Cancer Therapy Volume 4 Issue B by Cancer Therapy

CANCER THERAPY

Volume 4 Number 2 December, 2006

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-Gutenberg-University, 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., Universit채t 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., Universit채ts-Hautklinik Mannheim, Germany Schmitt, Manfred, Ph.D., Universit채t M체nchen, 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.cancertherapy.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 4-20 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 E-mail), 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-

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Table of contents Cancer Therapy Vol 4 Number 2, December 2006

Pages

Type of Article

Article title

Authors (corresponding author is in boldface)

171-182

Review Article

183-192

Review Article

Mustapha Aouida and Dindial Ramotar Leyla H. Sharaf

193-204

Research Article

205-222

Research Article

223-230

Research Article

Bleomycin transport holds the key for improved !anticancer therapy Current trends and recent advances in breast cancer !drug therapy Breast tumor cell clusters and their budding derivatives show different immunohistochemical profiles during stromal invasion: implications for hormonal and drug therapies Treatment of a number of cancer patients suffering from different types of malignancies by methylglyoxalbased formulation: A promising result Analysis of cytoplasmic polypeptides expression in gastric cancer and correlation with pathologic parameters

231-240

Review Article

Proton radiation therapy in oncology: Review of present clinical indications

241-248

Research Article

Muscle strength and functional ability in children during and after treatment for acute lymphoblastic leukemia or T-cell Non-Hodgkin lymphoma: a pilot study

249-252

Research Article

253-262

Review Article

263-270

Research Article

An analysis on the gene expression profiling of !lymphotoxin tumor necrosis factor-" in lymphoma Cardiac hormones: dramatic anticancer effects Prognosis of early invasive and locally advanced !(Stage I, II) uterine cervical adenocarcinomas

271-276

Research Article

277-282

Research Article

How could the light fluence rate influence the cure effect of photodynamic therapy? Surgical treatment of gastric cancer in South Western Greece

Yan-gao Man, Chengquan Zhao, Jin Wang

Dipa Talukdara, Subhankar Raya, Sanjoy Dasb, Ashok Kumar Jainb, Arvind !Kulkarnic and Manju Ray Panayiota K. Stroumbouli, Andreas Ch. Lazaris, Efstratios S. Patsouris, Anastasios Kalofoutis, Aphrodite Nonni, Sofia TseleniBalafouta Maurizio Amichetti, Augusto Lombardi, Carlo Algranati, Marco Schwarz Marja Schoenmakers, Tim Takken, Vincent AM Gulmans, Nico LU Van Meeteren, Marrie CA Bruin, Tom Revesz, Paul JM Helders Viroj Wiwanitkit

David L. Vesely Merih Hanhan, Mehmet Murat Inal, Deniz Oztekin, Nilgun Dicle, Zeynep !Ozsaran, Sivekar Tinar Tao Xu, Mingzhao Li Konstantinos Vagenas, Charalambos Spyropoulos, Pantelis Tsamalos, Panagiotis Papadopoulos, George Skroubis,

283-288

Review Article

Prevention of radiation enteritis

289-300

Research Article

Suppression of tumor metastasis by adenoviral-mediated gene transfer of Motility Related Protein –1 (MRP1/CD9)

John Spiliotis Mert Saynak, Vuslat YurutCaloglu, Gülden Bayir-Angin, Murat Caloglu, Rusen Cosar-Alas, Seden Ozbilen-Kucucuk, Cem Uzal, Isık Aslay Jun Zhang, Bogdan Ceacareanu, Haruhito Azuma, Aviv Hassid, Lisa K. Jennings and Yi Lu

Cancer Therapy Vol 4, page 171 Cancer Therapy Vol 4, 171-182, 2006

Bleomycin transport holds the key for improved anticancer therapy Review Article

Mustapha Aouida and Dindial Ramotar* Maisonneuve-Rosemont Hospital, Guy-Bernier Research Center, Montreal, Quebec, Canada

__________________________________________________________________________________ *Correspondence: Dindial Ramotar, Maisonneuve-Rosemont Hospital, Guy-Bernier Research Center, 5415, Boul. de l'Assomption, Montreal, Quebec, Canada, H1T 2M4; Tel: (514) 252-3400 ext 4684; Fax: (514) 252-3430; Email: dramotar.hmr@ssss.gouv.qc.ca Keywords: bleomycin, genome-wide screen, transport, cancer cells, yeast Abbreviations: apurinic/apyrimidinic, (AP); bleomycin hydrolase, (Blh1) Received: 03 February 2006; Revised: 17 April 2006 Accepted: 5 May 2006; electronically published: May 2006

Summary Bleomycin is a potent chemotherapeutic agent that can mediate cell killing by attacking the DNA. It is used in combination therapy for treating various cancers including testicular carcinomas, where it exhibits a striking cure rate of ~85%. However, the remaining fraction of testicular cancer patients relapses or resists bleomycin therapy. There is also clear evidence that certain human cancers, e.g., colon carcinoma, are highly resistant to bleomycin at the outset. Therefore, this raises an important question: What is the underlying mechanism that causes some tumors, and not others, to respond to bleomycin? Herein, we review our recent findings from the yeast model system showing that this mechanism involves a transporter, which actively transports the drug into the cell. We predict that a similar transporter exists in human cells, and that alteration of its activity may account for the differential responses of tumors to bleomycin therapy.

the destruction of more than one target (Carter et al, 1990; Huttenhofer et al, 1992; Holmes and Hecht, 1993, 1994; Morgan and Hecht, 1994; Keck and Hecht, 1995). However, accessing these targets is complicated by the fact that bleomycin is a large hydrophilic molecule that is first channel to the vacuoles, presumably for detoxification (Aouida et al, 2004a), and perhaps accumulated to a threshold level where it might cause oxidation of the vacuolar inner membrane and subsequent release into the cytoplasm (Ekimoto et al, 1985; Marnett, 1999). Bleomycin is administered systemically and it is used only in combination therapy with a number of other antineoplastic agents such as etoposide and cisplatin (Umezawa, 1971; Wharam et al, 1973; Jani et al, 1992ab; Einhorn, 2002). It is most effective against lymphomas, testicular carcinomas, and squamous cell carcinomas of the cervix, head, and neck (Povirk and Austin, 1991; Lazo et al, 1996). Like many other antitumor drugs, bleomycin also manifests clinical limitations. At high doses (>235 mg), bleomycin can induce pulmonary fibrosis, a condition that is triggered by lipid peroxidation (Ekimoto et al, 1985; Wang et al, 2000; Nagase et al, 2002), resulting in pulmonary insufficiency leading to fatal

I. Introduction Bleomycin is an antibiotic originally isolated four decades ago from Streptomyces verticillis (Umezawa, 1965; Umezawa et al, 1966). Subsequent studies clearly demonstrated that bleomycin can diminish the growth of experimentally induced tumors in mice and rats, and dramatically decrease the size of human tumors (Suzuki et al, 1968, 1970a; Ichikawa et al, 1969; Kanno et al, 1969; Oka et al, 1970; Terasima and Umezawa, 1970). Additional studies demonstrated that bleomycin possesses the ability to induce micronuclei formation and chromosome aberrations in human lymphocytes, as well as mitotic recombination and mutations in many organisms (Suzuki et al, 1970b; Terasima et al, 1970; Hoffmann et al, 1993). Collectively, these observations led to the suggestion that bleomycin mediates its chemotherapeutic effect by directly destroying the DNA and causing cell death. Following these findings, the detailed mechanism by which bleomycin induces DNA lesions began to unfold (Burger et al, 1981, 1982; Hecht, 1986; Kane and Hecht, 1994). Besides its action on DNA, bleomycin can also selectively destroy RNA raising the possibility that bleomycin-induced cell death might be a contribution from 171

Aouida and Ramotar: The transport of bleomycin hypoxemia (Sikic, 1986; Harrison and Lazo, 1987; Nagase et al, 2002). Another aspect that limits the clinical application of bleomycin is tumor resistance (Lazo et al, 1996). So far, no definitive mechanism, e.g., drug efflux or enhanced repair of bleomycin-induced DNA lesions or inactivation of bleomycin, explains the development of tumor resistance towards the drug (Miyaki et al, 1975; Akiyama et al, 1981; Sebti et al, 1991; Morris et al, 1992; Urade et al, 1992). Although, current findings from our laboratory raised the possibility that tumor resistance to bleomycin might be at the level of drug uptake, instead.

primary amines (e.g., histone amine) in vivo converts the bi-stranded lesions into a double strand break (Steighner and Povirk, 1990ab; Dedon and Goldberg, 1992; Absalon et al, 1995). In general, bleomycin produces structurallyand chemically-related lesions as those created by ionizing radiation, and therefore it is considered a radiomimetic agent (Steighner and Povirk, 1990ab; Dedon and Goldberg, 1992; Absalon et al, 1995; Hoehn et al, 2001). Bleomycin-induced DNA lesions are known to be highly mutagenic, and account for its potent antitumor ability (Steighner and Povirk, 1990ab; Bennett et al, 1993; Tates et al, 1994; Dar and Jorgensen, 1995; Pavon et al, 1995). Thus, if tumor cells can rapidly repair bleomycininduced lesions, then they are likely to become resistant to the genotoxic effects of the drug. Likewise, normal cells of cancer patients exposed to bleomycin must rely on DNA repair enzymes to process effectively the drug-induced lesions, in order to prevent lethal mutations that could lead to secondary tumors.

A. Properties and mechanism of action of bleomycin Bleomycin consists of three regions that include a carbohydrate moiety, a metal binding domain, and a DNA binding domain (Figure 1). The latter contains a chemical structure similar to the composition of polyamines (Hecht, 1986; Kane et al, 1994; Petering et al, 1996; Abraham, 1999; Leitheiser et al, 2000; Hoehn et al, 2001). Bleomycin also contains at least two accessible amino groups that could form covalent linkages with other compounds including fluorescein, thus providing a way to create a fluorescently labeled form of the drug (see below). Reduce Fe2+, as well as other metal ions, can bind to the metal pocket and trigger activation of bleomycin (Burger et al, 1979, 1981). During activation, the reduced Fe2+ becomes oxidized to Fe3+ resulting in the production of free radicals (Burger et al, 1979, 1981). The activated drug can intercalate with DNA to generate at least 4 types of DNA lesions, and the extent of formation of these lesions depends upon the oxygen content of the cell (Umezawa et al, 1966; Suzuki et al, 1970b; Terasima et al, 1970; Burger, 1998; Tounekti et al, 2001). For example, in the absence of oxygen bleomycin abstracts a hydrogen atom from the C4! position of the sugar moiety to create an unstable ring. This unstable sugar can ring open to form a mutagenic lesion, apurinic/apyrimidinic (AP) site, where the DNA strand is intact, but it lacks a base (Worth et al, 1993; Burger, 1998). In the presence of oxygen, bleomycin can damage the sugar moiety resulting in a single strand break blocked at the 3!-terminus with a fragment of the sugar (Giloni et al, 1981; Worth et al, 1993; Burger, 1998). This latter lesion, 3!phosphoglycolate, blocks DNA repair synthesis and therefore must be removed in order to promote cell division (Giloni et al, 1981; Worth et al, 1993; Burger, 1998). It is noteworthy that the remaining portion of the fragmented sugar exists in the free base propenal form, which has the ability to react with the DNA to form base adducts. For example, the base propenal bears a malondialdehyde moiety, which can react with guanine to form the most abundant adduct pyrimidopurinone of deoxyguanosine (Dedon et al, 1998). Bleomycin also produces bi-stranded DNA lesions at certain specific sequences, such as CGCC, which are generated when the Fe.bleomycin complex creates an AP site on one strand, and a directly opposed single strand break on the complementary strand (Steighner and Povirk, 1990ab; Dedon and Goldberg, 1992; Absalon et al, 1995; Hoehn et al, 2001). The spontaneous cleavage of the AP site by

B. Potential mechanisms leading to cellular bleomycin-resistance As pointed out above, tumor resistance is a major obstacle to bleomycin chemotherapy (Morris et al, 1991; Sebti et al, 1991; Jani et al, 1992ab). To date, no definitive mechanism(s) is known to explain how tumors acquire such resistance, although possible ones might include (i) enhanced repair of bleomycin-induced DNA lesions, (ii) increased drug efflux by multidrug transporters, (iii) inactivation of the drug by elevated levels of bleomycin hydrolase, and (iv) decreased drug uptake by plasma membrane permeases (Miyaki et al, 1975; Akiyama et al, 1981; Sebti et al, 1991; Morris et al, 1992; Urade et al, 1992; Sanz et al, 2002). These possibilities are discuss below, as well as striking findings from our laboratory indicating that bleomycin-resistance is due principally to altered drug uptake.

1. DNA repair enzymes Organisms exposed to bleomycin must recruit proteins to repair bleomycin-induced DNA lesions in order to avert the mutagenic effects of the drug (Ramotar and Wang, 2003). Over the years, we employed the yeast Saccharomyces cerevisiae as a model organism to search for enzymes that would repair bleomycin-induced DNA lesions, as such enzymes have not been characterized in mammalian cells (Ramotar, 1997; Sander and Ramotar, 1997; Jilani et al, 1999). S. cerevisiae provided a solid foundation for this study due to its multifaceted advantages that include (i) a powerful genetic system that permits rapid creation of gene nulls, (ii) a wealth of readily available technologies and a vast database information, and (iii) the ability to isolate clinically-relevant human disease genes by cross-species complementation (Phizicky and Fields, 1995; Bassett et al, 1996; Lashkari et al, 1997; Andrade et al, 1998; Pereira, 1998; Neff et al, 1999; Steinmetz and Davis, 2000; Steinmetz et al, 2002). To date, we and others have biochemically characterized three enzymes, i.e., Apn1, Apn2, and Tpp1, that clearly act in vitro to directly process bleomycininduced DNA lesions (Ramotar et al, 1991; Ramotar, 172

Cancer Therapy Vol 4, page 173 1997; Sander and Ramotar, 1997; Jilani et al, 1999; Vance and Wilson, 2001; Jilani and Ramotar, 2002). Genetic studies revealed that mutants lacking all three enzymes displayed severe hypersensitivity to bleomycin, while single mutants showed virtually no sensitivity to the drug, as compared to the parent (Ramotar, 1997; Vance and Wilson, 2001). These findings clearly indicate that more than one of these enzymes can compete to repair bleomycin-induced lesions in vivo (Ramotar et al, 1991; Ramotar, 1997; Sander and Ramotar, 1997; Jilani et al, 1999; Vance and Wilson, 2001; Jilani and Ramotar, 2002). Despite extensive searches, the Apn1 homologue has not been found yet in humans, although the counterparts of yeast Apn2 (hApe/ref-1) and Tpp1 (hPNKP) have been identified (Barzilay and Hickson, 1995; Jilani et al, 1999). Recent studies demonstrated that overproduction of hApe1/ref-1 in the testicular cancer cell line NT2/D1 resulted in ~3-fold increased protection against bleomycin (Robertson et al, 2001). However, it remains to be shown if hApe1 overexpression can account for the nearly 15% of patients that resist bleomycin-therapy (Robertson et al, 2001). Whether hApe or hPNKP, and other yet unidentified human DNA repair enzymes (e.g., human Apn1), plays a role in bleomycin-tumor resistance is currently under investigation.

(Umezawa et al, 1974; Sebti and Lazo, 1988; Nishimura et al, 1989; Schwartz et al, 1999). The characterized enzyme acts as a thiol protease and hydrolyzes the "-aminoalanine amide moiety near the DNA binding domain of bleomycin to generate the inactive deamido metabolite (Akiyama et al, 1981; Lazo and Humphreys, 1983; Sebti et al, 1991). The activity can be inhibited by the thiol protease specific inhibitor (E64), and mammalian cells exposed to E64 displayed sensitivity to bleomycin (Jani et al, 1992ab). These observations quickly led to the isolation of the corresponding bleomycin hydrolase gene from yeast and mammalian cells (Enenkel and Wolf, 1993; Magdolen et al, 1993; Bromme et al, 1996; O'Farrell et al, 1999; Ferrando et al, 1996). Expression of the yeast bleomycin hydrolase gene BLH1 in mouse NIH3T3 cells conferred a nearly 5-fold increase resistance to bleomycin, which could be blocked by E64 inhibitor (Pei et al, 1995). Notwithstanding this finding, independent studies showed conflicting data regarding the role of yeast Blh1 in the inactivation of bleomycin (Enenkel and Wolf, 1993; Magdolen et al, 1993 Kambouris, et al. 1992). While two studies showed that blh1 ! mutants are mildly sensitive to bleomycin, another study clearly established that these mutants exhibit no more sensitivity to the drug than parent strains (Kambouris et al, 1992; Enenkel and Wolf, 1993; Magdolen et al, 1993). In fact, we also demonstrated that blh1! mutants are not sensitive to bleomycin, and that overproduction of Blh1 in yeast cells does not protect bleomycin-hypersensitive mutants from the genotoxic effects of the drug (Wang and Ramotar, 2002). As such, we conclude that in vivo yeast Blh1 has no direct role in mediating cellular resistance to bleomycin, and that the role of the enzyme in producing tumor resistance remains unclear. However, it is worthy of noting that the yeast Blh1 protein, also called Gal6, is under the control of the Gal4 transcriptional activator (Zheng et al, 1997). Blh1/Gal6 binds specifically to the Gal4 transcription factor DNA binding site and acts as a repressor in a manner that negatively controls the pathway of galactose metabolism (Xu and Johnston, 1994; Joshua-Tor et al, 1995; Zheng et al, 1997). Equipped with this new function, it would appear that bleomycin hydrolase plays a more general role by degrading certain transcription factors in order to regulate gene expression, as well as ribosomal proteins (Zheng et al, 1998; Koldamova et al, 1999). If this is the case, the bleomycin resistance observed by expression of yeast Blh1 protein in mammalian cells could be explained, for example, by degradation of pro-apoptotic factors, thus preventing cell death.

2. Evidence against multidrug transporters as a mechanism of bleomycin-resistance In mammalian cells, elevated levels of plasma membrane ABC transporters, such as the multidrug resistant efflux pump, MDR1, and the multidrug resistantassociated protein, MRP1, are known to increase efflux of chemotherapeutic agents thereby allowing tumor (and normal) cells to evade drug-induced cytoxicity (Gottesman et al, 1995; Dean et al, 2001). So far, there is no convincing evidence for the involvement of either MDR or MRP efflux pumps in bleomycin resistance (Chen et al, 1994). Likewise in yeast, several well studied ABC transporters showed no involvement in the efflux of bleomycin (Ramotar and Masson, 1996; Decottignies and Goffeau, 1997). Consistent with this observation is that a yeast mutant strain, AD1-8, lacking seven of these transporters (Pdr5, Pdr10, Pdr11, Pdr15, Snq2, Yor1, and Ycf1), some of which share significant similarities to human ABC transporters and are involved in drug efflux, showed high level of sensitivities to numerous toxic compounds (Decottignies et al, 1998; Rogers et al, 2001). However, this mutant strain showed normal parental resistance to bleomycin (DR., unpublished). In other studies, the overexpression of each of these pumps did not confer upon parental yeast strains any additional resistance to bleomycin (DR., unpublished). Thus, it is unlikely that drug efflux pumps play a major role in bleomycin resistance.

4. Iron transport plays no role in bleomycin resistance So far, we found no evidence in yeast that altered iron metabolism leads to bleomycin resistance. For example, fre1! and fre2 ! mutants defective in the Fe/Cu reductase genes showed normal parental resistance to bleomycin (Georgatsou and Alexandraki, 1999).

3. Bleomycin-hydrolase confers no drug resistance to yeast It is demonstrated that bleomycin can be metabolically inactivated in normal and tumor tissues by an enzyme called bleomycin hydrolase (Blh1), and that such inactivation may play a role in bleomycin resistance 173

Aouida and Ramotar: The transport of bleomycin the vacuole, where this organelle might serve as a first line of defense by containing the drug and preventing its cytotoxicity and genotoxicity. To date, several approaches have been assessed to improve the genotoxicity of bleomycin that include exploiting chemical modifications, as well as directly transferring the drug into the cell via electroporation in order to bypass the vacuoles (Aouida et al, 2003). Although the latter technique seems promising in that it allows a defined number of bleomycin molecules into the cell and causes enhanced bleomycin cytotoxicity, this approach remains cumbersome (Orlowski et al, 1988; Kotnik et al, 2000). However, a more recent study employed the use of photochemical damage to the endocytic vesicles in order to trigger disruption of the organelles, with concomitant release of the accumulated bleomycin, in various cancer cells challenged with the drug (Berg et al, 2005). While this approach holds great promise to enhance the chemotherapeutic effects of the bleomycin (Berg et al, 2005), other rationale approaches are still forthcoming.

5. Evidence for bleomycin transport across the plasma membrane Previous evidence suggests that a protein exists on the plasma membrane of mammalian and yeast cells, which is believed to bind bleomycin and mediate its transport into the cell (Poddevin et al, 1991; Aouida et al, 2003). This uncharacterized putative protein has been identified by analyzing plasma membrane fractions, under non-denaturing gel conditions, for specific binding to bleomycin carrying labeled [57Co] cobalt in the metal ion pocket (Pron et al, 1993). This preliminary observation prompted us to examine the kinetic parameters of bleomycin uptake into yeast cells. However, this could not be readily achieved with the labeled [57Co]-bleomycin and instead we created a fluorescently labeled form of bleomycin to conduct the study (Mistry et al, 1992). The fluorescien conjugated bleomycin (F-bleomycin) has been purified and rigorously shown to retain nearly full capacity to act as a genotoxic agent, as well as to induce cell killing by creating endogenous DNA lesions analogous to the unmodified drug (Aouida et al, 2004a). We then used the F-bleomycin as a tool to show that the drug can be actively transported into parent strains in a concentration- and time-dependent manner (Aouida et al, 2004a). Moreover, these studies revealed that F-bleomycin transport may be dependent upon new protein synthesis, as uptake could be blocked by the protein synthesis inhibitor cycloheximide (Aouida et al, 2004a). Base on the above observations, we reasoned that a plasma membrane transporter must exist to permit bleomycin entry into the cell. If this is the case, mutants devoid of the transporter are expected to exhibit greater than parental resistance to bleomycin. In support of this prediction, a recent report documented that the copper transporter, Ctr1, is responsible for transporting the anticancer drug cisplatin into yeast and mammalian cells (Ishida et al, 2002). Yeast mutants lacking the Ctr1 transporter are resistant to cisplatin, and the overexpression of Ctr1 has been shown to cause increased sensitivity to the drug (Ishida et al, 2002). However, neither the Ctr1 nor other metal ion transporters in yeast is involved in bleomycin transport, favoring the possibility that the transporter in question is likely to exist in nature.

C. Genome-wide screen In the last five years, several genome-wide screens have been performed with different collections of yeast mutant cells exposed to various DNA damaging agents including #-rays, ultraviolet radiation, methyl methane sulfonate, and cisplatin (Bennett et al, 2001; Birrell et al, 2001; Chang et al, 2002; Desmoucelles et al, 2002; Giaever et al, 2002; Fry et al, 2005). These screens have allowed the identification of many previously reported gene functions, as well as uncovered new ones that are required to protect cells against DNA damaging agents, and which otherwise could not be rapidly isolated by other strategies that include the use of transposon-insertion mutagenesis and reverse genetics (Burns et al, 1994). The data generated from these genome-wide screens are being exploited to further establish, for example, those genes encoding proteins that participate in related biological processes (Fry et al, 2005). As discussed below, we have undertaken a similar genome-wide approach to unravel the gene functions that are involved in protecting cells against the genotoxic effects of bleomycin.

6. Bleomycin accumulates in the vacuoles following transport

1. Bleomycin-hypersensitive mutants revealed by a genome-wide screen in yeast

With the aid of fluorescent microscopy, we have shown that following F-bleomycin uptake into the parent strain the drug accumulated into the vacuoles (Aouida et al, 2004a). Since the vacuoles serve a function to degrade many macromolecules, it is reasonable to assume that bleomycin might be detoxified in this organelle. Interruption of the endocytotic pathway to the vacuoles caused F-bleomycin to be redistributed such that it is accumulated in the cytoplasm, where the drug can now readily diffuse into the nucleus and rapidly degrade the DNA (Aouida et al, 2004a). In general, all the mutants tested so far with defects in the endocytic pathway to the vacuole display marked hypersensitivity to bleomycin (Table 1). Thus, it would seem that bleomycin is actively transported across yeast plasma membrane and directed to

To directly complement our previous efforts to understand how cells provide resistance to bleomycin, we performed two independent robot-aided screens of the entire collection of haploid yeast mutants to identify all the bleomycin-hypersensitive mutants (Aouida et al, 2004b). The collection comprises single deletions in nearly 4,000 of the 6,000 yeast genes (www.unifrankfurt.de/fb15/mikro/euroscarf/)(Winzeler et al, 1999; Desmoucelles et al, 2002): The remaining 2,000 genes are essential for cell viability. The screens reproducibly identified 231 mutants that have been independently confirmed, and showing hypersensitivity to bleomycin (i.e., 4- to 20-fold more sensitive than the parent strain). Table 1 list all the genes deleted in the corresponding mutants. Among these genes, we previously identified 174

Cancer Therapy Vol 4, page 175 Table 1. BLM-hypersensitive and–resistant genes DNA repair Chromatin Structure CTF18* a CTF4* a + b CTF8 a + b

Cell Cycle CDC50* a CTK2 DOC1 b HTL1 a + b PHO85*

Mitochondria and ATP metabolism ADK1* b AFG3 ATP11

Vacuoles and vesicular transport AKR1 b APG17 APL2

Cell wall ANP1 CAX4 b CHS1 CWH36 b FKS1

EST2* FAB1* IWR1* b MRE11* a NAT3* a + b RAD27 a RAD54* a RAD57* a + b RAD6* a + b

RTS1* SFP1 b

ATP12 ATP14 ATP15 CAT5* ILM1 ISA1* MDJ1 MRPL51* MRPS8

CHC1* DID4* END3* GLO3 IES6 INP53* LCB4* LUV1 MON2*

FYV6 GAS1 HOC1 LAG2 MNN9 MNN10 OST4 RMD7 ROT2*

MSF1 MSY1* NHX1 OCT1 PDA1 PFK2 b PPA2 RML2 RSM19 RSM7 b RSM22 SNF1 SPF1* SSQ1 SWF3 SWS2

PEP5* PEP12* RIC1 RCY1* RVS161* b RVS167 b SHE4 SWF1* VAM6 VPH2 b VPS1 VPS3 VPS4 VPS8 VPS9 VPS15

SLG1*

TOM5 TUF1* UGO1* YDJ1 YHM1 YME1

VPS16* VPS20 VPS24 VPS25* VPS27 VPS45* VPS66 VPS67

RAI1* b REM50 a RNR1 RNR4* SNF6 SPT10 b SPT20 THP1* TRF4* VID31* a + b XRS2 a + b

Transcription ASF1* a + b BUR2 a + b b

RNA Metabolism ARC1* BRE5* BRF1 CDC40* a + b DBP7 DIA4 b KEM1* LOC1 b PAT1* b SAC3* SNT309 YER087W* Protein Synthesis ASC1 EAP1 EGD2 PDR13 b PFD1 b

CCR4 CTK1 CTK3 DHH1* b GAL11 IMP2 KCS1 POP2

RPL13B RPL1B RPL27A RPL35A RPL39 RPS0B* RPP1A TIF3

ROX3

TIF4631

RPB4

ZUO1*

RPB9* a + b RRN10 a SIN4 SPT21 SPT7* SRB2 a SRB5 a

Polarity and polarized growth BEM2

SPS4 TPS1

YIF2* b

BUD20* BUD23*

YPT6 YPT7

Protein Degradation GRR1* a + b UBP3

BUD25

BUD27 BUD31*

Cytoskeleton ARC18

UMP1*

BUD32* SAC2

CDC10* CNM67 b

SAC6 b

GCS1 HOF1* a + b

ARV1* ERG2 ERG4

BLMresistant AGP2* FES1* PTK2* SKY1* BRP1

YAF9*

SWI4 SWI6* a TAF14*

YMR031W-A YOR342C YPR044C

NPL6 OPT2 PLC1 PMP3 PRO1 REG1 RIB4 SHP1 SLX8*

BUD16*

Lipid metabolism

SWF5 YBR168W YBR267W* YDR049W* YDR532C YGL072C YGR237C YGR272C YLR374C

Miscellaneous ADE12 ADH1 APM1 ARP5* CYS4 DIA2 GLY1 GON1* GPH1 GUP1 MET22*

VPS69

SRB8* SSN8

Unknown Function APQ13 BAT2 GON7

SPC72

175

MMS sensitive

b IR sensitive * 88 genes conserved in human

Aouida and Ramotar: The transport of bleomycin IMP2, GAS1, SLG1, END3, and RAD6 by a different type of screen namely insertional-mutagenesis that causes functional disruption of genes (He et al, 1996; Masson and Ramotar, 1996; Leduc et al, 2003), thus validating the utility of our genome-wide approach. From the collection of genes, 88 encode proteins that share significant level of identity with a human protein (Table 1, shown with an asterisk), suggesting that yeast and human cells may conserve the same biological processes to combat the cytotoxic and genotoxic effects of bleomycin. The genes (Table 1) encode proteins belonging to several functional groups including DNA repair and chromatin structure, transcription, and cell cycle. Other groups participate in maintaining the vacuolar and mitochondrial functions. In fact, the largest number of genes identified belongs to the vacuolar pathway. Thus, it would appear that modulating the function of these gene products could likely affect how cancer cells respond to bleomycin. For example, blocking the detoxification pathway might effectively increase the cytoplasmic concentration of bleomycin (Aouida et al, 2004a; Berg et al, 2005). In addition, many chromatin-remodeling proteins such as Snf6, Spt20, and Spt10 might play a role in promoting specific repair of bleomycin-induced DNA lesions. These latter chromatin-remodeling proteins are under characterization in order to examine for relationships with DNA repair pathways and to determine the types of DNA lesions that are processed.

that agp2! mutants are unable to transport F-bleomycin into the cells causing a decreased accumulation in the vacuoles (Aouida et al, 2004b). However, reintroduction of the AGP2 gene into the agp2! reinstated F-bleomycin uptake and its accumulation into the vacuoles (Aouida et al, 2004b). If the Agp2 transporter is overproduced via increased gene dosage, it can greatly enhance the uptake of F-bleomycin and simultaneously sensitized the cells to killing by the drug, a consequence of increased damage to the chromosomal DNA (Aouida et al, 2004b). The overproduced Agp2 did not sensitize cells to various other DNA damaging agents including cisplatin, suggesting that Agp2 is selectively involved in bleomycin uptake (Aouida et al, 2004b). We reasoned that Agp2 is involved in the uptake of both bleomycin and L-carnitine as the bleomycin specie (bleomycin-A5) we used in the genomewide screen possesses a region with a chemical structure that is similar to L-carnitine and polyamine (Figure 1). In fact, preincubation of cells with L-carnitine blocked the uptake of bleomycin and protected the cells from bleomycin-induced cell death, further supporting the notion that Agp2 functions to transport the bleomycin-A5 specie (Aouida et al, 2004b). It is noteworthy that agp2! mutants showed only modest resistance to another specie of bleomycin, that is bleomycin-A2, which lacks the polyamine portion (M.A. and D.R., unpublished), indicating that this region plays a critical role in permitting entry of the drug into the cell. This observation prompted us to examine if the Agp2 transporter might in fact be the long sought high affinity polyamine transporter, particularly since a transporter of this nature remains unidentified in higher eukaryotic cells. Uptake studies performed with labeled spermidine revealed that Agp2 is indeed the high-affinity polyamine permease in yeast (Aouida et al, 2005). Deletion of the AGP2 gene dramatically reduces the initial velocity of spermidine and putrescine uptake and confers strong resistance to the toxicity of exogenous polyamines (Aouida et al, 2005). Reintroduction of the AGP2 gene from an expression vector restored polyamine transport into the agp2$ mutants (Aouida et al, 2005). Further analyses revealed that the transporter function of Agp2 is crucial to sustain the growth of cells (e.g., spe1$ mutant) lacking the ability to synthesize polyamine (Aouida et al, 2005). In fact, spe1$ agp2$ double mutants required more than 10-fold higher concentrations of exogenous putrescine to restore cell proliferation, as compared to the spe1$ single mutant (Aouida et al, 2005). Other genetic alterations, such as disruption of the END3 gene encoding a protein required for an early step of endocytosis, increase the abundance of Agp2 and causing a marked upregulation of spermidine transport velocity (Aouida et al, 2005). Collectively, these observations are consistent with the notion that (1) Agp2 is the first eukaryotic permease that preferentially uses spermidine over putrescine as a high-affinity substrate, and (2) Agp2 plays a central role in the uptake of polyamines into yeast cells. As such, we expect that agp2$ mutants would be resistant to other chemotherapeutic agents conjugated to polyamine, e.g., chlorambucilspermidine and difluoropolyamines (Hull et al, 1988; Cullis et al, 1994).

2. Bleomycin-resistant mutants revealed by the genome-wide screen in yeast In addition to the search for bleomycinhypersensitive mutants, we also exploited the screen to identify those mutants that would be resistant to the drug. At least five mutants that displayed nearly 500- to 3000fold more resistance to bleomycin than the parent have been identified (Table 1) (Aouida et al, 2004b). The mutants lacked the gene AGP2, PTK2, SKY1, FES1, and BRP1 (Table 1). Amongst these mutants, the agp2! mutants exhibited the greatest resistance (~3000-fold) to bleomycin, while the other four displayed at least 500-fold more resistance than the parent (Aouida et al, 2004b). Below, we highlighted the current knowledge of the five gene products involved in bleomycin-resistance. i. Agp2 Agp2 is a 67.2-kDa plasma membrane protein that belongs to the amino acid transporter family (Lee et al, 2002; Schreve and Garrett, 2004). Previous studies demonstrated that Agp2 is involved in the uptake of L-carnitine, which serves as a carrier to transport the end product of fatty acid "-oxidation, acetyl-CoA, from the peroxisome into the mitochondria for complete oxidation (van Roermund et al, 1999; Lee et al, 2002). However, it remains unclear if Agp2 has a direct role in L-carnitine transport, although there is a correlation showing that the expression level of the transporter is induced when cells are grown in the presence of fatty acid (van Roermund et al, 1999). We recently showed that Agp2 is responsible also for mediating the uptake of bleomycin into the cells (Aouida et al, 2004b). This observation is derived from the finding 176

Cancer Therapy Vol 4, page 177

Figure 1. Structure of bleomycin-A5 depicting several domains. The metal binding domain binds to reduce iron and in the presence of oxygen forms a free radical that attacks the DNA. While the polyamine-like region is involved in DNA binding, the function of the carbohydrate moiety is unknown. Reproduced from Leitheiser et al, 2000 with kind permission from Organic Letters.

ii. Ptk2 Ptk2 is a Ser/Thr protein kinase that belongs to the Npr1 kinase family, and which regulates the activity of permeases and transporters (Schmidt et al, 1998). We previously showed that Ptk2 is a key regulator of polyamine transport into yeast cells (Kaouass et al, 1997). Genetic studies revealed that ptk2$ mutants are unable to transport polyamine into the cells and thus these mutants are hyperresistant to toxic doses of spermine (Kaouass et al, 1997). Ptk2 has been shown to play a role in positively regulating the activity of the plasma membrane proton pump Pma1, which creates a voltage gradient that serves to provide energy for transporters (Goossens et al, 2000). Whether Ptk2 is required to directly phosphorylates Pma1 remains to be seen, but phosphorylation of Ser899 activates Pma1 activity (Portillo, 2000). In this context, we postulate that the voltage gradient created by Pma1 is also required to propel the function of the Agp2 transporter. Thus, it is logical to expect that mutants defective in Ptk2 function would display resistance to bleomycin and polyamines. As predicted from the functions of Ptk2 and Agp2, it is not surprising that agp2$ ptk2$ double mutant exhibits the same level of bleomycin and polyamine resistance as the single agp2$ mutant (unpublished data). From the above findings, it would appear that mammalian cells compromised for the maintenance of the voltage gradient is likely to display resistance to bleomycin.

bleomycin resistance, possesses a Ser/Arg rich domain that can serve as a phosphorylation acceptor site for the action of Sky1 kinase. Alternatively, Sky1 could affect the phosphorylation status of proteins with Ser/Arg rich domain that are involved in RNA binding activity and cellular location, and thus indirectly affecting the function of Agp2 (Gilbert et al, 2001). In the case of mammalian cells, a dominant negative form of the mammalian Sky1 kinase (SRPK1 serine kinase) has been shown to confer upon Chinese hamster lung fibroblast and HeLa cells resistance to bleomycin, but not to other DNA damaging agents (Sanz et al, 2002). This observation strongly suggests that a conserved pathway exists in eukaryotic cells to regulate the genotoxic effects of bleomycin (Sanz et al, 2002; Aouida et al, 2004b). Thus, unraveling the mechanism by which the dominant negative SRPK1 triggers bleomycin-resistance could be an important clue to understand the nature of this drug resistant pathway and implement better regimens. iv. Fes1 Fes1 is associated with the ribosomes and believed to play a role in protein translation (Kabani et al, 2002). fes1$ mutant is believed to have a translational defect, suggesting that Fes1 could signal translation of at least one of the components (Agp2, Sky1, Ptk2, and Brp1) involved in bleomycin resistance. So far, we found no evidence that the expression level of a functionally tagged form of Agp2, Agp2-cTAP (cTAP carrying protein domains for Tandem Affinity Purification), is diminished in the fes1$ mutant. Clearly, additional studies are needed to precisely determine the function of Fes1, and if it is involved in promoting the translation of the other members.

iii. Sky1 Sky1 is a Ser/Arg kinase also shown to regulate polyamine transport (Erez and Kahana, 2001), and that sky1$ mutants are hyperresistant to toxic doses of spermine (Erez and Kahana, 2001). Since the emerging scenario in yeast is that a family of protein kinases exists which is dedicated to regulate plasma membrane transporter activity, it is possible that Sky1 could control Agp2 activity by phosphorylation (Schmidt et al, 1998). However, neither Agp2 nor the other proteins involved in

v. Brp1 We designated the YGL007w gene as BRP1, which is predicted to encode a small 13-kDa bleomycin resistant protein of unknown function. Since the activity of some

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Aouida and Ramotar: The transport of bleomycin membrane transporters is regulated by small proteins, e.g., the small heat shock protein Hsp30 regulates the activity of the H+/ATPase, Pma1, it is possible that Brp1 might engage in a similar function (Braley and Piper, 1997; de la Fuente et al, 1997). Consistent with this notion, one group, which quickly reproduced our findings, provided preliminary evidence that Brp1 might be required to regulate the expression level of Pma1 (Porat et al, 2005).

II. Conclusions To date, a homologue of Agp2 has not yet been reported in humans, but two high affinity L-carnitine transporters CT2 and OCTN2, each sharing approximately 19% identity, have been identified (Tamai et al, 1998; Enomoto et al, 2002). Interestingly, hCT2 is expressed exclusively in human testis, whereas OCTN2 is expressed strongly in kidney, skeletal muscle, heart, and prostate (Tamai et al, 1998; Enomoto et al, 2002). The fact that hCT2 is expressed exclusively in the testis and that testicular cancers have a high cure rate with bleomycin therapy is striking, offering strong support for the notion that hCT2 could be the human transporter of bleomycin. If this is the case, hCT2 could play an important role in predicting the responses of patients to bleomycin. For example, mutations altering hCT2 transporter function are expected to be an important factor in tumors that gradually develop resistance to bleomycin. Extensive studies are in progress to examine if hCT2 is involved in both bleomycin and polyamine uptake (M.A and D.R., in preparation). We believe that seeking strategies to upregulate the transporter activity can lead to widening the application of bleomycin therapy to other cancers besides testicular.

D. Model It is clear from our studies that deletion of any of the following five yeast genes, AGP2, PTK2, SKY1, FES1, and BRP1 protects the organism against the cytotoxic and genotoxic effects of bleomycin (Aouida et al, 2004b). Thus, to explain the possible roles played by these gene products in bleomycin resistance, we propose the following model (Figure 2) that is most consistent with the current data. The Agp2 protein acts as the transporter of bleomycin and that its activity is likely regulated directly by the Sky1 kinase. In this model, the Ptk2 kinase acts to modulate the proton pumping activity of Pma1 to provide a voltage gradient needed to drive the activity of many transporters including Agp2. In contrast, the roles of Fes1 and Brp1 are less clear. However, we speculate that Fes1 could control the translation of either Sky1 or Ptk2 or Brp1, and that this latter protein in turn regulates the expression level of the proton pump Pma1. We believe that in depth functional analysis of these gene products will provide a much better understanding of the mechanism by which bleomycin enters the cell, and will lead to the identification of novel therapeutic targets aimed at enhancing the antitumor properties of the drug.

Acknowledgements The work described in this review is supported by a grant (MT-121391) to D.R. from the Canadian Institutes of Health Research. D.R. is awarded a senior fellowship from the Fonds de la Recherche en SantĂŠ du QuĂŠbec. M.A. is awarded a post-doctoral fellowship from the National Cancer Institute of Canada.

