopharyngeal carcinoma
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Cancer Therapy Vol 2, page 469 Cancer Therapy Vol 2, 469-474, 2004
High dose rate endobronchial brachytherapy for the management of non-small cell lung cancer with an endobronchial or peribronchial component Review Article
Jiade J. Lu1,*, Yadvindera S. Bains1, Aaron H. Wolfson1, Elio Donna2, Alfred H. Brandon1, William A. Raub1, Arnold M. Markoe1 1
Department of Radiation Oncology, University of Miami Medical Center/Jackson Memorial Hospital, 1475 N.W. 12th Ave, Miami, Florida, 33136, USA 2 Department of Pulmonary Medicine, University of Miami Medical Center/Jackson Memorial Hospital, 1475 N.W. 12th Ave, Miami, Florida, 33136, USA
__________________________________________________________________________________ *Correspondence: Jiade J. Lu, MD, Department of Radiation Oncology, National University Hospital, 5 Lower Kent Ridge Road, Singapore, 119074, Singapore; Tel: 011-65-6772-4869; Fax: 011-65-779-6320; Email: jlu@anderson.ucla.edu Key words: non-small cell lung cancer, radiation therapy, high dose rate, brachytherapy Abbreviations: Complete response, (CR); external beam radiation, (EBRT); high dose rate, (HDR); high-dose-rate endobronchial brachytherapy, (HDREB); intermediate dose rate, (IDR); low dose rate, (LDR); non-small cell lung cancer, (NSCLC); partial response, (PR) Received: 8 November 2004; Accepted: 22 November 2004; electronically published: December 2004
Summary The primary aim of this study was to determine whether a dose response exists for bronchial obstruction caused by non-small cell lung cancer (NSCLC) when treated with increasingly higher radiation doses delivered by a combination of external beam radiation (EBRT) and high-dose-rate endobronchial brachytherapy (HDREB). The secondary aim was to document the tolerability of EBRT combined with HDREB. Thirty patients with medically inoperable or surgically unresectable non-small cell lung cancer were treated in this phase I/II study with both EBRT and HDREB. All patients had a peribronchial or endobronchial component with bronchial obstruction of 50% or more on clinical examination. EBRT was delivered via megavoltage photons at standard fractionation schedules to a total dose of 66 Gy. The HDREB was delivered using the Nucleotron HDR remote after loading unit with a 10 Ci Ir-192 source. Each treatment of HDERB was delivered at 1 cm depth. The first group of 10 patients received 3 treatments of HDREB at 7 Gy per fraction. The second group of 10 patients received 3 treatments of 8 Gy per fraction. And the last group of 10 patients received 3 treatments of HDREB of 9 Gy per fraction. HDREB was delivered once weekly. Improvements of obstructions and acute adverse reactions were evaluated for each group of patients before escalating to the next higher-level dose level of HDREB. The medium follow-up time for all 30 patients was 14.5 months. The medium survival for all 30 patients was 13 months (Range 1 month to 38 months). There was no significant difference of survival among the 3 groups. Complete response or partial response of bronchial obstruction was seen in 60% of patients in Group 1 compared to 90% of patients in both group 2 and 3 (p=0.05). One patient in Group 1 and one patient in Group 3 developed grade IV toxicity, and 1 patient in Group 2 developed grade III toxicity. HDREB of 27 Gy in 3 divided weekly treatments following 66 Gy of external beam radiation is well tolerated for the treatment of NSCLC. A statistically significant dose response for bronchial obstruction from NSCLC is seen when treating with the combination of EBRT and HDREB beyond 24 Gy. 60-70 Gy due to the surrounding critical normal structures in the treatment field (Dosoretz et al, 1993; Hernandez et al, 1996; Nguyen et al, 1999). However, with this dose delivered using conventional fractionation, the proportion of patients obtaining partial or complete local response
I. Introduction Radiation is an effective treatment modality in the management of locally advanced non-small cell lung cancer (NSCLC), both in definitive or palliative settings. The dose of external beam radiation is usually limited to 469
Lu et al: High dose rate endobronchial brachytherapy for NSCLC After a total of 40 Gy delivered through the initial fields, patients were treated with boost field(s) include the tumor and enlarged lymph nodes defined by chest CT scan. The EBRT boost dose was 26 Gy. Dose to the spinal cord was limited to 45 Gy or less.
ranges from 20-60% only (Slawson and Scott, 1979; Majid et al, 1986; Chetty et al, 1989). This leaves considerable room for improvement. The use of high dose rate endobronchial brachytherapy (HDREB) in the treatment of non-small cell lung has been well documented (Macha et al, 1987; Cotter and Ellingwood, 1988; Lang et al, 1988). Combining external beam radiation with endobronchial brachytherapy allows for delivery of significantly higher dose to a limited area. By combining external beam radiotherapy (EBRT) and HDREB, higher partial or complete local response rates ranging between 67-99% can be achieved (Huber et al, 1987; Cotter and Ellingwood, 1988; Chang et al, 1994). Although HDREB has been frequently used to treat non-small cell lung cancer with an endobronchial or peribronchial component, ideal fractionation schemes and dosages have not yet been fully established. Doses and fractionation schemes are selected empirically because there is little prospective data on tolerance doses of the human bronchial wall after HDREB. Furthermore, the complications caused by the various dose and fractionation schemes using HDREB are not well characterized. This prospective phase I/II study was conducted for the following purposes: to determine whether a dose response exists for NSCLC when treated with a combination of 66 Gy external beam radiation and escalating doses of HDREB; and to establish a well tolerated dose schema in patients with NSCLC who undergo high dose rate endobronchial brachytherapy.
C. High-dose-rate brachytherapy High-dose-rate brachytherapy was delivered using the Nucleotron HDR remote afterloading unit with a 10 Ci Ir-192 source. HDREB were delivered prior to the EBRT if the endobronchial or peribronchial component could be fully covered by the HDR brachytherapy dose distribution. For patients with larger tumors, or with complete or near complete bronchial obstruction, HDREB were delivered after the completion of EBRT. The length of bronchus treated included gross tumor visualized on the bronchoscopy with 2 cm margins proximally and distally. Each treatment of HDREB was delivered at 1 cm depth from the central axis of the radiation source regardless of the size of the tumor at the time of brachytherapy. The first group of 10 patients received three treatments of HDREB at 7 Gy per fraction (Group1). The second group of ten patients received three treatments at 8 Gy per fraction (Group 2). The last group of ten patients received three treatments of HDREB at 9 Gy per fraction (Group 3). HDREB was delivered once weekly. Bronchoscopy was performed during placement of the brachytherapy catheters during each HDREB session to evaluate any acute side effects or complications. Dose escalation and thus the protocol would be terminated if there was treatment related death or more than 3 Grade 3 or 4 toxicities were observed in any treatment groups. Evaluation of tumor response was not performed until three months after the completion of the treatment.
II. Patients and methods
D. Chemotherapy
A. Patient selection
All patients in this study had locally advanced disease and received systemic chemotherapy in addition to external beam radiation and brachytherapy. All 30 patients received cisplatin or carboplatin based chemotherapy concurrently during external beam radiation but not during HDREB. Two patients in Group 1 also received a short course of neoadjuvant chemotherapy before entering this protocol. The pre-radiation baseline CT scan and bronchoscopy evaluations were performed for both patients after the completion of their neoadjuvant chemotherapy but within one week before the starting of radiation. Both patients had more than 50% obstruction at the primary sites in their baseline evaluations before the starting of radiation treatment.
Thirty patients with medically inoperable or surgically unresectable stage III NSCLC were treated in this institutional phase I/II study with both external beam radiotherapy and high dose rate endobronchial brachytherapy. Tissue diagnosis of NSCLC was mandatory for enrollment into the study. All patients had a peribronchial or endobronchial component with bronchial obstruction of 50% or more on bronchoscopy examination. Pre-radiotherapy evaluations included a complete history and physical examination, pulmonary function test, complete blood counts and serum electrolytes, chest X-ray, CT scan of chest, and bronchoscope. Patients with metastatic disease, malignant pleural effusion, history of any other malignancy other than skin cancer, poor performance status (Karnofsky performance status of less than 60), pathology other than non-small cell carcinoma, surgically treated lesions, prior history of radiotherapy to the chest, and those who refused to receive brachytherapy were excluded from the protocol. Written consent was obtained from all patients before starting the treatment.
E. Post-treatment Evaluation All patients were followed up by their radiation oncologists and pulmonologists after the completion of the treatment. All patients were required to receive monthly fiberoptic bronchoscopy for toxicity and response evaluation starting at two to four weeks after the treatment for at least 4 months. CT scan of the chest were required for all patients at 3 months time after the completion of treatment. After that, follow up clinical examinations were performed every three months in either the radiation oncology or pulmonology clinics. Response to treatment was graded by objective findings include both bronchoscopic and radiological studies at 3 months after the completion of radiotherapy. Subjective findings, including symptomatic changes, were not used to define the response to therapy. Complete response (CR) was defined as the complete disappearance of all clinical and radiological evidence of bronchial obstruction at the primary site; partial response (PR) as a 50% or greater decrease in the bronchial obstruction on both
B. External beam radiotherapy External beam radiation treatment and high-dose-rate endobronchial brachytherapy were delivered in a sequential manner. External beam radiotherapy was administered via megavoltage (6MVx) photons. The planned treatment dose of EBRT was 66Gy using a conventional fractionation (2 Gy/fraction) delivered 5 days per week over 33 days. The initial treatment portal included primary tumor, ipsilateral hilar lymph nodes, and mediastinum through opposed parallel AP and PA fields. Treatment of supraclavicular fossa was not mandatory.
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Cancer Therapy Vol 2, page 471 CT of the chest and bronchoscopy examination; stable disease as a response less than 50% decrease but not more than 25% increase of the bronchial obstruction on either chest CT or bronchoscopy examination; and progressive disease as more than 25% increase in the size of the obstruction in either examination. Acute and late toxicities from treatments were graded according to the Radiation Therapy Oncology Group criteria. Late toxicities were defined as symptoms first occurring 90 days after treatment or lasting beyond 90 days after the completion of treatment. Toxicities were evaluated weekly during the radiation treatment and at each follow-up after the completion of treatment (Cox et al, 1995).
requirement of the protocol including a CT scan of the chest at 3 months after the completion of treatment.
C. Response and survival Complete or partial response at the primary site was obtained in 6 cases (60%), 9 cases (90%), and 9 cases (90%) in the three groups of patients, respectively (Table 2). We compared the response data of patients in Group 1 with the response of patients in Group 2 and 3 combined because of the identical response rates (CR and PR combined), and found the difference reached statistical significance (p=0.05). The medial survival for all 30 patients was 13 months (range 1 to 36 months). Significant difference of survival among the three groups was not expected for this small group of patients with locally advanced disease.
III. Results A. Patient characteristics The median age of all 30 patients was 65 (range 5072). Twenty-six patients were male and 4 were female. All patients have either AJCC stage IIIa or IIIb non-small cell lung cancer with endobronchial or peribronchial components. Twenty-nine patients received both external beam radiotherapy and brachytherapy according to the protocol. One patient in Group 2 received three applications of HDR brachytherapy (total brachytherapy dose was 24 Gy) followed by external beam radiation but stopped external beam radiation at 60 Gy for non-medical reasons. Two patients in Group 1, 2 patients in Group 2, and 1 patient in Group 3 received HDREB prior to the starting of EBRT. Patientsâ&#x20AC;&#x2122; characteristics are listed in Table 1.
D. Toxicity Documentation of severe acute and late toxicities (defined as Grade 3 and Grade 4 toxicities according to RTOG toxicity criteria) was mandatory for all patients. Although documentation of Grade 1 and Grade 2 toxicities was required by our radiation oncology department policy, it was not required by this research protocol. One patient in Group 1 developed Grade 4 toxicity (hemorrhage from the bronchus at the HDR treatment site), 1 patient in Group 2 and 1 patient in Group 3 developed radiation bronchitis. All 3 patients developed toxicities within 90 days after the completion of their brachytherapy (Table 3). Although Grade 1 or 2 radiation induced toxicity of esophagus and bronchus was observed during follow-up examinations in multiple cases, no other substantial acute or late toxicities were observed and reported. There were no treatment-related deaths. The planned treatment with external beam radiation therapy with HDR boost was well tolerated in the majority of the patients.
B. Follow up The median follow up time for all 30 patients was 14.5 months (range 1 to 38 months). One patient in Group 1 died of local disease progression approximately 5 weeks after treatment. All other patients had more than 3 months follow-up and received examinations according to the
Table 1. Characteristics of eligible patients. Total patient number: 30 Characteristics
Number (%)
Sex Male Female
24 (80%) 6 (20%)
Age 50-60 60-70 >70 Median
2 (6.7%) 25 (83%) 3 (10%) 65
Stage (AJCC 1997) IIIa IIIb
13 (43.3%) 17 (56.7%)
KPS (minimum)
>60 (100%)
Table 2. Response and complications after external beam radiotherapy combined with HDR endobronchial brachytherapy.
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Lu et al: High dose rate endobronchial brachytherapy for NSCLC Median follow up time=14.5 months. Group
No. of Pt.
Brachytherapy Dose
Substantial Response *
1 2 3
10 10 10
21 Gy/3 weeks 24 Gy/3 weeks 27 Gy/3 weeks
6 (60%) 9 (90%) 9 (90%)
*Substantial response is defined as complete or partial response. Difference of response rate reached statistical significance when response rate of group 1 are compared to group 2 and 3 combined. (p=0.05)
Table 3. Acute complications after external beam radiotherapy combined with HDR endobronchial brachytherapy. Group
HDREB Dose
Severity
No. of Pt.
Complication
1 2 3
21 Gy/3 wks 24 Gy/3 wks 27 Gy/3 wks
Grade 4 Grade 3 Grade 4
1 (10%) 1 (10%) 1 (10%)
Hemoptysis Bronchitis Bronchitis
retrospectively reported that patients tolerated well with a single fraction of 15-20 Gy prescribed to 1 cm from the center of the source (Burt et al, 1990). Aliberti analyzed 9 series in the literature and reported 11.3% hemoptysis in 576 cases (Aliberti, 1995). In a retrospective study reported by Langendijk et al, external beam radiotherapy combined with endobronchial brachytherapy as primary treatment for NSCLC did not lead to a higher risk of massive hemoptysis as compared to EBRT alone when fraction sizes for brachytherapy of 7.5 or 10 Gy were used (Langendijk et al, 1998). Kotsianos et al, (2000) analyzed 48 biopsy specimens of three-dimensional miniorgans of the human bronchial wall histologically after high-doserate brachytherapy with escalating dosage. The range of the high-dose-rate irradiation was around 75 Gy. The authors found that there was no histologically apparent tissue damage regardless of the irradiation dose and concluded that normal bronchial epithelium has a high tolerance to early epithelial damage by irradiation (Kotsianos et al, 2000). Several other clinical studies attempted to define the optimum doses and fractionation schemes in brachytherapy for lung cancers (Huber et al, 1987; Speiser and Spratling, 1993; Gollins et al, 1994). These trials confirmed a high tolerance of bronchial epithelium to brachytherapy; however, the maximal tolerance dose has not been defined in any prospective studies. In our series, only 3 patients (one patient from each group) developed Grade 3 or Grade 4 toxicities. All patients developed treatment induced esophagitis with mild to moderate severity (Grade 1 to 3) during or shortly after the completion of external beam radiotherapy. However, no new symptoms of esophagitis were observed and no existing esophagitis worsened during the HDREB. We have confirmed that the total dose of up to 27 Gy in 3 weekly fractions of HDR endobronchial brachytherapy after a definitive course of external beam radiation treatment to the lung is well tolerated. Further dose escalation may be possible in light of our results. Most studies reported significant symptomatic improvement after HDREB (Burt et al, 1990; Chang et al,
IV. Discussion Patients with locally advanced non-small cell lung cancer are generally treated with external beam radiotherapy combined with chemotherapy; however, previous experience has confirmed that this combined treatment can only achieve limited clearance of the disease because the dose of the treatment is generally limited by other critical structures in the chest. Endobronchial brachytherapy for NSCLC has been well studied extensively previously. Palliative effects ranging from 50100% for different types of symptoms have been reported. Endobronchial brachytherapy can be delivered at low dose rate (LDR), intermediate dose rate (IDR), or high dose rate (HDR). HDR brachytherapy units, which deliver radiation at more than 12 Gy per hour, are now widely available in most cancer treatment centers. Because the radioactive sources are more precisely controlled and safety of the treatment delivery is improved, it is the more commonly utilized brachytherapy modality for lung cancer treatment. Even though HDREB is widely used to treat locally advanced NSCLC, no specific clinical guidelines have been established. The most commonly accepted dosage of HDREB used with external beam radiotherapy has been 35 fractions of 5 Gy HDR brachytherapy, but other HDR brachytherapy schedules have also been used. The brachytherapy dose and schedule are chosen mostly empirically. Theoretically, higher dose per fraction may increase the rate of complications, but it may also improve the rate of local control and/or palliation. Because of the rapid fall of dose with distance, the complication rates from brachytherapy treatments are generally tolerable given the relatively high doses delivered. Rates of severe complications from the HDREB are reported to be 3-11% (Burt et al, 1990; Aliberti, 1995; Langendijk et al, 1998; Kotsianos et al, 2000). Common complications from HDREB include hemoptysis, radiation bronchitis, and esophagitis. Fatal hemoptysis and tracheoesophageal fistula caused by HDREB have been reported but the occurrence is relative rare. Burt et al.
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Cancer Therapy Vol 2, page 473 1994; Gollins et al, 1994; Kelly et al, 2000). In a retrospective review reported by Kelly et al, significant and slight improvement of HDREB were reported in 32% and 34% patients, respectively (Kelly et al, 2000). However, repeated bronchoscopy after HDREB demonstrated that overall objective response rate correlated with subjective response and symptomatic relief in less than 80% of the cases. The response rates of our study were measured by both CT scan of the chest and follow up bronchoscopy examinations. All study results were confirmed by the treating pulmonologist and radiation oncologist. Subjective response information such as dyspnea, cough, amount of hemoptysis or other symptoms reported by patients, was not used to measure the response rate. Patients treated with higher dose HDREB (24 Gy or more) achieved significantly higher response rates compared to patients received 21 Gy of HDREB. Many patients with locally advanced NSCLC have relatively bulky primary disease; however, the therapeutic range of HDREB is limited and may not cover the primary tumor completely. Therefore, HDREB should be used after EBRT for the majority of patients unless the initial primary tumor volume can be covered by HDREB. In our study, only a few patients with small primary tumors were treated with HDREB first. Comparison between patients treated with HDREB before or after EBRT was not done because of the limited number of patients in each group. The response rate of patients in groups 2 and 3 is identical (90%). We do not know whether further increase of HDREB dose will further improve the local response or not. It was originally planned to escalate the HDREB dose to 30 Gy delivered in 3 weekly fractions; however, to detect any significant but small improvement of response over 90% would require a substantially larger sample size, and such study may not be feasible in a single institution. Patients with unresectable and locally advanced nonsmall cell lung cancer are generally treated with definitive radiotherapy with chemotherapy, either sequentially or concurrently (Perez et al, 1987; Dillman et al, 1990; Le Chevalier et al, 1991). All patients in this protocol received concurrent chemotherapy according to our institutional chemotherapy regimen. Two patients in Group 1 also received a short course of neoadjuvant chemotherapy. Both patients received brachytherapy after EBRT and had partial response after the completion of radiotherapy. The post-treatment evaluations for these 2 patients were compared to the chest CT scan and bronchoscopic findings after the completion of neoadjuvant chemotherapy. It is unlikely that this slight deviation from chemotherapy schedule would have any substantial effect on our overall conclusion. The prognosis of patients with locally advanced nonsmall-cell lung carcinoma is generally poor. The overall 2year survival rate is less than 25% (Perez et al, 1987; Hazuka and Turrisi, 1993), and the median survival is around 12 months with external beam radiation and concurrent chemotherapy (Sause et al, 1995). Distant metastasis is common in this group of patients and is the most common cause of death. The median survival time for our patients was 13 months, which is comparable to the
historical data. Because the end points of this study are the overall response rates and the rate of Grade 3 and 4 complications at different dose regimens, follow up for patientsâ&#x20AC;&#x2122; survival data was not required by this prospective protocol. Nevertheless, our clinical chart review data showed that the majority of patients deceased developed distant metastasis. Thus, improvements in survival will also depend on better systemic control.
V. Conclusion High-dose-rate brachytherapy of 27 Gy in three divided weekly treatments, plus 66 Gy of external beam radiation is well tolerated and effective for the treatment of obstruction from non-small cell lung cancer with endobronchial or peribronchial component. A statistically significant dose response for NSCLC is seen when treating with a combination of external beam radiation and high dose rate endobronchial brachytherapy with a dose of 24 Gy or higher in 3 weekly fractions. EBRT followed by fractionated adjuvant HDREB is a strategy that deserves to be optimized and then tested in a larger scale prospective trial to learn if it can improve outcome.
References Aliberti WE (1995) Endobronchial high dose rate brachytherapy. Int J Radiat Oncol Biol Phys 25, 753-5. Bastin KT, Mehta MP, Kinsela TJ (1993) Thoracic volume radiation sparing following endobronchial brachytherap, a quantitative analysis. Int J Radiat Oncol Biol Phys 25, 703707. Burt PA, Oâ&#x20AC;&#x2122;Driscoll BR, Notley HM, et al (1990) Intraluminal irradiation for the palliation of lung cancer with the high dose rate micro-Selectron. Thorax 45, 765-768. Chang LF, Horvath J, Peyton W, et al (1994) High dose rate afterloading intraluminal brachytherapy in malignant airway obstruction of lung cancer. Int J Radiat Oncol Biol Phys 28,589-596. Chetty KG, Moran EM, Sassoon CSH, et al (1989) Effect of radiation therapy on bronchial obstruction due to bronchogenic carcinoma. Chest 95, 582-584. Cotter GW, Ellingwood KE (1988) High dose rate brachytherapy treatment of malignant tumors. Ala Med 58, 13-15. Cox JD, Stetz J, Pajak TF (1995) Toxicity criteria of the Radiation Therapy Oncology Group (RTOG) and the European Organization for Research and treatment of Cancer (EORTC). Int J Radiat Oncol Biol Phys 31, 1341-6. Dillman RO, Seagren SL, Propert KJ, et al (1990) A randomized trial of induction chemotherapy plus high-dose radiation versus radiation alone in stage III non-small-cell lung cancer. N Engl J Med 323, 940-5. Dosoretz DE, Galmarini D, Rubenstein JH, et al (1993) Local control in medically inoperable lung cancer, an analysis of its importance in outcome and factors determining the probability of tumor eradiation. Int J Radiat Oncol Biol Phys 27, 507-516. Gollins SW, Burt PA, Barber PV, et al (1994) High dose rate intraluminal radiotherapy for carcinoma of the bronchus, Outcome of treatment of 406 patients. Radiother Oncol 33, 31-40. Hazuka MB, Turrisi AT (1993) The evolving role of radiation therapy in the treatment of locally advanced lung cancer. Semin Oncol 20, 173-84. Hernandez P, Gursahaney A, Roman T, et al (1996) High dose rate brachytherapy for the local control of endobronchial
473
Lu et al: High dose rate endobronchial brachytherapy for NSCLC carcinoma following external irradiation. Thorax 51, 354358. Huber RM, Rohloff R, Duft S, et al (1987) Treatment of tracheobronchial tumors by endoluminal irradiation with iridium 192 (afterloading technique). Clin Resp Phys 23, 389. Kelly JF, Delclos ME, Morice RC, et al (2000) High-dose-rate endobronchial brachytherapy effectively palliates symptoms due to airway tumors, the 10-year M.D. Anderson cancer center experience. Int J Radiat Oncol Biol Phys 48, 697702. Kotsianos D, Bach D, Gamarra F, et al (2000) High-dose-rate brachytherapy, dose escalation in three-dimensional miniograns of the human bronchial wall. Int J Radiat Oncol Biol Phys 46, 1267-73. Lang N, Maners A, Broadwater J, et al (1988) Management of airway problems in lung cancer patients using the neodymium-yttrium-aluminum-garnet laser and endobronchial radiotherapy. Am J Surg 156, 463-465. Langendijk JA, Tjwa MK, de Jong JM, et al (1998) Massive haemoptysis after radiotherapy in inoperable non-small cell lung carcinoma, is endobronchial brachytherapy really a risk factor? Radiother Oncol 49, 175-183. Le Chevalier T, Arriagada R, Quoix E, et al (1991) Radiotherapy alone versus combined chemotherapy and radiotherapy in nonresectable non-small cell lung cancer, first analysis of a randomized trial in 353 patients. J Natl Cancer Inst 83, 41723. Lu JJ, Bains YS, Abdel-Wahab M, et al (2002) High-dose-rate remote afterloading intracavitary brachytherapy for the treatment of extrahepatic biliary duct carcinoma. Cancer J 8, 74-78. Macha HN, Koch K, Stgadler M, et al (1987) New technique for treating occlusive and stenosing tumors of the trachea and main bronchi, Endobronchial irradiation by high dose Iridium-192 combined with laser canalization. Thorax 42, 511-515. Majid OA, Lee S, Khushalani S, et al (1986) The response of atlectasis from lung cancer to radiation therapy. Int J Radiat Oncol Biol Phys 12, 231-232.
Mazeron JJ, Noel G, Simon JM (2002) Head and neck brachytherapy. Semin Radiat Oncol 12, 95-108. Nguyen TD, Picavet B, Mallet F, et al (1999) Non-metastatic advanced non-small cell lung carcinoma, systemic high-dose brachytherapy following curative external radiotherapy. Cancer Radiother 3, 468-474. Perez CA, Pajak TF, Rubin P, et al (1987) Long tern observations of the patterns of failure in patients with unresectable non-oat cell carcinoma of the lung treated with definitive radiotherapy. Report by the RTOG. Cancer 59, 1847-81. Sause WT, Scott C, Taylor S, et al (1995) Radiation Therapy Oncology Group (RTOG) 88-08 and Eastern Cooperative Oncology Group (ECOG) 4588, preliminary results of a phase III trial in regionally advanced, unresectable nonsmall-cell lung cancer. J Natl Cancer Inst 87, 198-25. Slawson RG, Scott RM (1979) Radiation therapy in bronchogenic carcinoma. Radiology 132, 175-176. Speiser BL, Spratling L (1993) Remote afterloading brachytherapy for the local control of endobronchial carcinoma. Int J Radiat Oncol Biol Phys 25, 579-587.
Jiade J. Lu
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Cancer Therapy Vol 2, page 475 Cancer Therapy Vol 2, 475-500, 2004
Complementary alternative medicine for cancer: a review of effectiveness and safety Review Article
Ursula Werneke1,*, David Ladenheim2, Tim McCarthy3 1
Consultant Psychiatrist and Honorary Senior Lecturer, Homerton University Hospital and Institute of Psychiatry, London UK 2 Senior Pharmacist, Barts and the London NHS Trust, London, UK 3 Oncology Pharmacist, London, UK
__________________________________________________________________________________ *Correspondence: Dr Ursula Werneke, Consultant Psychiatrist, Homerton University Hospital, East Wing, Division of Psychiatry, Homerton Row, London E9 6SR, UK; e-mail: Ursula.Werneke@elcmht.nhs.uk Key words: alternative medicine for cancer, single or combined anticarcinogenic CAMs, anti-carcinogenics and immuno-stimulants, antioxidants â&#x20AC;&#x201C;vitamins and other remedies, remedies with endocrine properties, psychoactive remedies, Abbreviations: bovine spongioform encephalopathy, (BSE); complementary alternative medicines, (CAM); cytochrome P, (CYP); !linolenic acid, (GLA); diamino-N(10)-methylpteroic acid, (DAMPA); glomerular filtration rate, (GFR); methotrexate, (MTX); multidrug resistance, (MDR); Natural Medicines Comprehensive Database, (NMCD); Physiciansâ&#x20AC;&#x2122; Desk Reference, (PDR); prostaglandin-E1, (PGE1); prostate specific antigen, (PSA); randomised controlled trials, (RCTs); tumor necrosis factor, (TNF)
There is no conflict of interest. Ursula Werneke has received a grant form Pfizer Ltd and honoraria and speaker fees from Eli Lilly Ltd, all unrelated to this particular study and unrelated to the topic of CAMs in general. Received: 8 November 2004; Accepted: 29 November 2004; electronically published: December 2004
Summary The use of complementary alternative medicines (CAM) in cancer patients is well documented but their effectiveness is often not established. They may be less harmless than commonly assumed. We reviewed CAMs most commonly encountered in oncology practice, their effectiveness, potential adverse effects and interactions. These were divided into the following categories: i. Single or combined anti-carcinogenic remedies; ii. Anti-carcinogenics and immuno-stimulants; iii. Antioxidants; iv. Remedies with endocrine properties; v. Psychoactive remedies and vi. Other remedies used by cancer patients. In each category, potentially useful and potentially harmful substances were identified. Many of the potential side effects and interactions described are based on hypotheses about pharmacokinetic and pharmacodynamic properties, which frequently have only been demonstrated in vitro but could have significant clinical implications if found in vivo. Particularly, the potential significance of the cytochrome P (CYP) 450 system and the p-glycoprotein pump for drug interactions require further investigation. Currently, most advice available to patients taking CAMs depends on the plausibility of the hypothesized effects and side effects. Prospective systematic surveillance of CAMs is required to improve the evidence base. Meanwhile, the use of CAMs with potential for serious interactions reducing efficacy of conventional treatments should not be encouraged. range from 7% to 80% (Ernst and Cassileth, 1998; Risberg et al, 1998; Rees 2000, Richardson et al, 2000; Sparber et al, 2000; Bernstein and Grasso 2001; Ashikaga et al, 2002; Werneke et al, 2004a) (Table 1). CAMs can be grouped into herbal remedies, food supplements including vitamin preparations, trace elements and other substances such as omega-3 fatty acids. Cancer patients may take CAMs for a variety of reasons: to combat the cancer, strengthen the immune system, counter side effects of conventional treatments and treat psychological problems including low mood and anxiety.
I. Introduction The use of complementary alternative medicines (CAM) by patients suffering from chronic disorders, such as cancers and associated physical and psychological problems, is well documented (Eisenberg 1993; Ernst and Cassileth, 1999). CAMs are either used on their own (alternative) or in addition to conventional medicine (complementary) (Zimmerman and Thompson, 2002). Depending on the definition and inclusion criteria chosen, estimates of prevalence of CAM use in cancer patients
475
Werneke et al: Complementary alternative medicine for cancer Some patients may look for a more holistic approach to treatment, feeling that conventional cancer care tends to neglect their spiritual needs and leaves them out of control of their treatment (Sparber, 2000). Others hope that CAMs being “natural” have fewer or no side effects, while some cancer patients may feel disillusioned by the apparent ineffectiveness of conventional treatment. However, use of CAM and especially of herbal remedies and supplements is not without problems. Some may not be effective, and others have potentially dangerous side effects and interactions with conventional treatments. Particularly, the potential for interactions between CAMs and chemotherapeutic agents poses a challenge, since the number of novel antineoplastic agents exerting their effects by many different mechanisms is increasing. This reflects improved understanding of disease states and cellular makeup so that specific pathways can be targeted. Relatively new mechanisms of action include enzyme inhibition through protease inhibitors and protein-tyrosine kinase inhibitors and immuno-modulation, e.g. through monoclonal antibodies. Standard cytotoxic agents exert their effects in a number of ways, e.g. alkylation of cell DNA thereby impairing replication and causing cytotoxicity; enzyme inhibition preventing synthesis or action of cellular components such as pyrimidines and purines required for DNA and RNA synthesis; binding to DNA intercalating between base pairs and so upsetting the helical structure preventing cell replication; enzymic breakdown of products required for growth and replication; or production of intracellular free radicals resulting in cellular damage (Chabner and Longo, 2001). The aim of this review is to identify and categorize CAMs commonly encountered in clinical oncology practice, to review the evidence base for their purported effectiveness and to discuss potential adverse effects and interactions relating to their use.
but we also included other evidence including case reports, when the findings were relevant to our review. CAM names are usually given in English, and the scientific names are listed in Appendix 1.
III. Results CAMs can interact with chemotherapy in several ways, by either interfering with the pharmacokinetics or the mechanism of action, thereby altering the effectiveness of chemotherapy.
A. Pharmacokinetics The pharmacokinetics of most anticancer drugs are highly variable between patients and may be genetically determined. For instance, oxidative metabolism depends on the variability of the expression of the different cytochrome P450 enzymes, not only in the liver but also in extra-hepatic tissue including tumor tissue (Kivisto et al, 1995). Of the cytochrome system, CYP 3A4 seem to play the most important role. Sixty percent of all clinically used drugs are metabolized through CYP 3A4 including warfarin, ciclosporin, oral contraceptives, anticonvulsants, (e.g carbamazepine) and digoxin (Lake et al, 1992; Michalets 1998; Lill et al, 2000). Anticancer agents metabolized through CYP 3A4 include tamoxifen (Hukkanen et al, 1998), camptothecins (Sparreboom 2004), cyclophosphamide (Quintieri et al, 2000), ifosfamide (Quintieri et al, 2000), paclitaxel (Nallani et al, 2004), epipodophyllotoxins e.g teniposide, etoposide (Relling et al, 1994) and vinblastine (Zhou-Pan et al, 1993). Numerous CAMs, such as St John’s wort, goldenseal, quercitin, cat’s claw, licorice, red clover, soy, valerian, kava kava (NMCD, 2004) interact with CYP 3A4 and can alter the metabolism of other complementary or conventional medicines (Table 1-7). Foodstuffs can also affect the CYP system; e.g. grapefruit is a potent CYP 3A4 inhibitor (Ho and Saville, 2001). Some CAMs such as echinacea (Figure 1) and gingko may either inhibit or induce CYP 3A4, and the effect may depend on the extract composition, the duration of treatment and the dose used (Sparreboom 2004). Some of these and other CAMs also affect other P450 enzymes, e.g. CYP 2C9 and 2C19, which metabolize tamoxifen (Hukkanen et al, 1998), ifosfamide (Chang et al, 1997) or cyclophosphamide and teniposide (Sparreboom et al, 2004) respectively. However, CYP interactions observed in vitro may not necessarily assume clinical significance (Zuber et al, 2002; Sparreboom et al, 2004) and often no recommendations can be made where studies in humans are not available. CAMs can also influence the transmembraneous pglycoprotein pump, which participates in the transport of drugs out of cells. Increased levels of p-glycoprotein are found in multi-drug resistance (MDR). In consequence, tumor cells become resistant to a variety of drugs after exposure to a single drug. Drugs which are substrates to the CYP 3A enzymes are particularly affected. Examples include anthracyclines, epipodophyllotoxins, taxol and vinca alkaloids (Kivisto et al, 2004). Two psychoactive plants, St John’s wort (Figure 2) and ginkgo (Figure 3), may interfere with the p-glycoprotein pump. They may not only alter the effectiveness of chemotherapy but also
II. Method We searched the Medline and Cochrane databases for CAMs used in cancer treatment. We divided the substances into different categories: cancer treatments, anti-cancer remedies with immunostimulating effect, antioxidants, remedies with endocrine properties, psychotropic remedies and other substances frequently used by cancer patients. Search terms included the identified substances in each category, “effectiveness”, “trial”, “review”, “side effects”, “adverse drug reaction” and “interaction”. All recovered papers were reviewed for further relevant references. All evidence was collated and ranked as available. We also accessed web-based resources such as Natural Medicines Comprehensive Database (NMCD) 2004 and CAM formularies such as the Physicians' Desk Reference for Herbal Medicines (PDR) 2001 for further information on the identified substances. Where available, we used reviews summarising the proposed mechanism of action and effectiveness, since presenting all the evidence in detail would have been beyond the scope of this paper and duplicating existing work. Whenever possible, we gave meta-analyses and double blind randomised controlled trials (RCTs) priority, 476
Cancer Therapy Vol 2, page 477 change the results of MDR nuclear scans (Werneke and McCready, 2004). On the other hand some CAMs including selenium, quercitin and related flavonoids, sylmarin derived from milk thistle and curcumin contained in tumeric may inhibit the p-glycoprotein pump (Zhou et al, 2004, Bjorkhem-Bergman et al 2002). Catechins from green tea may exert differential effects (Zhou et al 2004).
grape seed, hydrazine, laertrile (apricot) and shark cartilage (Table 2A). For most of these remedies, the mechanism of action remains unclear and clinical studies are scarce. An exception is shark cartilage, which contains several macroproteins including U-995 and AE-941 (Neovastat), which have antiangiogenic and pro-apoptotic properties. AE-941 is currently being tested in clinical trials for renal, lung cancer and multiple myeloma (Bukowski 2003; Gringas et al, 2003). Previously, only parenteral formulations were effective, since oral formulations were not absorbed (Ernst and Cassileth, 1999)
B. Single or combined anticarcinogenic CAMs Single anticarcinogenic CAMs are extracted from one source, e.g. one plant, only. They include goldenseal,
Figure 2. St Johnâ&#x20AC;&#x2122;s wort
Figure 1. Echinacea
Figure 3. Ginkgo biloba
Table 1. Prevalence of CAM use in Cancer patients Study
Country
Werneke et al, 2004
UK
Sample size 318
All
52%
Ashikaga et al, 2002
USA
148
Breast
72%
Berstein and Grasso, 2001
USA
100
All
80%
Rees, 2000
UK
1023
Breast
32%
Richardson et al, 2000
USA
453
all
63%
Sparber et al, 2000 Ernst and Cassileth, 1998
USA 13 countries
100
all all
75% 7% to 64%
Risberg et al, 1998
Norway
252
all
45%
Table 2A. Cancer treatments: single agents 477
Cancer type
CAM use
Werneke et al: Complementary alternative medicine for cancer
Remedy
Postulated mechanism of action for oncology related indications
Effectiveness for oncology related indications
Potentially serious side effects
Potential drug interactions
Goldenseal: berberine
Inhibition of tumor promoters (Nishino et al, 1986)
In vitro only (Nishino et al, 1986)
" blood pressure and death from respiratory failure (NMCD 2004); # QTc interval (Xu, 1989); contraindicated in pregnancy (PDR, 2001)
Grape seed: proanthocyanidins
Antiproliferative, antiangiogenic, proapoptotic, antioxidant activity (Singh et al, 2004); synergistic with doxorubicin (Sharma et al, 2004);
No clinical evidence available
None identified
CYP 3A4 inhibition (Foster et al, 2003); QTc prolonging drugs including quinine and antipsychotics; caution with antihypertensives due to risk of # hypotensive effects (Covington, 1996) Anticoagulants: concomitant use with warfarin might increase warfarin's effects and the risk of bleeding (NMCD, 2004)
suppression of estrogen biosynthesis (Eng et al, 2004) Hydrazine
Unclear, also used for cancer induced cachexia
Tested in RCTs: not effective (Kosty et al, 1994; Loprinzi et al, 1994a, b)
Central and peripheral neurological effects; hypo-or hyperglycemia (NMCD, 2004)
Antidiabetics, CNS depressants; monoamineoxidase inhibitors (NMCD, 2004)
Laetrile (apricot): amygdalin
Unclear
Not effective (Moertel 1982; Foster et al, 1993; Newall et al, 1996)
Safety concern because of cyanide contents (NMCD, 2004)
None identified
Shark cartilage
Antiangiogenic activity and inhibition of tumor neovascularisation: inhibition of matrix proteinase and collagenase activity, induction of endothelial cell apoptosis (Gringas et al, 2003); two macro-proteins, U995 and AE-941 (Neovastat) implicated (Gonzales et al, 2001)
U-995 inhibits tumor growth in animal model when given intraperitoneally (Sheu et al, 1998); AE-941: clinical trials for renal, lung cancer and multiple myeloma ongoing (Bukowski, 2003; Gringas et al, 2003);
Anaphylaxis possible when given parenterally (Ernst and Cassileth, 1999); one case report of hepatitis (Ashar and Vargo, 1996)
None identified
effectiveness depends on bioavailability of remedy
No clinical data has been offered for the efficacy of amygdalin, which is the major component of laetrile. Instead, amygdalin can be broken down to hydrogen cyanide which is toxic, and its use should be actively discouraged. It was claimed that cancer tissues were rich in an enzyme that causes amygdalin to release cyanide thereby destroying cancer cells. Non-cancerous tissues would be protected from this fate by another enzyme rendering cyanide harmless. Later, it was marketed as a
vitamin (vitamin B17), possibly to evade its ban (Wilson, 2000). Combined CAMs are agents derived form several sources. They are combinations of different herbs or plant extracts with other substances. These may have synergistic effects or buffer the toxic effects of the other components (Vickers, 2002, Williamson 2001), but they may also increase the potential for side effects. Carctol, essiac and flor-essence are used for cancer in general (Table 2B). PC-SPES, PC-Hope and 714X are applied specifically to
478
Cancer Therapy Vol 2, page 479 prostate cancer (Table 2C). Carctol is an Ayurvedic preparation which is derived from eight different plants, some of which have antioxidant, anticarcinogenic and antiestrogenic properties. Clinical efficacy is unproven, and the potential for side effects and drug interactions may be much higher than claimed by its suppliers. Essiac is named after its inventor, Caisse, spelt backwards (Ernst and Cassileth, 1999). It is a combination of four herbs: burdoch, sheep sorrel, rhubarb and slippery elm. Sheep sorrel may act as an antioxidant since the leaves contain $carotene. No mechanism of action has been postulated for the anticarcinogenic properties of the other three herbs
involved. Flor-essence is a combination of essiac with four other herbs: water cress and kelp may act as antioxidants, blessed thistle has been attributed with some unspecified anti-carcinogenic activity and red clover is a phytoestrogen. The clinical efficacy is unproven, and high doses may be required. This theoretically increases the potential for side effects and possibly affects the nature of potential drug interactions. Watercress, sheep sorrel and rhurbarb can all compromise the kidney function, presumably since they contain oxalic acid.
Table 2B. Cancer treatments: combined agents used for all cancers Remedy
Postulated mechanism
Effectiveness
Potentially serious side effects
Potential drug interactions
No clinical evidence available
Indian sarsaparilla: # INR since contains coumarin (globalherbsupplies); cubebs: # gastric acid (NCMD, 2004); puncture vine: hypoglycemia in animal experiment (Li et al, 2002), one case report of pneumothorax through ingestion of spikes (Dudley, 1983); blistering ammania: skin irritant, topically can produce blisters (BoDD, 2004); Himalayan rhubarb: hypoglycaemia in vitro (Suresh et al, 2004)
Indian sarsaparilla: " of aminoglycoside induced nephrotoxicity (Kotnis et al, 2004); anticoagulants; cubebs: " effectiveness of antiulcer drugs (NCMD, 2004);
Burdoch: hypoglycemia, allergies; sheep sorrel and rhubarb: contain oxalate, caution in patients with kidney stones, hypokalaemia; rhubarb: arrhythmias, allergies (PDR, 2000; NMCD, 2004)
Burdoch: antidiabetic drugs; sheep sorrel and rhubarb: digitalis, diuretics corticosteroids due to potassium loss; slippery elm and rhubarb: possibly " absorption of other drugs (PDR, 2000; NMCD, 2004)
Watercress: kidney damage with large doses (NMCD, 2004); blessed thistle: (NMCD, 2004); cf. tables 4-7 for the other ingredients
Watercress: anticoagulants (NMCD, 2004); blessed thistle: antiacids, H2 blockers, protone pump inhibitors (NMCD, 2004);
of action Carctol: Indian sarsaparilla, blistering ammania, puncture vine (diosgenin) cubebs, Chinese sarsaparilla, garden cress thyme leaved gratiola, Himalayan rhubarb
Indian sarsaparilla: inhibits carcinogenesis of nitroso-amins (Iddamaldeniya, 2003); antioxidant (Mary et al, 2003); puncture vine: augmentation of estrogenic effect ( c.f. table 5); Chinese sarsaparilla, cubebs and Himalayan rhubarb: antioxidants (Karthikeyan and Rani, 2003, Krenn et al, 2003)Tripathi et al, 2003) garden cress: related to watercress (c.f. floressence);
puncture vine: # effect of antihyperglycemics, # effect of estrogen containing drugs (c.f. table 5); garden cress: related to water-cress: cf. floressence; Himalayan rhubarb: # effect of antihyperglycemics
Blistering ammania, thyme leaved gratiola: unclear Essiac:
Burdoch: unclear;
burdock, sheep sorrel, rhubarb, slippery elm
sheep sorrel: antioxidant, leaves contain $-carotene (NMCD, 2004); rhubarb: unclear; slippery elm: unclear
Flor-essence: as essiac + watercress, blessed thistle, red clover and kelp
Watercress: contains $carotene (NMCD, 2004); inhibits carcinogenesis of nitrosoamins (Hecht et al, 1995); induction of apoptosis (Chiao, 2004); blessed thistle: antitumour activity (NMCD, 2004);
In vitro: antiproliferative effects in high concentrations (Tai et al, 2004); no clinical data available (Kaegi 1998; Tamayo et al, 2000)
No clinical data available (Kaegi, 1998; Tamayo et al, 2000)
cf. tables 4-7 for the other ingredients
479
Werneke et al: Complementary alternative medicine for cancer Table 2C. Cancer treatments: combined agents used for prostate cancer Remedy
Postulated mechanism
Effectiveness
Potentially serious side effects
Potential drug interactions
Significant PSA decline in androgen-independent prostate cancer (Small et al, 2000; Pfeiffer et al, 2002)
Thrombosis, allergies (Small et al, 2000); contamination with diethyl-stilbestrol, indomethacin, and warfarin (Sovak et al, 2002)
Warfarin and hormonal treatments; cf. all other tables for individual agents
No clinical data available
Unclear
Quercitin: CYP3A4 and CYP 2C9 inhibition; CYP 3A4 induction also reported (Sparreboom et al, 2004)
No clinical data available
Oral camphor: significant toxicity including CNS depression and coma; hepatotoxic (NMCD, 2004)
Contains nitrates: nitrates are contraindicated with phosphodiesterase 5 inhibitors
of action PC-SPES: dyer’s woad, licorice, chrysanthemum, panax pseudo-ginseng, rabdosia, reishi, saw palmetto, Balkai scullcap
PC-HOPE: as PC-SPES + quercitine and sterolin
714X: various combinations of substances, e.g. (1) camphor, ammonium chloride and nitrate, sodium chloride, ethanol, and water; or (2) camphor, sodium chloride, calcium, copper, zinc and other elements
Dyers woad unclear; crysanthemum: unclear; licorice: phytoestrogen, cytotoxic in higher doses (c.f. table 6a); reishi: immune stimulating, antitumour; rabdosia, Balkai skullcap, saw palmetto: all cytotoxic; panax pseudoginseng: antioxidant, phytoestrogen (cf. Table 6a) Quercitin: up-regulation of tumor suppressor genes and reciprocal down-regulation of oncogenes and cell cycle genes (Nair et al, 2004); sterolins: balance Thelper 1 and T-helper 2 activity (Kidd, 2003) Unclear
Burdoch may interact with antidiabetic drugs, sheep sorrel with digitalis and diuretics and water cress with warfarin (NMCD, 2004). PC-SPES, PC standing for prostate cancer and SPES meaning hope in Latin, is a combination of eight herbs with antioxidant, cytotoxic and estrogenic properties. PCSPES has been shown to lead to a reduction in prostate specific antigen (PSA) in androgen independent prostate cancer (Pfeiffer et al, 2000; Small et al, 2000). Possibly, due to its phytoestrogen content, PC-SPES has been associated with thrombosis. It was withdrawn for the US market after contaminations with estrogens, warfarin and indomethacin were reported. The estrogen was presumably added to increase its efficacy, whereas warfarin and indomethacin may have been used to decrease the thrombosis risk. PC-SPES has reemerged as PC-HOPE, which extends the original combination with quercitin and sterolins. Whereas quercitin has anti-carcinogenic properties (Nair et al, 2004), sterolins are used as immune stimulants (Kidd, 2003). No clinical trials have been conducted. Quercitin is a CYP 3A4 inhibitor (Ho and
Saville, 2001) and may affect corresponding substrates as outlined above. 714X is also used for prostate cancer, but is unlikely to be effective (NMCD, 2004). The name reflects the inventor’s initials: 7th and 14th letter of the alphabet and his birth year 1924, X is the 24th letter in the alphabet (Barrett, 1999) 714X is a combination of camphor with several minerals including nitrates dissolved in water. Camphor can lead to significant hepatotxicity and CNS depression. Nitrates are contraindicated in patient using phosphodiesterase 5 inhibitors (e.g. sildenafil) for the oral treatment for erectile dysfunction.
C. Anti-carcinogenics stimulants
and
immuno-
Some CAMs are thought to be both anticarcinogenics and immunostimulants. The herbal remedies cat’s claw, echinacea, mistletoe and pau d’arco (Table 3A) are examples. Despite the theoretical understanding of their mechanism of action the evidence for the clinical effectiveness of these agents is sparse. A systematic
480
Cancer Therapy Vol 2, page 481 review of ten trials using mistletoe did not show any effectiveness (Ernst et al, 2003). However, improvement of quality of life has been noted (Ernst et al, 2003; Schuhmacher et al, 2003; Piao et al, 2004; Semiglasov et al, 2004). All these remedies may interfere with immunosuppressant therapies and corticosteroids. Echinacea may specifically interact with monoclonal antibodies targeting malignant B cells (Stimpel et al, 1984; Luettig et al, 1989).
Also, echinacea should only be taken intermittently and not long-term. It can have differential effects on the CYP 3A4 system leading either to inhibition or induction (Sparreboom, 2004). Three non-herbal remedies were identified with purported anticarcinogenic and immunostimulating action: $-glucans, kombucha tea and thymus extract (Table 3B).
Table 3A. Herbal anti-carcinogenics and immunostimulants Remedy
Postulated mechanism of action
Effectiveness
Potentially serious side effects
Potential drug interactions
Catâ&#x20AC;&#x2122;s claw
Stimulation of interleukin-1 and -6 production by alveolar macrophages (Lemaire, 1999);
In vitro evidence only
" blood pressure (NMCD, 2004)
CYP 3A4 inhibition (Budzinki et al, 2000);
Echinacea
apoptosis (Sheng, 1998); antimutagenic activity; apoptosis and inhibition of malignant leukocyte proliferation (NMCD, 2004) # production of complement properdin as a marker of immune system stimulation (Kim et al, 2002); stimulation of B lymphocytes which monoclonal antibodies are targeting (Stimpel et al, 1984; Luettig et al, 1989); stimulation of phagocytosis; # activity and mobility of leukocytes; induction of macrophages to produce cytokines (TNF%, IL-1, interferon$-2) (Stimpel et al, 1984; Luettig et al, 1989)
Mistletoe: lectins including viscumin
Stimulation of cytokins (Riberau-Gayon et al, 1997; Mansky et al, 2003); modulation of neutrophile response (Pelletier et al, 2001)
Pau dâ&#x20AC;&#x2122;arco: $-lapachone
Induction of apoptosis (Dubin et al, 2001; Choi et al, 2003)
immunosuppressants (NMCD, 2004); # effect of antihypertensives (NMCD, 2004)
Inconclusive for prevention and treatment for common cold /upper respiratory tract infections (Melchart et al, 2000; Ernst, 2002a; Goel et al, 2004); possibly effective in reducing chemotherapyinduced leucopenia (Melchart et al, 2000)
Long term use discouraged (PDR, 2000); allergic reactions including maculopapular rashes, asthma and anaphylaxis (Mullins and Heddle 2002; Taylor et al, 2003); # risk of allergic reaction in patients with atopy (Mullins and Heddle 2002); hepatotoxicity (Miller, 1998)
CYP 3A4 inhibition (Strandell et al, 2004); possibly differential effect on CYP 3A4: induction or inhibition depending on route of administration of substrate and composition of extract (Sparreboom, 2004); immunosuppressants and corticosteroids (Budzinski et al, 2000; NMCD, 2004); monoclonal antibodies targeting B lymphocytes (Stimpel et al, 1984; Luettig et al, 1989); # risk of hepato-toxicity with anabolic steroids, amiodarone, methotrexate, and ketoconazole (Miller, 1998)
Not effective on systematic review of ten RCTs (Ernst et al, 2003); improvement of quality of life (Ernst et al, 2003; Schuhmacher et al, 2003; Piao et al, 2004; Semiglasov et al, 2004) Insufficient evidence
481
Angioedema (Piao et al, 2004); # intracerebral pressure and allergic reactions (Ernst et al, 2003); hypotension (NMCD, 2004)
Immunosuppressants (NMCD, 2004)
Severe gastro-intestinal symptoms, dizziness, anemia; # bleeding risk as vitamin K antagonist (NMCD, 2004)
Anticoagulants, antiplatelets (NMCD, 2004)
Werneke et al: Complementary alternative medicine for cancer Table 3B. Other anticarcinogenics and immune-stimulants Remedy
Postulated mechanism of action
Effectiveness
Potentially serious side effects
Potential drug interactions
$-glucans: lentinan, schizophyllan (sizofiran)
Synergizes with monoclonal antibody therapy through activation of compliment factor CR3, (Hong et al, 2003) and carmustine through inhibition of glyoxalase I (Finkelstein et al, 2002): glyoxylase I inhibits anti-apoptosis signals in tumour cells (Fullerton et al, 2003; Tsuroro 2003) Unclear (Hauser 1990)
Adjuvant use with conventional cancer treatment Lentinan: # survival time for several cancers reported (Kidd, 2000); Schizophyllan: survival time for cervical cancer (Okamura et al, 1989) and head and neck cancer (Kimura et al, 1994)
Hypo / hypertension related to parenteral use (NMCD, 2004)
Immunosuppressant drugs (NMCD, 2004)
No evidence as treatment for cancer or immunostimulant (Ernst, 2003)
Since the culture must grow at room temperature for seven to ten days, contamination and growth of other organisms including aspergillus and anthrax (Gamundi and Valdivia 1995; Sadjadi 1998); hepatotoxic (Whiting et al, 2002); one death reported (CDC, 1995)
None reported
Activation of natural killer cells;
Insufficient evidence (Ernst, 1997)
Risk of infection in immunocompromised patients including a theoretical risk of BSE transmission (NMCD, 2004); risk of severe allergic reactions when injected (Ernst and Cassileth, 1999)
Immunosuppressants
Kombucha Tea (fungal infusion, of a mixture of bacteria, yeasts, tea and sugar)
Thymus therapy
# cytotoxic activity; # mitogen-induced interferon levels in human lymphocytes, inhibition of tumor growth (Ernst and Cassileth, 1999)
$ glucans are yeast components which are suggested to have ‘possible efficacy’ when used in conjunction with conventional therapy for different cancers (e.g. head and neck and cervical cancers) (NMCD, 2004).They are known to synergize with monoclonal antibodies and carmustine (Hong et al, 2002; Finkelstein et al, 2003). Initial RCTs have shown a significant increase of survival time for advanced cervical and gastric cancer (Fujimoto et al, 1984; Miyazaki et al, 1995). Kombucha tea is an infusion of bacteria, yeast, tea and sugar, and there is no evidence of effectiveness in the oncology setting. There are reports of contamination with aspergillus and anthrax (Gamundi and Valdivia 1995; Sadjadi 1998), and immuno-compromised patients may be particularly at risk. Equally, thymus therapy has not shown to be efficient in cancer, but carries a significant risk of contamination including a theoretical risk of bovine spongioform encephalopathy (BSE) transmission (NMCD, 2004).