. Figure 2. A model illustrating the transport and detoxification pathway of bleomycin. The drug enters the cell via a bleomycin transporter Agp2, whose activity might be influenced by the kinases Ptk2 and Sky1. These kinases are known to regulate the plasma membrane polyamine transporter. Following uptake, bleomycin is channeled to the vacuole for detoxification. Interruption of the endocytic pathway to the vacuoles resulted in mutants that are hypersensitive to bleomycin. Reproduced from Aouida et al 2004 with kind permission from Biochemical Journal and Cancer Research.

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Cancer Therapy Vol 4, page 183 Cancer Therapy Vol 4, 183-192, 2006

Current trends and recent advances in breast cancer drug therapy Review Article

Leyla H. Sharaf Faculty of Pharmacy, Kuwait University, Kuwait __________________________________________________________________________________ *Correspondence: Leyla H. Sharaf, Faculty of Pharmacy, Kuwait University, Kuwait. P.O Box: 24923 Safat 13110 Kuwait; Tel: 9655312300 (Ext: 6851/6861); Fax: 9655342807; e-mail: ls20504@hsc.edu.kw Key words: Breast cancer, Endocrine therapy, Estrogen-receptor modulators, Progestins, LHRH analogs, Aromatase inhibitors, Immunotherapy, Monoclonal antibodies, Adjuvant chemotherapy, Cytotoxic chemotherapy, Taxanes, Anthracycline regimens, CMF regimens, Novel targets Abbreviations: [Cyclophosphamide, Methotrexate, 5-Fluorouracil], (CMF); congestive heart failure, (CHF); epidermal growth factor receptor, (EGFR, also termed ErbB1); estrogen-receptor, (ER); farnesyltransferase inhibitors, (FTIs); Luteinizing hormone-releasing hormone, (LHRH); mammalian target of rapamycin, (mTOR); metastatic breast cancer, (MBC); phosphatidylinositol 3-kinase, (PI3K); Poly ADP Ribose polymerase, (PARP); progesterone receptor, (PR); vascular endothelial growth factor receptor, (VEGF)

Received: 17 January 2006; Revised: 31 March 2006 Accepted: 8 May 2006; electronically published: May 2006

Summary Considerable progress has been made in the understanding of the molecular basis of breast cancer. This presents an opportunity for the development of novel targeted drug therapies as well as the inclusion of new adjuvants in the classical regimens with the ultimate goal of enhancing efficacy and minimizing toxicity. Many endocrine agents proved to be beneficial as adjuvants and in advanced hormone-responsive breast cancer. These include selective estrogen receptor modulators, third-generation aromatase inhibitors, progestins, and LHRH analogs. Despite this, cytotoxic chemotherapy is still the mainstay of treatment especially in the metastatic setting. The most frequently used regimens are based on anthracyclines and/or taxanes. Abraxane, an albumin bound nanoparticle form of paclitaxel was formulated to enhance the therapeutic potential of taxane therapy while minimizing the drug's side effects and overcoming the need for toxic solvents. Capecitabine and gemcitabine have shown high activity and acceptable tolerability in a range of settings for metastatic breast cancer (MBC). The addition of trastuzumab has improved response rate and overall survival in patients whose tumors overexpress HER-2 compared to chemotherapy alone. Addition of bevacizumab to paclitaxel as first-line treatment of patients with metastatic breast cancer is showing promise. Although still under trial, novel targeted drug therapies including PARP inhibitors, farnesyl transferase inhibitors, mTOR antagonists, and tyrosine kinase inhibitors may give a new horizon for future management of breast cancer.

with cellular proliferation in a relatively non-specific manner.

I. Introduction Breast cancer is the second commonest cause of cancer-related death in women, both in Europe and in the USA (Fornier, 2005; Mouridsen, 2005). Despite significant advances in our understanding of the molecular basis of this disease, cure remains an elusive goal with most efforts focused on clinical management- a â&#x20AC;&#x2DC;damage limitation exerciseâ&#x20AC;&#x2122;. This article highlights current as well as well as some of the more recent drug treatment modalities for breast cancer. They are either targeted therapies aimed at inhibiting the action of defined growth modulatory genes or general cytotoxic agents that interfere

II. Endocrine therapy Endocrine therapy is an important systemic treatment for all stages of hormone receptor-positive breast cancer. The standard treatment for early, hormone-sensitive breast cancer is surgery and when breast-conserving surgery is performed, radiotherapy. This is generally followed by adjuvant endocrine therapy given in selected cases. Following disease progression or recurrence, second-line endocrine agents are employed. In advanced stages,

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Sharaf: Recent trends in breast cancer drug therapy treatment is essentially palliative, with the goal being disease control and maintenance of quality of life (Oâ&#x20AC;&#x2122;Shaughnessy, 2005). Sequential endocrine therapy continues as long as the patient remains hormonesensitive. Once hormone-resistant disease develops, chemotherapy is the current alternative (Howell, 2005).

C. LHRH agonists LHRH analogs such as goserelin (ZoladexR) and luprolide (LupronR) are a group of drugs that suppress ovarian estrogen production down to postmenopausal levels, essentially inducing a potentially reversible medical ovarian ablation. They are most effective in ER-positive early breast cancer in premenopausal women (von Minckwitz, 2004). Goserelin, a principal agent of this class, is a biodegradable sustained-release 3.6mg depot administered monthly by subcutaneous injection (Mitchell, 2004). The indirect comparison of goserelin with tamoxifen as a single drug in the adjuvant setting showed similar efficacy. Furthermore, goserelin alone or in combination with tamoxifen was shown to be as effective as cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) chemotherapy. Goserelin plus tamoxifen after cyclophosphamide, doxorubicin and 5-fluorouracil (CAF) chemotherapy resulted in improved disease-free survival compared with CAF alone. Data concerning taxane-based and dose-dense chemotherapy are still lacking. Moreover, duration of therapy with LHRH analogs (2-3 years or longer) is still a matter of debate (Rody, 2005). Early improvement in quality of life over the first 3-6 months of goserelin treatment supports its use as an alternative to chemotherapy in patients with early non-life threatening endocrine-responsive breast cancer (Mitchell, 2004). In general, LHRH analogs are well tolerated and associated with mild effects of estrogen withdrawal such as amenorrhea, hot flushes, and vaginal dryness.

A. Selective estrogen receptor modulators (SERMs) Endocrine therapy is based on the observation that estrogen is the major growth promoter for breast cancer cells. Tamoxifen (NovaldexR) is one of the oldest used SERMs. It inhibits the growth of breast tumors by competitive antagonism of estrogen at its receptor site. It also exhibits partial estrogen-agonist effects. These effects can be beneficial, since they may help prevent bone demineralization in postmenopausal women, but also detrimental, since they are associated with increased risks of uterine cancer, thromboembolism, and tamoxifen resistance (Fisher, 1996; Pritchard, 1997; Hortobagyi, 1998). In premenopausal patients, tamoxifen usage is often associated with bone loss in those who continue to menstruate after adjuvant chemotherapy. On the other hand, it decreased bone loss in women who developed chemotherapy-induced amenorrhea (Vehmanen, 2006). Tamoxifen is normally taken orally for five years, beyond which there seems to be little additional benefit. As adjuvant therapy postoperatively it is the current standard first-line agent for patients with early, estrogenreceptor (ER) positive and/or progesterone receptor (PR) positive breast cancer. It is also indicated as adjuvant therapy in patients with metastatic disease. In the chemoprevention setting, tamoxifen is the only available endocrine option for women at high risk of breast cancer but, given that these are healthy subjects, it is associated with an unacceptable rate of adverse events (Tobias, 2004). Toremifene (FarestonR), another antiestrogen closely related to tamoxifen may be an option for postmenopausal women with metastatic breast cancer. Newer SERMs, such as raloxifene (EvistaR), were initially approved to lower the risk of osteoporosis. Raloxifene's anti-cancer and chemopreventive effects are currently being investigated in the STAR (Study of Tamoxifen and Raloxifene) trial (Tobias, 2004).

D. Aromatase inhibitors In postmenopausal women, estrogen synthesis occurs in non-ovarian peripheral tissues. This mainly follows the route of conversion by aromatase, of the androgenic substrates androstenedione and testosterone to estrone and estradiol in the adrenal glands and adipose tissue, including that of the breast. Inhibitors of this enzyme have anti-proliferative effects presumably through suppression of this estrogen production (Smith, 2003). In the early 1990s, third-generation aromatase inhibitors (AIs) were developed exhibiting high specificity at clinical doses, with little or no effect on cortisol or aldosterone. This class includes the reversible nonsteroidal imidazole-base inhibitors (e.g. anastrozole (ArimidexR) and letrozole (FemaraR)), and the irreversible steroidal activators, exemestane (AromisinR) (Campos, 2004). AIs are of no value in premenopausal patients where the ovaries are the primary sites of estrogen production. Postmenopausal women with early hormonereceptor-positive breast cancer assigned to take letrozole after completing a five-year course of adjuvant tamoxifen (extended adjuvant therapy) were less likely than women on placebo to experience a recurrence, with improved four-year disease-free survival rates (Bryant, 2003; Goss, 2003). Letrozole has been shown to be superior to megestrol acetate and aminoglutethimide as second-line treatment for advanced breast cancer. Letrozole was also superior to

B. Progestins PR-positive advanced breast tumors can respond to the use of synthetic progesterone-like drugs such as megesterol acetate (MegaceR). Megestrol acetate was also shown to reduce the frequency of hot flushes in postmenopausal breast cancer patients (Wymenga, 2002). Owing to its steroidal nature, Megace induces a significant increase in appetite leading to weight gain. Sometimes, it is used to reverse weight loss in patients with advanced cancer. Progestins are usually restricted to second or thirdline therapies following aromatase inhibitors and/or antiestrogens.

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Cancer Therapy Vol 4, page 185 tamoxifen in first line-line treatment for advanced (Smith, 2003) as well as in systemic preoperative (neoadjuvant) treatment of locally advanced cancer (Mouridsen, 2005). A recent adjuvant trial demonstrated significant superiority of letrozole over tamoxifen in disease-free survival (Mouridsen, 2005). As first-line therapy, both anastrozole and letrozole have been shown to significantly prolong remission compared with tamoxifen in postmenopausal women with advanced breast cancer (Nabholtz, 2006). Anastrozole has very recently been granted fasttrack approval in the U.S and elsewhere for adjuvant treatment of early hormone-receptor positive breast cancer in postmenopausal women, particularly if tamoxifen is contraindicated (Bryant, 2003). In the adjuvant setting, results of the ATAC trial showed that women taking anastrozole over a 3-year period had a 17% better disease free survival rate, compared with women taking tamoxifen. The ATAC trial has already shown superior efficacy and a number of important tolerability benefits of anastrozole versus tamoxifen for time to recurrence and incidence of contralateral breast cancer (ATAC Trialistsâ&#x20AC;&#x2122; Group, 2002). The steroidal AI exemestane is highly active and well tolerated. Compared with megestrol acetate, exemestane treatment was shown to significantly prolong survival in women with progressive advanced breast cancer who experience failure of tamoxifen therapy, while at the same time, offering at least as much alleviation of pain and tumor-related signs and symptoms as megestrol acetate. Exemestane was associated with a significantly lower incidence of weight gain compared with megestrol acetate (Dixon, 2004). Exemestane has also shown superiority when compared with tamoxifen for objective response and clinical benefit. Furthermore, exemestane appears to provide additional tolerability benefits in terms of positive androgenic effects on bone metabolism and lipid cholesterol levels, an important consideration in the treatment of early stage breast cancer. In the first-line setting, data for exemestane have not been fully published and it is not yet approved for this indication (Nabholtz, 2006). Third-generation AIs are given orally and appear to be generally well tolerated, though some short-term mild side effects have been recorded. The most common ones are hot flushes, vaginal dryness, musculoskeletal pain, and headache. In contrast to findings with tamoxifen, there is no evidence to suggest an increased risk of uterine carcinoma or venous thromboembolism with AIs. Unlike tamoxifen which reduces bone demineralization through its agonist effects, AIs may enhance this process by lowering circulating estrogen levels. These undesired effects may be reduced with concurrent use of bisphosphonates such as pamidronate (ArediaR), aledronate (FosamaxR), or zoledronate (ZometaR). In light of their estrogen-lowering effects, nonsteroidal AIs are also expected to have adverse effects on blood lipids. One small study on postmenopausal women with breast cancer has reported an increase in total

and LDL cholesterol after 16 weeks of letrozole treatment (Elissaf, 2001). The long-term risks of AIs remain to be fully assessed. Currently, AIs are being evaluated for chemoprevention and in combination with chemotherapy and targeted therapies (Campos, 2004). The ASCO panel recommends the following general guidelines for the use of AIs (Winer, 2005): i. Postmenopausal women with ER-positive breast cancer may substitute an aromatase inhibitor for tamoxifen as initial adjuvant therapy to reduce the risk of recurrence. ii. Postmenopausal women who are currently taking tamoxifen may consider switching to an aromatase inhibitor after two to five years. iii. Women who switch to an aromatase inhibitor may continue this therapy for 2-3 more years, but no longer than 5 years. Women are advised that the result of treatment with an aromatase inhibitor for longer than this has not been studied and should best be taken in the context of a clinical trial. iv. There are no data to recommend taking tamoxifen after an aromatase inhibitor. v. In addition, women who develop invasive ER positive breast cancer while taking prophylactic tamoxifen for breast cancer risk reduction, and women who cannot take tamoxifen because of high risk of severe side effects, or who have tried tamoxifen and had to stop because of severe side effects, might be advised to consider using an aromatase inhibitor. The ASCO panel indicates that it is not known whether AIs could be used interchangeably in clinical practice and therefore favors using the agent with the most data relevant to each individual clinical setting.

E. Estrogen receptor antagonists An important addition to the armamentarium of endocrine therapies is the selective estrogen-receptor antagonist fulvestrant (FaslodexR), also termed "an estrogen receptor down-regulator". With no agonist effects, it competitively binds, blocks, and degrades the ER. It was FDA approved in 2002 for treatment of ERpositive metastatic breast cancer in postmenopausal women (www.infoaging.org/d-breast). Given by injection on a monthly basis, fulvestrant 250mg is effective in the treatment of postmenopausal women with advanced breast cancer following AI failure as well as tamoxifen failure. Fulvestrant is at least as effective as anastrozole following tamoxifen failure in terms of overall survival and shows activity after progression on AI (Tobias, 2004; Howell, 2005). As first-line therapy, fulvestrant has been shown to be of similar efficacy to tamoxifen in patients with hormone receptor-positive tumors (Pippen, 2003), although which patient population is the most appropriate for its use in the first-line setting has yet to be determined. Overall, the lower incidence of joint disorders compared to AIs, the absence of uterotrophic effects, the reduced incidence of hot flushes compared to tamoxifen, and the lack of cross-resistance with tamoxifen favors the use of fulvestrant in the sequential treatment of breast cancer (Howell, 2005). 185

Sharaf: Recent trends in breast cancer drug therapy

B. Bevacizumab

II. Monoclonal antibodies

Bevacizumab is a recombinant monoclonal antibody against vascular endothelial growth factor receptor (VEGF), a protein involved in the neovascularisation of multiple malignant tumors. In view of the known role of angiogenesis in breast cancer, two-phase III trials and three phase II trials were seeking to identify bevacizumab's effectiveness in various combinations with capecitabine, paclitaxel, and vinorelbine and as monotherapy. The only completed phase III trial compared capecitabine monotherapy with capecitabine plus bevacizumab in 462 patients with metastatic breast cancer. Inclusion criteria included prior treatment with an anthracycline and a taxane agent. No significant difference was found for time to disease progression. However, the overall response rate favored bevacizumab (19.8% versus 9.1% [p = 0.001]) (Miller, 2005). In view of the lack of extension of the time to progression in the completed phase III trial in patients with MBC, a second phase III trial is investigating bevacizumab for locally recurrent breast cancer. Six hundred eighty-five patients are being randomized to receive paclitaxel alone or in combination with bevacizumab (Miller and Wang, 2005). The adverse effect profile of bevacizumab includes hypertension, proteinuria, thrombosis, and epistaxis (Rugo, 2004). At this point, data on bevacizumab are too limited to be recommended as a treatment option for breast cancer outside of a clinical trial (Motl, 2005).

Significant advances in the use of targeted biological therapies with novel mechanisms of action over the past several years have changed, and continue to influence breast cancer treatment. The most studied are two monoclonal antibodies, trastuzumab and bevacizumab.

A. Trastuzumab About 25-30 % of all human breast cancer patients have ER at less than 10 fmol/mg tumor protein; these invariably have the worst prognosis. In these cases, endocrine agents are generally ineffective. This has led to development of therapies aimed specifically at inhibiting the HER2/neu gene, which appears to be overexpressed in these tumors. Trastuzumab (HerceptinR) is the first humanized monoclonal antibody approved for treatment of HER-2 positive metastatic breast cancer. This biologic response modifier selectively binds to the extracellular domain of the HER2/neu protein, causing down-regulation of HER2/neu and inhibiting the growth of HER2/neu overexpressing tumors. It is indicated as monotherapy for patients with refractory metastatic breast cancer and in combination with paclitaxel because of their synergistic effect (Colomer, 2001). When compared to chemotherapy alone, combinations of trastuzumab plus chemotherapy lead to higher response rates, longer time to disease progression, and improved overall survival (Slamon, 2001). A recent small study evaluated coadministration of trastuzumab and the cyclo-oxygenase (COX) 2-inhibitor celecoxib, which has shown chemoprotective and antineoplastic activity in rodent mammary carcinomas. Patients with HER-2 expression who had received trastuzumabâ&#x20AC;&#x201C;based therapy received celecoxib 400 mg twice a day and trastuzumab 2 mg/kg/week. To date, median time to disease progression was 9 weeks and one patient of nine (11%) had stable disease (Dang, 2002). Trastuzumab should be given for one year with cardiac monitoring, and by either the weekly or threeweekly schedule (NCCN, 2006). The recommended initial loading dose is 4 mg/kg administered as a 90-minute infusion. The recommended weekly maintenance dose is 2mg/kg that can be administered as a 30-minute infusion if the initial loading dose was well tolerated (Genentech, 2002). Trastuzumab, like other monoclonal antibodies is associated with infusion-related reactions, typically manifested as fever, chills, nausea, vomiting, diarrhea, and rare reports of pulmonary reactions. Trastuzumab has also been associated with an increased risk of cardiac dysfunction, primarily congestive heart failure (CHF) (Genentech, 2002). Trastuzumab is not advised to be given concurrently with an anthracycline because of cumulative cardiac toxicity. According to the National Comprehensive Cancer network (NCCN) guidelines, trastuzumab should be incorporated into the adjuvant therapy of node-positive breast cancer that over-expresses HER-2. It should be also considered for patients with node-negative tumors greater than or equal to 1 cm and that overexpress HER-2.

III. Cytotoxic chemotherapy Chemotherapy is administered in a series of cycles with intervals in between to minimize side effects as well as to allow time for cells in the patientâ&#x20AC;&#x2122;s normal tissues to recover. The number of cycles is agent dependent. Typically, 4-6 cycles for a total time of 3-6 months are employed depending on the agent used. Several types of cytotoxic agents are used to treat breast cancer. The most common ones are doxorubicin (AdriamycinR), pegylated liposomal doxorubicin, cyclophosphamide (CytoxanR), fluorouracil (5-FU), epirubicin (EllenceR), gemcitabine (GemzarR), and vinorelbine (NavelbineR). Docetaxel (TaxotereR), paclitaxel (TaxolR), or albumin-bound paclitaxel (AbraxaneR) are the preferred options for patients with metastatic breast cancer that does not respond to standard chemotherapy (NCCN, 2006). Capecitabine (XelodaR), an oral fluoropyrimidine carbamate, was developed as a prodrug of 5-FU with the goal of improving tolerability and intratumor drug concentrations through tumor-specific conversion to the active drug. Capecitabine is currently approved by the FDA for use (1) as a single agent in metastatic breast cancer patients who are resistant to both anthracyclineand paclitaxel-based regimens or in whom further anthracycline treatment is contraindicated and (2) in combination with docetaxel after failure of prior anthracycline-based chemotherapy. The most common dose-limiting adverse effects associated with capecitabine

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Cancer Therapy Vol 4, page 187 monotherapy are hyperbilirubinemia, diarrhea, and handfoot syndrome (Walko, 2005).

Chemotherapy is less commonly given to older women with early stage disease (lymph node-negative), as the absolute value of chemotherapy appears to decrease with advancing age. Neoadjuvant chemotherapy is employed to shrink the size of the tumor and allow more breast-conserving types of surgery to be undertaken or more complete excision of very large tumors. In the preoperative setting, doxorubicin, epirubicin, paclitaxel, or docetaxel-based regimens are recommended. Patients with tumors over-expressing HER2/neu should be considered for neoadjuvant chemotherapy incorporating trastuzumab (NCCN, 2006).

A. Chemotherapy in early breast cancer In early breast cancer, chemotherapy is usually applied as adjuvant treatment following local excision of the tumor, with the aim of preventing metastasis (Hennessy, 2005). Adjuvant combination chemotherapy is the systemic treatment of choice in lymph node-positive patients with ER negative tumors, irrespective of menopausal status, and may be considered as an option in addition to endocrine therapy in patients with ER positive tumors. For node-positive patients with no evidence of spread, anthracycline-based regimens are preferred (Table 1). Available data indicate that adjuvant chemotherapy with an anthracycline-containing regimen results in a small but statistically significant improvement in survival compared with regimens that do not contain an anthracycline (Early Breast Cancer Trialists' group, 1998). Taxanes were recently introduced in the adjuvant setting for node-positive breast tumors. Currently available phase III data with adjuvant paclitaxel-anthracycline combinations demonstrate their significant superiority in terms of clinical outcome when compared with doxorubicin-based, non-taxane-containing combinations (Henderson, 2003). Conventionally, lymph node-negative patients were not considered for chemotherapy, as it was widely believed that the disease was confined entirely to the breast and, therefore, would most likely be cured by local treatment. However, several large clinical trials (Early Breast Cancer Trialistsâ&#x20AC;&#x2122; Collaborative Group, 1998; Hennessy, 2005) showed significant improvement in disease-free survival in these patients. The adjuvant chemotherapy options for nodenegative patients include CMF (cyclophosphamide/methotrexate/5FU), FAC (5-FU, doxorubicin, cyclophosphamide), and AC (doxorubicin, cyclophosphamide).

B. Chemotherapy in advanced breast cancer Approximately 30% of women initially diagnosed with earlier stages of breast cancer eventually develop recurrent advanced or metastatic disease. Cytotoxic therapy tends to be used as first-line therapy in patients with ER-negative tumors, or at a later stage in patients with initially ER-positive tumors, which eventually fail to respond to endocrine interventions. In advanced breast cancer, the median duration of response to a chemotherapy regimen usually ranges from 6-12 months, which is generally less than that observed with hormonal therapies. In the metastatic setting, the use of combination therapy versus monotherapy or sequential single agents remains a controversial issue. Combination therapies generally result in higher response rates and times to disease progression than sequential single agents but usually at a cost of greater toxicity (Miles, 2002). With chemotherapy regimens, the taxanes were shown to exhibit a survival benefit of at least 20 to 30% in the majority of clinical trials. Capecitabine and gemcitabine, two antimetabolites, have shown high activity and acceptable tolerability in a range of settings for MBC. These include single-agent and combination regimens in patients with anthracycline- and/or taxanepretreated disease. Moreover, the introduction of targeted biologics such as trastuzumab and bevacizumab in

Table 1. Adjuvant chemotherapy regimens for early stage node-positive breast cancer1. Non-trastuzumab containing regimens FAC/CAF or FEC/CEF AC EC TAC with filgrastim support A! CMF E! CMF CMF ACx4 + sequential paclitaxel x4, every 2 weekly regimen with filgrastim support ! A! T! C every 2 weekly regimen with filgrastim support ! FEC! T ! ! ! ! ! ! ! !

Trastuzumab containing regimens Adjuvant: ! AC ! T + trastuzumab2 Neoadjuvant: ! T + trastuzumab ! CEF + trastuzumab

Adapted from NCCN practice guidelines in Oncology, 2006 Trastuzumab may be given beginning either concurrent with paclitaxel as part of the AC followed by paclitaxel regimen, or alternatively after the completion of chemotherapy. 2

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Sharaf: Recent trends in breast cancer drug therapy combination with traditional chemotherapeutics has substantially helped to improve the survival outcome in MBC (O’Shaughnessy, 2005). In patients with recurrent disease who have already had substantial anthracycline exposure, retreatment with an anthracycline is generally avoided. Taxane-based therapy is often considered if not included in the adjuvant setting. If time to recurrence is several years following adjuvant therapy, retreatment with prior active agents may be desirable. If the recurrence occurs in <12 months, the use of different classes is generally preferable. Four large, multicenter trials have evaluated single-agent capecitabine in patients with MBC that has progressed during or following anthracycline and taxane therapy, showing consistent efficacy and safety data (O’Shaughnessy, 2005). The most common cytotoxic agents included in recurrent or metastatic breast cancer treatment protocols are listed in Table 2.

Anthracycline-based polychemotherapy regimens are widely used, partly because they permit shorter treatment regimens. They are as follows: AC; EC; FAC/FEC [Epirubicin]; AC followed by CMF; doxorubicin or epirubicin followed by CMF; AC followed by paclitaxel; and docetaxel, doxorubicin, and cyclophosphamide. Misset et al showed that a course consisting of four cycles of doxorubicin and cyclophosphamide (4xAC) given at 3-weekly intervals is equivalent to 6xCMF (Misset, 1996). Randomized trials have demonstrated significantly improved survival for more intensive and longer duration anthracycline-based regimens; however, more research is needed to determine the optimal protocol. Meantime 6xAC/FAC/FEC may be considered for use in women at high risk of recurrence, or with tumors overexpressing Her2/neu, and in whom anthracyclines are not contraindicated (Hudis, 1999).

3. Taxanes

C. Frequently used cytotoxic agents

When used as monotherapy, paclitaxel and docetaxel produce good response rates of 30-50% in patients with advanced breast cancer refractory to standard chemotherapy. Their effects in polychemotherapy are also excellent, with response rates as high as 70-95% being achieved when used as first-line therapy (Perez, 2001). The role of taxanes in early breast cancer is still an area of active investigation and at present, they should only be considered for use in high-risk node-positive breast carcinomas. Weekly doses of AbraxaneR, an albumin-bound nanoparticle form of paclitaxel, enhance the therapeutic potential of taxane therapy in patients with advanced breast cancer. This biologically interactive nanoparticle, through its novel albumin receptor-mediated mechanism of action, provides an opportunity to realize the full therapeutic potential of chemotherapeutic agents, while minimizing the drug's side effects and overcoming the need for toxic solvents (www.fda.gov/cder/foi/label/2005). Recent studies have indicated that dose reduction during chemotherapy regimens may result in significant impairment of clinical outcome. The tolerability of this nanoparticle form was evident by the finding that 95% of cycles were given at the protocol specified dose of 100 mg/m!. Furthermore, 91% of patients were able to receive

Combination regimens are administered intermittently at intervals of 2-3 weeks. Treatment for 4-6 months is considered appropriate. Six cycles of FAC or FEC (duration, 18-24 weeks), six cycles of CMF (duration, 18-24 weeks), or four cycles of AC (duration, 12-16 weeks) are considered standard therapy. The addition of four cycles of AC improved both disease-free survival and overall survival rates (www.acor.org/cnet/october2001).

1. CMF regimens CMF [Cyclophosphamide, Methotrexate, 5Fluorouracil] regimens have been the most widely used form of polychemotherapy over the past 25 years and have been found to confer benefits in terms of both disease free and overall survival in both pre- and postmenopausal women (Fisher et al, 1990). The classical regimen involves six cycles of the drugs (6xCMF) administered at 4-week intervals. Whilst other regimens are now becoming more widely used, particularly those based on anthracyclines, 6xCMF remains a reasonable alternative, especially in women at increased risk of cardiotoxicity or with relatively low-risk breast carcinomas.

2. Anthracycline-based regimens Table 2. Preferred single-agent and combination regimens for metastatic / recurrent breast cancer3. Single agents Agent with Bevacizumab Combinations Combinations with Trastuzumab Other active agents

Doxorubicin, epirubicin, pegylated-liposomal doxorubicin, paclitaxel, docetaxel, capecitabine, vinorelbine, gemcitabine, albumin-bound paclitaxel, Paclitaxel CAF/FAC, FEC, AC, EC, AT, CMF, docetaxel/capecitabine, gemcitabine/paclitaxel Paclitaxel ± Carboplatin Docetaxel ± Carboplatin Vinorelbine Cisplatin, carboplatin, etoposide(po), vinblastine, fluorouracil continuous infusion

Adapted from NCCN practice guidelines in Oncology, 2006.

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Cancer Therapy Vol 4, page 189 100% of the planned dose of AbraxaneR at 100 mg/m2 administered weekly over 30 minutes with no dose reduction (www.fda.gov/cder/foi/label/2005). Paclitaxel was directly compared with albuminbound paclitaxel (ABI-007) in 460 patients with MBC in a randomized phase III trial (Gradishar, 2005). ABI-007 was associated with a significantly greater response rate and time to disease progression than paclitaxel, but median survival rates were similar in the two treatment groups.

A. PARP inhibitors Women who have inherited mutations in the BRCA1 and BRCA2 genes have about an eighty per cent risk of developing breast cancer, ovarian cancer or both. During replication, these genes are involved in DNA repair by a recombination mechanism. Cells with mutated BRCA genes cannot undergo recombination and therefore rely completely on another process involving a protein called PARP to fix the damage and continue replication. Bryant et al reported a novel approach to treating and preventing hereditary breast cancer. They described how the use of a PARP inhibitor could specifically kill tumor cells which have a defect in the gene causing hereditary breast cancer. These findings could also lead to a prophylactic treatment for women with identified high-risk mutations in the BRCA genes. The new treatment uses an agent that prevents PARP from repairing the DNA, making recombination essential. The breast cancer tumor cannot perform recombination and is therefore unable to replicate resulting in tumor necrosis. The beauty of this system is that only tumor cells lack BRCA genes and thus they completely rely on PARP. Normal cells are likely to be unaffected by the treatment and continue to use recombination to repair any mistakes that may occur. Since normal cells do not need the PARP backup system to survive, PARP inhibitors could be used as a prophylactic treatment to kill BRCA deficient cells and prevent tumor formation (Bryant, 2005).

D. Side effects of cytotoxics As with radiotherapy, cytotoxic agents affect all actively dividing cells in the body, both cancerous and healthy. This produces unpleasant side effects such as nausea, vomiting, alopecia, and occasionally more serious effects including neutropenia and cardiotoxicity (Partridge, 2001). Chemotherapy will also cause ovarian failure, resulting in amenorrhea in a substantial proportion of premenopausal women, depending upon the treatment regimen used. It is thought that at least part of the benefits achieved with chemotherapy in pre-menopausal women could be due to their additional ‘endocrine’ effects, i.e. manifested through chemical ovarian ‘ablation’. The side effects of chemotherapy regimens vary depending upon the particular drugs administered, the dose levels, routes of administration and duration of treatment. Some toxic effects are specific to particular agents. For instance, vincristine and vinorelbine can produce neurological effects (loss of reflexes, parasthesia, and neuropathy). Doxorubicin and epirubicin produce cumulative cardiac effects such as congestive heart failure while methotrexate causes renal and hepatic damage (NCCN, 2006).

B. Farnesyltransferase inhibitors (FTIs) Farnesyltransferase inhibitors (FTIs) are a new class of biologically active anticancer drugs. They inhibit farnesylation of a wide range of target proteins, including ras. It is thought that they block ras activation through inhibition of the enzyme farnesyl transferase, ultimately resulting in cell growth arrest (Appels, 2005). Although FTIs were originally designed to target the ras signal transduction pathway, it is now clear that several other intracellular proteins are also dependent on posttranslational farnesylation for their function. Studies with FTIs have shown promising activity in patients with breast cancer associated with ras mutations. Preclinical data revealed that although FTIs inhibit the growth of ras-transformed cells, they are also potent inhibitors of a wide range of cancer cell lines that contain wild-type ras, including breast cancer cells. Additive or synergistic effects were observed when FTIs were combined with cytotoxic agents (in particular the taxanes and gemcitabine) or endocrine therapies (tamoxifen). Phase I trials with FTIs have explored different schedules for prolonged administration, and dose-limiting toxicities included myelosuppression, gastrointestinal toxicity, peripheral neuropathy, and fatigue (Head, 2004). Clinical efficacy of the FTI tipifarnib (ZarnestraR) against breast cancer was seen in a phase II study (Johnston SRD, 2003). Seventy-six patients with advanced breast cancer were given tipifarnib orally either at a continuous dose of 300 mg twice daily (n=41) or an intermittent dose of 300 mg twice daily for 21 days

E. High-dose chemotherapy plus bone marrow/stem cell transplants The necessity of bone marrow rescue is a highly controversial issue. Some studies found that high-dose chemotherapy (i.e. 2-20 times standard doses) followed by stem-cell transplantation improved survival among women with widespread cancer (Rodenhuis, 2003). Others reported that although the treatment increased time to cancer recurrence, it did not improve overall survival (www.infoaging.org/d-breast).

IV. Novel targets for breast cancer therapy An increased understanding of the biology of breast cancer has led to the identification of novel therapeutic targets. New biological concepts may present an opportunity for the development of promising and innovative treatment paradigms that target multiple neoplastic pathways, with the goal of producing high efficacy and minimal toxicity.

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Sharaf: Recent trends in breast cancer drug therapy followed by 7 days of rest (n=35). The trial showed nine partial responses and nine cases of stable disease (of at least 24 weeksâ&#x20AC;&#x2122; duration). Although clinical efficacy was similar between patients treated intermittently and those receiving continuous dosing, the side effect profile was significantly improved by using an intermittent schedule. Although their true mechanism of action remains unclear, ongoing clinical trials are assessing the potential of FTIs to enhance the efficacy of current endocrine and cytotoxic therapies in breast cancer. Combinations with other signal transduction inhibitors may be an additional strategy that merits further research.

C. Small inhibitors

molecule

tyrosine

D. Rapamycin (mTOR antagonists) Mammalian target of rapamycin (mTOR) is a serinethreonine kinase member of the cellular phosphatidylinositol 3-kinase (PI3K) pathway, which is involved in multiple biologic functions such as transcriptional and translational control. mTOR is a downstream mediator in the PI3K/Akt signaling pathway and plays a critical role in cell survival. The mTOR signaling pathway is likely to be aberrantly activated in a substantial number of breast tumors, making mTOR an especially promising target for breast cancer therapy. In breast cancer, this pathway can be activated by membrane receptors, including the ErbB family of growth factor receptors, the insulin-like growth factor receptor, and the estrogen receptor. There is evidence suggesting that Akt promotes breast cancer cell survival and resistance to chemotherapy, trastuzumab, and tamoxifen. Rapamycin is a specific mTOR antagonist that targets the Akt/PI3K pathway and blocks the downstream signaling elements, resulting in cell cycle arrest in the G1 phase (Carraway, 2004). Rapamycin and rapamycin analogs have emerged as promising antitumor drugs for many cancer types, including breast cancer. When combined with other chemotherapeutic agents, rapamycin and rapamycin analogs have been reported to increase the efficacy of a variety of cytotoxic agents, including cisplatin, doxorubicin, 5-FU, and cyclophosphamide (Geoerger, 2001). Mondesire et al reported that in vitro rapamycin has a synergistic effect with paclitaxel, carboplatin, and vinorelbine and an additive effect with doxorubicin and gemcitabine. Furthermore, they showed that rapamycin in combination with paclitaxel leads to a significant reduction in tumor growth in vivo in a rapamycin-sensitive xenograft model. Their results demonstrated that rapamycin might be able to at least partially overcome resistance to paclitaxel and carboplatin in HER2/neu-overexpressing cells, suggesting a potential approach to these poorly behaving tumors. On the other hand, cell lines that were resistant to the growthinhibitory effect of rapamycin were also resistant to rapamycin-mediated chemosensitization. This suggests that combination therapy of cytotoxic agents with rapamycin may be effective in patients selected for aberrations in the PI3K/Akt pathway (Mondesire, 2004). In the next few years, and as trials with targeted agents mature, their role will be further defined and their mechanism of action will be more elaborated. This may help identify patients who will benefit the most from these novel therapeutic approaches.

kinase

A number of low molecularâ&#x20AC;&#x201C;weight tyrosine kinase inhibitors (TKIs) directed at members of the ErbB family (EGFR, HER2, and HER4) is now in clinical development. None has received US FDA approval for breast cancer treatment. These small molecules compete with ATP for binding to the kinase domain of the receptor. They are orally bioavailable and generally well tolerated. Knowing that EGFR is overexpressed in 16-48% of human breast cancers, several groups have reported an association between EGFR expression and poor prognosis. The reversible EGFR inhibitors gefitinib (IrissaR) and erlotinib (TarcevaR) are furthest in the course of development. Both gefitinib and erlotinib have activity against multiple breast cancer cell lines in vitro and in xenograft models. However, neither gefitinib nor erlotinib has demonstrated significant single-agent activity against refractory metastatic breast cancer (Albain 2002; Winer 2002). Several ongoing phase II and phase III trials utilize gefitinib and erlotinib in combination with chemotherapy in MBC. Alternatively, combining antibody-based therapy with TKIs may allow for more complete blockade of erbBmediated signal transduction pathways, and thereby delay or overcome drug resistance. This strategy is being employed in a phase II study of trastuzumab and gefitinib (Lin, 2004). Recent data also indicate that upregulation of the ErbB receptors may mediate endocrine resistance, due to crosstalk between the ErbB and estrogen receptor (ER) signal transduction pathways (Atalay, 2003). This crosstalk has been postulated to occur via multiple mechanisms, including upregulation of EGFR and HER2 expression by tamoxifen, ligand-independent signaling of ER via PI3K, and modulation of coactivators of ER via downstream effectors of the ErbB pathway (Johnston SR, 2003). In preclinical models, co-blockade of the EGFR and ER pathways with gefitinib and either tamoxifen or fulvestrant resulted in restoration of tamoxifen sensitivity and delay of resistance to estrogen deprivation in HER-2 overexpressing breast tumors. Based on these data, several phase II trials of gefitinib with hormonal therapy are ongoing in women with ER-positive metastatic breast cancer.

Acknowledgements I am highly indebted to Professor Yunus Luqmani for helpful comments and invaluable contribution to this study.

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opposite effects on bone mineral density in premenopausal patients depending on menstrual status. J Clin Oncol 24, 675-80. von Minckwitz G, Graf E, Geberth M, Eirmann W, Jonat W, Conrads B, Brunnert K, Gerber B, Zippel HH, Kaufmann M (2004) Goserelin versus CMF as adjuvant therapy for nodenegative, hormone receptor-positive breast cancer in premenopausal patients. The GABG IV-A-93 Trial (Abstract # 534). Proc ASCO 22, No 14S. Walko CM, Lindley C (2005) Capecitabine: a review. Clin Ther 27, 23-44. Winer E, Cobleigh M, Dickler M, Miller K, Jones C (2002) Phase II multicenter study to evaluate the efficacy and safety of Tarceva in women with previously treated locally advanced or metastatic breast cancer [abstract 445] Breast Cancer Res Treat 76 (Suppl 1), s115. Winer EP, Hudis C, Burstein HJ, Wolff AC, Pritchard KI, Ingle JN, Chlebowski RT, Gelber R, Edge SB, Gralow J, Cobleigh MA, Mamounas EP, Goldstein LJ, Whelan TJ, Powles TJ, Bryant J, Perkins C, Perotti J, Braun S, Langer AS, Browman GP, Somerfield MR (2005) American Society of Clinical Oncology technology assessment on the use of aromatase inhibitors as adjuvant therapy for postmenopausal women with hormone receptor-positive breast cancer: status report 2004. J Clin Oncol 23, 619-29. Wymenga AN, Sleijfer DT (2002) Management of hot flushes in breast cancer patients. Acta Oncol 4, 269-75. www.acor.org/cnet/october2001 www.fda.gov/cder/foi/label/2005 www.infoaging.org/d-breast

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Cancer Therapy Vol 4, page 193 Cancer Therapy Vol 4, 193-204, 2006

Breast tumor cell clusters and their budding derivatives show different immunohistochemical profiles during stromal invasion: implications for hormonal and drug therapies Research Article

Yan-gao Man1, Chengquan Zhao1, Jin Wang2 1

Department of Gynecologic and Breast Pathology, Armed Forces Institute of Pathology and American Registry of Pathology, Washington DC 2 Beijing Four-Ring Pharmaceutical Co, Ltd, Beijing, China

__________________________________________________________________________________ Correspondence: Yan-gao Man, MD., PhD, Director of Gynecological and Breast Research Laboratory, Department of Gynecologic and Breast Pathology, Armed Forces Institute of Pathology and American Registry of Pathology, 6825 16th Street, NW Washington DC 20306-6000, USA; Tel: 202-782-1612; Fax: 202-782-3939; E-mail: man@afip.osd.mil Key words: Breast tumor cell clusters, immunohistochemical profiles, stromal invasion, hormonal therapy, drug therapy Abbreviations: basement membrane, (BM); beta protein 1, (BP1); cytokeratins, (CK); ductal carcinoma in situ, (DCIS); epithelial specific antigen, (ESA); estrogen receptor negative cell clusters, (ER NCC); estrogen receptor, (ER); leukocyte common antigen, (LCA); matrix metalloproteinase-26, (MMP-26); myoepithelial, (ME); smooth muscle actin, (SMA); topoisomerase II alpha, (TOPOII!)