D. Antioxidants –vitamins and other remedies Certain nutrients in fruits and vegetables appear to protect the body against oxygen induced damage to tissues which occurs as part of the normal metabolism. These socalled anti-oxidants reduce oxidative stress by handling free-radicals and hydrogen-peroxide. However, antioxidants may affect chemotherapies which rely on oxidative action. For instance, alkylating agents, podphyllum agents and anthracycline antitumor antibiotics, create reactive oxygen species which are required to modify DNA. Doxorubicin creates free radicals through oxidation of nicotinamide adenine dinucleotide phosphate (Labriola and Livington, 1999). Clinically active anthracyclines such as doxorubicin have a number of mechanisms of cytotoxic action. They can bind to DNA and affect cell replication by inhibition of DNA topoisomerase II catalytic activity. They also create oxygen species (undergo electron reduction) to produce reactive compounds that can cause wide spread damage to intracellular components. This is also believed
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Cancer Therapy Vol 2, page 483 to play a role in the cardiotoxicity of the drug. One electron reduction is catalysed by flavin centred dehydrogenases or reductases e.g. CYP 450 reductase or nicotinamide adenine denucleotide reductase. These enzymes are distributed throughout human cells, allowing anthracyclines to produce activity in a number of organs. The reduction process produces the corresponding semiquinone free radical which donates its electron to oxygen creating the superoxide ion which produces hydrogen peroxide. Hydrogen peroxide is then cleaved by the semiquinone to produce the hydroxyl free radical which is responsible for cell destruction. In most cases, cells have defensive mechanisms to prevent cellular damage (Chabner and Longo, 2001). Vitamins with antioxidative properties include vitamin A and its precursor $-carotene, vitamin C and E (Table 4A). They have been shown to decrease the risk of several cancers, either in combination with other vitamins or supplements (Table 4A). Data on the anti-carcinogenic effectiveness are sparse. Vitamin C supplements may have no benefits (Moertel, 1985), and high dose supplements should usually be avoided (UK Food Standard Agency, 2003). Particularly, combination of high dose vitamin C with methotrexate (MTX) should be avoided, since
vitamin C acidifies urine leading precipitation of MTX and its less water soluable metabolites 7-OH-MTX and 2, 4-diamino-N(10)-methylpteroic acid (DAMPA), which cannot be excreted. This may lead to kidney damage and increased plasma levels of MTX (Sketris et al, 1984) resulting in reduced clearance and increased toxicity. $carotene may also be harmful, and increase the risk of lung and prostate cancer in smokers (The %-Tocopherol, $ Carotene Cancer Prevention Study Group, 1994; Heinonen et al, 1998; Patrick, 2000), but the mechanism of this adverse effect remains unclear. Frequently used other antioxidants include coenzyme Q10, green tea, selenium, tomato and tumeric (Table 4B). Apart from co-enzyme Q10, they are mostly used for cancer prevention. Selenium in particular has been associated with a decreased risk of lung, prostate and colorectal cancer (Clark, 1996). Selenium should only be taken in the recommended dosage, since the therapeutic/toxic index is low (NMCD, 2004). However, dose recommendations vary internationally. Also, all these other antioxidant remedies can interact with concomitant anticoagulants. Tumeric, additionally, interferes with antiplatelet drugs (NMCD, 2004)
Table 4A. Antioxidants (AOs): vitamins Remedy
Postulated mechanism of action
Effectiveness
Potentially serious side effects
Potential drug interactions
Vitamin A
AO
Dietary: " risk of premenopausal breast cancer in women with a positive family history (Russel, 2000)
Acute toxicity leads to neuro-psychiatric symptoms including delirium and coma; hepatotoxicity ranges from changes in liver function cirrhosis and death (Russel, 2000); hyper-vitaminosis A leads to skin, bone and nail abnormalities and blood dyscrasisas (Food standards agency, 2003); benign intracranial hypertension (Dhiravibulya, 1991; Visani, 1996); # INR (Watanabe et al, 1997b)
Benign intracranial hypertension in combination with tetracyclines (Walters and Gubbay, 1981; Dhiravibulya 1991); anticoagulants (Watanabe, 1997); retinoids # toxicity risk (NMCD, 2004); caution with drugs known for their potential hepatotoxicity
$-Carotene
AO
Dietary: " risk of premenopausal breast cancer (Zhang et al, 1999), prostate cancer (Cook et al, 1999), colon cancer (Nkondjock and Ghadirian, 2004), ovarian cancer (Cramer et al, 2001) Supplement: " risk of gastric cancer in combination with other antioxidants in malnourished
# risk of lung and prostate cancer in smokers (The %Tocopherol, $ Carotene Cancer Prevention Study Group 1994; Heinonen et al, 1998; Patrick 2000); potential # recurrence of colorectal adenomas in people who smoke or drink alcohol. (Druginfozone, 2003)
Potential " effect of simvastatin / niacin combination when used with a selenium / $carotene / vitamins C and E supplement (Brown et al, 2001); statins: potential " effect when used with $carotene (NMCD, 2004)
483
Werneke et al: Complementary alternative medicine for cancer populations (Blot et al, 1999) Vitamin C (Ascorbic acid)
AO
Dietary (200 mg daily): " risk of oral, oesophageal, stomach, colon and lung cancer (Levine 1999; Zhang et al, 1999) Supplement: no benefits (Moertel et al, 1985)
Gastro-intestinal problems with high doses (Food Standards Agency, 2003): 1000 mg or more daily can cause renal problems including hematuria due to oxalate accumulation; mega-doses associated with deep vein thrombosis
Vitamin E
AO
" risk of prostate cancer and prostate cancer mortality; alone or in combination with $carotene (Heinonen et al, 1998); " bladder cancer mortality with long term use (Jacobs et al, 2002); " risk of gastrooesophageal cancer in combination with selenium and $-carotene (Wang et al, 1994);
High doses risk of bleeding due to antagonism of vitamin K dependent clotting factors
" effect / plasma level of heparin, statins, aluminium protease inhibitors and warfarin; # effect/ plasma level of acetaminophen, salicylates, dubutamin (NMCD, 2004); high dose: acute renal failure in combination with methotrexate.; also # methootrexate plasma levels (Sketris, 1984) " of cisplatin induced neurotoxicity (Pace et al, 2003); topical treatment of anthracycline or mitomycin extravasation in combination with dimethylsulfoxide (Ludwig et al, 1987); anticoagulants, statins (Brown et al, 2001); " nitrate tolerance (Watanabe et al, 1997a)
" risk of colorectal cancer in combination with multivitamins (White et al, 1997)
Table 4B. Antioxidants: other substances Remedy
Postulated mechanism of action
Effectiveness
Potentially serious side effects
Potential drug interactions
Co-enzyme Q10 (Ubiquinone)
AO
None identified
" warfarin efficacy, antidiabetics and antihypertensives (NMCD 2004)
Green tea: catechins
AO: may activate multiple pathways (Hsu et al, 2003), including modification of mitogenic signals (Gouni-Berthold and Sachinidis, 2004)
Supplement:? advanced breast cancer in combination with surgery and other oxidants and omega 3 and omega 6 fatty acids (Lockwood et al, 1995) Possibly effective in prevention of various cancers including prostate (Klein and Thompson, 2004), bladder colorectal and pancreatic cancer (Ji et al, 1997); no clear evidence for prevention of breast (Suzuki et al, 2004) and stomach cancer (Borrelli et al, 2004); may protect against bladder cancer (Wakai et al, 2004), but one case-
CNS Stimulation in high doses (PDR, 2000)
Contains caffeine and interacts with other stimulating drugs; " warfarin efficacy (large amounts); # anti-platelet activity (NMCD, 2004); # theophylline levels (Sato et al, 1993); possible # lithium levels with abrupt caffeine withdrawal (Jefferson 1988); modulation of effects of doxorubicin Stammler and Volm 1997)
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Cancer Therapy Vol 2, page 485 control study found # risk of bladder cancer (Lu et al, 1999) Selenium
AO
Tomato: lycopene
AO: lycopene or combination of several constituents (Hwang and Bowen, 2002)
Tumeric: curcumin
AO: curcumin and related substances; inhibition of angiogenesis (Gao et al, 2003); inhibition of transcription (Aggarval et al, 2003)
" risk of lung, colorectal and prostate cancer (Clark et al, 1996, 1998);
Acute toxicity: nausea causes vomiting, nail changes irritability and weight loss;
" all cancer mortality (Clark et al, 1996)
chronic toxicity: resembles arsenic toxicity (NCMD, 2004).
Potential " risk of prostate cancer, effect dose dependent (Etminan et al, 2004); weak evidence for " risk of ovarian cancer (Cramer et al, 2001) and lung cancer (Arab et al, 2002) Preliminary evidence for cancer prevention and treatment (Aggarval et al, 2003); preliminary evidence for stabilization of colorectal cancer refractory to other treatments (Sharma et al, 2001)
Theoretically, cholinergic poisoning through high dietary intake possible (Krasowski et al, 1997)
E. Remedies with endocrine properties
Gall bladder contraction: do not use in patients with obstructed biliary drugs and gallstones (PDR, 2000)
Cf. $-carotene
# risk of bleeding when used with other anticoagulant / antiplatelet drugs (NMCD, 2004)
some effect on post-menopausal symptoms (Ernst and Huntley, 2003). Black cohosh may also inhibit proliferation of estrogen receptor positive and negative breast cancer cells (Einbond et al, 2004; Honstanska et al, 2004). There have been case reports of hepatotoxicity (Whiting et al, 2002). Evening primrose oil and its main constituent !linolenic acid has weak anti-estrogenic properties which may enhance the effect of tamoxifen (Ingram et al, 2002; Kenny et al, 2004). !-linolenic acid can also increase the toxicity of paclitaxel and vinorelbime in breast cancer (Menendez et al, 2002). The evidence of its effectiveness for mastalgia and post-menopausal symptom relief is inconclusive. It can decrease the effectiveness of antiepileptics including sodium valproate (Miller, 1989). !-linolenic acid is also found in other plants such as borage. Saw palmetto has androgenic and anti-estrogenic effects and is primarily used for the reduction of symptoms of benign prostate hypertrophy (PDR, 2002). It has a number of varied postulated drug interactions including anticoagulants, antiplatelet and estrogenic drugs. Saw-palmetto and evening primrose oil have coumarinic effects.
Remedies in this grouping are mostly concerned with effects on sex-steroids. Commonly used phyto-estrogens include dong quai, panax ginseng, licorice, red clover, soy and wild yam (Table 5A). Some of these may have anticarcinogenic activity, but most cancer patients may use them to decrease side effects of hormonal treatment such as long term treatment with tamoxifen and its postmenopausal symptoms. The evidence for reduction of such symptoms is inconclusive for most substances, although there is some preliminary evidence for the effectiveness of soy (Vincent and Fitzpatrick, 2000; Ernst and Huntley, 2004). All phytestrogens should be taken with caution in breast cancer, because they may stimulate the proliferation of estrogen receptor positive cancer cells (Abebe, 2002; Bodinet and Freudenstein, 2004). Naturally, they are likely to interact with all estrogenic drugs such as tamoxifen. Theoretically, all phyto-estrogens increase the thrombosis risk. On the other hand, some phyto-estogens such as dong quai, red clover and soy have coumarinic properties which decrease the INR (Abebe, 2002). Licorice and red clover inhibit CYP 3A4. Soy additionally affects other microsomal enzymes and can interact with corresponding substrates (Anderson et al, 2003; NMCD, 2004; Sparreboom, 2004). Other remedies with endocrine effects include black cohosh, evening primrose oil and saw palmetto (Table 5B). Black cohosh exerts its estrogenic effect through an unknown mechanism and may have
G. Psychoactive remedies In a recent study of CAM use among cancer patients, 32% used psychoactive remedies (Werneke et al, 2004a). Popular substances include anxiolytics and sedatives such as Bach flower remedies, kava kava and valerian (Table
485
Werneke et al: Complementary alternative medicine for cancer 6A). Bach flower remedies is a combination of 38 herbs. There is also a â&#x20AC;&#x153;rescue remedyâ&#x20AC;? version that contains five herbs. It has failed to show efficacy in a meta-analysis
(Ernst et al, 2002b). As this is similar to homeopathic formulations, the potential for side effects and drug interactions is low.
Table 5A. Remedies with endocrine properties: estrogen agonists Remedy
Postulated mechanism of ction
Effectiveness
Potentially serious side effects
Potential drug interactions
Dong Quai (Chinese Angelica)
Unclear, estrogenic properties suggested but not demonstrated (Oerter et al, 2003)
Not effective for reduction of hot flushes (Kronenberg and FughBergman, 2002)
NSAIDs, anticoagulants
Panax Ginseng: ginsenosides
Estrogen receptor agonist (Lee et al, 2003)
Not effective (North American Menopause Society, 2004);
May stimulate growth of breast cancer cells (Amato et al, 2002); # bleeding risk: antiplatelet (Abebe, 2002) and coumarinic activity (Heck et al, 2000) May stimulate growth of breast cancer cells (Amato et al, 2002; Lee et al, 2003); c.f. Table 7 Hyper-aldosteronism: hypokalemia and hypertension
CYP3A4 inhibition;
Licorice
Estrogen receptor agonist, but cytotoxic at higher doses (Maggiolini et al, 2002);
estrogenic activity may depend on formulation (Polan et al, 2004) In vitro evidence only
" testosterone levels in men, induction of apoptosis (Rafi et al, 2002) Red Clover
Soy (soya): genistein
Estrogen receptor agonist (Ernst and Huntley, 2003)
Estrogen agonist by itself, antagonist in the presence of estrogen (Ratna, 2002); antioxidant and antiangiogenic properties, may induce apoptosis (Sarkar and Li, 2003)
Augmentation of estrogenic effect (Aradhana, 1992)
hypokalemia: diuretics, digitalis and insulin; potentiation of corticosteroids, and negate the effect of antihypertensives (NMCD, 2004);
Inconclusive evidence for reduction of postmenopausal symptoms (Ernst and Huntley, 2003);
May induce cell proliferation in estrogen receptor positive breast cancer (Bodinet and Freudenstein, 2004);
preliminary evidence for effectiveness in the treatment of mastalgia, osteoporosis and benign prostatic hyperplasia (NMCD, 2004) Preliminary evidence for reduction of postmenopausal symptoms (Vincent and Fitzpatrick, 2000; Ernst and Huntley, 2004); possibly protective against breast cancer, however effect may be limited to women of Asian origin (Yamamoto et al, 2003);
# INR (Abebe, 2002)
possibly reduction of prostate cancer risk (Messina, 2003)
Wild Yam: diosgenin
C.f. table 7
No clinical evidence available
486
May induce cell proliferation in estrogen receptor positive breast cancer (Allred et al, 2004, Bodinet and Freudenstein, 2004); # risk of bladder cancer with high dietary intake (Sun et al, 2002); # risk of kidney stones in oxalate containing soy products (Massey et al, 2001); " INR (Cambria-Kiely, 2002) Picrotoxin like activity: seizures in overdose? (PDR, 2001)
CYP3A4 inhibition, anticoagulants, NSAIDs, contraceptives, tamoxifen (Abebe, 2002; NMCD, 2004)
NSAIDs, anticoagulants, estrogens (NMCD, 2004); " effect of tamoxifen (Jones et al, 2002); inhibition of CYP 1A, 2E1, 2A6, 2C9, 2D6, 3A4, 3A7 and UGT1A1, 2B15 (Sparreboom et al, 2004)
" effect of indomethacin (PDR, 2001); # effect of estrogen containing drugs (Aradhana, 1992)
Cancer Therapy Vol 2, page 487 Table 5B. Remedies with endocrine properties: other Remedy
Postulated mechanism of action
Effectiveness
Potentially serious side effects
Potential drug interactions
Black Cohosh
LH depression through binding to estrogen receptors and through an independent mechanism (LH) (Duker et al, 1991; Seidlova-Wuttke et al, 2003)
Possibly inhibits proliferation of estrogen receptor positive and negative breast cancer cells (Einbond et al, 2004; Honstanska et al, 2004); weak evidence for " postmenopausal symptoms (Ernst and Huntley, 2003); one RCT on hot flushes in breast cancer patients showed no effect (Jacobson et al, 2001) Cancer: only in vitro evidence available (Menendez et al, 2002)
Hepatotoxic (Whiting et al, 2002)
# effect of antihypertensives;
Evening primrose oil: !-linolenic acid
Cancer: potentiation of cyto-toxicity of paclitaxel and vinorelbime in breast cancer (Menendez et al, 2002); weak antiestrogenic properties may enhance the response to tamoxifen (Ingram et al, 2002; Kenny et al, 2004) Mastalgia: !-linolenic acid (GLA) is metabolized to dihomo!-linolenic acid (DGLA): a precursor of the prostaglandin-E1 (PGE1), which is inflammatory (Darlington and Stone, 2001) Menopause: as above
Saw palmetto
Inhibition of testosterone conversion to DHT (Prager et al, 2002); inhibition of DHT binding to the cytosolic DHT androgenic receptor and the %1adrenoreceptor in the prostate thereby inhibiting hyperplasia (Goepel, 1999), not confirmed in vivo (Goepel et al, 2001);
# toxicity of other hepatotoxic drugs such as acetaminophen (PDR, 2000; NMCD, 2004)
" seizure threshold; (Miller, 1989); # INR (NMCD, 2004); associated with obstetric complications (Dove and Johnson, 1999)
" effectiveness of antiepileptic medication, e.g. sodium valproate (Miller, 1989); anticoagulants; in vitro inhibition of CYP1A2, 2C9, 2C19, 2D6 and 3A4 (Sparreboom et al, 2004)
Mastalgia: evidence inconclusive (Gateley et al, 1992; Blommers et al, 2002; Ingram et al, 2002)
Menopause: inconclusive, reduction of episodes of night time flushing (Chenoy et al, 1994) Effective for symptom relief in benign prostate hypotrophy but may not reduce enlargement (PDR, 2002)
inhibition of cell growth in prostate cancer (Goldmann et al, 2001); antiestrogenic effect (PDR, 2000)
487
One case report of cholestatic hepatitis (NMCD, 2004)
" effectiveness of estrogens and oral contraceptives; # risk of bleeding when used with other anticoagulant antiplatelet drugs (NMCD, 2004)
Werneke et al: Complementary alternative medicine for cancer Valerian is a hypnotic, but evidence on its effectiveness remains inconclusive although a mechanism of action had been postulated (Stevinson and Ernst, 1999). Valerian inhibits CYP 3A4 and can potentiate the effect of other sedatives. Kava kava is an effective anxiolytic, but has been withdrawn from the UK market due to concerns about hepatotoxicity. Panax ginseng and ginkgo are popular cognitive enhancers (Table 6B). Both remedies have effects on the cerebral blood flow (Maclennan et al, 2002; Ahlemeyer and Kriegelstein 2003). They also have cholinergic properties (Tang et al, 2002), which are known to improve cognition (Lewis et al, 1999; Tang et al, 2002). They both interact with anti-thrombotic drugs and have effects on the CYP450 system. Ginkgo additionally may interfere with the p-glycoprotein pump (Sparreboom, 2004). St John’s wort is used as an antidepressant, however its efficacy may be limited to mild depressive episodes. Some recent larger studies including more severely depressed patients have not shown any effect (Werneke et al, 2004b). Patients taking St John’s wort should be reminded not to take the extract with other serotonergic antidepressants. Also, St John’s wort is an
inducer of CYP 3A4 and the p-glycoprotein pump. This can make pro-drugs, e.g. cyclophosphamide and ifosfamide, which are converted into the active forms through CYP3A4, more effective. Other chemotherapies, such as paclitaxel and epipodophyllotoxins may become less effective.
H. Other remedies used by cancer patients Clearly, patients may take many other CAMs, and it would be impossible to discuss all. For instance, in our recent study, 164 patients took 133 combinations of remedies (Werneke et al, 2004a). Here we discuss four CAMs, which are commonly used: cod liver oil, ginger, kelp and milk thistle. Cod liver oil is used for many indications including arthritis and depression. For arthritis, effectiveness has been demonstrated in several trials (Gruenewald et al, 2002; Curtiz et al, 2004) no added benefit, however, in conjunction with NSAIDs (Stammers et al, 1992).
Table 6A. Psychotropic substances: antidepressants and sedatives Remedy
Postulated mechanism of action
Effectiveness
Potentially serious side effects
Potential drug interactions
Bach Flower Remedies
38 herbs with postulated differential effects, rescue remedies for acute stress contain five different herbs
Not effective (Ernst, 2002b)
Unclear
Unclear
Kava Kava: kava lactones / kava pyrones
Sedative / anxiolytic
Meta-analysis of nine studies showed significant reduction of Hamilton anxiety score (Pittler and Ernst, 2003)
At least 68 cases of liver toxicity (NMCD, 2004); one case report of movement disorder (Meseguer et al, 2002)
CYP1A2, 2C9, 2C19, 2D6 and 3A4 inhibition (Mathews et al, 2002), CNS depressants and hepatotoxic drugs (Russmann et al, 2001)
St John’s wort: hyperforin
MAOI inhibition and GABAergic activity (Cott, 1997), monoamine re-uptake (Perovic and Muller, 1995; Neary and Bu, 1999), up-regulation of 5HT1A and 5HT2A receptors (Teufer-Mayer and Gleitz, 1997); modulation of cytokine production (Thiele et al, 1994)
Four meta-analyses with trend toward reduced effect size, possibly effective in mild depression (Werneke et al, 2004b)
Similar to serotonergic antidepressants; photosensitivity (Whiskey et al, 2001)
Serotonergic antidepressants;
GABAergic effects (Houghton, 1999)
Inconclusive evidence (Stevinson and Ernst, 1999)
Cognitive impairment and drowsiness; case reports of hepatotoxicity (Klepser and Klepser, 1999)
In vitro inhibition of CYP 2C19, 2D6 and 3A4 (Srandell et al, 2004), however clinical significance unclear (Donovamn et al, 2004); # effect of sedatives (NMCD, 2004):
Valerian
GABAergic effects (Jussofie, 1994; Dinh et al, 2001), D2 antagonist (Shelosky et al, 1995)
488
CYP 1A2, 2C9, 2C19, 2D6 3A4 and induction of p-glycoprotein pump (Peebles et al, 2001; Mannel 2004)
Cancer Therapy Vol 2, page 489 Table 6B. Psychotropic substances: cognitive enhancers Remedy
Postulated mechanism of action
Effectiveness
Potentially serious side effects
Potential drug interactions
Ginkgo Biloba
Cognitive enhancer antioxidant (Oken et al, 1998, Tabet et al, 2000);
No consistent positive effect on cognitive performance in healthy individuals shown (Canter and Ernst, 2002); possible improvement of cognitive function in patients with organic cognitive decline (Birks et al, 2002)
# bleeding time, case reports of intracerebral haemorrhage (Matthews, 1998; Benjamin 2001); possibly adverse effects on male and female fertility (Ondrizek, 1999); one case report of Stevens- Johnsons syndrome possibly attributed to Ginkgo (NMCD, 2004)
Antithrombolytic agents including warfarin (PDR, 2000); evidence for effects on CYP1A2, 2C9, 2D6 and 3A4 inconsistent: CYP2C19: inhibition (Sparreboom, 2004);
Improvement of mental arithmetic and abstraction; agedelaying properties unproven (Vogeler et al, 1999)
Insomnia, mania, hyperand hypotension, # vaginal bleeding (PDR, 2000);
Insulin and oral hyperglycemics, antithrombolytic agents including warfarin, MAOIs (phenelzine), loop diuretics, immunosuppressants (NMCD, 2004) (PDR, 2000);
# cerebral blood flow through platelet activation factor inhibition and nitric oxide pathways; (Maclennan et al, 2002) (Ahlemeyer and Kriegelstein, 2003); cholinergic effects (Tang et al, 2002)
Panax Ginseng: ginsenosides
Interference with platelet aggregation and coagulation (PDR, 2000); neuroprotection through nicotinic activity (Lewis et al, 1999); antioxidant effects (Chan and Tomlinson, 2000); possibly immunostimulating and antcarcinogenic activity (Xiaoguang et al, 1999; Shin et al, 2000); weak phyto-estrogenic effects (Lee, 1998)
Stevens-Johnsons syndrome (NMCD, 2004)
CYP2D6: potentially of no clinical significance (Markowitz et al, 2003); CYP3A4: either inhibition (Ohnishi et al, 2003; He and Edeki, 2004) or induction (Sugiyama et al, 2004); or of no clinical significance (Markowitz et al, 2003); potential interference with the p-glycoprotein pump (Sparreboom et al, 2004)
inhibition of CYP 3A4 may be extract specific (Sparreboom et al, 2004); in vitro CYP 3A4 induction without clinical correlate reported (Anderson et al, 2003); in vitro but no clinical evidence for inhibition of CYP 2C9, 2C19 and 2D6 (Henderson et al, 1999)
Table 7. Other remedies used by cancer patients Substance
Postulated mechanism of action
Effectiveness
Potentially serious side effects
Potential drug interactions
Cod liver oil
Cardiac: " triglycerides and, " VLDLs and # HDL (Jensen et al, 1989)
" triglycerides and hypertension demonstrated in RCTs (Vessby and Boberg, 1990; Toft et al, 1995; Dyerberg et al 2004)
# INR with high or changing doses (FughBergman, 2000);
# effect of warfarin, aspirin and non-steroidal anti-inflammatory drugs (Fugh-Bergman, 2000)
Arthritis: suppression of cytokines? (Darlington
Arthritis: effectiveness
489
contamination possible,e.g. with dioxin (Alvarez, 1991); # of blood sugar levels and insulin resistance
Werneke et al: Complementary alternative medicine for cancer and Stone, 2001)
demonstrated in several trials (Curtis et al, 2004, Gruenewald et al, 2002); no benefit in conjunction with NSAIDs (Stammers et al, 1992)
Depression: influences catecholaminergic serotonergic and cholinergic neurotransmission, modulation of signal transmission mechanisms in neuronal membranes, modulation of prostaglandins and ion channels (Haag, 2003)
Depression: results of small trials with short endpoints inconclusive (Marangell et al, 2003; Su et al, 2003); possibly effective when added to lithium in bipolar affective disorder (Bowden 2001)
Ginger: gingerols, gingeridone
Antiemetic, mechanism of action unclear, possibly centrally acting on serotonin receptors (Ernst and Pittler, 2000)
Nausea and vomiting including chemotherapy induced nausea (Ernst and Pittler, 2000)
Contraindicated in pregnancy (PDR, 2000); well tolerated in therapeutic doses; cardiac arrhythmias associated with large doses (NMCD, 2004)
Theoretically with antiulcer drugs (including H2-antagonists, protone pump inhibitors and antacids), anticoagulants, antihypertensives, antidiabetics and barbiturates (NMCD, 2004)
Kelp
Constituent of anticancer diets, mechanism of action unclear, possibly antioxidant (Maruyama et al, 1991)
Insufficient evidence
Hypo-and hyperthyroidism (Konno et al, 1994; Henzen et al, 1999)
# risk of hyperkalemia with potassium, supplements, potassium sparing diuretics, and ACE inhibitors; digoxin toxicity may be potentiated due to hyperkalaemia; thyroid hormones (NMCD, 2004)
Milk thistle: silymarin
Liver tonic, antioxidant, inhibitor of tumor necrosis factor (TNF) (NMCD, 2004);
In vitro evidence only
Usually well tolerated, mild laxative (NMCD, 2004)
CYP 2C9, 2D6, 2E1 and 3A4 inhibition, but clinical relevance unclear (Zuber et al, 2002); may affect the elimination of drugs which undergo glucuronidation as part of their metabolism (Kivisto et al, 1995; NMCD, 2004)
cell cycle arrest and apoptosis in human bladder transitional cell carcinoma (Tyagi et al, 2004); inhibition of telomerase activity and secretion of prostate specific antigen in prostate cancer cells (Thelen et al, 2004)
For depression, the evidence currently remains inconclusive (Marangell et al, 2003; Su et al, 2003). Cod liver oil can increase INR and interact with non-steroidal anti-inflammatory drugs and warfarin. Also, occasionally individual products may be contaminated. Ginger is
possible (Vessby and Boberg, 1990)
effective in chemotherapy induced nausea (Ernst and Pittler, 2000). Theoretically, it can interact with a variety of drugs including anticoagulants, and the PDR 2001 for herbal medicines suggests that ginger should be contraindicated in pregnancy, possibly due to an increased
490
Cancer Therapy Vol 2, page 491 bleeding risk. Kelp comprises various species such as laminaria and bladderwreck. It is a common product of anticancer diets, particularly macrobiotic diets. There is insufficient evidence for effectiveness, but possibly kelp acts as an antioxidant. Kelp can change thyroid function because it contains iodine (Konno et al, 1994; Henzen et al, 1999). In the case of radiotherapy for differentiated thyroid cancer, radioiodine uptake can be altered by exogenous iodine (Werneke and McCready, 2004). Kelp can contain potassium and lead to hyperkalemia in predisposed patients. Finally, milk thistle is used as a liver tonic. It also has antioxidant and pro-apoptotic properties (Tyagi et al, 2004). Milk thistle is generally well tolerated but can inhibit CYP 2C9 and 3A4.
may be possible to discontinue remedies during chemotherapy cycles or diagnostic procedures, but resume
APPENDIX 1 List of organic remedies and their scientific names Common name Baikal scullcap Black cohosh (black snakeroot) Blessed thistle Burdock Blistering ammania
IV. Discussion
Chinese sarsaparilla (Chobchini) Cat’s claw
We have attempted to review CAMs which cancer patients may be likely to use. There are many more remedies we could have considered, but we decided to limit our review to those remedies frequently discussed in the literature or come across in our own clinical work or in discussions with colleagues. In this article, we give an overview summarizing available evidence. Each remedy would warrant a review on its own, and that is why the list of side effects and interactions is not exhaustive. Our review highlights that many side effects and interactions are based on hypotheses about pharmacokinetic and pharmacodynamic properties, frequently only demonstrated in vitro. Also, effects may vary between different extracts of the same remedy, since products are usually not standardized, and there is no universal quality control. It is possible that in vitro effects will never assume clinical relevance and remain speculative. Conversely, adverse effects may emerge over time in remedies which have previously judged harmless. Thus, currently, much advice to patients taking CAM depends on the plausibility of hypothesized effects and side effects. Prospective systematic surveillance of CAMs is required to improve the evidence base. Such studies may be complicated by the fact that many patients take combinations of remedies rather than one single substance, so that it will not always be possible to attribute an adverse reaction unambiguously to one agent (Werneke et al, 2004c). Meanwhile, the use of CAMs with potentially serious interactions or a significant reduction in efficacy of the conventional treatment, should not be encouraged.In summary, clinicians and pharmacists need to be aware of CAM-induced side effects or interactions and should be able to identify hazards, advising patients accordingly and avoiding uncritical encouragement of potentially harmful use. Ignorance in this area, given the independent usage of CAMs, may lead to criticism and possibly litigation (Cohen and Eisenberg, 2002). Equally, patients should be encouraged to disclose information about CAMs to health care professionals. Such discussions need to be conducted sensitively in order to avoid alienating patients who may feel that they have not been taken seriously or have been criticised for using CAM. Also, even if remedies interact with conventional therapies, CAMs do not always have to be stopped. Often, it may suffice to monitor patients more frequently or adjust the doses of conventional drugs. It
Cubebs Crysantmum Dong quai Dyer’s woad Echinacea Garden cress Ginger Ginkgo Goldenseal Grape seed Green tea Himalayan rhubarb Indian sarsaparilla Kava Kava Kelp Laetrile (Apricot) Licorice Milk thistle Mistletoe Panax ginseng Panax pseudoginseng (SanQui ginseng) Pau d’arco Puncture vine Rabdosia Red clover Reishi Rhubarb Saw palmetto Shark cartilage Sheep sorrel (Yellow dock) Slippery elm Soy (Soya) St John’s wort Thyme leaved gratiola Tomato Tumeric Watercress Wild yam
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Scientific name Scutellaria baicalensis Actaea racemosa (Cimicifuga racemosa) Cnicus benedictus Arcticum lappa Ammani vesicatoria (Ammani bacciferra L.) Smilex china L. Uncaria tomentosa (Uncaria guianensi) Piper cubeba Dendranthema morifolium Angelica siniensis Isatis indigotica Echinacea angustifolia / Echinacea purpurea Lepidium sativum Zingiber officinale Ginkgo biloba Hydrastis canadensis Vitis vinifera (Vitis coignetiae) Camelia siniensis Rheumemodi Wall Hemidesmus indicus Piper methysticum. Laminaria digitata / Laminaria japonica Prunus armeniaca Glycyrrhiza uralensis (Glycyrrhiza glabra) Silybum marianum Viscum album Panax ginseng Panax pseudoginseng (Panax notoginseng) Tabebuia impetiginosa Tribulus terrestris Rabdosia rubescens Trifolium pratense Ganoderma lucidum Rheum officinale Serenoa repens Squalus acanthias Rumex crispus Ulmus rubra (Ulmus fulva) Glycine max (Glycine soja) Hypericum perforatum Blepharis edulis Lycopersicon esculentum Curcuma longa (Curcuma domestica) Naturstium officinale Dioscorea villosa (Dioscorea composite)
Werneke et al: Complementary alternative medicine for cancer Bjorkhem-Bergman L, Jonsson K, Eriksson LC, Olsson JM, Lehmann S, Paul C, Bjornstedt M (2004) Drug-resistant human lung cancer cells are more sensitive to selenium cytotoxicity. Effects on thioredoxin reductase and glutathione reductase. J Pharm Pharmacol 56, 1001-1005. Blommers J, de Lange-De Klerk ES, Kuik DJ, Bezemer PD, Meijer S (2002) Evening primrose oil and fish oil for severe chronic astalgia: a randomized, double-blind, controlled trial. Am J Obstet Gynecol 187, 1389-1394. Blot WJ, Li JY, Taylor PR (1993) Nutritional intervention trials in Linxian, China: supplementation with specific vitamin/mineral combinations, cancer incidence, and diseasespecific mortality in the general population. J Natl Cancer Inst 85, 1483-1492. Bodinet C, Freudenstein J (2004) Influence of marketed herbal menopause preparations on MCF-7 cell proliferation. Menopause. 11, 281-289. Borrelli F, Capasso R, Russo A, Ernst E (2004) Systematic review: green tea and gastrointestinal cancer risk. Aliment Pharmacol Ther 19, 497-510. Botanical Dermatology Database (BoDD) (2004) http:// www.bodd.cf.ac.uk. Bowden CL (2001) Novel treatments for bipolar disorder. Expert Opin Investig Drugs 10, 661-671. Brown BG, Zhao XQ, Chait A, Fisher LD, Cheung MC, Morse JS, Dowdy AA, Marino EK, Bolson EL, Alaupovic P, Frohlich J, Albers JJ (2001) Simvastatin and niacin, antioxidant vitamins, or the combination for the prevention of coronary disease. N Engl J Med 29, 1583-1592. Budzinski JW, Foster BC, Vandenhoek S, Arnason JT (2000) An in vitro evaluation of human cytochrome P450 3A4 inhibition by selected commercial herbal extracts and tinctures. Phytomedicine 7, 273-282. Bukowski RM (2003) AE-941, a multifunctional antiangiogenic compound: trials in renal cell carcinoma. Expert Opin Investig Drugs 12, 1403-1411. Cambria-Kiely JA (2002) Effect of soy milk on warfarin efficacy. Ann Pharmacother 36, 1893-1896. Canter PH, Ernst E (2002) A systematic review of controlled trials of the cognitive effects of ginkgo biloba extracts in healthy people. Psychopharmacology Bulletin 36, 108-123. CDC (1995) Unexplained severe illness possibly associated with consumption of Kombucha tea-Iowa, 1995. MMWR 44, 892-893, 899-900. Chabner BA and Longo DLI eds (2001) Cancer chemotherapy and Biotherapy. 3rd edition. Lippincott Williams and Wilkins, http://www.lww.com. Chan P, Tomlinson B (2000) Antioxidant effects of Chinese traditional medicine: focus on trilinolein isolated from the Chinese herb sanchi (Panax pseudoginseng). J Clin Pharmacol 40, 457-461. Chang TK, Yu L, Maurel P, Waxman DJ (1997) Enhanced cyclophosphamide and ifosfamide activation in primary human hepatocyte cultures: response to cytochrome P-450 inducers and autoinduction by oxazaphosphorines Cancer Res 57, 1946-1954. Chenoy R, Hussain S, Tayob Y, O'Brien PM, Moss MY, Morse PF (1994) Effect of oral gamolenic acid from evening primrose oil on menopausal flushing. BMJ 308, 501-503. Cheung NK, Modak S (2002) Oral (1-->3),(1-->4)-$-D-glucan synergizes with antiganglioside GD2 monoclonal antibody 3F8 in the therapy of neuroblastoma. Clin Cancer Res 8, 1217-1223. Chiao JW, Wu H, Ramaswamy G, Conaway CC, Chung FL, Wang L, Liu D (2004) Ingestion of an isothiocyanate metabolite from cruciferous vegetables inhibits growth of human prostate cancer cell xenografts by apoptosis and cell cycle arrest. Carcinogenesis 25, 1403-1408.
their use thereafter. In some cases, it may be better to continue the remedy. For instance, if chemotherapies are based on glomerular filtration rate (GFR) measurements, depending on the remedy, it may be advisable that the patients do not alter their pattern of intake after their GFR has been measured (Werneke and Mc Cready, 2004). Consultations about CAMs can be complex and may demand more time than is available in routine clinics. Service models need to be designed and tested to meet this challenge, with consideration even being given to specialist clinics providing regular updated advice to clinicians, pharmacists and patients.
Acknowledgements We would like to thank Don Albert who kindly provided the image of St Johnâ&#x20AC;&#x2122;s wort.
References Abebe W (2002) Herbal medication: potential for adverse interactions with analgesic drugs. J Clin Pharm Ther 27, 391-401. Aggarwal BB, Kumar A, Bharti AC (2003) Anticancer potential of curcumin: preclinical and clinical studies. Anticancer Res 23, 363-398. Ahlemeyer B, Krieglstein J (2003) Neuroprotective effects of Ginkgo biloba extract. Cell Mol Life Sci 60, 1779-92. Allred CD, Allred KF, Ju YH, Goeppinger TS, Doerge DR, Helferich WG (2004) Soy processing influences growth of estrogen-dependent breast cancer tumors in mice. Carcinogenesis 25, 1649-1657. Alvarez R (1991) Standard Reference Materials for dioxins and other environmental pollutants. Sci Total Environ 104, 1-7. Amato P, Christophe S, Mellon PL (2002) Estrogenic activity of herbs commonly used as remedies for menopausal symptoms. Menopause 9, 145-150. Anderson GD, Rosito G, Mohustsy MA, Elmer GW (2003) Drug interaction potential of soy extract and Panax ginseng. J Clin Pharmacol 43, 643-648. Arab L, Steck-Scott S, Fleishauer AT (2002) Lycopene and the lung. Exp Biol Med (Maywood) 227, 894-899. Aradhana, Rao AR, Kale RK (1992) Diosgenin--a growth stimulator of mammary gland of ovariectomized mouse. Indian J Exp Biol 30, 367-370. Ashar B, Vargo E (1996) Shark cartilage-induced hepatitis. Ann Intern Med 125, 780-1. Ashikaga T, Bosompra K, Oâ&#x20AC;&#x2122;Brian P, Nelson L (2002) Use of complementary and alternative medicine by breast cancer patients: prevalence, patterns and communication with physicians. Support Care Care 10, 542-548. Barrett S (1999) Fanciful claims for 714X. http://www.quackwatch.org. Benjamin J, Muir T, Briggs K, Pentland B (2001) A case of cerebral haemorrhage-can Ginkgo biloba be implicated? Postgrad Med J 77, 112-113. Bernstein BJ, Grasso T (2001) Prevalence of complementary and alternative medicine use in cancer patients. Oncology (Huntingt) 15, 1267-1272. Birks J, Grimley Evans J, Van Dongen M. (2003) Ginkgo Biloba for Cognitive Impairment and Dementia (Cochrane Review). In: The Cochrane Library, Issue 4, Chichester, UK: John Wiley and Sons, Ltd.
492
Cancer Therapy Vol 2, page 493 Choi BT, Cheong J, Choi YH (2003) $-Lapachone-induced apoptosis is associated with activation of caspase-3 and inactivation of NF-kappaB in human colon cancer HCT-116 cells. Anticancer Drugs 14, 845-850. Clark LC, Combs GF Jr, Turnbull BW, Slate EH, Chalker DK, Chow J, Davis LS, Glover RA, Graham GF, Gross EG, Krongrad A, Lesher JL Jr, Park HK, Sanders BB Jr, Smith CL, Taylor JR (1996) Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin. A randomized controlled trial. JAMA 276, 1957-1963. Clark LC, Dalkin B, Krongrad A, Combs GF Jr, Turnbull BW, Slate EH, Witherington R, Herlong JH, Janosko E, Carpenter D, Borosso C, Falk S, Rounder J (1998) Decreased incidence of prostate cancer with selenium supplementation: results of a double-blind cancer prevention trial. Br J Urol 81, 730734. Cohen MH and Eisenberg DM (2002) Potential physician malpractice liability associated with complementary and integrative medicinal therapies. Ann Intern Med 136, 596603. Cook NR, Stampfer MJ, Ma J, Manson JE, Sacks FM, Buring JE, Hennekens CH (1999) $-carotene supplementation for patients with low baseline levels and decreased risks of total and prostate carcinoma. Cancer 86, 1783-1792. Cott, JM (1997) In vitro receptor binding and enzyme inhibition by hypericum perforatum extract. Pharmacopsychiatry 30, 108-112. Covington TR (ed) (1996) Handbook of Nonprescription Drugs. 11th ed. Washington, DC: Am Pharmaceutical Association. Cramer DW, Kuper H, Harlow BL, Titus-Ernstoff L (2001) Carotenoids, antioxidants and ovarian cancer risk in pre- and postmenopausal women. Int J Cancer 94, 128-314. Curtis CL, Harwood JL, Dent CM, Caterson B (2004) Biological basis for the benefit of nutraceutical supplementation in arthritis. Drug Discov Today 9, 165-172. Darlington LG, Stone TW (2001) Antioxidants and fatty acids in the amelioration of rheumatoid arthritis and related disorders. Br J Nutr 85, 251-269. Dhiravibulya K, Ouvrier R, Johnston I, Procopis P, Antony J (1991) Benign intracranial hypertension in childhood: a review of 23 patients J Paediatr Child Health 27, 304-307. Dinh LD, Simmen U, Bueter KB, Bueter B, Lundstrom K, Schaffner W (2001) Interaction of various Piper methysticum cultivars with CNS receptors in vitro. Planta Med 67, 306311. Donovan JL, DeVane CL, Chavin KD, Wang JS, Gibson BB, Gefroh HA, Markowitz JS (2004) Multiple Nighttime Doses of Valerian (Valeriana officinalis) had Minimal Effects on CYP 3A4 activity and No Effect on CYP 2D6 Activity in Healthy Volunteers. Drug Metab Dispos [Epub ahead of print]. Dove D, Johnson P (1999) Oral evening primrose oil: its effect on length of pregnancy and selected intrapartum outcomes in low-risk nulliparous women. J Nurse Midwifery 44, 320324. Druginfozone (2003) Wednesday 21st May 2003. http://druginfozone.nhs.uk. Dubin M, Fernandez Villamil SH, Stoppani AO (2001) Cytotoxicity of $-lapachone, an naphthoquinone with possible therapeutic use. Medicina (B Aires) 61, 343-350. Dudley JP (1983) Bilateral pneumothorax resulting from the bronchoscopic removal of a puncture vine fruit. Ann Otol Rhinol Laryngol 92, 396-397. Duker EM, Kopanski L, Jarry H, Wuttke W (1991) Effects of extracts from Cimicifuga racemosa on gonadotropin release in menopausal women and ovariectomized rats. Planta Med 57, 420-424.
Dyerberg J, Eskesen DC, Andersen PW, Astrup A, Buemann B, Christensen JH, Clausen P, Rasmussen BF, Schmidt EB, Tholstrup T, Toft E, Toubro S, Stender S (2004) Effects of trans- and n-3 unsaturated fatty acids on cardiovascular risk markers in healthy males. An 8 weeks dietary intervention study. Eur J Clin Nutr 58, 1062-1070. Einbond LS, Shimizu M, Xiao D, Nuntanakorn P, Lim JT, Suzui M, Seter C, Pertel T, Kennelly EJ, Kronenberg F, Weinstein IB (2004) Growth inhibitory activity of extracts and purified components of black cohosh on human breast cancer cells. Breast Cancer Res Treat 83, 221-231. Eisenberg DM, Kessler RC, Foster C, Norlock FE, Calkins DR, Delbanco TL (1993) Unconventional medicine in the United States. Prevalence, costs and pattern of use. N Engl J Med 328, 246-252. Eng ET, Ye J, Williams D, Phung S, Moore RE, Young MK, Gruntmanis U, Braunstein G, Chen S (2003) Suppression of estrogen biosynthesis by procyanidin dimers in red wine and grape seeds. Cancer Res 63, 8516-8522. Ernst E (1997) Thymus therapy for cancer? A criteria-based, systematic review. Eur J Cancer 33, 531-535. Ernst E (2002a) The risk-benefit profile of commonly used herbal therapies: Ginkgo, St. John's Wort, Ginseng, Echinacea, Saw Palmetto, and Kava. Ann Intern Med 136, 42-53. Ernst E (2002b) "Flower remedies": a systematic review of the clinical evidence. Wien Klin Wochenschr 30, 963-966. Ernst E (2003) Kombucha: a systematic review of the clinical evidence. Forsch Komplementarmed Klass Naturheilkd 10, 85-87. Ernst E, Cassileth BR (1998) The prevalence of complementary/alternative medicine in cancer: a systematic review. Cancer 83, 777-782. Ernst E, Cassileth BR (1999) How useful are unconventional cancer treatments? Eur J Cancer 35, 1608-1613. Ernst E, Pittler MH (2000) Efficacy of ginger for nausea and vomiting: a systematic review of randomized clinical trials. Br J Anaesth 84, 367-371. Ernst E, Schmidt K, Steuer-Vogt MK (2003) Mistletoe for cancer? A systematic review of randomised clinical trials. Int J Cancer 107, 262-267. Etminan M, Takkouche B, Caamano-Isorna F (2004) The role of tomato products and lycopene in the prevention of prostate cancer: a meta-analysis of observational studies. Cancer Epidemiol Biomarkers Prev 13, 340-345. Finkelstein MP, Aynehchi S, Samadi AA, Drinis S, Choudhury MS, Tazaki H, Konno S (2002) Chemosensitization of carmustine with maitake $-glucan on androgen-independent prostatic cancer cells: involvement of glyoxalase I. J Altern Complement Med 8, 573-580. Food Standards Agency: Expert Group on Vitamins and Minerals: Safe upper levels for vitamins and Minerals. May 2003. http://www.foodstandards.gov.uk. Foster BC, Vandenhoek S, Hana J, Krantis A, Akhtar MH, Bryan M, Budzinski JW, Ramputh A, Arnason JT (2003) An in vitro inhibition of human cytochrome P450 mediated metabolism of marker substrates by natural products. Phytomedicine 10, 334-342. Foster S, Tyler VE (1993) Tyler's Honest Herbal: A Sensible Guide to the Use of Herbs and Related Remedies. 3rd ed., Binghamton, NY: Haworth Herbal Press. Fugh-Berman (2000) Herb-drug interactions. Lancet 355, 134138. Fullerton SA, Samadi AA, Tortorelis DG, Choudhury MS, Mallouh C, Tazaki H, Konno S (2000) Induction of apoptosis in human prostatic cancer cells with $-glucan (Maitake mushroom polysaccharide). Mol Urol 4, 7-13.