The opinions and assertions contained herein represent the personal views of the authors and are not to be construed as official or as representing the views of the Department of the Army or the Department of Defense. Received: 10 May 2006; Revised: 23 May 2006 Accepted: 09 June 2006; electronically published: June 2006

Summary Our previous studies in pre-invasive breast tumors suggested that estrogen receptor negative cell clusters (ER NCC) overlying focally disrupted myoepithelial cell layers represent the precursor of invasive breast lesions. Our hypothesis is in total agreement with previous findings that breast tumor progression is paralleled by a progressive hormonal independence, and that ER negative tumors have substantially more aggressive clinical behavior than ER positive tumors. Our hypothesis, however, is hard to reconcile with clinical reports that over 80% of invasive breast tumors are ER positive. This study attempted to identify the potential factors accounting for this discrepancy. Double immunohistochemistry was used to compare the expression profiles of ER and three tumor invasion- and drug resistance-related molecules in ER NCC and their derivatives in breast tumors with co-existing in situ and invasive components. Of 100 selected cases, 17 contained larger ER NCC with “budding” derivatives that were arranged as tongue-like projections, “puncturing” into the stroma, and 3 contained micro-invasive lesions that were immediately adjacent to ER NCC. All or nearly all the cells in each ER NCC completely lacked the expression of ER and tumor invasion- and drug resistance-related molecules, in sharp contrast to their adjacent counterparts within the same duct, which were strongly positive. The number of positive cells and intensity of immunostaining for these molecules in “budding” derivatives, however, increased linearly as cells invaded deeper into the stroma. The same pattern was seen in all three micro-invasive lesions. These findings suggest that ER NCC may belong to the population of tumor progenitor or stem cells that are not mature enough to express ER and other molecules. After a few divisions and invading the stroma, however, the derivatives of ER NCC could gain the expression of these molecules, probably due to a pre-programmed genetic sequence or stimulations from immunoreactive or stromal cells. Therefore, the derivatives of ER NCC might respond tomaxifen and other therapies, whereas ER NCC might resist the same treatment, representing “seeds” for drug resistant and recurrent tumors.

stroma by both the myoepithelial (ME) cell and basement membrane (BM). ME cells are joined by intercellular junctions and adhesion molecules, constituting a largely

I. Introduction The epithelium of normal and pre-invasive human breast tumor tissues is physically separated from the

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Man et al: Breast tumor cell clusters and their budding derivatives adjacent cells within the same duct but distant from disruptions, which were strongly and uniformly positive for ER. Compared to their adjacent ER positive counterparts, ER NCC displayed several unique features, including: [1] a significantly higher proliferation rate, [2] a significantly higher frequency of loss of heterozygosity at multiple chromosomal loci; [3] a significantly higher expression of cell cycle control and tumor invasion related genes; [4] physical signs of stromal and vascular invasion (Man et al, 2002b,c, 2003a,b, 2004a, 2005c; Man and Sang, 2004). Together, our findings suggest that focal ME cell layer disruptions substantially impact the biological presentations of the associated epithelial cells, and that ER NCC might represent the precursor of invasive lesions, which are in the process, or at the early stage of invasion. Our findings are in total agreement with previous reports that breast tumor progression is paralleled by a progressive hormonal independence, and that ER negative tumors have a substantially more aggressive clinical behavior and worse prognosis than ER positive tumors (Clarke et al, 1989; Schmitt, 1995; Sheikh et al, 1994, 1995; Rochefort et al, 2003). Our findings are also consistent with previous findings that breast tumor progression from one stage to another is driven by sequential expression of stage-specific molecules and the selection of biologically more aggressive cell clones (Pitot, 1993; Beckmann, 1997; Lakhani, 1999). Our findings, however, are hard to reconcile with the fact that over 80% of invasive breast cancers are reported to be ER positive (Luna-More et al, 2000; Swain et al, 2004). Our current study intended to identify potential factors accounting for this discrepancy. For the following three main reasons: [1] it has been well documented that most malignant tumors arise from a single cell (Nowell, 1976; Sell et al, 1994; Kordon and Smith et al, 1998; Middleton et al, 2000), [2] epithelial cell “budding” is a common event shared by both normal development and tumor invasion, which occurred exclusively at the site of focally degraded BM (Yang et al, 2003; Lu et al, 2005; Jourquin et al, 2006), and [3] our previous studies in both human breast and prostate tumors have revealed that a vast majority of the proliferating cells are located at the site of focal ME or basal cell layer disruptions, and that tumor cells “budding” from these focal disruptions are often in direct physical continuity with morphologically distinct invasive lesions (Man et al, 2002b,c, 2003a,b, 2004a, 2005d in press; Man and Sang, 2004; Yousefi et al, 2005), we have hypothesized that both ER NCC and their “budding” derivatives are derived from a monoclonal proliferation of the same tumor progenitor or stem cells. However, ER NCC and their “budding” derivatives are at different differentiation stages with different degrees of accumulations of genetic abnormalities. Thus, ER NCC and their “budding” derivatives might have a substantially different genetic and immunohistochemical profiles. In addition, our previous studies in surgically operated normal adult submandibular glands (which share the same myoepithelial cell population and other structural features) using the combination of immunohistochemistry and autoradiography (with multiple injections of tritiatedthymidine after the operation) had shown that newly

continuous sheet that encircles the epithelium (Murad and von Haam, 1986; Tsubura et al, 1988; Guelstein et al, 1993). The BM is composed of type IV collagens, laminins, and other molecules, forming a continuous lining surrounding and attaching to the ME cell layer (Nerlich, 1995; Damiani et al, 1999; Miosge, 2001). Since the epithelium is normally devoid of blood vessels and lymphatic ducts, its metabolism needed oxygen, nutrients, and growth factors must first pass through the BM, then the ME layer, in order to reach the epithelium. In contrast, epithelium-derived tumor cells must first pass through the ME layer, then the BM, in order to invade or metastasize. Breast cancer invasion has been attributed primarily, if not solely, to the over-production of proteolytic enzymes predominantly by tumor cells, which results in the degradation of the BM (Goldfarb and Liotta, 1986; Duffy et al, 2000). This theory alone, however, appears not to fully reflect the intrinsic mechanism of breast tumor invasion for three main reasons. First, the mechanism and process of the ME cell layer degradation are unknown. Second, results from all recent worldwide clinical trials with specific enzyme inhibitors have been very disappointing (Coussens et al, 2002; Matrisian et al, 2003). Third, our previous studies detected a significantly higher level of matrix metalloproteinase-26 (MMP-26), a key enzyme for BM degradation, and its mediated progelatinase B in ductal carcinoma in situ (DCIS) than in invasive carcinoma using immunohistochemistry and an integrated morphometry analysis (Zhao et al, 2004). Our recent studies further revealed that the invasive front of early invasive lesions in over 50 cases examined completely lacked expression of MMP-26, whereas cells within the same tumor and in adjacent micro-invasive lesions were strongly positive for MMP-26 (Man et al, 2005a,b). Since over 90% of breast cancer related death is caused by invasion related illness (Parker et al, 1997), there is an urgent need to disclose the intrinsic mechanism of, and to develop more effective approaches to prevent, breast tumor invasion. Promoted by the fact that several tumor suppressors are exclusively produced by the ME cells and that the absence of the ME cell layers is the most distinct sign of invasive or metastatic breast lesions (Murad et al, 1986; Tsubura et al, 1988; Guelstein et al, 1993; Zou et al, 1994; Barbareschi et al, 2001; Man et al, 2002a), our recent studies have attempted to identify the early signs of ME cell layer alterations and their impact on biological presentations of tumor cells. Using a double immunohistochemical method to simultaneously elucidate ME and tumor cells, our initial study detected 405 focal ME cell layer disruptions (the absence of ME cells resulting in a gap greater than the combined size of at least 3 ME cells) in 5,698 duct cross sections from 220 female patients with estrogen receptor (ER) positive, pre-invasive breast tumors (Man et al, 2003a). Of these disruptions, 367 (90.6%) occurred in malignant lesions and 38 (9.4%) in hyperplastic or normal appearing ducts. Focal disruptions in ME cell layers appeared to substantially impact the ER expression status in the overlying epithelial cells. A vast majority (86.4%) of the cell clusters overlying disruptions completely lacked ER expression, in sharp contrast to

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Cancer Therapy Vol 4, page 195 formed cell clusters by stem or progenitor cells not only morphologically differed from their adjacent normal adult counterparts, but also completely lacked the expression of several proteins that were heavily expressed in their adjacent adult counterparts (Man et al, 1995, 2001a). These newly formed cell clusters, however, gradually gained the expression of these molecules with time, and became morphologically and immunohistochemically indistinguishable from their adjacent normal adult counterparts two months after the operation. Based on these and other findings, we have further hypothesized that ER NCC might represent tumor progenitors or stem cells that are not mature enough to manufacture ER and other molecules, or are with “resting” genes for these molecules. The derivatives of ER NCC, however, might gradually gain the expression of these molecules after a few divisions and “budding” from focally disrupted ME layers and invading deeper into the adjacent stroma, probably due to a programmed genetic sequence or direct interactions with stromal or immunoreactive cells.

and topoisomerase II alpha (TOPOII!), which is an essential nuclear enzyme involved in DNA replication and is the target for many anti-cancer drugs used for cancer therapy (Houlbrook, 1995). The loss or reduction has been reported to be the main mechanism for drug resistance (Houlbrook et al, 1995). The expression status of TOPOII! in ER NCC and their derivatives was compared. To assess the possibility that ER NCC and their derivatives may have different expression profiles of tumor progression- and invasion-related molecules, sections were double immunostained for SMA and MMP26 (Zhao et al, 2004), or for SMA and beta protein 1 (BP1), a homobox gene product that was preferentially seen in over 80% of the invasive breast lesions (Man et al, 2005e). As our previous studies have consistently shown that focally disrupted ME cell layers have a significantly higher infiltration of immunoreactive cells (Man et al, 2005f; Man, 2005g; Yousefi et al, 2005), which have been reported to directly impact the gene expression and proliferation of associated tumor cells (Freeman et al, 1995; Takahashi et al, 1996; Asano-kato, et al 2005; Nienartowicz et al, 2006; Qu, 2006), selected sections were double immunostained for SMA and leukocyte common antigen (LCA). To assess the histological origin of ER NCC and their derivatives, sections were immunostained with three epithelial specific markers, cytokeratins (CK) AE1/AE3, epithelial specific antigen (ESA), and E-cadherin (Vector, Burlingame, CA). Sections were also immunostained with two stromal cell specific markers, SMA and vimentin (Vector, Burlingame, CA).

II. Materials and methods A total of 100 formalin-fixed, paraffin-embedded human mammary tumors with co-existing pre-invasive, invasive, or micro-invasive lesions were retrieved from the files of The Armed Forces Institute of Pathology and our own tissue bank. Consecutive sections at 4-5 "m thickness were made, placed on positively charged microscopic slides, and stained with H&E for morphological classification, based on our published criteria (Tavassoli and Man, 1995). To identify ER NCC and their potential derivatives (defined as cell clusters that directly “bud” or “sprout” from, but maintain direct physical continuity with, ER NCC overlying focally disrupted ME cell layers), sections were subjected to double immunohistochemical staining for smooth muscle actin (SMA) and ER (Vector, Burlingame, CA), as previously described (Man and Tavassoli, 1996). Immunostained sections were independently examined by the investigators to identify pure ER NCC and larger ER NCC (more than 15 cells/cluster) with “budding” derivatives. To compare the ER expression profiles between ER NCC and their potential derivatives, four technical approaches were used. First, the morphological and immunohistochemical profiles between pure ER NCC and ER NCC with “budding” derivatives were compared. Second, the derivatives of ER NCC were artificially divided into different parts based on their locations (see “Results” for detail), and the ER expression status in each part was compared. Third, sections were examined to identify microinvasive lesions that were immediately adjacent to ER NCC, and the number of ER positive cells and intensity of ER immunostaining in microinvasive lesions at different distances to focal ME cell layer disruptions were compared. Fourth, the morphological and cytological features of ER NCC and their derivatives were compared. To assess the possibility that ER NCC and their derivatives may respond differently to hormonal and drug therapies, sections were double immunostained for SMA

III. Results Of 100 selected cases, 52 (52%) contained pure ER NCC, and 17 (17%) contained a total of 26 larger ER NCC with “budding” derivatives. All or nearly all the cells in pure ER NCC seen in our current study were completely devoid of ER expression, identical to these seen in our previous studies (Figure 1). Compared to pure ER NCC, ER NCC with derivatives showed three unique features: [1] the size was substantially larger; [2] the clusters were more deeply “puncturing” into the stroma; [3] a variable number of ER positive cells were also present. The “budding” derivatives of ER NCC were generally arranged as tongue-like projections, “puncturing” into the stroma (Figure 2). These “buds” could be roughly divided into three parts: the base, tip, and shaft, which are in a direct continuity with the tumor core and disruption, within the stroma, and between the base and tip, respectively. All or nearly all the cells at the base were completely devoid of ER expression (Figure 2). A vast majority of the cells at the shaft were also completely devoid of ER expression, while ER positive cells were occasionally seen (Figure 2). The number of ER positive cells and intensity of ER immunostaining appeared to be linearly increasing as tumor cells invading deeper into the stroma (Figure 2). Over 75% of the budding ER NCC were seen in DCIS (Figures 2a-2f). About 20% of the budding ER NCC were seen in both normal or hyperplastic appearing ducts (Figure 2g-2h).

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Man et al: Breast tumor cell clusters and their budding derivatives In addition to “budding” ER NCC, three cases

contained

micro-invasive

breast

lesions

that

were

Figure 1. ER expression in pure ER NCC. Paraffin-embedded breast tissue sections were double immunostained for SMA (red) and ER (brown). Arrows identify pure ER NCC in DCIS (a & b), regular duct hyperplasia (c & d), and normal duct (e & f). a, c, and e: 150X. b, d, and f: a higher magnification (400X) of a, c, and e, respectively.

Figure 2. ER expression profiles in ER NCC and their “budding” derivatives Paraffin-embedded breast tissue sections were double immunostained for SMA (red) and ER (brown). a-f: ER NCC and “budding” derivatives in DCIS. g-h: ER NCC and “budding” derivatives in a hyperplastic appearing duct. a, c, e, and g: 150X; b, d, f, and h: a higher magnification of a, c, e, and g, respectively, 300X. Think arrows identify focal ME cell layer disruptions and overlying ER NCC. Thin arrows identify “budding” derivatives.

immediately adjacent to “budding” ER NCC (Figure 3). All or nearly all the cells overlying ME cell layer disruptions were completely devoid of ER expression. The number of ER positive cells and intensity of ER

immunostaining in these micro-invasive lesions appeared to increase linearly as they moved away from the tumor core and focal ME cell layer disruptions (Figure 3).

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Cancer Therapy Vol 4, page 197 whereas their adjacent counterparts within the same duct but distant from the focal ME cell layer disruption were strongly positive for TOPOII! (Figure 4). In contrast to ER NCC, some cells in the derivatives displayed distinct TOPOII! expression (Figure 4).

Of the 26 ER NCC with “budding” derivatives, 5 (19.5%) showed no distinct TOPOII! expression in either the tumor core, ER NCC, or ‘budding” derivatives. In the remaining 21 (80.7%), all or nearly all the cells in ER NCC were completely devoid of TOPOII! expression,

Figure 3. ER expression profiles in micro-invasive lesions Paraffin-embedded breast tissue sections were double immunostained for SMA (red) and ER (brown). a, c, and e: 150X; b, d, and f: a higher magnification of a, c, and e, respectively, 400X. Think arrows identify focal ME cell layer disruptions and overlying ER NCC. Thin arrows identify micro-invasive lesions.

Figure 4. TOPOIIa expression profiles in ER NCC and their “budding” derivatives Paraffin-embedded breast tissue sections were double immunostained for SMA (red) and TOPOIIa (brown). Circles identify ER NCC, which are negative for TOPOIIa. Arrows identify TOPOIIa positive cells in the derivatives of ER NCC. a & c: 150X. b & d: a higher magnification (400X) of a & c, respectively.

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Similar to ER, both MMP-26 and BP1 expression was totally absent in all the ER NCC that were informative for evaluation, in sharp contrast to adjacent cells within the same duct but distant from focal ME cell layer disruptions, which expressed high levels of MMP-26 and BP1 (Figure 5). A vast majority of the derivatives of ER NCC, however, were positive for these two molecules (Figure 5; Table 1). In sections double immunostained for LCA and SMA, all ER NCC and their “budding” derivatives showed a significantly higher infiltration of LCA positive cells, compared to their morphologically similar (the same histological type and grade, and similar size and architecture) counterparts with non-disrupted ME cell layers. Most infiltrated leukocytes were located at site of focal ME cell layer disruptions or within the derivatives of ER NCC (Figure 6). In H&E stained sections, tumors with these ER NCC and their derivatives or micro-invasive lesions showed no apparent architecturally and morphologically alterations, and were generally not appreciable under low magnification. Under high magnification, cells at the base

of a given ER NCC or among different ER NCC were generally identical or very similar, whereas they were readily distinguishable from their adjacent counterparts within the same duct by their elongated and condensed nuclei, much smaller size, much higher density, and the disorganized polarity (Figures 1-3). Cells of “budding” ER NCC seemed to increasingly undergo biochemical and morphological modifications as they invaded deep into the stroma. Most cells at the tips of ER NCC and microinvasive lesions immunohistochemically and morphologically resembled the cells in adjacent invasive lesions (not shown). Consistent with our previous findings (Man 2003a,b, 2005c; Man and Nieburgs 2006), all those ER NCC and their derivatives were negative for stromal phenotypic markers, but they were uniformly positive for all epithelial specific markers tested (not shown), suggesting that they are unlikely to represent cells that are undergoing epithelial-mesenchymal transition and may have acquired more mesenchymal features (Kang, 2004; Sato, 2004; Strizzi, 2004; Decraene, 2005; Martinez-Estrada, 2006;).

Figure 5. MMP-26 and BP1 expression in ER NCC and their derivatives Paraffin-embedded breast tissue sections were immunostained for MMP-26 (a & b) and BP1 (c & d). Circles identify ER NCC, which are negative for MMP-26 and BP1. Arrows identify “budding” derivatives and micro-invasive lesions, which are positive for MMP-26 and BP1. a & c: 150X. b & d: a higher magnification (400X) of a & c, respectively.

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Cancer Therapy Vol 4, page 199 Table 1. Comparison of expression of different molecules in ER NCC and their budding derivatives Cell Type ERNCC Derivatives

Total number 26 26

ER (+) 0 23

TOPOIIa (+) 0 21

MMP-26 (+) 0 20

BP1 (+) 0 21

Figure 6. Increased leukocyte infiltration in ER NCC with â&#x20AC;&#x153;buddingâ&#x20AC;? derivatives Paraffin-embedded breast tissue sections were double immunostained for SMA (red) and leukocyte common antigen (LCA; brown). Thick arrows identify LCA positive cells. Thin arrows identify the remaining ME cell layers. a, c, and e: 150X. b, d, and f: a higher magnification (400X) of a, c, and e, respectively.

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Man et al: Breast tumor cell clusters and their budding derivatives NCC and their derivatives is likely to reflect differences in the differentiation status. The evolution of breast cancer is believed to be a multistep process, sequentially or progressively undergoing normal, hyperplastic, in situ, invasive, and metastatic stages (Pitot, 1993; Beckmann et al, 1997; Lakhani, 1999). The progression from one stage to the other is believed to be driven by a sequential expression of stage-related signature genes and the selection of biologically more aggressive sub-clones, recapitulating the normal development process (Pitot, 1993; Beckmann et al, 1997; Lakhani, 1999). Our previous studies in both normal and surgically operated adult rat submandibular glands (which are structurally comparable to the human breast), using a combination of immunohistochemistry and autoradiograph, had shown that newly formed cell clusters by progenitor or stem cells not only differed morphologically from, but also completely lacked the expression of several proteins that were heavily expressed in their adjacent adult counterparts (Man et al, 1995, 2001a). The morphology and protein expression in these newly formed cell clusters, however, increasingly resembled their adult counterparts with time, and became indistinguishable from their normal adult counterparts two months after the operation (Man et al, 1995, 2001a). Similar changes had been observed in the human breast tissues (Nanba et al, 2001). Our findings are consistent with a number of previous reports in human breast tissues, which have well demonstrated the dissociation between ER expression and cell proliferation, and have suggested that some of the ER negative cells may represent normal or tumor stem cells of the human breast (Clarke, 1997, 2005; Schneider, 2006; Nanba, 2001). Alternatively, the discrepancy might represent the difference in the extent of interactions with the adjacent stromal and immunoreactive cells. Previous studies have well documented that the stroma not only exerts significant influences on epithelial cell proliferation, differentiation, and apoptosis, but also impacts the metastatic behavior of epithelial tumors (Fibach et al, 1972; Nakammura et al, 1997; Silberstein, 2001). On the other hand, under certain circumstances, normal stromal cells could convert malignant tumors, even highly aggressive acute leukemia into morphologically and functionally normal, or biologically less aggressive lesions (Fibach et al, 1972; Nakammura et al, 1997; Silberstein, 2001). A recent study revealed that microdissected cells from the periphery and center of the same DCIS had a substantially different frequency and pattern in the expression of 22 genes, assessed with Atlas human Cancer 1.2 Arrays containing 1176 known genes (Zhu et al, 2003). Our previous studies in human breast, cervical, and lung tumors had consistently shown that tumor and their adjacent stromal cells shared a high degree of concurrent immunohistochemical and genetic alterations (Man et al, 2000, 2001b, 2004b; Moinfar et al, 2000, 2005), although the mechanism is unknown. Our recent studies further revealed that both human breast and prostate tumors with focally disrupted ME or basal cell layers had a significantly higher (p<0.01) frequency of immunoreactive cell infiltration than their morphologically similar counterparts with an intact ME or basal cell layer (Yousefi

It is interesting to note that no distinct “budding” ER positive cell clusters were found in any of these selected cases. The lack of corresponding frozen tissues in these cases and the lack of previously published references on ER NCC and their potential derivatives, however, have hampered our efforts of further genetic assessments, including clonality analysis, on these cell clusters.

IV. Discussion Our previous studies revealed that a subset of preinvasive, ER positive mammary tumors contained focally disrupted ME cell layers (Man et al, 2003a). All or nearly all of the cells overlying these focal disruptions were uniformly devoid of ER expression, in a sharp contrast to their adjacent counterparts within the same duct but overlying the remaining non-disrupted ME cell layer, which were strongly positive for ER (Man et al, 2003a). The frequency and pattern of ME cell layer disruptions detected in our current study in tumors with co-existing in situ and invasive components were similar to those seen in our previous studies. The ER NCC seen in our current study, however, differed from those seen in our previous studies in three main expects: [1] the size was substantially larger; [2] the clusters were more deeply “puncturing” into the stroma; [3] a variable number of ER positive cells were also present. Despite these differences, ER NCC seen in our previous and current studies are likely to be derived from a monoclonal proliferation of the same progenitor for several reasons. First, they are exclusively located at focally disrupted ME cell layers. Second, cells within each or among different clusters share the same morphological and immunohistochemical features at the site of, or near the focal disruptions. Third, they are morphologically distinct from adjacent cells within the same duct in the cell density, size, nuclear shape, and polarity. More importantly, our previous studies have revealed that ER NCC from different cases share a very similar genetic and immunohistochemical profile, but they show a significantly higher rate of cell proliferation, genetic instabilities, and expression of tumor invasion and progression related genes than their adjacent counterparts within the same duct (Man et al, 2002b,c, 2003a, 2004a, 2005c; Man and Sang, 2004). Our findings and hypothesis are in total agreement with a number of previous reports: [1] a vast majority of malignant tumors are derived from monoclonal proliferation of a single tumor stem cell (Nowell, 1976; Sell, 1994; Kordon, 1998; Middleton, 2000), [2] epithelial cell “budding” is a common event shared by both normal development and tumor invasion, which occurred exclusively at the site of focally degraded BM (Yang, 2003; Lu, 2005; Jourquin, 2006), and [3] our previous studies in both human breast and prostate tumors have consistently shown that a vast majority of the proliferating cells are located at the site of focal ME or basal cell layer disruptions, and that tumor cells “budding” from these disruptions are generally in direct continuity with the adjacent invasive lesions (Man et al, 2002b,c, 2003a,b, 2004a, 2005c, in press; Man and Sang, 2004; Yousefi et al, 2005; Man and Nieburgs, 2006). The discrepancy seen in our current and previous studies in ER

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Cancer Therapy Vol 4, page 201 et al, 2005; Man et al, 2004d, 2005f, in press; Man, 2005g,h). Tumor cells overlying focal ME cell layer disruptions or adjacent to immunoreactive cells generally showed distinct morphological and immunohistochemical changes, and a vast majority of the proliferating cells were located at or near focal ME or basal cell layer disruptions (Yousefi et al, 2005; Man et al, 2004c; Man et al, 2005d, 2005f, 2005g, 2005h; Man, in press). Additionally, the discrepancy might result from the combination of multiple factors. Since the epithelium is normally devoid of both blood vessels and lymphatic ducts, and several tumor suppressors are exclusively produced by ME cells (Zou et al, 1994; Barbareschi et al, 2001; Man et al, 2002a), a focal disruption in the ME cell layer is likely to have several consequences. These consequences include: [1] a localized loss of tumor suppressors and paracrine inhibitory function; [2] a localized increasing of permeability for oxygen, nutrients, or growth factors; [3] a localized increasing of leukocyte infiltration; [4] the direct tumor-stromal cell contact, which could promote and facilitate tumor invasion through different mechanisms. The loss of tumor suppressors confers tumor cell growth advantages to escape the programmed cell death (Oliveira et al, 2005; Brummer et al, 2006; Yanochko and Eckhart, 2006). The change of oxygen to the physiologic level and the increase of growth factors could selectively favors the proliferation of progenitor or stem cells (Chakravarthy et al, 2001; Csete et al, 2001; Studer et al, 2001), or activate MAP kinases and protein kinase C that trigger the exit of cells from quiescence (Boulikas, 1994, 1995). The infiltrated leukocytes directly export growth factors to tumor cells (Freeman et al, 1995; Takahashi et al, 1996; Asano-kato, 2005; Nienartowicz et al, 2006; Qu, 2006). Direct tumorstromal cell contact augments the expression of stromal MMP or represses the expression of E-cadherin and other epithelial cell specific markers, which facilitates epithelialmesenchymal transition (Kang, 2004; Sato, 2004; Strizzi, 2004; DeCraene, 2005; Martinez-Estrada, 2006). The combined effects of these factors are likely to be able to significantly change the gene expression pattern, immunohistochemical profile, and the biological behavior in the derivatives of ER NCC, compared to the ER NCC that are in direct continuity with the tumor core. In either case, the unique genetic and immunohistochemical presentation and “budding” capability suggest that these ER NCC might represent tumor progenitor or stem cells that are not mature enough to produce ER and other molecules, or were with “resting” genes for these molecules. These clusters, however, could gain the expression of ER and other molecules after invading into the stroma, probably resulting from interactions with stromal and immunoreactive cells. Thus, it is very likely that ER NCC and their derivatives may have substantially different biochemical properties. Consequently, the derivatives of ER NCC might respond tomaxifen and other therapies, whereas ER NCC might resist the same treatment, representing “seeds” for drug resistant and recurrent tumors.

Acknowledgements This study was supported in part by grants DAMD17-01-1-0129, DAMD17-01-1-0130, PC051308 from Congressionally Directed Medical Research Programs, and 05AA from AFIP/ARP initiative fund to Yan-gao Man et al, MD., PhD.

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Cancer Therapy Vol 4, page 205 Cancer Therapy Vol 4, 205-222, 2006

Treatment of a number of cancer patients suffering from different types of malignancies by methylglyoxal-based formulation: A promising result Research Article

Dipa Talukdara, Subhankar Raya, Sanjoy Dasb, Ashok Kumar Jainb, Arvind Kulkarnic and Manju Raya,* a

Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Calcutta 700 032, India SVS Marwari Hospital, Calcutta 700 009, India c Lokmanya Medical Research Centre, Chinchwad, Pune 411 033 b

__________________________________________________________________________________ *Correspondence: Manju Ray Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Calcutta 700 032, India; Telephone: + 91 33 2473 4691; Fax: + 91 33 2473 2805; E-mail: bcmr@mahendra.iacs.res.in Key words: Methylglyoxal; Ascorbic acid; Cancer patients’ treatment; Chemotherapy Abbreviations: computerized tomography, (CT); Karnofsky performance scale, (KPS); methylglyoxal, (MG); ultrasonograms, (USG) Received: 12 April 2006; Accepted: 02 June 2006; electronically published: June 2006

Summary Previous in vitro and in vivo studies had shown remarkable anticancer effect of methylglyoxal. A recent toxicological study with four different species of animals has shown that methylglyoxal is potentially safe for human consumption (Ghosh et al, 2006). We have developed an anticancer formulation with methylglyoxal as the principal ingredient. To test the efficacy of this formulation, 46 patients suffering from different types of malignancies in different stages of the disease were randomly chosen: brain –2, head and neck –2, gastrointestinal –11, lung –6, gynecological –6, breast –3, urological –4, hematological –2, prostate –2, gall bladder –1, pancreas –2, others –5. The effect of the formulation on overall survival, regression of the tumours and general well being of the patients were analyzed. The follow-up of the patients ranged from 4–56 months. The results of the study show that 18 (39%) patients had complete remission, 18 (39%) patients had partial regression and/or stable disease condition, whereas 8 (17%) patients had progressive disease. In addition to the measurable improvement of the majority of the patients there was remarkable improvement in the quality of life of nearly all the patients. There was no significant adverse side effect in almost all the patients. The significant antitumour effect of methylglyoxal against a wide variety of cancer suggests that all the different types of cancer may have common altered site(s). Our next task will be to further improve this treatment and to evaluate its efficacy with a large number of patients.

Methylglyoxal is a normal metabolite (Ray and Ray, 1981, 1987, 1998; Cooper, 1984; Murata et al, 1986). It has the potential to act specifically against malignant cells (Szent-Györgyi, 1979; Ray and Ray, 1998). Recent research has indicated that methylglyoxal is tumouricidal (Ray et al, 1991; Halder et al, 1993; Biswas et al, 1997). It inhibits both glyceraldelyde-3-phosphate dehydrogenase (Halder et al, 1993; Ray et al, 1997a) and mitochondrial complex I (Ray et al, 1994; Ray et al, 1997b) of specifically malignant cells. Due to the catalytic activities of these two enzymes, nearly 85% of the cellular ATP is generated. The inhibitory effect of methylglyoxal on these

I. Introduction An ideal anticancer drug should either kill cancerous cells or totally arrest their growth and this effect should be specific, so that normal cells are not adversely affected. Then only, an anticancer drug will be truly effective to treat cancer patients without any adverse side effect. At present none of the anticancer drugs that is being used has effect specifically against cancerous cells. For this reason, these drugs are moderate to highly toxic and their efficacy to kill cancerous cells or arrest their growth is often variable.

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Talukdar et al: Treatment of malignancies by methylglyoxal-based formulation enzymes of malignant cells depletes their ATP pool rendering these cells non-viable (Ray et al, 1991; Halder et al, 1993; Biswas et al, 1997). In contrast, methylglyoxal has no inhibitory effect on these enzymes of normal cells (Biswas et al, 1997; Ray et al, 1997a, b). These findings also suggest the possibility of alternation of these two enzymes in malignant cells. The anticancer effect of methylglyoxal had also been tested in vivo (Apple and Greenberg, 1967; Apple and Greenberg, 1968; Együd and Szent-Györgyi, 1968). As early as 1967 it had been observed that methylglyoxal has significant curative effect on animals bearing a wide variety of cancers (Apple and Greenberg, 1967; Apple and Greenberg, 1968). Moreover, when mice, which were inoculated with malignant cells and then treated with methylglyoxal, no tumour developed and the mice remained completely healthy (Szent-Györgyi, 1968). Subsequent in vitro and in vivo studies from different laboratories had corroborated this remarkable anticancer effect of methylglyoxal (Conroy, 1979; Elvin and Slater, 1981). Despite these promising results, which had been documented in the literature, the efficacy of methylglyoxal in treating cancer patients had not been tested till recent past. This apathy probably stems from the widespread belief that methylglyoxal is toxic (Lee et al, 1999; MurataKamiya et al, 2000; Morgan et al, 2002; Bourajjaj et al, 2003; Kumar et al, 2004; Chang et al, 2005). However, we had tested in vivo, whether methylglyoxal has any toxic effect with four different species of animals. This study was primarily intended to evaluate whether methylglyoxal is potentially safe for consumption by cancer patients and also to have an idea of its safety margin. Besides monitoring the general physical and behavioural conditions of the animals, several biochemical, histological and pharmacokinetic parameters were studied. These studies had indicated that this compound is potentially safe for consumption by cancer patients (Ghosh et al, 2006). So, based on our toxicity study on animals, our in vitro studies on human and animal tissue samples as also the anticancer effect of methylglyoxal that had been documented, we had developed an anticancer formulation with methylglyoxal as the principal ingredient. A pilot study to test the efficacy of the formulation with 18 cancer patients had been done and the results were promising. A report of the study had been published (Ray et al, 2001). The present study was undertaken to further evaluate the formulation. So the objectives were to evaluate the following: 1.The formulation if taken orally in daily divided doses can lead to tumor shrinkage and/or no recurrence. 2. Whether the formulation can produce pain relief and improve quality of life in cancer patients. 3. Can the formulation prolong survival of cancer patients? 4. Moreover to assess if the formulation has any toxic effect. The present paper describes our studies of the treatment of forty-six patients who were suffering from different types of cancer. The results corroborating our earlier findings do indicate that the methylglyoxal-based formulation may be a truly effective, non-toxic anticancer drug.

II. Patients, Materials and Methods A. Patients 1. Selection criteria for inclusion 1.Histopathological proof of cancer. 2.Patients should have residual / recurrent tumor or high chance of recurrence of tumor. 3.Performance status in the Karnofsky scale, 30 and above. 4.Both male and female subjects to be enrolled. 5.Any type of cancer. 6.Patient has to agree not to enroll in any other cancer drug trial simultaneously. 7.Patient has to agree not to start simultaneous radiotherapy and/or chemotherapy.

2. Exclusion criteria 1.Female cancer patient who may be pregnant. 2.Patient under concurrent radiotherapy and/or chemotherapy. 3.Patient in terminal cancer stage Between November 2000 and March 2005 a total number of 46 patients were enrolled in the study. There were 20 males and 26 females. There median age was 60 years (range 37 to 83 years). The patients, chosen at random had different types of malignancy confirmed by radiological and histopathological evidences. A few of the patients received no prior treatment; some other were surgically treated and then received chemotherapy and/or radiotherapy; some patients were surgically treated but received no chemotherapy or radiotherapy. However the patients who received chemotherapy were chemo refractory and had developed further metastasis. The patients' performance status in the Karnofsky performance scale (KPS) was in the range of 30-100 (Cassidy et al. 2002a). All patients gave written informed consent to this treatment. Basal level evaluation included a complete history and physical and clinical examinations, histopathology reports, laboratory assessment by biochemical tests and microscopic observations of blood, radiological tests such as CT (computerized tomography) scan, USG (ultrasonography), MRI (magnetic resonance imaging) and also tumor markers. Other diagnostic procedures were according to patients’ symptoms.

B. Composition of the formulation and treatment schedule A stock solution of 0.45 M methylglyoxal is essentially the main component of the formulation to treat cancer patients. Methylglyoxal was obtained from either Sigma Chemical Company, St. Louis, Mo, U.S.A. or Fluka Chemic GmbH, Buchs, Switzerland. Each patient received orally at a time 8 ml of 0.45 M methylglyoxal diluted in 60 ml of water, followed by a tablet of chewable vitamin C containing 400 mg of sodium ascorbate. The patients received this treatment 4 times/day at regular interval. Taking the drug on an empty stomach is not recommended. This is equivalent to the ingestion of 30mg methylglyoxal /kg of body weight/day, considering a person of 60 kg body weight. Each patient also received orally a mixture of the B vitamins twice a day: B1 5mg, B6 2.5 mg, B12 5 µg and B5 7.5 mg. This mixture is usually a standard composition of vitamin B complex available. The duration of treatment at the same or at a reduced dose was determined by evaluating the response and general condition of the patient (see below). The dose of methylglyoxal for the treatment was determined based on the previous in vivo studies describing the treatment of cancer-bearing animals (Apple and Greenberg, 1967; Apple and Greenberg, 1968; Együd and Szent-Gyõrgyi, 1968) and also our previous pilot study on cancer patients (Ray et al, 2001). Institution’s Ethics Committee for the Tests on Human Subjects approved the protocol of treatment and Drugs Controller General of India also issued a clearance.

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Cancer Therapy Vol 4, page 207 56 months). So a statistical analysis of the response to treatment could not be performed. However we had compared the response of our treatment against the usual outcome by the conventional treatment.

C. Treatment evaluation Response was evaluated by different tests performed as well as quality of life, performance status according to Karnofsky scale. A complete response was defined as the complete disappearance of all objective parameters of the disease. A partial response was defined as more than 25% or greater decrease in the tumour mass of measurable lesions initially selected as targets, without progression at any other site with an improved quality of life. A stable disease was defined as less than 25% decrease or less than 25% increase in the tumour mass of all measurable lesions initially selected as targets. Progressive disease corresponded to an increase of 25% or more in all measurable lesions initially selected as targets, or appearance of any new lesion not previously identified. Toxicities were evaluated by different analyses performed as well as by clinical symptoms and patients' subjective feeling. These analyses include routine blood tests, biochemical evaluations of different organs such as liver, kidney, heart as well as ECG, echo ECG etc. The patients included in our study suffered from different types of cancer and were at different stages of the disease. Moreover they received treatment for different time periods (4-

III. Results A. Characteristics of the patients Between November 2000 and March 2005, total 46 patients, who were followed up for at least 10 weeks were enrolled in the study. Follow-up ranged from 4 to 56 months. Patients suffering from different types of cancer are as follows: Brain tumour-2, head and neck-2, gastronitestinal-11, lung-6, gynecological-6, breast-3, urological-4, hematological-2, prostate-2, gall bladder-1, pancreas -2, others-5, total - 46. Maximum performance status in the Karnofsky scale was100 and minimum was 30. The details of the patients' conditions and history of the disease and prior treatment, if any at the time of inclusion of methylglyoxal treatment are presented in Table 1.

Table 1. Patients, characteristics and history of the disease and treatment before the time of inclusion to methylglyoxal (MG) treatment (October 2000 to September 2005) *P N 1.

Age /Sex 61 M

Type of cancer (Diagnosis) Ca ethmoid Neuroendocrine Ca of ethmoid sinus, metastasis, recurrence

Other diseases

53 F

Ca breast Ca breast with bone metastasis.

Diabetic

October 1999

64 F

Diabetic, pneumobilia, Renal calculus (left), hypertensive.

December 2000

Operation on January 2001. Patient refused to take chemotherapy or radiotherapy

59 M

Ca gall bladder Moderately differentiated infiltrating papillary adenocarcinoma of gall bladder with metastasis to the lymph node Ca kidney Bilateral renal cell carcinoma,

February 2001

Renal angiography of right kidney March 2001(mass 5.6x5.2cm). radical nephrectomy of L-kidney- April 2001 (mass 7.9x7.8cm) Patient refused to take chemotherapy or radiotherapy (risk in radiotherapy)

Aortic calcification

Time of detection September 1997

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Previous Treatments Surgically treated. (Recurrence March 1999). Radiotherapy which did not help much Operation on December 1999 3 cycles of chemotherapy and 12 cycles of radiotherapy

Disease status and QOL at start Neuroendocrine Ca of ethmoid sinus (surgically treated) with calvarial metastasis. Nasal bleeding, difficult to open the eye of the affected side Ca breast (operated), Bone metastasis in the manubrium sternum and L4 and L5 vertebral segments with severe pain in whole body, pallor, difficult to walk and move Moderately differentiated infiltrating papillary adenocarcinoma of Gall Bladder (operated) with metastasis to the lymph node, too weak to walk, pallor, no appetite.

Started MG October 2000

Operated L-kidney, Huge mass in right kidney (5.6X 5.2 cm), high serum lactic dehydrogenase (693units/l) Severe weight loss and weakness.

May 2001

November 2000

February 2001

Talukdar et al: Treatment of malignancies by methylglyoxal-based formulation 5.