493
Werneke et al: Complementary alternative medicine for cancer Gamundi R, Valdivia M (1995) The Kombucha mushroom: two different opinions Sidahora Oct-Nov, 34-35. Gao C, Ding Z, Liang B, Chen N, Cheng D (2003) Study on the effects of curcumin on angiogenesis Zhong Yao Cai 26, 499-502. Gateley CA, Miers M, Mansel RE, Hughes LE (1992) Drug treatments for mastalgia: 17 years experience in the Cardiff Mastalgia Clinic. J R Soc Med 85, 12-15. Ghadirian P, Lacroix A, Maisonneuve P, Perret C, Potvin C, Gravel D, Bernard D, Boyle P (1997) Nutritional factors and colon carcinoma: a case-control study involving French Canadians in Montreal, Quebec, Canada. Cancer 80, 858864. Gingras D, Boivin D, Deckers C, Gendron S, Barthomeuf C, Beliveau R (2003) Neovastat--a novel antiangiogenic drug for cancer therapy. Anticancer Drugs 14, 91-96. Globalherbsupplies (2004) http://www.globalherbalsupplies.com /herb_information/hemidesmus_indicus.htm. Goel V, Lovlin R, Barton R, Lyon MR, Bauer R, Lee TD, Basu TK (2004) Efficacy of a standardized echinacea preparation (Echinilin) for the treatment of the common cold: a randomized, double-blind, placebo-controlled trial. J Clin Pharm Ther 29, 75-83. Goepel M, Dinh L, Mitchell A, Schafers RF, Rubben H, Michel MC (2001) Do saw palmetto extracts block human %1adrenoceptor subtypes in vivo? Prostate 46, 226-232. Goepel M, Hecker U, Krege S, Rubben H, Michel MC (1999) Saw palmetto extracts potently and noncompetitively inhibit human %1-adrenoceptors in vitro. Prostate 38, 208-215. Goldmann WH, Sharma AL, Currier SJ, Johnston PD, Rana A, Sharma CP (2001) Saw palmetto berry extract inhibits cell growth and Cox-2 expression in prostatic cancer cells. Cell Biol Int 25, 1117-1124. Gonzalez RP, Leyva A, Moraes MO (2001) Shark cartilage as source of antiangiogenic compounds: from basic to clinical research. Biol Pharm Bull 24, 1097-1101. Gouni-Berthold I, Sachinidis A. (2004) Molecular mechanisms explaining the preventive effects of catechins on the development of proliferative diseases. Curr Pharm Des 10, 1261-1271. Gruenwald J, Graubaum HJ, Harde A (2002) Effect of cod liver oil on symptoms of rheumatoid arthritis. Adv Ther 19, 101107. Haag, M (2003) Essential fatty acids and the brain. Can J Psychiatry 48,195-203. Hauser SP (1990) Dr. Sklenar's Kombucha mushroom infusion-a biological cancer therapy. Documentation No. 18 Schweiz Rundsch Med Prax 79, 243-246. He N, Edeki T (2004) The inhibitory effects of herbal components on CYP2C9 and CYP3A4 catalytic activities in human liver microsomes. Am J Ther 11, 206-212. Hecht SS, Chung FL, Richie JP Jr, Akerkar SA, Borukhova A, Skowronski L, Carmella SG (1995) Effects of watercress consumption on metabolism of a tobacco-specific lung carcinogen in smokers. Cancer Epidemiol Biomarkers Prev 4, 877-884. Heck AM, DeWitt BA, Lukes AL (2000) Potential interactions between alternative therapies and warfarin. Am J Health Syst Pharm 57, 1221-1227 Heinonen OP, Albanes D, Virtamo J, Taylor PR, Huttunen JK, Hartman AM, Haapakoski J, Malila N, Rautalahti M, Ripatti S, Maenpaa H, Teerenhovi L, Koss L, Virolainen M, Edwards BK (1998) Prostate cancer and supplementation with %-tocopherol and $-carotene: incidence and mortality in a controlled trial. J Natl Cancer Inst 90, 440-446. Henderson GL, Harkey MR, Gershwin ME, Hackman RM, Stern JS, Stresser DM (1999). Effects of ginseng components on c-
DNA-expressed cytochrome P450 enzyme catalytic activity. Life Sci 65, PL209-PL214. Henzen C, Buess M, Brander L (1999) Iodine-induced hyperthyroidism (iodine-induced Basedow's disease): a current disease picture. Schweiz Med Wochenschr 129, 658-664. Ho PC, Saville DJ (2001) Inhibition of human CYP3A4 activity by grapefruit flavonoids, furanocoumarins and related compounds. J Pharm Pharmaceut Sci 4, 217-227. Hong F, Hansen RD, Yan J, Allendorf DJ, Baran JT, Ostroff GR, Ross GD (2003) $-glucan functions as an adjuvant for monoclonal antibody immunotherapy by recruiting tumoricidal granulocytes as killer cells. Cancer Res 63, 9023-9031. Hostanska K, Nisslein T, Freudenstein J, Reichling J, Saller R (2004) Cimicifuga racemosa extract inhibits proliferation of estrogen receptor-positive and negative human breast carcinoma cell lines by induction of apoptosis. Breast Cancer Res Treat 84, 151-160. Houghton PJ (1999) The scientific basis for the reputed activity of Valerian. J Pharm Pharmacol 51, 505-512. Hsu S, Yu FX, Huang Q, Lewis J, Singh B, Dickinson D, Borke J, Sharawy M, Wataha J, Yamamoto T, Osaki T, Schuster G (2003) A mechanism-based in vitro anticancer drug screening approach for phenolic phytochemicals. Assay Drug Dev Technol 1, 611-618. Hukkanen J, Mantyla M, Kangas L, Wirta P, Hakkola J, Paakki P, Evisalmi S, Pelkonen O, Raunio H (1998) Expression of cytochrome P450 genes encoding enzymes active in the metabolism of tamoxifen in human uterine endometrium. Pharmacol Toxicol 82, 93-97. Huntley AL, Ernst E (2003) A systematic review of herbal medicinal products for the treatment of menopausal symptoms. Menopause 10, 465-476. Huntley AL, Ernst E (2004) Soy for the treatment of perimenopausal symptoms-a systematic review. Maturitas 47, 1-9. Hwang ES, Bowen PE (2002) Can the consumption of tomatoes or lycopene reduce cancer risk? Integr Cancer Ther 1, 12132; discussion 132. Iddamaldeniya SS, Wickramasinghe N, Thabrew I, Ratnatunge N, Thammitiyagodage MG (2003) Protection against diethylnitrosoamine-induced hepatocarcinogenesis by an indigenous medicine comprised of nigella sativa, hemidesmus indicus and smilax glabra: a preliminary study. J Carcinog. 2, 6. Induction and recovery of hepatic drug metabolizing enzymes in rats treated with Ginkgo biloba extract. Food Chem Toxicol. 42, 953-957. Ingram DM, Hickling C, West L, Mahe LJ, Dunbar PM (2002) A double-blind randomized controlled trial of isoflavones in the treatment of cyclical mastalgia. Breast 11, 170-174. Jacobs EJ, Henion AK, Briggs PJ, Connell CJ, McCullough ML, Jonas CR, Rodriguez C, Calle EE, Thun MJ (2002) Vitamin C and vitamin E supplement use and bladder cancer mortality in a large cohort of US men and women. Am J Epidemiol 156, 1002-1010. Jacobson JS, Troxel AB, Evans J, Klaus L, Vahdat L, Kinne D, Lo KM, Moore A, Rosenman PJ, Kaufman EL, Neugut AI, Grann VR (2001) Randomized trial of black cohosh for the treatment of hot flashes among women with a history of breast cancer. J Clin Oncol 19, 2739-45. Jefferson JW (1988) Lithium tremor and caffeine intake: two cases of drinking less and shaking more. J Clin Psychiatry 49, 72-73. Jensen T, Stender S, Goldstein K, Holmer G, Deckert T (1989) Partial normalization by dietary cod-liver oil of increased microvascular albumin leakage in patients with insulin-
494
Cancer Therapy Vol 2, page 495 dependent diabetes and albuminuria. N Engl J Med 321, 572-577. Ji BT, Chow WH, Hsing AW, McLaughlin JK, Dai Q, Gao YT, Blot WJ, Fraumeni JF Jr (1997) Green tea consumption and the risk of pancreatic and colorectal cancers. Int J Cancer 70, 255-258. Jones JL, Daley BJ, Enderson BL, Zhou JR, Karlstad MD (2002) Genistein inhibits tamoxifen effects on cell proliferation and cell cycle arrest in T47D breast cancer cells. Am Surg 68, 575-577. Jussofie A, Schmiz A, Hiemke C (1994) Kavapyrone enriched extract from Piper methysticum as modulator of the GABA binding site in different regions of rat brain. Psychopharmacology (Berl) 116, 469-474. Kaegi E (1998) Unconventional therapies for cancer: 1 Essaic. CMAJ 158, 897-902. Karthikeyan J, Rani P (2003) Enzymatic and non-enzymatic antioxidants in selected Piper species. Indian J Exp Biol 41, 135-140. Kenny FS, Pinder SE, Ellis IO, Gee JM, Nicholson RI, Bryce RP, Robertson JF (2000) ! linolenic acid with tamoxifen as primary therapy in breast cancer. Int J Cancer 85, 643-648. Kidd P (2003) Th1/Th2 balance: the hypothesis, its limitations, and implications for health and disease. Altern Med Rev 8, 223-246. Kidd PM (2000) The use of mushroom glucans and proteoglycans in cancer treatment. Altern Med Rev 5, 4-27. Kim LS, Waters RF, Burkholder PM (2002) Immunological activity of larch arabinogalactan and Echinacea: a preliminary, randomized, double-blind, placebo-controlled trial. Altern Med Rev 7, 138-149. Kimura Y, Tojima H, Fukase S, Takeda K (1994) Clinical evaluation of sizofilan as assistant immunotherapy in treatment of head and neck cancer. Acta Otolaryngol Suppl 511, 192-195. Kivisto KT, Kroemer HK, Eichelbaum M (1995) The role of human cytochrome P450 enzymes in the metabolism of anticancer agents: implications for drug interactions. Br J Clin Pharmacol 40, 523-530. Klein EA, Thompson IM (2003) Update on chemoprevention of prostate cancer. Curr Opin Urol 14, 143-149. Klepser TB, Klepser ME (1999) Unsafe and potentially safe herbal therapies. Am J Health Syst Pharm 56, 125-138. Konno N, Makita H, Yuri K, Iizuka N, Kawasaki K (1994) Association between dietary iodine intake and prevalence of subclinical hypothyroidism in the coastal regions of Japan. J Clin Endocrinol Metab 78, 393-397. Kosty MP, Fleishman SB, Herndon JE 2nd, Coughlin K, Kornblith AB, Scalzo A, Morris JC, Mortimer J, Green MR (1994) Cisplatin, vinblastine, and hydrazine sulfate in advanced, non-small-cell lung cancer: a randomized placebocontrolled, double-blind phase III study of the Cancer and Leukemia Group B. J Clin Oncol 12, 1113–1120. Kotnis MS, Patel P, Menon SN, Sane RT (2004) Renoprotective effect of Hemidesmus indicus, a herbal drug used in gentamicin-induced renal toxicity. Nephrology (Carlton) 9, 142-152. Krasowski MD, McGehee DS, Moss J (1997) Natural inhibitors of cholinesterases: implications for adverse drug reactions. Can J Anaestesiol 44, 525-534. Krenn L, Presser A, Pradhan R, Bahr B, Paper DH, Mayer KK, Kopp B (2003) Sulfemodin 8-O-$-D-glucoside, a new sulfated anthraquinone glycoside, and antioxidant phenolic compounds from Rheum emodi. J Nat Prod 66, 1107-1109. Kronenberg F, Fugh-Berman A (2002) Complementary and alternative medicine for menopausal symptoms: a review of randomized, controlled trials. Ann Intern Med 137, 805813.
Labriola D, Livingston R (1999) Possible interactions between dietary antioxidants and chemotherapy. Oncology (Huntingt) 13, 1003-1008. Lake KD, Nolen JG, Slaker RA, Reutzel TJ, Milfred SK, Solbrack DF, Hoffman FM (1992) Over-the-counter medication in cardiac transplant recipients: guidelines for use. Ann Pharmacotherapy 26, 1566-1575. Lee BM, Lee SK, Kim HS (1998) Inhibition of oxidative DNA damage, 8-OHdG, and carbonyl contents in smokers treated with antioxidants (vitamin E, vitamin C, $-carotene and red ginseng). Cancer Lett 132, 219-227. Lee YJ, Jin YR, Lim WC, Park WK, Cho JY, Jang S, Lee SK (2003) Ginsenoside-Rb1 acts as a weak phytoestrogen in MCF-7 human breast cancer cells. Arch Pharm Res 26, 5863. Lemaire I, Assinewe V, Cano P, Awang DV, Arnason JT (1999) Stimulation of interleukin-1 and -6 production in alveolar macrophages by the neotropical liana, Uncaria tomentosa (una de gato) J Ethnopharmacol 64, 109-115. Levine M, Rumsey SC, Daruwala R, Park JB, Wang Y (1999) Criteria and recommendations for vitamin C intake. JAMA 281, 1415-1423. Lewis R, Wake G, Court G, Court JA, Pickering AT, Kim YC, Perry EK (1999) Non-ginsenoside nicotinic activity in ginseng species. Phytother Res 13, 59-64. Li M, Qu W, Wang Y, Wan H, Tian C (2002) Hypoglycemic effect of saponin from Tribulus terrestris Zhong Yao Cai 25, 420-422. Lill J, Bauer LA, Horn JR and Hansten PD (2000) Cyclosporinedrug interactions and the influence of patient age. AM J Health-Syst Pharm 57, 1579-1584. Lockwood K, Moesgaard S, Yamamoto T, Folkers K (1995) Progress on therapy of breast cancer with vitamin Q10 and the regression of metastases. Biochem Biophys Res Commun 212, 172-177. Loprinzi CL, Goldberg RM, Su JQ, Mailliard JA, Kuross SA, Maksymiuk AW, Kugler JW, Jett JR, Ghosh C, Pfeifle DM (1994a) Placebo-controlled trial of hydrazine sulfate in patients with newly diagnosed non-small-cell lung cancer. J Clin Oncol 12, 1126–1129. Loprinzi CL, Kuross SA, O'Fallon JR, Gesme DH Jr, Gerstner JB, Rospond RM, Cobau CD, Goldberg RM (1994b) Randomized placebo-controlled evaluation of hydrazine sulfate in patients with advanced colorectal cancer. J Clin Oncol 12, 1121–1125. Ludwig CU, Stoll HR, Obrist R, Obrecht JP (1987) Prevention of cytotoxic drug induced skin ulcers with dimethyl sulfoxide (DMSO) and %-tocopherole. Eur J Cancer Clin Oncol 23, 327-329. Luettig B, Steinmuller C, Gifford GE, Wagner H, LohmannMatthes ML (1989) Macrophage activation by the polysaccharide arabinogalactan isolated from plant cell cultures of Echinacea purpurea. J Natl Cancer Inst 81, 669675. Maclennan KM, Darlington CL, Smith PF (2002) The CNS effects of Ginkgo biloba extracts and ginkgolide B. Prog Neurobiol 67, 235-257. Maggiolini M, Statti G, Vivacqua A, Gabriele S, Rago V, Loizzo M, Menichini F, Amdo S (2002) Estrogenic and antiproliferative activities of isoliquiritigenin in MCF7 breast cancer cells. J Steroid Biochem Mol Biol 82, 315-322. Mannel M (2004) Drug Interactions with St John's Wort: Mechanisms and Clinical Implications. Drug Saf 27, 773797. Mansky PJ, Grem J, Wallerstedt DB, Monahan BP, Blackman MR (2003) Mistletoe and gemcitabine in patients with advanced cancer: a model for the phase I study of botanicals
495
Werneke et al: Complementary alternative medicine for cancer and botanical-drug interactions in cancer therapy. Integr Cancer Ther 2, 345-352. Marangell LB, Martinez JM, Zboyan HA, Kertz B, Kim HF, Puryear LJ (2003) A double-blind, placebo-controlled study of the omega-3 fatty acid docosahexaenoic acid in the treatment of major depression. Am J Psychiatry 160, 996998. Markowitz JS, Donovan JL, Lindsay DeVane C, Sipkes L, Chavin KD(2003) Multiple-dose administration of Ginkgo biloba did not affect cytochrome P-450 2D6 or 3A4 activity in normal volunteers. J Clin Psychopharmacol 23, 576-581. Maruyama H, Watanabe K, Yamamoto I (1991) Effect of dietary kelp on lipid peroxidation and glutathione peroxidase activity in livers of rats given breast carcinogen DMBA. Nutr Cancer 15, 221-228. Mary NK, Achuthan CR, Babu BH, Padikkala J (2003) In vitro antioxidant and antithrombotic activity of Hemidesmus indicus (L) R.Br. J Ethnopharmacol 87, 187-191. Massey LK, Palmer RG, Horner HT (2001) Oxalate content of soybean seeds (Glycine max: Leguminosae), soyfoods, and other edible legumes. J Agric Food Chem 49, 4262-4266. Mathews JM, Etheridge AS, Black SR (2002) Inhibition of human cytochrome P450 activities by kava extract and kavalactones. Drug Metab Dispos 30, 1153-1157. Matsuoka H, Seo Y, Wakasugi H, Saito T, Tomoda H (1997) Lentinan potentiates immunity and prolongs the survival time of some patients. Anticancer Res 17, 2751-2755. Matthews MK Jr. (1998) Association of Ginkgo biloba with intracerebral hemorrhage Neurology 50, 1933-1944. Melchart D, Clemm C, Weber B, Draczynski T, Worku F, Linde K, Weidenhammer W, Wagner H, Saller R (2002) Polysaccharides isolated from Echinacea purpurea herba cell cultures to counteract undesired effects of chemotherapy--a pilot study. Phytother Res 16, 138-142. Melchart D, Linde K, Fischer P, Kaesmayr J (2000) Echinacea for preventing and treating the common cold. Cochrane Database Syst Rev (2):CD000530. Menendez JA, Ropero S, del Barbacid MM, Montero S, Solanas M, Escrich E, Cortes-Funes H, Colomer R (2002) Synergistic interaction between vinorelbine and !-linolenic acid in breast cancer cells. Breast Cancer Res Treat 72, 203-219. Meseguer E, Taboada R, Sanchez V, Mena MA, Campos V, Garcia De Yebenes J (2002) Life-threatening parkinsonism induced by kava-kava. Mov Disord 17, 195-196. Messina MJ (2003) Emerging evidence on the role of soy in reducing prostate cancer risk. Nutr Rev 61, 117-131. Michalets EL (1998) Review of Therapeutics, Update: Clinically significant cytochrome P-450 drug interactions. Pharmacotherapy 18, 84-112. Miller LG (1998) Herbal medicinals: selected clinical considerations focusing on known or potential drug-herb interactions. Arch Intern Med 158, 2200-2211. Moertel CG, Fleming TR, Creagan ET, Rubin J, O'Connell MJ, Ames MM (1985) High-dose vitamin C versus placebo in the treatment of patients with advanced cancer who have had no prior chemotherapy. A randomized double-blind comparison. N Engl J Med 312, 137-141. Moertel CG, Fleming TR, Rubin J, Kvols LK, Sarna G, Koch R, Currie VE, Young CW, Jones SE, Davignon JP (1982) A clinical trial of amygdalin (Laetrile) in the treatment of human cancer. N Engl J Med 306, 201-206. Mullins RJ, Heddle R (2002) Adverse reactions associated with echinacea: the Australian experience. Ann Allergy Asthma Immunol 88, 42-51. Nair HK, Rao KV, Aalinkeel R, Mahajan S, Chawda R, Schwartz SA (2004) Inhibition of prostate cancer cell colony formation by the flavonoid quercetin correlates with
modulation of specific regulatory genes. Clin Diagn Lab Immunol 11, 63-69. Nallani SC, Goodwin B, Buckley AR, Buckley DJ, Desai PB (2004) Differences in the induction of cytochrome P450 3A4 by taxane anticancer drugs, docetaxel and paclitaxel, assessed employing primary human hepatocytes. Cancer Chemother Pharmacol 54, 219-229. Natural Medicines Comprehensive Database (2004); http://naturaldatabase.com. Neary JT, Bu Y (1999) Hyperforin inhibits uptake of serotonin and norepinephrine in astrocytes. Brain Research 816, 358363. Newall CA, Anderson LA, Philpson JD (1996) Herbal Medicine: A Guide for Healthcare Professionals. London, UK: The Pharmaceutical Press, 1996 Nishino H, Kitagawa K, Fujiki H, Iwashima A (1986) Berberine sulfate inhibits tumor-promoting activity of teleocidin in two-stage carcinogenesis on mouse skin. Oncology 43, 131134. Nkondjock A, Ghadirian P (2004) Dietary carotenoids and risk of colon cancer: case-control study. Int J Cancer 110, 110-116. North American Menopause Society (2004) Treatment of menopause-associated vasomotor symptoms: position statement of The North American Menopause Society. Menopause 11, 11-33. Oerter Klein K, Janfaza M, Wong JA, Chang RJ (2003) Estrogen bioactivity in fo-ti and other herbs used for their estrogenlike effects as determined by a recombinant cell bioassay. J Clin Endocrinol Metab 88, 4077-4079. Ohnishi N, Kusuhara M, Yoshioka M, Kuroda K, Soga A, Nishikawa F, Koishi T, Nakagawa M, Hori S, Matsumoto T, Yamashita M, Ohta S, Takara K, Yokoyama T (2003) Studies on interactions between functional foods or dietary supplements and medicines. I. Effects of Ginkgo biloba leaf extract on the pharmacokinetics of diltiazem in rats. Biol Pharm Bull 26, 1315-1320. Okamura K, Suzuki M, Chihara T, Fujiwara A, Fukuda T, Goto S, Ichinohe K, Jimi S, Kasamatsu T, Kawai N (1989) Clinical evaluation of sizofiran combined with irradiation in patients with cervical cancer. A randomized controlled study; a five-year survival rate. Biotherapy 1, 103-107. Oken BS, Storzbach DM, Kaye JA (1998) The efficacy of ginkgo biloba on cognitive function in Alzheimer disease. Arch Neurol 55, 1409-1415. Ondrizek RR, Chan PJ, Patton WC, King A (1999) Inhibition of human sperm motility by specific herbs used in alternative medicine. J Assist Reprod Genet 16, 87-91. Pace A, Savarese A, Picardo M, Maresca V, Pacetti U, Del Monte G, Biroccio A, Leonetti C, Jandolo B, Cognetti F, Bove L (2003) Neuroprotective effect of vitamin E supplementation in patients treated with cisplatin chemotherapy. J Clin Oncol 21, 927-931. Patrick L (2000) $-carotene: the controversy continues. Alt Med Rev 5, 530-545 Peebles KA, Baker RK, Kurz EU, Schneider BJ, Kroll DJ (2001) Catalytic inhibition of human DNA topoisomerase II% by hypericin, a naphthodianthrone from St. John's wort (Hypericum perforatum). Biochem Pharmacol 62, 10591070. Pelletier M, Lavastre V, Savoie A, Ratthe C, Saller R, Hostanska K, Girard D (2001) Modulation of interleukin-15-induced human neutrophil responses by the plant lectrin Viscum album agglutinin-I. Clin Immunol 101, 229-236. Perovic S, M端ller WE (1995) Pharmacological profile of hypericin extract. Effect on serotonin uptake by postsynaptic receptors. Arzneimittelforschung 45, 1145-1148.
496
Cancer Therapy Vol 2, page 497 Pfeifer BL, Pirani JF, Hamann SR, Klippel KF (2000) PC-SPES, a dietary supplement for the treatment of hormone-refractory prostate cancer. BJU Int 85, 481-485. Physicians' Deck Reference (PDR) for Herbal Medicines 2nd edition (2000), Medical Economics Company, Montevale New Jersey. Physicians' Deck Reference (PDR) for Herbal Medicines 2nd edition (2001), Medical Economics Company, Montevale New Jersey. Piao BK, Wang YX, Xie GR, Mansmann U, Matthes H, Beuth J, Lin HS (2004) Impact of complementary mistletoe extract treatment on quality of life in breast, ovarian and non-small cell lung cancer patients. A prospective randomized controlled clinical trial. Anticancer Res 24, 303-309. Pittler MH, Ernst E (2003) Kava extract for treating anxiety (Cochrane Review). In: The Cochrane Library, Issue 4, Chichester, UK: John Wiley and Sons, Ltd. Polan ML, Hochberg RB, Trant AS, Wuh HC (2004) Estrogen bioassay of ginseng extract and ArginMax, a nutritional supplement for the enhancement of female sexual function. J Womens Health (Larchmt) 13, 427-430. Prager N, Bickett K, French N, Marcovici G (2002) A randomized, double-blind, placebo-controlled trial to determine the effectiveness of botanically derived inhibitors of 5-%-reductase in the treatment of androgenetic alopecia. J Altern Complement Med 8, 143-152. Quintieri L, Rosato A, Napoli E, Sola F, Geroni C, Floreani M, Zanovello P (2000) In vivo antitumor activity and host toxicity of methoxymorpholinyl doxorubicin: role of cytochrome P450 3A. Cancer Res 60, 3232-3238. Rafi MM, Vastano BC, Zhu N, Ho CT, Ghai G, Rosen RT, Gallo MA, DiPaola RS (2002) Novel polyphenol molecule isolated from licorice root (Glycrrhiza glabra) induces apoptosis, G2/M cell cycle arrest, and Bcl-2 phosphorylation in tumor cell lines. J Agric Food Chem 50, 677-684. Ratna WN (2002) Inhibition of estrogenic stimulation of gene expression by genistein. Life Sci 71, 865-877. Relling MV, Nemec J, Schuetz EG, Schuetz JD, Gonzalez FJ, Korzekwa KR (1994) O-demethylation of epipodophyllotoxins is catalyzed by human cytochrome P450 3A4. Mol Pharmacol 45, 352-358. Ribereau-Gayon G, Jung ML, Frantz M, Anton R (1997) Modulation of cytotoxicity and enhancement of cytokine release induced by Viscum album L. extracts or mistletoe lectins. Anticancer Drugs 8, S3-S8. Richardson MA, Sanders T, Palmer JL, Greising A, Singletary SE (2000) Complementary/alternative medicine use in a comprehensive cancer centre and the implications for oncology. J Clin Oncol 18, 2505-2014. Risberg T, Lund E, Wist E, Kaasa S, Wilsgards T (1998) Cancer patients use nonproven therapy: 5-year follow-up study. J Clin Oncol 16, 1-2. Ross GD, Vetvicka V, Yan J, Xia Y, Vetvickova J (1999) Therapeutic intervention with complement and $-glucan in cancer. Immunopharmacology 42, 61-74. Russell RM (2000) The vitamin A spectrum: from deficiency to toxicity. Am J Clin Nutr 71, 878-884. Russmann S, Lauterberg BH, Hebling A (2001) Kava hepatotoxicity. Ann Intern Med 135, 68. Sadjadi J (1998) Cutaneous anthrax associated with the Kombucha "mushroom" in Iran. JAMA 280, 1567-1568. Sarkar FH, Li Y (2003) Soy isoflavones and cancer prevention. Cancer Invest 21, 744-757. Sato J, Nakata H, Owada E, Kikuta T, Umetsu M, Ito K (1993) Influence of usual intake of dietary caffeine on single-dose kinetics of theophylline in healthy human subjects. Eur J Clin Pharmacol 44, 295-298.
Schelosky L, Raffauf C, Jendroska K, Poewe W (1995) Kava and dopamine antagonism. Neurol Neurosurg Psychiatry 58, 639-640. Schumacher K, Schneider B, Reich G, Stiefel T, Stoll G, Bock PR, Hanisch J, Beuth J (2003) Influence of postoperative complementary treatment with lectin-standardized mistletoe extract on breast cancer patients. A controlled epidemiological multicentric retrolective cohort study. Anticancer Res 23, 5081-5087. Seidlova-Wuttke D, Hesse O, Jarry H, Christoffel V, Spengler B, Becker T, Wuttke W (2003) Evidence for selective estrogen receptor modulator activity in a black cohosh (Cimicifuga racemosa) extract: comparison with estradiol-17$. Eur J Endocrinol 149, 351-362. Semiglasov VF, Stepula VV, Dudov A, Lehmacher W, Mengs U (2004) The standardised mistletoe extract PS76A2 improves QoL in patients with breast cancer receiving adjuvant CMF chemotherapy: a randomised, placebo-controlled, doubleblind, multicentre clinical trial. Anticancer Res 24, 12931302. Sharma G, Tyagi AK, Singh RP, Chan DC, Agarwal R (2004) Synergistic anti-cancer effects of grape seed extract and conventional cytotoxic agent doxorubicin against human breast carcinoma cells. Breast Cancer Res Treat 85, 1-12. Sharma RA, McLelland HR, Hill KA, Ireson CR, Euden SA, Manson MM, Pirmohamed M, Marnett LJ, Gescher AJ, Steward WP (2001) Pharmacodynamic and pharmacokinetic study of oral Curcuma extract in patients with colorectal cancer. Clin Cancer Res 7, 1894-1900. Sheng Y, Pero RW, Amiri A, Bryngelsson C (1998) Induction of apoptosis and inhibition of proliferation in human tumor cells treated with extracts of Uncaria tomentosa. Anticancer Res 18, 3363-3368. Shin HR, Kim JY, Yun TK, Morgan G, Vainio H (2000). The cancer-preventive potential of Panax ginseng: a review of human and experimental evidence. Cancer Causes Control 11, 565-576. Singh RP, Tyagi AK, Dhanalakshmi S, Agarwal R, Agarwal C (2004) Grape seed extract inhibits advanced human prostate tumor growth and angiogenesis and upregulates insulin-like growth factor binding protein-3. Int J Cancer 108, 733-740. Small EJ, Frohlich MW, Bok R, Shinohara K, Grossfeld G, Rozenblat Z, Kelly WK, Corry M, Reese DM (2000) Prospective trial of the herbal supplement PC-SPES in patients with progressive prostate cancer. J Clin Oncol 18, 3595-3603. Sovak M, Seligson AL, Konas M, Hajduch M, Dolezal M, Machala M, Nagourney R (2002) Herbal composition PCSPES for management of prostate cancer: identification of active principles. J Natl Cancer Inst 94, 1275-1281. Sparber A, Bauer L, Curt G, Eisenberg D, Levin T, Parks S, Steinberg SM, Wootton J (2000) Use of complementary medicine by adult patients participating in cancer clinical trials. Onc Nurs Forum 27, 623-630. Sparreboom A, Cox MC, Archarya MA, Figg WD (2004) Herbal remedies in the United States: potential adverse interactions with anticancer agents. J Clin Oncol 22, 2489-2503. Stammers T, Sibbald B, Freeling P (1992) Efficacy of cod liver oil as an adjunct to non-steroidal anti-inflammatory drug treatment in the management of osteoarthritis in general practice. Ann Rheum Dis 51, 128-129. Stammler G, Volm M (1997) Green tea catechins (EGCG and EGC) have modulating effects on the activity of doxorubicin in drug-resistant cell lines. Anticancer Drugs 8, 265-268. Stevinson C, Ernst E (1999) Valerian for insomnia: a systematic review of randomized clinical trials. Sleep Medicine 1, 9199.
497
Werneke et al: Complementary alternative medicine for cancer Stimpel M, Proksch A, Wagner H, Lohmann-Matthes ML (1984) Macrophage activation and induction of macrophage cytotoxicity by purified polysaccharide fractions from the plant Echinacea purpurea. Infect Immune 46, 845-849 Strandell J, Neil A, Carlin G (2004) An approach to the in vitro evaluation of potential for cytochrome P450 enzyme inhibition from herbals and other natural remedies Phytomedicine 11, 198-104. Strandell J, Neil A, Carlin G (2004) An approach to the in vitro evaluation of potential for cytochrome P450 enzyme inhibition from herbals and other natural remedies. Phytomedicine 11, 98-104. Su KP, Huang SY, Chiu CC, Shen WW (2003) Omega-3 fatty acids in major depressive disorder. A preliminary doubleblind, placebo-controlled trial. Eur Neuropsychopharmacol 13, 267-71. Sugiyama T, Kubota Y, Shinozuka K, Yamada S, Yamada K, Umegaki K (2004) Induction and recovery of hepatic drug metabolizing enzymes in rats treated with Ginkgo biloba extract. Food Chem Toxicol 42, 953-7. Sun CL, Yuan JM, Arakawa K, Low SH, Lee HP, Yu MC (2002) Dietary soy and increased risk of bladder cancer: the Singapore Chinese Health Study. Cancer Epidemiol Biomarkers Prev 11, 1674-1677. Suzuki Y, Tsubono Y, Nakaya N, Suzuki Y, Koizumi Y, Tsuji I (2004) Green tea and the risk of breast cancer: pooled analysis of two prospective studies in Japan. Br J Cancer 90, 1361-1363. Tabet N, Birks J, Grimley Evans J (2004) Vitamin E for Alzheimer's disease. In: The Cochrane Library, Issue 1, Chichester, UK: John Wiley and Sons, Ltd. Tai J, Cheung S, Wong S, Lowe C (2004) In vitro comparison of Essiac and Flor-Essence on human tumor cell lines. Oncol Rep 11, 471-476. Tamayo C, Richardson MA, Diamond S, Skoda I (2000) The chemistry and biological activity of herbs used in flor essence herbal tonic and essiac. Phytother 14, 1-14. Tang F, Nag S, Shiu SY, Pang SF (2002) The effects of melatonin and Ginkgo biloba extract on memory loss and choline acetyltransferase activities in the brain of rats infused intracerebroventricularly with $-amyloid. Life Sci 71, 26252631. Taylor JA, Weber W, Standish L, Quinn H, Goesling J, McGann M, Calabrese C (2003) Efficacy and safety of echinacea in treating upper respiratory tract infections in children: a randomized controlled trial. JAMA 290, 2824-2830. Teufel-Mayer R, Gleitz J (1997) Effects of long-term administration of hypericum extracts on the affinity and density of the central serotonergic 5-HT1 A and 5-HT2 A receptors. Pharmacopsychiatry 30 Suppl 2, 113-116. The %-Tocopherol, $ Carotene Cancer Prevention Study Group (1994) The effect of vitamin E and $ carotene on the incidence of lung cancer and other cancers in male smokers. N Engl J Med 330, 1029-35. Thelen P, Wuttke W, Jarry H, Grzmil M, Ringert RH (2004) Inhibition of telomerase activity and secretion of prostate specific antigen by silibinin in prostate cancer cells. J Urol 171, 1934-1938. Thiele B, Brink I, Ploch M (1994) Modulation of cytokine expression by hypericin extract. J Geriatric Psych Neurol 7, S60-S62. Toft I, Bonaa KH, Ingebretsen OC, Nordoy A, Jenssen T (1995) Effects of n-3 polyunsaturated fatty acids on glucose homeostasis and blood pressure in essential hypertension. A randomized, controlled trial. Ann Intern Med 123, 911-918. Tripathi YB, Upadhyay AK, Chaturvedi P (2001) Antioxidant property of Smilex china Linn.Indian J Exp Biol 39, 1176-9.
Tsuruo T (2003) Molecular cancer therapeutics: recent progress and targets in drug resistance. Intern Med 42, 237-243. Tyagi A, Agarwal C, Harrison G, Glode LM, Agarwal R (2004) Silibinin causes cell cycle arrest and apoptosis in human bladder transitional cell carcinoma cells by regulating CDKICDK-cyclin cascade, and caspase 3 and PARP cleavages. Carcinogenesis [Epub ahead of print] van Schaik RH (2004) Implications of cytochrome P450 genetic polymorphisms on the toxicity of antitumor agents. Ther Drug Monit 26, 236-240. Vessby B, Boberg M (1990) Dietary supplementation with n3 fatty acids may impair glucose homeostasis in patients with non-insulin-dependent diabetes mellitus. J Intern Med 228, 165-171. Vickers A (2002) Botanical medicines for the treatment of cancer: rationale, overview of current data, and methodological considerations for phase I and II trials. Cancer Invest 20, 1069-1079. Vincent A, Fitzpatrick LA (2000) Soy isoflavones: are they useful in menopause? Mayo Clin Proc 75, 1174-1184. Visani G, Manfroi S, Tosi P, Martinelli G (1996) All-transretinoic acid and pseudotumor cerebri. Leuk Lymphoma 23, 437-442. Vogeler BK, Pittler MH, Ernst E (1999) The efficacy of ginseng. A systematic review of randomized controlled trials. Eur J Clin Pharmacol 55, 567-575. Wakai K, Hirose K, Takezaki T, Hamajima N, Ogura Y, Nakamura S, Hayashi N, Tajima K (2004) Foods and beverages in relation to urothelial cancer: case-control study in Japan. Int J Urol 11, 11-19. Walters BN, Gubbay SS (1981)Tetracycline and benign intracranial hypertension: report of five cases. Br Med J (Clin Res Ed) 282, 19-20. Wang GQ, Dawsey SM, Li JY, Taylor PR, Li B, Blot WJ, Weinstein WM, Liu FS, Lewin KJ, Wang H (1994) Effects of vitamin/mineral supplementation on the prevalence of histological dysplasia and early cancer of the esophagus and stomach: results from the General Population Trial in Linxian, China. Cancer Epidemiol Biomarkers Prev 3, 161-166. Watanabe H, Kakihana M, Ohtsuka S, Sugishita Y (1997a) Randomized, double-blind, placebo-controlled study of supplemental vitamin E on attenuation of the development of nitrate tolerance. Circulation 96, 2545-2550. Watanabe R, Murata M, Takayama N, Tokuhira M, Kizaki M, Okamoto S, Kawai Y, Watanabe K, Murakami H, Kikuchi M, Nakamura S, Ikeda Y (1997b) Long-term follow-up of hemostatic molecular markers during remission induction therapy with all-trans retinoic acid for acute promyelocytic leukemia. Keio Hematology-Oncology Cooperative Study Group (KHOCS). Thromb Haemost 77, 641-645. Werneke U, Earl J, Seydel C, Horn O, Crichton P, Fannon D (2004a) Potential health risks of complementary alternative medicines in cancer patients. Br J Cancer 90, 408-413. Werneke U, Horn O, Taylor D (2004b) How effective is St Johnâ&#x20AC;&#x2122;s wort? â&#x20AC;&#x201C; The evidence revisited. J Clin Psych 65, 611617. Werneke U, Earl J, Seydel C, Horn O, Crichton P, Fannon D (2004c) Reply: Assessing health risks of complementary alternative medicines in cancer patients. Br J Cancer 91, 996-997. Werneke U, McCready VR (2004) Complementary alternative medicine and nuclear medicine. Eur J Nucl Med Mol Imaging 31, 599-603. Whiskey E, Werneke U, Taylor D (2001) A systematic review of hypericum perforatum in depression. Int Clin Psychopharmacol 16, 239-252.
498
Cancer Therapy Vol 2, page 499 White E, Shannon JS, Patterson RE (1997) Relationship between vitamin and calcium supplement use and colon cancer. Cancer Epidemiol Biomarkers Prev 6, 769-774. Whiting PW, Clouston A, Kerlin P (2002) Black cohosh and other herbal remedies associated with acute hepatitis. Med J Aust 177, 440-443. Williamson EM (2001) Synergy and other interactions in phytomedicines. Phytomedicine 8, 401-409. Wilson B (2000) The rise and fall of Laetrile. http://www.quackwatch.org Xiaoguang C, Hongyan L, Xiaohong L, Zhaodi F, Yan L, Lihua T, Rui H (1998) Cancer chemopreventive and therapeutic activities of red ginseng. J Ethnopharmacol 60, 71-78. Xu Z, Cao HY, Li Q (1989) Protective effects of berberine on spontaneous ventricular fibrillation in dogs after myocardial infarction. Zhongguo Yao Li Xue Bao 10, 320-324. Yamamoto S, Sobue T, Kobayashi M, Sasaki S, Tsugane S (2003) Japan Public Health Center-Based Prospective Study on Cancer Cardiovascular Diseases Group. Soy, isoflavones, and breast cancer risk in Japan. J Natl Cancer Inst 95, 906913. Zhang S, Hunter DJ, Forman MR, Rosner BA, Speizer FE, Colditz GA, Manson JE, Hankinson SE, Willett WC (1999) Dietary carotenoids and vitamins A, C, and E and risk of breast cancer. J Natl Cancer Inst 91, 547-56. Zhou S, Lim LY, Chowbay B (2004) Herbal modulation of Pglycoprotein. Drug Metab Rev 36, 57-104. Zhou-Pan XR, Seree E, Zhou XJ, Placidi M, Maurel P, Barra Y, Rahmani R (1993) Involvement of human liver cytochrome P450 3A in vinblastine metabolism: drug interactions. Cancer Res 53, 5121-5126.
Zimmerman RA, Thompson IM Jr (2002) Prevalence of complementary medicine in urologic practice. A review of recent studies with emphasis on use among prostate cancer patients. Urol Clin North Am 29, 1-9. Zuber R, Modriansky M, Dvorak Z (2002) Effect of silybin and its congeners on human liver microsomal cytochrome P450 activities. Phytother Res 16, 632-638.
Ursula Werneke, M.D.
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Inhibition of telomerase improves chemosensitivity in cisplatin resistant ovarian cancer cells Research Article
Brenda L. Shoup1,3, Nancy E. Lowell2 and Patricia A. Kruk2,3* 1
Departments of Obstetrics and Gynecology Pathology and Laboratory Medicine, University of South Florida 3 H. Lee Moffitt Cancer Center, Tampa, FL 2
__________________________________________________________________________________ *Correspondence: Patricia A. Kruk, Ph.D., Department of Pathology and Laboratory Medicine, MDC 11, University of South Florida, 12901 Bruce B. Downs Blvd., Tampa, FL 33612, USA; Tel: 1-(813)-974-0548; Fax: 1-(813)-974-5536; e-mail: pkruk@hsc.usf.edu Key words: telomerase, ovarian cancer, cisplatin, drug resistance Abbreviations: Cisplatin, (CP); DNA fragmentation factor 45, (DFF45); Dulbeccoâ&#x20AC;&#x2122;s phosphate buffered saline, (DPBS); Green fluorescent protein, (GFP); Oligonucleotide, (ODN); Polymerase chain reactionâ&#x20AC;&#x201C;enzyme linked immunosorbant assay, (PCR-ELISA); reverse transcriptase-polymerase chain reaction, (RT-PCR); Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (SDS-PAGE) Received: 8 November 2004; Revised: 24 November 2004 Accepted: 1 December 2004; electronically published: December 2004
Summary Telomerase is expressed in 95% of gynecologic malignancies and in over 90% of epithelial ovarian carcinomas, contributing to the maintenance of the malignant phenotype by conferring immortality and enhanced cell survival. Since the development of drug resistance contributes to the poor prognosis associated with ovarian cancer, the objective of the current study was to determine if inhibition of telomerase could impact platinum resistance in epithelial ovarian cancer cells. The cisplatin (CP)-sensitive, OV2008, and its CP-resistant daughter, C13, ovarian cancer cell lines were subjected to telomerase inhibition using telomere sequence specific DNA oligonucleotides and a 2-O-(methyl) RNA oligomer, in the presence and absence of CP. Though treatment with the RNA oligomer resulted in the greatest degree of telomerase inhibition, both the DNA oligonucleotides and 2-O-(methyl) RNA oligomer significantly decreased cell viability when combined with CP in CP-resistant cells. Increased CP-sensitivity associated with telomerase inhibition was related to increased caspase 3-dependent apoptosis. Therefore, cisplatin sensitivity can be enhanced in ovarian cancer cells by concomitant treatment with a telomerase inhibitor. This suggests that telomerase inhibitors may have clinical utility as chemo-sensitizing agents in patients with platinumresistant ovarian cancer. et al, 1994; Creemers et al, 1996; Gordon et al, 1997; Kauffman and von Minckwitz, 1997; Muggia et al, 1997; Ozols, 1997; ten Bokkel Huinink et al, 1997; Bookman et al, 1998; Rose et al, 1998). Telomerase is a multimeric ribonucleoprotein that adds telomeric repeats to chromosome ends (Morin, 1989). Thus, it stabilizes chromosomal DNA and is thought to confer immortality to cells (Shay and Bacchetti, 1999). Telomerase activity is generally limited to stem cell populations and tumor cells and is suppressed in normal somatic cells (Kim et al, 1994). Telomerase consists of a RNA component (hTR) that serves as an internal telomeric template, telomerase associated proteins and hTERT, a reverse transcriptase (Kim et al 1994; Weinrich et al, 1997). While both the protein catalytic hTERT subunit and the hTR RNA component have been identified and cloned, reports suggest that the reverse transcriptase catalytic
I. Introduction Ovarian cancer is the fifth most common cancer in women with an estimated 23,100 new cases in the U.S. annually (Greenlee et al, 2000). Ovarian cancer has the highest mortality rate among gynecological cancers (5year survival is approximately 35%). Optimal cytoreduction followed by platinum based chemotherapy is the gold standard of therapy. While the response rate to primary chemotherapy can be as high as 76%, response rate is dramatically reduced after relapse of disease (Kigawa et al, 1993). Platinum resistance, defined as disease recurrence less than six months from completion of therapy is an important prognostic predictor. Patients with platinum-resistant tumors have a response rate of less than 10% when retreated with platinum compounds (Blackledge et al, 1989). Alternative options also have poor response rates of 18-30% (Kohn et al, 1994; Thigpen 501
Shoup et al: Inhibition of telomerase improves chemosensitivity in cisplatin resistance subunit is the limiting determinant of telomerase activity (Feng et al, 1995; Meyerson et al, 1997; Weinrich et al, 1997; Counter et al, 1998). We have previously shown that a number of exogenous stresses can enhance telomerase activity in ovarian cancer cells as well as activate de novo telomerase activity in non-malignant ovarian surface epithelial cells (Alfonso-De Matte et al, 2001). Further, stress-induced telomerase activity is regulated in a phosphatidylinositol triphosphate kinase/c-Jun NH2terminal kinase-dependent manner (Alfsono-De Matte et al, 2001, 2002a,b, 2004). Over 90% of tumors examined to date, including ovarian cancers, express telomerase activity (Hastie et al, 1990; Counter et al, 1994; Kyo et al, 1996, 1998a,b; Summerfeld et al, 1996; Hoos et al, 1998). In the ovary, studies have shown an absence of telomerase activity in normal ovarian surface epithelium and pre-malignant lesions, while tumor cells from both acites fluid and ovarian carcinomas express telomerase activity (Counter et al, 1994; Kyo et al, 1996, 1999; Murakami et al, 1997, 1998a,b; Duggan et al, 1998; Datar et al, 1999; Oishi et al, 1999; Park et al, 1999). Telomerase activity has been directly linked to ovarian tumor stage and aggressiveness (Murakami et al, 1998a,b; Hahn et al, 1999; Yan et al, 1999). While the primary function of telomerase is the maintenance of telomeric integrity, several recent reports suggest an association between telomerase with reduced apoptosis and increased chemotherapeutic resistance (Asai et al, 1998; Kondo et al, 1998a,b; Herbert et al, 1999; Tian et al, 1999; Zhang et al 1999; Kiyozuka et al, 2000; Villa et al, 2000). Iida et al, (2000) showed that telomerase activity was significantly higher in colorectal cancers concurrently expressing the anti-apoptotic protein, Bcl-2. A similar relationship was demonstrated in cervical and colorectal cancer cell lines (Mandal et al, 1997). Likewise, reduced apoptosis in pancreatic cancer cells after exposure to etoposide was associated with elevated telomerase activity (Sato et al, 2000). Lastly, recent reports suggested that hTERT suppressed apoptosis prior to mitochondrial dysfunction and caspase activation in cultured cells (Holt et al, 1999; Fu et al, 2000). Although there have been very few similar studies in the ovary, Faraoni et al, (1999) showed that telomerase activity in primary cultures of ovarian cancer cells was inversely related to chemosensitivity. In a pilot study, Takahashi et al, (2000) showed that ovarian cancer patients responding to platinum therapy had low levels of telomerase whereas 50% of non-responders demonstrated elevated telomerase activity. Given its clinical importance, telomerase has been investigated as a potential diagnostic or prognostic tool and especially as a therapeutic target (Murakami et al, 1998b; Hahn et al, 1999; Yan et al, 1999). Telomerase inhibitors include both synthetic and naturally occurring compounds (Naasani et al, 1998; Perry et al, 1998; Togashi et al, 1998; Lian et al, 2001; Lin et al, 2003; Kraveka et al, 2003; Yokoyama et al, 2004) and fall into two general categories. The first involves compounds that bind telomeric DNA, preventing telomerase access to the telomere and predominately include small linear
molecules that intercalate the G-quadruplex telomeric structure (Perry et al, 1998; Gavathiotis et al, 2003; Shammas et al, 2003, 2004; Yin et al, 2003). This group of inhibitors also includes modified oligonucleotides with sequence homology to telomeric DNA (Gavathiotis et al, 2003; Mata et al, 1997). The second group of telomerase inhibitors abrogate the enzymatic function of telomerase. These include reverse transcriptase inhibitors, most notably AZT (Kitagawa et al, 2000; Brown et al, 2003; Yamahuchi et al, 2003), RNA interference directed towards the hTERT catalytic component (Kosciolek et al, 2003), expression of dominant-negative hTERT mutants (Delhommeau et al, 2002), peptide nucleic acids (Herbert et al, 1999), as well as anti-sense or modified oligonucleotides complementary to the telomerase hTR RNA template (Kushner et al, 2000). Accessibility and easy design of oligonucleotides makes hTR an ideal target for telomerase inhibition. In addition, oligonucleotide modification, often with oligomer methylation or phosphorothioate linkages between bases (Levin, 1999; Chen et al, 2003), enhances oligomer stability from nuclease digestion to dramatically increase oligomer halflife (Geary et al, 2001). To date, there have been many promising and favorable reports regarding anti-telomerase chemotherapeutic strategies. Anti-sense inhibition of telomerase increased the susceptibility of glioblastoma cells to cisplatin-induced apoptosis (Kondo et al, 1998a). Inhibition of telomerase with oligonucleotides resulted in enhanced pheochromocytoma cell apoptosis (Fu et al, 1999). Hahn et al, (1999) showed that complete inhibition of telomerase activity using a mutant form of human telomerase resulted in the death of tumor cells and in vivo expression of mutant telomerase eliminated tumorigenicity. Clearly, chemosensitivity is a crucial prognostic factor for ovarian cancer and levels of or changes in telomerase may predict therapeutic outcome. Because of the presence of telomerase in ovarian tumor cells and the propensity for therapeutic failure from the development of drug resistance, we sought to determine whether inhibition of telomerase improved sensitivity to cisplatin in vitro in ovarian cancer cell lines OV2008 and C13, parent and daughter, CP-sensitive and CP-resistant cell lines, respectively (Asselin et al, 2001). Two telomerase inhibitors were chosen, a hexameric phosphorothioate oligonucleotide with sequence homology identical to the repeat sequence of the mammalian telomere, 5â&#x20AC;&#x2122;d(TTAGG)-3â&#x20AC;&#x2122; (Mata et al, 1997) and a 2â&#x20AC;&#x2122;-O-(2methoxyethyl) RNA oligomer complementary to the telomerase hTR RNA component (Pitts and Corey, 1998, Herbert et al, 1999). In the present study, we show that inhibition of telomerase activity dramatically improved sensitivity to cisplatin in CP-resistant ovarian cancer cells.
II. Materials and methods A. Cell Culture Two ovarian cancer cell lines, OV2008 and C13 were used. The cell lines were selected because OV2008 is CP sensitive and its daughter cell line, C13 is resistant to CP (Asselin et al, 2001). Cells were maintained in medium 199/MDCB 105 (1:1) (Sigma,
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Cancer Therapy Vol 2, page 503 7.5x104 cells per 60 mm2 dish in serum free media for 24 hours. C13 cells were transfected with 34 µl Lipofectamine (Life Technologies, Inc., Grand Island, NY), and 8 µl RNA [100nMol dissolved in 469 µl MW 4691.8]. OV2008 cells were transfected in the same manner except 17 µl Lipofectamine was used after optimization with Green Fluorescent Protein (GFP) cDNA to establish transfection conditions favoring the most oligomer uptake. In all transfection experiments, parallel cultures transfected with GFP were used as a control for transfection efficiency. After 24 hours the transfection media was removed ± 25 µM CP was added for 2 hours followed by serum containing media. Cells were then collected and assayed for telomerase activity, cell viability and levels of DNA fragmentation factor 45 (DFF45) at various time intervals up to 72 hours.
St. Louis, MO) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), and 10 µg/ml gentamicin (GIBCO BRL, Grand Island, NY) in a humidified 5% CO2/ 95% air atmosphere. Cell counts were performed using a hemocytometer and cellular viability determined by Trypan blue exclusion assay. Cell growth was determined by the MTS colorimetric assay (Promega, Madison, WI). The assay was performed in 96 well microtiter plates according to manufacturer’s instructions and is based on soluble formazan production by dehydrogenase enzymes found in metabolically active cells. Samples were seeded six at 5x103 cells per well. Absorbance was determined at 490nm using a Dynex MRX plate reader (Dynex Technologies, Chantilly, VA).
B. Telomerase assay To quantitatively detect changes in telomerase levels, cells were assayed for telomerase activity using the telomerase polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA) (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer’s directions and as performed previously (Alfonso-De Matte et al, 2001). This assay has been demonstrated to be as sensitive as the radioactive, telomere repeat amplification protocol (TRAP) assay. Briefly, cells were washed with Dulbecco’s phosphate buffered saline (DPBS), lysed in CHAPS lysis buffer and then assayed for protein using the Bio-Rad DC Protein Assay (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. C13 and OV2008 cell extracts equivalent to 3 µg of protein were used. Following PCR-ELISA, telomerase activity was detected using a DynexMRX plate reader and recorded as absorbance units.
E. SDS-PAGE and western blot analysis In order to determine the extent to which apoptosis was affected by telomerase inhibition, cell lysates were assayed for DFF45 cleavage or caspase-3 activation. Fifteen micrograms of protein were added to 4x loading buffer (250 mM Tris pH 6.8, 8% SDS, 20% glycerol, 0.012% bromophenol blue, 4% !mercaptoethanol), heated to 95 °C for 5 minutes, electrophoresed in 12.5% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Amersham, Piscataway, NJ) via semidry transfer. Membranes were blocked with 3% nonfat milk in DPBS or 5% nonfat milk in Tris buffered saline plus 0.1% Tween-20 for DFF45 and caspase 3, respectively. All membranes were incubated overnight at 4°C in primary antibody. Polyclonal antibodies for caspase-3 and DFF45 were purchased from Cell Signaling (Beverly, MA) and BDPharMingen (San Diego, CA), respectively. Membranes were incubated and developed according to the Enhanced Chemiluminescent Protocol, according to manufacturer’s instructions (Amersham). After initial blotting, membranes were reprobed for actin to ensure even loading. Immunoblots were scanned and analyzed with ImagQuant software. Values reported for target proteins were normalized to the immunoblots’ respective actin levels.
C. RT-PCR To examine the contribution of transcriptional control for telomerase activity in OV2008 and C13 cells, cells were incubated ± 5 µg/ml Actinomycin D and RT-PCR for hTERT mRNA was performed as described previously (Alfonso-De Matte et al, 2001). Total RNA was collected using Trizol reagent (GIBCO BRL). One microgram total RNA, oligo(dT) and reverse transcriptase were used to generate single-stranded cDNA. The cDNA samples were amplified using the PerkinElymer GeneAmp kit (Palo Alto, CA). !-actin was used as an internal control. The amplified products were separated by electrophoresis on a 9% polyacrylamide gel, stained with 1X SyberGreen (FMC Bioproducts, Rockland, MD) and analyzed with the Kodak EDAS 120 Digital Analysis System. Net hTERT mRNA intensities were normalized to their corresponding !actin mRNA levels and expressed as a percentage of !-actin mRNA expression.
F. Statistics Samples for telomerase activity, MTS and trypan blue viability counts were run at least in triplicate and the data subjected to Student t test analysis for determination of statistical significance for telomerase activity or inhibition between treated and untreated samples. The results were expressed as mean ± standard error.
III. Results D. Treatment with 5’-d(TTAGG)-3’ oligonucleotides and 2’-O-(2-methoxyethyl) RNA oligomers
A. Telomerase activity is increased in C13 cells. In contrast to primary cultures of normal ovarian surface epithelial cells (MF10 and XD cells) that are telomerase negative (Kruk et al, 1999), both OV2008 and C13 ovarian cancer cells expressed telomerase activity (Figure 1A). However, telomerase activity in the CP-sensitive OV2008 cells was consistently lower by approximately 25% than in C13 cells. RT-PCR analysis revealed at least three times more of the 145-bp hTERT RNA product in C13 cells compared with OV2008 cells when normalized to their respective 98-bp !-actin product (Figure 1B) confirming increased telomerase in C13 cells. In addition, hTERT mRNA stability was at least 2.5-fold greater in C13 cells compared to OV2008 cells with 40%
To examine the effect of interfering with telomerase activity on drug sensitivity, exponentially growing cells were treated with phosphorothioate DNA oligonucleotides (ODNs) complementary to the human telomeric repeat sequence or composed of a scramble sequence (Oncogene, Boston, MA) as described previously (Mata et al, 1997) at 2.5 µM in serum free media for 24 hours. Parallel cultures were also treated with 25 µM CP for two hours after incubation with the ODNs. Cells were then collected and assayed for telomerase activity, cell growth and caspase 3 levels at various time intervals up to 72 hours. Controls were untreated cells in serum free media. To examine the effect of telomerase inhibition using the 2’O-(2-methoxyethyl) RNA oligomer targeting the hTR template (Oncogene, Boston, MA) on CP sensitivity, cells were plated at
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Figure 1. Enhanced telomerase expression in CP-resistant ovarian cancer cells. (A) Telomerase activity was determined by PCRELISA from lysates of C13 and OV2008 ovarian cancer cells as well as from lysates of primary cultures of normal ovarian surface epithelial cells (MF10 and XD). Results are expressed as the average absorbance at 450nm ± SE. (B) C13 (grey bars) and OV2008 (open bars) cells were treated with 5 µg/ml Actinomycin D for 2 hours. The 145-bp hTERT product seen following RT-PCR and PAGE was normalized to the respective 98-bp !-actin product.
O-methyl-RNA ± CP (*p<0.004 and **p<0.005 compared to control values, respectively) (Figure 2D). However, telomerase inhibition appeared to be reversible in C13 cells as demonstrated by the restoration to baseline telomerase activity at 48 hours following transfection.
loss of transcript in OV2008 cells compared to 15% transcript loss in C13 cells following incubation with Actinomycin D (Figure 1B).
B. Telomerase activity is inhibited by 2’O’methyl-RNA, but not by 5’-d(TTAGG)-3’
C. 5’-d(TTAGG)-3’ ODN and 2’O’methyl-RNA increase CP-mediated cytotoxicity and apoptosis in C13 cells
OV2008 and C13 cell lines were assayed for telomerase activity by PCR-ELISA after treatment with 5’d(TTAGG)-3’ ODN or 2’-O’methyl-RNA oligomers. As expected, telomerase activity was not significantly lower at 24 (p<0.8) or 48 hours (p<0.67) in OV2008 cells when treated with the 5’d(TTAGG)-3’ (T) ODN alone (Figure 2A) since 5’d(TTAGG)-3’ blocks access of telomerase to the telomere and does not directly inhibit telomerase activity, but contributes to increased telomeric instability. Similarly, the negative control antisense scramble oligonucleotide (TN) did not cause a significant change in telomerase activity (Figure 2A). While a 2 hour treatment with CP did not immediately affect telomerase activity, a 46% decrease in telomerase activity was observed 48 hours after the 2 hour CP exposure in OV2008 cells regardless of ODN treatment (*p<0.02 and **p<0.01 when compared to controls or to 5’d(TTAGG)-3’ alone). This reduction in telomerase activity appeared to correlate with increased cell mortality as determined by morphologic examination. Likewise, ODNs did not cause a significant decrease in the telomerase activity in C13 cells (Figure 2B). Further, CP failed to have any significant effect telomerase activity in these cells. Cells were transfected with 2’-O-methyl-RNA after optimization with GFP. Transfection efficiency was 70% in C13 cells and 50% in OV2008. There was no significant change in telomerase activity in OV2008 cells by 24 hours, however, telomerase activity was reduced by 40% at 48 hours following transfection (Figure 2C) (*p"0.04 and **p"0.02 2’-O-methyl-RNA or 2’-O-methyl-RNA + CP compared to controls, respectively). In contrast, in the faster growing C13 cells, telomerase activity was reduced by up to 90% at 24 hours following transfection with 2’-
In order to determine the effect of 5’-d(TTAGG)-3’mediated telomerase inhibition on ovarian cancer cell growth, MTS assays were performed. We noted that OV2008 cells grew slower than the C13 cells with an average doubling time approximately half that of C13 cells. Though 5’-d(TTAGG)-3’ (T) and its scramble negative control oligomer (TN) had no effect on CPsensitive OV2008 cell growth, CP, in the absence or presence of either T or TN ODNs, abrogated all cell growth and reduced cell numbers to well below starting values (*p" 0.0005) as would be expected of CP-sensitive ovarian cancer cells (Figure 3A). ODNs or CP treatments alone also had little effect on C13 cell growth, reducing cellular proliferation less than 20% (*p" 0.001) (Figure 3B). However, combined treatment of 5’-d(TTAGG)-3’ with CP completely abolished C13 cell growth, indicating that such a combined treatment improved sensitivity to CP (**p" 0.00004 and ***p" 0.0003 compared to controls and CP-treated cells) (Figure 3B). To determine the role of apoptosis for improved chemosensitivity by 5’-d(TTAGG)3’, cell lysates were assayed for cleaved caspase-3 using SDS-PAGE and western immunoblot. The levels of active caspase 3 among controls, samples collected immediately after a 2 hr treatment with 25 µM CP or 5’-d(TTAGG)3’ oligomertreated samples were negligible in both OV2008 and C13 cells (Figure 3C,D). However, densitometric analysis revealed at least an 85x and 44x increase of caspase 3 activity in OV2008 and C13 samples, respectively, collected 48 hours after treatment with both 5’-
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Figure 2. 2’-O-(methyl)RNA oligomers inhibit telomerase activity in OV2008 and C13 cells. Telomerase activity was determined from triplicate samples of OV2008 (A, C) and C13 (B, D) cells following treatment with ODNs (A, B) or transfection with 2’-O(methyl)RNA (C,D). A, B) Cells were treated with 5’-d(TTAGG)3’ (T) or a scramble sequence (TN) ODN. Parallel cultures were treated for 2 hours with 25 µM CP after which time the medium was refreshed and the cultures maintained for up to 48 hours while controls were untreated samples. (C, D) Cells were transfected with 2’-O-(methyl)RNA (T) and similarly treated ± CP as in (A, B). Controls consisted of untreated samples collected at 24 hours. Results are expressed as fold increase or decrease in telomerase activity compared to control samples ± SE.
d(TTAGG)3’ and 2 hours CP (Figure 3C,D). Trypan blue exclusion cell counts were used to determine the number viable, adherent cells after transfection with 2’-O-(2-methoxyethyl) RNA (Figure 4 A,B). In agreement with our results presented above, sensitivity to CP in OV2008 cells was apparent. Treatment of OV2008 cells with CP for 2 hours, with or without 2’O-(2-methoxyethyl) RNA, caused a 99% decrease in the number of living cells by 72 hours following treatment (*p" 0.001) (Figure 4 A). 2’-O-(2-methoxyethyl) RNA alone did not affect OV2008 cell viability (Figure 4 A). As expected, C13 cells were only modestly sensitive to CP with only a 34% decrease in adherent living cells by 72 hours when treated with CP alone (*p" 0.05) (Figure 4B). Similarly, 2’-O-(2-methoxyethyl) RNA did not significantly affect the number of viable C13 cells (Figure 4B). However, the combined treatment of CP and 2’-O-(2-
methoxyethyl) RNA caused a 61% reduction in adherent, living cells as compared to the control group (**p" 0.006) and further increased CP-mediated cytotoxicity by an additional 40% (***p" 0.04) (Figure 4B). To determine whether telomerase inhibition by 2’-0(2-methoxyethyl) RNA also enhanced apoptosis, SDSPAGE was performed on cell lysates and western immunoblots were probed for DFF45, a known cleavage substrate of caspase-3 (Widlak, 2000). While treatment with 2’-O-(2-methoxyethyl) RNA alone did not affect DFF45 expression in either OV2008 or C13 cells, combined treatment of 2’-O-(2-methoxyethyl) RNA with CP greatly enhanced DFF45 degradation (Figure 4C,D). Densitometric analysis revealed approximately 2x and 3x increased DFF45 cleavage in OV2008 and C13 cells, respectively, when CP was combined with RNA treatment.
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Figure 3. 5’-d(TTAGG)-3’ enhances CP-mediated cytotoxicity and caspase 3 activation in C13 cells. Cell growth was measured by MTS assays and expressed as average absorbance at 490 nm ± SE in OV2008 (A) and C13 (B) cells after treatment with 5’-d(TTAGG)3’ (T) or the scrambled negative control (TN) ± 2 hour treatment with CP while controls consisted of untreated samples. Parallel cultures of OV2008 (C) and C13 (D) cells were subjected to SDS-PAGE. Following transfer, blots were probed for (active) cleaved caspase-3 (17-20kDa). Actin served as a loading control.
Figure 4. 2’-O-(methyl) RNA enhances CP-mediated cytotoxicity and DFF45 cleavage in C13. Cell viability was determined by trypan blue exclusion counts and expressed as the number of cells x 104 ± SE in OV2008 (A) and C13 (B) cells after transfection with the 2’-O(methyl) RNA inhibitor (I) ± 2 hour treatment with CP while controls consisted of untreated samples. Parallel cultures of OV2008 (C) and C13 (D) cells were subjected to SDS-PAGE. Following transfer, blots were probed for the caspase 3 cleavage substrate, DFF45. Actin served as a loading control.
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Cancer Therapy Vol 2, page 507 In the classical model of telomerase activity, inhibition of the enzyme alone would require multiple cell doublings to cause significant telomere shortening to signal cell death. Our investigation, as well as others (Kondo et al, 1998; Fu et al, 1999, Kushner et al, 2000), suggests that it is unlikely that telomerase inhibitionmediated cell death is related to telomere erosion alone although telomeric and, hence, chromosomal distortions due to inhibition of telomerase function could signal DNA damage responses, including increased apoptosis. Our results support an anti-apoptotic contribution by telomerase, since inhibition of telomerase resulted in increased caspase 3-dependent apoptosis. This is in agreement with studies reporting an association between telomerase activity and enhanced cancer cell survival consistent with poor prognosis in colon, breast, gastric, cervical, uterine and lymphoid cancers (Asai et al, 1998; Kiyozuka et al, 2000; Villa et al, 2000) in which telomerase appears to mediate its protective effect by conferring resistance to apoptosis (Herbert et al, 1999; Iida et al, 2000; Kondo et al, 1998a; Mandel and Kumar, 1997; Tian et al, 1999; Zhang et al, 1999). Oligonucleotides as telomerase inhibitors have many benefits. These oligonucleotides are commercially available or can be readily synthesized which makes them attractive experimental agents. They bind the hTR template and prevent the addition of telomeric repeats and mismatch negative controls can be used to delineate proof of the mechanism of inhibition. There are methods for large-scale oligo synthesis, their pharmacokinetic properties are well characterized and the toxicity is low (Geary et al, 2001). There are also indications that adequate oral bioavailability can be achieved (Khatsenko et al, 2000). However, telomerase as a therapeutic target still raises many questions since not all tumors exhibit telomerase activity (Shay, 1995). Further, some normal cells, such as germ cells and hematopoietic cells, express low levels of telomerase activity and an effective inhibitor may impair the function of these cells (Morin, 1995). Subsequently, telomere loss resulting from prolonged telomerase inhibition in normal cells could result in chromosomal instability that can promote mutations required for cellular transformation (de Lange and Jacks, 1999). Nevertheless, our results are promising in that both telomerase inhibitors employed significantly increased CP-mediated cytotoxicity in CP-resistant ovarian cancer cells, possibly in a caspase 3-dependent manner. The possibility of improving the sensitivity of such cells to current chemotherapeutic treatments is exciting and, clearly, warrants further study as an effective adjuvant treatment for recurrent platinum resistant ovarian cancer. Therefore, by understanding the relationship between telomerase activity and apoptotic pathways in ovarian tumor progression, abrogation of telomerase in conjunction with conventional platinum regimes could improve current therapeutic efficacy and clinical outcome by increasing cellular sensitivity to chemotherapeutic agents.
IV. Discussion Since telomerase is expressed in >90% of human tumors and absent from most normal somatic cells and its expression correlates with tumor aggressiveness, telomerase is an attractive target for therapeutic intervention. Telomerase inhibition has been successfully achieved by several mechanisms including antisense technology, ribozymes directed against telomerase, and Gquadruplex binding agents that focus on either preventing access of telomerase to telomeric ends or abrogating hTERT and/or hTR sites (Rowley and Tabler, 2000). Inhibiting the addition of telomeric repeats to chromosome ends does not necessarily cause immediate cell death and can require substantial time to induce telomeric instability with subsequent growth arrest because the rate of telomeric erosion is approximately 50-100 bases per cell population doubling (Hastie et al, 1990; Shay, 1995; Shammas et al, 2004). In contrast, inhibition of telomerase by directly abrogating hTERT or hTR function may disrupt telomerase associated anti-apoptotic properties as well as promote telomeric instability. Nonetheless, disruption of telomerase activity appears to increase sensitivity to DNA damaging chemotherapeutic agents in breast cancer cells (Ludwig et al, 2001), glioblastoma cells (Kondo et al, 1998), and in neoplastic cells derived from telomerase RNA null mice (Lee et al, 2001). In agreement, we found that platinum resistance in C13 ovarian cancer cells was associated with increased telomerase activity that could contribute to enhanced cancer cell survival. In contrast, prior to the development of drug resistance, CP is an extremely effective chemothapeutic agent as demonstrated by the exceptional cytotoxic capacity of CP for OV2008 cells. Therefore, since chemoresistance, especially to platinum agents, is well documented in ovarian cancer and presents a serious therapeutic challenge, we sought to improve chemotherapeutic response in CP-resistant ovarian cancer cells by employing telomerase inhibition as a novel adjuvant therapy. 5’-d(TTAGG)3’ has been shown to inhibit telomerase activity in cell lysates, lengthen cell doubling time and induce apoptosis in Burkitts lymphoma cells (Mata et al, 1997). In the present study, 5’-d(TTAGG)3’ did not significantly lower the telomerase activity though it did dramatically improve chemosensitivity to CP in C13 cells. A scrambled negative control did not affect telomerase activity or improve sensitivity to CP though partial activity of the nonsense negative controls was described in Mata et al, (1997). While Lee et al, (2001) suggested that telomere dysfunction rather than telomerase itself is the principle determinant governing chemosensitivity, unlike previous studies, we did not see changes in MTL likely because of the short time frame of our experiments. 2-O-methyl RNA was an effective inhibitor of telomerase in both OV2008 and C13 cells. This inhibitor has already been characterized in animal and early human clinical trials (Pitts and Corey, 1998). Our investigation demonstrated a significant reduction in cell viability in cisplatin resistant cells when treated with both the 2-Omethyl RNA and cisplatin.
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Shoup et al: Inhibition of telomerase improves chemosensitivity in cisplatin resistance Datar R, Naritoku WY, Li P, Tsao-Wei D, Groshen S, Taylor CR, and Imam SA (1999) Analysis of telomerase activity in ovarian cystadenomas, low-malignant-potential tumors, and invasive carcinomas. Gynecol. Oncol. 74, 338-345. de Lange T, and Jacks T (1999) For better or worse? Telomerase inhibition and cancer. Cell 98, 273-5. Delhommeau F, Thierry A, Feneux D, Lauret E, Leclercq E, Courtier MH, Sainteny F, Vainchenker W, and BennaceurGriscelli A (2002) Telomere dysfunction and telomerase reactivation in human leukemia cell lines after telomerase inhibition by expression of a dominant-negative hTERT mutant. Oncogene 21, 8262-8271. Duggan BD, Aan M, Yu MC, Roman LD, Muderspack LI, Delgadillo E, Li W-Z, Martin SE, and Dubeau L (1998) Detection of Ovarian Cancer Cells: Comparison of a telomerase assay and cytologic examination. J. Natl. Cancer Instit. 90, 238-242. Faraoni I, Graziani G, Turriziani M, Masci G, Mezzetti M, Testori A, Veronesi U, and Bonmassar E (1999) Suppression of telomerase activity as an indicator of drug-induced cytotoxicity against cancer cells: in vitro studies with fresh human tumor samples. Lab. Invest. 79, 993-1005. Feng J, Funk W, Wang S, Weinrich S, Avilion A, Chiu C-P, Adams R, Chang E, Yu J, Le S, West M, Harley CB, Andrews W, Greider CW, and Villeponteau B (1995) The human telomerase RNA component. Sci. 269, 1236-1241. Fu W, Begley JG, Killen MW, and Mattson MP (1999) Antiapoptotic role of telomerase in pheochromocytoma cells. J. Biol. Chem. 274, 7264-7271. Fu W, Killen M, Culmsee C, Dhar S, Pandita TK, and Mattson MP (2000) The catalytic subunit of telomerase is expressed in developing brain neurons and serves a cell survival-promoting function. J. Mol. Neurosci. 14, 3-15. Gavathiotis E, Heald RA, Stevens MF, and Searle MS (2003) Drug recognition and stabilization of the parallel-stranded DNA quadruplex d(TTAGGGT)4 containing the human telomeric repeat. J. Mol. Biol. 334, 25-36. Geary RS, Yu RZ, and Levin AA (2001) Pharmacokinetics of phosphorothioate antisense oligodeoxynucleotides Curr. Opin. Investigat. New Drugs 2, 562-573. Gordon A, Carmichael J, and Malfetano J (1997) Final analysis of a phase III randomized study of topotecan vs paclitaxel in advanced epithelial ovarian cancer. Intl. Topotecan Study Group 17, 356a. Greenlee RT, Murray T, and Bolden S (2000) Cancer statistics, 2000. Can. Cancer J. 50, 7-33. Hahn WC, Stewart SA, Brooks MW York SG, Eaton E, Kurachi A, Beijersbergen RL, Knoll JH, Meyerson M, and Weinberg RA (1999) Inhibition of telomerase limits growth of human cancer cells. Nat. Med. 5, 1164-1170. Hastie ND, Dempster M, Dunlop MG, Thompson AM, Green DK, and Allshire RC (1990) Telomere reduction in human colorectal carcinoma and with ageing. Nat. 346, 866-868. Herbert BS, Pitts AE, Baker SI, Hamilton SE, Wright WE, Shay JW, and Corey DR (1999) Inhibition of human telomerase in immortal human cells leads to progressive telomere shortening and cell death. Proc. Natl. Acad. Sci. USA 96, 14276-81. Holt SE, Glinsky VV, Ivanova AB, and Glinsky GV (1999) Resistance to apoptosis in human cells conferred by telomerase function and telomere stability. Mol. Carcinogenesis 25, 241248. Hoos A, Hepp HH, Kaul S, Ahlert T, Bastert G, and Wallwiener D (1998) Telomerase activity correlates with tumor aggressiveness and reflects therapy effect in breast cancer. Intl. J. Cancer 79, 8-12.
Acknowledgements We thank Dr. Nicole C. Johnson and Ms. Yira Bermudez for help with densitometric analysis. This work was supported, in part, by a United States Army Department of Defense New Investigator Award DAMD 17-00-1-0565 (P.A.K.).
References Alfonso-De Matte MY, and Kruk PA (2004) Phosphatidylinositol triphosphate kinase-dependent and cJun NH2-terminal kinase-dependent induction of telomerase by calcium requires Pyk2. Cancer Res. 64, 23-26. Alfonso-De Matte MY, Cheng JQ, and Kruk PA (2001) Ultraviolet irradiation- and dimethyl sulfoxide-induced telomerase activity in ovarian epithelial cell lines. Exp. Cell Res. 267, 13-27. Alfonso-De Matte MY, Moses-Soto H, and Kruk PA (2002a) Calcium-mediated telomerase activity in ovarian epithelial cells. Arch. Biochem. Biophys. 399, 239-244. Alfonso-De Matte MY, Yang H, Evans MS, Cheng JQ, and Kruk PA (2002b) Telomerase is regulated by c-Jun NH2-terminal kinase in ovarian surface epithelial cells. Cancer Res. 62, 4575-4578. Asai A, Kiyozuka Y, Yoshida R, Fujii T, Hioki K, and Tsubura A (1998) Telomere length, telomerase activity and telomerase RNA expression in human esophageal cancer cells: correlation with cell proliferation, differentiation and chemosensitivity to anticancer drugs. Anticancer Res. 18, 1465-1472. Asselin E, Mills GB, and Tsang BK (2001) XIAP regulates Akt activity and caspase-3-dependent cleavage during cisplatininduced apoptosis in human ovarian epithelial cancer cells. Cancer Res 61, 1862-68. Blackledge G, Lawton F, Redman C, and Kelly K (1989) Response of patients in phase II studies of chemotherapy in ovarian cancer: Implications for patient treatment and the design for phase II trials. Br. J. Cancer 59, 650-3. Bookman MA, Malmstrom H, Bolis G, Gordan A, Lissoni A, Krebs JB, and Fields SZ (1998) Topotecan for treatment of advanced epithelial ovarian cancer: an open label phase II study in patients treated after prior chemotherapy that contained cisplatin or carboplatin and paclitaxel. J. Clin. Oncol. 16, 3345-52. Brown, T, Sigurdson E, Rogatko A, and Broccoli D (2003) Telomerase inhibition using Azidothymidine in the HT-29 colon cancer cell line. Ann. Surg. Oncol. 10, 910-915. Chen Z, Koeneman KS, and Corey DR (2003) Consequences of telomerase inhibition and combination treatments for the proliferation of cancer cells. Cancer Res. 63, 5917-5925. Corey DR (2002) Telomerase inhibition, oligonucleotides, and clinical trials. Oncogene 21, 631-637. Counter CM, Hirte HW, Bacchetti S, and Harley CB (1994) Telomerase activity in human ovarian carcinoma. Proc. Natl. Acad. Sci. USA 91, 2900-2904. Counter CM, Meyerson M, Eaton EN, Ellisen LW, Caddle SD, Haber DA, and Weinberg RA (1998) Telomerase activity is restored in human cells by ectopic expression of hTERT (hEST2), the catalytic subunit of telomerase. Oncogene 16, 1217-1222. Creemers GJ, Bolis G, Gore M, Scarfone G, Lacave AJ, Guastalla JP, Despax R, Favalli G, Kreinberg R, Van Belle S, Hudson I, Verweij J, and Ten Bokkel Huinink WW (1996) Topotecan, an active drug in the second line treatment of epithelial ovarian cancer: results of large European phase II study. J. Clin. Oncol. 14, 3056-61.
508
Cancer Therapy Vol 2, page 509 Iida A, Yamaguchi A, and Hirose K (2000) Telomerase activity in colorectal cancer and its relationship to bcl-2 expression. J. Surg. Oncol. 73, 219-223. Kauffman M, and von Minckwitz G (1997) Gemcitabine in ovarian cancer an overview of safety and efficacy. Eur. J. Cancer 33, S31-3. Khatsenko O, Morgan R, Truong L, York-Defalco C, Sasmor H, Conklin B, and Geary RS (2000) Absorption of antisense oligonucleotides in rat intestine: effect of chemistry and length. Antisense Nuc. Acid Drug Dev. 10, 35-44. Kigawa J, Minagawa Y, Ishihara H, Kanamori Y, and Terakawa N (1993) Tumor DNA ploidy and prognosis with serous cystadenocarcinoma of the ovary. Cancer 72, 804-808. Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PLC, Coviello GM, Wright WE, Weinrich SL, and Shay JW (1994) Specific association of human telomerase activity with immortal cells and cancer. Sci. 266, 2011-2015. Kitagawa Y, Kyo S, Takakura M, Kanaya T, Koshida K, Namiki M, and Inoue M (2000) Demethylating reagent 5-azacytidine inhibits telomerase activity in human prostate cancer cells through transcriptional repression of hTERT. Clin. Cancer Res. 6, 2868-2875. Kiyozuka Y, Yamamoto D, Yang J, Uemura Y, Senzaki H, Adachi S, and Tsubura A (2000) Correlation of chemosensitivity to anticancer drugs and telomere length, telomerase activity and telomerase RNA expression in human ovarian cancer cells. Anticancer Res. 20, 203-212. Kohn EC, Sarosy G, Bicher A, Link C, Christian M, Steinberg SM, Rothenberg M, Adamo DO, Davis P, and Ognibene FP (1994) High response rate in patients with platinum resistant recurrent ovarian cancer. J. Natl. Cancer Inst. 86, 18-24. Kondo S, Tanaka Y, Kondo Y, Hitomi M, Barnett GH, Ishizaka Y, Liu J, Haqqi T, Nishiyama A, Villeponteau B, Cowell JK, and Barna BP (1998) Antisense telomerase treatment: induction of two distinct pathways, apoptosis and differentiation. FASEB J. 12, 801-811. Kondo Y, Kondo S, Tanaka Y, Haqqi T, Barna BP, and Cowell JK (1998) Inhibition of telomerase increases the susceptibility of human malignant glioblastoma cells to cisplatin-induced apoptosis. Oncogene 16, 2243-2248. Kosciolek BA, Kalantidis K, Tabler M, and Rowley PT (2003) Inhibition of telomerase activity in human cancer cells by RNA interference. Mol. Cancer Therap. 2, 209-216. Kraveka JM, Li L, Bielawski J, Obeid LM, and Ogretmen B (2003) Involvement of endogenous ceramide in the inhibition of telomerase activity and induction of morphologic differentiation in response to all-trans-retinoic acid in human neuroblastoma cells. Arch. Biochem. Biophys. 419, 110119. Kruk PA, Godwin AK, Hamilton TC, and Auersperg N. (1999) Telomeric instability and reduced proliferative potential in ovarian surface epithelial cells from women with a family history of ovarian cancer. Gynecol. Oncol. 73, 229-236. Kushner DM, Paranjape JM, Bandyopadhyay B, Cramer H, Leaman DW, Kennedy AW, Silverman RH, and Cowell JK (2000) 2-5A Antisense directed against telomerase RNA produces apoptosis in ovarian cancer cells. Gynecol. Oncol. 76, 183-192. Kyo S, Kanaya T, Ishikawa H, Ueno H, and Inoue M (1996) Telomerase activity in gynecological tumors. Clin. Cancer Res. 2, 2023-2028. Kyo S, Kanaya T, Takakura M, Tanaka M, Yamashita A, Inoue H, and Inoue M (1999) Expression of human telomerase subunits in ovarian malignant, borderline and benign tumors. Intl. J. Cancer 80, 804-809. Kyo S, Takakura M, Tanaka M, Kanaya T, and Inoue M (1998b) Telomerase activity in cervical cancer is quantitatively
distinct from that in its precursor lesions. Intl. J. Cancer 79, 66-70. Kyo S, Takakura M, Tanaka M, Murakami K, Saitoh R, Hirano H, and Inoue M (1998a) Quantitative differences in telomerase activity among malignant, premalignant, and benign ovarian lesions. Clin. Cancer Res. 4, 399-405. Lee KH, Rudolph KL, Ju YJ, Greenberg RA, Cannizzaro L, Chin L, Weiler SR, and Depinho RA (2001) Telomere dysfunction alters the chemotherapeutic profile of transformed cells. Proc. Natl. Acad. Sci. USA 98, 3381-3386. Levin AA (1999) A review of the issues in the pharmacokinetics and toxicology of phosphorothioate antisense oligonucleotides. Biochim. Biophys. Acta 1489, 69-84. Lian Z, Fujimoto J, Yokoyama Y, Niwa K, and Tamaya T (2001) Herbal complex suppresses telomerase activity in chemoendocrine resistant cancer cell lines. Eur. J. Gynaec. Oncol. 22, 347-349. Lin X, Cai YJ, Li ZX, Chen Q, Liu ZL, and Wang R (2003) Structure determination, apoptosis induction, and telomerase inhibition of CFP-2, a novel lichenin from Cladonia furcata. Biochim. Biophys. Acta 1622, 99-108. Ludwig A, Saretzki G, Holm PS, Tiemann F, Lorenz M, Emrich T, Harley CB, and von Zglinicki T (2001) Ribozyme cleavage of telomerase mRNA sensitizes breast epithelial cells to inhibitors of topoisomerase. Cancer Res. 61, 30533061 Mandal M and Kumar R (1997) Bcl-2 modulates telomerase activity. J. Biol. Chem. 22, 14183-14187. Mata JE, Joshi SS, Palen B, Pirruccello SJ, Jackson JD, Elias N, Page T, Medlin KL, and Iversen PL (1997) A hexameric phosphorothioate oligonucleotide telomerase inhibitor arrests growth of Burkittâ&#x20AC;&#x2122;s lymphoma cells in vitro and in vivo. Toxicol. Appl. Pharmacol. 144, 189-97. Meyerson M, Counter CM, Eaton EN, Ellisen LW, Steiner P, Caddle SD, Ziaugra L, Beijersbergen RL, Davidoff MJ, Liu Q, Bacchetti S, Haber DA, and Weinberg RA (1997) hEST2, the putative human telomerase catalytic subunit gene, is upregulated in tumor cells and during immortalization. Cell 90, 785-795. Morin GB (1989) The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats. Cell 59, 521-529. Morin GB (1995) Is telomerase a universal cancer target? J. Natl. Cancer Inst. 87, 859-860. Muggia FM, Hainsworth JD, Jeffers S, Miller P, Groshen S, Tan M, Roman L, Usiely B, Muderspach L, Garcia A, Burnett A, Greco FA, Morrow CP, Paradiso LJ, and Liang LJ (1997) Phase II study of liposomal doxorubicin in refractory ovarian cancer: antitumor activity and toxicity modification by liposomal encapsulation. J. Clin. Oncol. 15, 987-93. Murakami J, Nagai N, and Ohama K (1998a) Telomerase activity in body cavity fluid and peritoneal washings in uterine and ovarian cancer. J. Intl. Med. Research 26, 129-139. Murakami J, Nagai N, and Ohama K (1998b) Telomerase activity and telomere length as diagnostic tumor marker for ovarian tumors. Jpn. J. Clin. Med. 56, 1310-1315. Murakami J, Nagai N, Ohama K, Tahara H, and Ide T (1997) Telomerase activity in ovarian tumors. Cancer 15, 1085-1092. Naasani I, Seimiya H, and Tsuruo T (1998) Telomerase inhibition, telomere shortening, and senescence of cancer cells by tea catechins. Biochem. Biophys. Res. Commun. 249, 391-396. Oishi T, Kigawa J, Minagawa Y, Shimada M, Takahashi M, and Terakawa N (1999) Alteration of telomerase activity associated with development and extension of epithelial ovarian cancer. Obstet. Gynecol. 91, 568-571.
509
Shoup et al: Inhibition of telomerase improves chemosensitivity in cisplatin resistance Ozols RF (1997) Treatment of recurrent ovarian cancer: increasing options – recurrent results. J Clin Oncol 15, 2177-80. Park T-W, Reithdorf S, Reithdorf L, Loning T, and Janicke F (1999) Differential telomerase activity, expression of the telomerase catalytic sub-unit and telomerase-RNA in ovarian tumors. Intl. J. Cancer 84, 426-431. Perry PJ, Gowan SM, Reszka AP, Polucci P, Jenkins TC, Kelland LR, and Neidle S (1998) 1,4- and 2,6-Disubstituted amidoanthracene-9,10-dione derivatives as inhibitors of human telomerase. J. Med. Chem. 41, 3253-3260. Pitts AE, and Corey DR (1998) Inhibition of human telomerase by 2’-O-methyl-RNA. Proc. Natl. Acad. Sci. USA 95, 11549-11554. Rose PG, Blessing JA, Mayer AR, and Homesley HD (1998) Prolonged oral etoposide as second line therapy for platinum resistant and platinum sensitive ovarian carcinoma. Clin. Oncol. 16, 405-10. Rowley PT, and Tabler M (2000) Telomerase inhibitors. Anticancer Res. 20, 4419-4430 Sato N, Mizumoto K, Kusumoto M, Nishio S, Maehara N, Urashima T, Ogawa T, and Tanaka M (2000) Up-regulation of telomerase activity in human pancreatic cancer cells after exposure to etoposide. Brit. J. Cancer 82, 1819-1826. Shammas MA, Koley H, Beer DG, Li C, Goyal RK, and Munshi NC (2004) Growth arrest, apoptosis, and telomere shortening of Barrett’s-associated adenocarcinoma cells by a telomerase inhibitor. Gastroenterol. 126, 1337-1146. Shammas MA, Shmookler Reis RJ, Akiyama M, Koley H, Chauhan D, Hideshima T, Goyal RK, Hurley LH, Anderson KC, and Munshi NC (2003) Telomerase inhibition and cell growth arrest by G-quadruplex interactive agent in multiple myleoma. Mol. Cancer Therap. 2, 825-833. Shay JW (1995) Aging and cancer: Are telomeres and telomerase the connection? Mol. Med. Today 1, 378-384. Shay JW, and Bacchetti S (1999) A survey of telomerase activity in human cancer. Eur. J. Cancer 33, 787-791. Summerfeld H-J, Meeker AK, Piatyszek MA, Bova GS, Shay JW, and Coffey DS (1996) Telomerase activity: a prevalent marker of malignant human prostate tissue. Cancer Res. 56, 218-222. Takahashi M, Kigawa J, Oishi T, Itamochi H, Shimada M, Sata S, Kamazawa S, Akeshima R, and Terakawa M (2000) Alteration of telomerase activity in ovarian cancer after chemotherapy. Gynecol. Obstet. Invest. 49, 204-208. ten Bokkel Huinink W, Gore M, Carmichael J, Gordon A, Malfetano J, Hudson I, Broom C, Scarabelli C, Davidson N, Spanczynski M, Bolis G, Malmstrom H, Coleman R, Fields SC, and Heron JF (1997) Topotecan vs. paclitaxel for treatment of recurrent epithelial ovarian cancer. J. Clin. Oncol. 15, 2183-93. Thigpen JT, Blessing JA, Ball H, Hummel SJ, and Banett RJ (1994) Phase II trial of paclitaxel in patients with progressive ovarian carcinoma after platinum based therapy: a Gynecologic Oncology Group Study. J. Clin. Oncol. 12, 1748-53. Tian X-X, Pang JCS, To SST, and Ng H-K (1999) Restoration of wild-type PTEN expression leads to apoptosis, induces differentiation, and reduces telomerase activity in human glioma cells. J. Neuropathol. Exp. Neurol. 58, 472-479.
Togashi K, Kakeya H, Morishita M, Song Y-X, and Osada H (1998) Inhibition of human telomerase activity by alterperylenol. Oncol. Res. 10, 449-453. Villa R, Folini M, Perego P, Supino R, Setti E, Daidone MG, Zunino F, and Zaffaroni N (2000) Telomerase activity and telomere length in human ovarian cancer and melanoma cell lines: correlation with sensitivity to DNA damaging agents. Intl J Oncol. 16, 995-1002. Wan M, Li W-Z, Duggan BD, Felix JC, Zhao Y, and Dubeau L (1997) Telomerase activity in benign and malignant epithelial ovarian tumors. J. Natl. Cancer Instit. 89, 437441. Weinrich SL, Pruzan R, Ma L, Ouellette M, Tesmer VM, Holt SE, Bodnar AG, Lichtsteiner S, Kim NW, Tragewr JB, Taylor RD, Carlos R, Andrews WH, Wright WE, Shay JW, Harley CB, and Morin GB (1997) Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTERT. Nat. Genet. 17, 498-502. Widlak P (2000) The DRR40/CAD endonuclease and its role in apoptosis. Act. Bioc. Polonica 47, 1037-1044. Yamaguchi T, Takahashi H, Jimmei H, Takayama Y, and Saneyoshi M (2003) Inhibition of vertebrate telomerases by the triphosphate derivatives of some biologically active nucleosides. Nucleos. Nucleot. Nuc. Acids. 22, 1575-1577. Yan P, Coindre JM, Benhattar J, Bowman FT, and Guillou L (1999) Telomerase activity and human telomerase reverse transcriptase mRNA expression in soft tissue tumors: correlation with grade, histology and proliferative activity. Cancer Res. 59, 3166-3170. Yin F, Liu J, and Peng X (2003) Triethylene tetraamine: a novel telomerase inhibitor. Bioorgan. Med. Chem. Letters 13, 3923-3926. Yokoyama M, Noguchi M, Nakao Y, Pater A, and Iwasaki T (2004) The tea polyphenol, (-)-epigallocatechin gallete effects on growth, apoptosis and telomerase activity in cervical cell lines. Gynecol. Oncol. 92, 197-204. Zhang X, Mar V, Zhou W, Harrington L, and Robinson MO (1999) Telomere shortening and apoptosis in telomeraseinhibited human tumor cells. Genes Dev. 13, 2388-2399.
Patricia A. Kruk
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Cancer Therapy Vol 2, page 511 Cancer Therapy Vol 2, 511-518, 2004
Current treatment strategies in locally advanced non-small cell lung cancer Review Article
H. Cuneyt Ulutin* and Gorkem Aksu Gulhane Military Faculty of Medicine, Radiation Oncology Department
__________________________________________________________________________________ *Correspondence: Dr. Cuneyt Ulutin; Kizilcik sok. Kizilcikapt. 10/10 Anittepe Ankara Turkey; Telephone: 90-312-3044684; Fax: 90312-3044150; e-mail: culutin@yahoo.com Key words: locally advanced non-small cell lung cancer, chemotherapy, radiotherapy, Abbreviations: American Society of Clinical Oncology, (ASCO); Cancer and Leukemia Group B, (CALGB); compared continuous hyperfractionated accelerated radiotherapy, (CHART); European Organization for Research and Treatment of Cancer, (EORTC); NonSmall Cell Lung Cancer, (NSCLC); radiotherapy, (RT) Received: 30 November 2004; Revised: 6 December 2004 Accepted: 7 December 2004; electronically published: December 2004
Summary Lung cancer is the leading cause of cancer death in both men and women. By the time the patients seek medical attention, the disease is usually locally advanced or metastatic since symptoms of early-stage, localized disease is nonspecific. Complete surgical resection is possible in less than 30% of all lung cancers and the overall 5- year survival rate is less than 15%. 10–15% of lung cancer patients are stage IIIB at the time of initial diagnosis with a median survival of 8 months and a 5-year survival rate of < 5%. Surgery may be indicated in very selected patients for stage IIIB disease and the majority of patients are best managed with chemotherapy plus radiotherapy or with radiotherapy alone. In this review, we evaluated the current treatment strategies in locally advanced lung cancer by regarding the recent results of both phase III and phase II trials. median survival time of 12 months and a 5-year survival rate of 15%, while stage IIIB disease is associated with survival time of 8 months and 5-year survival rate of < 5% (Ries et al, 1983; Mountain, 1997).
I. Introduction Lung cancer is the leading cause of cancer death in both men and women. In the United states alone, it was estimated that, approximately 170,000 new cases of lung cancer is seen per year and that lung cancer will account for 31% of cancer deaths in men and 25% cancer deaths in women, a total of nearly 160,000 deaths (Landis et al, 1999). Cigarette smoking is the main risk factor accounting for about 90% of the lung cancer deaths in men and about 85% in women. The other major occupational and enviromental carcinogenic agents are arsenic, asbestosis, beryllium, chloromethyl ethers, chromicin, hydrocarbons, mustard gas, nickel and radiation (Saccomanno et al, 1976; US Departmant of Health & Human Services, 1988). Symptoms of early-stage, localized disease is insidious and nonspecific, so by the time the patients seek medical attention, the disease is usually locally advanced or metastatic. Complete surgical resection is possible in less than 30% of all lung cancers and the overall 5- year survival rate is less than 15% (Ries et al, 1983). Stage IIIA disease appears to be associated with a better prognosis than stage IIIB disease. According to the results of many studies, stage IIIA is associated with a
II. Surgery in locally advanced lung cancer Surgery may be indicated for stage IIIB disease only in carefully selected situations. Analyzing the patients who had T4N0–1 disease due to a satellite tumor nodule(s) within the primary tumor lobe it was seen that; such patients had a 5-year survival of approximately 20% with surgery alone (Deslauriers et al, 1989; Urschel et al, 1998). In a different study, 5-year survival of patients with T4N0–1 disease due to main carinal involvement who has been treated with carinal resection with or without pulmonary resection was approximately 20% (Darteville et al, 1988). Neoadjuvant chemotherapy, or chemoradiotherapy followed by surgical resection, has been used in patients with N2 (IIIA) disease. A study by the Southwestern Oncology Group employed concurrent chemoradiotherapy in 51 patients with IIIB disease that excluded superior
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Ulutin and Aksu: Current treatment strategies in locally advanced non-small cell lung cancer vena cava syndrome and malignant effusions. The results of this study demonstrated a resectability rate of 80%, with a median survival time of 17 months and a 3-year survival rate of 24% (Albain et al, 1995). These results were similar for patients with IIIA disease reported in the same trial. However, up-to-date there is no phase III trial data that demonstrate, neoadjuvant treatment followed by surgery in patients with IIIB disease results in prolonged survival compared with combination chemoradiotherapy. Still, there are no phase III trial data available to document that surgery adds to survival in patients with stage IIIB Non-Small Cell Lung Cancer (NSCLC) due to T4 (excluding Pancoast tumors) or N3 disease who are treated with neoadjuvant chemotherapy or chemoradiotherapy followed by surgery. The results of limited phase II trials are available so this approach should not be considered as standard therapy. The role of surgery following induction chemotherapy or chemoradiotherapy for patients with initially unresectable cancer is being explored. In phase II testing, the use of concomitant chemoradiotherapy has led to improved resectability and overall survival compared with historical controls in patients with T3 to T4 NSCLC tumors of the superior sulcus (Rusch et al, 2001). The data from surgical series demonstrate that; induction chemotherapy does not result in increased risk of anastomotic complications in bronchoplastic or angioplastic surgical procedures (Roth et al, 1994, 1998; Rosell et al, 1999; Veronesi et al, 2002) but these findings shall be tested in randomized trials that will investigate the effect of induction chemoradiotherapy followed by surgical resection on survival. Depending on sites of tumor involvement and performance score status, the majority of patients with IIIB disease does not benefit from surgery and are best managed with chemotherapy plus radiotherapy or with radiotherapy alone.
favorable performance status (Eastern Cooperative Oncology Group performance status, 0 or 1) and minimal weight loss (< 5% of body weight in the preceding 3 months) were randomized to receive 60 Gy of radiation in 6 weeks or two cycles of induction chemotherapy with cisplatin and vinblastine followed by identical RT. An update of this trial has been published which included 7-year follow-up (Dillman et al, 1996). A total of 78 patients were randomized to chemoradiation, while 77 were randomized to RT alone. The objective response rate was 56% to combined treatment and 43% to RT alone (p = 0.092). The group randomized to induction chemotherapy achieved a significant improvement in median survival compared to the group randomized to RT alone (13.7 vs 9.6 months, respectively; p = 0.012) as well as improvement in the proportion of patients surviving 1, 2, 3, 5, and 7 years (54%, 26%, 24%, 17%, and 13% vs 40%, 13%, 10%, 6%, and 6%, respectively). In The Radiation Therapy Oncology Group and Eastern Cooperative Oncology trial 452 eligible patients were randomized to the same two treatment arms employed in study CALGB 84â&#x20AC;&#x201C;33 and a third arm that included hyper fractionation RT to a total dose of 69.6 Gy (Sause et al, 1995). The hyper fractionation RT arm had been demonstrated to produce a survival advantage for favorable patients in Radiation Therapy Oncology Group protocol 83â&#x20AC;&#x201C;11 (Cox et al, 1990). Preliminary results of the confirmatory trial indicate that the 1-year survival rate and median survival time were superior in the group randomized to receive induction chemotherapy compared to the other two groups (p = 0.03). The 1-year survival rate and median survival time for the three groups are as follows: induction chemotherapy and RT, 60% and 13.8 months, respectively; hyper fractionation RT, 51% and 12.3 months and standard RT, 46% and 11.4 months, respectively. In the European Organization for Research and Treatment of Cancer (EORTC) trial which randomized 331 patients to three arms of 50-Gy thoracic RT in one arm and two other arms in which concurrent cisplatin and RT were utilized on one of two schedules (SchaakeKoning et al, 1992) Cisplatin was administered daily (6 mg/m2/d) along with radiation or weekly (30 mg/m2/wk). A significant survival advantage was obtained in daily cisplatin/radiation therapy arm compared with RT alone arm. 2- and 3-year survival rates were 26% and 16% vs. 13% and 2%, respectively (p = 0.009). For weekly cisplatin and radiotherapy arm, 2- and 3-year survival rates were 19% and 13% with no significant difference from either of the other arms. Improved local control seems to be the cause of the survival benefit observed in daily cisplatin and radiotherapy group (p = 0.003). In a different multicenter study, 353 patients were randomized to receive 65-Gy radiation or to three cycles of induction chemotherapy with cisplatin, vindesine, cyclophosphamide, and lomustine given prior to RT followed by three additional cycles of chemotherapy (Le Chevalier et al, 1991, 1992). Although there was a very high rate of local-regional failure in both groups and local control was 17% in the RT arm and 15% in the combinedmodality arm, the distant failure rate was significantly
III. Induction chemotherapy The use of induction chemotherapy is based on several theoretic considerations. It has been suggested that the early use of the chemotherapy lowers the systemic tumor burden and prevents the growth of microscopic systemic disease, while bulky locoregional macroscopic disease is decreased and addressed more easily by surgery, radiotherapy or both. The role of induction chemotherapy combined with thoracic radiation has been investigated in patients with stage III NSCLC. The main advantage of induction chemotherapy is stage reduction to facilitate improved local control by radiation, surgery, or both. Micrometastases are addressed early in the course of treatment and the response rates to identical chemotherapy regimens appear to be higher when utilized for stage III disease than for stage IV disease. (Green, 1994). Finally, some studies suggest that induction chemotherapy is better tolerated than chemotherapy administered later in the course of treatment (Dillman et al, 1990, 1996; Sause et al, 1995). CALGB 84â&#x20AC;&#x201C;33 phase III trial in stage III NSCLC is one of the most important studies about induction chemotherapy (Dillman et al, 1990). Patients with 512
Cancer Therapy Vol 2, page 513 reduced in the combined-modality arm compared to the RT arm (22% vs. 46% failure at 1 year, respectively; p < 0.001). The response rate to induction chemotherapy was 27%. One, 2-, and 3-year survival rates for the combined chemoradiotherapy arm were 50%, 21%, and 11%, respectively. The one, 2- and 3 year survivals for the radiotherapy arm were 41%, 14%, and 5%, respectively (p = 0.08). With a mean follow-up of 61 months, there was a statistically significant improvement in survival time associated with combined-modality treatment (p < 0.02). A meta-analysis of totally 2589 patients from 14 randomized trials comparing chemotherapy and RT to radiotherapy alone in regionally advanced stage III NSCLC revealed that the use of combination chemotherapy and RT reduces the risk of death by 12% at 1 year, 13% at 2 years, and 17% at 3 years (Pritchard and Anthony, 1996). This means a gain of life expectancy about 2 months. Similarly, the magnitude of benefit was independent of whether sequential or concurrent chemotherapy and RT were utilized. Local-regional recurrence remains a major problem and most of the patients continue to have recurrences and metastatic disease, despite the use of chemoradiation for regionally advanced NSCLC. Green has emphasized that patterns of failure despite induction chemotherapy and RT mandate better control of both macroscopic intrathoracic disease and distant micrometastatic disease in the setting of regionally advanced NSCLC (Green, 1995). In study CALGB 84â&#x20AC;&#x201C;33, the group treated with chemoradiation had an 80% incidence of local-regional failure, while the group treated with RT alone had a 90% incidence of local-regional failure. Phase II trials of induction chemotherapy and surgical resection in regionally advanced stage III NSCLC varied considerably with respect to many factors. The reasons of these variations are the usage of concomitant or sequential chemoradiotherapy in the studies, the differentiation in the choice and doses of chemotherapeutic agents, usage of different techniques in surgical staging of the mediastinum, different radiotherapy schedules, and also differences in the definition of resectable disease. Such inconsistencies have led to considerable difficulties in the interpretation of these trials (Skarin et al, 1989; Weiden and Piantadosi, 1991; Burkes et al, 1992; Reddy et al, 1992; Strauss et al, 1992; Martini et al, 1993; Elias et al, 1994, 1997; Jeremic et al, 1995; Sugarbaker et al, 1995; Choi et al, 1997). Seven of these trials used preoperative RT (in six trials, RT was administered concurrently with chemotherapy, and in one trial it was administered sequentially), two trials used only postoperative RT, and two trials did not employ RT. Cisplatin-based combination chemotherapy regimens were used in all of the trials. Response rates to induction therapy varied from 39 to 77%. Resectability rates exceeded 50% in each of these trials. The highest resectability rate (93%) was noted in a trial that used hyperfractionated RT concurrently with induction chemotherapy (Choi et al, 1997) and the other trials reported pathologic complete response rates in the range of 10 to 20% following induction chemotherapy.
Analyzing the studies it was seen that, only a minority of patients had long-term survival and possible cure. Two multi-institutional trials showed that patients who were found to have negative N2 lymph nodes at the time of resection had a significantly better survival rate than those with persistent N2 positive disease at resection (Albain et al, 1995; Sugarbaker et al, 1995). Albain et al, (2003) reported the first outcome analysis of North American Intergroup trial 0139 (RTOG 93-09) which is a phase III study of concurrent chemotherapy and full course radiotherapy (CT/RT) versus CT/RT induction followed by surgical resection for stage IIIA(pN2) non-small cell lung cancer. 392 patients were evaluated. All patients had induction with cisplatin 50 mg/m2 d1,8 and etoposide 50 mg/m2 and daily RT to 45 Gy starting day 1. Arm 1 underwent resection if no progression (PD) foolowed by two cycles of cisplatin and etoposide. Arm 2 received uninterrupted RT to 61 Gy and 2 cycles of cisplatin-etoposide. Induction cemoradiotherapy compliance was excellent (95%). Third anf fourth cycles of cisplatin and etoposide were not received in 42% of patients in arm 1 and 21% of patients in arm 2 (p<0.0001). RT was per protocol in 97% of patients in arm 1 and 82% of patients in arm 2 (p= 0.002). Most common grade 3/4 toxicities from cemoradiotherapy were neutropenia, emesis and esophagitis (9% in arm 1 and 20% in arm 2, p= 0.001). Arm 1 had 14 deaths from treatment (6.9%), most of which were ARDS (postop, 4; during/after consolidation, 10). In Arm 2, 3 deaths (1.6%) occurred with/after consolidation. Patterns of failure were similar. The pathologic complete response rate was 36% in arm 1 and progression free survival was also superior in arm 1 (median, 14.0 vs 11.7 monhs; 3-year 29% vs 19%) (p= 0.02). The median survival for each arm was 22 months (OS p=0.51). These first results showed that; the pathologic complete response rate in this trial was significantly higher than previous trials and chemoradiotherapy followed by surgery results in superior progression free survival. Final report is expected to determine if surgery significantly prolongs survival in stage IIIA(pN2) NSCLC (Albain et al, 2003). None of these trials were designed to evaluate the therapeutic role of surgery in the context of regionally advanced disease, but the addition of surgery to the localregional treatment regimen almost certainly accomplishes an improvement in local control. Local recurrence has generally been observed in < 50% of patients who undergo trimodality therapy. This contrasts with an 80 to 90% rate of persistent or recurrent local-regional disease among those patients who do not undergo resection. Thus, these phase II trials employing chemotherapy, RT, and surgery demonstrate a shift in recurrence patterns from both local and distant to predominantly distant. Furthermore, improved local control rates may result in significant gains in overall survival times as has been demonstrated in some other studies (Strauss et al, 1992; Jeremic et al, 1995). Reports of small-randomized trials comparing induction chemotherapy followed by surgical resection to resection without systemic treatment have been influential in modifying the perception of the role of chemotherapy in the management of regionally advanced NSCLC.
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Ulutin and Aksu: Current treatment strategies in locally advanced non-small cell lung cancer A study from Spain, randomized patients to an induction chemotherapy regimen of cisplatin, mitomycin C, and ifosfamide followed by resection and postoperative RT (50 Gy) or to resection and the same postoperative RT. There was a dramatic threefold survival advantage for those patients randomized to receive induction chemotherapy (Rosell et al, 1994). In the group randomized to chemotherapy, the median survival time was 26 months, while in the group randomized to surgery plus radiotherapy, survival time was 8 months, which was lower than expected (p < 0.001). In a similar trial conducted at MD Anderson Cancer Center, patients were randomized to induction chemotherapy consisting of three cycles of cyclophosphamide, etoposide, and cisplatin followed by resection or to surgical resection alone (Roth et al, 1994). Of note, RT was given to > 50% of patients in both arms of the study. There was a great difference in median survivals between two groups since while the group that received chemotherapy achieved an estimated median survival time of 64 months, the patients who were randomized to surgery alone had a median survival of 11 months (p < 0.008). Similarly, the 3-year survival rate was 56% for the induction chemotherapy group compared with 15% for the surgery-alone group. In randomized trial from the National Cancer Institute; the experimental group was treated with induction cisplatin and etoposide chemotherapy followed by resection and postoperative chemotherapy. The control group underwent immediate surgical resection and postoperative RT (54 to 60 Gy). Patients treated with induction chemotherapy had a superior survival time, but the difference was not statistically significant (28.7 vs. 15.6 months, respectively) (Pass et al, 1992). Preliminary results of CALGB 9314 TRIAL which used induction chemotherapy with cisplatin and etoposide for two cycles followed by resection in one arm, two additional cycles of chemotherapy, and subsequent RT (54 or 60 Gy) in the second arm and a control arm of preoperative RT (40 Gy) followed by resection and postoperative RT (to a total dose of 54 or 60 Gy), show that the median overall survival time was 19 months for the group undergoing induction chemotherapy compared with 23 months for the group receiving preoperative RT (Elias et al, 1997). Although three of these four randomized trials showed a survival benefit with the use of induction chemotherapy (including two trials in which the differences were statistically significant), these studies have significant limitations such as having small number of patients (totally 204 patients), early stoppings and possible imbalance of prognostic factors between the arms (Pass et al, 1992; Roth et al, 1994). Despite the limitations of the randomized induction chemotherapy and surgery trials in stage IIIA NSCLC, the results of these trials lend some support to the conclusion that for favorable patients with stage IIIA NSCLC, induction chemotherapy followed by resection (with or without radiotherapy (RT)) may enhance disease outcome compared with that achieved with resection (with or without RT). While the magnitude of the survival
advantages added by chemotherapy are very likely to have been exaggerated, particularly in the MD Anderson Cancer Center trial, numerous phase II trimodality studies support a similar conclusion, though with a modest degree of effectiveness. In addition, these results for induction chemotherapy with surgery are consistent with numerous phase III studies that have demonstrated that induction chemotherapy with definitive RT improves outcome when compared with thoracic RT alone. The recent results of both phase III and phase II trials provide a basis for optimism that real therapeutic progress is finally being achieved in regionally advanced NSCLC. Further study of therapeutic strategies that incorporate aggressive systemic treatment and maximal local-regional therapy in stage III NSCLC is clearly warranted. In a prospective trial by Radiation Therapy Oncology Group (RTOG), ECOG, and the Southwest Oncology Group (SWOG), patients were randomized to receive 2 months of cisplatin + vinblastine chemotherapy followed by 60 Gy of radiation at 2 Gy per fraction; or 1.2 Gy / fraction radiation twice daily to a total dose of 69.6 Gy, or 2 Gy / fraction of radiation once daily to 60 Gy. Overall survival was statistically superior for the patients receiving chemotherapy and radiation versus the other two arms of the study (13.2 months v 12 months, v 11.4 months, respectively; p = 0.04) (Sause et al, 2000). But; the survival benefit in this trial was less than that seen in the original Cancer and Leukemia Group B (CALGB) trial (Dillman et al, 1996). Distant metastasis was less for patients who received chemotherapy (Komaki et al, 1997). In a review of chemotherapy in NSCLC patients there was also a survival benefit in favour of chemotherapy (Nonsmall Cell Lung Cancer Collaborative Group Anonymous, 2000).