66 M

Ca stomach Well differentiated adenocarcinoma of stomach infiltrating in the serosal coats, multiple lymph nodes involved

March 2001

61 F

May 2001

74 M

Ca stomach Moderately differentiated infiltrating signet cell carcinoma (adenocarcinoma) of stomach, with metastasis in peri gastric and coliac lymph nodes Ca skin Primary well differentiated squamous cell carcinoma of skin (nodular growth in left leg), Secondary (adenocarcinoma) in pancreas and in liver.

Diabetic and Cardiac patient, simple renal cysts- bilateral kidneys, degenerative change in dorso-lumber vertebrae

November 2000

54 M

Multiple myeloma, with bone metastasis

Chronic Bronchitis

January 2001

83 F

Ca urinary bladder, multiple papillary transition cell carcinoma-grade II

October 2000

10.

56 F

Ca tongue Squamous cell carcinoma at base of the tongue.

August 2001

Partial gastrectomy and feeding jejunoscopy on April 2001 Patient refused to take chemotherapy or radiotherapy. Partial gastrectomy May 2001 Patient refused to take chemotherapy or radiotherapy

Well-differentiated adenocarcinoma of stomach (partial gastrectomy), involvement of multiple lymph nodes, occult blood in stool.

May 2001

Moderately differentiated infiltrating adenocarcinoma of stomach (partial gastrectomy) with metastasis in peri gastric and coliac lymph nodes

June 2001

Excision of lesion of left legNovember 2000 Radiotherapy 30 cyclesâ&#x20AC;&#x201C; (December 2000 to January. 2001) After that secondary (adenocarcinoma) in pancreas, in liver & metastasis in abdominal nodule (retro peritoneal mass). Patient refused to take chemotherapy 6 cycles of chemotherapy then patient refused to take further chemotherapy for severe side effect without benefit

Primary well differentiated squamous cell carcinoma of skin (excision of nodular growth of left leg), Secondary (adenocarcinoma) in pancreas,& in liver, raised LFT and CPK value (basal).

August 2001

Multiple myeloma, bone metastasis with severe pain in head and whole body, pallor, difficult to walk and move, no appetite (Hb 5.5g/dl, platelet 80,000, WBC 6000), at start two unit of blood transfusion. Heamaturia (sometimes clotted blood), severe pain, heaviness in lower abdomen

September 2001

A mass lesion at base of tongue extending to the vallecula and right pyriform sinus. Very poor quality of life, could not walk, eat, severe pain, very weak, voice chocked, feeding gastrostomy suggested.

November 2001

TURBT in December 2000. Patient was asymptomatic for 3 months 4 cycles of chemotherapy and 23 cycles of radiotherapy without any benefit, progressive disease, .

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July 2001

Cancer Therapy Vol 4, page 209 11.

51 F

Acute myeloid leukemia

Piles, right ovarian cyst

January 2002

Oral chemotherapy 2 cycles, then patient refused further chemotherapy due to toxicity.

12.

55 F

Ca ovary Moderately differentiated serous papillary adenocarcinoma of ovary with metastasis in omentum

Diabetic, gall bladder stone, pleurapulmonary inflammatory changes with effusion, mild hydronephrotic changes in both kidneys

December 2000

13.

70 F

Ca breast,

Diabetic and Cardiac patient, degenerative changes in knee, ankles, hands, feet & dorsal vertebrae.

August 1999

14.

60 M

Ca kidney

Prostatomegaly Grade II, prostatic urethral collapse

April 2002

15.

46 F

16.

75 M

Ca ovary Adenocarcinoma of ovary, endometriosis in other ovary, uterus leiomyoma, adenomyosis uteri Ca lung Squamous cell carcinoma of lung, rib metastasis, in-operable.

Laparotomy of tumours Dec. 2000, 6 cycles chemotherapy May 2001, recurrence in right adnexa with loculated collection in the pelvis April 2002, and patient refused to take further chemotherapy. Operated September 2000, radiotherapy completed November 2001, then on herbal treatment, multiple skeletal metastasis – bone scan on May 2002 - collapsed L1 vertebra Stone removal from left kidney on March 2002, nephrectomy on right kidney on April 2002, patient refused to take chemotherapy or radiotherapy. Hysterectomy April 2002 Refused chemotherapy

March 2002

Pulmonary Koch’s treated for 6 months

May 2002

209

Hb 9.1 gm/dl, Total WBC 35,000, 84% blast cells , 5% neutrophil, Nomoblast C occasional, severe weakness, too weak to walk, no appetite, sleeplessness, feels heart palpitation, cardiomegaly, ECG within normal, admitted on trial after 2 units of blood transfusion Ca Ovary (laparotomy), Recurrence in right adnexa with loculated collection in the pelvis April 2002, Bleeding / discharge per vagina granulomatous growth vaults – bleeds on touch, ascites, pleural effusion, high CA-125 value.

April 2002

Ca Breast (operated) with multiple skeletal metastasis with collapsed L1 vertebra, severe pain , breathing problem, sleeplessness, no appetite.

June 2002

Ca kidney (nephrectomy of right kidney with prostatomegaly Grade II)

June 2002

Ca Ovary, anorexia, weight loss, Ca 125- normal range

June 2002

At start irregular mass lesion in right upper lobe, ribs erosion, severe excruciating pain, severe weakness, unable to walk, increased urea, breathing problem, loss of appetite. Patient had feature of prostatism.

September 2002

May 2002

Talukdar et al: Treatment of malignancies by methylglyoxal-based formulation 17.

75 F

Ca oesophagus Invasive moderately differentiated squamous cell carcinoma of esophagus, in-operable

Cortical cyst in right kidney

May 2000

18.

50 F

Ca buccal mucosa well differentiated infiltrating Squamous cell carcinoma of buccal mucosa (right)

19.

70 F

Cardiac patient, high cholesterol

August 2001

20.

37 M

Ca ovary Endometroid adenocarcinoma of ovary Ca lung Extensive adenocarcinoma of right lung, bilateral cerebral and cerebellar metastasis, also some polyps in both maxillary antrum

Diabetic, Multiple cystic lesions in brain

October 2002

No chemotherapy or radiotherapy due to poor general condition of patient

21.

46 M

Unknown Metastatic deposit of adenocarcinoma in right lung with pleural effusion, lymph node involvement, primary unknown

Diabetic, hypertensive, fundus gastritis

April 2001

22.

51 F

Ca breast Adenocarcinoma of left breast

23.

75 M

Ca colon Adenocarcinoma of colon infiltrating through musculature into the outer serosa, lymph nodes with reactive changes, ascites, bilateral pleural effusion.

Chemotherapy 8 cycles, radiotherapy full course December 2002 â&#x20AC;&#x201C; whole body bone scintigraphy shows disease progression Modified radical mastectomy done followed by local radiotherapy and chemotherapy (6 cycles) then recurrence Exploratory laparotomy done. Then hemicolectomy and ileotranverse end to end anastomists with 4 units of blood transfusion, no chemotherapy or radiotherapy

March 2002

April 1999

Prostatomegaly, lumber spondylosis

March 2003

210

Chemotherapy and radiotherapy completed. Recurrence in August 2002 with lung and bronchial metastasis Buccal mucosal growth and retro molar growthExcised April 2002, followed by radiotherapy 30 cycles, again local recurrence with nodal metastasis, modified radical neck dissection and wide excision and skin grafting done. Refused to take chemotherapy Operated, chemotherapy taken

Irregular narrowing of esophagus with distortion of mucosa, dysphagia, pain, weight loss, loss of appetite, increased alkaline phosphatase value

September 2002

Well differentiated infiltrating squamous cell carcinoma with local recurrence and nodal metastasis. Alkaline phosphatase level high.

October 2002

Metastasis in left inguinal lymph node, weakness, sleeplessness, loss of appetite Extensive adenocarcinoma of right lung, bilateral cerebral and cerebellar metastasis, also some polyps in both maxillary antrum, blurred vision, imbalance, ataxic gait, vertigo, severe neck pain, cough, hemoptysis, fatigue liver, pleural effusion, high SGOT and SGPT value Multiple bone and brain metastasis, raised alkaline phosphatase and cholesterol, pallor, breathing problem, malignant pleural effusion

October 2002

Developed multiple bone metastasis- November 2002, severe pain in whole body, weakness

April 2003

Adenocarcinoma of colon, laparotomy done, infiltrating through musculature into the outer serosa, lymph nodes with reactive changes , Very poor general condition , high grade fever, hospitalized for 4 days, patient was stabilized with supportive treatment

April 2003

November 2002

January 2003

Cancer Therapy Vol 4, page 211 24.

73 M

Leiomyosarcoma Hemangiopericytoma, lung metastasis, intraperitoneal (Leiomyosarcoma)

25.

68 F

Ca brain Bilateral corpus callosal glioma (SOL) in frontal region, inoperable

26.

70 M

Ca kidney Transition cell carcinoma, grade IIIII of renal pelvis with invasion to adjacent renal parenchyma, metastasis in lymph node and liver

Bilateral pneumonitis, diabetic, hypertrophy of prostrate, bronchitis

May 1997

Exploratory laparotomy followed by chemotherapy in May1997, huge intraperitoneal tumour from right omentum inherent to sigmoid colon and bladder peritoneal fold, recurrence 2002, again chemotherapy, March 2003recurrent lesion, (mass in right pelvis, close contact with sigmoid colon and urinary bladder) and lung metastasis (two nodular lesion in left lower lobe of lung)

May 2003

Prostate enlarged

May 2002

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Nephrectomy (right) May 2002, second operation September 2002metastatic lymph node over right Iliac vessels. Then 4 cycles chemotherapy (November 2002February 2003), severe bone marrow depression, heavy bleeding from nose, occasional black out, 4 units of blood transfusion, platelet 21,500, stopped further chemotherapy, after 4 monthsenlarged lymph nodes in both upper and lower part of neck

Recurrent hemangiopericytoma (in abdomen) metastasis in right pelvis and metastatic infection etiology (two well defined nodular lesions in left lower lobe) lung metastasis.

April 2003

Bilateral corpus callosal glioma (SOL) in frontal region. Behavioral changes, incontinence of sphincters occasionally observed for last 45 days, slowing of activity Ca kidney with metastasis in lymph node over right Iliac vessels. Severe bone marrow depression and enlarged lymph nodes in both upper & lower part of neck

June 2003

Talukdar et al: Treatment of malignancies by methylglyoxal-based formulation 27.

45 F

Leiomyosarcoma Leiomyosarcoma of uterus with liver (right lobe) metastasis

Chemotherapy done but no improvement

28.

52 F

Ca Ovary Lung metastasis with effusion and ascites,

April 2003

Laparotomy on April 2003, May 2003Chemotherapy, patient gradually deteriorated

66 M

Ca lung Moderately differentiated adenocarcinoma in left lung, solid lump (7.6 x 7.2 cm)

February 2003

48 F

Ca breast Infiltrating ductal carcinoma (grade II) metastasis in axillary lymphnodes.

November 2002

70 F

Ca stomach Moderately differentiated adenocarcinoma of stomach

March 2003 to May 2003 – 3 cycles of chemotherapy, 6 units of blood transfusion, lump increased from 7.6 X 7.2 cm to 9 x 9.2 x 6.7 cm, June 2003 to July 2003, radiotherapy (30 cycles) November 2002 modified radical mastectomy, December 2002 to May 2003 – 8 cycles chemotherapy, May 2003 to August 2003 radiotherapy completed November 2003 partial gastrectomy with oesophageal gastrostomy with feeding jejunostomy, developed luminal narrowing, difficulty in taking food, repeated oesophageal dilation done, no specific benefit.

Cortical cyst in left kidney

October 2003

212

Leiomyosarcoma of uterus with liver (right lobe) metastasis, two large and one small masses in right lobe of liver, high level SGOT and alkaline phosphatase CA-125 raised Ca Ovary with lung metastasis. Effusion and ascites, poor general condition, severe headache, pallor, left eyelid-dropping, no appetite, no movement, bowels after 7 days, high CA-125 value (357U/ml) Ca lung with pleural effusion, very weak, pallor, bowels not clear, hard breathing, occasional gastritis

June 2003

Ca breast (operated), Infiltrating ductal carcinoma (grade II) metastasis in axillary lymph nodes, estrogen receptor–weakly positive, progesterone receptor – positive

Nov ember 2003

Moderately differentiated adenocarcinoma of stomach (gastrectomy), oesophageal gastrostomy with feeding jejunostomy, repeated oesophageal dilation, difficulty in taking food

December 2003

July 2003

August 2003

Cancer Therapy Vol 4, page 213 32

75 M

Ca prostate Bone metastasis

Patient with cerebral attack and hyperinflatted both lungs.

July 2003

Second prostate operation in July 2003 followed by an orchidectomy in August 2003, haematuria started December 2003, and operation could not be conducted due to blood pressure fluctuation, bone metastasis. 1 cycle of chemotherapy in January 2004, severe health deterioration, stopped chemotherapy Patient strongly against surgery and chemotherapy.

Ca prostate (operated) with bone metastasis, very weak, haematuria, bed ridden, no appetite, irregularities in stool and urine, pain in whole body

February 2004

70 F

Dilated oesophagus – achalasia, cyst in left lower lobe of apical segment

February 2004

55 F

Diabetic, cardiac and hyper-tensive patient

54 F

Ca oesophagus Infiltrating adenocarcinoma in oesophagus with occasional focus of squamous metaplasia, lung metastasis (?) Ca stomach Diffused infiltrating grade III adenocarcinoma of stomach with metastasis to lesser curvature lymph node, small fluid in pouch of Douglas Ca Uterus Adenocarcinoma, endometrial involvement of myometrium

Difficulty in taking food, low appetite, weakness, nausea.

February 2004

Partial gastrectomy on February 2004

Ca stomach, (partial gastrectomy) with metastasis to lesser curvature lymph node, pallor, enlarge cardiac size, high glucose, urea and creatinine level in blood

February 2004

December 2003

Hysterectomy, of uterus in December 2003, then 50 cycles of radiotherapy

April 2004

Diabetic, tiny calculi in gall bladder, a simple cortical cyst in left kidney

April 2004

Laparotomy for obstruction. right hemicolectomy, April 2004

A large calculus in lumen of gall bladder

May 2004

Diabetic patient with bi-pedal oedema

April 2004

Hysterectomy of uterus on May 2003, leiomyoma of uterus. May 2004 -huge ascites with endomatous transverse mesocolon and mesentery – percicerlasis done Lower radical gastrectomy

Ca uterus (operated) followed by 50 cycles of radiotherapy. Hypo echoic small area on right side of bladder (enlarged lymphnode?) Ca in pancreas (hemicolectomy) with metastasis to liver and lymph nodes, high SGPT, SGOT and alkaline phosphatase levels in blood, marker high (HIAA) – 14mg/24 hrs Adenocarcinoma in ascites fluid with liver metastasis, unknown primary. Very high CA-125 value (600.00 U/ml), ESR – high, SGOT – marginally raised.

47 M

63 F

Ca pancreas Carcinoid carcinoma in pancreas with metastasis to liver and lymph nodes, obstruct at ileocaecal junction by concentric mass. Primary in colon Unknown Adenocarcinoma in ascites fluid with liver metastasis, origin unknown

66 F

Ca stomach Adenocarcinoma of stomach with metastasis in perigastric and omental lymph nodes

Adenocarcinoma of stomach (operated) with metastasis in perigastric and omental lymph nodes, weakness by hyper acidity

May 2004

213

April 2004

May 2004

Talukdar et al: Treatment of malignancies by methylglyoxal-based formulation 39

52 F

45 M

57 M

50 M

43 M

51 F

Ca oesophagus Infiltrating squamous cell carcinoma in oesophagus, infrahilar para oesophageal nodes, lymph nodes of celiac group metastatic deposit of squamous cell carcinoma Ca pancreas Infiltrating malignant glands in the stroma indicating adenocarcinoma (grade III) in pancreas as likely primary Ca prostate Adenocarcinoma â&#x20AC;&#x201C; Grade III prostate

Ca colon Well differentiated adenocarcinoma of rectosigmoid colon with serosal and mesenteric and lymph node involvement with metastasis in liver Ca brain Glioblastoma multiforma (astrocytoma, Grade IV)

Ca stomach Poorly differentiated (Grade IV) adenocarcinoma of stomach with metastasis in perigastric lymph nodes (inoperable), metastatic lesion in left lobe of liver , extensive locally advanced growth in distal stomach with pancreatic involvement. Pancreatic/ sub pyloric lymphadenopathy, locally unresectable

Calculi in gallbladder

July 2004

Lower end esophagectomy and partial gastrectomy, 3 units of blood transfusion

Infiltrating squamous cell carcinoma in esophagus (esophagectomy and partial gastrectomy)

July 2004

Diabetic, pancreatic calcifica-tion, pneu-mobilia

January 2004

Pancreatic gastrostomy on January 2004 then 6 cycles of chemotherapy up to June 2004

Ca head of pancreas, (gastrostomy), high SGPT, SGOT, alkaline phosphatase values, severe pain

Aug ust 2004

Diabetic (insulin- dependent), carbuncle operation on September 2004 followed by cerebral attack Hernia with anemia

August 2003

Bilateral Orchidectomy on November 2003,

Adenocarcinoma of prostrate (bilateral orchidectomy), weight loss, weakness, difficulty in urination with burning sensation, PSA (34.3 ng/ml) increased.

September 2004

December 2004

Laparotomy on November 2004

Adenocarcinoma of rectsigmoid colon (laparotomy) with serosal and mesenteric involvement, lymph node involvement, with metastasis in liver, rise in SGPT, SGOT and alkaline phosphatase values

December 2004

Diabetic, hypertensive

November 2004

Glioblastoma multiforma (astrocytoma, Grade IV) (operated) and completed radiotherapy, rise in SGPT, SGOT and alkaline phosphatase values

February 2005

Calcification in right lobe of liver, dilated left renal pelvis

January 2005

November 2004left temporal craniotomy and decompression of tumor. February 2005radiotherapy completed (34 cycles) January 2005exploratory laparotomy for locally advanced Ca-distal stomach, anterior gastrojejunostomy done with feeding jejunostomy

Ca- stomach (laparotomy) with metastasis in perigastric lymph nodes, liver and pancreas. Raised SGOT and alkaline phosphatase values

February 2005

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Cancer Therapy Vol 4, page 215 45

57 F

Ca colon Moderately differentiated adenocarcinoma of recto sigmoid colon with serosal invasion and nodal metastasis

Renal cyst in left upper pole, psychiatric patient

April 2003

73 M

Ca lung Small cell adenocarcinoma of left lung

Hiatus Hernia, Erosive fundal gastritis, bilateral renal cyst and right sided Para pelvic cyst, wall calcified in both carotid arteries, calcified nodules in left parietal and frontal regions

September 2003

May 2003- high anterior resection May 2003 to October 2003- 6 cycles of chemotherapy. December 2003enlarged left pelvic lymph nodes and small para aortic lymph nodes noted. December 2004persistent enlarged lymphadenopathy, bilateral nodular lesion in lung, January 2005oral chemotherapy, after 2 cycles developed hand foot syndrome and severe toxicity, stopped further chemotherapy October 2003- 3 cycles of chemotherapy December 2003complete pneumonectomy of left lung, post operative 3 cycles of chemotherapy, June 2004- left sided pleural effusion January 2005recurrence in bronchogenic mass, metastasis in right lung again started chemotherapy but without benefit, patient refused further chemotherapy

Ca- recto sigmoid colon (resection) with serosal invasion and nodal metastasis in para aortic lymph nodes and bilateral lungs (both lungs fields) patient developed severe toxicity for chemotherapy and hand foot syndrome, weakness, breathing problem, pain, high CEA and alkaline phosphatase values

Febuary 2005

Ca- left lung (complete pneumonectomy) metastasis in right lung with pleural effusion, breathing problem, bronchogenic mass

March 2005

PN, patient number; QOL, quality of life

as CT scan, USG, routine blood tests, biochemical evaluations of different organs of all the 46 patients, Tables 1 and 2 qualitatively summarize the patients, conditions at the time of inclusion to the treatment and response to the treatment respectively. We have all the relevant data in our records. From Table 2 it appears that of all the different types of malignancies that have been tested our formulation appears to be most effective in the case of gastrointestinal cancers. Moreover, for carcinoma of gall bladder there is at present no chemotherapy available ( Cassidy et al,

B. Response to treatment Table 2 shows the outcome of treatment by methylglyoxal to the patients. Of the 46 patients, 7 died due to progressive malignant disease, whereas 3 patients died due to causes other than malignancy. Some patients who have at present no sign of the disease however continuing the treatment but at a reduced dose. Most of these patients strongly desire to continue the treatment for fear of recurrence. The KPS of the patients improved. Because it is very difficult to present quantitative results of the different tests that had been performed such

215

Talukdar et al: Treatment of malignancies by methylglyoxal-based formulation 2002b). The recurrence after surgery is also high. The one case of carcinoma of gall bladder (patient no3) that we have treated is free of recurrences for the last fifty six months after surgery. In addition to the measurable improvement as observed such as through computerized tomography (CT) scan, ultrasonograms (USG), blood test, the quality of life dramatically improved almost from starting the methylglyoxal treatment. The most remarkable improvement in this regard is the relief from pain. There were no mentionable side effects in almost all the patients. The only observable side effect was acidity in the stomach of some of the patients. This is probably due to the fact that both methylglyoxal and ascorbic acid, the main ingredients of the formulation are acidic in nature. However this mild adverse effect could be managed by standard antacid formulations available in the market.

There are many reports in the literature, which suggest that methylglyoxal may be responsible for many adverse effects related to diabetes. However our study shows that methylglyoxal has no adverse effect on the blood glucose level of all patients some of them are diabetic also. In addition to Tables 1 and 2, which summarize the patients' conditions before and after the treatment we present a very brief summary of the outcome of the treatment in Table 3. Seventy eight percent of the patients are greatly benefited. Especially effective in cancer of gastrointestinal tract (colon, stomach, oesophagus), ovary, kidney, gall bladder and tongue. Quality of life improved and life span increased for most of the patients with partial regression and/or stable disease condition. Even for some patients who had progressive disease, their quality of life improved.

Table 2. Outcome on quality of life and disease status of the patients after methylglyoxal (MG) treatment PN

Date of Last follow up / Medication

Outcome of treatment on quality of life and disease status

Duration of MG treatment (months) 38

December 2003, then lost contact

August 2003

Continuing with maintenance dose

March 2001 and July 2002 biopsy reports of nasal exudates showed inflamed Good regression, mucosa with inflammatory exudates and necrotic tissues, no viable tumour. normal life till June 2002 CT scans of brain and para nasal sinus â&#x20AC;&#x201C; regression, no other last medication. abnormality. September 2003 CT guided biopsy of vertebra - no tumour seen. *ND, LN September 2003- radiotherapy due to back pain on spine. July 2001- CT scan of chest showed no metastasis. October 2001 USG of Excellent whole abdomen showed no metastasis. Bone scan January 2002 â&#x20AC;&#x201C; remission, normal Improvement of the lesions at the manubrium sterna and lower lumber life till last vertebrae. Radionuclide whole body bone scan December 2003 - No bony medication. involvement /metastasis. ND, LN August 2001- Echocardiography report normal, October 2001- CT scan of Excellent whole abdomen normal, CEA- normal. August 2002, September 2003 and remission, normal July 2004- USG reports and October 2002- CT scan report showed no life till date. evidence of tumour. ND, LN July 2004 -teleradiogram of chest normal. July 2004- blood report normal except plasma glucose value.

Continuing with maintenance dose

August 2003 then lost contact

Continuing with maintenance dose

CT scan and USG reports showed mass in right kidney slightly decreased after 3 months, then remained static about 7 months, again started to decrease, after 5 months it again remained static for about 29 months. During that time it changed from solid mass to hypo echoic sol. December 2004- no detectable mass in right kidney and no recurrence in left kidney. June 2002 and November 2002 USG showed normal, then patient complained of abdominal pain., August 2003 -biopsy report of designated gastroscopic mucosal bits from gastrojejunal stomal ulcer showed no malignancy , benign stomal ulcer with chronic gastritis. CT scan and USG of whole abdomen showed no recurrence, no further metastasis , no ascites up to 40 months. Then right sided pleural effusion and bilateral cystic ovary adherent to uterus was developed. October 2004operation, histology report showed metastatic adenocarcinoma deposit in both ovaries (Krukenberg tumour) with spread to the body of the uterus and wall of the cervix but peritoneal fluid had no malignancy. March 2005 -USG showed minimal pleural effusion, minimal ascites, cystilis bilateral mild hydronephrosis, CA-125 report - normal . Reports September 2005 (Biochemical / Marker / Radiological) showed no abnormality.

216

Status at last follow-up and comments

Excellent remission, normal life till date. ND, LN Excellent remission, normal life till last date of medication. ND, LN Partial response, normal life without any major complaint. RD, LN

Cancer Therapy Vol 4, page 217 7.

September 2003 then lost contact

February 2002-CT scan showed reduction in retro peritoneal mass and static in liver lesion. May 2002, August 2002, April 2003 -blood report within normal. April 2003- CT scan data showed static in retroperitoneal mass but reduction in liver lesion

April 2003 then opted for surgery and chemotherapy

Hemoglobin percentage and platelet count gradually increased from 8.6 to 9.9 gm/dl from 80,000 to 2,60,000 respectively within 5 months and remained almost constant till last date of medication. On April 2003 MRI of lumbosacral spine showed osteoporosis, L4-L5 and L3-L4 degenerative disc disease with bulging annulus causing neuroforaminal narrowing and L4-L5 central canal stenosis and focal lesion of myeloma in right half of L4 body and in right iliac bone

Continuing with maintenance dose

10.

January 2003 then lost contact

11.

April 2003

After 3 months USG report showed in urinary bladder wall thickened and adherent soft tissue mass. Then after 12 months 2 sessile mass (25 x 24 mmleft side, 14 x 12 mm- right side). After 17 months normal wall thickness and one nodulated mass over right lower border of urinary bladder wall. Recent radiological data shows normal wall thickness and no mass in urinary bladder, no lymphadenopathy, vesical sol (17 X 17mm) in left posterolateral wall Symptomatic improvement, good quality of life, pain reduced, can walk, good appetite, no burning feeling within one month and gradually improved and maintained till last date of medication. After 5 months CT scan of the neck showed mild focal thickening at the base of the tongue with involvement of epiglottis and periepiglottic region, no cervical lymphadenopathy. After 13 months thickness reduced, no lymphadenopathy (checked by ENT specialist) Symptomatic improvement, could walk and sleep, gained appetite. Fluctuation of hemoglobin percentage (13.6-7.2gm/dl) required blood transfusion during treatment. USG report: January 2003 normal.

12.

December 2002

13.

March 2004

Period

Hb (gm/d)

Total Count(%)

Blood report: Neutrophil Lymphphocyte (%) (%)

May-02 Jun-02 Jul-02 Aug-02 Sep-02 Oct-02 Jan-03 Apr-03

11.9 9.6

3700 6900 34100 75000 110000 8400 5500 4900

29 18 4 4 15 67 24 30

7.8 7.6 7.2 8.2 10.1 9.6

63 76 6 9 11 30 46 52

Blast cell (%) not done not done 8 8 70 2 20 16

Expired June 2003 probably due to septicaemia Symptomatic improvement, bleeding stopped, gained appetite. After 3 months oesophago-gastro-duodenoscopy reports -hiatus hernia, peritoneal fluid contained RBC. After 6 months USG report showed ascites (slight), leftsided pleural effusion, bilateral dilated renal pelvis, heterogeneous sol in uterine fossa (secondary?). Expired, February 2003 Symptomatic improvement, pain reduced, could walk, good appetite normal sleep within a short period. December 2003 radiological data of dorso-lumbar spine showed - scoliosis with diffuse ostopenia, multiple partial compression collapse, spondylotic changes with marginal osteophytes. March 2004 CT scan of brain-normal. Blood â&#x20AC;&#x201C;normal Expired, March 2004 due to heart attack.

217

Good regre-ssion, stable disease, normal life till last date of medication. RD, LN Excellent remission, normal life upto March 2003 without any complaint. Initial good response. On April 2003 patient complained back pain. MRI done, patient opted for chemotherapy SD Good regression, normal life till date. RD, LN

Good response, normal life till last date of medication. RD, LN Progressive disease but hematologically improved, partial regression, good quality of life, occasional anemia. PD, DC

Progressive disease, symptomatic improvement, ascites reduced. PD, DC Excellent response, normal life till last date of medication. ND, DN

Talukdar et al: Treatment of malignancies by methylglyoxal-based formulation 14.

May 2003 then lost contact

After 6 months USG report showed normal left kidney, clear renal fossa on right kidney, prostatomegaly (grade II). After 13 and 18 months, both USG showed similar results with previous. Teleradiogram chest –normal throughout the medication. No recurrence or metastasis.

Excellent response, normal life till last date of medication. ND, LN

15.

June 2004

Recent CT scan and USG of whole abdomen, CA 125, liver function test reports – normal. No recurrence, no metastasis.

January 2003

Symptomatic improvement. Pain, weakness and breathing problem reduced, gained appetite within a short period. Expired February 2003

April 2003 opted again for chemotherapy

Symptomatic improvement. Pain reduced, gained appetite within a short period. After 5 months CT scan showed oesophageal wall thickening for a length of 6 cm.

May 2004 then lost contact.

April 2004 – all radiological (USG, X ray) and all biochemical reports were within normal, No recurrence or metastasis.

January 2004

June 2003

Symptomatic improvement, no weakness, gained appetite, normal sleep within a short period. After 3 months blood and CA125 reports - normal. After 10 months USG - lymph nodes slightly decreased (from 3.6 X 2.5 cm to 3.3 X 2.3 cm). Expired February2004 due to heart attack. Symptomatic improvement, pain reduced, normal gait, no hemoptysis, normal vision within short period. After 4 months- clinically status quo, blood reportwithin normal except high SGPT, SGOT and plasma glucose. After 4 months X-ray chest, after 6 months USG - whole abdomen showed- right sided pleural effusion. After 5 months brain scan - lesion & nodes slightly decreased in size. Expired July 2003.

September 2004

Excellent remission, normal life till date ND, LN Progressive disease, symptomatic improvement PD, DC Progressive disease, symptomatic improvement PD Excellent remission, normal life till last date of medication ND, LN Stable disease, normal life till last date of medication ND, DN Stable disease, symptomatic improvement and clinically status quo for 4 months. Reduction of brain lesion. SD, DC Partial remission, normal life till last date of medication RD, LN

May 2004 opted again for chemotherapy

Continuing

Symptomatic improvement , normal life within a short period. . After 9 and 17 months- CEA and blood reports - within normal. After 17 months - USG report – normal, no recurrence.

April 2004

After 3 months CT scan of whole abdomen and chest showed status quo except slight increase in size of left lower lobe pulmonary nodule. After 9 months CT scan of whole abdomen showed minimal increase in size of lesion.

Symptomatic improvement. Non-evaluable.

November 2003 then lost contact October 2003

Symptomatic improvement within short period. April 2003- MRI showed large cystic lesion with haemorrhage in the right parietooccipital region and cystic lesion with separations in left parietooccipital region. Also focal enhancing lesion involving dura of left parietooccipital region. After 9 months MRI of brain on September 2003 showed marked regression of parenchymal and dural lesion as compared to MRI of April 2003. Symptomatic improvement, pain reduced, no weakness within a short period. Bone scan report showed improvement.

After 3 months USG showed hepatic secondary while no other appreciable change, increase in the size of nodes in neck. Expired November 2003

218

Partial regression, normal life till last date of medication PD Excellent remission, normal life till date ND, LN Stable disease, normal life till last date of medication SD, LN Quality of life improved.

Progressive disease. No improvement PD, DC

Cancer Therapy Vol 4, page 219 27

December 2003 opted again for chemotherapy March 2004

After 3 months - decrease in SGOT, alkaline phosphatase values, USG - multiple large hypoechoic lesion and few small hypoechoic lesions in both lobes, others normal.

April 2004 opted again for chemotherapy

August 2004

No recurrence, good quality of life.

Continuing with maintenance dose

No difficulty in taking food. After 6 months USG whole abdomen, after 10 months barium meal X-ray follow up, after 13 months USG whole abdomen showed- normal. Recent barium meal X-Ray report and blood report shows normal. No evidence of recurrence or metastasis or ascites.

December 2004

Symptomatic and clinical improvement and this improvement maintained for 10 months. Almost no pain, stopped haematuria, could walk, gained appetite , PSA reduced. Biochemical report – normal, radiological report – nonevaluable Expired January 2005 due to old- age cardiac problem.

Symptomatic and clinical improvement till last date of medication. RD, DN

Symptomatically improved, gained appetite. After 4 months CT scan showed identical with initial results (February 2004). Maintained status quo.

Stable disease, symptomatic improvement SD

May 2004 opted for operation followed by chemotherapy and lost contact May 2004

Continuing

Symptomatic improvement. Expired June 2004 due to heart failure during endoscopy. Recent CT scan of lower abdomen and biochemical reports - normal , no recurrence, no metastasis.

Continuing

After 14 months chest X Ray - normal. Recent CT scan of whole abdomen – no abnormalities, biochemical report – normal, Markers: Chromogranin A, Elisa – normal, 24 hrs. urinary 5-hydroxy, 3-indole acetic acid (5HIAA) excretion - normal

Symptomatic improvement Excellent remission, normal life till date ND, LN Excellent remission, normal life till date RD, LN

Symptomatic improvement. Reduction of CA 125 value (from 600 to 6.9U/ml) within 1 month, required occasional ascites removal, others remained status quo. After 4 months CA 125 began to rise.

September 2004 opted for chemotherapy Continuing

Continuing

Recent USG of whole abdomen - normal, blood report - normal, no problem in taking food, no recurrence

November 2004

Symptomatic improvement, pain reduced,. Expired December 2004

Symptomatic improvement, no weakness, gained appetite, normal sleep, normal vision within a short period. Reduction in CA 125 value. Radiological data showed reduction of pleural effusion and ascites. Expired May 2004. Symptomatic improvement, gained appetite, reduced breathing problem within a short period. Significant reduction of lesion in left lower lobe. Improvement - solid mass changed to soft tissue mass. After 8 months patient was suffering from fever, cough and weakness due to tuberculosis infection (Koch’s infection) in pleural fluid. Patient improved gradually after treatment of tuberculosis.

Recent radiological report showed no recurrence, no metastasis, biochemical report – normal

219

Stable disease. Gradually improved SD Partial regression. SD, DC Good response. Clinically , radiologically and symptomatically improved till last date of medication SD Excellent remission, normal life till last date of medication ND, LN Excellent remission, normal life till date. ND, LN

Progressive disease, quality of life improved. PD Excellent remission, normal life till date ND, LN Excellent remission, normal life till date ND, LN Progressive disease, symptomatic improvement PD, DC

Talukdar et al: Treatment of malignancies by methylglyoxal-based formulation 41

Continuing

Within 3 months symptomatic improvement in urinary obstruction, feeling almost normal. PSA reduced.

Continuing

Symptomatic improvement. Within 2 months SGPT, SGOT and alkaline phosphatase values - within normal. Recent CT scan of whole abdomen normal, endoscopy study - moderate degree of antral gastritis, blood reports within normal, no recurrence, no other abnormalities.

Continuing

September 2005 CT scan of brain suggested no abnormal enhancement of residual/ recurrent mass, others normal. Blood report - SGPT, alkaline phosphatase, SGOT- normal.

Continuing

Symptomatic improvement. Biochemical and clinical reports – normal, radiologically not evaluated.

Continuing

Symptomatic improvement. Weakness reduced, no pain, breathing problem reduced, normal skin in palm and foot within a short period. Recent X-Ray report of chest – status quo, USG report – normal. Biochemical report – normal except high alkaline phosphatase values (maintained status quo), Marker CEA – normal

Continuing

Symptomatic improvement, no breathing problem. After 4 months CT scan of thorax and neck showed status quo.

Good response, normal life till date without any specific complaint. ND, LN Excellent remission, normal life till date ND, LN Excellent remission, normal life till date RD, LN Normal life till date, without any specific complaint. SD Stable disease, quality of life almost normal, with some complaint. SD Stable disease, normal life till date. SD

*ND – no tumour, LN – living normally, RD – regressive disease, SD – stable disease, PD – progressive disease, DC – died of cancer, DN – died due to other disease

Table 3. A summary of the effect of methylglyoxal-based formulation on 46 patients suffering from different types of cancer

Period of observation: October 2000- September 2005 Longest follow-up: 56 months Average follow-up: 18 months Complete remission: 18 (39%) patients Partial regression/stable: 18(39%) patients Progressive: 8 (17%) patients Not evaluated: 2 Total: 46 patients

human tissue materials had suggested that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase and mitochondrial complex I might be specifically altered in malignant cells (Halder et al, 1993; Ray et al, 1994, 1997a, b; Biswas et al, 1997). As mentioned in the Introduction that despite the documented anticancer property of methylglyoxal it had not been tested till recently for treating cancer patients. On the other hand, methylglyoxal bisguanylhydrazone, a derivative of methylglyoxal had been used to treat human medulloblastoma xenografts in nude mice with no success (Friedman et al, 1986). This compound when tested to treat to cancer patients had been found to be toxic and with little beneficial effect to the host (Gastaut et al, 1987).

IV. Discussion The results presented in this paper show that methylglyoxal in combination with ascorbic acid could deliver significant beneficial effects to a group of cancer patients. Total forty-six patients were included in the study and these patients were suffering from different types of cancer. The study period for the patients was 4 to 56 months. The remarkable finding of this drug regimen is that it is effective against a wide variety of cancer. This is in sharp contrast to other anticancer drugs now widely used. This remarkable effect of methylglyoxal over a wide range of cancer type suggests that all the different types of cancer may have common altered site(s) and which is targeted by methylglyoxal. In fact as mentioned before previous in vitro studies in our laboratory with animal and 220

Cancer Therapy Vol 4, page 221 This apathy of using methylglyoxal possibly stems from the widespread belief that, methylglyoxal is toxic. Recently, numerous papers have appeared in the literature, which mostly with in vitro studies under non-physiological conditions have shown that methylglyoxal reacts with arginine, lysine and free terminal amino groups in proteins resulting in AGE (advanced glycation end products) formation. The possibilities of many deleterious effects of methylglyoxal in the body have been extrapolated based mostly on these in vitro studies. The notable complications are related to diabetes, hypertension and cataract formation (Lee et al, 1999; Morgan et al, 2002; Bourajjaj et al, 2003; Kumar et al, 2004). Evidences have also been put forward that methylglyoxal is mutagenic (MurataKamiya et al, 2000) and induces reactive oxygen species formation (Chang et al, 2005). On the other hand as mentioned before several in vivo and in vitro studies suggest that methylglyoxal may have some beneficial effects too particularly to higher animals. It inhibits mitochondrial respiration and glycolysis of malignant cells, whereas these functions of normal cells are unaffected (Halder et al, 1993; Ray et al, 1994, 1997a,b; Biswas et al, 1997; Ray and Ray, 1998). There is a report that methylglyoxal rapidly suppresses the mitochondrial permeability transition thereby preventing from the possible deleterious effect of high Ca2+ and ganglioside (Speer et al, 2003). Methylglyoxal can also protect gastric mucosa against different necrotizing agents (Al-Shabanah et al, 2000). The most remarkable beneficial effect of methylglyoxal had originated from the in vivo studies by Szent Györgyi and colleagues and Apple and Greenberg, which showed the curative effect of methylglyoxal on cancer-bearing animals (Apple and Greenberg, 1967; Apple and Greenberg, 1968; Együd and Szent-Györgyi, 1968; Conroy, 1979). From the results of all these studies it is logical to conceive that many of the purported in vitro toxic effects of methylglyoxal may be counteracted by many countervailing reactions in intact living animals. Our previous investigation on the possible toxic effect of methylglyoxal supports this conjecture. Pharmacokinetic studies had indicated that methylglyoxal is appropriately cleared from the body (Ghosh et al, 2006). The apparent lack of toxicity of methylglyoxal-based treatment and also the wide applicability deserve that it should be further studied. Because methylglyoxal is a normal metabolite, the presence of several active catabolyzing enzymes such as glyoxalase I may diminish its efficacy (Ray and Ray, 1998; Ghosh et al, 2006; Cooper, 1984). There are several synthetic inhibitors of glyoxalase I (Creighton et al, 2003). By in-vivo experiments it should at first be ascertained whether these compounds are non-toxic and effective in augmenting the anticancer effect of methylglyoxal. Then some of these compounds in combination with methylglyoxal could be used to treat cancer patients. Mode of treatment should be another consideration. It is expected that intravenous and/or peritumoural injection may significantly improve the efficacy. Similar to all form of cancer treatment early diagnosis is advantageous. Because methylglyoxal acts specifically

against malignant cells it has the potential to destroy metastasis also. But if vital organ(s) are irreversibly damaged then it is very difficult to bring back the patients to normalcy. At present combination chemotherapy is widely used in the treatment of cancer. Recently besides our work a paper has appeared describing a study of treatment of nonHodgkin lymphoma by two different combinations of drugs, one of which contained methylglyoxal. However, the drug combination, which contained methylglyoxal showed no better results (Chamorey et al, 2005). In our present work, we show that only methylglyoxal in combination with ascorbic acid is quite effective in arresting tumour growth and in some cases could completely eliminate the malignant tissues. Previous in vitro study from our laboratory with human tissue materials had shown that the anticancer effect of methylglyoxal was significantly augmented in presence of ascorbic acid (Ray et al, 1991). Finally, our next task will be to further evaluate the efficacy of methylglyoxal and ascorbic acid treatment using a large number of patients and different types of cancers at different stages of the disease. If found effective then to further improve the treatment.