IV. Concurrent chemoradiotherapy
and
sequential
Administration of chemotherapy concurrently with radiation therapy theoretically improves local control by sensitizing the tumor to radiation, while simultaneously treating systemic disease, albeit at the expense of greater local toxicity. Two large phase III studies suggest improvement in both local control and survival with concurrent chemoradiotherapy as compared with sequential chemotherapy followed by radiation for patients with stage III NSCLC. According to Furuse et al, (1999) randomized patients received either concurrent or sequential chemoradiotherapy. The concurrent arm demonstrated statistically significant superiority in response rate (84% v 66%, P = .0002) and median survival time 16.5 v 13.3, P = .040). But myelosuppression was greater in the concurrent arm (Furuse et al, 1999). In RTOG 94-10 trial which randomized patients to three arms: sequential cisplatin + vinblastine followed by thoracic radiation of 60 Gy; concurrent cisplatin + vinblastine with radiation to a total dose of 60 Gy; or cisplatin + etoposide concurrent with radiation to a total dose of 69.6 Gy delivered in twice-a-day fractions., median survival was superior in the concomitant
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Cancer Therapy Vol 2, page 515 chemotherapy plus daily radiation arm compared with the sequential arm (17 months v 14.6 months; P = 0.08). The concomitant chemotherapy plus twice-daily radiation arm demonstrated intermediate survival compared with the other study arms. Nonhematologic toxicity was also higher on the concurrent arms (Curran et al, 2000). A French cooperative group performed a phase III randomized trial of sequential versus concurrent chemoradiotherapy in unresectable IIIA/IIIB patients. The chemotherapy was cisplatin and vinorelbine for 3 cycles, followed by thoracic radiotherapy (66 Gy/33 Fx), or concurrent cisplatin/etoposide, with thoracic radiotherapy followed by cisplatin and vinorelbine. Seventy-five percent of patients had IIIB disease, and over 100 patients were enrolled in each arm. Incidence of grade 3/4 esophagitis was 26% in patients in the concurrent therapy arm vs 0% in the sequential arm. The median survival time was 13.8 months with sequential therapy and 15 months with concurrent therapy. The 2-year survival rates were 23% and 35%, respectively. While there was a trend in favor of concurrent therapy, it was not statistically significant at the time of this preliminary report (Pierre et al, 2001). These studies suggest improvement in both local control and survival with concurrent chemoradiotherapy as compared with sequential chemotherapy followed by radiation for patients with stage III NSCLC. Therefore, based on these large phase III randomized trials, concurrent chemoradiotherapy appears to result in better survival than sequential therapy. It is associated with some increased toxicity, mainly acute esophagitis, and should be reserved for patients with PS 0 or 1 and minimal weight loss.
receiving CHART, but the incidence at 3 months was similar to patients receiving standard radiotherapy, and there was no difference in late morbidity. Recently, a phase III trial of HART (same as CHART except for no treatments on weekends) versus standard radiotherapy after induction chemotherapy had to be closed due to poor accrual in the Eastern Cooperative Oncology Group (Saunders et al, 1997, 1999). Accordingly, at this time we have one phase III trial of CHART that showed superior survival over standard radiotherapy, but it had only 61% of patients with stage IIIA and IIIB disease, and no data are available concerning the combination of CHART and chemotherapy. Additionally, the schedule of radiotherapy 3 times per day seems to have been rejected by radiotherapists in North America. Therefore, at this time, neither CHART nor HART can be recommended as standard therapy. Optional dose, volume, and fractionation schedules are evolving. An RTOG randomized phase III trial in the 1980s demonstrated that 60 Gy produced a nonstatistically significant survival improvement compared with 50 Gy or two different schedules of 40 Gy (Perez et al, 1980). Accordingly, the dose of 60 Gy in 6 weeks has been widely used for 20 years. However, this dose and fractionation schedule results in local control rates of 15 to 30% (Perez et al, 1980; Le Chevalier et al, 1992; Saunders et al, 1997, 1999; Stuschke and Thames, 1997; Sause et al, 2000; Stella et al, 2001; Cancer Care Ontario Practice Guideline Initiative, 2002). Therefore, higher doses may yield better results. Guidelines of the American Society of Clinical Oncology (ASCO), (2004) advise that definitive dose thoracic radiotherapy should be at least 60 Gy in 1.8 to 2.0 Gy fractions. For stage IIIB NSCLC patients, there is no convincing data that hyperfractionated (two or more fractions daily) radiotherapy is superior to standard once daily treatment. Two randomized prospective phase III trials (RTOG 9410 and NCCTG 94-24-52) compared chemotherapy plus either QD RT vs. BID RT. Neither found a survival advantage to BID RT. (Schild, curan). Continuous hyperfractionated accelerated radiotherapy (CHART) was demonstrated in one small trial to be superior to standard once daily therapy. Although incompleted, the findings of the ECOG trial and some other trials which evaluated hyperfractionated radiotherapy and chemotherapy were quite provocative and more investigation of TID fractionation is warranted (Oral et al, 1999; Kirkbride et al, 2002; Schild et al, 2002; Belani et al, 2003). With the use of 3-D-Conformal radiotherapy it is now possible to give higher doses to the tumor volume than standard RT doses. In a recent study from Memorial Sloan-Kettering Cancer Center, seventy-two patients with Stage III NSCLC and gross tumor volumes (GTV) of greater than 100 cc were treated with three-dimensional conformal radiotherapy (3D-CRT). Patients were divided into two groups: those treated to less than 64 Gy (37 patients) and those treated to 64 Gy or higher (35 patients). The 1-year and 2-year local failure rates were 27% and 47% for patients who received 64 Gy or higher, and 61% and 76% for those treated to less than 64 Gy (p = 0.024).
V. Different radiotherapy schedules and fractionation Different radiotherapy schedules and doses are used in some of the trials instead of conventional radiotherapy. In order to reduce long-term normal tissue toxicity by smaller fraction size and to reduce repopulation in rapidly proliferating tumors accelerated radiotherapy schedules are used. Accelerated radiotherapy is defined as the use of two or more fractions of standard fraction size daily to the same conventional total dose as standard radiotherapy, but increasing the number of fractions per week and shortening the overall treatment time. Hyperfractionated accelerated radiotherapy combines the features of accelerated and hyperfractionated regimen. It uses two or three fractions of smaller fraction size daily, delivered over a shorter period of time than conventional therapy. A phase III trial compared continuous hyperfractionated accelerated radiotherapy (CHART) to standard radiotherapy (60 Gy/30 Fx). CHART consisted of three treatments per day (1.5 Gy/Fx), at least 6 h apart, for 12 days without a break (54 Gy). Sixty-one percent of patients were IIIA or IIIB. The 1- and 2-year survival rates for the CHART arm were 63% and 29%, respectively, vs 55% and 20% for standard radiotherapy. Overall, there was a 22% reduction in the relative risk of death (p = 0.008). Acute esophagitis was more severe for patients
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Ulutin and Aksu: Current treatment strategies in locally advanced non-small cell lung cancer The median survival time for patients treated to 64 Gy or higher was 20 months vs. 15 months for those treated to less than 64 Gy (p = 0.068). Multivariate analysis revealed that dose and GTV are predictors of local failure-free survival. A 10 Gy increase in dose resulted in a 36.4% decreased risk of local failure (Rengan et al, 2004). Rosenmann et al. reported the reesults of a phase I/II clinical trial in which 62 Stage IIIA/IIIB inoperable nonsmall-cell lung cancer (NSCLC) patients were treated with two cycles of induction carboplatin/paclitaxel chemotherapy, followed by concurrent weekly carboplatin/paclitaxel with radiation doses escalated from 60 to 74 Gy. 48 patients completed the entire course of treatment. Eight patients (13%) suffered locoregional relapse as the only site of failure. Only 1 patient had Grade 2 radiation pneumonitis. Five patients (8%) had RTOG Grade 3 or 4 esophagitis; 40 (65%) had a Grade 1 or 2 esophagitis. The median survival was 24 months. The survival rate was 50% at 2 years and 38% at 3 years. The results showed that doses of 74 Gy appear to be safe and may possibly contribute to increased survival in patients with inoperable Stage IIIA/IIIB NSCLC (Rosenman et al, 2002). Evaluating the results of these and some other studies it is suggested that administration of higher doses using 3D-CRT improves local control in Stage III NSCLC patients.
symptom relief as second- and third-line treatment in these patients. Having a favorable adverse effect profile at 250 mg/day, Gefitinib is recommended for the treatment of patients with locally advanced or metastatic nonâ&#x20AC;&#x201C;small-cell lung cancer after failure of both platinum-based and docetaxel chemotherapies in the guidelines of ASCO (2004).
VIII. Conclusion As a result for stage IIIB NSCLC patients with PS 0 or 1 and minimal weight loss, concurrent chemoradiotherapy is still recommended as the standard therapy since randomized studies show that concurrent therapy appears to be associated with improved survival vs. sequential therapy. When radiotherapy is combined with chemotherapy, there is no convincing data that hyperfractionated (two or more fractions daily) radiotherapy is superior to standard once daily treatment. Although further study of therapeutic strategies that incorporate aggressive systemic treatment and maximal local-regional therapy in stage III NSCLC is clearly warranted; by regarding the recent results of both phase III and phase II trials it can be hopefully declared that real therapeutic progress is about to be achieved in locally advanced NSCLC.
References
VI. Duration of chemotherapy
Albain KS, Rusch VW, Crowley JJ et al (1995) Concurrent cisplatin/etoposide plus chest radiation therapy followed by surgery for stage IIIA (N2) and IIIB non-small cell lung cancer, mature results of Southwest Oncology Group Phase II Study 8805. J Clin Oncol 13, 1880-1892. Albain KS, Scott CB, Rusch VR, et al (2003) Phase III study of concurrent chemotherapy and full course radiotherapy (CT/RT) versus CT/RT induction followed by surgical resection for stage IIIA(pN2) non-small cell lung cancer (NSCLC): First outcome analysis of North American Intergroup trial 0139 (RTOG 93-09) Lung Cancer 41,Supplement 2, 4. American Society of Clinical Oncology (2004) Treatment of Unresectable Non-Small-Cell Lung Cancer, Guideline, Update 2003. J Clin Oncol 22, 2, 330-353. Belani CP, Wang W, Johnson DH et al (2003) Induction chemotherapy followed by standard thoracic radiotherapy (Std.TRT) vs. hyperfractionated accelerated radiotherapy (HART) for patients with unresectable stage III A & B nonsmall cell lung cancer (NSCLC): Phase III study of the Eastern Cooperative Oncology Group (ECOG 2597). Proc Am Soc Clin Oncol 22, 622. Burkes RL, Ginsberg RJ, Sheperd FA et al (1992) Induction chemotherapy with mitomycin, vindesine, and cisplatin for stage III unresectable non-small cell lung cancer, results of a Toronto phase II trial. J Clin Oncol 10, 580-586. Cancer Care Ontario Practice Guideline Initiative (2002) Altered fractionation of radical radiation therapy in the management of unresectable non-small cell lung cancer. (GPG 7â&#x20AC;&#x201C;12) Choi NC, Carey RW, Daly W et al (1997) Potential impact on survival of improved tumor downstaging and resection rate by preoperative twice-daily radiation and concurrent chemotherapy in stage IIIA non-small cell lung cancer. J Clin Oncol 15, 712-722. Cox JD, Azarnia N, Byhardt RW et al (1990) A randomized phase I-II trial of hyperfractionated radiation therapy with
The optimal duration of chemotherapy for patients with unresectable stage III NSCLC being treated with combined-modality therapy is still unclear and since no benefit was seen in studies when chemotherapy was continued until progression of disease, the duration of initial chemotherapy is recommended to be between two and four cycles of platinum-based therapy, with an upper limit of four cycles in the Guidelines of the ASCO (2004).
VII. Gefitinib in locally advanced lung cancer The use of epidermal growth factor receptor tyrosine kinase inhibitor, gefitinib in patients with advanced NSCLC with disease progression or intolerance to cisplatin or carboplatin and docetaxel is investigated in two phase II trials (Kris et al, 2002; Fukuoka et al, 2003). Two hundred ten patients with advanced NSCLC who were previously treated with one or two chemotherapy regimens (at least one containing platinum) were randomized to receive either 250-mg or 500-mg oral doses of gefitinib once daily. Efficacy was similar for the 250and 500-mg/day groups. Objective tumor response rates were 18.4 and 19.0%. Symptom improvement rates were 40.3% and 37.0%, median progression-free survival times were 2.7 and 2.8 months and median overall survival times were 7.6 and 8.0 months, respectively. Adverse events at both dose levels were generally mild (grade 1 or 2) and consisted mainly of skin reactions and diarrhea. Drugrelated toxicities were more frequent in the higher-dose group. The results showed that; Gefitinib showed clinically meaningful antitumor activity and provided 516
Cancer Therapy Vol 2, page 517 total doses of 60.0 Gy to 79.2 Gy, possible survival benefit with > 69.6 Gy in favorable patients with radiation therapy oncology group stage III non-small cell lung carcinoma, report of radiation therapy oncology group 83–11 J Clin Oncol 8, 1543-1555. Curran WJ Jr, Scott C, Langer C et al (2000) Phase III comparison of sequential vs concurrent chemoradiation for patients with unresected stage III non-small cell lung cancer (NSCLC), Initial report of radiation therapy oncology group (RTOG) 9410. Proc Am Soc Clin Oncol 19, 484. Darteville PG, Khalife J, Chapelier A et al (1988) Tracheal sleeve pneumonectomy for bronchogenic carcinoma, a report of 55 cases. Ann Thorac Surg 46, 68-72. Deslauriers J, Brisson J, Cartier R et al (1989) Carcinoma of the lung, evaluation of satellite nodules as a factor influencing prognosis after resection. Thorac Cardiovasc Surg 97, 504 512. Dillman RO, Herndon J, Seagren SL et al (1996) Improved survival in stage III non-small cell lung cancer, seven-year follow-up of Cancer and Leukemia Group B (CALGB) 8433 trial. J Natl Cancer Inst 88, 1210-1215. Dillman RO, Herndon J, Seagren SL et al (1996), Improved survival in stage III non-small-cell lung cancer, Seven-year follow-up of cancer and leukemia group B (CALGB) 8433 trial. J Natl Cancer Inst 88, 1210-1215. Dillman RO, Seagren S, Propert K et al (1990) A randomized trial of induction chemotherapy plus high-dose radiation versus radiation alone in stage III non-small cell lung cancer. N Engl J Med 323, 940-945. Elias AD, Herndon J, Kumar P et al (1997) A phase III comparison of "best local-regional therapy" with or without chemotherapy for stage IIIA T1–3N2 non-small cell lung cancer (NSCLC), preliminary results. Proc Am Soc Clin Oncol Annu Meet 16, 448a. Elias AD, Skarin AT, Gonin P et al (1994) Neoadjuvant treatment of stage IIIA non-small cell lung cancer, long-term results. Am J Clin Oncol 17, 26-36. Elias AD, Skarin AT, Leong T et al (1997) Neoadjuvant therapy for surgically staged IIIA N2 non-small cell lung cancer (NSCLC). Lung Cancer 17, 147-161. Fukuoka M, Yano S, Giaccone G, et al (2003) Multi-institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small cell lung cancer. J Clin Oncol 21, 2237–46. Furuse K, Fukuoka M, Kawahara M et al (1999) Phase III study of concurrent versus sequential thoracic radiotherapy in combination with mitomycin, vindesine, and cisplatin in unresectable stage III non-small-cell lung cancer. J Clin Oncol 17, 2692-2699. Green MR (1994) Multimodality therapy for solid tumors. N Engl J Med 330, 206-207. Green MR (1995) Multimodality therapy in unresected stage III non-small cell lung cancer, the American Cooperative Groups’ experience. Lung Cancer 12 (suppl 1), 87-94. Jeremic B, Shibamoto Y, Acimovic L et al (1995) Randomized trial of hyperfractionated radiation therapy with or without concurrent chemotherapy for stage III non-small cell lung cancer. J Clin Oncol 13, 452-458. Kirkbride P, Hatton M, Lorigan P et al (2002) Fatal Pulmonary Fibrosis Associated with Induction Chemotherapy with Carboplatin and Vinorelbine Followed by CHART Radiotherapy for Locally Advanced Non-small Cell Lung Cancer. Clinical Oncology 14, 361–366. Komaki R, Scott CB, Sause WT et al (1997), Induction cisplatin/vinblastine and irradiation vs. irradiation in unresectable squamous cell lung cancer, Failure patterns by cell type in RTOG 88-08/ECOG 4588—Radiation Therapy
Oncology Group Eastern Cooperative Oncology Group. Int J Radiat Oncol Biol Phys 39, 537-544. Kris MG, Natale RB, Herbst RS, et al (2002) A phase II trial of ZD1839 (‘Iressa’) in advanced non-small cell lung cancer (NSCLC) patients who had failed platinum- and docetaxelbased regimens (IDEAL 2). Proc Am Soc Clin Oncol, 21, 292a. Landis SH, Murray T, Bolden S, Wingo PA (1999) Cancer statistics 1999. CA Cancer J Clin 49, 8-31. Le Chevalier T, Arriagada R, Quoiz E et al (1991) Radiotherapy alone versus combined chemotherapy and radiotherapy in nonresectable non-small-cell lung cancer, first analysis of a randomized trial in 353 patients. J Natl Cancer Inst 83, 417423. Le Chevalier T, Arriagada R, Tarayre M et al (1992) Correspondence-significant effect of adjuvant chemotherapy on survival in locally advanced non-small cell lung carcinoma [letter]. J Natl Cancer Inst 84, 58. Martini N, Kris M, Fhelinger B et al (1993) Preoperative chemotherapy for stage IIIA (N2) lung cancer, the SloanKettering experience with 136 patients. Ann Thorac Surg 55, 1365-1374. Mountain CF (1997) Revisions in the International System for Staging Lung Cancer. Chest 111, 1710 -1717. Non-small Cell Lung Cancer Collaborative Group Anonymous (2000) Chemotherapy for non-small cell lung cancer. Cochrane Database of Systematic Reviews CD002139. Oral EN, Bavbek S, Kizir A, et al (1999) Preliminary Analysis of a Phase II Study ofPaclitaxel and Continuous Hyperfractionated Accelerated Radiotherapy in Locally Advanced Non-Small Cell Lung Cancer. Lung Cancer 25, 191-198. Pass HI, Pogrebnick HW, Steinberg SM et al (1992) Randomized trial of neoadjuvant therapy for lung cancer, interim analysis. Ann Thorac Surg 53, 992-998. Perez CA, Stanley K, Rubin P et al (1980) A prospective randomized study of various irradiation doses and fractionation schedules in the treatment of inoperable non-oat cell carcinoma of the lung. Cancer 45, 2744-2753. Pierre F, Maurice P, Gilles R et al (2001) A randomized phase III trial of sequential chemoradiotherapy in locally advanced non-small cell lung cancer. Proc Amer Soc Clin Oncol 20, 312a. Pritchard RS, Anthony SP (1996) Chemotherapy plus radiotherapy compared with radiotherapy alone in the treatment of locally advanced, unresectable, non-small cell lung cancer. Ann Intern Med 125, 723-729. Reddy S, Lee MS, Bonomi P et al (1992) Combined modality therapy for stage III non-small cell lung cancer, results of treatment sand patterns of failure. Int J Radiat Oncol Biol Phys 24, 17-32. Rengan R, Rosenzweig KE, Venkatraman E et al (2004) Improved local control with higher doses of radiation in large-volume stage III non-small-cell lung cancer. Int J Radiat Oncol Biol Phys 60, 741-7. Ries LG, Pollack ES, Young JL (1983) Cancer patient survival, Surveillance, Epidemiology and End results program. J Natl Cancer Inst 70, 693 -709. Rosell R, Gomez-Codina J, Camps C et al (1994) A randomized trial comparing preoperative chemotherapy plus surgery with surgery alone in patients with non-small cell lung cancer. N Engl J Med 330, 153-158. Rosell R, Gomez-Codina J, Camps C, et al (1999) Preresectional chemotherapy in stage IIIA non-small-cell lung cancer, A 7year assessment of a randomized controlled trial. Lung Cancer 26, 7-14. Rosenman JG, Halle JS, Socinski MA et al (2002) High-dose conformal radiotherapy for treatment of stage IIIA/IIIB non-
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Ulutin and Aksu: Current treatment strategies in locally advanced non-small cell lung cancer small-cell lung cancer: technical issues and results of a phase I/II trial. Int J Radiat Oncol Biol Phys 54, 348-56. Roth JA, Atkinson EN, Fossella F, et al (1998) Long-term follow-up of patients enrolled in a randomized trial comparing perioperative chemotherapy and surgery with surgery alone in resectable stage IIIA non-small-cell lung cancer. Lung Cancer 21, 1-6. Roth JA, Fossella F, Komaki R et al (1994) A randomized trial comparing preoperative chemotherapy and surgery with surgery alone in resectable stage IIIA non-small cell lung cancer. J Natl Cancer Inst 86, 673-680. Rusch VW, Giroux DJ, Kraut MJ, et al (2001) Induction chemoradiation and surgical resection for non-small cell lung carcinomas of the superior sulcus, Initial results of Southwest Oncology Group Trial 9416 (Intergroup Trial 0160). J Thorac Cardiovasc Surg 121, 472-483. Saccomanno G, Archer VE, Saunders RP et al (1976) Early indices of cancer risk among uranium miners with reference to modifying factors. Ann NY Acad Sci 271, 377-383. Saunders M, Dische S, Barrett A et al (1997) Continuous hyperfractionated accelerated radiotherapy (CHART) vs conventional radiotherapy in non-small cell lung cancer, a randomized multicentre trial. Lancet 350, 161-165. Saunders M, Dische S, Barrett A et al (1999) Continuous, hyperfractionated, accelerated radiotherapy (CHART) vs conventional radiotherapy in non-small cell lung cancer, mature data from the randomized multicentre trial. Radiother Oncol 52, 137-148. Sause W, Kolesar P, Taylor S IV et al (2000) Final results of phase III trial in regionally advanced unresectable non-small cell lung cancer. Chest 117, 358-364. Sause W, Kolesar P, Taylor SI et al (2000), Final results of phase III trial in regionally advanced unresectable non-small cell lung cancer, Radiation Therapy Oncology Group, Eastern Cooperative Oncology Group, and Southwest Oncology Group. Chest 117, 358-364. Sause W, Scott C, Taylor S et al (1995) Radiation Therapy Oncology Group (RTOG) 88–08 and Eastern Cooperative Oncology Group (ECOG) 4588, preliminary results of a phase III trial in regionally advanced, unresectable non-small cell lung cancer. J Natl Cancer Inst 87, 198-205. Schaake-Koning C, Van Den Bogaert W, Dalesio O et al (1992) Effects of concomitant cisplatin and radiotherapy on inoperable non-small cell lung cancer. N Engl J Med 326, 524-530. Schild SE, Stella PJ, Geyer SM et al (2002) Phase III trial comparing chemotherapy plus once-daily or twice-daily radiotherapy in Stage III non-small-cell lung cancer. Int J Radiat Oncol Biol Phys 5, 370-8. Schild SE, Wong WW, Vora SV et al (2002) Phase I study of hyperfractionated accelerated radiotherapy and escalating doses of daily cisplatin for patients with locally advanced non–small-cell lung cancer. Int J Radiat Oncol Biol Phys 54, 729–734. Skarin A, Jochelson M, Sheldon T et al (1989) Neoadjuvant chemotherapy in marginally resectable stage III M0 nonsmall cell lung cancer, long-term follow-up in 41 patients. J Surg Oncol 40, 266-274. Stella P, Marks R, Schild S et al (2001) Phase III trial of chemotherapy either standard radiotherapy or accelerated hyperfractionated thoracic radiotherapy for stage III nonsmall cell lung cancer. Proc Amer Soc Clin Oncol 20, 312a (1245). Strauss GM, Herndon JE, Sherman DD et al (1992) Neoadjuvant chemotherapy and radiotherapy followed by surgery in stage IIIA non-small cell carcinoma of the lung, report of a Cancer and Leukemia Group B phase II study. J Clin Oncol 10, 1237-1244.
Stuschke M, Thames HD (1997) Hyperfractionated radiotherapy of human tumors, overview of the randomized clinical trials. Int J Radiat Oncol Biol Phys 37, 259-267. Sugarbaker DJ, Herndon J, Kohman, LJ et al (1995) Results of Cancer and Leukemia Group B Protocol 8935, a multiinstitutional phase II trimodality trial for stage IIIA (N2) non-small cell lung cancer. J Thorac Cardiovasc Surg 109, 473-485. Urschel JD, Urschel DM, Anderson TM et al (1998) Prognostic implications of pulmonary satellite nodules, are the 1997 staging revisions appropriate? Lung Cancer 21, 83-87. US Departmant of Health & Human Services (1988) The health consequences of smoking, nicotine addiction, a report of the Surgeon General. USHHS, CDC # 88-8406. Veronesi G, Solli PG, Leo F, et al (2002) Low morbidity of bronchoplastic procedures after chemotherapy for lung cancer. Lung Cancer 36, 91-97. Weiden P, Piantadosi S (1991) Preoperative chemotherapy (cisplatin and flourouracil) and radiation therapy in stage III non-small cell lung cancer, a phase II study of the Lung Cancer Study Group. J Natl Cancer Inst 83, 266-272.
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Cancer Therapy Vol 2, page 519 Cancer Therapy Vol 2, 519-524, 2004
Skull base chondrosarcoma Review Article
William M. Mendenhall1,*, Stephen B. Lewis2, Douglas B. Villaret3, and Nancy P. Mendenhall1 1
Radiation Oncology Department Neurosurgery Department 3 Otolaryngology Department, University of Florida College of Medicine, Gainesville, Florida 2
__________________________________________________________________________________ *Correspondence: William M. Mendenhall, M.D., Department of Radiation Oncology, University of Florida Health Science Center, P.O. Box 100385, Gainesville, FL 32610-0385; Telephone: (352) 265-0287; Fax: (352) 265-0759; e-mail: mendewil@shands.ufl.edu Key words: Chondrosarcomas, Base of skull, Radiotherapy, Proton radiotherapy, Treatment outcomes Abbreviations: American Joint Committee on Cancer, (AJCC); cobalt gray equivalents, (CGE); Computed tomography, (CT); epithelial membrane antigen, (EMA); loss of heterozygosity, (LOH); magnetic resonance imaging, (MRI); radiotherapy, (RT); retinoblastoma, (Rb) Received: 18 November 2004; revised: 6 December 2004 Accepted: 7 December 2004; electronically published: January 2005
Summary The purpose of this paper is to discuss the natural history, optimal treatment, and outcomes for skull base chondrosarcomas. The pertinent literature was reviewed. The majority of skull base chondrosarcomas are low grade and exhibit an indolent growth pattern. A small subset of patients present with mesenchymal or dedifferentiated chondrosarcomas and have a poor prognosis. Although extensive skull base resections may result in long disease-free survival, the likelihood of a complete resection with negative margins is low, and the permanent morbidity of these operations is significant. Proton radiotherapy after subtotal resection or biopsy results in a high rate of cure with a relatively low probability of late complications. The preferred treatment for skull base chondrosarcomas is proton radiotherapy alone or combined with less aggressive surgical procedures.
1998; Hug et al, 1999; Rosenberg et al, 1999). Rosenberg et al, (1999) observed a 1:1.3 male to female ratio and an age range of 10 to 79 years (mean, 39 years). Korten et al, (1998) reported on 15 patients with skull base chondrosarcomas treated in the Netherlands, and 177 patients reported in the literature and observed a 1:1.1 male to female ratio and an age range of 3 months to 76 years (mean, 37 years). Patients often present with cranial nerve deficits (usually the abducens nerve), headaches, and symptoms related to temporal bone invasion (Volpe et al, 1993; Korten et al, 1998; Crockard et al, 2001; Raghu et al, 2004). Korten et al, (1998) reported the following presenting symptoms: oculomotor dysfunction, 51%; headaches, 31%; and diminished hearing, dizziness, and tinnitus, 21%. Volpe et al, (1993) evaluated the neuroophthalmologic findings in 48 patients with skull base chordomas and 49 patients with skull base chondrosarcomas and observed abnormal visual examinations in 67% and 94%, respectively. The duration of symptoms before presentation is variable. Korten et al, (1998) observed a range of 1 month to 12 years (mean, 27 months; median, 15 months).
I. Introduction Chondrosarcoma is a relatively rare tumor that may arise in any bone that is preformed by cartilage (Brown et al, 1994). It accounts for 11% to 19% of all primary bone tumors and may arise de novo or in preexisting conditions including Pagetâ&#x20AC;&#x2122;s disease, enchondromas, osteocartilaginous exostoses, Ollierâ&#x20AC;&#x2122;s disease, and osteochondromas (Brown et al, 1994). Skull base chondrosarcomas usually arise de novo and account for approximately 0.15% of all intracranial tumors (Brown et al, 1994; Crockard et al, 2001). Approximately 75% of chondrosarcomas occur in the trunk, femur, or humerus, 5% to 12% arise in the head and neck, and roughly 1% are found in the skull base (Brown et al, 1994). The majority of skull base chondrosarcomas arise in synchondroses near the temporooccipital junction (Rosenberg et al, 1999; Raghu et al, 2004). Rosenberg et al, (1999) reported on 200 patients treated at the Massachusetts General Hospital (Boston) for skull base chondrosarcomas and observed the following site distribution: temporooccipital, 66%; clivus, 28%; and sphenoethmoid complex, 6%. Skull base chondrosarcomas exhibit a roughly equal gender distribution and a wide age range. (Korten et al,
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Mendenhall et al: Skull base chondrosarcoma whereas chondroid chordomas stained positively for both. Eisenberg et al, (1997) analyzed loss of heterozygosity (LOH) of the retinoblastoma (Rb) gene (a tumor suppressor gene found in a number of malignancies) in 7 patients with skull base chordomas and 2 patients with skull base chondrosarcomas. Two of 7 chordomas exhibited LOH compared with 0 of 2 chondrosarcomas; both chordomas with LOH behaved very aggressively (Eisenberg et al, 1997).
II. Pathology Chondrosarcomas may be stratified as conventional, mesenchymal, and dedifferentiated and are graded based on cellularity, nuclear pleomorphism, and mitotic activity (Brown et al, 1994). In contrast to conventional chondrosarcomas, mesenchymal, and dedifferentiated chondrosarcomas exhibit aggressive behavior and portend a poor prognosis (Brown et al, 1994). Conventional chondrosarcomas are composed of round or oval cartilaginous cells with single or multiple nuclei and may contain myxoid changes, calcifications, and/or ossification (Brown et al, 1994; Rosenberg et al, 1999). Mesenchymal chondrosarcomas are composed of islands of cartilage and sheets of undifferentiated small stromal cells with hyperchromatic nuclei (Brown et al, 1994). Differentiated chondrosarcomas exhibit anaplastic foci within a low-grade cartilaginous matrix (Brown et al, 1994). Korten et al, (1998) observed the following histologic distribution: grade 1, 51%; grade 2, 11%; mesenchymal, 30%; and myxoid, 8%. The following grade distribution was reported by Rosenberg et al, (1999) in 200 patients with conventional chondrosarcomas: grade 1, 50.5%; mixed grade 1 and grade 2, 28.5%; and grade 2, 21%. Chondrosarcomas must be distinguished from chondroid chordomas that tend to behave more aggressively and have a worse prognosis. Rosenberg et al, (1999) observed that 96 of 97 chondrosarcomas (99%) stained positively for Sâ&#x20AC;&#x201C;100; none exhibited keratin positivity. Seven of 88 patients (8%) stained faintly for epithelial membrane antigen (EMA) (Rosenberg et al, 1999). Ishida and Dorfman, (1994) analyzed 9 patients with skull base chondrosarcomas and 7 patients with skull base chondroid chordomas and found that chondrosarcomas did not stain for cytokeratin or EMA,
III. Diagnostic evaluation Computed tomography (CT) and magnetic resonance imaging (MRI) are employed to evaluate the primary tumor; chest CT should be obtained in patients with poorly differentiated tumors. CT generally demonstrates a lytic lesion and is used to demonstrate the extent of bone invasion and tumor mineralization (Brown et al, 1994; Crockard et al, 2001; Maleuz et al, 1996). Contrast-enhanced CT often shows moderate enhancement (Maleuz et al, 1996). MRI is useful to demonstrate the soft tissue extent of the tumor. T1-weighted gadolinium DTPA enhanced MRI shows a hyperintense mass in the portions of the tumor that are non-calcified; the calcified part of the tumor exhibits a mixture of hypo- and hyperintensity (Maleuz et al, 1996). T2-weighted contrast-enhanced MRI reveals a hyperintense tumor with areas of inhomogeneity corresponding to the calcified portions of the mass (Maleuz et al, 1996).
IV. Staging Patients are staged according to the recommendations of the American Joint Committee on Cancer (AJCC) (2002) staging system (Table 1).
Table 1. American Joint Committee on Cancer Staging System, 2002 DEFINITION OF TNM Primary Tumor (T) TX
Primary tumor cannot be assessed
T0
No evidence of primary tumor
T1
Tumor 8 cm or less in greatest dimension
T2
Tumor more than 8 cm in the greatest dimension
T3
Discontinuous tumors in the primary bone site
Regional Lymph Nodes (N) NX
Regional lymph nodes cannot be assessed
N0
No regional lymph node metastasis
N1
Regional lymph node metastasis
Distant Metastasis (M) MX
Distant metastasis cannot be assessed
M0
No distant metastasis
M1
Distant metastasis
M1a
Lung
M1b
Other distant sites
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Cancer Therapy Vol 2, page 521 Stage Grouping Stage 1A
T1
N0
M0
G1, 2 Low grade
Stage 1B
T2
N0
M0
G1, 2 Low grade
Stage IIA
T1
N0
M0
G3, 4 High grade
Stage IIB
T2
N0
M0
G3, 4 High grade
Stage III
T3
N0
M0
Any G
Stage IVA
Any T
N0
M1a
Any G
Stage IVB
Any T
N1
Any M
Any G
Any T
Any N
M1b
Any G
Histologic Grade (G) GX
Grade cannot be assessed
G1
Well differentiated - Low grade
G2
Moderately differentiated - Low grade
G3
Poorly differentiated - High grade
G4
Undifferentiated - High grade
patient died postoperatively after a fourth resection 3 years after the first surgical procedure. Crockard et al, (2001) reported on 17 patients with low-grade (15 patients) or mesenchymal (2 patients) skull base chondrosarcomas treated surgically at St. Bartholomewâ&#x20AC;&#x2122;s Hospital (London) between 1986 and 1998. Two patients had previous surgery, 3 patients had previous surgery and RT, and 2 patients had previous surgery and chemotherapy. Survival was calculated from the time of tissue diagnosis, leading to significant leadtime bias in the 7 previously treated patients. All 17 patients underwent resection; none of the resections resulted in complete tumor removal. The 2 patients with mesenchymal chondrosarcomas died at 1.7 and 3 years, respectively. The 5-year overall survival rate for the 15 patients with low-grade chondrosarcomas was 93%. However, some of those patients were previously treated and, as previously stated, the method of outcomes analysis resulted in significant lead-time bias. Gay et al, (1995) reported on 60 patients with skull base chondrosarcomas (14 patients) and chordomas (46 patients) treated surgically at the University of Pittsburgh (PA) between 1984 and 1993. Thirty of 60 patients (50%) were previously treated. Sixty-seven percent of the 60 patients had a total or near total resection and 20% received postoperative RT. The 5-year recurrence-free survival rate for the 14 patients with chondrosarcomas was 90%. Raghu et al, (2004) reported on 3 patients with temporal bone chondrosarcomas treated with surgery and postoperative RT at Addenbrookes Hospital (Cambridge, UK). All 3 patients were alive and disease-free at 5, 6, and 8 years, respectively.
V. Treatment The treatment of skull base chondrosarcoma is controversial and varies from skull base resection alone or combined with conventional radiotherapy (RT) or charged particle RT to less aggressive operations combined with proton RT (Ruark et al, 1992; Stapleton et al, 1993; Gay et al, 1995; Berson et al, 1988; Hug et al, 1999; Rosenberg et al, 1999, Crockard et al, 2001). There are few data pertaining to the efficacy of stereotactic radiosurgery (Muthukumar et al, 1998). It is difficult to compare the outcomes of various treatment strategies because skull base chondrosarcomas are uncommon, the majority are low grade and exhibit an indolent growth pattern, and the outcomes data are sometimes combined with those of chordomas which have a more unfavorable prognosis.
VI. Localâ&#x20AC;&#x201C;regional survival
control
and
A. Surgery Stapleton et al, (1993) reported on 8 patients with skull base chondrosarcomas treated surgically at Atkinson Morleyâ&#x20AC;&#x2122;s Hospital (London) between 1985 and 1991. Two patients were operated on after treatment failures from previous RT (1 patient) or surgery (1 patient). No patient received proton RT. One patient underwent an operation; the remaining 7 patients underwent 2 operations (4 patients), 4 operations (2 patients), and 5 operations (1 patient), respectively. One patient received postoperative RT. Three patients were alive and disease-free at 7, 9, and 10 years, respectively. One patient was alive with the disease at 4.8 years, 2 patients died with disease at 2 and 8 years, 1 patient was lost to follow-up at 1 year, and 1
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Mendenhall et al: Skull base chondrosarcoma
B. Radiotherapy
patients (3%); meningitis, 6 patients (10%); brain infarct, 2 patients (3%); and death, 2 patients (3%). Forty-eight patients (80%) had a new cranial nerve deficit (usually the 6th cranial nerve) after the operation, 15% had hearing loss (usually partial), 8% had 7th cranial nerve paralysis or paresis, and 8% had visual loss or decline. Overall, 24 of 60 patients (40%) had a permanent functional decline after the operation, usually 10 points on the Karnofsky performance scale. Raghu et al, (2004) reported on 3 patients treated with surgery and postoperative RT for temporal bone chondrosarcomas; all 3 had cranial nerve injuries after the operation.
Berson et al, (1988) reported on 13 patients with chondrosarcomas of the skull base or cervical spine treated with subtotal resection and charged particle RT alone or combined with photons at the University of California Lawrence Berkeley Laboratory between 1977 and 1986. The 5-year local control and survival rates were approximately 77% and 72%, respectively. Three patients (23%) developed distant metastases; all 3 had grade 2 chondrosarcomas. Castro and co-workers, (1994) reported an update of their experience that included 27 patients with skull base chondrosarcomas: the 5-year local control and survival rates were 78% and 83%, respectively. Hug et al, (1999) reported on 25 patients treated with proton RT at Loma Linda University Medical Center (CA) between 1992 and 1998 for skull base chondrosarcomas. Two of 25 patients (8%) were previously treated, 9 patients (36%) had brainstem invasion, and 21 patients (84%) had gross tumor present at the time of proton RT. Twenty-three of 25 patients (92%) were locally controlled and remained disease-free after treatment. Noël et al, (2003) reported on 67 patients treated with proton RT for skull base or cervical spine chondrosarcomas (18 patients) or chordomas (49 patients) between 1995 and 2000 at the Centre de Protonthérapie d’Orsay (Orsay, France). Median follow-up was 20 months. Two-thirds of the treatment was delivered with photons and one-third with protons. Four of 18 patients with chondrosarcomas underwent gross total resection, 11 had a subtotal resection, and 3 had a biopsy before proton RT. Thirteen patients had no previous therapy and 5 patients were treated for locally recurrent disease. The 3year local control and survival rates after proton RT were 85% and 75%, respectively. Rosenberg et al, (1999) reported on 200 patients with grade 1 and 2 skull base chondrosarcomas treated at the Massachusetts General Hospital and Harvard Cyclotron (Boston, MA) between 1978 and 1997. Five percent of patients had a gross total resection, 74% had a subtotal resection, and 21% had a partial resection or biopsy before proton RT. Follow-up ranged from 2 months to 18.5 years (mean, 65 months). Patients received a median dose of 72.1 cobalt gray equivalents (CGE) in 38 fractions (range, 64.2 to 79.6 CGE). The 10-year local control and causespecific survival rates were 98% and 99%, respectively. No patient experienced hematogenous dissemination. Austin et al, (1993) analyzed the cause of failure in 3 patients treated with proton RT at the Massachusetts General Hospital; two recurrences were marginal and likely due to low doses in regions where the dose was constrained because of the risk of normal tissue toxicity, and one recurrence was within the high-dose volume.
B. Radiotherapy Hug et al, (1999) reported on 58 patients treated with proton RT for skull base chondrosarcomas (25 patients) and chordomas (33 patients); 3 patients (5%) experienced late symptomatic complications including severe unilateral hearing loss (1 patient), a single focal seizure (1 patient) and significant bilateral loss of hearing and vision (1 patient). Noël et al, (2003) reported on 67 patients treated with proton RT for skull base and cervical spine chondrosarcomas (18 patients) and chordomas (49 patients). Sixteen patients (24%) experienced total (14 patients) or partial (2 patients) hypopituitarism, 12 patients (18%) had mild hearing loss, and 4 patients (6%) experienced severe late complications including oculomotor impairment (2 patients), severe hearing loss (1 patient), and near complete bilateral visual loss (1 patient).
VIII. Conclusion Skull base chondrosarcoma is rare, usually low grade, and exhibits an indolent growth pattern. Although patients can be treated with aggressive resection, the probability of complete resection with negative margins is low and the permanent morbidity of these procedures is often significant. Charged particle RT (such as protons or carbon ions) alone or combined with subtotal resection results in a high probability of cure and relatively low risk of toxicity.
References American Joint Committee on Cancer (2002) Bone. In: American Joint Committee on Cancer. AJCC Cancer Staging Manual. New York: Springer, 187-92 Austin JP, Urie MM, Gardenosa G, Munzenrider JE (1993) Probable causes of recurrence in patients with chordoma and chondrosarcoma of the base of skull and cervical spine. Int J Radiat Oncol Biol Phys 25, 439-44 Berson AM, Castro JR, Petti P, Phillips TL, Gauger GE, Gutin P, Collier JM, Henderson SD, Baken K (1988) Charged particle irradiation of chordoma and chondrosarcoma of the base of skull and cervical spine: The Lawrence Berkeley laboratory experience. Int J Radiat Oncol Biol Phys 15, 559-65 Brown E, Hug EB, Weber AL (1994) Chondrosarcoma of the skull base. Neuroimaging Clin N Am 4, 529-41 Castro JR, Linstadt DE, Bahary JP, Petti PL, Daftari I, Collier JM, Gutin PH, Gauger G, Phillips TL (1994) Experience in
VII. Complications A. Surgery Gay et al, (1995) reported on 60 patients treated surgically at the University of Pittsburgh (PA) for skull base chondrosarcomas (14 patients) and chordomas (46 patients). Postoperative complications for the overall group of 60 patients included cerebrospinal fluid leak, 18 522
Cancer Therapy Vol 2, page 523 charged particle irradiation of tumors of the skull base: 19771992. Int J Radiat Oncol Biol Phys 29, 647-55. Crockard HA, Cheeseman A, Steel T, Revesz T, Holton JL, Plowman N, Singh A, Crossman J (2001) A multidisciplinary team approach to skull base chondrosarcomas. J Neurosurg 95, 184-9 Eisenberg MB, Woloschak M, Sen C, Wolfe D (1997) Loss of heterozygosity in the rhinoblastoma tumor suppressor gene in skull base chordomas and chondrosarcomas. Surg Neurol 47, 156-61 Gay E, Sekhar LN, Rubinstein E, Wright DC, Sen C, Janecka IP, Snyderman CH (1995) Chordomas and chondrosarcomas of the cranial base: Results and follow-up of 60 patients. Neurosurgery 36, 887-97 Hug EB, Loredo LN, Slater JD, DeVries A, Grove RI, Schaefer RA, Rosenberg AE, Slater JM (1999) Proton radiation therapy for chordomas and chondrosarcomas of the skull base. J Neurosurg 91, 432-9 Ishida T, Dorfman HD (1994) Chondroid chordoma versus lowgrade chondrosarcoma of the base of the skull: Can immunohistochemistry resolve the controversy? J NeuroOncol 18, 199-206 Korten AGGC, ter Berg HJW, Spincemaille GH, van der Laan RT, Van de Wel AM (1998) Intracranial chondrosarcoma: Review of the literature and report of 15 cases. J Neurol Neurosurg Psychiatry 65, 88-92 Maleux G, Demaerel P, Vanslambrouck K, Aerts P, Brijs S, Tanghe W (1996) Chondrosarcoma of the skull base: CT, MR and pathological features. ROFO (or Fortschr Rontgenstr) 165, 599-601 Muthukumar N, Kondziolka D, Lunsford LD, Flickinger JC (1998) Stereotactic radiosurgery for chordoma and chondrosarcoma: Further experiences. Int J Radiat Oncol Biol Phys 41, 387-92 NoĂŤl G, Habrand JL, Jauffret E, de Crevoisier R, Dederke S, Mammar H, Haie-Meder C, Pontvert D, Hasboun D, Ferrand R, Boisserie G, Beaudre A, Gaboriaud G, Guedea F, Petriz L, Mazeron JJ (2003) Radiation therapy for chordoma and chondrosarcoma of the skull base and cervical spine.
Prognostic factors and patterns of failure. Strahlenther Onkol 179, 241-8 Raghu M, Moumoulidis I, De R, Moffat D (2004) Chondrosarcomas of the temporal bone: Presentation and management. J Laryngol Otol 118, 551-5 Rosenberg AE, Nielsen GP, Keel SB, Renard LG, Fitzek MM, Munzenrider JE, Liebsch NJ (1999) Chondrosarcoma of the base of the skull. A clinicopathologic study of 200 cases with emphasis on its distinction from chordoma. Am J Surg Pathol 23, 1370-8 Ruark DS, Schlehaider UK, Shah JP (1992) Chondrosarcomas of the head and neck. World J Surg 16, 1010-5 Stapleton SR, Wilkins PR, Archer DJ, Uttley D (1993) Chondrosarcoma of the skull base: A series of eight cases. Neurosurgery 32, 348-56 Volpe NJ, Liebsch NJ, Munzenrider JE, Lessell S (1993) Neuroophthalmologic findings in chordoma and chondrosarcoma of the skull base. Am J Ophthalmol 115, 97-104
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Lymphokine-activated killer (LAK) cell activity is not a continuously inducible activity: implications for LAK cell and IL-2 immunotherapy Research Article
David T. Harris* Dept. Microbiology & Immunology, 1501 N. Campbell Avenue, Rm. 643, Life Sciences North, University of Arizona, Tucson, AZ 85724
__________________________________________________________________________________ *Correspondence: Dr. David T. Harris, Dept. Microbiology & Immunology, 1501 N. Campbell Avenue, Rm. 643 Life Sciences North, University of Arizona, Tucson, AZ 85724, Tel: (520) 626-5127; FAX: (520) 626-2100; davidh@u.arizona.edu Key words: LAK cells, Immunotherapy, IL-2 Abbreviations: Interleukin-2, (IL-2); Lymphokine-activated killer, (LAK); natural killer, (NK); Peripheral blood, (PBL) Received: 3 December 2004; Accepted: 14 December 2004; electronically published: January 2005
Summary Despite advances in conventional cancer therapy, many patients continue to succumb to their malignancies. In part, this failure is due to the difficulty in successfully treating metastatic disease present at or after the discovery of the primary tumor. Augmentation of the patient’s own immune system to combat the malignancy through the use of immunotherapy has been proposed to supplement conventional therapy. The use of lymphokines (e.g., IL-2 and IFN-!) and cellular therapy (e.g., LAK cells) have been the methods most commonly utilized, although with limited success. We have investigated the mechanisms of LAK cell/IL-2 therapy in order to understand its limitations and to investigate methods by which to overcome these problems. In cultures of peripheral blood lymphocytes stimulated with IL-2 it was observed that despite the continual presence of IL-2, LAK cell activity diminished with time in culture even if the cells were washed and recultured in fresh media plus IL-2. LAK cell activity never reattained initial levels of activity upon subsequent IL-2 stimulation. Further, despite continual restimulation with IL-2 (every third day or once every 2 weeks) LAK cell activity continued to decline. LAK cell activity diminished at a faster rate than conventional NK cell activity. Loss of cell viability was not a factor in the loss of lytic activity as cultures maintained at least 70% viability, and all assays were adjusted for viable cell numbers. Flow cytometric analyses revealed that CD3-16+ cell numbers decreased with time in culture, while CD3+8+ cells increased. Cells expressing activation markers increased with time (i.e., CD56+, HLA-DR+, CD45RA-), but quickly reached a plateau and then declined. Thus, one potential reason why LAK cell/IL-2 immuunotherapy may not be as successful as expected in the treatment of cancer is that LAK cell activity is in actuality an activity that has limited inducibility, and that this activity rapidly declines despite the continual presence of IL-2 and viable effector cells. adjunct to the therapy of acute leukemia (Klein et al, 1990; Taylor and Hersh, 1990). Although it was hoped that the immune system would be activated to kill the cancer and limit metastatic disease, success was limited (Taylor and Hersh, 1990). Investigations have been primarily focused upon the role of nonspecific immune augmentation in devising immunotherapeutic clinical protocols (e.g., BCG, C. parvum, thymic hormones, etc) (Taylor and Hersh, 1990). More recently, emphasis has shifted to the use of cytokines as a means to activate nonspecific immune mechanisms such as natural killer (NK) cells (Rosenberg et al, 1985; Brunda et al, 1987; Winkelhake et al, 1987), particularly since these cells display an innate cytotoxicity towards a variety of tumor cells (Grimm et al, 1982). NK
I. Introduction Despite formidable advances in conventional cancer therapy (i.e., chemotherapy, surgery and radiation), many patients succumb to either recurrence or metastatic spread of their primary malignancy. It has been proposed that if it were possible to stimulate the patient’s own immune system to recognize the cancer it should be possible to overcome these problems (Klein, 1967, 1968, 1969). Attempts to utilize the patient’s immune system to combat primary and metastatic disease has a long history, dating back to the use of intralesional BCG injections to treat malignant melanoma (Klein et al, 1990; Taylor and Hersh, 1990), and the use of BCG and tumor cell vaccines as an
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Harris: Limited LAK cell responses to IL-2 LAK cell cultures were performed using the following two conditions. PBL were cultured in 100mm petri dishes for the indicated periods of time at 1x106 cells/ml in RPMI-1640 media supplemented with antibiotics, nonessential amino acids, glutamine and 10% fetal bovine serum (Hyclone, Logan, UT). In culture condition “A”, IL-2 was added to the cultures at 1000 U/ml, the cultures were harvested and washed and viable cells replated/restimulated as above every two weeks in the presence of additional IL-2. In culture condition “B”, IL-2 was added to the cultures at 1000 U/ml every 3 days. At the end of two weeks of culture, the cells were harvested, washed and viable cells replated/restimulated as above with IL-2. At each of the indicated timepoints a constant 5ml was removed from the cultures and used for the assays. All assays were performed based on viable cell numbers.
cells are thought to be intimately involved in tumor surveillance and have been shown in animal models to prevent experimental tumors and metastases (Mule et al, 1984, 1985; Lafreniere and Rosenberg, 1985). Rosenberg et al, (1985) were the first group to take advantage of the lytic properties of NK cells in the clinical situation by incubating patient lymphocytes in IL-2 (to generate LAK cells), and then reinjecting the effector cells in combination with the lymphokine (Rosenberg et al, 1985). This type of immunotherapy has shown limited success, and has been complicated by the often serious side-effects of high-dose IL-2 injections (Rosenberg et al, 1987; Rosenberg, 1988). Although NK/LAK cells work well in vitro and in vivo in animal models (Mule et al, 1984, 1985; Lafreniere and Rosenberg, 1985), a possible explanation for its limited clinical success may be that these effector cells act differently in humans. Alternatively, there could be a problem in effector localization, and the loss of lytic activity in vivo also must be considered. In the present study an investigation has been made to analyze the mechanisms of LAK cell activity in an attempt to understand why LAK cell therapy is not as successful as expected in the clinical setting. It was observed that LAK cell activity (i.e., cytolytic activity versus NK cell-resistant tumor cells) was not an activity that could be maintained for long periods of time, and was not reinducible to any significant extent after the initial stimulation with IL-2, although typical NK cell activity persisted for longer periods of time. Further, typical NK cells were rapidly lost from the LAK cell cultures, being replaced by T cells. By a variety of analyses, activated T cells capable of LAK cell activity were also lost with time in culture and replaced by T cells incapable of LAK cell cytolysis. These observations were found whether IL-2 was continuously present (i.e., exogenous IL-2 added every 3 days) or if IL-2 was added every other week. Thus, LAK cell activity is not a continuously inducible activity, and this finding may explain why this type of immunotherapy has not been more successful clinically.
D. Cytotoxicity assays Cytotoxic effector cell responses were determined in a standard 4h 51Cr-release assays (Koren et al, 1981). Data were calculated as lytic units at the 20% specific lysis level (LU20) by computer-assisted regression analysis according to Pross et al (1981). The following tumor cell lines were used in the experiments: K562 (human erythroleukemia, HLA-negative, sensitive to NK cell lysis) and IM9 (human B cell leukemia, HLA-positive, resistant to NK cell lysis).
E. Flow cytometric analyses Phenotypic analyses of PBL samples and cultures were performed by two-color flow cytometry using a BectonDickinson FACStar Plus flow cytometer (Mountain View, CA). A minimum of 10,000-20,000 gated events were analyzed for each sample. Data were analyzed as previously described (Harris et al, 1992).
III. Results A. Kinetics of NK and LAK cell activity in long-term IL-2 cultures Long-term LAK cell cultures (40 days) were set up as described and assessed for NK and LAK cell lytic activity at various timepoints. As shown in Figure 1, NK cell cytolysis as assessed by lysis of K562 target cells, was rapidly augmented (by day 2) upon IL-2 addition at day 0. IL-2 addition every 3 days augmented NK cell activity above that of IL-2 addition every 2 weeks. NK cell cytolysis peaked at day 12-14 of culture regardless of the schedule of IL-2 addition, rapidly declined, and could not be reactivated to initial levels by either schedule of IL-2 stimulation. Minimal NK cell activity was detected by the end of the culture period (day 40). It should be noted that all cytolytic assays were conducted based on viable cell numbers, thus loss of NK cell activity was not attributable to recovery of nonviable cells. Interestingly, IL-2 addition every 3 days did not alter these observations and in fact, seemed to accelerate the decline in lytic activity with time in culture. These results indicated that IL-2 concentrations did not become limiting during the culture period. When the cultures were tested for LAK cell cytolytic activity (as measured by the ability to lyse the NKresistant target cell IM9, Figure 2) it was observed that LAK cell lytic activity was augmented more slowly by the presence of IL-2, peaking at day 16 of culture. The addition of fresh exogenous IL-2 every 3 days augmented the level of lytic activity to a greater extent than did IL-2
II. Materials and methods A. Reagents Recombinant human Interleukin-2 (IL-2) was the kind gift of Dr. E. Akporiaye (University of Arizona) and Amgen (Thousand Oaks, CA). All monoclonal antibodies (mAbs) used in the experiments (anti-CD3, anti-CD16, anti-CD8, anti-CD4, anti-CD56, anti-HLA-DR) were obtained from Becton-Dickinson (Mountain View, CA) and were used as suggested by the manufacturer.
B. Collection and separation of peripheral blood Peripheral blood (PBL) was collected by venipuncture using heparinized syringes from healthy adults of both sexes (aged 21-45 years). Peripheral blood was separated by centrifugation over Ficoll-Hypaque density gradients according to Boyum (1968).
C. LAK cell culture conditions
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Cancer Therapy Vol 2, page 527 addition every 2 weeks. However, unlike the results shown in Figure 1, the kinetics of peak LAK cell cytolytic activity and the kinetics of decline in LAK cell activity were identical for both types of cultures. Restimulation of the cultures with IL-2 (either every 2 weeks or every 3 days) reinduced marginal levels of LAK cell activity that
were far below the observed peak levels, which again rapidly declined to insignificant levels by day 32-40 of culture. Once again, it should be remembered that all assays were performed based on viable cell numbers recovered from the cultures.
Figure 1. Kinetics of NK cytolytic activity in long-term IL-2 cultures. Peripheral blood effector cells wee isolated as described and cultured for a period of 40 days in the presence of IL-2. At various times the cultures were tested for NK lytic activity versus K562 tumor target cells. The data are presented as the mean (+/- SEM) of the LU20 obtained from 7 independent experiments. Circles represent culture condition “A”, while Squares represent culture condition “B”.
Figure 2. Kinetics of LAK cell cytolytic activity in long-term IL-2 cultures. Peripheral blood effector cells were isolated as described and cultured for a period of 40 days in the presence of IL-2. At various times the cultures were tested for LAK cell lytic activity versus IM9 tumor target cells. The data are presented as the mean (+/- SEM) of the LU20 obtained from 7 independent experiments. Circles represent culture condition “A”, while Squares represent culture condition “B”.
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B. Loss of cytolytic activity is not due to loss of viable effector cells
C. Flow cytometric analysis of effector cell populations present in long-term LAK cell cultures
In an attempt to understand why NK and LAK cell activity declined with time in culture despite the continual presence of IL-2, the cultures were analyzed at each of the above timepoints for cell numbers and viabilities (Figure 3). Not unexpectedly, cell viabilities decreased immediately after commencement of the cultures. However, the cultures reached a plateau of 70-80% viability throughout the majority of the culture period, exhibiting greater than 80% viability even at 5 weeks of culture. A constant sampling technique was employed to assess cell numbers during this period of time. Similar to cell viability, cell numbers initially decreased upon culture initiation. Cell numbers then increased up to day 18 of culture, followed by a drop in recovered cells up to day 24, and then an additional increase in cell numbers up to day 32. A drop in cell numbers was again observed at day 40 of culture indicative of the cyclical nature of the cell numbers. It should be noted that throughout the culture period that absolute cell numbers increased. However, there was no correlation between cell numbers, cell viabilities and the levels of either NK cell or LAK cell lytic activity. These results seemed to indicate that cell proliferation occurred during the entire culture period. The cyclical nature of the cell numbers recovered implied that different subpopulations of cells might be overgrowing the cultures at different times, each of which had a limited lifespan.
Experiments were performed to determine the types of effector cells present in the longterm cultures. Flow cytometric analyses revealed that typical CD3-CD16+ NK cells rapidly declined with time in culture reaching a plateau after approximately 1 week, which was constant for an additional period of several weeks. These effector cell then declined to negligible levels thereafter (Figure 4, panel A), indicating that typical NK cells died off during the culture period. During this time in culture, CD3+ T cells rapidly increased over time, as did CD3+CD8+ T cells (Figure 4, panel B). By the end of the culture period essentially 100% of the effector cells were typical T cells, and the CD8+ T cells had increased two-fold to constitute approximately 50% of the cultured cells. By using the data shown in Figures 3 and 4, it was possible to calculate that the absolute numbers of CD3+ T cells and CD8+ T cells increased significantly with time in culture with IL-2, while absolute numbers of CD3-CD16+ NK cells decreased. Analyses were then conducted to examine for the presence of activated T cells and T cells capable of performing LAK cell cytolysis (Figure 5). Activated T cells were identified by the expression of HLA-DR molecules (Lanier and Phillip, 1986; Phillips and Lanier, 1986; Harris et al, 1993; Sala et al, 1993; Westermann and Pabst, 1990). It was observed that activated T cells increased with time in culture, reaching peak levels by day 24 of culture and then rapidly declined thereafter to initial levels.
Figure 3. Effect of long-term IL-2 culture on effector cell viability and cell numbers. Peripheral blood effector cells were isolated as described and cultured for a period of 40 days in the presence of IL-2. At various times the cultures were sampled and assayed for cell numbers and viability. The data are presented as the mean (+/- SEM) for 7 independent experiments. Open circles represent cell numbers, while darkened circles represent cell viability.
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Figure 4. Levels of CD16+, CD3+ and CD8+ cells during long-term IL-2 cultures. Peripheral blood effector cells were isolated as described and cultured for a period of 40 days in the presence of IL-2. At various times the cultures were assayed for levels of CD16+, CD3+ and CD8+ effector cells by flow cytometry. The data are presented as the mean (+/- SEM) for 7 independent experiments. Panel A: CD16+ cells; Panel B: open circles represent CD3+ cells, while darkened circles represent CD8+ cells.
Figure 5. Flow cytometric analysis of effector cell activation during long-term IL-2 culture. Peripheral blood effector cells were isolated as described and cultured for a period of 40 days in the presence of IL-2. At various times the cultures were assayed for levels of CD56+ and HLA-DR+ effector cells by flow cytometry. The data are presented as the mean (+/- SEM) for 7 independent experiments. Circles represent CD56+ cells, while Squares represent HLA-DR+ cells.
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Harris: Limited LAK cell responses to IL-2 Nonspecific cytolytic T cells were identified by the expression of the CD56 molecule (Lanier and Phillip, 1986; Phillips and Lanier, 1986; Harris et al, 1993; Sala et al, 1993; Westermann and Pabst, 1990). These nonspecific T effector cells also increased during the first 3 weeks of culture and exhibited similar kinetics of decline as activated T cells over the rest of the culture period. Although not shown, cells expressing the CD45RA molecule (representing naïve T cells) also decreased with time in culture.
It was observed that although both NK and LAK cell cytotoxicity was rapidly augmented by the addition of IL2, lytic activity peaked early in culture and rapidly declined. This decline in activity was observed whether IL-2 was added every two weeks or every 3 days. In fact, more frequent addition of IL-2 to the cultures seemed to accelerate the decline in lytic activity. These results indicated that NK and LAK cells have limited potential for continuous restimulation of lytic activity by IL-2, and that the continued presence of high concentrations of IL-2 are not able to reactivate these effector cells once the effector cells are in the decline phase of the culture. Possibly, these effector cells need extended periods of “rest” prior to being once again responsive to IL-2 stimulation. Rapidly declining cell numbers and cell viabilities in the cultures could not explain the decrease in lytic activity as the cells continued to proliferate throughout the culture period and cell viabilities never fell below 70%. It may be that the cultures are overgrown with (T) cells either unable to perform LAK cell function or (T) cells anergic to IL-2 stimulation. This observation may be of particular clinical relevance. It should be noted that other doses of IL-2 (i.e., 100-10,000 U/ml) produced similar results (data not shown). While T cells and CD8+ T cells increased with time in culture, typical NK cells disappeared from the cultures. Although these T/LAK cells were cytolytically active for a short period of time, it is unknown whether thee effector cells are as active as NK cells that have been activated to become LAK cells. Regardless, T cells capable of performing LAK cell cytolysis were rapidly lost in culture. The activation of T cells capable of nonspecific lytic activity was confirmed by the expression of HLA-DR and CD56 antigens (Lanier and Phillip, 1986; Phillips and Lanier, 1986; Harris et al, 1993; Sala et al, 1993; Westermann and Pabst, 1990). Undoubtedly, the CD8+ T cells were responsible for the majority (if not all) of the LAK cell activity observed. However, these activated and LAK-like T cells were rapidly lost from the cultures, correlating with the loss of lytic activity. It appeared that the cultures were overgrown by T cells either anergic to or unable to respond to IL-2. The loss of CD45RA+ cells may indicate that only these particular T cells were capable of mediating LAK cell activity, or that CD45RA- cells were able to overgrow the cultures rapidly. It is interesting to speculate that IL-2 by itself may initiate the conversation from a CD45RA+ to a CD45RA- phenotype. Whatever the explanation, it appeared that when CD45RA+ cells disappeared from the cultures (as well as HLA-DR+ and CD56+ cells), there were no longer cells capable of responding to IL-2 and performing LAK cell activities. In terms of the clinical situation, these results imply that short-term in vitro expansion of patient lymphocytes may be preferable to long-term expansion prior to use in LAK cell/IL-2 immunotherapy. Further, injection of IL-2 at very high doses with the aim of elevating systemic IL-2 levels longterm may actually be detrimental to this type of therapy. It would seem that in order for the standard LAK cell/IL-2 immunotherapy to be more successful, the procedure would need to be repeated with longer periods of “rest” between injections due to problems of
IV. Discussion Immunotherapy offers the hope of being able to complement conventional cancer therapy and possibly treat metastatic disease that is not amenable to these standard therapies. Immunotherapy offers the potential advantage of being less toxic to the patient and inducing lifelong immunity to subsequent tumor recurrence. Although many investigators have developed methods of immunotherapy to treat a variety of cancer types, the work of Rosenberg et al (Rosenberg et al, 1985, 1987; Rosenberg, 1988) has been of particular interest in this area. Rosenberg et al combined the availability of cloned, recombinant IL-2 with the ability to expand lymphocytes in vitro in its presence to develop the LAK cell/IL-2 immunotherapy regime. This type of immunotherapy was designed to take advantage of two immunological mechanisms. One, the ability of IL-2 to activate NK cells and generate LAK cells after IL-2 injection in vivo. And two, injection of large numbers of LAK cells capable of killing tumor cells whose activity would be sustained by the presence of IL-2. Although this approach has shown effectiveness in vitro and in animal models (Mule et al, 1984, 1985; Lafreniere and Rosenberg, 1985), this particular mode of immunotherapy has met with limited success in clinical trials (Rosenberg et al, 1987; Rosenberg, 1988). It is not understood why LAK cell/IL-2 immunotherapy has not been more successful, although effector cell localization to sites other than the tumor site (e.g., liver, lung and spleen) has been suggested as a possible explanation. Inefficient effector cell localization cannot be entirely at fault as large numbers of LAK cells localize to the lung (Felgar and Hiserodt, 1990; Basse et al, 1991), but this mode of therapy is not particularly effective for lung cancer (Rosenberg et al, 1987; Rosenberg, 1988). It is possible that systemic levels of IL2 decrease too rapidly to maintain the activity of injected LAK cells and/or to in vivo activate NK/LAK cells. Further, the side-effects of high levels of systemic IL-2 have also limited its use in the clinic (Rosenberg et al, 1987; Rosenberg, 1988). As this particular therapeutic idea has found its way into gene therapy of cancer whereby IL2 genes are injected or transferred into tumors in situ or IL-2 secreting tumors are given as vaccines in the hope of bypassing the side-effects of IL-2 while activating T/NK/LAK cells to kill tumor cells, we sought to analyze the reasons for the previous limited success of this therapy. We hypothesized that even in the presence of continuous levels of IL-2 that LAK cells became inactive both after in vivo activation and after in vitro expansion/activation and injection. 530
Cancer Therapy Vol 2, page 531 Klein E (1969) Hypersensitivity reactions at tumor sites Cancer Res. 29, 2351-2362. Klein E, Holtermann O, Milgrom H, Case RW, Klein D, Rosner D, Djerassi I (1976) Immunotherapy for accessible tumors utilizing hypersensitivity reactions and separated components of the immune system. Med Clin North Am 60, 389-418. Koren HS, Anderson SJ, Fischer DG, Copeland CS, Jensen PJ (1981) Regulation of human natural killing. I. The role of monocytes, interferon and prostaglandins. J Immunol 127, 2007-2013. Lafreniere R and Rosenberg SA (1985) Successful immunotherapy of murine experimental hepatic metastases with lymphokine-activated killer cells and recombinant interleukin-2. Cancer Res 45, 3735-3741. Lanier LL and Phillip JH (1986) Evidence for three types of human cytotoxic lymphocytes. Immunol. Today 7, 132-134. Mule JJ, Shu S, Rosenberg SA (1985) The anti-tumor efficacy of lymphokine-activated killer cells and recombinant interleukin-2 in vivo. J Immunol 135, 646-652. Mule JJ, Shu S, Schwarz SL, Rosenberg SA (1984) Adoptive immunotherapy of established pulmonary metastases with LAK cells and recombinant inerleukin-2. Science 225, 14871489. Phillips JH and Lanier LL (1986) Dissection of the lymphokineactivated killer phenomenon. Relative contribution of peripheral blood natural killer cells and T lymphocytes to cytolysis. J. Exp. Med. 164, 814-825. Pross HF, Baines MG, Rubin P, Shragge P, Patterson MS (1981) Spontaneous human lymphocyte-mediated cytotoxicity against tumor target cells. IX. The quantitation of natural killer cell activity. J Clin Immunol 1, 51-62. Rosenberg SA (1988) Editorial, Cancer therapy with interleukin2, Immunologic manipulations that can mediate the regression of tumors in humans. J Clin Oncol 6, 403-406. Rosenberg SA, Lotze MT, Muul LM, Chang AE, Avis FP, Leitman S, Linehan WM, Robertson CN, Lee RE, Rubin JT, et al (1987) A progress report on the treatment of 157 patients with advanced cancer using lymphokine-activated killer cells and interleukin-2 or high-dose interleukin-2 alone. N Engl J Med 316, 889-897. Rosenberg SA, Lotze MT, Muul LM, Leitman S, Chang AE, Ettinghausen SE, Matory YL, Skibber JM, Shiloni E, Vetto JT, et al (1985) Observations on the systemic administration of autologous lymphokine-activated killer cells and recombinant interleukin-2 to patients with metastatic cancer. N Engl J Med 313, 1485-1492. Rosenberg SA, Mule JJ, Spiess PJ, Reichert CM, Schwarz SL (1985) Regression of established pulmonary metastases and subcutaneous tumor mediated by the systemic administration of high-dose recombinant IL-2. J Exp Med 161, 1169-1188. Sala P, Tonutti E, Feruglio C, Florian F, Colombatti A (1993) Persistant expansion of CD4+CD8+ peripheral blood T cells. Blood 82, 1546-1552. Taylor CW and Hersh EM (1990) Immunotherapy and biological therapy of cancer, Current clinical status and future prospects Immunopharmacol. Rev. Vol. I, J.W. Hadden and A. Szentivanyi (Eds.), Plenum Publishing Corp., p. 89-157. Westermann J and Pabst R (1990) Lymphocyte subsets in the blood, a diagnostic window on the lymphoid system? Immunol Today 11, 406-410. Winkelhake JL, Stampfl S and Zimmerman RJ (1987) Synergistic effects of combination therapy with human recombinant interleukin-2 and tumor necrosis factor in murine tumor models. Cancer Res 47, 3948-3953.
maintaining significant LAK cell activity. In actuality it may be more preferable to either use IL-2 gene therapy (either by in situ gene transfer or be tumor cell vaccine), or a combination of IL-2 gene therapy with immunotherapy (i.e., LAK cell transfer) to maximize the efficacy of this approach. Further, typical NK cells (CD3-16+56+) may be more useful as LAK cells for immunotherapy than are T cells after IL-2 expansion and stimulation, although at present we have no conclusive evidence for this hypothesis. In conclusion, the present study has investigated possible reasons for the limited success of LAK cell/IL-2 immunotherapy in the treatment of cancer. It was observed that LAK cell activity is limited in terms of its duration and in terms of the potential of effector cells to be continually induced to display this cytolytic activity. The continual presence of IL-2 and proliferating, viable effector cells had no effect on these results. These results implied that in its standard form LAK cell/IL-2 immunotherapy will be of limited clinical usefulness.
Acknowledgements I wish to acknowledge Patti Parker for her expert technical assistance, and Barb Carolus for assistance with the flow cytometric analyses. Further, helpful discussions of this work with Dr. Evan Hersh are greatly appreciated. This work was supported in part by a grant from the National Cancer Institute, National Institutes of Health, CA48085-06.
References Basse P, Herberman RB, Nannmark U, Johansson BR, Hokland M, Wasserman K, Goldfarb RH (1991) Accumulation of adoptively transferred adherent, lymphokine-activated killer cells in murine metastases. J Exp Med 174, 479-488. Boyum A (1968) Isolation of mononuclear cells and granulocytes from human blood. Scand J Clin Invest 21 (Suppl), 77-89. Brunda MJ, Bellatoni D and Sulich V (1987) In vivo anti-tumor activity of combinations of interferon-" and interleukin-2 in a murine model. Correlation of efficacy with the induction of cytotoxic cells resembling natural killer cells. Int J Cancer 40, 365-371. Felgar RE and Hiserodt JC (1990) In vivo migration and tissue localization of highly purified lymphokine-activated killer cells (A-LAK) in tumor-bearing rats. Cell Immunol 129, 288-298. Grimm EA, Mazumder A, Zhang HZ, Rosenberg SA (1982) Lymphokine-activated killer cell phenomenon, Lysis of natural killer-resistant fresh solid tumors by interleukin-2activated autologous human peripheral blood lymphocyte. J Exp Med 155, 1823-1841. Harris DT, Jaso-Friedmann L, Evans DL (1993) A novel target cell antigen involved in the NK-like lytic activity of antigenspecific cytotoxic T lymphocytes. Immunol Lett 38, 11-18. Harris DT, Schumacher MJ, Locascio J, Besencon FJ, Olson GB, DeLuca D, Shenker L, Bard J, Boyse EA (1992) Phenotypic and functional immaturity of human umbilical cord blood T lymphocytes. Proc Natl Acad Sci U S A 89, 10006-10010. Klein E (1967) Differential immunologic reactions in normal skin and epidermoid neoplasms Fed Proc 26, 430. Klein E (1968) Tumors of the skin. X. Immunotherapy of cutaneous and mucosal neoplasms N.Y. State J Med. 68, 900-911.
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Abscopal regression of subcutaneously implanted N29 rat glioma after treatment of the contra-lateral tumours with pulsed electric fields (PEF) or radiation therapy (RT) and their combinations (PEF+RT) Research Article
Bertil R.R. Persson1,3,*, Catrin Baureus Koch1,2,3, Gustav Grafström1,3, Crister Ceberg1,3 and Leif Salford2,3 1
Dept. of Medical Radiation physics Dept. of Neurosurgery 3 Rausing Laboratory, Biomedical Centre, Lund University, 221 85 LUND, Sweden 2
__________________________________________________________________________________ *Correspondence: Bertil R.R. Persson, PhD, MD h.c., professor, Dept. of Medical Radiation Physics, University Hospital in Lund, Klinikgatan 7, S-221 85 LUND, Sweden; Tel: +4646173110; Fax: +4646134249; e-mail: bertil.persson@radfys.lu.se Key words: Abscopal effect, Fischer rat, glioma, N29 tumour, electric pulses, PEF, radiation therapy, 60Co-! radiation Abbreviations: Abscopal enhancement ratio, (AER); control, (Ctrl); intraperitoneally, (i.p.); Pulsed Electric Field, (PEF); radiation therapy, (RT); source-skin distance, (SSD); Specific abscopal effect ratio, (SAER); Specific abscopal effect, (SAE); Specific therapeutic effect, (STE); Therapeutic enhancement ratio, (TER); tumor volume, (TV); tumour volume growth rate, (TGR); unexposed controls, (UC); unexposed tumour, (UE) Received: 8 November 2004; Accepted: 26 November 2004; electronically published: January 2005
Summary The aim of the present work is to study the Abscopal regression of subcutaneously implanted contra-laterally tumors treated with pulsed electric fields and/or radiation therapy. The study was performed on rats of the Fischer344 strain with N29 rat glioma tumors implanted subcutaneously on both the hind legs. Only the tumours on the right hind legs were treated with pulsed electric field "PEF" (16 exponentially decaying pulses with a maximum electric field strength of 1300 V/cm and tl/e = 1 ms) or/and radiation therapy "RT" (60Co-! radiation given in daily fractions of 5 Gy, to a total absorbed dose of 20 Gy). The animals were arranged into one group of untreated controls and 3 groups of various treatments: PEF only, RT only, and in combination PEF + RT. The tumour growth rate of the right-lateral treated tumours was significantly decreased for independent treatment with pulsed electric fields (PEF, p<0.005) or radiation therapy (RT, p<0.001) as well as combined treatments (PEF+RT, p<0.005). For the left-lateral untreated tumours the growth rate was significantly decreased in rats with right-lateral tumours treated with RT (p<0.00 1) and the combination PEF+RT (p<0.00l). In rats treated with PEF only, there was no significant decrease in tumour growth rate of the left-lateral tumour. The specific therapeutic effect "STE" of the right-lateral treated tumours were all significantly different from zero (p<0.00l) STE = 0.28 ± 0.02 for PEF; 0.44 ± 0.02, for RT and 0.3 ± 0.10 for the combined treatment PEF+RT. Corresponding quantity for the left-lateral unexposed tumours is named the specific abscopal effect "SAE", which is highly significantly different from zero p<0.00l for animals treated on the opposite side with RT, SAE = 0.22 ± 0.02, or with the combined treatment PEF+RT 0.20 ± 0 03. In rats treated with PEF on the opposite side the SAE= 0.01 ± 0.08 that is not significantly different from zero (p=0.8). Therapeutic enhancement ratio (TER) and Abscopal enhancement ratio (AER) at combined treatment with PEF and RT is 0.43 ± 0.05 and 1.1 ±1 0.1 respectively. It is interesting to note that the AER is about twice the Therapeutic enhancement ratio (TER). Thus by combining radiation therapy with pulsed electric fields and there is an enhancement therapeutic effect of the treated tumour as well as an effect on the distant non-treated tumours.
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Persson et al: Abscopal regression of tumours (N29 rat glioma) 1998; Mir et al, 1998; Rols et al 1998). A series of clinical trials of this treatment modality has been performed and with encouraging results in cancer therapy (Heller 1996b, 1997, 1998, 1999; Mir et al, 1998; Sersa et al, 1998, 1999, 2000). Pulsed Electric Field (PEF) treatment can by itself create cellular and sub cellular lesions by inducing lipid per-oxidation associated with the membrane area being permeabilized, and appears to be correlated to cell survival (Maccarrone et al, 1995a, b; Danfelter et al, 1998). In biological tissue peroxide can form hydroxyl radicals OH•, which enable single strand breaks in DNA (MelloFilho and Meneghini, 1984). DNA damage from pulsed electric fields has thus been reported, where the number of DNA strand breaks was correlated to the field strength and duration of the electric pulses (Meaking, et al, 1995; Meldruni et al, 1999). Pulsed electric fields have also been found to induce apoptosis and activation of caspases (Pinero et al, 1997; Hofmann et al, 1999).
I. Introduction A. Radiation therapy and the Abscopal effect Radiation therapy is beside surgery still the most widely used therapy modality for cancer treatment and in developed countries it is given to one out of two patients with cancer (Knöös 1991, SBU 2003). Although a lot of progress is made in development of new methods and techniques for radiotherapy about half of the curatively intended treatments fail as a result of either distal or local recurrences (DeVita 983). Improvements of local radiotherapy are focused on fractionation and higher absorbed dose to the clinical target volume without exceeding the tolerance of surrounding normal tissue (Suit and Miralbell 1989; Suit et al, 1988, 2002). The therapeutic effect of radiation therapy refers to the direct exposed tumours and effects on tumours outside the treated target area are mostly not considered in radiation therapy. Effects of radiation therapy on cancer tumors outside of the radiation field have, however, been reported in many malignancies (Nobler et al, 1969; Ehlers et al, 1973; Kingsley 1975; Antoniades et al, 1977; Rees et al, 1981; Rees and Ross 1983; Sham et al, 1995; Obba et al, 1998). This phenomenon was originally described as abscopal effect by R. J. Mole in 1953. The definition of abscopal effect comes from the Latin ab (position away from) and scopus (mark or target). The abscopal mechanism of action remains still unexplained, although a variety of under-lying biologic events can be hypothesized, including a possible role for the immune system (Uchida et al, 1989; Macklis et al, 1992). The Abscopal effect studied in mice with 67NR tumor after RT with 2 or 6 Gy, was recently proven to be an immune mediated effect (DeMaria 2004). The abscopal effect is, however, not often observed clinically, possibly because many tumor-bearing hosts develop immune suppression (Kusmartsev and Gabrilovich 2002). Nagasawa and Little (1992) observed that cells hit by "-particles and neighbouring cells without hit both exhibit same type of damage. The phenomenon was called “bystander effect” borrowed from the gene therapy field. Since then several reports and reviews have appeared dealing with this kind of nonlinear dose-response relationship that is called both bystander effect and abscopal effect (Mothersill and Seymour 2001, Azzam et al 2004).
C. Effect of pulsed electric fields in combination with radiation therapy Pulsed Electric fields used in combination with radiation therapy has recently been found to increase the differential response between tumour and normal tissue (Engström et al, 2001b; Persson et al, 2003). The therapeutic effect of pulsed electric fields combined with radiation therapy was first studied in rats of the Fischer344 strain with glioma tumors implanted subcutaneously on the thigh (Engström et al, 2001b). Exponentially decaying pulsed electric fields of 1400 V/cm and tl/e= 1ms were applied to the tumors with 16 pulses in the 4 consecutive days combined with radiation therapy of total absorbed radiation dose of 20 Gy, in 4 fractions of 5 Gy each day in four consecutive days. The combined treatment with pulsed electric fields was found to have a sensitizing effect on radiation therapy and resulted in a high fraction of complete remissions (6/9) and increased survival compared to the controls (60% at 100 days) (Engström et al, 200lb). Fitting the data obtained from consecutive measurements of tumor volume (TV) of each individual tumor to an exponential model TV = TV0 !exp [TGR! t] estimated the tumor growth rate (TGR %per day) after the first day of treatment (t=0). The TGR of N32 tumors treated with the combination of 4PEF+4RT are significantly decreased compared to the controls p<0.0001) compared to RT alone p<0.0001) and compared to PEF alone p<0.001). The combined treatment of N32 gives significant effect on the tumor growth rate after 2, 3 and 4 treatment session while RT alone seems to be most efficient after one treatment of 5 Gy and PEF alone is most efficient after 2 treatments at 2 consecutive days (Persson et al, 2003). The specific therapeutic effect STE is defined as the difference between the average tumor growth rates of controls and exposed tumors divided by the average tumor growth rate of the controls. With 4 PEF treatments alone the average STE value was 0.32 for N32 tumors and 0 for N29; for 4RT alone the STE values were 0.29 and 0.42 respectively, and for combined treatments 4PEF+4RT 0.67
B. The effect of pulsed electric fields Pulsed Electric Field (PEF) treatment is the application of short, intense electric pulses of high field strength that cause transient permeabilization of the cell membrane, thus allowing extra cellular administered compounds, e.g. DNA plasmids, cytotoxic drugs or other pharmaceuticals to reach the interior of the cell. This method has been employed in vivo to introduce anticancer agents into tumors (Engström et al, 1998, 2001a; Jaroszeski et al, 1997a, b, 1999; Salford et al, 1993) and recently also DNA-plasmids and proteins into various tissues (Heller et al; 1996a, 2001; Nishi et al, 1996; Aihara
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Cancer Therapy Vol 2, page 535 thickness measured with a calliper. When the tumours reached a volume of 9 cm3, the animal was sacrificed of ethical reasons.
and 0.17 respectively. For the N29 tumors treated with 2PEF+4RT the STE value was 0.53. The values of the therapeutic enhancement ratio, TER = STEExperimental /STE Independent , increase with the number of treatment sessions and the TER of the combined treatments is above 1 in two of the N32 series, which indicates a synergistic effect of 4PEF+4RT (Persson et al, 2003). So far, there are no studies performed of the abscopal effect of pulsed electric fields or its combination with radiation therapy. In the present study we investigate the abscopal effect in Fischer rats with a N29 tumor inoculated on both the right leg and on the left leg. The right tumor was treated at about 30 days after inoculation with either RT or PEF and their combination (PEF+RT) while the left tumor was untreated. The size of the tumors on the flank was measured daily and growth of tumor was evaluated in terms of the change in tumor growth rate of exposed tumor relative to the control.
B. Procedure of tumour treatment The effect of pulsed electric fields and radiation therapy and their combination was investigated in male rats of the Fischer-344 strain with rat glioma N29 tumours implanted subcutaneously on the flank or on the thigh of the hind leg. The animals were arranged into five groups, which received electric pulses followed by radiation (PEF+RT) radiation treatment followed by electric pulses (RT+ PEF) radiation treatment only (RT) electric pulses only (PEF) or no treatment (Ctrl). The delay between electro pulsation and radiation treatment was kept as short as possible (4-5 minutes). The animals were arranged into groups of various treatments as shown in Table 1.
1. Radiation treatment Animals were given fractionated radiation treatment using a 60Co radiotherapy unit (Siemens Gammatron S) with a sourceskin distance (SSD) of 80 cm and the maximum absorbed dose rate 0.65-0.70 Gy/min. A 0.5 cm thick, tissue-equivalent bolus (Super Flab, Mike Radio-nuclear instruments inc. NY, USA) was placed over the tumor to achieve full dose buildup and a more homogeneous dose distribution in the tumor. The radiation field size was collimated to cover the tumor area with a margin of at least 1 cm (Figure 1). The absorbed dose to the exposed right-lateral tumour was 5 Gy/day x 4 (consecutive) days, in all 20 Gy, which in preparatory experiments showed to be a suitable, suboptimal and non-curative dose. While the absorbed dose to the left-lateral tumour was negligible.
II. Materials and methods A. Experimental inoculation
animals
and
tumour
1. Animals Rats of the Fischer 344 strain were used. The strain was maintained by continuous, single line, brother/sister mating in our laboratory.
2. Tumour cell cultures All tumour cells were cultivated in antibiotic-free RPMI 1640 supplemented with 10% fetal calf serum, 2 mM Lglutamine, 10 mM Hepes, 0.5 mM pyruvate, and 0.096% NaHCO3. Cell cultures were maintained in culture flasks Nunc, Denmark) and harvested with trypsin-EDTA.
Table 1. Number of animals in each group of various treatments.
3. Subcutaneous tumours The rat glioma N29 was induced in our laboratory by subcutaneous administration in the hind legs. 200 000 cells were inoculated into the right leg, whilst 50 000 cells were inoculated into the left leg in order to simulate a secondary smaller tumour. Tumour volume is estimated as an ellipsoid by length, width and
Group
Treatment
Ctrl RT PEF PEF+RT
Controls with no treatment Radiation therapy 4x5 Gy Pulsed Electric Fields Pulsed Electric Fields + Radiation Therapy
Total
Figure 1. Experimental set-up for radiation treatment and electric impulse treatment
535
Number of rats 40 15 25 15 95
Persson et al: Abscopal regression of tumours (N29 rat glioma) 2. PEF treatment
C
"
TGR ! TGRiE C
;
TGR
i=1
C
TGR The average of the individual tumour growth rate constant (day-1) in the group of unexposed control rats. The STE is equal to 0 when the average of tumour growth rate constant of the exposed group, is equal to the average of the tumour growth rate constant of the control The STE is equal to 1 when the average tumour growth rate constant of the exposed group, is equal to 0, which means arrested tumour growth. The STE is larger than 1 when the average tumour growth rate constant of the exposed group, is less than 0, which means a declining tumour volume.
3. Specific abscopal effect (SAE) The "specific abscopal effect" SAE is defined as the tumour growth rate difference between the left-lateral unexposed tumour and corresponding control divided by tumour growth rate of the controls.
The tumours are treated with PEF radiation or a combination of these two. The question is how to express the enhancement of the therapeutic effect of the combined treatment with the single treatment and a hypothetical independent combination of the two single treatments.
SAE =
1. Tumour growth rate
1 N
N
UC
" i=1
TGR
! TGRiUE UC
;
TGR
where UE The average of the individual tumour growth rate TGRi constant of the unexposed (UE) left-lateral tumours in the group of N exposed rats.
The tumour volume measurements of each tumour fitted to a model of exponential growth made the tumour volume growth rate (TGR) according to the following equation.
where TV TGR TV0
N
where E The average of the individual tumour growth rate TGRi constant (day-1) in the group of N exposed rats.
C. Model for tumour growth analysis and synergistic enhancement
!TV = TGR "TV; dt
1 N
STE =
Two rectangular flat electrodes were mounted on a slide calliper and connected to an exponential pulse generator (Figure 1). In the first series of experiment we used a BTX6OO (Genetronics, San Diego, USA) and since year 2000 a CytorExp 2000 (Aditus Medical AB, Lund Sweden) was used. The pulsed delivered was monitored by an oscilloscope and the load was adjusted so that the time constant of the exponential pulse was 1 ms. The hair over the tumor was shaved off and the skin was carefully covered with electrocardial paste to ensure good electrical contact between electrodes and skin. The paste also moistened the skin, reducing the transdermal impedance and limited the risk of skin necrosis from the pulse treatment. The tumours were gently fixed in position between the two electrodes, and the voltage was adjusted to the distance between the electrodes to deliver pulses of identical field strength to all tumours. Sixteen pulses of approximately 1400 V/cm with a time constant of 1 ms were delivered transdermally to the whole tumor during approx. 20 seconds. This treatment was repeated daily for four days.
UC
TGR " t
TGR The average of the individual tumour growth rate constant in the left-lateral tumours in the group of unexposed controls (UC).
Tumour volume Tumour growth rate constant day-1 Tumour volume at the time of treatment
4. The therapeutic enhancement ratio and abscopal effect enhancement ratio
TV = TV " e
The enhancement effect the combined treatments (PEF+RT) is the ratio of the effect of the experimental combination of PEF and RT and the therapeutic effect the hypothetical independent combination of the two agents. The therapeutic enhancement ratio of the exposed turnours is thus defined as:
The therapeutic effect is defined as the ratio of the tumour volume between the treated tumour and the control group.
2. Specific therapeutic effect (STE) The ratio of the tumour volume of the exposed tumour and corresponding control is a measure of surviving fraction, SF, of the cells in the treated tumour:
TER =
SF = TVExposed / TVControl
STE Experimental STE Independent
and the abscopal enhancement ratio of the left-lateral unexposed tumour is defined as:
The therapeutic effect, TE, is a measure of the number of lethal events that has occurred in the cells of the treated tumour volume and thus defined as:
AER =
TE = - ln (SF) = [TGRControl - TGRExposed ] ! t
SAE Experimental SAE Independent
where the hypothetical effect by independent (additive) action of ionizing RT and PEF is given by
In order to get a therapeutic effect measure independent of time a quantity named "specific therapeutic effect" STE is defined. That is the tumour growth rate difference between the control and exposed tumour divided by tumour growth rate of the controls.
STEIndependent = STERT + STEPEF for the exposed tumours
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Cancer Therapy Vol 2, page 537 SAEIndependent = STERT + STEPEF
not quite correct to draw quantitative conclusions directly out of the growth curves of the averaged data. In order to perform a more correct analysis of the results, the tumour growth rate is estimated from the volume measurements of each individual tumour. Thus the tumour volume data after the time of treatment of each individual tumour were fitted to a model of exponential growth TV = TV0 ![TGR !t] . Where "TV" is tumour volume, 99TGR" is tumour growth rate constant (day-1) and time "t" is the number of days after the first treatment. The results thus obtained from the growth rate of right lateral tumours tumours in all series-A, -B, -C, and D with average of treated with either PEF or RT and their combinations (PEF+RT) are displayed in Figure 3 in blue columns. The tumour growth rate results of the corresponding untreated left-lateral tumours are displayed in red. The p-values of significant difference to the controls are displayed in the columns. Series-A include controls only and series-C controls and tumours treated with pulse electric fields (PEF) only. In the series-D the right-lateral tumours were treated with two fractions of exponential pulsed electric fields (PEF) and 4 fractions of radiation therapy (RT) and their combination (PEF+RT). The PEF treatment was performed on days 40 and 44 after inoculation of the tumours by applying 16 exponential pulses at electric field strength of 1400 V/cm, and 1.0 ms time constant. The radiation therapy was performed at days 33, 34, 36 and 37 after inoculation with four daily fraction of 5 Gy (total 20 Gy). The combined treatment was performed with less than 1 h between the PEF and RT treatments performed as above at days 33, and 36, while at days 34 and 37 were given RT only.
for the left-lateral unexposed tumours The enhancement ratios are measures of any synergistic or diminishing effect obtained in the combination of the two agents. It may be due to interaction of sub lethal lesions induced by both agents to produce lethal events that cause the enhancement ratio> 1. If the individual therapies are highly aggressive by themselves there might, however, also be an "over killing" effect that reduce the effect compared to the additive action, so that enhancement ratios <1. It is thus important to investigate the effect of combined treatments at various dose levels to find the maximum value of enhancement ratios.
III. Results A. TGR The tumour growth of each individual tumour was measured during the entire lifetime of all the animal in the following experimental groups (Table 2). The female Fischer-344 rats had N29 glioma tumours implanted on both thighs and only the tumours on the right side were treated. In the series B the right-lateral tumours were treated with four fractions of exponential PEF, RT and their combination (PEF+RT). The PEF treatment was performed at days 42, 43, 45 and 46 after inoculation of the tumours by applying 16 exponential pulses at electric field strength of 1400 V/cm, and 1.0 ms time constant with plate electrodes over the tumours. The radiation therapy was performed at days 29, 30, 32 and 33 after inoculation with four daily fractions of 5 Gy (total 20 Gy). The combined treatment was performed with less than 1 h between the PEF and RT treatments performed as above at days 30, 31, 33 and 34 after the day of inoculation. The tumour volume was estimated by more or less daily measurements. At those occasions the rats were also observed for symptoms from the tumour growth. The Figures 2 display the average tumour volume at each time of measurement of tumours in the animals of the series-B. At about 30 days after inoculation and thereafter the tumour growth data fit well to an exponential growth model. The fitted curves for all tumours in each group are displayed in the Figure 2 as solid lines. The time the tumour reached a volume of about 9 cm3, when the animal was sacrificed, varied within the group and at the end only few or a single rat survived more than 50 or 60 days in the control groups. It is, thus,
Table 2. Number of animals in each experimental series and groups of treatment. Exp. series A B C D All
537
Controls
PEF
RT
PEF+RT
8 17 8 7 40
9 9 7 25
8
7
7 15
8 15
Persson et al: Abscopal regression of tumours (N29 rat glioma)
Figure 2. Average tumour volume at each time of measurement of tumours in the animals of the series-B. The left stack of diagrams show the tumour volume of the left-lateral untreated tumours and the right stack of diagrams shows the tumour volume of the rightlateral treated tumour. The solid lines show the fitted exponential growth model with the growth rate constant â&#x20AC;&#x153;Râ&#x20AC;? given in the lower right corner of each diagram. Figure 3A. Tumour growth rate of control tumours from the experimental series-A, -B, -C and D. Controls for the treated right side in blue and untreated left side in red.
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Cancer Therapy Vol 2, page 539 Figure 3B. Tumour growth rate in rats treated with PEF from the experimental series-B, -C and -D. The PEF treated right side in blue and untreated left side in red. The dashed lines are the average of the controls for the treated right side in blue and untreated left side in red.
Figure 3C. Tumour growth rate in rats treated with RT from the experimental series-B and -D. The RT treated right side in blue and untreated left side in red. The dashed lines are the average of the controls for the treated right side in blue and untreated left side in red
539
Persson et al: Abscopal regression of tumours (N29 rat glioma) Figure 3D. Tumour growth rate in rats treated with RT from the experimental series-B and -D. The PEF+RT treated right side in blue and untreated left side in red. The dashed lines are the average of the controls for the treated right side in blue and untreated left side in red.
Figure 4A. Results of the specific therapeutic effect STE (blue) and specific abscopal effect SAE (red) from pooled data (All) of series-B, C, and -D.
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Cancer Therapy Vol 2, page 541 Figure 4B. Tumour growth rate in rats treated with PEF from the experimental series; B, C, D. The PEF treated right side in blue and untreated left side in red. The dashed lines are the average of the controls for the treated right side in blue and untreated left side in red.
Figure 4C. Tumour growth rate in rats treated with RT from the experimental series; B, D. The RT treated right side in blue and untreated left side in red. The dashed lines are the average of the controls for the treated right side in blue and untreated left side in red.
B. STE and SAE
This quantity is independent of time and individual variations in the tumour growth characteristics of each experiment. The corresponding quantity "specific abscopal effect" SAE is obtained by normalizing the difference in tumour growth rate to the tumour growth rate of each individual unexposed tumour (UE) of the average of its corresponding UC at the same period of time.
The therapeutic effect is equivalent to the difference in tumour growth rate between the controls and the exposed tumours calculated from the day of treatment. The "specific therapeutic effect" STE is obtained by normalizing the difference in tumour growth rate to the tumour growth rate of each individual exposed tumour (F) of the average of its controls at the same period of time. STE =
1 N
N
" 1
C
TGR ! TGRiE C
SAE =
;
TGR
541
1 N
N
" 1
UC
TGR
! TGRiUE UC
TGR
;
Persson et al: Abscopal regression of tumours (N29 rat glioma)
AER = SAE Experimental / SAE Independent
Since the STE and SAE values are normalized to the controls of each experimental series they can be pooled and treated as one population. The results the pooled data of series B, C, D are displayed in Figure 5.
TER and AER at combined treatment with PEF and RT are given in Figure 7 with data from the experimental series; B, and D to perform a statistical analysis the results the tumour growth rate of each individual tumour is estimated from the tumour volume measurements. In the experimental series-B the tumour on the left side was implanted 8 days after the tumour was implanted on the right side, in order to simulate the occurrence of a distant smaller secondary tumour. The right tumour was considered as the main tumour and was treated with PEF, RT and their combination. The tumour growth rate of the treated tumour was significantly decreased for independent treatments with PEF (p<0.05) and RT (p<0.001). But there was no significantly decrease (p=0.2) in the tumour growth rate with combined treatment (PEF+RT). Although there is a large effect in some tumours as seen in Figure 3, the variation is large between individual tumours. The tumour growth rate of the untreated left-lateral tumour, however, is most significantly reduced (p<0.0005) in the group of rats with right-lateral tumours treated with the combination PEF+RT. In the groups with independent treatments of the right-lateral tumour, the tumour growth rate was significantly reduced for RT (p<0.01) but no significant effect (p=0.08) with PEF treatment. In the experimental series D the tumour on the left side was simultaneously inoculated with the tumour on the right side, but with 1/4 fewer cells to get a smaller tumour at the time of treatment. The right lateral tumours were treated with PEF, RT and their combination PEF+RT. The tumour growth rate of the right-lateral treated tumour in this series was significantly decreased for independent treatments with PEF (p<005) and RT (p<0.005) and with combined treatment PEF+RT (p<0.005).
C. Specific abscopal effect ratio (SAER) The Specific Abscopal Effect Ratio of various types of treatments is evaluated by comparing the specific therapeutic effect growth of the exposed tumour to the corresponding untreated left- lateral tumours in the same group of treatment.
SAER = [SAE Left / STE Right ] Where SAELeft is the average of the specific abscopal effect evaluated for the untreated left tumours and SAERight is the average of the specific therapeutic effect of the treated right tumours. The "Specific abscopal effect ratio" SAER of tumours in all series with animals treated with PEF, RT and their combinations (PEF+RT) are given in Figure 6, which includes data from the experimental series, B, C, D.
D. Therapeutic enhancement ratio (TER) and AER The therapeutic enhancement ratio of the combined treatment is defined as the ratio of the specific therapeutic effect of the combined treatment and the sum of the independent treatments.
TER = STE Experimental / STE Independent The abscopal enhancement ratio of the combined treatment is defined as the ratio of the specific therapeutic effect of the untreated tumours on rats with combined treatment of the left-lateral tumour and the sum of the corresponding independent treatments.
Figure 5. The “Specific abscopal effect ratio” SAER of tumours in all series with animals treated with pulsed electric field (PEF) radiation therapy (RT) and their combinations (PEF+RT). “All” includes data from the experimental series-B, -C and -D.
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Cancer Therapy Vol 2, page 543 Figure 6. Therapeutic enhancement ratio (TER) and Abscopal enhancement ratio (AER) at combined treatment with PEF and RT. Data from the experimental series-B and -D.
measurements of each tumour. The results of the tumour growth rate thus obtained are summarized in Table 3.
The tumour growth rate of the left-lateral untreated tumour is most significantly reduced in the group of rats independently treated with RT on the right-lateral tumour. In the groups with independent treatments with PEF and with combined treatments PEF+RT of the right-lateral
B. Specific therapeutic effect (STE) and specific abscopal effect (SAE) The therapeutic effect is the difference in tumour growth rate between the controls and the exposed tumours. The “specific therapeutic effect” STE is obtained by normalizing the difference in tumour growth rate to the tumour growth rate of the controls. This quantity is independent of time and the tumour growth characteristics of each experiment. As a measure of the Abscopal effect the “specific abscopal effect” SAE was evaluated as the difference in tumour growth rate between the controls and the untreated left tumour. The results of the “specific therapeutic effect” STE are summarized in Table 4. In the experimental series-B the tumour on the left side was implanted 8 days after the tumour was implanted on the right side, in order to simulate the occurrence of a distant smaller secondary tumour. The right tumour was considered as the main tumour and was treated with PEF, RT and their combination. The tumour growth rate of the treated tumour was significantly decreased for independent treatments with PEF (p<0.05) and RT (p<0.001). But there was no significantly decrease (p=0.2) in the tumour growth rate with combined treatment (PEF+RT). Although there is a large effect in some tumours as seen in Figure 3, the variation is large between individual tumours. The tumour growth rate of the untreated left lateral tumour, however, is most significantly reduced (p<0.0005) in the group of rats with right-lateral tumours treated with the combination PEF+RT.
tumour, there was no significant reduction of tumour growth rate on the left-lateral tumour. By combining the data from all comparable experimental series (A, B, C, D) the tumour growth rate of the right-lateral treated tumour was significantly decreased for independent treatments with PEF (p<0.005) in 25 tumours and RT (p<0.001) in 15 tumours and with combined treatment PEF+RT (p<0.005) in 15 tumours. The tumour growth rate of the left-lateral untreated tumour, however, is significantly reduced in the group of rats treated with RT (p<0.000l) in 15 tumours and the combination PEF + RT (p<0.000l) in 15 tumours. In the groups with independent treatment of right-lateral tumours with PEF, there was no significant reduction of the tumour growth rate of the left-lateral tumours.
IV. Discussion A. Tumour growth rate (TGR) It is quite difficult to draw any quantitative conclusions out of the average growth curves because of the variations in the growth of the individual tumour and the time of death. In order to perform a statistical analysis the results the tumour growth rate of each individual tumour is estimated from the tumour volume
543
Persson et al: Abscopal regression of tumours (N29 rat glioma) Table 3. Average mean tumour growth-rate (% per day) of all experiments t-test Series of Type of TGR vs Ctrl TGR Experiment treatment Right SE N Right Left All Controls 8.4 ± 0.3 40 1.0 9.1 A Controls 9.4 ± 1.1 8 1.0 10.6 B Controls 7.7 ± 0.3 17 1.0 8.7 C Controls 9.1 ± 0.7 8 1.0 9.6 D Controls 8.2 ± 0.8 7 1.0 7.8
Series of Experiment
Type of treatment
All A B C D
PEF PEF PEF PEF PEF
Series of Experiment
Type of treatment
All A B C D
RT RT RT RT RT
Series of Experiment All A B C D
Type of treatment PEF+RT PEF+RT PEF+RT PEF+RT PEF+RT
SE ± 0.3 ± 0.6 ± 0.3 ± 0.6 ± 0.7
N
t-test vs Ctrl left
40 8 17 8 7
1.0 1.0 1.0 1.0 1.0
N
N
25
0.003
8.1
SE ± 0.4
t-test vs Ctrl left
5.9
SE ± 0.6
t-test vs Ctrl Right
25
NS
5.1 6.7 5.7
± 0.9 ± 1.4 ± 0.6
9 9 7
0.03 NS 0.03
7.2 9.9 7.1
± 0.7 ± 0.6 ± 0.6
9 9 7
NS NS NS
N
N
TGR Right
4.5
SE ± 0.3
15
t-test vs Ctrl Right < 0.0001
4.4
± 0.5
8
4.5
± 0.3
TGR Right
TGR Left
6.1
SE ± 0.4
15
t-test vs Ctrl left < 0.0001
< 0.0001
6.6
± 0.6
8
0.01
7
0.002
5.6
± 0.3
7
0.02
TGR Right 5.4
SE ± 0.7
N 15
t-test vs Ctrl Right 0.003
6.0
± 1.3
7
4.9
± 0.4
8
In the groups with independent treatments of the rightlateral tumour, the tumour growth rate was significantly reduced for RT (p<0.01) but no significant effect (p=0.08) with PEF treatment. In the experimental series-D the tumour on the left side was simultaneously inoculated with the tumour on the right side, but with fewer cells to get a smaller tumour at the time of treatment. The right lateral tumours were treated with PEF, RT and their combination PEF+RT. The tumour growth rate of the right-lateral treated tumour in this series was significantly decreased for independent treatments with PEF (p<0.05) and RT (p<0.005) and with combined treatment PEF+RT (p<0.005). The tumour growth rate of the left-lateral untreated tumour is most significantly reduced in the group of rats independently treated with RT on the right-lateral tumour. In the groups with independent treatments with PEF and with combined treatments PEF+RT of the right-lateral tumour, there was no significant reduction of tumour
TGR Left
TGR Left 6.69
SE ± 0.18
N 15
t-test vs Ctrl left < 0.0001
NS
6.71
± 0.32
7
0.0004
0.004
6.67
± 0.21
8
NS
growth rate on the left-lateral tumour. By combining the data from all experimental seriesA,- B, -C and -D) the tumour growth rate of the rightlateral treated tumour was significantly decreased for independent treatments with PEF (p<0.005) in 25 tumours and RT (p<0.001) in 15 tumours and with combined treatment PEF+RT (p<0.005) in 15 tumours. The tumour growth rate of the left-lateral untreated tumour, however, is significantly reduced in the group of rats treated with RT (p<0.0001) in 15 tumours and the combination PEF + RT (p<0.0001) in 15 tumours. In the groups with independent treatment of right-lateral tumours with PEF, there was no significant reduction of the tumour growth rate of the left-lateral tumours. In series-D the combined treatment PEF+RT resulted in significant decrease in the tumour growth rate (p<0.005) and high specific therapeutic effect 0.41. But the tumour growth rate of the left-lateral untreated tumour was not significantly different from the controls (p=0.2) and the
544
Cancer Therapy Vol 2, page 545 Table 4. Average Specific Therapeutic effects STE and Specific Abscopal effect SAE of all experiments Series of Type of STE t-test SAE Experiment treatment Right SE N STE > 0 Left SE ± 0.08 All PEF 0.28 ± 0.02 27 0.0005 0.01 ± 0.1 B PEF 0.33 ± 0.1 9 0.17 ± 0.1 C PEF 0.26 ± 0.2 9 -0.03 ± 0.1 D PEF 0.26 ± 0.1 9 -0.10 Series of Experiment
Type of treatment
All B C D
RT RT RT RT
Series of Experiment
Type of treatment
All B C D
PEF+RT PEF+RT PEF+RT PEF+RT
STE Right
SE 0.44 ± 0.02 0.42 ± 0.06
15 8
0.47 ± 0.06 STE Right
SE 0.32 ± 0.10 0.22 ± 0.17
t-test STE > 0
N
< 0.0001
7
15 7
0.41 ± 0.05
8
specific abscopal effect was as low as 0.17. In a previous investigation it was shown that the therapeutic response of the combined treatment increased with the number of PEF treatments (Persson et al 2003). In the series-D only two PEF treatments were given in comparison to 4 in series-B. Thus the difference in the number of PEF treatments in the two series might be the reason for the difference in therapeutic response.