References Al-Shabanah OA, Qureshi S, Al-Harbi MM, Al- Bekairi AM, AlGharably NM, Raza M (2000) Inhibition of gastric mucosal damage by methylglyoxal pretreatment in rats. Food Chem Toxicol 38, 577-584. Apple MA, Greenberg DM (1967) Arrest of cancer in mice by therapy with normal metabolites. I. 2- Oxopropanal (NSC 79019). Cancer Chemother Rep 51, 455-464. Apple MA, Greenberg DM (1968) Arrest of cancer in mice by therapy with normal metabolites. II. Indefinite survivors among mice treated with mixtures of 2-oxopropanal (NSC79019) and 2, 3-dihydroxypropanal (NSC-67934). Cancer Chemother Rep 52, 687-696. Biswas S, Ray M, Misra S, Dutta DP, Ray S (1997) Selective inhibition of mitochondrial respiration and glycolysis in human leukemia leukocytes by methylglyoxal. Biochem J 323, 343-348. Bourajjaj M, Stehouwer CD, van Hinsbergh VW, Schalkwijk CG (2003) Role of methylglyoxal adducts in the development of vascular complications in diabetes mellitus. Biochem Soc Trans 31, 1400-1402. Cassidy J, Bissett D, Spence RAJ (2002a) Performance status. In: Oxford Handbook of Oncology. Oxford University Press, New Delhi India, p 80. Cassidy J, Bissett D, Spence RAJ (2002b) Cancer of the biliary tract. In: Oxford Handbook of Oncology. Oxford University Press, New Delhi India, pp 266-269. Chamorey E, Gressin R, Peyrade F, Rossi JF, Lepeu G, Foussard C, Harrousseau JL, Fabbro M, Richard B, Delwail V, Maisonneuve H, Vilque JP, Thyss A (2005) Prospective randomized study comparing MEMID with a Chop-like regimen in elderly patients with aggressive non-Hodgkin’s lymphoma. Oncology 69, 9-26. Chang T, Wang R, Wu L (2005) Methylglyoxal-induced nitric oxide and peroxynitrite production in vascular smooth muscle cells. Free Radic Biol Med 38, 286-293 . Conroy PJ (1979) Carcinostatic activity of methylglyoxal and related substances in tumor-bearing mice. Ciba Found. Symp 67, 271-300.

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Talukdar et al: Treatment of malignancies by methylglyoxal-based formulation Cooper RA (1984) Methylglyoxal metabolism in microorganisms. Annu Rev Microbiol 38, 49-68. Creighton DJ, Zheng ZB, Holewinski R, Hamilton DS, Eiseman JL (2003) Glyoxalase I inhibitors in cancer chemotherapy. Biochem Soc Trans 31, 1378-1382. Együd LG, Szent-Györgyi A (1968) Cancerostatic action of methylglyoxal. Science 160, 1140. Elvin P, Slater TF (1981) Anti-tumor activity of novel adducts of ascorbic acid with aldehydes. Eur J Cancer Clin Oncol 17, 759-765 . Friedman HS, Schold SCJr, Bigner DD (1986) Chemotherapy of subcutaneous and intracranial human medulloblastoma xenografts in athymic nude mice. Cancer Res 46, 224-228. Gastaut JA, Tell G, Schechter PJ, Maraninchi D, Mascret B, Carcassonne Y (1987) Treatment of acute myeloid leukemia and blastic phase of chronic myeloid leukemia with combined eflorinthine (alpha difluoromethylornithine) and methylglyoxal-bisguanyl hydrazone (methyl- GAG). Cancer Chemother Pharmacol 20, 344-348. Ghosh M, Talukdar D, Ghosh S, Bhattacharyya N, Ray M, Ray S (2006) In vivo assessment of toxicity and pharmacokinetics of methylglyoxal. Augmentation of the curative effect of methylglyoxal on cancer-bearing mice by ascorbic acid and creatine. Toxicol Appl Pharmacol 212, 45-58. Halder J, Ray M, Ray S (1993) Inhibition of glycolysis and mitochondrial respiration of Ehrlich ascites carcinoma cells by methylglyoxal. Int J Cancer 54, 443-449. Kumar MS, Reddy PY, Kumar PA, Surolia I, Reddy GB (2004) Effect of dicarbonyl- induced browning on alpha-crystallin chaperon-like activity: physiological significance and caveats of in vitro aggregation assays. Biochem J 379, 273-282. Lee KW, Simpson G, Ortwerth B (1999) A systematic approach to evaluate the modification of lens proteins by glycationinduced cross-linking. Biochim Biophys Acta 1453, 141151 . Morgan PE, Dean RT, Davies MJ (2002) Inactivation of cellular enzymes by carbonyls and protein-bound glycation/ glycoxidation products. Arch Biochem Biophys 403, 259269. Murata K, Saikusa T, Fukuda Y, Watanabe K, Inoue Y, Shimosaka M, Kimura A (1986) Metabolism of 2oxoaldehydes in yeasts. Possible role of glycolytic bypath as a detoxification system in L-threonine catabolism by Saccharomyces cerevisiae. Eur J Biochem 157, 297-301. Murata-Kamiya N, Kamiya H, Kaji H, Kasai H (2000) Methylglyoxal induces G:C to C:G and G:C to T:A transversions in the supF gene on a shuttle vector plasmid replicated in mammalian cells. Mutat Res 468, 173-182. Ray M, Basu N, Ray S (1997a) Inactivation of glyceraldehyde-3phosphate dehydrogenase of human malignant cells by methylglyoxal. Mol Cell Biochem 177, 21-26.

Ray M, Ghosh S, Kar M, Datta S, Ray S (2001) Implication of the bioeletronic principle in cancer therapy: treatment of cancer patients by methylglyoxal-based formulation. Indian J Phys 75B, 73-77. Ray M, Halder J, Dutta SK, Ray S (1991) Inhibition of respiration of tumor cells by methylglyoxal and protection of inhibition by lactaldehyde. Int J Cancer 47, 603-609. Ray M, Ray S (1987) Aminoacetone oxidase from goat liver. Formation of methylglyoxal from aminoacetone. J Biol Chem 262, 5974-5977. Ray M, Ray S (1998) Methylglyoxal: From a putative intermediate of glucose breakdown to its role in understanding that excessive ATP formation in cells may lead to malignancy. Current Sci 75, 103-113. Ray S, Biswas S, Ray M (1997b) Similar nature of inhibition of mitochondrial respiration of heart tissue and malignant cells by methylglyoxal. A vital clue to understand the biochemical basis of malignancy. Mol Cell Biochem 171, 95-103. Ray S, Dutta S, Halder J, Ray M (1994) Inhibition of electron flow through complex I of the mitochondrial respiratory chain of Ehrlich ascites carcinoma cells by methylglyoxal. Biochem J 303, 69-72. Ray S, Ray M (1981) Isolation of methylglyoxal synthase from goat liver. J Biol Chem 256, 6230-6233. Speer O, Morkunaite-Haimi S, Liobikas J, Franck M, Hensbo L, Linder MD, Kinnunen PK, Wallimann T, Eriksson O (2003) Rapid suppression of mitochondrial permeability transition by methylglyoxal. Role of reversible arginine modification. J Biol Chem 278, 34757-34763 . Szent-Györgyi A (1979) The living state and cancer. Ciba Found Symp 67, 3-18.

Manju Ray

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Cancer Therapy Vol 4, page 223 Cancer Therapy Vol 4, 223-230, 2006

Analysis of cytoplasmic polypeptides expression in gastric cancer and correlation with pathologic parameters Research Article

Panayiota K. Stroumbouli1, Andreas Ch. Lazaris1, Efstratios S. Patsouris1, Anastasios Kalofoutis2, Aphrodite Nonni1, Sofia Tseleni-Balafouta1 1

Department of Pathology, Department of Biochemistry, School of Medicine, National and Kapodistrian University of Athens, 75 Mikras Assias Str., GR-115 27 Athens, Greece 2

__________________________________________________________________________________ *Correspondence: Panayiota Stroumbouli, PhD, 42 Dodekanisou str., GR-15234 Vrilissia, Greece; Tel: 2108101161; Fax: 2108102690; E-mail: ny11@tellas.gr Key words: 2D eletrophoresis, cytoplasmic polypeptides, gastric cancer, pathologic parameters, prognostic markers Abbreviations: 3-(3-cholamidotropyl) dimethylammonio)-1-prop-anesulphonate, (CHAPS) dithiothreitol, (DTT); ethyleneldinitrinotetraacetic acid, (EDTA); isoelectric focusing, (IEF); isoelectric point, (PI); molecular weight, (MW); phenylmethylsulfonyl fluoride, (PMSF); sodium dodecylsulfate, (SDS); two-dimensional electrophoresis, (2D-electrophoresis) Received: 13 March 2006; Revised: 26 May 2006 Accepted: 1 June 2006; electronically published: July 2006

Summary Although there have been many studies including analysis of prognostic factors in gastric cancer, there are currently very few markers that are clinically in use. The aim of this study is to relate polypeptide expression with pathologic characteristics of patients with gastric cancer in order to determinate potential prognostic markers. Using high-resolution two-dimensional electrophoresis (2D-electrophoresis), 154 stomach tissues were examined. Seventy-seven of the specimens were histologically diagnosed as primary adenocarcinomas. The other 77 specimens represented mirror biopsies from each patient, obtained far from the tumor area, from endoscopically and histologically normal gastric mucosa. Significant polypeptide expression differences had been noted comparing each cancer tissue sample with the respective mirror biopsy of the same patient. More specifically, 24 cytoplasmic polypeptides had been studied. Six of them were detected in all mirror biopsy tissues and not detected in the majority of gastric cancer tissues ( Pa, Pb, Pc, Pk, Pl, Pm), 14 polypeptides were over-expressed ( P2, P3, P4, P5, P7, P8, P9, P10, P11, P13, P15, P16, P17, P18) and 4 were under-expressed ( P1, P6, P12, P14) in cancer tissues. Tumor samples were classified in groups according to pathologic prognostic variables. Polypeptidesâ&#x20AC;&#x2122; densities were normalized and the results were evaluated statistically. Statistical analysis indicated that several polypeptides were associated with pathologic characteristics. Polypeptides P4 (29/6.0), P16 (17/6.9) showed an extremely significant density increase (p<0.0001) in tumor samples with 7-15 metastatic lymph nodes and in those samples with tumor invasion to the muscular layer. Polypeptide Pc (46/2.6) was detected only in poorly differentiated tumor samples and so may serve as a marker of poor prognosis. Polypeptides P4, P16, Pc may have prognostic value and their further analysis may provide useful information to the direction of developing markers of stomach cancer.

reported. The identification of prognostic factors is important for the establishment of therapeutic strategies. Of the many factors relevant to survival, depth of invasion, lymph node metastasis and degree of differentiation have been considered major prognostic factors in gastric cancer (Ryu et al, 2003). There have been many studies including analysis of prognostic factors but there are currently very few markers that are clinically in use.

I. Introduction Although gastric cancer mortality has declined markedly around the world the last twenty years, gastric cancer still remains the second most common cancer next to lung cancer worldwide (Roder, 2002). The results of gastric cancer treatment have been markedly improved and considerable progress in diagnostic methods is being

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Stroumbouli et al: Analysis of cytoplasmic polypeptides in gastric cancer 2D-PAGE electrophoresis analysis of cytosolic polypeptides was performed according to the procedure of O’ Farrel in 1975) and modified by Hochstrasser and colleagues in 1988 using the BioRad 2D-PAGE system (BioRad Lab Inc., USA). Lyophilised protein samples were dissolved in sample solution (9M urea, 4 % w/v 3-(3-cholamidotropyl) dimethylammonio) -1-prop-anesulphonate (CHAPS), 1% DTT, ampholytes 3.5-10 (1%, 4-7 (4%). Polypeptide content was determined using the Ramagli and Rodriguez (1985) modification of the Bradford protein assay (Bradford, 1976).

High-resolution two-dimensional electrophoresis (2Delectrophoresis) is widely used in comparative studies of protein expression levels between healthy and diseased states with the purpose of developing diagnostic markers. This method can readily separate a number of proteins based on differences in their molecular weight (MW) and isoelectric point (PI) properties (Nagase et al, 1991). Highresolution 2D-electrophoresis combines first dimension isoelectric focusing (IEF) with the conventional sodium dodecylsulfate (SDS) electrophoresis in the second dimension (Whilson, 1977). Whether 2D-electrophoresis is followed by computational image analysis and protein identification with tumor samples could also lead to the defining of cancer–specific protein markers which will be the basis for developing new methods for the early diagnosis of cancer (Kim et al, 1998). 2D-electrophoresis has also been applied in the study of gastrointestinal track cancers (Isoda et al, 1990). More specifically, in relation to colon cancer, five major polypeptide spots were detected in tumor samples which were not detected in normal specimens. Researchers (Okuda, 1989; Tracy et al, 1982) detected two hightmolecular weight polypeptides in colon cancer tissues which were absent in normal mucosa. In relation to gastric cancer, 140 proteins were identified in cancer stomach tissues, 7 proteins were over-expressed and 7 were under-expressed in stomach cancer (Ryu et al, 2003). An acid proteinase was detected in gastric tumor samples and was not detected in normal specimens (Aoki, 1994). Other researchers, using 2D-electrophoresis, were studied two proteins, annexin I and thioredoxin, which were over-expressed in cancer cells (Sinha et al, 1998). The aim of this study is to relate polypeptide expression with pathologic characteristics of patients with gastric cancer in order to determine their prognostic impact.

B. First dimension (IEF) First dimension (IEF) was performed in a 180 x 1.5 mm tube gel consisting of 30% acrylamide solution, containing 10 M urea and a 4:1 ratio of 4-7 and 3.5-10 carrier ampholytes (final concentration 5 %) 75 !g of polypeptide were loaded onto the basis end of each IEF gel. The cathode chamber was filled with freshly degassed NaOH solution (0.1M) and the proteins were focused 700V for the approximately 19h. Each sample was electrofocused in dublicate with and without internal 2D-SDSPAGE. Standards (Bio Rad Laboratories) and pI calibration kit (BDH, labs Supplies, Poole, England). The gels were gently extruded from the glass tubes and stored in extended form on Parafilm strips at –70° C until use. The IEF gels were allowed to soak in equilibration buffer (0.04M Tris-HCL, 3.2 SDS, 0.034M DTT) for 5 min before being loaded on at the second dimensional gel and sealed with a small amount of overlay agarose (0.025M Tris base, 0.192M glycine 0.1% SDS, 0.5% agarose).

C. Two D- SDS-PAGE Two- D- SDS-PAGE electrophoresis was performed at constant current of 35 mA/gel at 8° C. Gels were fixed in ethanol water (30%) and acetic acid (10%). Proteins were visualized on 2D gel preparations by silver staining with Sigma Silver Stain Kit (Sigma Chemicals Co.,St. Louis, USA). For increased sensitivity the staining prosedure was repeated. Gels were photographed and scanned, using a GS-700 BioRad Imaging Densitometer. The analysis was carried out by BioRad PDQuest-2D software, based on a modification of the grid system used by Narayan et al, 1986. Spots were numbered in descending order of molecular mass and were identified by their location relative to (known) protein markers (ie, albumin, actin, transferrin). Spot densities were normalized using the above software, prior to performing comparisons between different gels. The statistical analysis was performed using student's-t-test with the package STATA (STATA Co, USA). Tumor samples were classified in groups according to pathologic prognostic variables (Table 1).

II. Materials and methods A total of 154 stomach tissues deriving from 77 gastric cancer patients were surgically obtained at the Surgical Department of the "Hippocration" Hospital of Athens, over a period of 6 years (1994-2000). Seventy-seven of the specimens were histologically diagnosed as primary adenocarcinomas. The other 77 specimens represented mirror biopsies from each patient, obtained far from the tumor area, from endoscopically and histologically normal gastric mucosa.

III. Results Two-dimensional electrophoresis of the stomach cancer tissue produced about 1500 spots for each sample, every spot representing a cytosolic polypeptide. In this study we compared each cancer tissue sample with the respective mirror biopsy of the same patient in order to evaluate any differential polypeptide expression between them. While most of the polypeptides were present in both cancerous tissues and mirror biopsy tissues, several qualitative and quantitative polypeptide differences were noted between cancerous and noncancerous samples. As far as qualitative differences are concerned, the comparison between stomach cancer tissue and mirror biopsy of each patient showed six polypeptides in the acid

A. Preperation of stomach tissue sambles Fresh specimens were collected directly by the surgeon in the operating room, rinsed immediately in isotonic saline to remove external blood contamination and were frozen instantly in liquid nitrogen. Samples were stored at -70° C until further use. On the day of analysis, frozen tissues were sliced with a scalpel, washed in a hogenasation buffer consisting of 10mM Tris HCL, pH 7.4, 1.5mM ethyleneldinitrinotetraacetic acid (EDTA), 0.5 mM dithiothreitol (DTT), 0.2 mM phenylmethylsulfonyl fluoride (PMSF) as a protease inhibitor and homogenized at 0°C using a Virtis homogenizer for 3x12 sec. Homogenates were centrifuged for 1h at 105.000xg and the supernatant fraction was collected. This fraction referred hitherto as cytosolic fraction was lyophilized to dryness immediately after preparation and stored at -70° C prior to electrophoresis.

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Cancer Therapy Vol 4, page 225 area of PI between 3.0-5.0 and MW 40-46 kDa which were detected in all mirror biopsy tissues (Figure 1a) and not detected in the majority of adenocarcinomas (Figure 1b). These polypeptides were identified as follows (MW/PI): Pa (46/3.3), Pb (46/2.8), Pc (46/2.6), Pk (40/4.2), Pl (40/4.0), Pm (40/3.8).

In addition to the above significant qualitative differences, quantitative differences in polypeptide concentration were also observed, highlighting 18 polypeptides significantly altered in malignant specimens (Figure 1a, 1b).

Table 1. Classification of tumor samples according to histological characteristics of prognostic potential Histological characteristics

Depth of tumor invasion mucosa and submucosa layers muscular layer Lymph nodes metastasis negative lymph nodes 1-6 positive lymph nodes 7-15 positive lymph nodes Lauren's classification Intestinal type Diffuse type Degree of differentiaiton Well differentiated Moderately differentiated Poorly differentiated

34 43 12 21 44 62 15 9 30 38

Figure 1. Electrophoretic patterns of cytosolic polypeptides from stomach adenocarcinoma (A) and the respective mirror biopsy (B). Missing spots are illustrated with open circles (Pa, Pb, Pc, Pk, Pl, Pm spots) (A). All quantitative differences are indicated with small arrowheads in both figures (Alb: albumin, Actin: actin, Tran: transferin).

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Stroumbouli et al: Analysis of cytoplasmic polypeptides in gastric cancer Fourteen polypeptides were significantly increased in tumor samples (ie, P2, P3, P4, P5, P7, P8, P9, P10, P11, P13, P15, P16, P17, P18) while four polypeptides (ie, P1, P6, P12, P14) were decreased in malignant specimens. Statistical analysis of the polypeptide densities showed highly significant differences (p<0.001) with regard to polypeptides Pa, Pb, Pc, Pl qualitative expression in normal tissues compared to the respective densities in adenocarcinomas as well as with regard to polypeptides P9 and P10, as far as their quantitative expression is concerned. All other polypeptides showed a merely significant difference in their quantitative expression (p<0.05). We attempted a correlation between the above mentioned polypeptide expression with clinicopathologic characteristics such as presence of lymph node metastasis,

depth of tumor invasion, degree of differentiation, Lauren’s histologic type classification, in order to determine any prognostic value for the examined polypeptides. As far as lymph nodes metastasis and depth of invasion is concerned, statistical analysis (student’s t-test) showed that polypeptides P2, P4, P10, P16, P17 were over-expressed in tumor samples with metastasis in 7-15 lymph nodes (Figure 2, Table 2) and polypeptides P3, P4, P10, P16, P17 were over-expressed in tumor samples with invasion to the muscular layer (Figure 3, Table 3). Moreover, polypeptides P4, P16 showed an extremely significant density increase (p<0.0001) in both above mentioned tumor groups while all other polypeptides showed a merely significant increase in their expression in the above tumor groups (p<0.05).

Table 2. Correlation between polypeptide densities and lymph nodes metastasis

Negative lymph nodes* P P2 P4 P10 P16 P17

0.64±0.02 1.32±0.44 0.32±0.04 1.08±0.63 0.47±0.05

Lymph nodes metastasis Positive lymph nodes 1-6 lymph nodes* 7-15 lymph nodes* 0.967±0.23 1.251±0.14 2.093±0.85 2.73±1.37 1.093±0.57 1.63±1.02 1.92±0.94 2.53±1.187 0.83±0.46 2.84±1.06

p- value 0.039 0.0076 0.045 0.0052 0.015

C% 110 90 190 80 240

P: polypeptide, * mean density of the respective tumor samples ± statistical error, C:% change

Figure 2. Electrophoretic patterns of cytosolic polypeptides from tumor sample with metastasis in 7-15 lymph nodes where polypeptides P2, P4, P10, P16, P17 were over-expressed (A) and tumor sample with metastasis in 1-6 lymph nodes (B).

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Cancer Therapy Vol 4, page 227 Table 3. Correlation between polypeptide densities and depth of tumor invasion

P P3 P4 P10 P16 P17

Depth of tumor invasion mucosa or submucosa layer* 0.79±0.37 2.15±2.61 1.12±1.47 1.67±.0.12 0.95±0.045

muscular layer* 1.63±0.404 3.93±2.31 1.38±1.025 3.18±1.45 1.605±0.34

p- value 0.032 0.0029 0.027 0.007 0.041

C% 110 90 30 90 80

P: polypeptide, *: mean density of tumor samples ± statistical error, C: % change

Figure 3. Electrophoretic patterns of cytosolic polypeptides from tumor sample with invasion to muscular layer, where polypeptides P3, P4, P10, P16, P17 were over-expressed (A) and tumor sample with tumor invasion to mucosa or submucosa layer (B).

With regard to tumor grade, statistical analysis of the polypeptide expression (student’s t-test) have provided the following results: i) the majority of polypeptides densities were over-expressed in moderately differentiated tumors samples, except for the polypeptides (molecular weight/isoelectric point) P9(25/7.3), P11(22/6.7), P13(20/5.6); the latter were significantly increased in poorly differentiated tumor samples (p<0.05) ii) polypeptides: Pa (46/3.3), Pb(46/2.8), Pc (46/2.6), were not detected in well differentiated tumor samples; moreover, the expression of polypeptides Pa, Pb showed a significant increase in moderately and poorly differentiated tumor samples (p<0.05). iii) polypeptide Pc was detected only in poorly differentiated tumor samples (Figure 4, Table 4). With regard to Lauren’s classification of histologic type, no significant differences were noticed. Multivariate statistical analysis indicates polypeptides P4, P16 as independent prognostic factors.

IV. Discussion In the present study, using 2D-electrophoresis, we observed 6 cytosolic polypeptides being detected in all mirror biopsy tissues and not detected in the majority of gastric cancer tissues, as well as 14 cytosolic polypeptides being over-expressed and 4 being under-expressed in cancer tissues. Our results are in agreement with those of other researchers who have also observed several polypeptides to be significantly altered in human gastric cancer tissues, by comparison to their mirror biopsies (Ryu et al, 2003). As it has been reported, changes in cytosolic polypeptides expressed in tumors of gastrointestinal tract may occur as a result of the expression of silent genes, the arrest of immature cells at an early stage of their normal differentiation or the presence of an unusual cell-cycle in actively proliferating groups of cells (Natly et al, 1998). We have evaluated our 2D- data based on a recent 2D-map of human stomach tissue (18) in an effort to provide potential identifications. The polypeptides 227

Stroumbouli et al: Analysis of cytoplasmic polypeptides in gastric cancer Table 4. Correlation between polypeptide densities and degree of differentation

P Pa Pb Pc

Poorly differentiated 1.36±0.64 1.23±0.99 0.82±0.02

Degree of differentiation Moderately differentiated 1.8±0.69 1.49±0.05 -

Well differentiated -

p-value 0.015 0.042 0.003

P2 P3 P4

1.93±0.44 0.95±0.44 1.02±0.88

2.89±0.32 2.12±1.55 1.45±0.65

".65±0.21 0.45±0.07 0.92±0.51

0.041 0.032 0.006

3.44±2.11

1.04±0.06

0.77±0.33

0.022

P10 P11

2.38±1.98 2.48±1.67

4.14±2.36 2.02±1.38

0.78±0.42 0.89±0.63

0.055 0.011

P13

1.64±0.55

1.48±0.81

1.55±0.73

0.028

P16 P17

1.36±0.57 2.13±1.69

3.02±2.01 2.57±1.11

0.28±0.02 0.34±0.05

0.051 0.048

P: polypeptide, *: mean density of the respective tumor samples ± statistical error

Figure 4. Electrophoretic patterns of cytosolic polypeptides from tumor sample poorly differentiated where polypeptide Pc is detected (A) and tumor sample moderately differentiated where polypeptide Pc is absent (B).

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Cancer Therapy Vol 4, page 229 Hepatogastroenterology 48, 1523-30. Ha GH, Lee SU, Kang DG, Ha NY, Kim SH, Kim J, Bae JM, Kim JW, Lee CW (2002) Proteome analysis of human stomach tissue: separation of soluble proteins by twodimensional polyacrylamide gel electrophoresis and identification by mass spectrometry. Electrophoresis 23, 2513-24 Hochstrasser DF, Harrington MG, Hochstrasser AC, Miller MJ, Merril CR (1988) Methods for increasing the resolution of two-dimensional protein electrophoresis. Anal Biochem 173, 424-35. Isoda N, Kajii E, Ikemoto S, Kimura K (1990) Two-dimensional polyacrylamide gel electrophoretic pattern of duodenal tumour proteins. J Chromatogr 534, 47-55. Kennedy JA, Kirk SJ, McCrory DC, Halliday MI, Barclay GR, Rowlands BJ (1994) Modulation of immune function and weight loss by L-arginine in obstructive jaundice in the rat. Br J Surg 81, 1199-201 Kim JP, Jung SE (1987) Patients with gastric cancer and their prognosis in accordance with number of lymph node metastases. Scand J Gastroenterol 22, 33-5. Kim JP, Lee JH, Kim SJ, Yu HJ, Yang HK (1998) Clinicopathologic characteristics and prognostic factors in 10 783 patients with gastric cancer. Gastric Cancer 1, 125-133. Nagase T, Sugiyama T, Kawata S, Tarui S, Deutsch HF, Taniguchi N (1991) Analyses of polypeptides in the liver of a novel mutant (LEC rats) to hereditary hepatitis and hepatoma by two-dimensional gel electrophoresis: identification of P29/6.8 as carbonic anhydrase III and triosephosphate isomerase. Comp Biochem Physiol B 99, 193-201. Nalty TJ, Taylor CW, Yeoman LC (1988) Variation in cytosolic protein expression between human colon tumors that differ with regard to differentiation class. Clin Chem 34, 71-5. Narayan RK, Heydorn WE, Creed GJ, Jacobowitz DM (1986) Protein patterns in various malignant human brain tumors by two-dimensional gel electrophoresis. Cancer Res 46, 468594. O'Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250, 4007-21. Okuda K (1989) Biological and immunological studies on human tumor-associated cellular proteins detected by twodimensional gel electrophoresis. Nippon Geka Gakkai Zasshi 90, 1697-705. Ramagli LS, Rodriguez LV (1985) Quantitation of microgram amounts of protein in two-dimentional polyacrylamide gel electrophoresis sample buffer. Electrophoresis 6, 559-563. Roder DM (2002) The epidemiology of gastric cancer. Gastric Cancer 5 (suppl 1), 5-11. Ryu JW, Kim HJ, Lee YS, Myong NH, Hwang CH, Lee GS, Yom HC (2003) The proteomics approach to find biomarkers in gastric cancer. J Korean Med Sci 18, 505-9. Sinha P, Hutter G, Kottgen E, Dietel M, Schadendorf D, Lage H (1998) Increased expression of annexin I and thioredoxin detected by two-dimensional gel electrophoresis of drug resistant human stomach cancer cells. J Biochem Biophys Methods 37, 105-16. Tracy RP, Wold LE, Currie RM, Young DS (1982) Patterns for normal colon mucosa and colon adenocarcinoma compared by two-dimensional gel electrophoresis. Clin Chem 28, 9159 Wilson DL, Hall ME, Stone GC, Rubin RW (1977) Some improvements in two-dimensional gel electrophoresis of proteins. Protein mapping of eukaryotic tissue extracts. Anal Biochemistry 83, 33-44.

(MW/PI): P5 (27/6.2), P6 (26/6.0), P18 (15/5.3) correspond to the antioxidants glutathione-S-transferase, peroxiredoxin-2 and thioredoxin, respectively. These antioxidants protect cells from oxidative damage caused by various oxidative stimuli and have been related with chemoresistance of tumor cells, especially to the oxidative stress that anticancer drugs produce (Chung et al, 2001). We also showed that several polypeptidesâ&#x20AC;&#x2122; expression is associated with the most significant prognostic factors in gastric cancer. As other researchers have reported, the presence of lymph node metastasis, the depth of tumor invasion and the tumor degree of classification have prognostic significance in gastric cancer (Kim and Jung, 1987; Adachi et al, 1994) while Laurenâ&#x20AC;&#x2122;s classification seems to have no significant value (Fiocca et al, 2001). Apart from the clinical and pathological prognostic factors in gastric cancer, there is a great research interest in new prognostic factors such as growth factors and receptors, oncogenes and suppressor genes, cell adhesion molecules (Ryu et al, 2003). The polypeptides P4(29/6.0), P16(17/6.9) showed an extremely significant density increase (p<0.0001) in tumor samples with 7-15 metastatic lymph nodes and tumor invasion to the muscular layer and may serve as prognostic factors. Multivariate statistical analysis reinforces the above results. Taking into account their presence in a small number of tumor samples, polypeptides Pa (46/3.3), Pb (46/2.8), which were not detected in well differentiated tumor samples, could be further investigated as potential markers for the biological aggressiveness because their expression showed a significant increase in moderately and poorly differentiated tumor samples polypeptides. Polypeptide Pc (46/2.6) was detected only in poorly differentiated tumor samples and so were, though to a lesser extend, polypeptides P9(25/7.3), P11(22/6.7), P13(20/5.6); the latter polypeptides may be associated with poor prognosis of gastric cancer because of their significant density increase in poorly differentiated tumor samples. In conclusion, we propose that polypeptides P4, P16, Pc may be significant biomarkers of poor prognosis. Therefore, their further molecular identification may provide useful information to the direction of developing markers for stomach cancer.

References Aoki T, Takasaki T, Morikawa J, Yano T, Watabe H (1994) Electrophoretic analysis of a gastric cancer-associated acid proteinase using a highly sensitive detection system. Biol Pharm Bull 17, 1358-6 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248-54. Chung YM, Yoo YD, Park JK, Kim YT, Kim HJ (2001) Increased expression of peroxiredoxin II confers resistance to cisplatin. Anticancer Res 21, 1129-33. Fiocca R, Luinetti O, Villani L, Mastracci L, Quilici P, Grillo F, Ranzani GN (2001) Molecular mechanisms involved in the pathogenesis of gastric carcinoma: interactions between genetic alterations, cellular phenotype and cancer histotype.

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Cancer Therapy Vol 4, page 231 Cancer Therapy Vol 4, 231-240, 2006

Proton radiation therapy in oncology: Review of present clinical indications Review Article

Maurizio Amichetti*, Augusto Lombardi, Carlo Algranati, Marco Schwarz ATreP, Provincial Agency for Proton Therapy, Trento-Italy

__________________________________________________________________________________ *Correspondence: Maurizio Amichetti M.D., ATreP, Provincial Agency for Proton Therapy, Via Perini, 181, 38100 Trento-Italy; Tel: 39 0461 390409; Fax: 39 0461 391648; e-mail: amichett@ect.it Key words: proton therapy, radiotherapy, cancer Abbreviations: “quality adjusted life years”, (QALY); 3D-conformal radiation therapy, (3D-CRT); Gray equivalent, (GyE); industrial provider, (IBA); Intensity-Modulated Radiation Therapy, (IMRT); organ at risk, (OAR); proton therapy, (PT); relative biological effectiveness, (RBE); stereotactic radiotherapy, (SRT) Received: 7 July 2006; Accepted: 11 July 2006; electronically published: July 2006

Summary Radiotherapy has been rapidly changing in recent years moving towards the ability to reduce the radiation exposure of normal tissue and to increase treatment conformality, i.e. the ability to concentrate radiation dose in the intended target area. Due to their physical properties, proton beams are a very attractive radiation source to address this issue. Protons have a unique advantage over photons because of the superior ability of confining the high-dose region to the target while minimizing the dose to the surrounding normal tissues. These characteristics translate in an improved therapeutic ratio and have been exploited in treating more and more frequently several tumors in adults and children with very promising results. The availability of protons in hospital-based facilities currently planned and built in Europe, Asia, and United States will soon increase the role of protons in the multimodal approach to cancer therapy.

extensively studied in the 70’s and 80’s mainly in research laboratories dedicated to nuclear physics where the accelerators (cyclotrons or synchrotrons) were located. Only recently, on the basis of positive clinical results, there is a growing interest in hospital-based clinical facilities. Beginning with the first patients treated in the fifties more than 40.000 patients have now been treated worldwide (Table 1), (Sisterson, 2005). Several centers are proposed or under construction and the clinical indications increasingly expand following the pioneering treatments of ocular and base of skull tumors.

I. Introduction Radiotherapy is based on the principle of using ionizing radiation to cause irreparable damage to the DNA of tumor cells, thus inhibiting their duplication. From the point of view of physical interaction with radiation, tumor cells are not different from normal cells, so the irradiation is not causing per se a selective damage in tumors. Consequently, methods have to be devised to maximize the effect of radiation on tumor cells and to spare the surrounding healthy tissues as much as possible. Charged particle beams of accelerated protons accomplish the function of an almost ideal external radiation source for cancer therapy. The main property is that fast moving protons, once stopped in a body, can be directed to deliver most of their energy in an uniform manner in the target. None or very little energy is left for potential damages beyond the target volume. In clinical terms, that means depositing a high radiation dose in the tumor and a low dose outside. This property allows irradiating tumor targets located next to critical structures with high doses avoiding or reducing important side effects in organ at risk (OAR). After the first proposal of their clinical utility in the seminal article by Wilson in 1947, protons have been

II. Physical properties External radiotherapy treatments are usually delivered with photon beams. The physical interaction of photons with biological tissues is such that for all practical purposes it is impossible to control the penetration depth of photons in tissue. In other words, photons give a radiation dose significantly different from zero over a large thickness of tissue (Figure 1), thus delivering dose to the patient not only in the tumor volume but also in the surrounding tissue. The technique typically used in photon radiotherapy to minimize the dose to the healthy tissues consists in

231

Amichetti et al: Proton radiation therapy in oncology irradiating the tumor from many different directions, thus creating a high dose region on the target and a low dose region elsewhere. Conventional radiotherapy plans are typically designed by defining a number of beam directions (typically two to seven), each beam direction having a single portal. In other words, for a given beam

direction, a uniform intensity profile is applied. While in the past field sizes and shapes were defined on 2-D anatomy projection obtained with fluoroscopy, in the 90’s the so-called 3D-conformal radiation therapy (3D-CRT) was introduced, where CT data were available to define

Table 1. Patients treated with protons worldwide until December 2004 WHO

Berkeley 184 Berkeley Uppsala (1) Harvard Dubna (1) ITEP, Moscow Los Alamos St. Petersburg Berkeley Chiba TRIUMF PSI (SIN) PMRC (1), Tsukuba PSI (72 MeV) Uppsala (2) Clatterbridge Loma Linda Louvain-la-Neuve Nice Orsay iThemba LABS MPRI (1) UCSF - CNL HIMAC, Chiba TRIUMF PSI (200 MeV) G.S.I Darmstadt H. M. I, Berlin NCC, Kashiwa Dubna (2) HIBMC, Hyogo PMRC (2), Tsukuba NPTC, MGH HIBMC, Hyogo INFN-LNS, Catania WERC Shizuoka MPRI (2) Wanje, Zibo

WHERE

CA. USA CA. USA Sweden MA. USA Russia Russia NM. USA Russia CA. USA Japan Canada Switzerland Japan Switzerland Sweden England CA. USA Belgium France France South Africa IN USA CA USA Japan Canada Switzerland Germany Germany Japan Russia Japan Japan MA USA Japan Italy Japan Japan IN USA China

WHAT

p He p p p p pp ion p ! ! p p p p p p p p p p p C ion p p C ion p p p p p p C ion p p p p p

DATE FIRST RX 1954 1957 1957 1961 1967 1969 1974 1975 1975 1979 1979 1980 1983 1984 1989 1989 1990 1991 1991 1991 1993 1993 1994 1994 1995 1996 1997 1998 1998 1999 2001 2001 2001 2002 2002 2002 2003 2004 2004

DATE LAST RX — 1957 — 1992 — 1976 — 2002 — 1996 — 1982 — 1992 — 1994 — 1993 — 2000

– 1993

– 1999

TOTAL

232

RECENT PATIENT TOTAL 30 2054 73 9116 124 3785 230 1145 433 145 367 503 700 4182 418 1372 9585 21 2555 2805 468 34 632 1796 89 209 198 546 300 296 483 492 973 30 82 19 100 21 1 1100 4511 40801 46412

DATE OF TOTAL

Dec, 2004 Apr, 2004 Apr, 2002

Jan, 2004 Dec, 2004 Nov, 2004 Apr, 2004 Dec, 2003 Nov, 2004 Jun, 2004 Feb, 2004 Dec, 2003 Dec, 2004 Dec, 2003 Dec, 2004 Oct, 2004 Dec, 2004 Dec, 2004 Jul, 2004 Dec, 2004 Dec, 2002 Oct, 2004 Oct, 2004 Dec, 2004 Jul, 2004 Dec, 2004 pions ions protons all particles

Cancer Therapy Vol 4, page 233

Figure 1. Energy deposition plots comparing various radiation sources.

treatment fields based on the 3D anatomy of targets and organs at risk. In 3D-CRT the dose distributions are therefore shaped by defining beam number and orientation, field shape and weight. In the last decade, the possibility of achieving additional dose conformality was significantly increased with the introduction of Intensity-Modulated Radiation Therapy (IMRT) (Webb, 2001). IMRT is a technical improvement of CRT, aiming at an increased capability of applying non–uniform (modulated) intensity of radiation over the treatment field allowing the best compromise between tumor irradiation and OAR sparing. The irradiation patterns of IMRT are characterized by a large number of fields, each with its own shape and weight, which are obtained with to a highly computerized treatment planning procedure. Thanks to that, and to the technical improvements of the delivery methods at the linear accelerator, with IMRT it is possible to generate dose distributions that follow more accurately the shape of the target volume, thus reducing the volume of organs at risk irradiated at high doses As we said earlier, with photons it is possible to modulate the dose only in two-dimension, while modulation in the third dimension (i.e. along the beam axis) is impossible due to the properties of physical interactions between photons and matter. With protons, on the contrary, the penetration depth can be controlled within very few millimeters by just properly ‘tuning’ the energy (Figure 1). The physics of interaction of protons with tissues is such that protons deliver little dose in their path and then, just before stopping, lose most of their energy, thus delivering most of the dose at the point referred to as the ‘Bragg peak’. As a

consequence, while photon fluence can be controlled just in the plan orthogonal to the beam direction by properly shaping the beam apertures, proton fluence can be controlled in all three dimensions of space, including along the beam directions. The most evident effect of the sharp distal dose fall of is that it allows to spare those OARs located just downstream of the target along the beam direction. In addition, proton beams used for the clinical treatments have very good ballistic and geometrical qualities. The geometrical definition is of the order of a millimeter and also the precision for spotting the target is of the same magnitude. The energy deposition depth, as shown in the picture (Figure 1), is very well controlled specially in the beam direction with a sharp distal dose fall off. Finally, from a radiobiological point of view, protons show a small benefit compared to X-rays, having a relative biological effectiveness (RBE) of 1.1 (Paganetti et al, 2002).

III. Equipment and beam delivery Figure 2 schematically demonstrates the basic features that constitute a proton therapy (PT) apparatus: the high energy proton source which is an accelerator delivering protons at predetermined velocity (cyclotron or synchrotron) the beam transport line, which brings the protons to a certain space position preserving their geometrical properties the beam delivery line. This is the most demanding part from the technology point of view. It can be designed as a fixed beam delivery line or as a rotating system (gantry). In the second option, the beam can be delivered to the patient from any angle (360°) 233

Amichetti et al: Proton radiation therapy in oncology similar to the conventional X-ray radiotherapy linear accelerator. The patient couch for the proper positioning and centering of the body on the axis Figure 3 shows a possible layout.