SE ± 0.02 0.22 ± 0.07 0.24 0.20
t-test STE > 0
N
SAE Left
0.003
± 0.07
SAE Left
SE ± 0.03 0.20 ± 0.04 0.23 0.17
± 0.03
N
t-test SAE > 0
27 NS 9 9 9
N 15 8
t-test SAE > 0 0.0003
7
N 15 7
t-test SAE > 0 < 0.0001
8
the values Abscopal enhancement ratio (AER) are about twice the Therapeutic enhancement ratio (TER) in both experimental series. In a previous study of the therapeutic effect of PEF+RT on rat glioma the TER values were >1 in a few cases (Persson, 2003). Thus by combining tumour treatment with pulsed electric fields and radiation therapy there is an enhancement effect of the treated tumour as well as on the distant non-treated tumours that is more than additive. The average of the abscopal enhancement value AER>1 might indicate that the combination PEF+RT has a synergistic effect. After PEF- treatment only there is no abscopal effect, while it is significantly increased after RT and PEF+RT treatment. This might indicate that RT produces specific factors responsible for the abscopal effect. Emerit et al, (1995) observed radiation-induced clastrogenic factors in plasma samples from RT - patients. It has also been observed that cells exposed with 60Co !-radiation produced a factor that mediates cell death in cells never exposed to radiation (Mothersill 2001). Other studies suggest that ionizing radiation induces the release of cytokines which mediate a systemic anti-tumour effect by activation of immune activity (Uchida 1989). The existence of radiation induced factors in vivo is now well accepted and they are likely to be tissue and patient specific (Mothersill 2002). The mechanisms explaining this phenomenon might be either a molecular or an immunological effect which will be further considered in future investigations.
C. Therapeutic (TER) and abscopal enhancement ratio (AER) The therapeutic enhancement ratio of the combined treatment is defined as the ratio of the specific therapeutic effect of the combined treatment and the sum of the independent treatments.
TER = STEExperimental / STEIndependent The abscopal enhancement ratio of the combined treatment is defined as the ratio of the specific therapeutic effect of the left-lateral untreated tumours on rats at combined treatment of the right-lateral tumour and the sum of the corresponding independent treatments.
AER = SAEExperimental / SAEIndependent Therapeutic enhancement ratio (TER) and Abscopal enhancement ratio (AER) at combined treatment with PEF + RT are 0.29 ± 0.07 and 0.55 ± 0.05(SE) respectively in series-B, and 0.57 ± 0.08 and 1.7± 0.2 respectively in series-D. The average of the enhancement ratios of the two experimental series-B, -D are TER =0.43 ± 0.07 and AER=1.1 ± 0.1 respectively. It is interesting to note that
V. Conclusions The tumour growth rate of the right-lateral treated tumours was significantly decreased for independent treatment with pulsed electric fields (PEF, p<0.005) or
545
Persson et al: Abscopal regression of tumours (N29 rat glioma) new antitumor treatment combining radiation and electric pulses. Anticancer Res 21, 1809-1816. Heller L and Coppola D (2002) Electrically mediated delivery of vector plasmid DNA elicits an antitumour effect. Gene Therapy 9, 1321-1325. Heller R (1995) Treatment of cutaneous nodules using electrochemotherapy. J Florida Med Assoc 82, 147-150. Heller R, Jaroszeski M, Atkin A, Moradpour D, Gilbert R, Wands J and Nicolau C (1996a) In vivo gene electroinjection and expression in rat liver. FEBS Lett 389, 225-228. Heller R, Jaroszeski MJ, Glass LF, Messina JL, Rapaport DP, DeConti RC, Fenske NA, Gilbert RA, Mir LM and Reintgen DS (1996b) Phase I/Il trial for the treatment of cutaneous and subcutaneous tumors using electrochemotherapy. Cancer 77, 964-971. Heller R, Jaroszeski M, Perrott R, Messina J and Gilbert R (1997) Effective treatment of B 16 melanoma by direct delivery of bleomycin using electrochemotherapy. Melanoma Res 7, 10-18. Heller R, Jaroszeski MJ, Reintgen DS, Puleo CA, DeConti RC, Gilbert RA and Glass LF (1998) Treatment of cutaneous and subcutaneous tumors with electrochemotherapy using intralesional bleomycin. Cancer 83, 148-157. Reller R, Gilbert R and Jaroszeski MJ (1999) Clinical applications of electrochemotherapy. Adv Drug Deliv Rev 35, 119-129. Heller R, Gilbert R and Jaroszeski MJ (2000) Clinical trials for solid tumors using electrochemotherapy, in Electrochemotherapy, electrogenetherapy and transdermal drug delivery. Heller R, et al, eds, Humana Press, Totowa, NJ, 137-156. Heller R, Schultz J, Lucas LM, Jarosze ski MJ, Heller LC, Gilbert C, Moelling K and Nicolau C (2001) Intradermal delivery of IL-12 Plasmid by in vivo electroporation. DNA Cell Biol 20, 21-26. Hofmann F, Ohnimus H, Scheller C, Strupp W, Zimmermann U and Jassoy C (1999) Electric field pulses can induce apoptosis. J Membr Biol 169, 103-109. Jaroszeski MJ, Gilbert R and Heller R (1997a) Electrochemotherapy - An Emerging Drug Delivery Method for the Treatment of Cancer [Review]. Adv Drug Deliv Rev 26, 185-197. Jaroszeski MJ, Gilbert RA and Heller R (1997b) In vivo antitumor effects of electrochemotherapy in a hepatoma model. Biochim Biophys Acta 1334, 15-18. Jaroszeski MJ, Illingworth P, Pottinger C, Hyacinthe M and Heller R (1999) Electrically mediated drug delivery for treating subcutaneous and orthotopic pancreatic adenocarcinoma in a hamster model. Anticancer Res 19, 989-994. Kingsley DP (1975) An interesting case of possible effect in malignant melanoma. Br J Radiol 48, 863-866. Knöös T (1991) Dose Planning and Dose Delivery in Radiation Therapy, PhD thesis, ISBN 91-628-0276-3 Ma1mö, Lund University, Lund, Sweden. Kusmartsev S and Gabrilovich DI (2002) Immature myeloid cells and cancer-associated immune suppression. Cancer Immunol Immunother 51, 293-298. Maccarrone M, Rosato N and Finazzi-Agr‰ A (1995b) Electroporation enhances cell membrane peroxidation and luminescence. Biochem Biophys Res Commun 206, 238245. Macklis RM, Mauch PM, Burakoff SJ et al (1992) Lymphoid irradiation results in long-term increases in natural killer cells in patients treated for Hodkin's disease. Cancer 778-783. Meaking WS, Edgerton J, Wharton CW and Meldrum RA (1995) Electroporation-induced damage in mammalian cell DNA. Biochim Biophys Acta 1264, 357-362.
radiation therapy (RT, p<0.001) as well as combined treatments (PEF+RT, p<0.001). The tumour growth rate of the left-lateral untreated tumour was significantly reduced in the group of rats independently treated with RT (p<0.001) and the combination PEF+RT (p<0.001) of the right-lateral tumours. In the groups with independent treatments with PEF of the right-lateral tumour, there was no significant reduction of tumour growth rate of the left-lateral tumours. The specific therapeutic effect “STE” of the rightlateral treated tumours were significant different from zero (p<0.001) for all type of treatments. Corresponding quantity for the left-lateral unexposed tumours, the specific abscopal effect “SAE”, is highly significantly different from zero (p<0.001) for animals treated on the opposite side with RT, or with the combined treatment PEF+RT. But in rats treated with PEF on the right-lateral side the SAE is not significantly different from zero. Abscopal enhancement ratio (AER) at combined treatment with PEF + RT is about twice the Therapeutic enhancement ratio (TER). Thus by combining tumour treatment with pulsed electric fields and radiation therapy there is an enhanced therapeutic effect of the treated tumour as well as on the distant non-treated tumours.
Acknowledgements We thank Susanne Strömblad and Catarina Blennow for excellent technical assistance. Financial support from Swedish Cancer Society, Hedvig Foundation, John and Augusta Persson Foundation for Medical Research, Lund University Hospital's donation funds and Faculty of Medicine at Lund University is gratefully acknowledged.
References Aihara H and Miyazaki J (1998) Gene transfer into muscle by electroporation in vivo. Nat Biotechnol 16, 867-870. Ahtoniades J, Brady LW and Lightfoot DA (1977) Lymphangiographic demonstration of the abscopal effect in patients with malignant lymphomas. Int J Radiat Oncol Biol Phys 2, 141-147. Danfelter M, Engström P, Persson BRR and Salford LG (1998) Effect of high voltage pulses on survival of Chinese hamster V79 lung fibroblast cells. Bioelectrochemistry and Bioenergetics 47, 97-101. DeMaria S, Ng B, Devitt ML, Babb JS, Kawashima N, Liebes L and Formenti SC (2004) Ionizing Radiation Inhibition of Distant Untreated Tumors (Abscopal Effect) is Immune Mediated. Int J Radiation Oncology Biol Phys 58, 862870. DeVita V (1983) Progress in cancer management: Keynote address. Cancer 51, 2401-2409. Ehiers G and Fridman M (1973) Abscopal effect of radiation in papillary adenocarcinoma. Br J Radiol 46, 220-222. Engström P, Salford LG and Persson BRR (1998) Dynamic ! camera studies of 111In-Bleomycin Complex in normal and glioma bearing rats after in vivo electropermeabilization using exponential high-voltage pulses. Bioelectrochemistry and Bioenergetics 46, 241-248. Engström PE, Ivarsson K, Tranberg K-G, Stenram U, Salford LG and Persson RBR (2001a) Electrically mediated drug delivery for treatment of an adenocarcinoma transplanted into rat liver. Anticancer Res 21, 1817-1822. Engström PE, Persson RBR, Brun A and Salford LG (2001b) A
546
Cancer Therapy Vol 2, page 547 Meldrum RA, Bowl M, Ong SB, Richardson S. (1999) Optimisation of electroporation for biochemical experiments in live cells. Biochem Biophys Res Commun 256, 235-239. Mello-Filbo AC and Meneghini R (1984) In vivo formation of single-strand breaks in DNA by hydrogen peroxide is mediated by the Ilaber-Weiss reaction. Biochim Biophys Acta 781, 56-63. Mir LM, Glass LF, Sersa G, Teissie J, Domenge C, Miklavcic D, Jaroszeski MJ, Orlowski S, Reintgen DS, Rudolf Z, Belebradek M, Gilbert R, Rols MP, Belehradek 3, Bachaud JM, DeConti R, Stabuc B, Cemazar M, Coninx P and Heller R (1998) Effective treatment of cutaneous and subcutaneous malignant tumours by electrochemotherapy. Br J Cancer 77, 2336-2342. Mole RJ (1953) Whole body irradiation-radiology or medicine? Br J Radiol 26, 234-241. Nishi T, Yoshizato K, Yamashiro S, Takeshima H, Sato K, Ramada K, Kitamura I, Yoshimura T, Saya H, Kuratsu J and Ushio Y (1996) High-efficiency in vivo gene transfer using intraarterial plasmid DNA injection following in vivo electroporation. Cancer Res 56, 1050-1055. Nobler MP (1969) The abscopal effect in malignant lymphoma and its relationship to lymphocyte circulation. Radiology 93, 410-412. Obha K, Omagari K, Nakamura T et al (1998) Abcopal regression of hepatocellular carcinoma alter radiotherapy for bone metastatis. Gut 43, 575-577. Persson BRR, Bauréus Koch C, Grafström Cl, Engström PE and Salford LG (2003) A Model for Evaluating Therapeutic Response of Combined Cancer Treatment Modalities. Applied to treatment of subcutaneous implanted brain tumours (N32 and N29) in Fischer rats with Pulsed electric fields (PEF) and 60Co-! radiation (RT). Technol Cancer Res Treat 2, 459-470. Pinero J, LopezBaena M, Ortiz T and Cortes F (1997) Apoptotic and necrotic cell death are both induced by electroporation in HL60 human promyeloid leukaemia cells. Apoptosis 2, 330336. Rees OJ (1981) Abscopal regression in lyphomas. A mechanism in common with total body irradiation? Clin Radiol 32, 475480. Rees GJ and Ross C (1983) Abscopal regression following
Bertil R.R. Persson,
radiotherapy for adenocarcinoma. Br J Radiol 56, 63-66. Rols MP, Delteil C, Golzio M, Dumond P, Cros S and Teissie J (1998) In vivo electrically mediated protein and gene transfer in murine melanoma. Nat Biotechnol 16, 168-171. Salford LO, Persson RBR, Brun A, Ceberg CP, Kongstad PC and Mir LM (1993) A new brain tumour therapy combining bleomycin with in vivo electropermeabilization. Biochem Biophys Res Commun 194, 938-943. SBU (2003) The Swedish Council on Technology Assessment in Health Care. Radiation therapy for cancer. SBU Report 162/1, www.sbu.se, Box 5650, SE-114 86 Stockholm. Sersa G, Stabuc B, Cemazar M, Jancar B, Miklavcic D, Rudolf Z (1998) Electrochemotherapy with cisplatin: potentiation of local cisplatin antitumour effectiveness by application of electric pulses in cancer patients. Eur J Cancer 34, 12131218. Sersa G, Cemazar M, Rudolf Z and Fras AP (1999) Adenocarcinoma skin metastases treated by electrochemotherapy with cisplatin combined with radiation. Radiol Oncol 33, 291-296. Sersa G, Kranjc S and Cemazar M (2000) Improvement of combined modality therapy with cisplatin and radiation using electroporation of tumors. Int J Radiat Oncol Biol Phys 46, 1037-1041. Sham RL (1995) The abscopal effect and chronic lymphocytic leukaemia. Am J Med 98, 307-308. Suit HD (2002) The Clray Lecture 2001: Coming technical advances in radiation oncology. Int J Radiat Oncol Biol Phys 53, 798-809. Suit HD, Becht J, Leong J, Stracher M, Wood WC, Verhey L and Cloitein M (1988) Potential for improvement in radiation therapy. Int J Radiat Oncol Biol Phys 14, 777-786. Suit HD and Miralbell R (1989) Potential impact of improvements in radiation therapy on quality of life and survival. Int J Radiat Oncol Biol Phys 16, 891-895. Uchida A, Mizutani Y, Nagamuta M et al (1989) Effects of Xray irradiation on natural killer (NK) cell system. I. Elevation of sensitivity of tumour cells and lytic function of NK cells. Immunopharmacol Immunotoxicol 11, 507-519.
Catrin Baureus Koch,
Crister Ceberg
Leif Salford
547
Gustav Grafström,
Persson et al: Abscopal regression of tumours (N29 rat glioma)
548
Cancer Therapy Vol 2, page 553 Cancer Therapy Vol 2, 553-560, 2004
Occult node metastasis in cervical cancer Research Article
Sumonmal Manusirivithaya1,*, Sumalee Siriaungkul2, Surapan Khunamornpong2, Sunida Rewsuwan2, Siriwan Tangjitgamol1, Manit Sripramote1, Jatupol Srisomboon3, Somnuek Jesadapatarakul4 1
Department of Obstetrics and Gynecology, Bangkok Metropolitan Administration (BMA) Medical College and Vajira Hospital, 2 Department of Pathology, Faculty of Medicine, Chiangmai University, 3Department of Obstetrics and Gynecology, Faculty of Medicine, Chiangmai University, 4 Department of Pathology, BMA Medical College and Vajira Hospital
__________________________________________________________________________________ *Correspondence: Sumonmal Manusirivithaya M.D., Department of Obstetrics and Gynecology, Bangkok Metropolitan Administration Medical College and Vajira Hospital, 681 Samsen Road, Dusit, Bangkok 10300. Tel: 66-2-2443405. Fax 66-2-2437907. Email: sumonmal@vajira.ac.th Key words: occult node metastasis, micrometastasis, cervical cancer, radical hysterectomy, recurrence Abbreviations: Bangkok Metropolitan Administration, (BMA); Hematoxylin-Eosin staining, (H/E staining); lymphovascular space invasion, (LVSI); phosphate buffer saline, (PBS)
This study is supported by the Medical Research Fund of the BMA Medical College and Vajira Hospital Received: 21 December 2004; Accepted: 10 January 2005; electronically published: February 2005
Summary We evaluated the rate of immunohistochemical occult positive lymph nodes, which were negative by conventional Hematoxylin-Eosin staining (H/E staining) in 34 cervical cancer patients who had recurrence after treatment with radical hysterectomy and pelvic nodes dissection during the period of 1992 to 1998. The paraffin embedded tissue blocks of all 1,012 dissected lymph nodes were stained immunohistochemically with AE1/AE3 cytokeratin antibody. The median age of the patients at the time of their primary surgery was 38 years (range, 33-67 years). Local recurrence occurred in 23 patients (68%), five (15%) had isolated distant recurrence, and six (17%) experienced both sites of recurrences. The median number of nodes evaluated in each patient was 26 nodes (range, 6-70 nodes). Obturator nodes comprised nearly half the number of lymph nodes studied as 424 nodes (42%); the other groups of nodes were external iliac, common iliac and paraaortic groups as 304 nodes (30%), 236 nodes (23%), and 48 nodes (5%) respectively. From the immunohistochemical study, all nodes were negative for cytokeratin. In conclusion, occult node metastasis was not detected by AE1/AE3 immunohistochemical staining in any of the negative nodes from H/E staining in the cervical cancer patients who had recurrence post radical hysterectomy. Manusirivithaya et al, 2001b). Its presence necessitates postoperative adjuvant therapy. Despite the adjuvant treatment, recurrences of disease in patients with positive nodes are found as high as 33-42% (Burke et al, 1987; Larson et al, 1988; Manusirivithaya et al, 2001b). However, patients without pelvic lymph node metastases are not absolutely free from tumor recurrences, albeit at lower rate, it still occurs in 7-11% (Burke et al, 1987; Larson et al, 1988; Manusirivithaya et al, 2001b). One possible explanation of this finding is the cancer cells might have already been existing in these negative pelvic nodes, but are not recognized from the routine Hematoxylin-Eosin staining (H/E staining). In recent years, the advantage of immunohistochemical techniques using anticytokeratin
I. Introduction Cervical cancer is the most common cancer, which causes a significant health problem in Thai women. Approximately 5,500 Thai women develop invasive cervical cancer per year (Deerasamee et al, 1999). Among these, 15-25% are in early stage of disease (stage Ib or IIa) (Manusirivithaya, 2001a). These patients are usually treated by radical hysterectomy and pelvic lymphadenectomy, yielding a 5-year survival rate of 8090% (Landoni et al, 1997; Kim et al, 2000). Among various prognostic factors, lymph node metastasis is the most significant predictor of survival, and also influences on the rate of recurrence (Burke et al, 1987; Larson et al, 1988; Wilailak et al, 1993;
553
Manusirivithaya et al: Occult node metastasis in cervical cancer no. K0675 bottle 1). After being rinsed in PBS, the sections were coated with streptavidin-HRP (Dako LSAB code no. K0675 bottle 2) for 10 minutes. The reaction product was developed with diaminobenzidine solution for 10 minutes. Sections were then counterstained with Harry hematoxylin, dehydrated through 95% alcohol and absolute alcohol, and were mounted. Primary cervical cancer specimen was used as positive control. The negative control consisted of sections that were treated with the same technique with the primary antibody omitted. Occult node positive or micrometastasis was defined as a single tumor cell or cluster of tumor cells that were not evidenced on conventionally H/E staining but were detected by anticytokeratin immunohistochemical staining.
antibodies has been demonstrated in identification of micrometastases in histologically negative lymph node on routine H/E staining. Multiple types of cancer have been reported with the positive result of this technique, such as breast cancer (Trojani et al, 1987; McGuckin et al 1996; Reed et al, 2004), colorectal cancer (Cutait et al, 1991; Greenson et al, 1994; Broll et al, 1997; Oberg et al, 1998; Isaka et al, 1999; Yasuda et al, 2001), gastric cancer (Fukagawa et al, 2001; Nakajo et al, 2001; Siewert et al, 1996), esophageal cancer (Natsugoe et al, 1998; Glickman et al, 1999; Komukai et al, 2000; Matsumoto et al, 2000; Mueller et al, 2000; Sato et al, 2001), gall bladder cancer (Yokoyama et al, 1999; Nagakura et al, 2001), lung cancer (Osaki et al, 2002), endometrial cancer (Yabushita et al, 2001; Bosquet et al 2003), prostate cancer (Wilcox et al, 1998), thyroid cancer (Qubain et al, 2002), oral and oropharyngeal cancer (Stoecki et al, 2002). However, the benefit of immunohistochemical staining in detection of occult lymph node metastasis is not consistent in all studies. One study in vulvar cancer did not find any positive node from immunohistochemical staining in addition to H/E staining (Leys et al, 2000). To our knowledge, there have been few studies reported on occult positive node by immunohistochemical staining in cervical cancer (Auger and Cogan, 1990; Juretzka et al, 2004; Lentz et al, 2004). The objective of our study was to examine the rate of occult node positive detected by immunohistochemical staining in cervical cancer patients, who had undergone radical hysterectomy with pelvic lymphadenectomy, and had negative nodes from H/E staining, who eventually developed tumor recurrence.
III. Results During the study period, 39 recurrent cervical cancer patients met all other eligible criteria. Five cases with unavailable pathological paraffin blocks of lymph nodes were excluded. Totals of 34 patients with 1,012 nodes were included in the study. The median age of the patients at the time of surgery was 38 years (range, 33-67 years). Other clinical and pathological characteristics of the patients and tumors are demonstrated in Table 1. Approximately 94% of the patients were in stage Ib. Approximately 62% of the tumors were squamous cell carcinoma; and at the same percentages, the tumor were grade 2. Lymph-vascular space invasion were present in 68%, and depth of invasion were more than half of cervical thickness in 77%. The primary tumor size ranged from no definite gross lesion to 6 cm in maximal diameter with the median of 2.7 cm. Local recurrences occurred in 23 patients (68%), five patients (15%) had isolated distant recurrence, and six patients (17%) had both local and distant recurrences. The recurrence-free interval from primary surgery ranged from 5-67 months with the median of 15 months. About 70% of the recurrence were evidenced within 2 years after surgery, and 94% were diagnosed within 5 years. The treatment for tumor recurrences were radiation alone in 15 cases (44%), radiation and chemotherapy in seven cases (20%), and chemotherapy alone in two cases (6%). Two patients underwent surgery followed by chemotherapy alone or chemotherapy and radiotherapy in each of them. Eight patients declined any treatment. By the time of this report, 18 patients (52%) were dead, eight (24%) were lost to follow up, and eight (24%) were doing well without evidence of disease at their last follow-up visit. The overall 5-year survival rate after recurrence was 44.0% (95%CI: 18.3%, 62.3%). From the total number of 1,012 negative lymph nodes from 34 patients, most were from the obturator group as 424 nodes (42%); the others were external iliac, common iliac and paraaortic nodes as 304 nodes (30%), 236 nodes (23%), and 48 nodes (5%) respectively. The number of nodes evaluated in each patient ranged from 670 nodes with the median of 26 nodes. Approximately 74% of patients had more than 20 lymph nodes dissected from each of them. All of these negative nodes from H/E staining were also negative for cytokeratin from AE1/AE3 immunohistochemical staining.
II. Materials and Methods A. Patients The medical records including personal data, tumor characteristics and follow-up information of all stage Ib and IIa cervical cancer patients, who were treated by radical hysterectomy with pelvic lymph node dissection between January 1992 and December 1998, in Maharaj Nakorn Chiangmai Hospital and Bangkok Metropolitan Administration (BMA) Medical College and Vajira Hospital were reviewed. The inclusion criteria were: 1) patients with tumor histology of squamous cell carcinoma, adenocarcinoma, or adenosquamous cell carcinoma 2) patients who had negative lymph node reported from H/E staining 3) developed tumor recurrence. The exclusion criteria were: 1) past or present history of other cancers (two primary cancers) 2) unavailable paraffin blocks of lymph nodes.
B. Immunohistochemistry study Paraffinâ&#x20AC;&#x201C;embedded tissue blocks of all dissected nodes from the eligible patients were identified. Single section of 3micrometer in thickness was cut from each paraffin block. Paraffin sections were dewaxed with xylene and treated with 95% alcohol. Sections were then treated with 3%H2O2 in phosphate buffer saline (PBS) to block endogenous peroxidase activity. For antigen retrieval, they were immersed in 10 mM/L citrate buffer (pH 6.0), and microwaved at 750 W power for 10 minutes. After the buffer had cooled, sections were treated with anticytokeratin antibody (AE1/AE3) diluted 1:300 for 60 minutes at room temperature. Sections were treated for another 10 minutes with the biotinylated link antibody (Dako LSAB code
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Cancer Therapy Vol 2, page 555 Table 1. Basic characteristics and number of nodes evaluated (n=34) Number
%
Stage Ib IIa
32 2
94.1 5.9
Histology squamous adenocarcinoma adenosquamous
21 9 4
61.8 26.4 11.8
Tumor grade 1 2 3
8 21 5
23.5 61.8 14.7
Lymph-vascular space invasion presence absence
23 11
67.6 32.4
Depth of invasion <1/2 of cervical thickness >1/2 of cervical thickness
8 26
23.5 76.5
Number of nodes evaluated in each patient <10 nodes 11-20 nodes 21-30 nodes 31-40 nodes 41-50 nodes >50 nodes
1 8 12 5 5 3
3.0 23.5 35.3 14.7 14.7 8.8
occult foci which might not be evidenced from the conventional H/E staining. Immunohistochemical staining with anticytokeratin antibody is practically a sensitive and specific method for detecting occult node metastasis. Its application has been reported in many types of cancers. Although its clinical significance could not be demonstrated in some studies (Cutait et al, 1991; Oberg et al, 1998; Fukagawa et al, 2001; Sato et al 2001), most authors (Trojani et al, 1987; Broll et al, 1997; Isaka et al, 1999; Matsumoto et al, 2000; Komukai et al, 2000; Yabushita et al, 2001; Yasuda et al, 2001; Osaki et al, 2002) reported the significant association between microscopic metastasis and higher recurrent rate or shorter survival in various types of cancer (Table 2). In cervical cancer, only few studies (Auger and colgan, 1990; Juretzka et al, 2004; Lentz et al, 2004) reported on occult node metastasis and its clinical significance. In 1990, Auger and Colgan studied the prevalence of occult node micrometastases in cervical cancer by immunohistochemical staining using polyclonal antibody directed against keratin. Only a single metastatic focus was seen in 209 pelvic lymph nodes from 15 radical hysterectomized cervical cancer patients. The authors concluded that the immunohistochemical staining with
IV. Discussion Lymph node metastasis is one of the most important prognostic factors in various types of cancer. Conventionally, the identification of lymph node metastases is based on H/E staining. Recently, with the novel emerging technologies such as immunohistochemical staining or genetic study, the negative lymph nodes that have been evaluated by H/E staining are found to be positive from these techniques. Immunohistochemical staining using anticytokeratin antibody is a simple method which is widely used for the detection of occult node positive in various cancers. Cytokeratin proteins are the essential constituents of the cytoskeleton of epithelial cells (Moll et al, 1982). Approximately 19 different molecular forms of cytokeratin (acidic and basic types) have been identified in both normal and malignant epithelial cell lines, and serve as reliable markers for the cells of epithelial origin (Moll et al, 1982). Usually, these cytokeratins are not present in lymph nodes, unless they are involved with the metastatic tumors. Therefore, the immunohistochemical staining using antibodies that recognize cytokeratin would supplementarily enable the pathologists to detect foci of tumor cells in the lymph nodes, especially the minute or
555
Manusirivithaya et al: Occult node metastasis in cervical cancer Table 2. Relationship between lymph node micrometastases and tumor recurrence in the patients with Hematoxylin-Eosinnegative node Authors Year
Type and Number of Follow up** Cytokeratin positive RR stage of [% (N)] (95% CI) cancer Patients Nodes* Recurrent Nonrecurrent Study that demonstrated the relationship between lymph node micrometastases and tumor recurrence
Trojani, 1986
breast N0M0
122
NA 12 (4-29)
10 yr (6-15 yr)
30.8 (4/13)
8.3 (9/109)
3.7 (1.3-10.4)
Broll, 1997
Colorectal Stage I-III
32
NA
84 mo (2-102mo)
40.0 (2/5)
14.8 (4/27)
2.9 (0.6-13.7)
Isaka, 1999
Rectal Duke B
42
644 15.3 (3-40)
5.2 yr (97-6303d)
50.0 (5/10)
12.5 (4/32)
3.6 (1.3-9.9)
Colorectal Duke B
42
1013 18, (3-94)
18 mo (3-94 mo)
91.7 (11/12)
70 (21/30)
3.4 (0.5-23.5)
Komukai, 2000
Esophagus Squamous, N0
37
2774 75 (38-175)
At least 5 years
77.8 (7/9)
25.0 (7/28)
5.8 (1.4-23.9)
Matsumoto, 2000
Esophagus Squamous, N0
59
NA
NA
94.1 (16/17)
54.8 (23/42)
8.2 (1.2-57.5)
Osaki, 2002
Lung St I, NSCLC
115
2432 21
35.8 mo (0.1-90.6mo)
50.0 (11/22)
22.6 (21/93)
2.6 (1.3-5.4)
Yabushit, 2001
Endometrium Stage I
36
225
> 5 yrs
35.7 (5/14)
0.0 (0/22)
26.1 (1.3519.5)
Yasuda, 2001
Study that could not demonstrate the relationship between micrometastases and tumor recurrence Cutait, 1991
Colorectal Duke A/B
46
603 7 (1-37)
NA (64-135 mo)
29.4 (10/34)
16.7 (2/12)
1.2 (0.9-1.7)
Oberg, 1998
Colorectal Duke A/B
147
609 4 (1-15)
54 mo (18-111mo)
34.8 (8/23)
31.5 (39/124)
1.13 (0.5-2.5)
Fukagawa, 2001
Colorectal T2N0M0
107
4484
120 mo (71-185 mo)
33.3 (2/6)
35.6 (36/101)
0.9 (0.2-4.7)
Sato, 2001
Esophagus Squamous
50
1840
67 mo (7-136 mo)
55.6 (5/9)
36.6 (15/41)
1.9 (0.6-6.1)
Abbreviations: NA, not available; RR, relative risk *node : total in the study; mean or median,( range) in each patient **follow up: median (range)
polykeratin antibody was unlikely to increase the sensitivity in detection of lymph node metastases in cervical cancer. The other study of Juretzka et al (2004) attempted to explore further on the association between the clinical outcome and the positive staining. Their study
used the AE1/ CAM 5.2 immunohistochemical staining to detect the occult node micrometastasis, not initially identified by H/E staining in 976 nodes from 49 stage Ia2Ib2 cervical cancer patients who underwent radical hysterectomy. Two sections from each paraffin tissue 556
Cancer Therapy Vol 2, page 557 block were immunohistologically studied. They could identify micrometastasis in only four nodes from four patients. All of these four patients had other poor prognostic features including lymph-vascular space invasion (n=3), >4 cm in size of primary cancer (n=2). With the mean follow up of 39 months, higher proportion of patients with micrometastasis had recurrences compared to those with negative micrometastasis (2 of 4 patients versus 3 of 45 patients respectively). However, the numbers of patients in this study was too small to draw any conclusion regarding the clinical implications of the immunohistochemically occult node metastasis in cervical cancer. Recently, Lentz et al (2004) studied the prevalence of micrometastasis in 3,106 histologically (H/E staining) negative lymph nodes from 132 cervical cancer patients who were in stage Ia-Ib2. Micrometastases were present in only 29 nodes (1%) from 19 patients (15% [95%CI; 9%, 22%]). The patients who had > 20 resected nodes had higher detection rate than those with <20 nodes (26% compared to 2%). Since the rate of the patients with micrometastatic lymph node (15%) was remarkably approximate to the rate of patients who eventually experience recurrence in the apparently negative node patients, the authors proposed that these patients with micrometastatic node should be the same group of patients who would experience recurrence. However, their study did not provide any follow up information, so the hypothesis remained to be proven. If the hypothesis of Lentz et al is valid, immunohistochemical staining with anticytokeratin antibody should lead us to a more precise identification of the patients who are at risk of recurrence, and this particular group of patients would certainly gain benefit from the adjuvant postoperative treatment. Our study was limited to stage Ib to IIa cervical cancer patients, who had negative node by conventional H/E staining but eventually developed tumor recurrence. We expected that these patients would probably have high rate of occult node positive. Unexpectedly, we could not demonstrate any occult positive node in all 1,012 nodes from the 34 recurrent patients. Our result was different from the previous reports (Juretzka et al, 2004; Lentz et al, 2004). Juretzka et al, (2004) although found only 1% prevalence of occult node positive but they found obviously higher recurrences in the occult node positive patients compared to those with occult node negative, 50% versus 7% respectively. While Lentz et al, (2004) reported the prevalence of micrometastasis as 15% in stage Ia2-Ib2 cervical cancer irrespective of recurrence. We can hardly postulate the total negative occult node in our study despite the high-risk condition of the patients. All of our patients had tumor recurrence; most had other poor prognostic factors as presence of lymphovascular space invasion (LVSI) (68%) and deep tumor invasion (76%). Regarding the number of lymph nodes retrieved from each patient, which was found to be a significant factor associated with the rate of occult node metastasis (Isaka et al, 1999; Matsumoto et al, 2000; Lentz et al, 2004), approximately 74% of our patients had more than 20 nodes harvested.
The limitation of our study was the only single number of section from each lymph node block was evaluated by immunohistochemical staining. Theoretically, in order to detect all possible metastases, all nodes should be serially cut and stained. However, this process could not be applied in general practice because it is impractical, labor intense, and costly. Nevertheless, this factor may not be the rationale to our negative result because the studies which demonstrated the prognostic significance of occult node positive in other cancers also evaluated only single section of each block of lymph node (Greenson et al, 1994; Siewert et al, 1996; Natsugoe et al, 1998; Yokoyama et al, 1999; Mueller et al, 2000; Nagakura et al, 2001; Nakajo et al, 2001; Yabushita et al, 2001). Even the study of McGuckin et al, who conducted their study with four-level sections did not find a significant increase of the positive detection rate for cytokeratin immunohistochemical staining over one-level section (McGuckin et al, 1996). The other difference between our study and the study of Juretzka et al, (2004) and Lentz et al, (2004) is the type of primary antibody for cytokeratin. We used AE1/AE3 antibody while they used AE1/CAM 5.2 Antibody (Juretzka et al, 2004; Lentz et al, 2004). AE1/AE3 is a mixture of two antibodies (AE1 and AE3), which react to a broad spectrum of human keratins. AE1 reacts to most acidic keratin (type I), while AE3 reacts with most basic (type II) cytokeratin (Moll et al, 1982). CAM 5.2 is a mouse monoclonal antibody specialized for cytokeratin 8 and 18 cytokeratin (Moll et al, 1982). We select AE1/AE3 for this study based on the result from Fukagawaâ&#x20AC;&#x2122;s study, which found that AE1/AE3 is the most sensitive antibody for the detection of micrometastasis compared to KL-1 and CAM 5.2 (Fukagawa et al, 2001). However, this statement might be questioned from the negative result of our study whether it is a truly sensitive antibody. On the other hand, the negative result of our study would be affirmed. One could question that the aging of the paraffin blocks might be the reason for negative micrometastasis node in our study. However, both the primary tumor and the positive node of other patients who were operated during the same period clearly demonstrated positive cytokeratin immunohistochemical staining. Hence, the aging of the blocks was unlikely to be the reason for our negative result. We studied the tissue blocks from patients who were operated during the period of 1992 to1998 because this retrospective descriptive study was destined to be the first phase trial. The forthcoming case-control study was intended, if the immunohistochemical staining for cytokeratin could demonstrate a high incidence of occult node metastasis in patients with tumor recurrence. In this circumstance, the controls of the planned study should be those who are free of recurrence for at least 5 years after surgery. In conclusion, in this study, immunohistochemical stiaining with AE1/AE3 antibody did not have any advantage over the conventional staining in demonstration of an occult positive node in any of the early stage cervical cancer patients, even in the high-risk group of patients who eventually developed tumor recurrences.
557
Manusirivithaya et al: Occult node metastasis in cervical cancer Landoni F, Maneo A, Colombo A, Placa F, Milani R, Perego P, Favini G, Ferri L, Mangioni C (1997) Randomised study of radical surgery versus radiotherapy for stage Ib-IIa cervical cancer. Lancet 23, 535-540. Larson DM, Copeland LJ, Stringer CA, Gershenson DM, Malone JM Jr, Edwards CL (1988) Recurrent cervical carcinoma after radical hysterectomy. Gynecol Oncol 30, 381-387. Lentz SE, Muderspach LI, Felix JC, Ye W, Groshen S, Amezcua CA (2004) Identification of micrometastases in histologically negative lymph nodes of early-stage cervical cancer patients. Obstet Gynecol 103,1204-1210. Leys CM, Hartenbach EM, Hafez GR, Mahvi DM (2000) Screening for occult node metastasis in squamous cell carcinoma of the vulva. Int J Gynecol Pathol 19, 243-247. Manusirivithaya S (2001a) Gynecologic Oncology Annual Report of BMA Medical College and Vajira Hospital No 3, p. 64. Manusirivithaya S, Charoeniam V, Isariyodom P, Pantusart A (2001b) Risk for radical hysterectomy failure. J Med Assoc Thai 84, 791-797. Matsumoto M, Natsugoe S, Nakashima S, Sakamoto F, Okumura H, Sakita H, Baba M, Takao S, Aikou T (2000) Clinical significance of lymph node micrometastasis of pN0 esophageal squamous cell carcinoma. Cancer Lett 153, 189197. McGuckin MA, Cummings MC, Walsh MD, Hohn BG, Bennett IC, Wright RG (1996) Occult axillary node metastases in breast cancer: their detection and prognostic significance. Br J Cancer 73, 88-95. Moll R, Franke WW, Schiller DL, Geiger B, Krepler R (1982) The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell 31, 20021124. Mueller JD, Stein HJ, Oyang T, Natsugoe S, Feith M, Werner M, Rudiger Siewert J (2000) Frequency and clinical impact of lymph node micrometastasis and tumor cell microinvolvement in patients with adenocarcinoma of the esophagogastric junction. Cancer 89, 1874-1882. Nagakura S, Shirai Y, Yokoyama N, Hatakeyama K (2001) Clinical significance of lymph node micrometastasis in gallbladder carcinoma. Surgery 129, 704-713. Nakajo A, Natsugoe S, Ishigami S, Matsumoto M, Nakashima S, Hokita S, Baba M, Takao S, Aikou T (2001) Detection and prediction of micrometastasis in the lymph nodes of patients with pN0 gastric cancer. Ann Surg Oncol 8, 158-162. Natsugoe S, Mueller J, Stein HJ, Feith M, Hofler H, Siewert JR (1998) Micrometastasis and tumor cell microinvolvement of lymph nodes from esophageal squamous cell carcinoma: frequency, associated tumor characteristics, and impact on prognosis. Cancer 83, 858-866. Osaki T, Oyama T, Gu CD, Yamashita T, So T, Takenoyama M, Sugio K, Yasumoto K (2002) Prognostic impact of micrometastatic tumor cells in the lymph nodes and bone marrow of patients with completely resected stage I nonsmall-cell lung cancer. J Clin Oncol 20, 2930-2936. Oberg A, Stenling R, Tavelin B, Lindmark G (1998) Are lymph node micrometastases of any clinical significance in Dukes Stages A and B colorectal cancer? Dis Colon Rectum 41, 1244-1249. Qubain SW, Nakano S, Baba M, Takao S, Aikou T (2002) Distribution of lymph node micrometastasis in pN0 welldifferentiated thyroid carcinoma. Surgery 131, 249-256. Reed W, Bohler PJ, Sandstad B, Nesland JM (2004) Occult metastases in axillary lymph nodes as a predictor of survival in node-negative breast carcinoma with long-term follow-up. Breast J 10, 174-180. Sato F, Shimada Y, Li Z, Watanabe G, Maeda M, Imamura M (2001) Lymph node micrometastasis and prognosis in
Acknowledgements The authors would like to thank Ms. Aree Pantusart for her kindly help in gathering the clinical data, Ms. Lakana Eienleng for doing all the immunohistochemical stainings, the staffs in the Department of Pathology of both hospitals for their co-operation. The authors also appreciate the Medical Research Fund of the BMA Medical College and Vajira Hospital for the grant support of this research.
References Auger M, Colgan TJ (1990) Detection of metastatic vulvar and cervical squamous carcinoma in regional lymph nodes by use of a polyclonal keratin antibody. Int J Gynecol Pathol 9, 337-342. Bosquet JG, Keeney GL, Mariani A, Webb MJ, Cliby WA (2003) Cytokeratin staining of resected lymph nodes may improve the sensitivity of surgical staging for endometrial cancer. Gynecol Oncol 91, 518-525. Broll R, Schauer V, Schimmelpenning H et al (1997) Prognostic relevance of occult tumor cells in lymph nodes of colorectal carcinomas: an immunohistochemical study. Dis Colon Rectum 40, 1465-1471. Burke TW, Hoskins WJ, Heller PB, Bibro MC, Weiser EB, Park RC (1987) Prognostic factors associated with radical hysterectomy failure. Gynecol Oncol 26, 153-159. Cutait R, Alves VA, Lopes LC et al (1991) Restaging of colorectal cancer based on the identification of lymph node micrometastases through immunoperoxidase staining of CEA and cytokeratins. Dis Colon Rectum 134, 917-920. Deerasamee S, Martin N, Sontipong S, Sriamporn S, Sriplung H, Srivatanakul P, Vatanasapt V, Parkin DM, Ferlay J (1999) Cancer in Thaliland. IARC technical report No 34, p.56. Fukagawa T, Sasako M, Mann GB, Sano T, Katai H, Maruyama K, Nakanishi Y, Shimoda T (2001) Immunohistochemically detected micrometastases of the lymph nodes in patients with gastric carcinoma. Cancer 92, 753-760. Glickman JN, Torres C, Wang HH, Turner JR, Shahsafaei A, Richards WG, Sugarbaker DJ, Odze RD (1999) The prognostic significance of lymph node micrometastasis in patients with esophageal carcinoma. Cancer 85, 769-778. Greenson JK, Isenhart CE, Rice R, Mojzisik C, Houchens D, Martin EW Jr (1994) Identification of occult micrometastases in pericolic lymph nodes of Duke's B colorectal cancer patients using monoclonal antibodies against cytokeratin and CC49. Correlation with long-term survival. Cancer 73, 563-569. Isaka N, Nozue M, Doy M, Fukao K (1999) Prognostic significance of perirectal lymph node micrometastases in Dukes' B rectal carcinoma: an immunohistochemical study by CAM5.2. Clin Cancer Res 5, 2065-2068. Juretzka MM, Jensen KC, Longacre TA, Teng NN, Husain A (2004) Detection of pelvic lymph node micrometastasis in stage Ia1 â&#x20AC;&#x201C; Ib2 cervical cancer by immunohistochemical analysis. Gynecol Oncol 93, 107-111. Kim SM, Choi HS, Byun JS (2000) Overall 5-year survival rate and prognostic factors in patients with stage IB and IIA cervical cancer treated by radical hysterectomy and pelvic lymph node dissection. Int J Gynecol Cancer 10, 305-312. Komukai S, Nishimaki T, Watanabe H, Ajioka Y, Suzuki T, Hatakeyama K (2000) Significance of immunohistochemically demonstrated micrometastases to lymph nodes in esophageal cancer with histologically negative nodes. Surgery 127, 40-46.
558
Cancer Therapy Vol 2, page 559 patients with oesophageal squamous cell carcinoma. Br J Surg 88, 426-432. Siewert JR, Kestlmeier R, Busch R, Bottcher K, Roder JD, Muller J, Fellbaum C, Hofler H (1996) Benefits of D2 lymph node dissection for patients with gastric cancer and pN0 and pN1 lymph node metastases. Br J Surg 83, 1144-1147. Stoeckli SJ, Pfaltz M, Steinert H, Schmid S (2002) Histopathological features of occult metastasis detected by sentinel lymph node biopsy in oral and oropharyngeal squamous cell carcinoma. Laryngoscope 112, 111-115. Trojani M, de Mascarel I, Bonichon F, Coindre JM, Delsol G (1987) Micrometastases to axillary lymph nodes from carcinoma of breast: detection by immunohistochemistry and prognostic significance. Br J Cancer 55, 303-306. Wilailak S, Tangtrakul S, Srisupundit S, Bullangpoti S, Linasmita V, Bhamarapravati Y (1993) Prognostic factors associated with recurrence in stage IB cervical carcinoma after radical hysterectomy and pelvic lymphadenectomy. J
Med Assoc Thai 76 Suppl 1, 74-77. Wilcox GE, Kattan MW, Slawin KM, Scardino PT, Wheeler TM (1998) Occult pelvic node (PLN) metastases in patients with â&#x20AC;&#x153;pT3N0â&#x20AC;? prostate cancer (Pca) Lab invest 78, 99-102. Yabushita H, Shimazu M, Yamada H, Sawaguchi K, Noguchi M, Nakanishi M, Kawai M (2001) Occult lymph node metastases detected by cytokeratin immunohistochemistry predict recurrence in node-negative endometrial cancer. Gynecol Oncol 80, 139-144. Yasuda K, Adachi Y, Shiraishi N, Yamaguchi K, Hirabayashi Y, Kitano S (2001) Pattern of lymph node micrometastasis and prognosis of patients with colorectal cancer. Ann Surg Oncol 8, 300-304. Yokoyama N, Shirai Y, Hatakeyama K (1999) Immunohistochemical detection of lymph node micrometastases from gallbladder carcinoma using monoclonal anticytokeratin antibody. Cancer 85, 1465-1469.
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Cancer Therapy Vol 2, page 561 Cancer Therapy Vol 2, 561-570, 2004
FAS ligand gene transfer for cancer therapy# Review Article
Jaime F. Modiano1,2,3,*, Angela R. Lamerato-Kozicki1,2, Cristan M. Jubala1,2, David Coffey1, Michelle Borakove1, Jerome Schaack3,4, Donald Bellgrau1,3 1
Integrated Department of Immunology, University of Colorado Health Sciences Center; AMC Cancer Center; 3 Program in Immunology and Immunotherapy, University of Colorado Cancer Center; 4 Department of Microbiology, University of Colorado Health Sciences Center, Denver, CO, USA 2
__________________________________________________________________________________ *Correspondence: Jaime F. Modiano, VMD, PhD, AMC Cancer Center and Integrated Department of Immunology, University of Colorado Health Sciences Center, 2-Diamond Research Bldg., 1600 Pierce Street, Denver CO 80214, USA; Phone: 303-239-3408; Fax: 303-239-3560; E-mail: modianoj@amc.org Key words: Fas ligand, cancer therapy, gene transfer, Apoptosis, Inflammation, antitumor immune responses, antigen load Abbreviations: activation-induced cell death, (AICD); cyclin dependent kinase, (CDK) death-inducing signaling complex, (DISC); Fas ligand, (FasL); Fas-associated death domain, (FADD); IL-1-converting enzyme, (ICE); Lewis Lung carcinoma, (LL); Lung Krupple-like factor, (LKLF or KLF-2); tumor necrosis factor, (TNF); TNF-related apoptosis inducing ligand, (TRAIL); #
Supported by grants from The Cancer League of Colorado, Inc., grants P30CA46934, R01DK58722, R21DK63410, and PO1HD38129 from the National Institutes of Health, and by a grant from the Monfort Family Foundation to the University of Colorado Cancer Center Received: 18 December 2004; Accepted: 11 February 2005; electronically published: February 2005
Summary Fas ligand (FasL) gene therapy has been explored in various clinical settings. This versatility can be traced to pleotropic effects elicited by interactions of Fas and FasL in different tissues, to the context within which FasL is expressed, and to the persistence of FasL in the system. When used to treat cancer, FasL expression is achieved on tumor cells by exogenous gene delivery. In susceptible tumors, ectopic FasL expression leads to Fas-dependent apoptosis, in Fas-resistant tumors, it initiates robust, local inflammatory responses that result in tumor cell death. This minimizes persistence of FasL in the system, but along with the inflammation, tumor cell death promotes specific, protective antitumor immune responses. In this review, we discuss mechanisms that underlie the ability of FasL gene therapy to promote or sustain antitumor responses, as well as its potential as a contemporary clinical tool to treat minimal residual disease and microscopic metastases. cells, FasL promotes tumor cell killing directly and indirectly, and induces reliable antitumor immune responses that can protect animals against subsequent tumor challenge (Figure 1). This approach could be applicable to most cancers, since it relies on the native tumor to induce a systemic antitumor immune response. Conceptually, this may allow development of tumor vaccines without the need to identify or enrich specific tumor antigens.
I. Introduction: Rationale to use Fas ligand gene transfer for cancer therapy Advances in early detection and in conventional tumor therapies have made many cancers treatable, chronic diseases. However, suffering and death rates remain unacceptable for cancers of the lung, breast, prostate, bone, malignant melanoma, and others that are metastatic or inaccessible to conventional treatments. Immunotherapy is an appealing modality to treat cancer because many tumors that are resistant to conventional treatment such as radiation and chemotherapy or that are inaccessible to a surgical approach can be treated using immunologic approaches. One such approach that has shown preclinical promise is the use of Fas ligand (FasL) gene transfer. When expressed in the context of tumor
II. Apoptosis, Fas, and Fas Ligand Apoptosis death program trophic growth death receptor
561
is an evolutionarily conserved, innate cell that can be secondary to withdrawal of factor signals or to signals mediated by pathways (Boise and Thompson, 1996).
Modiano et al: Fas ligand gene transfer for cancer therapy
Figure 1. Antitumor effects of FasL gene transfer. Ectopic expression of FasL can induce antitumor immunity against both Fas-sensitive and Fas-resistant tumors. In Fas sensitive tumors, the interaction between Fas and FasL leads to apoptosis, which in turn can provide priming antigens for an immune response (Bianco et al, 2003). In both cases, ectopic expression of FasL promotes inflammation via interaction with Fas on host leukocytes (neutrophils and macrophages). The inflammatory response kills the tumor cells, making tumor antigens available to prime the immune system.
Death due to trophic factor withdrawal is an evolutionary adaptation seen in multicellular organisms where “death of the one benefits the whole” (Plas et al, 2001; Vander Heiden et al, 2001). The mechanisms involve glucose sensors and the energy producing machinery of the mitochondria. Death receptor-dependent cell death occurs upon ligation of the receptors by their cognate ligands. Death receptors are closely related proteins of the tumor necrosis factor (TNF) receptor family and include TNF receptors I and II, Fas (CD95 or Apo-1) and TNF-related apoptosis inducing ligand (TRAIL) receptors (DR4 and DR5) (Schulze-Osthoff et al, 1998). In particular, Fas is a 45-kDa cell surface glycoprotein that is expressed widely in tissues, although its expression is highest in the liver and in cells of the immune system. Its natural ligand, FasL, is a 55-kDa, TNF-related type II transmembrane protein (Nagata and Golstein, 1995; Krammer, 2000). In
contrast to Fas, expression of FasL is largely restricted to activated T cells and natural killer (NK) cells, although it also is present in some tumors (Nagata and Golstein, 1995; Krammer, 2000; Restifo, 2000; O'Connell et al, 2001). The roles of Fas and FasL in immune homeostasis were first revealed by the lpr and gld mice, which show pathological lymphoproliferation and autoimmunity that is mediated by dysfunctional Fas receptor/ligand interactions (Watanabe-Fukunaga et al, 1992; Suda et al, 1993). The lpr/gld models have been widely interpreted to indicate that Fas receptor/ligand interactions are important to contract clonally expanded populations of activated lymphocytes. This ‘activation-induced cell death’ (AICD) mediated by the interaction of Fas and FasL is essential to terminate immune responses and eliminate unneeded and potentially hostile lymphocytes in normal animals (Nagata
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Cancer Therapy Vol 2, page 563 and Golstein, 1995; Schulze-Osthoff et al, 1998; Holtzman et al, 2000). Most studies on Fas and FasL have focused on their pro-apoptotic functions, although it is noteworthy that these proteins transduce proliferative and activating signals through pathways that remain incompletely understood (Wajant, 2002; Desbarats et al, 2003). Fasmediated cell death can result by a self-propagating caspase cascade (in so called “type I” cells), or by mitochondrial amplification (in “type II” cells). The mechanisms controlling Fas-dependent apoptosis have been reviewed extensively elsewhere (Nagata and Golstein, 1995; Schulze-Osthoff et al, 1998; Krammer, 2000; Savill and Fadok, 2000; Wajant, 2002) and will only be discussed here briefly. In type I cells, ligation of Fas by FasL or by anti-Fas antibodies that promote Fas receptor multimerization leads to formation of a death-inducing signaling complex (DISC) that contains the Fas-associated death domain (FADD) protein and procaspase 8 (FLICE). Procaspase 8 is activated proteolytically in the DISC to form active caspase-8, which in turn cleaves and activates caspase-3 and caspase-9 to culminate in apoptosis (Krammer, 2000). Type II cells are refractory to DISC formation; in these cells, caspase-8 cleaves the BH3 protein Bid, which in turn promotes release of Cytochrome c and SMAC/DIABLO from the mitochondria with consequent activation of downstream caspases. The apoptotic effects of FasL can be propagated locally by cleavage of the protein into a soluble form (FasL-sol) by the action of matrix metalloproteases. Tumors that express Fas receptor and retain intact downstream signaling pathways are susceptible to FasLinduced apoptosis. In such cells, ectopic FasL is an efficient inducer of apoptosis in vitro (Hedlund et al, 1999; Bianco et al, 2003), even in tumor cells that are resistant to Fas ligation with anti-Fas antibodies. In vivo, ectopic expression of FasL can prevent tumor growth and induce tumor regression in both transplantable and naturally occurring tumors (Hedlund et al, 1999; Bianco et al, 2003). More importantly, FasL gene therapy has been shown to be safe in tumor-bearing dogs that approximate human cancer patients more closely than experimental rodents, and the disease-free interval and survival in dogs that received adjuvant FasL was equal to or greater than that seen in historical controls treated with standard-ofcare (Bianco et al, 2003). While FasL-induced tumor cell death and regression in Fas-sensitive tumors was predictable, a more curious observation was that FasL could produce similar effects in Fas-resistant tumors. For example, we and others showed that murine EL-4 thymoma cells (Leon et al, 1998), Lewis Lung carcinoma (LL) cells (Modiano et al, 2004), CT26 human colon carcinoma cells (Arai et al, 1997), and some naturally occurring canine melanomas (Bianco et al, 2003) are resistant to Fas-dependent apoptosis, but remain sensitive to the antitumor effects of ectopic FasL expression in vivo. These and other studies underscore that one or more downstream effects of FasL that are independent of its pro-apoptotic function are important for its antitumor activity.