PT has been delivered for more than 40 years, the accumulated experience in the clinical setting can still be considered limited and PT is probably far from having reached its best performances. However, many types of tumors have been treated and interesting results obtained. A review of the results obtained for selected tumor sites is reported below.

IV. Clinical indications The attractiveness of proton beam from a clinical point of view is based on its capability to deposit its energy at precise and controllable depths and to stop in the target. Proton therapy has been historically used in quite rare tumors located close to critical normal tissues and, in particular, close to serially organized tissues (i.e. spinal cord) where it is difficult to achieve an adequate radiation dose and avoid fatal complications even with a small local overdose or unwanted small dose deposition. Even though

A. Eye Uveal melanoma is the most frequently observed ocular tumor. Enucleation has been for years the standard treatment method of cure for this tumor, but it has been recently reassessed due to other therapeutic developments. Conservative methods such as trans-scleral local resection, transretinal resection, diode laser phototherapy and radiotherapy techniques (brachytherapy with radioactive plaques, stereotactic radiotherapy (SRT), proton

Figure 2. Schematic of the basic equipment involved in the proton therapy.

Figure 3. Possible layout proposed by an industrial provider (IBA) with one cyclotron, two gantries, and one fixed beam delivery systems.

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Cancer Therapy Vol 4, page 235 irradiation) are an adequate alternative to enucleation depending on tumor size, location and growth rate (Damato, 2006). Radiotherapeutic treatments induce growth arrest within the tumour and its slow involution over several years. Given the proximity to the tumor of critical structures of the eye, including cornea, lens, optic nerve and fovea, conventional radiotherapy is not able to preserve useful vision and special techniques as the above mentioned are needed. Although ocular retention rates are excellent, regrowth of tumors due to resistance and neovascular glaucoma can lead to enucleation of up to 10% of affected eyes. The goal of irradiation techniques is to maintain an useful vision by employing a treatment with a few side effects as possible. Protons and other charged particles offer significant advantages for tumors near the optic nerve and macula and large tumors in comparison with radioactive plaques (Gragoudas and Lane, 2005) and seem favourable in term of side effects development rates (Puusaari et al, 2004). In comparison with SRT, protons can result in similar levels of dose conformation but with better dose homogeneity and with a better sparing of the homolateral structures most important for visual acuity, lacrimal gland, and of all the contralateral OAR (Weber et al, 2005, Hocht et al, 2005). Ocular tumors are treatable with protons in nonhospital designed facilities in a relatively simple fashion with fixed fields of low energies (60-70 MeV), due to the intrinsic dose localization properties of the beam and the relative ease of head immobilization. The most significant experiences in the definitive treatment of uveal melanoma are those of the Massachusetts General Hospital (Munzenrieder, 1999), Paul Scherrer Institute (Egger et al, 2003), Orsay (Dendale et al, 2006), Nice (Courdi et al, 1999), Clatterbridge (Damato et al, 2005) and Berlin (Hocht et al, 2004) where thousands of patients have been treated obtaining local control rates between 89% and 97% and eye preservation in 87.5-92% of cases (Table 2) with limited side effects. These results indicate that the conservative treatment with PT can be considered the treatment of choice for uveal melanoma, especially if deeply located and large in size, having resulted in a comparable risk of incidence of metastasis respect to enucleation. Very good results are reported also in the treatment of retinoblastoma approaching a local control of 100%

(Krengly et al, 2005), orbital rhabdomyosarcoma (Yock et al, 2005), and choroidal metastases (Tsina et al, 2005). Another field of interest is that of macular degeneration disease of the retina, a typical nononcological disease of the old age where PT can reach a stabilization or an improvement in 75% of the cases (Yonemoto et al, 1996). However, the discussion of the appropriate treatment and of the definitive role of PT in this disease remains still open (Ciulla et al, 2002).

B. Skull base and paraspinal tumors Tumors of the base of the skull are typically located very close to dose-limiting tissues (brain, brainstem, optic chiasm, optic nerves, spinal cord, pituitary) and are represented mainly by chordomas and chondrosarcomas; these often slow-growing lesions have a low potential of metastatization and therefore local tumor control can result in cure of the patient (Mendenhall et al, 2005). These tumors cannot generally be resected completely and the dose traditionally safely deliverable with conventional radiotherapy was often limited to typical levels of 60-65 Gy (Catton et al, 1996; Zorlu et al, 2000). The results of PT are clearly superior in comparison with those obtained by conventional radiotherapy, even though other new radiotherapeutic modalities such as SRT with Cyberknife (Gwack et al, 2006) or IMRT and tomotherapy (Soisson et al, 2006) could be a potentially valuable treatment option once the long-term results are defined. Results of the literature show that with protons it is possible to deliver high doses of radiation (up to 70-74 Gy) to the tumor while observing a low risk of damage to critical organs. Specifically for chondrosarcomas and subgroups of chordomas postoperative PT after maximal surgery can be now considered the treatment of choice (Table 3 for a summary of the results). Benign meningiomas in general do not require radical dose levels for long term tumor control and can well be treated with modern photons radiotherapy (Metellus et al, 2005; Milker-Zabel et al, 2005). Here proton radiation therapy is reserved for hard-to-treat unresectable disease in order to minimize risks of side effects since these patients can live for a long time. However, for Atypical and Malignant Meningiomas the results after conventional RT are still disappointing and high dose RT as delivered by PT has demonstrated some encouraging results (Table 3). Prospective studies in this field are needed.

Table 2. Five-year results of proton therapy in uveal melanoma Institutio / (Authors) MGH / (Munzenrider et al, 1999) Nice / (Courdi et al, 1999) PSI / (Egger et al, 2003) Clatterbridge / (Damato et al, 2005) Berlin / (Hocht et al, 2004) CPO / (Dendale et al, 2006)

years 1975-98 1991-96 1984-99 1993-03 1998-03 1991-01

N. 2586 538 2645 349 245 1406

LC 96% 89% 95% 96.5% 째95.5% 96%

Eye ret. 90% -89% 90.6% 째87.5% 92%

Surv. 80% 77% 73% 90% 79%

Abbreviations: MGH: Massachussets General Hospital; PSI: Paul Sherrer Institute; CPO: Centre de Protontherapie de Orsay; LC: local control; Surv.: survival; N.: number; ret.: retention 째 at three years

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Amichetti et al: Proton radiation therapy in oncology Table 3. Results* (five-year local control rate) achieved with protons in tumors of the base of the skull

Meningioma Chordoma Chondrosarcoma

MGH 88% 73% 98%

NAC 89% 76% 92%

^PSI 91.7% 87.5% 100%

째CPO 98% 53.5% 85%

Tsukuba 46%

Abbreviations: MGH: Massachussets General Hospital; PSI: Paul Sherrer Institute; CPO: Centre de Protontherapie de Orsay; LLUMC: Loma Linda University Medical Center; NAC: National Accelerator Centre, South Africa *data obtained from the reports of Munzenrider and Liebsch 1999; Hug et al, 1999; Wenkel et al, 2000; Vernimmen et al, 2001; Noel et al, 2003, 2005a,b, 2005; Weber et al, 2004a,b; Igaki et al, 2004. ^: data at 3 years; 째 data at 4 years obtained with mixed beams (protons/photons)

Another very similar clinical situation is represented by the juxtaspinal cord tumors that are rarely completely resectable. Usually they invade or adhere to spinal cord, vertebrae or peripheral nerve roots. Moreover, spinal cord represents a dose limiting organ for conventional radiotherapy, as the maximum doses safely deliverable to this organ are in the range of 45-55 Gy, and therefore inadequate specifically for macroscopic residual disease. The dose given to residual or compressing masses could be safely increased in comparison with three-dimensional conformal photon treatment (Isaccson et al, 1997). High rates of local control can be observed with the use of protons (Fagundes et al, 1995).

high-grade acute or late morbidity. The published series are however limited and even if they compare well with those of radical surgery, brachytherapy, and of the most modern radiotherapeutic approaches (3-D conformal radiotherapy and IMRT), they have relatively short follow-up periods and require further confirmation in larger, possibly controlled, trials.

E. Head and neck Some tumors of the head and neck, such as the maxillary sinus tumors, represent a challenge in radiotherapy for their shape, dimensions and location that make it very difficult to deliver curative doses (Miralbell et al, 1992). PT can offer potential improvement in the outcome of these tumors. Many tumors have been treated in this anatomic area with encouraging results and quite limited toxicity (Tokuuye et al, 2004). PT has been used also in nasopharyngeal recurrent carcinoma (Lin et al, 1999) oropharyngeal tumors (Slater et al, 2005) with mixed photon-protons therapy and with a multimodal approach in neuroendocrine tumors of the sinonasal tract (Fitzek et al, 2002). Excellent results can be obtained in the treatment of acoustic neuroma (Bush et al, 2002) particularly in large and irregularly shaped tumors with radiosurgical methods or with a fractionated course.

C. Arteriovenous malformations of the brain Arteriovenus malformations can be treated with interesting results and a high rate of obliteration particularly in large sized lesions (Miralbell and Urie, 1993; Amin-Hanjani, 1999; Vernimmen et al 2005). Non resectable lesions or not fully embolizable ones are usually treated at MGH and LLUMC with two fractions of 10 Gy (Laramore and Phillips, 2004).

D. Prostate The treatment of prostate cancer could benefit from an improved dose distribution (Sandler et al, 1992). This is the only site until now where protons have been used in a randomized trial to test the effect of dose escalation (Shipley et al, 1995). After a pelvic dose of 50.4 Gy with photons, a 25.2 Gy equivalent proton boost was compared with 16.8 Gy photon boost. Results of proton boost were favorable only in a subgroup of patients with poorly differentiated tumors but late toxicity was increased (Benk et al 1993). It is however to note that in this trial, due to the limitation of the beam, simple techniques were used. Further studies have been developed with more modern proton facilities showing favorable results with more than 70% of biochemical control in an unselected population (Slater et al, 2004, Rossi et al, 1998, Hara et al, 2004) with minimal morbidity. A recently published phase III trial (Zietman et al, 2004) performed a dose escalation with a conformal proton boost of 19.8 or 29.8 Gray equivalent (GyE) followed by a 3D X-ray pelvic treatment to total doses of 70.2 GyE and 79.2 GyE, respectively. Higher radiation doses resulted in an increase of biochemical failure-free survisal without any comparable increase in

F. Lung The current standard dose for definitive radiotherapy is 60-66 Gy, which is significantly lower than the dose required for more than 50% local control; a dose of about 80 Gy is required for a probability of LC of 90% (Martel et al, 1999; Murshed et al, 2004). Unfortunately, significant toxicities at normal lung, spinal cord, esophagus, and heart limit the possibility of dose escalation for conventional fractionated RT. At LLUMC patients with inoperable (for medical reasons) non-small cell lung cancer were treated with hypofractionated protons obtaining particularly favorable response in stage I patients (86% disease free at 2 years) (Bush et al, 1999). Recent data of the same group (Bush et al, 2004) and (Shioyama et al, 2003) confirm these favorable results in this stage. These results have to be compared with recent data of stereotactic hypofracionated RT (McGarry et al, 2005; Beitler et al, 2006; Nyman et al, 2006) where, however, results seems to be inferior and toxicity greater, particularly in larger tumors.

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Cancer Therapy Vol 4, page 237 radiotherapy in the near future (Goitein and Jerman, 2003). This cost should be evaluated in a cost/effectiveness analysis in the frame of the efficacy that this treatment modality will be able to show. It is possible that higher treatment costs will be acceptable in certain subsets of patients (Lundqvist et al, 2005a) taking into consideration of the â&#x20AC;&#x153;quality adjusted life yearsâ&#x20AC;? gained (QALY). We should not restrict our thinking to the mere cost of the treatment but consider the total cost for the health care system of a patient cured without severe side effects. This is valid mainly in oncology where radiotherapy has to be considered substantially cheaper in comparison to many other forms of treatment (Lievens and den Bogaert, 2005).

G. Pediatric tumors The greatest challenge for the treatment of pediatric tumors is to attain the highest probability of cure with the least morbidity. It is essential in radiating children with cancer to limit the high-dose treatment area to the tumor only and minimize the radiation dose to the surrounding tissues (Lin et al, 2000). Even though the clinical relevance of the integral dose is not well known, the reduction of the dose (low-dose region and median-dose region) widespread outside the target is of utmost importance in pediatric tumors. In these patients it is very important not only to deliver the dose with high conformality but also to spare normal tissues in the development stages, minimizing the risk of late sideeffects and radiation-induced malignancies. Many examples of therapy with proton beam are available in the literature such as the treatment of patients with medulloblastoma, retinoblastoma, rhabdomyosarcoma, craniopharyngioma (Archambeau et al, 1992; Yuh et al, 2004; Krengli et al, 2005; Luu et al, 2006) where protons have demonstrated their capability of superior target dose coverage and sparing of normal structures (Lee et al, 2005). In particular in the treatment of medulloblastoma, PT shows the advantage to reduce toxicity (Yuh et al, 2004) and to be cost-effective (Lundkvist et al, 2005). A strong additional argument supporting the use of PT in young patients is the potential significant reduction of secondary cancers (Miralbell et al, 2002; Hall and Wuu, 2003)

H. tumors

Gastrointestinal

and

VI. Conclusions During a period of about 40 years PT has been available mainly in research institutions where particle accelerators built for physics research were used to treat patient, thus limiting the take full clinical benefit of this technology. The current trend, however, sees an increased interest in protons from clinically oriented centers. Increasing popularity, acceptance, and availability of protons will result in increasing use. Additional indications are being currently actively explored and under investigation. Due to their physical properties (absence of exit dose), the high-energy protons can deliver a highly conformal dose distribution in comparison to x-rays, when comparable techniques are used, allowing the ultimate expression of conformal irradiation. This technology can reduce the irradiation of normal tissues and improve the possibility of dose escalation with greater chances of local control. It is of utmost importance in the near future to demonstrate that these potential advantages will result in better outcome for cancer patients by conducting appropriate clinical trials and comparing this technology with other competitive X-ray treatment modalities.

abdominal

In many mucosal and glandular sites of the enteric apparatus, proton beam therapy showed to be feasible and well tolerable. Many data are reported in the literature based on the experiences made primarily in Japan on esophageal tumors (Koyama and Tsujii, 2003; Sugahara et al, 2005), and pancreatic cancer (Hsiung-Stripp et al, 2001). In the context of a renewed interest in the irradiation of hepatocellular carcinoma (Ben-Josef and Lawrence, 2005), proton therapy showed to be efficacious and well tolerated in the treatment of these lesions (Bush et al, 2004; Chiba et al, 2005; Hata et al, 2005; Kawashima et al, 2005).

Aknowledgements The authors thanks Eugene B. Hug for his critical reading of the manuscript and for the helpful suggestions.

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Wenkel E, Thornton AF, Finkelstein D, Adams J, Lyons S, De La Monte S, Ojeman RG, Munzenrider JE (2000) Benign meningioma, partially resected, biopsied, and recurrent intracranial tumors treated with combined proton and photon radiotherapy. Int J Radiat Oncol Biol Phys 48, 1363-1370. Wilson RR (1946) Radiological use of fast protons. Radiology 47,487-491. Yock T, Schneider R, Friedmann A, Adams J, Fullerton B, Tarbell N (2005) Proton radiotherapy for orbital rhabdomyosarcoma: clinical outcome and a dosimetric comparison with protons. Int J Radiat Oncol Biol Phys 63, 1161-1168. Yonemoto LT, Slater JD, Friedrichsen EJ, Loredo LN, Ing J, Archambeau JO, Teichman S, Moyers MF, Blacharski PA, Slater JM (1996) Phase I/II study of proton beam irradiation for the treatment of subfoveal choroidal neovascularization in age-related macular degeneration: treatment techniques and preliminary results. Int J Radiat Oncol Biol Phys 36, 867-871. Yonemoto LT, Slater JD, Rossi CJ Jr, Antoine JE, Loredo L, Archambeau JO, Schulte RW, Miller DW, Teichman SL, Slater JM (1997) Combined proton and photon conformal radiation therapy for locally advanced carcinoma of the prostate: preliminary results of a phase I/II study. Int J Radiat Oncol Biol Phys 37, 21-29. Yuh GE, Loredo LN, Yonemoto LT, Bush DA, Shahnazi K, Preston W, Slater JM, Slater JD (2004) Reducing toxicity from craniospinal irradiation: using proton beams to treat medulloblastoma in young children. Cancer J 10, 386-390. Zietman AL, DeSilvio M, Slater JD, Rossi CJ, Yonemoto LT, Slater JM, Miller DW, Berkley B, Adams JA, Shipley WU (2004) randomized trial comparing conventional dose (70.2 GyE) and high-dose (79.2 GyE) conformal radiation in early stage adenocarcinoma of the prostate: results of an interim analysis of PROG 95-09. Int J Radiat Oncol Biol Phys 60, 131. Zorlu F, Gurkaynak M, Yildiz F, Oge K, Atahan IL (2000) Conventional external radiotherapy in the management of clivus chordomas with overt residual disease. Neurol Sci 21, 203-207.

Maurizio Amichetti

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Cancer Therapy Vol 4, page 241 Cancer Therapy Vol 4, 241-248, 2006

Muscle strength and functional ability in children during and after treatment for acute lymphoblastic leukemia or T-cell Non-Hodgkin lymphoma: a pilot study Research Article

Marja Schoenmakers1, Tim Takken1,*, Vincent AM Gulmans1, Nico LU Van Meeteren2, 3, Marrie CA Bruin4, Tom Révész4, Paul JM Helders1 1

Department of Pediatric Physical Therapy & Exercise Physiology, Wilhelmina Children’s Hospital, University Medical Center, Utrecht, The Netherlands, 2 Rudolf Magnus Institute of Neuroscience, Department of Neuroscience, Section Rehabilitation, University Medical Center, Utrecht, The Netherlands, 3 Department of Physiotherapy, Academy of Health Sciences, Utrecht, The Netherlands, 4 Department of Pediatric Hematology, Wilhelmina Children’s Hospital, University Medical Center, Utrecht, The Netherlands

__________________________________________________________________________________ *Correspondence: Tim Takken, Department of Pediatric Physical Therapy & Exercise Physiology, University Medical Center, Wilhelmina Children’s Hospital, RM KB 02. 056.0, P.O. Box 85090, 3508 AB Utrecht, The Netherlands; Phone: +31302504030; Fax: +31302505333 ; E-mail: t.takken@umcutrecht.nl Key words: leukemia; children; muscle strength; functional skills; motor performance Abbreviations: 6 months after diagnosis, (T3); 6 months after the end of treatment, (T5); 6-mercaptopurine, (6MP); 6-thioguanine, (6TG); acute lymphoblastic leukemia, (ALL); at diagnosis, (T1); at the end of treatment, (T4); cytosine-arabinoside, (Ara-C); daunorubicine, (DNR); dexamethasone, (dexa); dose of intrathecal medications is based on age, (*); doxorubicine, (Adria); Dutch Childhood Leukemia Study Group, (DCLSG); event-free survival, (EFS); following induction, (T2); intrathecal, (i.th)); L-asparaginase, (L-Asp); methotrexate, (MTX); Pediatric Evaluation of Disability Inventory, (PEDI); prednisone, (Pred); T-cell non-Hodgkin Lymphoma, (T-NHL); vincristine, (VCR) Received: 15 March 2006; Revised: 24 March 2006 Accepted: 31 July 2006; electronically published: August 2006

Summary The aim of this pilotstudy was to explore the course of muscular strength, functional skills, and motor performance in children during and after treatment for acute lymphoblastic leukemia (ALL) or T-cell non-Hodgkin Lymphoma (T-NHL). Eighteen children and adolescents, aged between 0-18 years, with standard-risk ALL or with T-NHL were included in this prospective descriptive study. Nine were treated according to a BFM-based protocol (ALL-8), and 9 with protocol ALL-9 which is an antimetabolite-based treatment and uses high doses of dexamethasone and vincristine. Since there were no statistically significant differences between these two groups, the data were pooled for analysis. Muscle strength and functional skills were measured during and after treatment. Motor performance was measured only after treatment. Muscle weakness occurred in all patients, and was most severe during the first two months of treatment. After cessation of treatment, muscle strength recovered towards normal for most muscle groups, although knee- and foot extensors were still decreased as compared to reference values. Similarly, functional skills were also deficient in the first two months, mainly concerning transfers, walking, and going up- and down stairs. After cessation of treatment, these basic skills normalized. Six months after treatment, fine motor problems were present in 2 patients, and gross motor problems in 4 of the 18 patients. Muscle weakness and mobility problems were most severe in the first two months of treatment. These problems were reversible in most patients. However, in some children muscle weakness and fine and motor deficits were present after treatment.

lymphoblastic leukemia (ALL) are much improved due to the use of intensive combination chemotherapy. However,

I. Introduction The

treatment

results

childhood

acute 241

Schoenmakers et al: Functional outcome in children with leukemia vincristine during the whole treatment period of two years (Table 2). In protocol 8 both dexamethasone and prednisone were used as corticosteriod-therapy, in protocol 9 this was dexamethasone. The equivalent doses of steroids were also 5-6 times higher in protocol 9 than protocol 8. On the other hand, the latter protocol contained more cystostatic agents (daunorubicine, ara-C, 6-thioguanine). Both protocols did not include cranial irradiation.

these results come at a cost: short and long-term side effects of chemotherapy. Among other complications, muscle weakness is increasingly recognized as a problem in children treated for ALL (Hovi et al, 1993) In 1984 the Dutch Childhood Leukemia Study Group (DCLSG) developed a relatively non-toxic, antimetabolite-based treatment protocol (ALL-6) with high cumulative doses of vincristine and dexamethasone for standard-risk ALL patients (Veerman et al, 1996). The 8-year event-free survival (EFS) on this protocol was 80%. During and after treatment with this protocol, many children had significant gross- and fine motor difficulties, thought to be due to a vincristine-induced neuropathy (Ryan and Emami, 1983; Postma et al, 1993; ReindersMesselink et al, 1996). Between 1991 and 1996 the DCLSG followed a slightly modified BFM protocol (DCLSG-ALL 8) which included lower doses of vincristine and steroids (Kamps et al, 1999). Since 1997 all Dutch children with ALL are treated with protocol ALL-9, which again is based on the ALL-6 protocol that includes vincristine and dexamethasone pulses during the whole treatment period of two years. Vainionpää, (1993) and Reinders-Messelink et al, (1996, 1999) described motor problems in children during and after treatment for ALL. In these studies, motor problems like gait disturbances, clumsiness and fine motor problems are mainly attributed to peripheral neuropathy, which is thought to be a neurotoxic side effect of vincristine. Although Reinders-Messelink and colleagues, systematically investigated in 1999 the relationship between motor problems and vincristine, possible toxic effects of other medications could not be excluded. To our knowledge, the course of muscular weakness, motor performance and activities of daily living, has sparsely been explored before in children treated for ALL. The aim of this pilotstudy was to explore the clinical course of muscle strength, functional skills and motor performance as well as the reversibility of these symptoms in children treated according to protocol ALL-8 or ALL-9.

B. Measurements Muscle strength and functional skills were evaluated prospectively at the time of diagnosis (T1=week 0), twice during treatment (T2 = week 7 and T3 = week 28), at the end of treatment (T4 = week 105) and 6 months after treatment (T5 = week 131) (Table 2). Motor performance and hand-held myometry were evaluated 6 months after treatment (T5). All measurements were performed by the same pediatric physical therapist (MS).

C. Muscle strength Strength of the upper and lower extremity muscles was scored according to the manual muscle testing criteria of the Medical Research Council, using a 6-point scale (range 0-5) (Medical Research Council, 1950). As manual muscle testing is a less reliable method measuring strength for grade 4 or 5 (Wadsworth et al, 1987), a hand-held myometer (Penny and Giles Biometrics Ltd., Blackwood, Gwent, UK.) was used in children with strength ! grade 4, to evaluate upper and lower extremity muscles on the non-dominant side, according to Bäckman et al (1989). The following muscles were assessed: shoulder abductors, elbow flexors, wrist extensors, hip flexors, hip abductors, hip extensors, knee extensors, and dorsal extensors of the foot. The scores of these muscle groups were compared with published reference values for healthy children (Backman et al, 1989).

D. Functional skills Functional skills were measured with the adapted Dutch version of the ‘Pediatric Evaluation of Disability Inventory’ (PEDI) (Haley et al, 1992; Custers et al, 2002). The PEDI is a validated and reliable parental questionnaire which measures functional skills and the amount of caregiver assistance in three domains: self-care, mobility and social function (Haley et al, 1992). With this reference-based instrument, discrimination can be made between disabled and non-disabled children (Haley et al, 1992). The domain of self-care includes eating, grooming, dressing, bathing and toileting skills. The mobility domain includes transfers, indoor and outdoor mobility, and capability to climb stairs, walking distance and walking speed. The social domain includes communication, social-interaction, and household and community tasks. Reference values are provided for children between 0.5-7.5 years. Normal values are defined in the range of 2 SD score (50 ± 20). In children over the age of 7.5 years, all functional skills should be mastered leading to a scale score of 100, which is considered to be normal (Haley et al, 1992).

II. Methods and Patients A. Patients Nineteen patients, aged between 0-18 years, who started treatment for ALL or T-NHL at the Pediatric HematologyOncology Clinic of the Wilhelmina Children’s Hospital, University Medical Center Utrecht, the Netherlands from January 1996 to December 1998, were included in this study. Informed consent was obtained from their parents and also from the children if they were older than 12 years of age. Of these 19 children, one was excluded, because in this child complete remission of the disease was not achieved. Of the remaining 18 children, 9 were treated according to protocol ALL-8 including three children with T-cell Non-Hodgkin lymphoma, and 9 according to protocol ALL-9. There was a change of treatment protocols on 1-1-1997. The last 9 consecutive patients treated prior to this date and according to the DCLSG protocol ALL-8 were included in the study, along with the first 9 consecutive patients on the new ALL-9 protocol. All patients were evaluated during and after treatment. The outline of treatment according to the two protocols is shown in Table 1. Children treated with protocol ALL-8 received 8 x 1.5 mg/m2 vincristine in two periods of four weeks. Children treated with protocol ALL-9 received 34 x 2.5 mg/dose

E. Motor performance In children from 4 years of age and up, motor performance was measured with the Movement Assessment Battery for Children (Henderson and Sugden, 1992). The Movement-ABC is a valid and reliable instrument that has been developed to evaluate gross and fine motor function in children from 4 years of age (Henderson and Sugden, 1992). Percentile scores of the child’s motor abilities can be compared with a normative agematched sample of children. A score below the 5th percentile

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Cancer Therapy Vol 4, page 243 indicates that the child has significant movement difficulties. In scores between the 5th and 15th percentile, the child is at risk for these difficulties. The motor performance is adequate in scores above the 15th percentile (Henderson and Sugden, 1992).

III. Results Eighteen patients were examined in this study. Data concerning gender, age and type of leukemia are presented in Table 1. The percentage of muscle groups with impaired muscle strength (MRC " grade 4) during and after treatment is shown in Figure 1. One patient has already received 1 dose of vincristine at the time of first examination (T1); therefore his scores were not included. One four year old girl did not co-operate on muscle testing, while 5 children were less than four years old and were too young to be cooperative for adequate muscle testing. At T2 and T3 there are missing data due to the fact that some children were too ill to co-operate. At T4, 2 patients did not show up for the follow-up examination. As can be appreciated from Figure 1, muscle weakness was most apparent 7 weeks following chemotherapy induction (T2). All patients had muscle strength " 4 in at least one muscle group. Muscle weakness occurred both in upper and lower limbs, and proximal as well as distal. Six months after the end of

F. Statistical analysis Our original intention was to compare the results of the two groups. However, due to the small number of patients and the individual variability in response, there were no statistically significant differences between the two groups with respect to any of the variables. Therefore, the data of both groups were pooled and used together in the statistical analysis. Descriptive statistics were used to explore the course of the measures. Z-scores or percentile scores were calculated when reference values in a healthy population were available. The nonparametric Friedman test (Friedman, 1937) was used for analyzing repeated measurements. When the differences between the groups appeared to be significant (P < 0.05), the Wilcoxon signed-rank test was used to detect the significant differences. Differences in percentages were tested using Fishers exact test. All data were analyzed using SPSS 11.5 for Windows.

Table 1. Patient characteristics and outline of treatment according to protocol ALL-8 and ALL-9 ALL-8 9 5:4 8.7 (1-16)

ALL-9 9 4:5 7.5 (2-15)

VCR/Pred/DNR/L-Asp + MTX/Ara-C/Pred i.th.

VCR/Pred/L-Asp + MTX/Ara-C/Pred i.th.

Intensification

MD-MTX/6MP + MTX/ Ara-C/Pred i.th.

MD-MTX/6MP + MTX/Ara-C/Pred i.th.

Reinduction

VCR/Dexa/Adria/L-Asp/ 6MP, Ara-C, 6TG +

none

Maintenance

MTX/Ara-C/Pred i.th.

6MP/MTX + Q5 weeks: VCR/Dexa

Number Male to female ratio Age, mean (range) Medication Induction

6MP/MTX Abbreviations: 6-mercaptopurine, (6MP); 6-thioguanine, (6TG); cytosine-arabinoside, (Ara-C); daunorubicine, (DNR); dexamethasone, (dexa); doxorubicine, (Adria); intrathecal, (i.th)); L-asparaginase, (L-Asp); methotrexate, (MTX); prednisone, (Pred); vincristine, (VCR)

Table 2. Test periods and cumulative doses of vincristine and steroids Assessments Weeks after diagnosis Cumulative dose vincristine (mg/m2) ALL-8 ALL-9 Cumulative dose of corticosteroids ALL-8: prednisone (mg/m2) prednisone intrathecal (mg/age)* ALL-9: dexamethasone (mg/m2) prednisone intrathecal (mg/age)*

T1 0

T2 6-8

T3 28

T4 105

T5 131

0 0

6 21.25

12 42.5

12 85

0 0

1600 12-24

1600 36-72

1600 48-84

0 0

210 16-24

462 64-96

1386 104-156

Abbreviations: 6 months after diagnosis, (T3); 6 months after the end of treatment, (T5); at diagnosis, (T1); at the end of treatment, (T4); dose of intrathecal medications is based on age, (*); following induction, (T2)

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Schoenmakers et al: Functional outcome in children with leukemia

Figure 1. Percentage of muscle groups with impaired strength (MRC " grade 4) during and after treatment (T1-T5). Legend; hip: hip muscles (hip flexors, hip abductors, hip extensors); leg: lower extremity (knee extensors, dorsal extensors of the foot), ue: upper extremity (shoulder abductors, elbow flexors, wrist extensors,). The number behind the muscle region denotes the measurement; 1: at diagnosis (N=11), 2: following induction (N=14), 3: 6 months after diagnosis (N=13), 4: at the end of treatment (N=16), 5: 6 months after the end of treatment (N=18).

treatment (T5), muscle weakness was improved to grade 5 in almost all patients (15/18). One adolescent still had muscle weakness in his hip- and shoulder abductors. Two children had weakness of hip-abductor or hip-extensor muscles. They where younger than 4 years of age at the start of treatment. In Figure 2 the Z-scores of muscle strength are displayed using hand-held myometry in 13 patients 6

month after the end of treatment. Almost all muscle groups were within the normal limits, however, the knee extensors and foot extensor were still significantly decreased in the patients. Myometry data of children under the age of 5.5 years (n=5) were excluded for this analysis, because these measurements were not reliable.

Figure 2. Z-scores (and 95% confidence intervals) of muscle strength with hand-held myometry 6 months after the end of treatment (T5).

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Cancer Therapy Vol 4, page 245 Data concerning functional skills measured on the PEDI during and after treatment are shown in Figure 3. Most problems were seen in the mobility domain at T2, which indicated problems with transfers, in- and outdoor locomotion, walking up- and down stairs, and restricted walking distance and walking speed. Six months after the end of treatment (T5), recovery was seen in all domains, except for the domain of social function, which was deviant in one eight-year-old boy with attention deficit disorder. Mean normative scores on the PEDI from T1 to

T5 were within the normal ranges (between 30-70) in the domains of self-care and social function. Deviant scores (< 30) were only seen in the domain of mobility following induction (T2). Data concerning motor performance as measured on the Movement-ABC six months after the end of treatment (T5) are shown in Figure 4. Eleven to 28 percent of the patients experienced problems in all three domains and in the total score. P-values were 0.24 for manual dexterity, 0.052 for Balance and Total score and 0.022 for ball skills.

Figure 3. Percentage of patients with deviant functional skills (PEDI scores < 30 in children < 7.5 years, and PEDI scores < 100 in children > 7.5 years) during and after treatment. Legend for measurement: 1: at diagnosis, 2: following induction, 3: 6 months after diagnosis, 4: at the end of treatment, 5: 6 months after the end of treatment. Only PEDI mobility was significantly increased at T1 and T2.

Figure 4. Percentage of patients with impaired motor performance (scores <15th percentile on the Movement-ABC) 6 months after the end of treatment (T5).

245

Schoenmakers et al: Functional outcome in children with leukemia Five children were younger than 4 years of age at the time of diagnosis. Six months after the end of treatment (T5), weakness of hip-extensor or hip-abductor muscles (MRC grade 4) was seen in 2 of these children. All five had normal functional skills in all the PEDI-domains, ranging from 35.1-72.1. Scores on motor performance were normal in 4 of these children, ranging from the 38th to the 93rd percentile. One boy, the youngest patient at time of diagnosis, had significant fine and gross motor difficulties after treatment (1st percentile). Severe vincristine neurotoxicity occurred in 1 patient. This child was younger than 4 years of age at time of diagnosis. She was treated according to protocol ALL9. She showed severe ptosis and very severe constipation after a few weeks of treatment. Her vincristine dose had to be lowered temporarily. At T5, her PEDI-scores on selfcare, mobility and social function were within normal ranges (48.1-54.5), as well as her motor performance (46th percentile score). This pilot study shows that muscle weakness, and mobility problems occurred in almost all patients during treatment for ALL. These problems were reversible in most patients; whereas gross and as well as fine motor problems were present in some patients after treatment. Although muscle strength recovered towards normal for most muscle groups, knee- and foot extensors were still decreased as compared to reference values. Over the last decade more attention has been paid to the consequences of chemotherapy on motor performance in children during and after treatment for ALL (Vainionpaa, 1993; Reinders-Messelink et al, 1996, 1999; Wright et al, 1998). In these studies, motor problems and muscle weakness, are mainly attributed to peripheral neuropathy. However, during intensive vincristine/ corticosteroid-based treatment protocols, steroid induced myopathy may also occur (DeAngelis et al, 1991). DeAngelis et al, (1991) described the course of muscle weakness due to either vincristine neuropathy or steroid myopathy in 27 adults during treatment for NonHodgkinâ&#x20AC;&#x2122;s lymphoma. In their study, all patients had moderate to severe signs of muscle weakness. They suggested that muscle weakness due to steroid use was typically proximal in location and primarily affected lower extremities. Weakness caused by vincristine was in their opinion mostly distal in location and affected hands and feet equally, resulting in problems with fine manipulative abilities. Our data on muscle strength in children are in part in agreement with DeAngelis et al, (1991). In the first two months of treatment, we found muscle weakness in at least one muscle group in all patients. It occurred in both proximal as well as distal muscle groups. After treatment, muscle strength improved to scores > 4 in almost all of our patients. Since manual muscle testing is less reliable in scores ! 4, hand-held myometry was also performed in these patients. Mean Z-scores for muscle strength using myometry were within normal ranges (> 2SD). However, knee extensors and foot extensors were still significantly decreased compared to references values. Recently, Gocha Marchese and colleagues also found a decreased muscle strength in ALL patients during treatment (Gocha Marchese et al, 2003). Moreover, Hovi et al, (1993)

investigated the late sequelae of leukemia treatment and found a decreased muscle strength in ALL patients off therapy for 1 to 19 years. They also found a decreased muscle endurance in these patients (Hovi et al, 1993), which is a frequent complaint in ALL patients after therapy (Jenney et al, 1995; Turner-Gomes et al, 1996; Warner et al, 1997). The observed muscle weakness might be due to a combination of steroid myopathy and vincristine myopathy and neuropathy. Wright and colleagues, studied in 1998 the long-term effects of cancer treatment on musculoskeletal function and gross motor skills in 36 children and compared their outcome with healthy peers. They found that survivors of ALL were able to perform most basic motor functions, such as walking, running and climbing stairs, but their levels of gross motor proficiency and performance (balance and running speed) were significantly lower than those of their school-aged peers. We found in fraction of our patients, deviant scores on PEDI before as well as during treatment for ALL. Initially, patients had deviant scores in the mobility domain (transfers, walking abilities) of the PEDI. The scores in self-care domain were also low at this time, but they were still within normal ranges. This might be biased due to the fact that 5 children in our study were younger than 4 years of age, and 3 were aged between 4-5 years. Caregiver-assistance in self-care skills is normal at this age. Eight of 18 children were unable to perform functional skills in the mobility domain during treatment. After treatment PEDI-scores normalized, however motor performance as measured on the Movement-ABC, was still deviant in 5 children, mainly concerning ball-handling and balance skills. Reinders-Messelink and colleagues, studied in 1999 motor performance in 17 children during and after chemotherapy. They found balance problems to be most severe during treatment: 50% (7/14) at the start of treatment, 69% (11/16) during treatment, and 27% (4/15) after treatment. An opposite pattern was seen in fine motor skills. The percentage of patients with fine motor problems was higher after treatment then at start, 33% (5/15) and 14% (2/14) respectively. Reinders-Messelink and colleagues, (1996, 1999, 2000) stated that a relation between these motor problems and vincristine-induced neurotoxicity seemed plausible but the effect of other neurotoxic drugs, like methotrexate and steroids could not be ruled out. The number of patients with fine motor problems in our study (2/18) is not significantly different from the number of patients (5/15) reported by ReindersMesselink and colleagues, (1996, 1999). We found gross motor disturbances more frequently than fine motor problems. This is in agreement with VainionpĂ¤Ă¤, (1993), who found gross motor disturbances in 30% (10/33) and fine motor problems in 18% (6/33) of the patients. Why some children seem never fully able to recover after cytostatic/corticosteroid treatment might depend more on their individual sensitivity to these agents and their pharmacokinetic variability than the actual doses of vincristine and corticosteroids. It is generally thought that children treated for ALL benefit from programs that promote physical activity to

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Cancer Therapy Vol 4, page 247 Hovi L, Era P, Rautonen J and Siimes MA (1993) Impaired muscle strength in female adolescents and young adults surviving leukemia in childhood. Cancer 72, 276-81.

improve gross motor function (Wright et al, 1998; Reinders-Messelink et al, 1999). Further studies are needed to clarify to what extent physical therapy can prevent, or improve disturbed muscle and motor function. Marchese et al, (2004) found significant improvement of muscle strength in children who received physical therapy intervention early in treatment for ALL. In an other small pilot study, it was found that exercise improved endurance, mood state and body fat levels (Shore and Shepard, 1999).

Jenney ME, Faragher EB, Jones PH and Woodcock A (1995) Lung function and exercise capacity in survivors of childhood leukaemia. Med Pediatr Oncol 24, 222-30. Kamps WA, Bokkerink JP, Hahlen K, Hermans J, Riehm H, Gadner H, Schrappe M, Slater R, van den Berg-de Ruiter E, Smets LA, de Vaan GA, Weening RS, van Weerden JF, van Wering ER and den der Does-van den Berg A (1999) Intensive treatment of children with acute lymphoblastic leukemia according to ALL-BFM-86 without cranial radiotherapy: results of Dutch Childhood Leukemia Study Group Protocol ALL-7 (1988-1991). Blood 94, 1226-36.

IV. Conclusions Severe muscle weakness and mobility problems (such as transfers, walking, navigating stairs) occurred in children treated for standard risk ALL or T-NHL. These problems were most severe in the first two months of treatment, but they were mainly reversible, whereas gross and fine motor deficits were still present six months after treatment in some patients. Although muscle strength recovered towards normal for most muscle groups, kneeand foot extensors were still decreased as compared to reference values. Muscle weakness occurred in both proximal and distal muscles and could be due to a combination of steroid myopathy, vincristine myopathy and neuropathy and inactivity. However, a more precisely planned prospective multi-center study with sufficient numbers of patients is indicated to study the effects of and recovery after chemotherapy in this patient group. Further studies are needed to clarify to what extent physical therapy can prevent, or improve disturbed muscle function during and after treatment for childhood leukaemia.

Marchese VG, Chiarello LA and Lange BJ (2004) Effects of physical therapy intervention for children with acute lymphoblastic leukemia. Pediatr Blood Cancer 42, 127-33. Medical Research Council (1950) Aids to the Investigation of Peripheral Nerve Injuries. War Memorandum No. 7, Report of the Council for the years 1939-45, Her Majesty Stationery Office, London. Postma TJ, Benard BA, Huijgens PC, Ossenkoppele GJ and Heimans JJ (1993) Long-term effects of vincristine on the peripheral nervous system. J Neurooncol 15, 23-7. Reinders-Messelink H, Schoemaker M, Snijders T, Goeken L, van Den Briel M, Bokkerink J and Kamps W (1999) Motor performance of children during treatment for acute lymphoblastic leukemia. Med Pediatr Oncol 33, 545-50. Reinders-Messelink HA, Schoemaker MM, Hofte M, Goeken LN, Kingma A, van den Briel MM and Kamps WA (1996) Fine motor and handwriting problems after treatment for childhood acute lymphoblastic leukemia. Med Pediatr Oncol 27, 551-5. Reinders-Messelink HA, Van Weerden TW, Fock JM, Gidding CE, Vingerhoets HM, Schoemaker MM, Goeken LN, Bokkerink JP and Kamps WA (2000) Mild axonal neuropathy of children during treatment for acute lymphoblastic leukaemia. Eur J Paediatr Neurol 4, 225-33.

References Backman E, Odenrick P, Henriksson KG and Ledin T (1989) Isometric muscle force and anthropometric values in normal children aged between 3.5 and 15 years. Scand J Rehabil Med 21, 105-14.

Ryan JR and Emami A (1983) Vincristine neurotoxicity with residual equinocavus deformity in children with acute leukemia. Cancer 51, 423-5.

Custers JW, Wassenberg-Severijnen JE, Van der Net J, Vermeer A, Hart HT and Helders PJ (2002) Dutch adaptation and content validity of the 'Pediatric Evaluation Of Disability Inventory (PEDI)'. Disabil Rehabil 24, 250-8.

Shore S and Shepard RJ (1999) Immune responses to exercise in children treated for cancer. J Sports Med Phys Fitness 39, 240-3.

DeAngelis LM, Gnecco C, Taylor L and Warrell RP, Jr. (1991) Evolution of neuropathy and myopathy during intensive vincristine/corticosteroid chemotherapy for non-Hodgkin's lymphoma. Cancer 67, 2241-6.

Turner-Gomes SO, Lands LC, Halton J, Hanning RM, Heigenhauser GJ, Pai M and Barr R (1996) Cardiorespiratory status after treatment for acute lymphoblastic leukemia. Med Pediatr Oncol 26, 160-5.

Friedman M (1937) The use of ranks to avoid the assumption of normality implicit in the analysis of variance. Journal of the American Statistical Association 32, 675-701.

Vainionpaa L (1993) Clinical neurological findings of children with acute lymphoblastic leukaemia at diagnosis and during treatment. Eur J Pediatr 152, 115-9.

Gocha Marchese V, Chiarello LA and Lange BJ (2003) Strength and functional mobility in children with acute lymphoblastic leukemia. Med Pediatr Oncol 40, 230-2.

Veerman AJ, Hahlen K, Kamps WA, Van Leeuwen EF, De Vaan GA, Solbu G, Suciu S, Van Wering ER and Van der DoesVan der Berg A (1996) High cure rate with a moderately intensive treatment regimen in non-high-risk childhood acute lymphoblastic leukemia. Results of protocol ALL VI from the Dutch Childhood Leukemia Study Group. J Clin Oncol 14, 911-8.

Haley SM, Coster WJ and Ludlow LH (1992) Pediatric Evaluation of Disability Inventory' (PEDI). Development, Standardization and Administration Manual., New England Medical Center-PEDI Research Group, Boston, MA.

Wadsworth CT, Krishnan R, Sear M, Harrold J and Nielsen DH (1987) Intrarater reliability of manual muscle testing and hand-held dynametric muscle testing. Phys Ther 67, 1342-7.

Henderson SE and Sugden DA (1992) Movement Assessment Battery for Children, The Psychological Corporation, Kent.

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Schoenmakers et al: Functional outcome in children with leukemia Warner JT, Bell W, Webb DK and Gregory JW (1997) Relationship between cardiopulmonary response to exercise and adiposity in survivors of childhood malignancy. Arch Dis Child 76, 298-303. Wright MJ, Halton JM, Martin RF and Barr RD (1998) Longterm gross motor performance following treatment for acute lymphoblastic leukemia. Med Pediatr Oncol 31, 86-90.

Tim Takken

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Cancer Therapy Vol 3, page 249 Cancer Therapy Vol 3, 249-252, 2005

An analysis on the gene expression profiling of lymphotoxin tumor necrosis factor-! in lymphoma Research Article

Viroj Wiwanitkit Department of Laboratory Medicine, Faculty of Medicine, Chulalongkorn University, Bangkok Thailand 10330

__________________________________________________________________________________ *Correspondence: Viroj Wiwanitkit, M.D., Department of Laboratory Medicine, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand 10330; Tel: 662 256 4136; Fax: 662 218 3640; e-mail: Viroj.W@Chula.ac.th Key words: Lymphotoxin tumor necrosis factor-!, gene expression, profiling Abbreviations: adult T cell leukemia, (ATL); focal adhesion kinase, (FAK); Gene Expression Pattern Analysis Suite, (GEPAS); human T cell leukemia virus type I, (HTLV-I); osteoclast activating factor, (OAF); Phosphatidylinositol 3-kinase, (PI3K); Self-Organising Tree Algorithm,, (SOTA); tumor necrosis factor-!, (TNF-!) Received: 12 April 2006; Revised: 10 June 2006 Accepted: 8 August 2006; electronically published: August 2006

Summary Lymphotoxin tumor necrosis factor-! (TNF-!) is believed to relate to hypercalcemia in patients with lymphoma. It is also found that the extent of TNF-! gene expression was correlated with the histopathological features of neovascularization and there was also a relationship between the extent of TNF-! gene expression and the presence of B-symptoms in those lymphoma patients, which could imply for the prognostic factor prediction. Here, the author performed a bioinformatics analysis on the gene expression pattern of lymphotoxin TNF-!. Based on transciptomic technology, a clustering gene expression of lymphotoxin TNF-! gene and other closed relate lymphoma genes was generated and presented. The DNA array data from the published report concerning lymphoma was extracted and further analyzed by Sotarray analysis. The result of the algorithm is a hierarchical cluster, genes with similar expression profiles are clustered together. According to this study, the node for lymphotoxin TNF-! gene and other closed relate lymphoma genes was derived at cluster â&#x20AC;&#x153;node 690â&#x20AC;?. The genes corresponding to focal adhesion kinase (FAK) and Phosphatidylinositol 3-kinase p110 catalytic gamma are the two known genes detected as closed related genes to lymphotoxin TNF-! gene. According to the closed related in gene expression profiling of the three genes, the concerns on the prognostic factor prediction as well as cancer treatment based on lymphotoxin TNF-! might be applied for the other two genes, FAK and PI3K. with human T cell leukemia virus type I (HTLV-I) (Kato et al, 1994). They also found that the extent of TNF-! gene expression was correlated with the histopathological features of neovascularization and there was also a relationship between the extent of TNF-! gene expression and the presence of B-symptoms in those lymphoma patients, which could imply the prognostic factor prediction (Kato et al, 1994). The antitumor effect of lymphotoxin and the underlying cellular mechanism have been mentioned (Qin and Blankenstein, 1995). The cancer treatment outcome might have some clinical correlated to the lymphotoxin TNF-!. However, the knowledge on the gene expression of lymphotoxin TNF-! gene is limited. Here, the author performed a bioinformatics analysis on the gene expression pattern of lymphotoxin TNF-!. Based on transciptomic technology, a clustering gene expression of

I. Introduction Hypercalcemia in hematological malignancy is frequently encountered in lymphoid malignancies such as adult T cell leukemia (ATL) and multiple myeloma and is difficult to manage (Ishibashi et al, 1992). Lymphotoxin tumor necrosis factor-! (TNF-!) is believed to relate to hypercalcemia in patients with lymphoma (Ishibashi et al, 1992). Ishibashi et al noted that the TNF-! secreted from ATL cells might be one of the factors contributing to the hypercalcemia in patients with ATL functioning as an osteoclast activating factor (OAF) (Ishibashi et al, 1992). In 1994, Kato et al investigated the levels of TNF-! mRNA in the tumorous tissues of a series of Japanese patients with lymphoma, to assess the contribution of the expression of this gene to the features of the disease and found the level of TNF-! mRNA was semiquantified against that in MT-2 cells, a line of human T cells infected 249

Wiwanitkit: Gene expression profiling of lymphotoxin TNF-! in lymphoma

Figure 1. Clustering gene expression of lymphotoxin TNF-! gene and other closed relate lymphoma genes

lymphotoxin TNF-! gene ! other closed relate lymphoma genes was generated and presented.

together in this study, these two genes should have closed related expressions. Concerning FAK, it is is activated by TNF ", UV light and increases in intracellular calcium levels (Chauhan et al, 1999). The correlation to calcium metabolism can have some linkage to the expression of lymphotoxin TNF-!. In addition, transient overexpression of FAK induces apoptosis (Chauhan et al, 1999), which might also correlate to the tumor necrosing of TNF-!. Concerning Phosphatidylinositol 3-kinase (PI3K), Ward performed an analysis of neutrophil migration from mice deficient in the gamma-isoform of the p110 catalytic subunit (Ward, 2004). Ward found that PI3K activation can be a dispensable signal for directed cell migration in T lymphocytes. This expression is similar to lymphotoxin TNF-! (Ward, 2004). Ward noted that the non-universal role of PI3K in directional cell migration and the existence of cell-specific signalling pathways for chemotactic responses had important implications for the validation of effective new targets for inflammation, where one aim was to block migration of leukocytes to the site of inflammatory lesion (Ward, 2004). According to the closed related in gene expression profiling of the three genes, the concerns on the prognostic factor prediction as well as cancer treatment based on lymphotoxin TNF-! might be applied for the other two genes, FAK and PI3K. Some limitations of the study should be mentioned. Co-expression is a suggestion of similar functionality, not a proof. To demonstrate coexpression and form a practical point of view, suggest other molecules as markers, more datsets should be used. However, there is no more available public accessible dataset on B-cell lymphoma at present, future use of more datasets when available is planned for clearly proof.

II. Materials and Methods A. DNA array data The DNA array data was extracted from the report of Alizadeh et al (2000). In that study, Alizadeh et al used DNA microarrays to conduct a systematic characterization of gene expression in lymphoma (Alizadeh et al, 2000). The data in GenePix GPR file format was used for further analysis.

B. Data processing In data processing, the author used the Gene Expression Pattern Analysis Suite (GEPAS) program (Herrero et al, 2003) for all in silico simulation. The author performed Sotarray analysis on the published data to get the cluster of the lymphotoxin TNF-! gene and other closed relate lymphoma genes (Herrero et al, 2003). Normalization of the data was performed using Diagnosis and Normalization for Microarray Data (DNMAD) tool (Vaquerizas et al, 2004). Concerning the Sotarray analysis, it is a new approach to the analysis of gene expression data coming from DNA array experiments, using an unsupervised neural network, the Self-Organising Tree Algorithm, (SOTA), that grows adopting the topology of a binary tree (Dopazo and Carazo, 1997). In this study, the algorithm parameters were “error threshold = 0.0001, actualization factors = 0.01, 0.005, 0.001 and maximum number of epoch in a cycle = 1000” and the distance between genes was set as “correlation coefficiency (linear)”. The result of the algorithm is a hierarchical cluster obtained with the accuracy and robustness of a neural network (Herrero et al, 2001). Then the derived cluster was viewed by TreeView (Herrero et al, 2003).

References

III. Results

Agostinis P, Buytaert E, Breyssens H, Hendrickx N (2004) Regulatory pathways in photodynamic therapy induced apoptosis. Photochem Photobiol Sci 3, 721-9. Ali SM, Chee SK, Yuen GY, Olivo M (2002) Photodynamic therapy induced Fas-mediated apoptosis in human carcinoma cells. Int J Mol Med 9, 257-70. Ali SM, Olivo M, Yuen GY, Chee SK (2001) Photodynamicinduced apoptosis of human nasopharyngeal carcinoma cells using Hypocrellins. Int J Oncol 19, 633-43. Almeida RD, Manadas BJ, Carvalho AP, Duarte CB (2004) Intracellular signaling mechanisms in photodynamic therapy. Biochim Biophys Acta 1704, 59-86. Betz CS, Lai JP, Xiang W, Janda P, Heinrich P, Stepp H, Baumgartner R, Leunig A (2002) In vitro photodynamic therapy of nasopharyngeal carcinoma using 5-aminolevulinic acid. Photochem Photobiol Sci 1, 315-9. Chan AT, Teo PM, Huang DP (2004) Pathogenesis and treatment of nasopharyngeal carcinoma. Semin Oncol 31, 794-801.

The results from the Sotarray analysis showed 625 clusters. Of those 625 clusters, the node for lymphotoxin TNF-! gene and other closed relate lymphoma genes was derived at cluster “node 690”. This cluster was selected and viewed with TreeView and the result was shown in Figure 1.

IV. Discussion According to this study, the author can generate the clustering gene expression of lymphotoxin TNF-! gene and other closed relate lymphoma genes. The genes corresponding to FAK and Phosphatidylinositol 3-kinase p110 catalytic gamma are the two known genes detected as closed related genes to lymphotoxin TNF-! gene. Since genes with similar expression profiles are clustered 250

Cancer Therapy Vol 3, page 251 Chang JT, Ko JY, Hong RL (2004) Recent advances in the treatment of nasopharyngeal carcinoma. J Formos Med Assoc 103, 496-510. Demidova TN, Hamblin MR (2004) Photodynamic therapy targeted to pathogens. Int J Immunopathol Pharmacol 17, 245-54. Dragieva G, Scharer L, Dummer R, Kempf W (2004) Photodynamic therapy--a new treatment option for epithelial malignancies of the skin. Onkologie 27, 407-11. Du H, Olivo M, Mahendran R, Bay BH (2004a) Modulation of Matrix metalloproteinase-1 in nasopharyngeal cancer cells by photoactivation of hypericin. Int J Oncol 24, 657-62. Du HY, Bay BH, Mahendran R, Olivo M (2002) Endogenous expression of interleukin-8 and interleukin-10 in nasopharyngeal carcinoma cells and the effect of photodynamic therapy. Int J Mol Med 10, 73-6. Du HY, Bay BH, Olivo M (2003a) Biodistribution and photodynamic therapy with hypericin in a human NPC murine tumor model. Int J Oncol 22, 1019-24. Du HY, Olivo M, Tan BK, Bay BH (2003b) Hypericin-mediated photodynamic therapy induces lipid peroxidation and necrosis in nasopharyngeal cancer. Int J Oncol 23, 1401-5. Du HY, Olivo M, Tan BK, Bay BH (2004b) Photoactivation of hypericin down-regulates glutathione S-transferase activity in nasopharyngeal cancer cells. Cancer Lett 207, 175-81. Hendrickx N, Volanti C, Moens U, Seternes OM, de Witte P, Vandenheede JR, Piette J, Agostinis P (2003) Up-regulation of cyclooxygenase-2 and apoptosis resistance by p38 MAPK in hypericin-mediated photodynamic therapy of human cancer cells. J Biol Chem 278, 52231-9. Kulapaditharom B, Boonkitticharoen V (1999) Photodynamic therapy for residual or recurrent cancer of the nasopharynx. J Med Assoc Thai 82, 1111-7. Lai J, Tao Z, Xiao J, Yan Y, Wang X, Wang C, Zhou S, Tian Y (2001) Effect of photodynamic therapy (PDT) on the expression of pro-apoptotic protein Bak in nasopharyngeal carcinoma (NPC). Lasers Surg Med 29, 27-32.

Lai JP, Tao ZD, Xiao JY, Zhao SP, Tian YQ (1997) Effect of photodynamic therapy on selected laboratory values of patients with nasopharyngeal carcinoma. Ann Otol Rhinol Laryngol 106, 680-2. Lofgren LA, Hallgren S, Nilsson E, Westerborn A, Nilsson C, Reizenstein J (1995) Photodynamic therapy for recurrent nasopharyngeal cancer. Arch Otolaryngol Head Neck Surg 121, 997-1002. Mak NK, Kok TW, Wong RN, Lam SW, Lau YK, Leung WN, Cheung NH, Huang DP, Yeung LL, Chang CK (2003) Photodynamic activities of sulfonamide derivatives of porphycene on nasopharyngeal carcinoma cells. J Biomed Sci 10, 418-29. Sun ZQ (1990) Hematoporphyrin derivative (HPD) plus laser photodynamic therapy for nasopharyngeal carcinoma-analysis of 57 cases. Zhonghua Zhong Liu Za Zhi 12, 1202. Sun ZQ (1992) Photodynamic therapy of nasopharyngeal carcinoma by argon or dye laser--an analysis of 137 cases. Zhonghua Zhong Liu Za Zhi 14, 290-2. Tong MC, van Hasselt CA, Woo JK (1996) Preliminary results of photodynamic therapy for recurrent nasopharyngeal carcinoma. Eur Arch Otorhinolaryngol 253, 189-92. Yee KK, Soo KC, Bay BH, Olivo M (2002) A comparison of protoporphyrin IX and protoporphyrin IX dimethyl ester as a photosensitizer in poorly differentiated human nasopharyngeal carcinoma cells. Photochem Photobiol 76, 678-82. Yow CM, Mak NK, Szeto S, Chen JY, Lee YL, Cheung NH, Huang DP, Leung AW (2000) Photocytotoxic and DNA damaging effect of temoporfin (mTHPC) and merocyanine 540 (MC540) on nasopharyngeal carcinoma cell. Toxicol Lett 115, 53-61. Zhao SP, Tao ZD, Xiao JY, Peng YY, Yang YH, Zeng QS, Liu ZW (1988) Clinical use of hematoporphyrin derivative and photoradiation therapy in nasopharyngeal carcinoma. Chin Med J (Engl) 101, 86-91.

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Cardiac hormones: dramatic anticancer effects Review Article

David L. Vesely Departments of Internal Medicine, Physiology & Biophysics, Cardiac Hormone Center, University of South Florida Health Sciences Center and James A. Haley Veterans Medical Center, Tampa, Florida USA

__________________________________________________________________________________ *Correspondence: David L. Vesely, M.D., Ph.D., Director, Cardiac Hormone Center, University of South Florida Medical School, 13000 Bruce B. Downs Blvd., Tampa, Florida 33612; Telephone: (813) 972-7624; Fax: (813) 972-7623; Email: david.vesely@med.va.gov Key words: Peptide hormones, guanosine-3’,5’-cyclic monophosphate, Extracellular Receptor Kinase 1,2, Nuclear Factor !", breast, colon, pancreatic and prostate adenocarcinomas, angiosarcomas, small-cell and squamous cell lung carcinomas. Abbreviations: Atrial natriuretic peptide, (ANP); Extracellular receptor kinase, (ERK) 1,2; Guanosine-3’5’-cyclic monophosphate, (cyclic GMP); long acting natriuretic peptide, (LANP); Nuclear factor kappa beta, (NF!B) Received: 13 February 2006; Accepted: 16 August 2006; electronically published: August 2006

Summary Cardiac natriuretic hormones consist of a family of six peptide hormones that are synthesized by three different genes and then stored as three different prohormones. Within the 126-amino acid atrial natriuretic peptide (ANP) prohormone are four peptide hormones i.e., long-acting natriuretic peptide (LANP), vessel dilator, kaliuretic peptide, and ANP, whose main known biologic properties are blood pressure regulation and maintenance of plasma volume. The newest discovered property of these peptide hormones is their anticancer effects. These four peptide hormones decrease up to 97% of human prostate, breast, pancreatic and colon adenocarcinoma as well as small-cell and squamous cell lung cancer cells within 24 hours in vitro. Their effects are specific as demonstrated with their respective antibodies blocking all of their anticancer effects. These cardiac hormones mechanism(s) of action involve localizing to the nucleus of cancer cells where they inhibit 83% or greater of DNA synthesis mediated via the intracellular messenger cyclic GMP. In addition to strong DNA inhibitory properties, they inhibit the activation of Extracellular Receptor Kinase (ERK) 1,2, a cancer growth-promoting peptide, and inhibit activation of an intracellular mediator of growth, Nuclear Factor Kappa Beta (NF! B). These peptide hormones effects are even more impressive in vivo. When given subcutaneously via osmotic pumps to athymic mice with human pancreatic adenocarcinomas they completely stop the growth of these adenocarcinomas after one week. Vessel dilator, LANP, and kaliuretic peptide within one week decrease the volume of human pancreatic adenocarcinomas by 49%, 28% and 11%.

I. Introduction: hormones

cardiac

numbered by their a.a. sequences beginning at the Nterminal end of the ANP prohormone, consist of the first 30 a.a. of the prohormone i.e., long acting natriuretic peptide (LANP), a.a. 31-67 (vessel dilator), a.a. 79-98 (kaliuretic peptide) and a.a. 99-126 (ANP) (Vesely 1992; 2002). The BNP and CNP genes, on the other hand, appear to each synthesize only one peptide hormone within their respective prohormones, i.e., BNP and CNP (Gardner et al, 1997; Lainchbury et al, 1997; Scotland et al, 2005). Each of these peptide hormones circulates in healthy humans with vessel dilator and LANP’s concentrations in plasma being 17- to 24-fold higher than ANP, 33 to 48fold higher than BNP and 124 to 177-fold higher than CNP (Winters et al, 1989; Vesely et al, 1989; Hunter et al, 1998; DePalo et al, 2000; Franz et al, 2000, 2001; Vesely, 2003).

peptide

Cardiac natriuretic peptide hormones consist of a family of peptide hormones that are synthesized by three different genes and then stored as three different prohormones [i.e., 126 amino acid (a.a.) atrial natriuretic peptide (ANP), 108 a.a. brain natriuretic peptide (BNP), and 103 a.a. C-type natriuretic peptide (CNP) prohormones] (Brenner et al, 1990; Gardner et al, 1997). Within the 126 a.a. ANP prohormone are four peptide hormones (Figure 1) whose main known biologic properties are blood pressure regulation and maintenance of plasma volume in animals (Martin et al, 1990; Gunning et al, 1992; Benjamin and Peterson, 1995; Ziedel, 1995; Villarreal et al, 1999; Dietz et al, 2001) and humans (Vesely et al, 1994 a,b, 1998). These peptide hormones, 253

Vesely: Cardiac hormones: dramatic anticancer effects ANP has growth-regulatory properties (Toshimori et al, 1987; Abell et al, 1989; Itoh et al, 1990, 1992). In vascular smooth muscle, ANP inhibits cell proliferation (hyperplasia) as well as smooth muscle cell growth (hypertrophy) (Toshimori et al, 1987; Abell et al, 1989; Itoh et al, 1990, 1992). Atrial natriuretic peptide has growth-regulatory properties in a variety of other tissues

including brain, bone, myocytes, red blood cell precursors, and endothelial cells (Johnson et al, 1998; Appel, 1988, 1990, 1992; Haneda et al, 1993; Pedram et al, 1997; Yu et al, 1997; Calderone et al, 1998). In the kidney, ANP causes antimitogneic and antiproliferative effects in glomerular mesangial cells (Appel 1988, 1990, 1992; Haneda et al, 1993).

Figure. 1. Atrial natriuretic peptide (ANP) gene synthesizes a 126 amino acid (a.a.) prohormone which contains four peptide hormones consisting of amino acids 1-30 (i.e., long acting natriuretic peptide, LANP) a.a. 31-67 (vessel dilator), a.a. 79-98 (kaliuretic peptide) and atrial natriuretic peptide (ANP, a.a. 99-126 of this prohormone). Reproduced from Vesely et al, 2006 with kind permission from Atrial Natriuretic Hormones, 1st Edition 1992, Pearson Education, Inc., Upper Saddle River, NJ.

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II. Cardiac natriuretic hormones decrease the number and DNA synthesis of human pancreatic adenocarcinomas

III. Cardiac natriuretic hormones stop the growth of human pancreatic adenocarcinomas in vivo

The first cancer studied both in vitro and in vivo was human pancreatic adenocarcinomas which have the lowest 5-year survival rate of all common cancers (Pitchumoni, 1998; Wolff et al, 2000). The 5-year survival rate of persons with adenocarcinoma of the pancreas is 1% with a median survival of only four months (Pitchumoni, 1998; Wolff et al, 2000). Current cancer chemotherapy and surgery prolong survival by a few months but the abovementioned survival rates are for persons treated with surgery and/or current cancer chemotherapeutic agents (Pitchumoni, 1998; Wolff et al, 2000). Vessel dilator, LANP, kaliuretic peptide and ANP (each at a relatively low concentration of 1 µM) decrease the number of human pancreatic adenocarcinoma cells in culture by 65% (P<0.001), 47% (P<0.01), 37% and 34% (both at P<0.05), respectively, within 24 hours (Vesely et al, 2003). This decrease was sustained without any proliferation of the adenocarcinoma cells occurring in the three days following this decrease in number (Vesely et al, 2003). Thus, when exposed to vessel dilator, LANP, kaliuretic peptide and ANP for 48 hours the inhibition of the number of cancer cells compared to controls was 68% (P<0.001), 43%, 40% and 33% (P<0.05 for these three peptides), respectively (Vesely et al, 2003). At both 72 hours and 96 hours, the decrease in number of adenocarcinoma cells secondary to vessel dilator was 70% (P<0.001) (Vesely et al, 2003). LANP for 72 and 96 hours resulted in the number of adenocarcinoma cells being reduced 47% and 48% (P<0.01 for both), respectively (Vesely et al, 2003). At 72 and 96 hours, the number of cancer cells with kaliuretic peptide present was decreased by 39% and 42% compared to untreated control cells (P<0.05 for each) (Vesely et al, 2003). The number of adenocarcinoma cells at 72 and 96 hours was decreased secondary to ANP by 37% and 35% (P<0.05 for both) (Vesely et al, 2003). At least part of the mechanism of these peptide hormones’ decrease in cancer cell number and antiproliferative effects was a 83% or greater inhibition of DNA synthesis (Vesely et al, 2003). Thus, vessel dilator, LANP, kaliuretic peptide and ANP each at their 1 µM concentrations inhibited DNA synthesis when incubated with adenocarcinoma cells for 24 hours by 91%, 84%, 86% and 83%, respectively (P<0.001 for each). One of the known mediators (Waldman et al, 1984; Vesely 1997) of these peptide hormones’ mechanism(s) of action, i.e., cyclic GMP, inhibited DNA synthesis in these adenocarcinoma cells by 51%. Dose-response curves revealed that 8-bromo- cyclic GMP, the cell permeable analog of cyclic GMP, decreased DNA synthesis in these cancer cells 46%, 42%, 39%, and 34% (all P<0.05) at its 3 mM, 1 mM, 100 µM, and 1 µM concentrations, respectively (Vesely et al, 2003). Even at 1 nM (i.e., 10-9 M) of 8-bromo-cyclic GMP there was a 25% decrease in DNA synthesis in the adenocarcinoma cells (P<0.05) (Vesely et al, 2003). At 100 pM of 8-bromo cyclic GMP, its effects on DNA synthesis in these adenocarcinoma cells became not significant (14% decrease).

In vivo, these peptide hormones’ effects as anticancer agents were even more impressive. Vessel dilator (139 ng/min/kg of body weight) infused subcutaneously for 14 days via osmotic pumps completely stopped the growth of human pancreatic adenocarcinomas in athymic mice (n=14) with a decrease in their tumor volume, even when the tumor volume was large i.e., 60-fold increase in size over basal palpable tumor before peptide infusion was begun, to mimic what occurs in humans, i.e., the pancreatic adenocarcinomas in humans are usually large before they are discovered (Vesely et al, 2004). The tumor volume increased 69-fold in this two-week period (P<0.001) when measured with electronic Vernier calipers in the placebo (n=30)-treated mice (Vesely et al, 2004). Dose-response studies revealed that at concentrations as low as 1.7 ng/ min/20 gram mouse, vessel dilator could completely stop the growth of the human pancreatic adenocarcinomas, but at this concentration there was no decrease in the volume of the tumor by vessel dilator (Vesely et al, 2004). The tumor volume of the untreated human pancreatic adenocarcinoma increased 172-fold in three weeks and was almost 300-fold increased four weeks after the tumors first became palpable (Vesely et al, 2004). After two months, the volume of this untreated aggressive adenocarcinoma was 1306-fold greater than when the tumors first became palpable (Vesely et al, 2004). When these peptide hormones at 10-fold higher concentrations (i.e., at 1.4 µg/min/kg body weight) were infused for four weeks, in addition to completely stopping the growth of this aggressive adenocarcinoma, vessel dilator, long acting natriuretic peptide and kaliuretic peptide decreased human pancreatic adenocarcinomas’ tumor volume after one week by 49%, 28%, and 11%, respectively, with a oneand 20-fold increase in the tumor volume in ANP- and placebo-treated mice (Vesely et al, 2004). Cyclic GMP (0.05 µg/ min/20 gram mouse body weight) inhibited after one week the growth of this cancer 95% (Vesely et al, 2004). There was no evidence of cytotoxicity in any of the normal tissues during the infusion of these peptide hormones.

IV. Localization of the cardiac natriuretic hormones within the human pancreatic adenocarcinomas Immunocytochemical evaluation after removal of the human pancreatic adenocarcinomas revealed that vessel dilator, LANP, kaliuretic peptide and ANP each localized to the nucleus and cytoplasm of the cancer cells and to the endothelium of the capillaries growing into these tumors (Figure 2) (Saba et al, 2005). These cardiac hormones also localized to the fibroblasts within the adenocarcinomas (Figure 2) (Saba et al, 2005). This investigation was the first demonstration of any anti-growth peptide hormone localizing to the nucleus where they could directly inhibit DNA synthesis (Saba et al, 2005). It is, thus, of interest that all four of these peptide hormones, which inhibit

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Vesely: Cardiac hormones: dramatic anticancer effects DNA synthesis, localized to the nucleus. Growth promoting peptides such as Extracellular Receptor Kinase (ERK)-1 have been shown to move from the plasma membrane to the nucleus to cause proliferation. A slightly modified kaliuretic peptide for nanotechnology can substantially decrease the activation ERK-1/2 (Mohapatra

et al, 2004). These peptide hormones may, thus, inhibit the growth of cancer cells not only by directly inhibiting DNA synthesis in the nucleus but by also decreasing the activation of growth promoting substances like ERK-1/2 which promote the growth of cancer cells (Mohapatra et al, 2004).

Figure 2. Immunoperoxidase localization of vessel dilator, LANP, kaliuretic peptide, and ANP within human pancreatic adenocarcinomas with each of these peptide hormones strongly localizing to cytoplasm (Cy), nucleus (N), endothelium (E), and fibroblasts (F). The light blue stain in the fibroblasts is the nuclei of the fibroblasts. (A) Vessel dilator treated, (B) LANP treated, (C) kaliuretic peptide treated, and (D) ANP treated. Primary antibody of each peptide was diluted 1:800. Original magnification x 60. The inset in (A) is an isolated nuclei illustrating that vessel dilator has immunoperoxidase staining within the nucleus. The inset in (D) is a negative control using the human pancreatic adenocarcinoma with substitution of the primary antibodies with normal rabbit serum. Reproduced from Saba et al, 2006 with kind permission from Journal of Histochemistry and Cytochemistry.

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Cancer Therapy Vol 4, page 257 side effects of the 100 µM or 1 mM concentrations of these peptide hormones in vivo during their one month of infusion or in the two months post-infusion follow-up (BA Vesely, et al, unpublished observation).

V. Four cardiac hormones eliminate up to 97% of human prostate, breast and colon adenocarcinoma cells as well as small-cell and squamous carcinoma of the lung cells

VII. Cell-cycle adenocarcinoma cells hormones

Dose-response curves have revealed that there is a significantly greater (P<0.05) decrease in the number of cancer cells at each 10-fold increase in concentration of the four peptide hormones synthesized by the ANP gene in human breast, colon, and prostate adenocarcinomas as well as in small-cell and squamous carcinoma of the lung cells (Vesely et al, 2005a,b,c, 2006; Gower et al, 2005). Table 1 is an example of this with colon adenocarcinoma cells. At 1 mM of these respective hormones up to 97% of the above human cancer cells were eliminated, i.e., only 3 to 4 ± 0.6 cells were not killed. Thus, with vessel dilator nearly all of the human cancer cells were eliminated (Vesely et al, 2005a,b,c, 2006; Gower et al, 2005). In several fields there were no cancer cells at all, i.e., vessel dilator had killed 100% of the cancer cells within 24 hours. In none of the human cancers is there a proliferation of the remaining cancer cells after the initial decrease in cancer cell number secondary to these peptide hormones. Dose-response investigations have further revealed that brain natriuretic peptide (BNP) has no anticancer effects in any concentration and that CNP, from the third cardiac gene, has anticancer effects only at 100-fold higher concentrations than that observed with the above four cardiac hormones (Vesely et al, 2005a,b,c, 2006; Gower et al, 2005).

VI. Elimination of adenocarcinomas in vivo

human

arrest of with cardiac

Cell-cycle progression was directly affected by several of the cardiac hormones. The majority of the cardiac hormones had their strongest modification of cellcycle progression in the synthetic (S) phase of the cell cycle. Vessel dilator, long acting natriuretic peptide, kaliuretic peptide and 8-bromo-cyclic GMP (each at 1 µM) decreased the number of breast cancer cells in the S phase of the cell cycle by 62%, 33%, 50%, and 39%, respectively (all P<0.05) (Vesely et al, 2005b). ANP caused a 40% decrease in the G2-M proliferative phase the cell cycle. There was an accumulation of cells in the resting G0-G1 phase secondary to LANP, vessel dilator, kaliuretic peptide and ANP (Vesely et al, 2005b). Vessel dilator, which caused the largest decrease in cells in the S phase, had the largest accumulation of cells in the G0-G1 phase (Vesely et al, 2005b). BNP had no effect on the S phase or any other portion of the cell cycle (Vesely et al, 2005b).

VIII. Natriuretic peptide receptors (NPR) A and C are present in human cancers Each of the human cancer cells listed above have natriuretic peptide receptors to mediate these peptide hormones’ effects (Vesely et al, 2005a,b,c, 2006; Gower et al 2005). Thus, when human breast adenocarcinoma cells were evaluated by Western blots, natriuretic peptide receptors (NPR)-A and -C were demonstrated to be present (Vesely et al, 2005b). It is of interest that the breast adenocarcinoma cells have developed an NPR-Aand -C receptors to mediate ANP’s effects via membrane-

breast

We have infused subcutaneously 100 µM and 1 mM of vessel dilator and LANP for four weeks with a weekly change in the osmotic pumps in athymic mice harboring human breast adenocarcinomas. After three weeks, in the majority of the mice, the human breast cancers disappeared and no evidence of breast cancer was present in any tissue or organ at necropsy two months later (BA Vesely, et al. unpublished observation). There were no

Table 1. Percent Decrease in Human Colon Adenocarcinoma Cells Within 24 Hours: Dose-Response Relationships. % Decrease Dose 1 mM 100 µM 10 µM 1 µM

Vessel Dilator 97 90 72 60

LANP 89 68 55 31

Kaliuretic Peptide 89 72 55 30

ANP 89 69 54 35

BNP 5 4 2 1

CNP 6 4 3 1

Cyclic GMP 84 65 57 33

LANP = Long acting natriuretic peptide; ANP = Atrial natriuretic peptide, BNP = Brain natriuretic peptide, CNP = C-natriuretic peptide; Cyclic GMP = cyclic 3’,5’-guanosine monophosphate. The decrease in colon cancer cells with 1 mM of vessel dilator, LANP, kaliuretic peptide, ANP and cyclic GMP was significant at P<0.001 while BNP and CNP’s effects were not significant when evaluated by repeated analysis of variance (ANOVA), (n = 60 for each group). Reproduced from Gower et al Jr, 2005 with kind permission from International Journal of Gastrointestinal Cancer.

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Vesely: Cardiac hormones: dramatic anticancer effects bound guanylate cyclase which is part of the NPR-A receptor and via NPR-C receptor mediated mechanisms, respectively. The NPR-C receptor does not contain guanylate cyclase which catalyzes the formation of the intracellular cyclic GMP. ANP’s signaling via the NPR-C receptor is thought to involve a cascade of Ca2+ influx, activation of endothelial nitric oxide synthase with resulting formation of nitric oxide activating cytosolic guanylate cyclase, which in turn, increases the concentration of cyclic GMP (Murthy et al, 1998). The presence of these receptors helps to explain why ANP, but not BNP and CNP, has effects at its 1 µM concentration as ANP binds to both receptors with a stronger affinity than BNP or CNP and, thus, a lower concentration of ANP is needed to have effects (Vesely et al, 2005a). When the concentrations of CNP and BNP are increased 100-fold, in dose-response curves, CNP but not BNP has effects of decreasing the number of cancer cells (Vesely et al, 2005c). This is consistent with CNP’s binding to NPR-C receptor with a stronger affinity than BNP but not as strong as ANP i.e., binding to NPR-C receptors is ANP > CNP > BNP (Suga et al, 1992).

and 1% decrease in prostate cancer cell number at 48, 72, and 96 hours, respectively (Vesely et al, 2005a). When ANP’s antibody plus ANP were incubated for 48, 72, and 96 hours there was a 0%, 0%, and 0% decrease in prostate cancer cells at each time period (Vesely et al, 2005a). When the antibodies alone (same concentration) were incubated for 24 hours without the addition of any of the peptide hormones, the vessel dilator antibody resulted in a 5% increase (rather than decrease) (i.e., 93.4 ± 1.5 cells vs 88 ± 6 control cancer cells) in prostate cancer cell number while the ANP antibody resulted in a 3% increase (92 ± 2 cells) in prostate adenocarcinoma cells (Vesely et al, 2005a). With the LANP and kaliuretic peptide antibodies alone for 24 hours there was a 0% (89 ± 2 cells) and 2% increase (91 ± 2 cells), respectively versus 89 ± 2 untreated prostate cancer cells (Vesely et al, 2005a). Thus, there was no significant decrease in cancer number with the antibodies alone, but rather 3 of the 4 antibodies caused a small increase in cancer cell number within 24 hours.

X. These cardiac hormones’ ability to inhibit DNA synthesis is specifically mediated by cyclic GMP

IX. Cardiac hormones anticancer effects are specific

With respect to the mechanism of how these peptide hormones inhibit DNA synthesis, one of the second messengers of their biologic effects i.e., cyclic GMP was found using 8-bromo-cyclic GMP (1 µM) to inhibit DNA synthesis 57% in the human colon cancer cells (Gower et al, 2005). Cyclic GMP’s mimicking the effects of these peptide hormones on DNA synthesis in the same cells suggests that cyclic GMP is one of the mediators of these peptide hormones’ ability to decrease cancer cell number and to inhibit DNA synthesis in colon adenocarcinoma cells. This was further defined when utilizing a cyclic GMP antibody it was demonstrated that this antibody could essentially block all of cyclic GMP’s effects on decreasing colon cancer cell number and DNA synthesis (Gower et al, 2005). Utilizing this antibody suggests that cyclic GMP’s effects on cancer cells are specific, i.e., not due to some other mediator. Further, the cyclic GMP antibody almost completely blocked each of the cardiac peptide hormones’ ability to inhibit DNA synthesis which strongly suggests that their ability to inhibit DNA is almost exclusively mediated by cyclic GMP (Gower et al, 2005).

To determine if the effects of these peptide hormones to decrease the number of human cancer cells were specific these peptides hormones’ respective antibodies (Ab) were utilized in a 1:5 concentration of peptide hormone to their respective antibody in the examination of human prostate adenocarcinomas. When these peptide hormones (each at 1 µM) were incubated with their specific antibodies (5 µM) the decrease in cancer cell number secondary to vessel dilator alone of 63% (89 ± 2 to 33 ± 2 cancer cells) was reduced to 2% only (89 ± 2 cells in control vs 87 ± 2 cancer cells with Ab plus vessel dilator) (Vesely et al, 2005a). There was no decrease in cell number with LANP plus its antibody (89 ± 2 control cancer cells vs 89 ± 2 cells with LANP and Ab) (Vesely et al, 2005a). Kaliuretic peptide plus its antibody resulted in a 0.4% (89 ± 2 control cells vs 88 ± 2 cancer cells with kaliuretic peptide plus Ab) decrease versus a 30% decrease (62 ± 3 cells vs 89 ± 2 control cells) with kaliuretic peptide alone (Vesely et al, 2005a). These antibodies studies also indicated that ANP’s effects were specific with the 37% decrease (to 56 ± 3 cells) in cell number with ANP alone decreased to 2% (87 ± 2 cells vs 89 ± 2 control cancer cells) when its antibody was added (Vesely et al, 2005a). The addition of specific antibody blocked each of these peptides ability to decrease cancer cells at P<0.0001. When these specificity experiments were extended to 48, 72, and 96 hours of incubation of antibody plus peptide hormones, for vessel dilator plus antibody, the decrease in number of cancer cells was 1%, 0%, and 1%, respectively (versus untreated control prostate cancer cells at these three time points) (P<0.0001). LANP plus its antibody resulted in 0%, 1%, 1% decrease in cancer cells at 48, 72, and 96 hours (Vesely et al, 2005a). With kaliuretic peptide plus its antibody there was a 0%, 0%,

XI. Mechanism of action of cardiac hormones decreasing the number of cancer cells When the cyclic GMP antibody was incubated with vessel dilator, LANP, kaliuretic peptide and ANP it blocked 75 to 80% of their effects on decreasing colon cancer number (Gower et al, 2005). This would suggest that these peptide’s mechanism(s) of action as anticancer agents is mediated partially but not completely by cyclic GMP (Gower et al, 2005). This information is compatible with the information that one of these peptides, i.e., kaliuretic peptide’s ability to decrease activation of

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Cancer Therapy Vol 4, page 259 extracellular receptor kinase (ERK) 1/2, a cancer growth promoting peptide which translates from the extracellular membrane to the nucleus of the cell to promote growth (Mohapatra et al, 2004). Thus, one additional mechanism of these peptides’ antigrowth effects is their ability to decrease the activation of growth promoting peptides (Mohapatra et al, 2004) as well as directly inhibiting DNA synthesis within the nucleus (Saba et al, 2005). Kaliuretic peptide and ANP also significantly decrease the activation of NF!B, an intracellular mediator of growth (Mohapatra et al, 2004).