III. Inflammation induced by Fas ligand A hallmark of apoptosis is cell death in the absence of inflammation (Savill and Fadok, 2000). Especially in higher vertebrates, this benefits the organism by allowing organogenesis, removal of effete cells and tissue remodeling without triggering “danger signals” that activate innate immune responses and damage tissues. This concept of quiescent cell death led to the hypothesis that FasL might contribute to peripheral tolerance by destroying activated T cells that ventured into sites of “immune privilege”, such as the anterior chamber of the eye and other tissues (Bellgrau et al, 1995; Griffith et al, 1995; Uckan et al, 1997). In fact, several reports suggested that ectopic expression of FasL by solid and hematopoietic tumors contributed to tumor growth and survival by making the tumor an immune privileged site (Hahne et al, 1996; O'Connell et al, 1996; Greil et al, 1998; Houston et al, 2003). This notion of immune evasion and tumor counterattack mediated by FasL remains controversial, as it has not been universally reproducible (Chappell et al, 1999; Restifo, 2000, 2001). Rather, a large number of studies showed that transplantation of tissues expressing ectopic FasL led to vigorous suppurative inflammation at the transplant site, with consequent tissue rejection (Restifo, 2000). It remains possible that, in some cases, FasL mediates immune privilege when it is expressed in tissues that produce large amounts of anti-inflammatory cytokines such as transforming growth factor-! (TGF-!) (O'Connell et al, 2001), and that inducing inflammation that overcomes immune privilege is a quantitative, rather than a qualitative feature of FasL expression (Gregory et al, 2002). As noted above, a series of experiments designed to test the concept of immune privilege conferred by ectopic expression of FasL showed, somewhat surprisingly, that ectopic expression of FasL had proinflammatory properties and unanticipated effects (Allison et al, 1997; Kang et al, 1997). This is attributable to release of chemotactic factors and inflammatory cytokines upon apoptosis of macrophages or neutrophils (see below). However, it now appears that the magnitude of the proinflammatory properties also is dependent on the context of FasL expression. In the case where ectopic FasL expression occurs endogenously (i.e., as a transgene), the organism can mitigate its inflammatory effects. For example, one study showed that transgenic expression of FasL in heart muscle led to mild leukocyte infiltration with increased levels of TNF-", IL-1!, IL-6, and TGF-!, but no tissue destruction (Nelson et al, 2000). On the other hand, the inflammatory effects of ectopic expression of exogenous FasL such as that seen when the gene is expressed in tumors by DNA delivery or adenovirus transduction, are unmitigated. This could be because the gene is delivered exogenously, or because higher levels of expression are achievable in this context. Many laboratories, including our own (Modiano et al, 2004) showed that ectopic expression of FasL in transplanted tumors caused neutrophils to infiltrate the tumor injection site (Arai et al, 1997; Seino et al, 1997;
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Modiano et al: Fas ligand gene transfer for cancer therapy Shimizu et al, 1999, 2001; Behrens et al, 2001; Shudo et al, 2001; Gregory et al, 2002; Simon et al, 2002). The nature of the inflammatory response elicited by ectopic FasL expression is becoming clearer. It appears to begin with activation of a caspase cascade, including caspase-1, which is also called IL-1-converting enzyme (ICE). In addition to IL-1, ICE also cleaves and activates IL-18; these cytokines act to recruit neutrophils and macrophages (O'Connell et al, 1996; Restifo, 2000; Shudo et al, 2001; Hohlbaum et al, 2002). High levels of FasL expression in a concentrated area, such as a tumor mass, lead to extensive apoptosis of neutrophils and macrophages (Hohlbaum et al, 2001; Shimizu et al, 2001), which in turn perpetuates the inflammatory response by recruiting additional leukocytes. Although adenoviruses themselves activate innate immunity and promote inflammation, tumors transduced with control adenoviruses (e.g., Ad-GFP) do not elicit either tumor cell killing or protective antitumor responses. This indicates that the inflammatory response that promotes antitumor immunity is specifically initiated by FasL. This can be advantageous in the context of adenoviral gene therapy, given the concerns of persistent adenovirus transduction of human or animal patients. Replication deficient adenoviruses can persist in the body for weeks to months (>70 days), but the adenovirus-mediated expression of FasL is extinguished in a relatively short time (~14 days) because the transduced cells are killed as a consequence of the inflammatory response (Regardsoe et al, 2004). However, the possibility that adenovirus DNA, and hence the ectopic gene, persists in the system and help maintain the immune response cannot not be discounted.
therapy â&#x20AC;&#x201C; mechanisms that generate protective antitumor immune responses It is useful to understand the properties of FasLmediated antitumor responses before we can clarify their underlying mechanisms. First, the generation of protective antitumor responses in experimental animals is wholly dependent on the initial inflammatory response (Shimizu et al, 1999), and it can be blocked by administration of neutralizing anti-FasL antibodies that prevent neutrophils from infiltrating the tumor site. The response is also dependent on the interaction of ectopic FasL with endogenous Fas, as evidenced by the observation that lpr mice do not reject FasL-expressing tumors, nor do they mount protective antitumor responses (Shimizu et al, 1999). Conversely, this response is independent of the tumor cellsâ&#x20AC;&#x2122; susceptibility to apoptosis mediated by death receptors, and it does not require secretion of perforin granules (Behrens et al, 2001). The kinetics of the protective antitumor immune response generated by FasL gene transfer resembles those seen in primary immune responses to antigen. Figure 2 shows that, in the transplantable Lewis Lung carcinoma model of B6 mice, systemic protective antitumor immunity against wild type, FasL-negative tumors arises within ~1 week after the first exposure to tumor antigens (generated by tumor destruction during the initial rejection of the FasL-expressing tumor). After 14 days, 100% of mice reject a wild type tumor challenge. The kinetics of the response probably reflect the time required to produce sufficient tumor-specific T cells, as immunization of mice with FasL-expressing tumors on the contralateral flank at the same time or subsequent to wild type tumor challenge can delay, but not completely prevent growth of wild type tumors (Figure 3).
IV. FasL gene transfer in cancer
Figure 2. Kinetics of FasL-Mediated Antitumor Responses. Five groups of 8 mice were vaccinated with LL cells engineered to express FasL by adenovirus transduction. Groups were challenged with unmodified, wild type LL cells on the day of vaccination, or 7, 10, 15, or 21 days later. The data show the percent of mice from each group that developed tumors after challenge. Asterisks indicate that protection was statistically significant (P<0.01) when mice were challenged at day +10 or later; and trend was present as early as day +7 (P=0.07).
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Figure 3. FasL Gene Transfer Delays Growth of Transplantable Tumors. Groups of mice were challenged with unmodified, wild type LL cells in the lateral flank, and vaccinated with LL cells engineered to express FasL by adenovirus transduction in the contralateral flank on the same day, or 3, 7, or 10 days later. All of the mice developed tumors, but the tumor burden in mice vaccinated on Day 0 and Day +3 was significantly smaller than controls (P<0.005, asterisks).
antigens can undergo limited clonal expansion in vivo, these cells are largely ineffective to mount antitumor responses (Lee et al, 1999). Thus, antitumor immune responses seem to be subject to the same set of rules that maintain autoimmune responses in check, that is, the immune system may be largely tolerant to tumor antigens. When we consider that robust inflammation induced by ectopic FasL induces protective antitumor immunity despite the fact that lymphocytes see cancer cells as “self”, it raises a series of testable, non-mutually exclusive hypotheses. The first is that “autoimmune prone” cells that recognize tumor cells are present in normal naïve animals, but their activation is repressed by intrinsic and/or extrinsic negative regulatory pathways that are overcome by the consequences of ectopic FasL expression (increased antigen load and inflammation). The second is that the inflammatory response initiated by the interaction of FasL with host neutrophils and macrophages provides ‘danger signals’ that break self-tolerance. And the third is that proteolysis of tumor-derived antigens within the inflammatory milieu formed in response to ectopic FasL expression generates novel ‘non-self’ peptides that can be recognized by T cells. Specifically, peptides produced by extracellular proteolysis would be distinct from both native “self” peptides and peptides contained within apoptotic bodies. Peptides generated extracellularly would likely be processed through Class II MHC pathways, whereas peptides contained within apoptotic bodies could be processed through either Class I or Class II MHC pathways (Bellone et al, 1997; Albert et al, 1998; Henry et al, 1999). Tumors that are resistant to FasL-dependent apoptosis, but that are rejected upon ectopic expression of FasL offer viable systems to distinguish among these possibilities (Table 1).
Protective antitumor immunity is only achieved with ectopic expression of membrane-bound isoforms of FasL (Hohlbaum et al, 2001, 2002; Shudo et al, 2001; Gregory et al, 2002; Simon et al, 2002). In fact, ‘outside-in’ signaling by FasL is not necessary as evidenced by the observation that an intracellular truncated form of FasL (Modiano et al, 2004) can induce a protective antitumor response that is as effective as wild type FasL and follows similar kinetics (Figure 4). Rejection of FasL-expressing tumors can occur in the absence of T cells, although it is delayed in athymic nude mice (Shimizu et al, 1999) and in lymphocyte-deficient SCID mice (our unpublished results). On the other hand, the generation of protective antitumor immunity requires CD8 T cells as evidenced by the fact that depletion of CD8 T cells prevents the development of such a response (Shimizu et al, 1999) and by our observation that this response also does not occur in MHC class-I knockout mice (that lack peripheral CD8 T cells). The protective antitumor response generated by FasL gene transfer is specific, as it cannot protect mice from challenge with a distinct tumor (Shimizu et al, 1999). The data presented above indicate that antitumor immunity induced by ectopic FasL expression in tumors is similar to any other immune response to antigen. However, the reasons why most tumors by themselves do not induce robust antitumor responses remain the subject of intense investigation. A few cases are documented where tumor-associated antigens are generated de novo through mutational events, producing proteins that the immune system recognizes as foreign (“non-self”) (Wolfel et al, 1995). However, most tumor-associated antigens are, in fact, normal “self” proteins to which the immune system is tolerant (Sotomayor et al, 1996; Ganss et al, 1999), and although T cells that recognize these tumor
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Figure 4. Outward Signaling by Membrane-Bound FasL is Responsible for Antitumor Response. Four groups of mice were vaccinated as in Figure 2 with LL cells transduced using a replication-defective adenovirus encoding wild type FasL (FasL-wt), a membrane-bound, intracellular truncated FasL mutant (FasL-ict), or a soluble form of FasL (FasL-sol) (Modiano et al, 2004). Groups were challenged with wild type LL cells 10 days after vaccination and followed for tumor growth. The data show the percent of mice from each group that developed tumors after challenge. Asterisks indicate that FasL-wt and FasL-ict afforded significant protection (P<0.02) as compared to the control group.
Table 1. Possible mechanisms for antitumor responses initiated by ectopic FasL gene transfer and consequent inflammation 1. Release of lymphocytes from negative regulation • Increased antigen presentation upon tumor cell death overcomes threshold of intrinsic negative regulation • Cytokines produced by phagocytic cells signal to disable negative regulatory molecules • Expansion of self-reactive tumor-specific cells, or non-self reactive cells with low affinity for antigen promotes antitumor responses 2. Danger signals that break tolerance • Maturation of dendritic cells • Increased antigen load with recognition by B cells (self-antigen) and presentation to T cells • T cell help leads to antitumor responses (humoral, cellular) 3. Generation of novel peptides • Primary tumor cell killed by inflammatory cells • Inflammatory milieu promotes proteolytic digestion of tumor-derived antigens • Novel (non-self) tumor-associated peptides generated from proteolytic digestion are presented to T cells • T cell help leads to cellular antitumor responses
A. Negative regulation and inhibition of antitumor immune responses
proliferation resemble memory cells (Goldrath et al, 2000), and that their requirements for activation are quite distinct from those of naïve T cells, as they are less sensitive to the influence of negative regulation. This suggests that in normal animals (absent homeostatic proliferation), tumor-specific cells might be restrained by the tolerogenic effects of negative regulation. Peripheral tolerance can be mediated by regulatory T cells (extrinsic) (Shevach 2002), as well as by intrinsic negative regulatory factors (Buckley et al, 2001; Tzachanis et al, 2001; Yusuf and Fruman 2003; Lang et al, 2004). A growing body of evidence supports an important role for regulatory T cells to suppress antitumor responses (Sakaguchi et al, 2001; Gavin and Rudensky 2003).
The possibility that “autoimmune prone” cells that recognize tumor cells are present in normal naïve animals is supported by the observation that, under some experimental conditions, homeostatic proliferation is sufficient to overcome tolerance and reject transplanted tumors without a need for a priming response (Hu et al, 2002). In these studies, expansion of naïve cells transferred into Rag1-deficient mice generated specific, type I CD4 and CD8 tumor-specific T cells when mice were challenged with B16 melanoma cells. It has been shown that naïve T cells undergoing homeostatic
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Cancer Therapy Vol 2, page 567 Therefore, it is possible that antitumor responses initiated by FasL selectively eliminate regulatory T cells. Yet, this is unlikely as CD4+, CD25+ regulatory T cells are largely resistant to Fas-dependent apoptosis (Banz et al, 2002). Hence, the alternative possibility is that the inflammatory response initiated by FasL provides impetus for signals that disable intrinsic negative regulation. At least four interrelated pathways of lymphocyte negative regulation have been described. These include transcriptional repression by Lung Krupple-like factor (LKLF or KLF-2) (Kuo et al, 1997; Buckley et al, 2001), Tob (Tzachanis et al, 2001), Forkhead Box proteins (Yusuf and Fruman 2003), and NFATc2 (Baksh et al, 2002; Frazer-Abel et al, 2004). These proteins all share the functional maintenance of proteins such as the cyclin dependent kinase (CDK) inhibitor p27Kip-1 (p27), which prevent cell cycle entry and consequently increase the threshold of T cell activation. Intriguingly, the phenotypes of mice harboring targeted deletion of these molecules are distinct. Homozygous deletion of LKLF resulted in embryonic lethality; however, conditional deletion of LKLF in the lymphoid compartment using the Rag-deficient model led to viable mice whose peripheral T lymphocytes had a spontaneously activated cell surface phenotype and underwent premature apoptosis (Buckley et al, 2001). Tob-deficient mice are viable (Tzachanis et al, 2001), but have hyper-responsive T cells that exist in a state of partial activation. Mice deficient in FoxP3 lack regulatory T cells and develop autoimmunity (Fontenot et al, 2003). Finally, NFATc2-deficient mice have marked splenomegaly and hyper-responsive T cells (Xanthoudakis et al, 1996; Baksh et al, 2002; Frazer-Abel et al, 2004). These models will be useful tools to explore the relationship between immune activation that follows FasL-dependent inflammatory responses and disengagement of intrinsic negative T cell regulation, as well as the significance of these events in the generation of antitumor immune responses.
between these experiments and others showing protection that was mediated by T cells is unclear, but it may be peculiar to the growth patterns and resistance to T celllysis by B16 melanoma rather than due to the route of inoculation (subcutaneous) or other experimental conditions. Most importantly, the antibodies that mediated protective antitumor responses in these animals were clearly self-reactive, directed against melanocyte differentiation antigens, and non cross-reactive with antigens derived from other tumors. These results show that FasL can mimic the ‘danger signals’ that promote dendritic cell maturation into antigen presenting cells, and that these cells can then initiate T cell-dependent, antitumor responses against self-antigens. That is, ectopic expression of FasL in a tumor environment can break peripheral tolerance and produce antitumor responses that resemble autoimmunity. Yet, a fine balance may remain between this ‘autoimmune’ antitumor response and unregulated autoimmunity, as the authors of this study reported the occurrence of a predictable autoimmune response against normal melanocytes (depigmentation) in only 20% of their experimental animals (Simon et al, 2002).
C. Tumor cell death and antigen load in antitumor immune responses A limitation of tumor vaccines and treatments directed against specific tumor antigens is that expression of such antigens can be variable in cancer patients. To circumvent this limitation a treatment approach can be used that relies on inducing immunity against self-antigens or novel antigens generated from the patient’s own tumor. In principle, the ‘danger signals’ associated with ectopic FasL expression that result in tumor cell death and inflammation should enhance the load of intact tumor antigens that are processed and presented by antigen presenting cells, therefore leading to greater recognition of endogenous tumor antigens. On the other hand, proteolytic activity at the site also could generate novel antigens that are distinct from self-antigens. Immunologic priming by apoptosis has been documented in various systems, including uptake and presentation of viral antigens (Bellone et al, 1997; Albert et al, 1998) and tumor antigens (Henry et al, 1999). We showed that apoptotic cell priming increased the capacity of peripheral lymphocytes to kill viable melanoma cells in the presence of IL-2, indicating that there was recruitment of additional IL-2-responsive cells upon presentation of apoptotic cell antigens (Bianco et al, 2003). This was true in vivo, as apoptosis induction also offered a remarkable advantage to prime cytolytic activity of autologous cells from patients with naturally occurring tumors. Adoptive transfer experiments such as those described above could be used to formally distinguish if protective antitumor responses initiated by ectopic expression of FasL-priming require generation of distinct antigens that cannot be recognized by naïve cells even if negative regulation is disengaged and tolerance is broken. If this were the case, such experiments would show that the only mice that acquired protective immune responses were those adoptively transferred with T cells from mice
B. Danger signals that break tolerance to self-antigens promote antitumor immunity The observations that naïve cells can recognize tumors when activation thresholds are reduced by homeostatic proliferation or by inactivation of negative regulatory factors do not distinguish if these cells are selfreactive cells restrained by tolerance, or simply non-self reactive cells with low affinity for antigen. In a series of elegant experiments using FasL-expressing B16 melanoma cells, Simon et al showed that the generation of protective antitumor immunity required inflammation associated with FasL, and that the response was directed against selfantigens (Simon et al, 2002). The mechanisms responsible for protective immunity appeared to require traditional T cell dependent responses: FasL-induced inflammation led to maturation of dendritic cells that not only expressed higher levels of co-stimulatory molecules (CD80, CD86, MHC Class II), but also were more efficient antigen presenting cells; depletion of CD4 T cells abrogated the response. Intriguingly, depletion of CD8 T cells did not affect the response and adoptive transfer of these cells to naïve mice did not protect them from tumor challenge, but the response was transferable by antibody. The difference 567
Modiano et al: Fas ligand gene transfer for cancer therapy Dow SW, Bellgrau D, Cutter GR, Helfand SC and Modiano JF (2003) Enhancing anti-melanoma immune responses through apoptosis. Cancer Gene Ther 10, 726-736. Boise LH and Thompson CB (1996) Hierarchical control of lymphocyte survival. Science 274, 67-68. Buckley AF, Kuo CT and Leiden JM (2001) Transcription factor LKLF is sufficient to program T cell quiescence via a c-Myc-dependent pathway. Nat Immunol 2, 698-704. Chappell DB, Zaks TZ, Rosenberg SA and Restifo NP (1999) Human melanoma cells do not express Fas (Apo-1/CD95) ligand. Cancer Res 59, 59-62. Desbarats J, Birge RB, Mimouni-Rongy M, Weinstein DE, Palerme JS and Newell MK (2003) Fas engagement induces neurite growth through ERK activation and p35 upregulation. Nat Cell Biol 5, 118-125. Fontenot JD, Gavin MA and Rudensky AY (2003) Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol 4, 330-336. Frazer-Abel AA, Baksh S, Fosmire SP, Willis D, Pierce AM, Meylemans H, Linthicum DS, Burakoff SJ, Coons T, Bellgrau D and Modiano JF (2004) Nicotine activates NFATc2 and prevents cell cycle entry in T cells. J Pharmacol Exp Ther 311, 758-769. Ganss R, Limmer A, Sacher T, Arnold B and Hammerling GJ (1999) Autoaggression and tumor rejection: it takes more than self-specific T-cell activation. Immunol Rev 169, 263272. Gavin M and Rudensky A (2003) Control of immune homeostasis by naturally arising regulatory CD4+ T cells. Curr Opin Immunol 15, 690-696. Goldrath AW, Bogatzki LY and Bevan MJ (2000) Naive T cells transiently acquire a memory-like phenotype during homeostasis-driven proliferation. J Exp Med 192, 557-564. Gregory MS, Repp AC, Holhbaum AM, Saff RR, MarshakRothstein A and Ksander BR (2002) Membrane Fas ligand activates innate immunity and terminates ocular immune privilege. J Immunol 169, 2727-2735. Greil R, Egle A and Villunger A (1998) On the role and significance of Fas (Apo-1/CD95) ligand (FasL) expression in immune privileged tissues and cancer cells using multiple myeloma as a model. Leuk Lymphoma 31, 477-490. Griffith TS, Brunner T, Fletcher SM, Green DR and Ferguson TA (1995) Fas ligand-induced apoptosis as a mechanism of immune privilege. Science 270, 1189-1192. Hahne M, Rimoldi D, Schroter M, Romero P, Schreier M, French LE, Schneider P, Bornand T, Fontana A, Lienard D, Cerottini J and Tschopp J (1996) Melanoma cell expression of Fas(Apo-1/CD95) ligand: implications for tumor immune escape. Science 274, 1363-1366. Hedlund TE, Meech SJ, Srikanth S, Kraft AS, Miller GJ, Schaack JB and Duke RC (1999) Adenovirus-mediated expression of Fas ligand induces apoptosis of human prostate cancer cells. Cell Death Differ 6, 175-182. Henry F, Boisteau O, Bretaudeau L, Lieubeau B, Meflah K and Gregoire M (1999) Antigen-presenting cells that phagocytose apoptotic tumor-derived cells are potent tumor vaccines. Cancer Res 59, 3329-3332. Hohlbaum AM, Gregory MS, Ju ST and Marshak-Rothstein A (2001) Fas ligand engagement of resident peritoneal macrophages in vivo induces apoptosis and the production of neutrophil chemotactic factors. J Immunol 167, 6217-6224. Hohlbaum AM, Saff RR and Marshak-Rothstein A (2002) Fasligand--iron fist or Achilles' heel? Clin Immunol 103, 1-6. Holtzman MJ, Green JM, Jayaraman S and Arch RH (2000) Regulation of T cell apoptosis. Apoptosis 5, 459-471.
that were originally immunized with FasL-expressing tumors for >1 week. A failure of these adoptive transfer experiments to offer protection from challenge with wild type tumor, would suggest that additional cells (e.g., dendritic cells or B cells) are required for the effect, or that other essential events required for the response occur during presentation of tumor antigens in the host that is vaccinated with the FasL-expressing tumor. A similar experiment could be used to test the efficacy of this therapy to treat minimal residual disease and metastasis, by evaluating protection afforded by the adoptively transferred cells to reject transplantable tumors that are inoculated systemically and seed distant sites.
V. Conclusion In this review, we present a preponderance of evidence to support the generation of specific, protective antitumor responses by ectopic expression of FasL in distinct tumor types. Understanding the mechanisms that mediate these responses will allow us to continue developing this therapy as an integral component of the armamentarium to manage cancer patients and improve their outcomes.
Acknowledgements The authors thank Drs. Juan Sun and Ashley FrazerAbel for experimental assistance and helpful discussions.
References Albert ML, Sauter B and Bhardwaj N (1998) Dendritic cells acquire antigen from apoptotic cells and induce class Irestricted CTLs. Nature 392, 86-89. Allison J, Georgiou HM, Strasser A and Vaux DL (1997) Transgenic expression of CD95 ligand on islet ! cells induces a granulocytic infiltration but does not confer immune privilege upon islet allografts. Proc Natl Acad Sci U S A 94, 3943-3947. Arai H, Gordon D, Nabel EG and Nabel GJ (1997) Gene transfer of Fas ligand induces tumor regression in vivo. Proc Natl Acad Sci U S A 94, 13862-13867. Baksh S, Widlund HR, Frazer-Abel AA, Du J, Fosmire S, Fisher DE, DeCaprio JA, Modiano JF and Burakoff SJ (2002) NFATc2-mediated repression of cyclin-dependent kinase 4 expression. Mol Cell 10, 1071-1081. Banz A, Pontoux C and Papiernik M (2002) Modulation of Fasdependent apoptosis: a dynamic process controlling both the persistence and death of CD4 regulatory T cells and effector T cells. J Immunol 169, 750-757. Behrens CK, Igney FH, Arnold B, Moller P and Krammer PH (2001) CD95 ligand-expressing tumors are rejected in antitumor TCR transgenic perforin knockout mice. J Immunol 166, 3240-3247. Bellgrau D, Gold D, Selawry H, Moore J, Franzusoff A and Duke RC (1995) A role for CD95 ligand in preventing graft rejection. Nature 377, 630-632. Bellone M, Iezzi G, Rovere P, Galati G, Ronchetti A, Protti MP, Davoust J, Rugarli C and Manfredi AA (1997) Processing of engulfed apoptotic bodies yields T cell epitopes. J Immunol 159, 5391-5399. Bianco SR, Sun J, Fosmire SP, Hance K, Padilla ML, Ritt MG, Getzy DM, Duke RC, Withrow SJ, Lana S, Matthiesen DT,
568
Cancer Therapy Vol 2, page 569 Houston A, Bennett MW, O'Sullivan GC, Shanahan F and O'Connell J (2003) Fas ligand mediates immune privilege and not inflammation in human colon cancer, irrespective of TGF-! expression. Br J Cancer 89, 1345-1351. Hu HM, Poehlein CH, Urba WJ and Fox BA (2002) Development of antitumor immune responses in reconstituted lymphopenic hosts. Cancer Res 62, 39143919. Kang SM, Schneider DB, Lin Z, Hanahan D, Dichek DA, Stock PG and Baekkeskov S (1997) Fas ligand expression in islets of Langerhans does not confer immune privilege and instead targets them for rapid destruction. Nat Med 3, 738-743. Krammer PH (2000) CD95's deadly mission in the immune system. Nature 407, 789-795. Kuo CT, Veselits ML and Leiden JM (1997) LKLF: A transcriptional regulator of single-positive T cell quiescence and survival. Science 277, 1986-1990. Lang JA, Kominski D, Bellgrau D and Scheinman RI (2004) Partial activation precedes apoptotic death in T cells harboring an IAN gene mutation. Eur J Immunol 34, 23962406. Lee PP, Yee C, Savage PA, Fong L, Brockstedt D, Weber JS, Johnson D, Swetter S, Thompson J, Greenberg PD, Roederer M and Davis MM (1999) Characterization of circulating T cells specific for tumor-associated antigens in melanoma patients. Nat Med 5, 677-685. Leon RP, Hedlund T, Meech SJ, Li S, Schaack J, Hunger SP, Duke RC and DeGregori J (1998) Adenoviral-mediated gene transfer in lymphocytes. Proc Natl Acad Sci U S A 95, 13159-13164. Modiano JF, Sun J, Lang J, Vacano G, Patterson D, Chan D, Franzusoff A, Gianani R, Meech SJ, Duke R and Bellgrau D (2004) Fas ligand-dependent suppression of autoimmunity via recruitment and subsequent termination of activated T cells. Clin Immunol 112, 54-65. Nagata S and Golstein P (1995) The Fas death factor. Science 267, 1449-1456. Nelson DP, Setser E, Hall DG, Schwartz SM, Hewitt T, Klevitsky R, Osinska H, Bellgrau D, Duke RC and Robbins J (2000) Proinflammatory consequences of transgenic Fas ligand expression in the heart. J Clin Invest 105, 1199-1208. O'Connell J, Houston A, Bennett MW, O'Sullivan GC and Shanahan F (2001) Immune privilege or inflammation? Insights into the Fas ligand enigma. Nat Med 7, 271-274. O'Connell J, O'Sullivan GC, Collins JK and Shanahan F (1996) The Fas counterattack: Fas-mediated T cell killing by colon cancer cells expressing Fas ligand. J Exp Med 184, 10751082. Plas DR, Talapatra S, Edinger AL, Rathmell JC and Thompson CB (2001) Akt and Bcl-xL promote growth factorindependent survival through distinct effects on mitochondrial physiology. J Biol Chem 276, 12041-12048. Regardsoe EL, McMenamin MM, Charlton HM and Wood MJ (2004) Local adenoviral expression of Fas ligand upregulates pro-inflammatory immune responses in the CNS. Gene Ther 11, 1462-1474. Restifo NP (2000) Not so Fas: Re-evaluating the mechanisms of immune privilege and tumor escape. Nat Med 6, 493-495. Restifo NP (2001) Countering the 'counterattack' hypothesis. Nat Med 7, 259. Sakaguchi S, Sakaguchi N, Shimizu J, Yamazaki S, Sakihama T, Itoh M, Kuniyasu Y, Nomura T, Toda M and Takahashi T (2001) Immunologic tolerance maintained by CD25+ CD4+ regulatory T cells: their common role in controlling
autoimmunity, tumor immunity, and transplantation tolerance. Immunol Rev 182, 18-32. Savill J and Fadok V (2000) Corpse clearance defines the meaning of cell death. Nature 407, 784-788. Schulze-Osthoff K, Ferrari D, Los M, Wesselborg S and Peter ME (1998) Apoptosis signaling by death receptors. Eur J Biochem 254, 439-459. Seino K, Kayagaki N, Okumura K and Yagita H (1997) Antitumor effect of locally produced CD95 ligand. Nat Med 3, 165-170. Shevach EM (2002) CD4+ CD25+ suppressor T cells: more questions than answers. Nat Rev Immunol 2, 389-400. Shimizu M, Fontana A, Takeda Y, Yagita H, Yoshimoto T and Matsuzawa A (1999) Induction of antitumor immunity with Fas/APO-1 ligand (CD95L)-transfected neuroblastoma neuro-2a cells. J Immunol 162, 7350-7357. Shimizu M, Fontana A, Takeda Y, Yoshimoto T, Tsubura A and Matsuzawa A (2001) Fas/Apo-1 (CD95)-mediated apoptosis of neutrophils with Fas ligand (CD95L)-expressing tumors is crucial for induction of inflammation by neutrophilic polymorphonuclear leukocytes associated with antitumor immunity. Cell Immunol 207, 41-48. Shudo K, Kinoshita K, Imamura R, Fan H, Hasumoto K, Tanaka M, Nagata S and Suda T (2001) The membrane-bound but not the soluble form of human Fas ligand is responsible for its inflammatory activity. Eur J Immunol 31, 2504-2511. Simon AK, Gallimore A, Jones E, Sawitzki B, Cerundolo V and Screaton GR (2002) Fas ligand breaks tolerance to selfantigens and induces tumor immunity mediated by antibodies. Cancer Cell 2, 315-322. Sotomayor EM, Borrello I and Levitsky HI (1996) Tolerance and cancer: a critical issue in tumor immunology. Crit Rev Oncog 7, 433-456. Suda T, Takahashi T, Golstein P and Nagata S (1993) Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family. Cell 75, 1169-1178. Tzachanis D, Freeman GJ, Hirano N, van Puijenbroek AA, Delfs MW, Berezovskaya A, Nadler LM and Boussiotis VA (2001) Tob is a negative regulator of activation that is expressed in anergic and quiescent T cells. Nat Immunol 2, 1174-1182. Uckan D, Steele A, Cherry, Wang BY, Chamizo W, Koutsonikolis A, Gilbert-Barness E and Good RA (1997) Trophoblasts express Fas ligand: a proposed mechanism for immune privilege in placenta and maternal invasion. Mol Hum Reprod 3, 655-662. Vander Heiden MG, Plas DR, Rathmell JC, Fox CJ, Harris MH and Thompson CB (2001) Growth factors can influence cell growth and survival through effects on glucose metabolism. Mol Cell Biol 21, 5899-5912. Wajant H (2002) The Fas signaling pathway: more than a paradigm. Science 296, 1635-1636. Watanabe-Fukunaga R, Brannan CI, Copeland NG, Jenkins NA and Nagata S (1992) Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 356, 314-317. Wolfel T, Hauer M, Schneider J, Serrano M, Wolfel C, Klehmann-Hieb E, De Plaen E, Hankeln T, Meyer zum Buschenfelde KH and Beach D (1995) A p16INK4ainsensitive CDK4 mutant targeted by cytolytic T lymphocytes in a human melanoma. Science 269, 12811284. Xanthoudakis S, Viola JP, Shaw KT, Luo C, Wallace JD, Bozza PT, Luk DC, Curran T and Rao A (1996) An enhanced immune response in mice lacking the transcription factor NFAT1. Science 272, 892-895.
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Modiano et al: Fas ligand gene transfer for cancer therapy Yusuf I and Fruman DA (2003) Regulation of quiescence in lymphocytes. Trends Immunol 24, 380-386.
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Cancer Therapy Vol 2, page 571 Cancer Therapy Vol 2, 571-574, 2004
Primary peritoneal malignant mixed mullerian tumor (MMMT): a case report Case Report
Marcelo Carraro Nascimento1, Poh See Choo2, Judy Bligh3, Andreas Obermair1,* 1
Queensland Centre for Gynaecological Cancer, Royal Brisbane and Womenâ&#x20AC;&#x2122;s Hospital, 6th Floor, Ned Hanlon Building, Butterfield Street, Herston QLD 4029, Australia 2 Greenslopes Specialist Centre, Greenslopes Hospital, Newdegate Street, Greenslopes QLD 4120, Australia 3 Sullivan Nicolaides Pathology, 134 Whitmore Street, Taringa, QLD 4068, Australia
__________________________________________________________________________________ *Correspondence: A/Prof. Andreas Obermair, Queensland Centre for Gynaecological Cancer, Royal Brisbane and Womenâ&#x20AC;&#x2122;s Hospital, 6th Floor, Ned Hanlon Building, Butterfield Street, Herston QLD 4029, Australia; Phone ++61 7 3636 8501; Fax ++61 7 3636 5289; Email: andreas_obermair@health.qld.gov.au Key words: Primary peritoneal malignant mixed mullerian tumor, Histopathologic findings Abbreviations: malignant mixed mullerian tumor (MMMT); Received: 2 December 2004; Accepted: 28 January 2005; electronically published: March 2005
Summary Primary Peritoneal Malignant Mixed Mullerian Tumours (MMMT) are extremely rare and our knowledge about the effectiveness of surgery and postoperative treatment is limited. A case of Primary Peritoneal MMMT in an 86year old patient is reported. She presented with ascites, abdominal mass and elevated CA125 serum level. Due to medical impairment she received neoadjuvant single agent carboplatin, which was ineffective. After adding paclitaxel she had a remarkable response with improvement of symptoms and decline of CA125 levels. Subsequent optimal debulking revealed primary peritoneal MMMT. We believe that the addition of paclitaxel to carboplatin was crucial to both, improve the symptoms and minimise the disease preoperatively. This case should encourage investigators to evaluate the role of paclitaxel as part of combination chemotherapy of MMMT. demonstrated a large intra-abdominal mass, disseminated disease and ascites. Her Chest X-Ray was clear. Her CA125 was 720 U/mL and CEA was 1.3 mcg/L at presentation. Her surgical history included removal of a squamous cell carcinoma of her nose and a total hip replacement. Medically she was troubled by ischaemic heart disease, hypertension, high cholesterol levels, and osteoporosis. She had no history of lung disease. On general examination Mrs. W.B. was short of breath on very minor levels of exertion. Her abdomen was distended due to ascites. A small umbilical hernia was noted and on internal examination a mass could be felt in the pelvis, and nodularity was noted in the pouch of Douglas. A paracentesis revealed cells highly suspicious of adenocarcinoma. The diagnosis of advanced ovarian cancer was assumed. The patient was felt not to be fit for major pelvic surgery. Neo-adjuvant chemotherapy with single-agent carboplatin (360 mg/m2) was administered for two cycles. During this treatment her CA 125 increased to 1311 U/mL. Chemotherapy was changed to carboplatin (360
I. Introduction First described in 1955, Primary Peritoneal Malignant Mixed Mullerian Tumours (MMMT) are extremely rare (Ober and Black, 1955). To date, approximately 30 cases with MMMT arising from the pelvic peritoneum, the pelvic wall, or omentum have been reported (Shen et al, 2001). Little is known about its histogenesis and pathogenesis. A possible association between endometriosis and primary peritoneal MMMT was described (Rose, 1997). There is no standard treatment and our knowledge about the effectiveness of surgery and postoperative treatment is limited. The prognosis is extremely poor and most patients die of disease within a year from diagnosis (Garamvoelgyi, 1994). We describe a case of primary peritoneal MMMT treated with neoadjuvant chemotherapy followed by surgery.
II. Case report Mrs W.B., 86 years of age, presented in October 2003 with increasing abdominal distension and discomfort. CT and ultrasound of the pelvis/abdomen 571
Nascimento et al: Primary peritoneal malignant mixed mullerian tumor (MMMT) mg/m2) plus paclitaxel (175 mg/m2) for another 5 cycles. During this combination chemotherapy her general health improved dramatically and her CA125 levels dropped to 67 U/mL in January 2004. Surgical debulking in February 2004 revealed an umbilical metastasis of 2 cm and several pelvic masses of up to 11 cm, which were firmly attached to the rectosigmoid colon. There was miliary disease on the peritoneal surfaces of the pelvic peritoneum but the peritoneum of the upper abdomen was clear. The uterus and the ovaries looked macroscopically uninvolved. A total hysterectomy, bilateral salpingo-oophorectomy, pelvic and aortic lymph node dissection, resection of pelvic tumour and resection of the umbilical metastasis were performed. The post-operative macroscopic residual tumour was less than 0.5 cm.
disease was primarily peritoneal and very minimal involvement of the surface of only the right adnexa was consistent with a primary peritoneal carcinosarcoma (Figure 2). Mrs W.B. completed another three cycles of carboplatin and paclitaxel. Currently she is asymptomatic and her CA125 is 9 U/ml.
IV. Discussion We report a case of primary peritoneal MMMT who responded to paclitaxel-based chemotherapy. Primary peritoneal MMMT is extremely rare. So far, approximately 30 cases of primary peritoneal MMMT have been reported in the literature with the majority of patients being postmenopausal. Compared to other tumours in the female genital tract, primary peritoneal MMMT holds an extremely poor prognosis with most of the patients dying of their disease in less than twelve months (Garamvoelgyi, 1994). For the present case, the primary therapy was chosen based on the clinical presentation, levels of CA125, imaging results and cytopathologic findings. The assumed diagnosis was advanced epithelial ovarian cancer. Due to the patientâ&#x20AC;&#x2122;s age and co-morbidities treatment commenced with single agent carboplatin chemotherapy. However, the CA125 levels increased and symptomatically the patient did not improve. paclitaxel was added to carboplatin for five more cycles. During this treatment the CA125 levels substantially decreased and the patientâ&#x20AC;&#x2122;s symptoms improved dramatically. Subsequent surgical debulking revealed the diagnosis of a primary peritoneal MMMT. Cytoreduction was successfully performed.
III. Histopathologic findings Macroscopically, both ovaries were unremarkable. The sigmoid tumour mass was 110x95x60mm. The rectal nodule measures 35x23x18mm. The bladder nodule measured 10mm and the pelvic node specimen 40x23 mm. On microscopic examination, the left ovary showed no evidence of malignancy. No sarcoma was seen in the ovary. Within the atrophic cortex of the right ovary, there was a 3.5mm diameter deposit of adenocarcinoma. The carcinomatous component included serous carcinoma and squamous carcinoma, with an associated foreign body giant cell reaction to free keratin. The carcinoma was present with poorly differentiated sarcomatous stroma. Malignant cartilaginous nodules with focal ossification were scattered throughout the sarcomatous stroma in keeping with heterologous sarcomatous differentiation (Figure 1). The distribution of
Figure 1. The peritoneal MMMT. Section shows the sarcomatous and carcinomatous components
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Figure 2. Absence of disease in the ovary mullerian tumors of the ovary (OMMMT). Gynecol Oncol. 93, 506-512. Curtin JP, Blessing JA, Soper JT, DeGeest K. (2001) Paclitaxel in the Treatment of Carcinosarcoma of the Uterus: A Gynecologic Oncology Group Study. Gynecol Oncol. 83, 268-270. Garamvoelgyi E, Guillou L, Gebhard S, Salmeron M, Seematter RJ, Hadji MH (1994) Primary Malignant Mixed Mullerian Tumor (Metaplastic Carcinoma) of the Female Peritoneum. Cancer 74, 854-863. Ober WB, Black MB (1955) Neoplasm of the subcoelomic mesenchyme. AMA Arch Pathol. 59, 698. Rose PG, Rodriguez M, Abdul-Karim FW (1997) Malignant Mixed Mullerian Tumor of the Female Peritoneum: Treatment and Outcome of Three Cases. Gynecol Oncol. 65, 523-525. Shen DH, Khoo US, Xue WC, Ngan HYS, Wang JL, Liu VWS, Chan YK, Cheung ANY (2001) Primary Peritoneal Malignant Mixed Mullerian Tumors. Cancer 1, 1052-1060. Sutton GP, Blessing JA, Rosenshein N, Photopulos G, DiSaia PJ (1989) Phase II trial of ifosfamide and mesna in mixed mesodermal tumors of the uterus (a Gynecologic Oncology Group study). Am J Obstet Gynecol.161, 309-312. van Rijswijk RE, Vermorken JB, Reed N, Favalli G, Mendiola C, Zanaboni F, Mangili G, Vergote I, Guastalla JP, ten Bokkel Huinink WW, Lacave AJ, Bonnefoi H, Tumulo S, Rietbroek R, Teodorovic I, Coens C, Pecorelli S (2003). Cisplatin, doxorubicin and ifosfamide in carcinosarcoma of the female genital tract. A phase II study of the European Organization for Research and Treatment of Cancer Gynaecological Cancer Group (EORTC 55923). Eur J Cancer 39, 481-487.
Ovarian and primary peritoneal MMMT are extremely rare tumours and their prognosis is poor when compared to epithelial cancers (Barnholtz-Sloan et al, 2004). Traditionally, patients with advanced or recurrent uterine MMMT receive ifosfamide, cisplatin and/or doxorubicin. Treatment with single agent ifosfamide produced five complete and four partial responses among 28 patients with advanced uterine MMMT (Sutton et al, 1989). Recently, a GOG study showed a moderate 18% total response rate to single-agent paclitaxel in 44 patients with recurrent or advanced uterine carcinosarcoma who had radiotherapy previously (Curtin et al, 2001). An EORTC phase II study reported an overall response rate of 56% and a median survival of 26 months in a cohort of 48 patients with genital carcinosarcoma treated with concomitant doxorubicin, cisplatin and ifosfamide (van Rijswijk et al, 2003). The aim of this case report is to share our experience, in which the addition of paclitaxel to carboplatin was crucial to both, improve the symptoms and minimise the disease. Our neoadjuvant chemotherapy approach made optimal surgical debulking possible. Almost one year after treatment, our patient is asymptomatic and well. This case also should encourage investigators to evaluate the role of paclitaxel as part of combination chemotherapy of MMMT.
References Barnholtz-Sloan JS, Morris R, Malone Jr. JM, Munkarah AR (2004) Survival of women diagnosed with malignant, mixed
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Complete remission of an unusual location of metastatic gestational trophoblastic neoplasia GTN:a case report Case Report
Nadereh Behtash1,*, Malihe Hasanzadeh1, Parviz Hanjani2 1 2
Fellow of Gynecology Oncology, Tehran University of Medical Sciences. Professor of Gynecology Oncology, Gynecology Oncology Department, Temple University, Philadelphia, Pennsylvania.
__________________________________________________________________________________ *Correspondence: Nadereh Behtash, Associate Professor, Gynecologist Oncologist, Tehran University of Medical Sciences. Gynecology Oncology Department, Vali-e-Asr Hospital, Imam Khomeini Hospital Complex, Keshavarz Blvd., Tehran 14194, Iran; Phone: #98-21-6939320; Fax: #98-21-6937321; E-mail: valrec2@yahoo.com, nadbehtash@yahoo.com Key words: Gestational trophoblastic neoplasia, Rectovaginal septum, Pelvic metastasis, Vaginal metastasis, Actinomycin-D. Abbreviations: gestational trophoblastic neoplasia, (GTN); gestational trophoblastic tumor, (GTT) Received: 13 December 2004; Revised: 23 February 2005 Accepted: 2 March 2005; electronically published: March 2005
Summary Metastatic gestational trophoblastic neoplasia, (GTN) occurs in 4% of patients, after evacuation of a complete mole and infrequently after other pregnancies. The most common metastatic sites are the lung (80%), vagina (30%), brain (10%), liver (10%). We couldn't find any report of metastasis in rectovaginal septum in literature. To our knowledge this is the first case of metastatic GTN in rectovaginal septum. Four weeks after evacuation of a molar pregnancy in a 28-year-old woman, a 45-55 mm solid mass was palpable deep in rectovaginal septum. Rising titer of serum B-HCG and pelvic MRI, confirmed metastatic lesion. After 5 courses of single agent chemotherapy, serum BHCG returned to normal, and pelvic MRI showed no abnormality. Although very rare, GTN can metastases to rectovaginal septum. Careful pelvic examination can help detection of metastatic site in the absence of metastasis in other common sites. This large deep seated pelvic metastasis responded to single and alternate agent chemotherapy.
anterior wall and 25% were in the fornices (Yingna et al, 2002). A search of MEDLINE data base using the PUBMED retrieval service, with the keywords "Gestational Trophoblastic Neoplasia" (GTN), "rectovaginal septum", "vaginal metastasis" and "pelvic metastases" for the period from 1966 to July 2004, showed no previously described case of metastatic GTN to rectovaginal septum. To our knowledge, the present case is the first case of GTN with involvement of rectovaginal septum. Also, we found all reported vaginal metastatic of GTN have mucosal surface involvement.
I. Introduction Metastatic gestational trophoblastic neoplasia (GTN) occurs in 4% of patients after evacuation of a complete mole and infrequently after other pregnancies (Berkowitz and Goldstein, 1996) The most common metastatic sites are the lung (80%), vagina (30%), and brain (10%) (Berek and Hacker, 2000). Following evacuation of a hydatiform mole, about 20% of women will subsequently undergo further treatment for suspected persistent gestational trophoblastic tumor (GTT) (Lurain et al, 1985). Vaginal metastases are usually highly vascular and may appear reddened or violaceous. They can bleed vigorously if sampled for biopsy (Berek and Hacker, 2000) Metastases to the vagina may occur in the fornices or suburethra and may produce irregular bleeding or a purulent discharge (Berek and Novak, 2002). Yingna et al, (2002) reported that 73% of vaginal metastasis were located in lower part and 59.05% were located on the
II. Case report A 28- year-old, Gravid 2, Para 1, was referred to Gynecology oncology services of Vali-Asr hospital in Oct. 2003 due to molar pregnancy. At 8 weeks of gestation, following persistent bleeding, pelvic ultrasonography revealed, molar pregnancy. 575
Behtash et al: Complete remission of an unusual location of metastatic GTN After complete blood chemistry and chest X-ray, abdominal sonography, she underwent suction curettage at September 2003.Pathology report was mole hydatiform. Weekly serum B-HCG titer showed decreasing level for 3 weeks, then it began to rise. In pelvic and rectovaginal exam a 45-55 mm soft rounded mass in the rectovaginal space was detected. The inferior border of the tumor was at 4 cm from the vaginal introitus and 4 cm from the anus.The tumor was deeply seated in rectovaginal septum and it was definitely separate from uterus and adenexa. Pelvic MRI revealed a lesion in the septum rectovaginal (Figure 1). Chest X-ray, abdominal CT scan and brain CTscan were normal. There was no other abnormal finding in metastatic workup. The total WHO scores for the patient obtained: 5, as a low risk patient (Kohorn, 2001) )(hcg >100000 :score 4 + tumor size 45 -55 mm : score 1 ). We started single agent chemotherapy (MTX&FA/8 days) for the patient:
if possible) At the end of the 1st course of chemotherapy, B-HCG titer decreased to <5 mIu/ml.Two additional courses of actinomycine-D administrated as consolidation therapy (Figure 2). Pelvic MRI revealed no metastatic lesion in pelvis (Figure 3). In regular follow up visit, there were no abnormal finding in physical exam, chest X-ray and pelvic MRI. Serial serum B-HCG remained < 5 at 20 weeks after treatment.
III. Discussion We report a rare case of metastatic GTN which has been presented as a tumor on the rectovaginal septum. The vagina and pelvic, next to the lungs is the second most common metastatic site in trophoblastic tumors (Song et al, 1983). The vaginal lesion is replete with abundant venous plexus without valva.This puts the patients at high risk for significant, repeated, and uncontrolled hemorrhage (Yingna et al, 2002) In spite of numerous cases of GTN in our hospital during last 15 year, we had no such patient with a metastatic lesion in rectovaginal septum. In our search we found, all reported vaginal metastases of GTN have mucosal surface involvement, but in this case, the mucosal of vagina was intact and tumor was deeply seated in rectovaginal septum. This case illustrates that GTN could be considered in a woman of reproductive age with rectovaginal mass .Serum concentration of human chorionic gonadotropin may reveal the exact final diagnosis.
Methotrexate 1 mg/gk IM on day 1,3,5,7 Folinic acid 0/1 mg/m2 IM on days 2,4,6,8 Repeated every 7 days if possible She received 4 courses of MTX&FA regimen, HCG titer, had persistent declining titer. After the end courses of chemotherapy, serum B-HCG titer began rise. The chemotherapy regimen switched actinomycine-D: Actinomycine
1/25 mg/m2
B4th to to
IV (repeat every 14 days
Figure 1. pelvic MRI with rectovaginal metastatic lesion (There is one signal area posterior of endocervical)
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Figure 2. B-HCG regression curve and chemotherapy courses Figure 3. pelvic MRI after treatment without metastatic lesion
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Behtash et al: Complete remission of an unusual location of metastatic GTN Trophoblastic neoplasia is known to be extremely responsive to appropriate chemotherapy. Cure rates are high with chemotherapy even in widely disseminated disease. In low risk patient even stage II and III, single agent regimen ,Methotrexate or actinomycin-D has been recommended (Lurain, 2002). MTX with Folinic acid had 95% remission in non metastatic patients compared with 56% success rate in low risk metastatic disease. Toxicity is low, particularly in comparison with the standard methods of giving MTX (Disaia and Creasman, 2002). Our patient was treated with MTX &FA because this regimen is safe and hepatic toxicity is lower compared other regimen. Traditionally, the patients who fail to respond to MTX have been switched to actinomycin-D and vice versa. All the patients with low risk disease cured by alternate secondary chemotherapy, regardless of which regimen was used. Actinomycin-D, given as a single bolus every 14 days also has an extremely high therapeutic ratio in women with nonmetastatic GTN and has been successfully used in the therapy of a few patients with low risk metastatic disease (Petrilli and Morrow, 1980). We reported a case of rectovaginal septum metastasis in a patient with GTN, which is very rarely seen. In this case, mucosa of the vagina and rectum were intact and the mass was deeply seated in rectovaginal septum. Regardless of the large size of the tumor and deep pelvic
site, single agent chemotherapy was effective in remission of the disease in this patient.
References Berek JS, Hacker NF (2000) Gestational trophoblastic neoplasia chapter 15, Practical Gynecologic oncology, third edition, by Lippincott Williams and Wilkins p: 615-35. Berek JS, Novak S (2002) Gynecology, thirteenth Edition, Lippincott Williams and Wilkins. Berkowitz RS, Goldstein D (1996) Chorionic tumors. N Engl J Med 335, 1740. Disaia PJ, Creasman WT (2002) Gestational trophoblastic disease in Disaia PJ, Creasman WT, editors. Clin Gynecol Oncol.6th ed. St.Louis, Mosby-year book, 185-206. Kohorn EI (2001) The new FIGO 2000 staging and risk factor scoring system for gestational trophoblastic disease: description and critical assessment. Int J Gynecol Cancer 11, 73-77. Lurain JR (2002) Treatment of gestational trophoblastic tumors. Curr Treat Options Oncol 3, 113-24. Lurain JR, Brewer JI, Torek EE, Halpern B (1985) Natural history of hydatiform mole after primary evacuation. Am J Obstet Gynecol 145, 591. Petrilli ES and Morrow CP (1980) Actinomycin D toxicity the treatment of trophoblastic disease: A comparison of the five day course to single-dose administration. Gynecol Oncol 9, 18-22. Song HZ, Wu PC, Wang YH (1983) Trophoblastic disease: diagnosis and treatment. Beijing: Medical publishing House, 12-168. Yingna S, Yang X, Xiuyu Y, Hongzhao S (2002) Clinical characteristics and treatment of gestational trophoblastic tumor with vaginal metastasis. Gynecol Oncol 84, 416-19.
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