Acknowledgements I thank Darren Manelski and the Darren Manelski Foundation, New York, New York and the United States Department of Veterans Affairs for support of this research. I also thank Charlene Pennington for excellent secretarial assistance.

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XII. Cardiac hormones’ effects on sarcomas These cardiac hormones decrease the number of sarcoma cells and their DNA synthesis (Vesely et al, 2006) as well as in the cancer cells outlined above. About one-fourth of all primary cardiac tumors are malignant and usually are invasive (Colucci and Schoen, 2001). These malignant tumors of the heart can occur at any age but are most common in the third to fifth decades of life (Colucci and Schoen, 2001). The malignant tumors of the heart have an equal incidence in women and men and can occur in children and infants (Chahinian et al, 2003). The most common primary malignant cardiac tumors are angiosarcomas (Chahinian et al, 2003). From a clinical standpoint, primary malignant tumors of the heart cause a rapid downhill course with death occurring from a few weeks to two years after the onset of symptoms (Donsbeck et al, 1999; Colucci and Schoen, 2001). Complete surgical resection of cardiac angiosarcomas is usually impossible (Chahinian et al, 2003). The combination of surgery followed by radiotherapy and chemotherapy based upon doxorubicin has resulted in no cures and usually a failure to alter the course leading to death (Colucci and Schoen, 2001). Within 24 hours, vessel dilator, LANP, kaliuretic peptide, ANP and their intracellular mediator cyclic GMP each at relatively low concentration of 1 µM decreased the number of angiosarcoma cells in vitro 61%, 30%, 29%, 36%, and 32%, respectively, and its DNA synthesis 68% to 85% (Vesely et al, 2006). BNP and CNP had no effect(s) at the same concentration (Vesely et al, 2006).

XIII. Natriuretic peptides do not have side effects of current anticancer agents The above four cardiovascular peptide hormones do not cause nausea, vomiting, alopecia, or myleosuppression that is common with current cancer chemotherapy or the more severe toxicity of permanent ovarian dysfunction and leukemia and/or secondary tumors that occurs with currently utilized anticancer agents (Pitchumoni 1998; Wolff et al, 2000). The lack of these side effects and their beneficial effects of decreasing the number of cancer cells suggests that the cardiovascular hormones synthesized by the ANP prohormone gene may have use in the future as anticancer agents.

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Cancer Therapy Vol 4, page 263 Cancer Therapy Vol 4, 263-270, 2006

Prognosis of early invasive and locally advanced (Stage I, II) uterine cervical adenocarcinomas Research Article

Merih Hanhan1, Mehmet Murat Inal1,*, Deniz Oztekin1, Nilgun Dicle2, Zeynep Ozsaran3, Sivekar Tinar1 q

SSK Aegean Obstetrics and Gynecology Training and Research Hospital, Oncology Department SSK Aegean Obstetrics and Gynecology Training and Research Hospital, Pathology Department 3 Ege University Faculty of Medicine Radiation Oncology Department, Izmir, Turkey 2

__________________________________________________________________________________ *Correspondence: Mehmet Murat Inal, MD, Department of Gynecological Oncology, Aegean Obstetrics and Gynecology Training and Research Hospital, 669 sokak Ata sitesi No: 4/8, Gaziemir-35410 Izmir/Turkey; Tel: +90 532 6281350; Fax: +90 232 4224144; E-mail: inal21@hotmail.com Key words: Uterine cervical adenocarcinoma, prognosis, prognostic criterias Abbreviations: cervical adenocarcinomas, (CAC); lympho-vascular area involvement, (LVAI) Received: 1 March 2006; Accepted: 25 September 2006; electronically published: November 2006

Summary The objective was to evaluate the prognosis, clinicopathologic factors affecting the prognosis and management modalities of early invasive and locally advanced cervical adenocarcinomas (CAC). Early invasive and locally advanced (stage I, II) cervical adenocarcinoma cases diagnosed at our department in between January 1988 and December 2001 were treated with surgery+adjuvant radiotherapy (20%), whereas 19 were treated with radical radiotherapy. Cervical adenocarcinoma cases were revealed as 10.2% (70 cases) of 686 cervix malignancy cases. Thirty-nine out of these 70 cases were with early invasive and locally advanced (FIGO Stage I, II) types, who were evaluated for management modalities and effect of clinicopathologic factors to prognosis. Mean age of this series was 50.4 years and stage distribution of these cases was as follows; 12 cases were Ib1 (30.7%), 3 cases Ib2 (7.9%), 11 cases were IIa (30.7%) and 13 cases were IIb (30.7%). Mean tumor diameter was 27.6 mm, lymphovascular area involvement was 47%, and pelvic and paraaortic lymph node involvement was 23% and 7.6%, respectively, in surgery applied cases. When the progression observed three cases (7.6%) were excluded, recurrence was observed in 12/36 cases. Most frequently observed surgical morbidity was urinary retention (20%). Mean follow-up period of the cases was 49.2 months, but exitus was observed in 14 cases (35.6%). Cervical adenocarcinomas have worse prognosis than squamous cell carcinomas of the cervix, especially when the spread of cancer cells observed out of the cervix (Stage II and over). Consequently, establishment of management modalities of primary and metastatic lesions requires study of more larger series together with a longer period of time.

in the incidence (Ferenczy and Blaustein, 1982). The incidence of cervical adenocarcinomas is continuously increasing in relative to squamous cell carcinoma of cervix especially in cases of 35 years of age or younger (Kjorstad and Bond, 1984; Weiss and Lucas, 1986; Ruey-Jien et al, 1998). Lymph node metastasis, lympho-vascular area involvement (LVAI), depth of stromal invasion, diameter of the tumor, grade and tissue sub-type are being reported as effective prognostic factors on survival and recurrences for cervical adenocarcinomas (Mc Lellan et al, 1994; Look et al, 1996). It is assumed that these tumors invade the lymph nodes in very early periods and they are less

I. Introduction Cervical cancer is the most frequently observed cancer of female genital system in our country (Aydinli et al, 1987). Cervical adenocarcinomas (CAC), are the second most frequently observed cervical cancer type with a mean rate of 10% after squamous cell carcinoma type (Syrjanen, 2000). The incidence of CAC increased from 5% to 27% in time, and the prognosis is generally worser than that of the squamous cell carcinoma (Smith et al, 2000). Unavailability of reliability and adequacy of tissue characteristics which can provide the diagnosis at premalignant stage like squamous cell carcinomas in adenocarcinomas, might be responsible from this increase 263

Manhan et al: Prognosis of early invasive and locally advanced (Stage I, II) uterine CACs sensitive to radiation and chemotherapy than the squamous cell carcinomas (Berek et al, 1985). Although radical surgery is the primary choice in management of these tumors, it can not provide sufficient survival rates especially in advanced and recurrent cases (Moberg et al, 1986). To evaluate the prognosis, clinicopathologic factors affecting the prognosis and management modalities of early invasive and locally advanced cervical adenocarcinomas.

III. Results Mean age of the cases at time of the diagnosis was 50.4 years (range 32-72 years) (Figure 1). Eighteen cases (46.1%) were applied to the hospital with postmenopausal vaginal bleeding, whereas 14 cases (35.8%) were with intermittent and post-coital vaginal bleedings. Staging of the cases were as follows; 12 cases with Stage Ib1 (30.7%), 3 cases with Ib2 (7.9%), 11 cases with IIa (28.1%) and 13 cases with IIb (33.3%). There was a statistically significant difference in between stage I and stage II cases according to the mean ages of the cases of which were 44.9 years and 52.1 years, respectively (p<0.01). Prognostic data, tumor recurrence and death rates of the cases were given in Table 1. Mean diameter of tumor mass was observed to be 27 mm (range 15-60 mm), less than or equal to 39 mm in 11 cases (64.7%) and more than or equal to 40 mm in 6 cases (35.3%). LVAI positivity was 27.2% in cases with tumor diameter less than or equal to 39 mm, whereas the incidence was increased to 83.3% significantly in cases with diameter more than or equal to 40 mm (p<0.05). (Table 2). In case of LVAI positivity observed in 47% of the cases, lymph node metastasis was observed to be 28.5%, whereas this rate was 16.5% in case of LVAI negativity, but due to small number of the cases, statistical significance could not be obtained (p=1.00) (Table 3). When the LVAI positivity was taken into account according to the stages, the positivity rates were 27.2% and 83.3% in stages I and II, respectively. These rates were found to be statistically significant (p<0.05) (Table 4). But no significant effect of the LVAI positivity was observed on survival rates (p>0.05). Para-aortic lymph node metastasis was not observed in Stage I, whereas pelvic lymph node metastasis was observed in only one case (12.5%). In stage II, one case with pelvic and another case with pelvic and paraaortic lymph node metastasis together was observed (40%), but due to small number of the cases, statistical significance could not be obtained (p>0.05) (Table 5). Mean follow-up period of our series was 49.2 months (range 9-153 months), but progressive disease was observed in primary radiotherapy applied three cases (7.6%). One of these cases developed liver metastasis at third month of the follow-up and the other two cases developed brain metastasis at 8th and 9th months of their follow-up; and all three cases were lost at 9th, 13th and

II. Patients and Methods Tissue type of 70 cases (10.2%) out of 686 cervical cancer cases recorded in Oncology Department of SSK Aegean Obstetrics and Gynecology Training and Research Hospital in between January 1988 and December 2001 were established to be adenocarcinoma. Thirty-nine out of these 70 cases were with early invasive and locally advanced (FIGO Stage I, II) types, who were evaluated for management modalities and effect of clinicopathologic factors to prognosis. Twenty of these cases were treated with surgery + adjuvant radiotherapy (20/39) and the rest 19 cases were treated with primary radical radiotherapy. Type I (7 cases) and Type III (13 cases) hysterectomy, bilateral salphingo-oopherectomy, bilateral pelvic and paraaortic lymphadenectomy (TAH+BSO+PPLA) was applied in surgical approach, and adjuvant radiotherapy (external pelvic 5040 cGy and brachitherapy 1700 cGy) was administered to all of these cases after surgery. Type I hysterectomy and BSO applied cases had been operated in different centers and then referred to our department, the stages of these cases were Stage Ib1 (3 cases), Ib2 (1 case) and IIb (3 cases), but no lymphadenectomy was applied to these case. All of the Type III hysterectomy + BSO + PPLA applied cases were operated in our department and the stages of these cases were Stage Ib1 (8 cases), IIa (4 cases) and IIb (1 case). Primary radical radiotherapy was applied in Ege University Faculty of Medicine Department of Radiation Oncology as external pelvic radiotherapy (a total of 5040 cGy divided into 180 cGy daily fractions) and brachitherapy (a total of 1700 cGy with weekly applied two fractions of 850 cGy in two weeks). After completion of therapies, clinical follow-up of cases were designed as once in three months for the first two years, once in six months for the following three years and after that each year. Statistical analysis of the data was performed by using Fischerâ&#x20AC;&#x2122;s Exact test, and Kaplan-Meier survival analysis was used in order to calculate the five year disease free survival and five year life expectancy.

Figure 1. Age distribution of CAC cases

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Cancer Therapy Vol 4, page 265 Table 1. Prognostic data, tumor recurrence and death rates of the cases

Stage

Ib1* Ib1* Ib1* Ib1 Ib1 Ib1 Ib1 Ib1 Ib1 Ib1 Ib1 Ib2* IIa IIa IIa IIa IIb* IIb* IIb* IIb n.20

15 15 17 15 20 20 15 24 30 15 50 40 60 20 40 45 45 27

SURGERY AND RADIOTHERAPY LVAI Pelvic Para Recur RecRx LAP aortic (m) LAP No 64 No No No No No No No Yes No No No No No Yes No No No Yes No 25 kt Yes No No No No No No 22 Yes No No Yes No No No No No Yes Yes No 49 kt Yes 73 rt 35 kt Yes Yes Yes 36 kt 8/17 3/13 1/13 7/20

Ex (m)

Alive (m)

67 34 23 58 99 49 6/20

93 69 104 153 89 37 66 31 28 48 95 65 46 37 (+) 14/20

RADIOTHERAPY Stage Recur Ex Alive (m) (m) (m) Ib1 Ib2 Ib2 IIa** IIa IIa IIa IIa IIa** IIa IIb IIb IIb IIb IIb IIb IIb IIb IIb** n.19

29 9** 11 10 8** 18 12 3** 8/19

* Type I hist.BSO applied cases ** Progressive disease (+) Alive with disease (Recur: Recurrence), (Rx: treatment), (m: month), (kt: chemotherapy), (rt: radiotherapy)

Table 2. Relationship in between tumor mass diameter and LVAI (p:0.04) Tumor Diameter

LVAI Positive (%) 3 (%27.2) 5 (%83.3)

Tumor ! 39 mm (n.11) Tumor " 40 mm (n.6)

Negative (%) 8 (%72.8) 1 (%15.7)

Table 3. Relationship in between LVAI - pelvic-paraaortic lymph node involvement (p:1.00) LVAI positivity according to stages I II Total

Lymph node metastasis negative positive 5 1 5 2 10 3

Total(%) 1/6 (%16.5) 2/7 (%28.5) 3/13 (%23)

Table 4. Relationship in between stage and LVAI (p: 0.04) Stage III Total

LVAI negative 8 1 9

Total(%) positive 3 5 8

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3/11 (%27.2) 5/6 (%83.3) 8/17 (%47)

11 28 19 13 25 12

117 53 (+) 47 32 38 70 44 36 63 31 30 51 9 8/19 11/19

Manhan et al: Prognosis of early invasive and locally advanced (Stage I, II) uterine CACs Table 5. Relationship in between tumor diameter and Pelvic-paraaortic LN involvement (p: 0.20) Tumor diameter ! 40 mm > 40 mm Total

Lymph node involvement negative positive 8 1 2 2 10 3

11th months of their follow-up, respectively. If these three cases with progressive disease were excluded, tumor recurrence was observed in 12/36 cases (33.3%) during the follow-up, and local recurrence rate was found to be 13.8% (5/36 cases), and distant metastasis rate was found to be 19.4% (7/36 cases). Clinical and prognostic characteristics of recurrent cases were given on Table 6. Recurrence periods of the cases according to the treatment modalities were given on Table 7. Increase in recurrence and mortality rates was observed with the

Total(%) 1/9(%11.1) 2/4(%50) 3/13(%23)

increase in stage, whereas according to the treatment modalities, no mortality was observed in stage I cases treated with radical radiotherapy, but no statistical significance was observed (p>0.05) (Table 8). Recurrence rates were similar in both treatment modalities. Recurrence rate distribution was not statistically significant according to the stages (p=1.00 and p=0.64) and treatment modalities (p=0.35 and p=1.00) (Table 9).

Table 6. Clinical and prognostic characteristics of recurrent cases Age

Stage

61 65 45 56

IIa* IIa IIb Ib1

65 48

IIa Ib2

Ib2

Tumor Treatment diameter RT RT RT ? Type I His + RT RT RT > 4 cm

Type I His + RT 36 Ib1 < 4 cm Type 3 His + RT 58 IIb ? Type I His + RT 37 IIb ? Type 1 His + RT 61 IIa* RT 33 IIb > 4 cm Type I His + RT 41 IIa RT 46 IIb > 4 cm Type 3 His + RT 68 IIb* RT * Progressive disease (3 cases)

LVAI

LN met. Recur(m)

9* 23 12 64

Recur localization Brain Liver Abdomen Pelvis

Survival(m)

Now

11 * 28 12 67

Ex Ex Ex Ex

10 29

Lung Pelvis

19 53

Liver

Ex Tumor + alive Ex

Yes

Lung

Bone

Yes

8* 73

Brain Vagina

13 * 99

Ex Ex

Yes

18 36

Pelvis Lung

25 37

Ex Tumor + alive

Liver

Table 7. Recurrence periods according to treatment modalities Trt / Recur time (m) 0-12 m Surgery+Adj.RT Pelvic (n.20) Distant Exitus Radiotherapy Pelvic 1 (n.16)* Distant 2 Exitus 3 *Progressive three cases were excluded

13-24 m 1 1 1 3

25-36 m 4 1 1 2

266

> 37 m 2 4 -

Total 2 5 6 3 2 8

Cancer Therapy Vol 4, page 267 Table 8. Recurrence-mortality rates according to the treatment modalities and stages Surgery + Adjuvant RT Stage I Stage II 3/12 (%25) 4/8 (%50) 3/12 (%25) 3/8 (%37.5)

Recurrence Mortality

Radical radiotherapy Stage I Stage II 1/3 (%33.3) 7/16 (%43.7) 0/3 (%0) 8/16 (%50)

Table 9. Tumor recurrences according to the treatment modalities and stages Surgery+Radiotherapy Pelvic Distant 1/12 (%8.3) 2/12 (%16.6)

Radiotherapy Pelvic 1/3 (%33.3)

Distant 0/3

4/15(%26.6)

1/8 (%12.5)

3/8 (%37.5)

2/13 (%15.3)

8/21(%38)

2/20 (%10) 5/20 (%25) 7/20 (%35) *Progressive 3 cases were excluded

3/16 (%18.7) 5/16 (31.2)

2/16 (%12.5)

12/36(%33.3)

Recurrence Stage I(n.15) Stage II(n.21) Total (36)*

Recurrence and mortality rates of lymph node positive cases after type III hyst + BSO + PPLA applied 13 cases, were observed to be 66.6% and 33.3%, respectively, and median survival rate was 39 months, whereas no recurrence or mortality was observed in lymph node negative cases and median survival rates was 71.6 months. Recurrence and mortality rates of the type I hysterectomy + BSO alone applied cases (without lymphadenectomy) were 71.4% (5/7 cases, at 22nd, 35th, 49th, 64th and 73rd months), all these five cases were lost even after the therapies against the recurrences (23rd, 49th, 58th, 67th, and 99th months, respectively), median survival period was 65.4 months. The places of the tumor recurrences observed in surgery-adjuvant radiotherapy applied cases were intrapelvic in 3/7 cases (42.8%) and extrapelvic in 4/7 cases (57.2%). Type I hysterectomy without lymph node dissection had been applied to 75% of the cases with intrapelvic recurrences and 66.6% of the cases with extrapelvic recurrences.

Total

If the three cases who developed progressive disease were excluded, recurrence and mortality rates of radical radiotherapy applied cases were observed to be 31.2% (5/16 cases) and 25% (4/16 cases), respectively, median survival period was 43.5 months (Table 10). Disease-free 5 year survival and life expectancy rates of the cases according to the treatment modalities for stage I and stage II cases were given in Table 11, but when Long Rank analysis test revealed no statistically significant difference. When all of the cases (stage I+II) were evaluated according to the treatment modalities; disease-free five year survival and five year life expectancy rates were 65% and 70%, respectively, in surgery-adj.RT group, whereas 52.6% and 57.8% in radical RT group (Table 12, Figures 2, 3), but Long Rank analysis test revealed no statistically significant difference. Disease-free five year survival and five year life expectancy rates only according to the stages were found to be 73.3% and 80%, respectively, in stage I and 54.1% in stage II (Table 13)

Table 10. Recurrence, mortality and survival according to the treatment modalities n.36 Recurrence Mortality Mean survival Localization of recurrence

(m) Local Distant

Type III hyst.PPLA+adj.RT Node (+) n.3 Node (-) n.10 2/3 (%66.6) 1/3 (%33.3) 39 71.6 1/3 (%33.3) 1/3 (%33.3) -

Type I hyst+adj.RTn.7 5/7 (%71.4) 5/7 (%71.4) 65.4 2/7 (%28.5) 3/7 (%42.8)

Radical RT (n.16) 5/16 (%31.2) 4/16 (%25) 43.5 3/16 (%18.7) 2/16 (%12.5)

Table 11. Disease-free survival and life expectancy according to the stages and treatment modalities

Disease free survival Life expectancy

Surgery + adj.RT Stage I Stage II %75 %50 %75 %62.5

267

RRT Stage I %66.6 %100

Stage II %50 %50

Manhan et al: Prognosis of early invasive and locally advanced (Stage I, II) uterine CACs Table 12. Disease-free survival and life expectancy according to the treatment modalities (p=0.11) Stage I + II Disease free survival Life expectancy

Surgery + adj.RT %65 %70

RRT %52.6 %57.8

Table 13. Disease-free survival and life expectancy according to the stages Stage Disease-free survival Life expectancy

stage I %73.3 %80

stage II %54.1 %54.1

Figure 2. Cumulative disease-free survival graphics of cases (stage I+II) according to the treatment modalities.

Figure 3. Cumulative life expectancy graphics of cases (stage I+II) according to the treatment modalities.

268

Cancer Therapy Vol 4, page 269 When radical surgery-PPLA applied cases were compared with radical surgery-PPLA un-applied cases; it was observed that radiotherapy added to both of the modalities led prevention of early recurrences without causing a significant decrease in life expectancy. Chen et al reported better results with surgery when compared with radiotherapy (surgery; 63%-radiotherapy; 37%), and also reported that stage, lymph node involvement, bulky tumor presence and treatment modalities were significantly effective and independent factors on prognosis (Chen et al, 1998). In our study, we also observed that LVAI positivity increased with the increase in stage and tumor mass diameter, and consequently an increase in lymph node involvement, but we could not observe a significant effect of LVAI positivity on survival rates. We also observed that late recurrences (>2 years) were less in radical radiotherapy group, but all of the mortalities were observed in first 36 months. Within the same period, the mortality rate was 33.3% in surgeryadjRT group. Recurrence, mortality and median survival periods were as follows; radical surgery-adjRT group; 66.6%, 33.3%, 39 months; Radical RT group; 31.2%, 25%, 43.5 months and type I hysterectomy-adjRT group; 71.4%, 71.4% and 65.4 months, respectively. The results of the first two treatment modalities revealed no statistical significance, whereas high recurrence and mortality rates were observed in type I hysterectomy applied and (referred cases) adjRT added cases, but mean survival period (disease positive) was found to be longer. We believe that adjRT added to the treatment together with presence of early stage (Stage Ib1) cases in this last group, supplied positive effect on survival time. Although no statistically significant difference was observed in surgery-adjRT and RRT groups according to five year disease-free survival and life expectancy, the same comment could be made for different stages. There is still controversy on which treatment modality can provide better prognosis in adenocarcinomas. Authors like McLellan and Kilgore, reported that surgery had better prognosis than the radiotherapy (Mc Lellan et al, 1994; Kilgore et al, 1998). Some believe that both surgery and radiotherapy have equal prognostic affectivity (Hopkins et al, 1988), and some believe that radical hysterectomy causes a high rate of insufficient treatment (Larson et al, 1988).

IV. Discussion This study establishes the presence of independent prognostic factors for cervical adenocarcinomas affecting the survival like clinical stage, lymph node involvement and presence of bulky tumor. Cervical adenocarcinomas were observed with a rate of 10% in cervical cancers after squamous cell type. In our series this rate was also 10.2%, similar to the rates reported by Syrjanen, 2000. Mean age of the cases in CACs was 50 years according to the literature data (Brand et al, 1988) which is similar to mean age (50.4 years) in our series. Erzen et al reported peak age group as 31-40 years range (31.9% of cases) (Erzen et al, 2002), we found it as 40-49 years age (35.6% of cases). Hopkins et al found the effect of age on prognosis statistically non-significant (Hopkins et al, 1987). But Shingleton and Berkowitz established a relation in between young age and early stages of the tumor (Berkowitz et al, 1979; Shingleton et al, 1981). Attanoos et al reported the young age as an independent good prognostic factor in relation to early stages of the tumor (Attanoos et al, 1995). A relationship in between the stages and age was also found in our series, revealing mean ages of 44.9 years and 52.1 years for stage I and stage II, respectively. But recent studies by Chen et al reported that age as a prognostic factor for squamous cell carcinomas had no independent significant meaning for adenocarcinomas (Chen et al, 1998). Evaluation of stage distribution of all cases revealed that 44.8% of cases were diagnosed in stage I similar to the literature which comprises stage Ib1 cases with a rate of 34.5% (Table 14). Early stage diagnosis chance of cases seems to be lower than the rates reported in the literature. LVAI positivity was 27.2% in cases with tumor diameter !39 mm, whereas the incidence was increased to 83.3% significantly in cases with diameter "40 mm (p<0.05). In case of LVAI positivity observed in 47% of the cases, lymph node metastasis was observed to be 28.5%, whereas this rate was 16.5% in case of LVAI negativity, but due to small number of the cases, statistical significance could not be obtained (p=1.00). LVAI positivity revealed no significant effect on survival. In a study accepting the tumor diameter cut-off as 20 mm, pelvic lymph node involvement found to be 16.6% and 50%, respectively (p<0.05) (Ayhan et al, 1989). But in our series, lymph node involvement was 11.1% in tumors with diameter !39 mm, whereas this rate was found to be 50% in tumors with diameter "40 mm, but this difference was not statistically significant which might be due to small number of the cases. Sabol et al, 1999 and Meydanli et al, 2001 reported lymph node involvement as 34% and 31.4%, whereas this was 23% (3/13) in our series.

V. Conclusion Cervical adenocarcinomas have worse prognosis than squamous cell carcinomas especially when the cancer cells observed at out of the cervix (stage II and higher).

Table 14. Distribution of cervical adenocarcinoma cases according to the stages Martel 2000 Stage I %69.4 Stage II %14.3 Stage III + IV %16.3

al, Ishikawa et al, Meydanli et al, Aegean Hospital 1999 2001 %74.6 %32.5 %44.8 %21.2 %60.4 %34.6 %4.1 %6.9 %20.6 269

Manhan et al: Prognosis of early invasive and locally advanced (Stage I, II) uterine CACs Look KY, Brunetto VL, Clarke-Pearson DL, Averette HE, Major FJ, Alvarez RD, Homesley HD, Zaino RJ (1996) An analysis of cell type in patients with surgically staged stage IB carcinoma of the cervix a Gynecologic Oncology Group study. Gynecol Oncol 63, 304-311. Martel P, Connan L, Bonnet F, Dellannes M, Farnarier J, El Ghaoui A (2000) Cervical adenocarcinomas Diagnostic, prognostic and therapeutic aspects in a 49 case-control study. J Gynecol Obstet Biol Reprod 29, 48-54. Mc Lellan R, Dillon MB, Woodruff JD, Heatley GJ, Fields AL, Rosenshein NB (1994) Long-term follow-up of stage I cervical adenocarcinoma treated by radical surgery. Gynecol Oncol 52, 253-9. Meydanli MM, Tulunay G, Çalı#kan E, Özgül N, Köse MF (2001) Serviks adenokarsinomunda prognostik faktörler. Klinik Bilimler ve Doktor 5, 702-707. Moberg PJ, Einhorn N, Silverward C (1986) Adenocarcinoma of the uterine cervix. Cancer 57, 407-410. Ruey-Jien C, Daw-Yuan C, Men-Luh Y, Evelyn FL, Su-Cheng H (1998) Prognostic factors of primary adenocarcinoma of the uterine cervix. Gynecol Oncol 69, 157-164. Sabol A, Emerich J, lapinska S, Stankowska B, Starnawski M (1999) Clinical aspects of patients operated for adenocarcinoma and adenosquamous carcinoma of the uterine cervix. Ginekol Pol 70, 75-80. Shingleton HM, Gore H, Bradley DH, Soong SJ (1981) Adenocarcinoma of the cervix. Clinical evaluation and pathologic features. Am J Obstet Gynecol 139, 799-814. Smith HO, Tiffany MF, Qualls CR, Key CR (2000) The rising incidence of adenocarcinoma relative to squamöz cell carcinoma of the uterine cervix in the United States-a 24 year population-based study. Gynecol Oncol 78, 97-105. Syrjanen K (2000) HPV and cervical adenocarcinoma In Syrjanen K, Syrjanen S editors Papillomavirus infections in human pathology New York Wiley, p 189-206 [chapter 8]. Weiss RJ, Lucas WE (1986) Adenocarcinoma of the uterine cervix. Cancer 57, 1996-2001.

This characteristic was tried to be defined by early lymph node metastasis capability and less sensitivity to radiation. Consequently, the primary treatment modality for primary and metastatic lesions is still controversial. High recurrence and mortality rates can be observed even after administration of postoperative adjuvant radiotherapy if insufficient surgical procedures are applied to these type of cases.

References Attanoos R, Nahar K, Bigrigg A, Roberts S, Newcombe RG, Ismail SM (1995) Primary adenocarcinoma of the cervix. A clinicopathologic study of prognostic variables in 55 cases. Int J Gynecol Cancer 5, 179-186. Aydinli K, Bozkurt S, Erkün E, Atasü T (1987) adin Genital Kanser Insidansi. Jinekoloji ve Obstetrik Dergisi 1, 33. Ayhan A, Yüce K, Tuncer ZS, Urman B, Ayhan A (1989) Stage I Adenocarcinoma of the Uterine Cervix Tumour size, grade, lymph node metastases and 5-year survival. Aust- NZ J Obstet Gynaecol 29, 443-44. Berek JS, Hacker NF, Fu Y, Sokale JR, Leuchter RC, Lagasse LD (1985) Adenocarcinoma of the uterine cervix histologic variables associated with lymph node metastasis and survival. Obstet Gynecol 65, 46-52. Berkowitz RS, Ehrmann RL, Lavizzo-Mourey R, Knapp RC (1979) Invasive cervical carcinoma in young women. Gynecol Oncol 8, 311-6. Brand E, Berek JS, Hacker NF (1988) Controversies in the management of cervical adenocarcinoma. Obstet Gynecol 71, 261-269. Chen RJ, Chang DY, Yen ML, Lee EF, Huang SC, Chow SN, Hsieh CY (1998) Prognostic factors of primary adenocarcinoma of the uterine cervix. Gynecol Oncol 69, 157-164. Erzen M, Mozina A, Bertole J, Syrjanen K (2002) Factors predicting disease outcome in early stage adenocarcinoma of the uterine cervix. Eur J Obstet Gynecol Reprod Biology 101, 185-191. Ferenczy A in A Blaustein A (1982) (ed) Pathology of the Female Genital tract New York, Springer-Verlag, 2nd ed, pp 200 Hopkins MP, Schmidt RW, Roberts JA, Morley GW (1988) The prognosis and treatment of stage I adenocarcinoma of the cervix. Gynecol Oncol 72, 915-921. Hopkins MP, Sutton P, Roberts JA (1987) Prognostic features and treatment of endocervical adenocarcinoma of the cervix. Gynecol Oncol 27, 69-75. Ishikawa H, Nakanishi T, Inoue T, Kuzuyaka K (1999) Prognostic factors of adenocarcinoma of the uterine cervix. Gynecol Oncol 73, 42-46. Kilgore LC, Soong SJ, Gore H, Shingleton HM, Hatch KD, Partridge EE (1998) Analysis of prognostic features in adenocarcinomas of the cervix. Gynecol Oncol 31, 137-148. Kjorstad KE, Bond B (1984) Stage Ib adenocarcinoma of the cervix metastatic potential and patterns of dissemination. Am J Obstet Gynecol 150, 297-299. Larson D, Copeland L, Stringer C, Gersheson D, Malone J, Edwards C (1988) recurrent cervical carcinoma after radical hysterectomy. Gynecol Oncol 30, 381-387.

Mehmet Murat Inal

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Cancer Therapy Vol 4, page 271 Cancer Therapy Vol 4, 271-276, 2006

How could the light fluence rate influence the cure effect of photodynamic therapy? Research Article

Tao Xu*, Mingzhao Li, Academy of Metrology and Quality Inspection of Shenzhen, GuangDong Pro, China, 510085

__________________________________________________________________________________ *Correspondence: Tao Xu, Academy of Metrology and Quality Inspection of Shenzhen, GuangDong Pro, China, 5100; Phone: +86755-26941691; Fax: +86-755-26941524; E-mail: xutao780606@126.com Keywords: photodynamic therapy, fluence rate, microvasculature damage, RBCs column diameter!!Krogh tissue model Abbreviations: bovine serum albumin, (BSA); haematoporphyrin derivative, (HpD); optical density, (OD); Photodynamic therapy, (PDT); RBCs column diameter, (RBCCD) Received: 11 May 2006; Revised: 16 November 2006 Accepted: 01 December 2006; electronically published: December 2006

Summary Some works have been done in this paper to study the role of light fluence rate in the photodynamic therapy (PDT), which focuses on the influence of light fluence rate on the microvasculature damage, the cell killing and the photodynamic reaction impetus. The microvasculature damage is studied through observing the values of RBCs column diameter during the process of the HpD mediated PDT. It is found less microvasculature damage is induced by 75mW/cm2 illumination than that by 150mW/cm2, indicating that under 75mW/cm2 illumination tumor oxygen can be better preserved than 150mW/cm2. The cell killing experiment is performed in vitro and designed in the manner that cell killing rate was only influenced by light characters. We find PDT with 75mW/cm2 illumination can cause higher cell killing rate than with 150mW/cm2 illumination. In addition a PDT model of Krogh tumor is built to analyze the promotion of photodynamic reaction. We find under a same initial oxygen environment (18.4!M) the PDT stops at oxygen concentration of 3.54!M when the illuminating light is 75mW/cm2 and 5.2!M when the illuminating light is 150mW/cm2. The calculation results indicate a more powerful PDT impetus under low fluence rate than high one. The oxygen concentration in 28.4 percent of the tumor volume is higher than 5.2!M and 59.8 percent of the tumor volume is higher than 3.54!M, indicating a larger therapy range under lower fluence rate illuminating.

It is noticed at the same time that at similar oxygen environment low fluence rates cause more tumor cure than high fluence rates for the same total fluence (Sitnik et al 1998; Foster et al 1993; Veenhuizen and Stewart 1995). Are there any other reasons that cause the higher cure rate except oxygen? Maybe the fluence rate can influence the tumor cell killing ability, but it needs to be verified. Some experiments observing the microscopic cell killing rate at different fluence rates must be performed. PDT impetus is also a guide to evaluate the efficiency of therapy. The relationship between the PDT impetus and the fluence rate should be observed. We can build a model to simulate the photodynamic reaction process. As photodynamic reaction will stop under some oxygen level (Nichols and Foster 1994), the threshold can be calculated to represent the PDT impetus. By this attempt, we can find out how the fluence rate influences the reaction process.

I. Introduction Photodynamic therapy (PDT) is believed to act through the cytotoxic singlet oxygen, which forms when the photosensitizer is excited by light and transfers its energy to the molecular oxygen in tissues. It may be the principle way to destroy the tumor cell in PDT that the singlet oxygen will oxidize some elements of the cell. Photosensitizer, tissue oxygen and irradiation light are considered to be the three element factors of PDT (Dougherty ea al 1998; Rosenthal and Glatstain 1994). Oxygen in tissues can be easily depleted at high fluence rates while it can be preserved at low fluence rates (Henderson et al 2000). And a rule has been concluded that the higher fluence rates, the more decreasing in the oxygen concentration (Foster et al 1991; Schunck and Poulet 2000). While how oxygen can be preserved at low fluence rates is a puzzling problem. It is believed to have something to do with the microvasculature.

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Xu and Li: How could the light fluence rate influence the cure effect of photodynamic therapy? Shanghai, China). The fraction cell surviving was defined as the ratio of the mean OD of experiment groups to the control group.

Works mentioned above can help us to recognize how the fluence rate can influence the photodynamic therapy effect.

C. Study on the PDT impetus The theory of oxygen diffusion from capillaries has been well established since Krogh, 1919 presented his analytical prediction of how the delivery from vessels was governed. Since that time, researchers have tried to exploit this model to better interpret the oxygen diffusion and consumption processes in tissues. It is accepted that oxygen diffuses away from the high relative concentration in the vessel to regions of tissue with lower concentration. According to these models, The equation describing PDT process can be developed as follow: "C(r) = #(D#C(r)) + s(C(r))$ %met (C(r)) $ %pdt (C(r)) (1) "t where C (r) is the oxygen concentration at position r, D is the diffusion coefficient for oxygen in tissue, which is generally taken to be 2!10-5cm 2s-1(Pogure et al, 2001). !met, !pdt, s are the metabolic oxygen consumption rate, the PDT oxygen consumption rate and the supply of oxygen at each point r respectively, and all vary with C (r). According to the MichaelisMenten model (Tang, 1933), we have deduced the expression of !met (Xu, 2004): 9.8C "met (C) = (2) C + 1.408

II. Materials and Method A. Microvasculature damage in PDT against light fluence rate

Wistar rats weighted 200Âą10g (Experimental Animal Center of PLA General Hospital, Beijing, China) were obtained at least 1 week before the experimentation. They were housed in a room with subdued lighting and fed with a standard pellet diet and water. Approval for this project was obtained from the Animal Experiment Center of Tianjin Medical University. A chondrosarcoma tumor cell line was transplanted into the right flank of each rat. The tumors that grew to be approximately 6-7 mm in size within about 5 days after transplantation were used in this experiment. ! The animals were given the haematoporphyrin derivative -1 (HpD) via tail vein injection at a dose of 2.5 mg kg . About 24 hours after the injection the rats were anaesthetized with a 50 mg kg-1 sodium pentobarbital. The RBCs column diameter (RBCCD) observation method described in (Fingar et al 1992, 1999; Xu et al 2005) was employed to investigate the tumor microvasculature. The distribution of the vascular was observed by unaided eyes and little fatty regions with arteriole and venule !pdt has been given in the fore studies by: in pairs were chosen. The RBCCD was measured through the % t ( k ot C k os playback of the recorded images every 4 min for 32 min for the ! "pdt = "0 exp&# "pdt dt) (3) 75 mW/cm2 illuminating group. For the 150 mW/cm2 k ot C + k p ' k oa [ A] 0 * illuminating group it is 2 min for 16 min. The relative RBCs column diameter RBCCDrel at time t kot is the bimolecular rate of triplet quenching by triplet was defined as RBCCD (t)/RBCCD (0). Ten animals were used in oxygen, koa is the rate of chemical reaction between singlet each different experimental group and the mean relative RBCCD ! oxygen and unspecified substrate [A], kos is the rate of chemical at time t was thought to reflect the status of the microvasculature reaction between singlet oxygen and the photosensitizer, which after some dose of illumination. always seems to be 0. And kp is the decay rates of the photosensitizer triplet, respectively. !0 is a constant that B. Cell killing in PDT against light fluence describes the initial rate of photochemical oxygen consumption rate and changes linearly with the light fluence rate (Nichols and Mammary cancer cell MDAMB 543 was obtained from the Foster 1994, Geogakoudi et al 1997). Setting values of all the tumor laboratory of PLA General Hospital of China and was parameter can be found in the paper of Mitra et al, 2000. cultured in vitro with 90% RPMI 1640 incubation media Equation (3) indicates that PDT will stop at a quite low oxygen (GIBCOBRL Lifetechnologies, Rockville, MD, USA), 10% concentration (<<kp / kot). bovine serum albumin (BSA) and 1% penicillin and streptomycin There is a little trouble in defining s. First, during PDT at 37!. The cancer cell was laid in incubator with 5% CO2. The there is a similar arterial blood oxygen level with that before cultured MDAMB 543 cells were made suspension of single cell PDT, while the venous blood oxygen concentration decreases with 0.25% trypsase (Sigma, St Louis, MO, USA). The cell much after the beginning of PDT (Guyton 1981, Slonim and concentration of the suspension was 1.0"105/ml. Seven culture Hamilton, 1981). So there is a higher oxygen supply during PDT plates with 96 wells (Costar, Cambridge, MA, USA) were used than before. Second, the higher oxygen concentration at the outer to inoculation the suspension in a manner of 0.2 ml per well and vascular wall, the higher blood oxygen supply occurs. placed in the 5% CO2 incubator at 37! for 16 hours. Then the Considering these two factors, we define s by (Xu, 2004): supernatant culture media was removed. HpD as the +photosensitizer, which was diluted by RPMI 1640 incubation t 9.8C % C (t) (/s= " ,1 +[# $ 1] " '1$ out *0dt (4) media to a concentration of 0.004 mg/ml, was added to the wells. 0 C + 1.408 & C out (0) )-1 . The culture plates was then placed in the 5% CO2 incubator at 37! and completely protected from light. About 4 hours later Cout(t) is the oxygen concentration at the outer vascular six of the plates were illuminated by 150mW/cm2 laser for 10s, wall at time t after the beginning of PDT. " is a constant which 20s, 30s and 75 mW/cm2 laser for 20s, 40s, and 60s respectively ! can be set as 1~3. while the other one plate was still kept in protection of light as a control group. After illuminating the culture plates were placed back in the incubator for 24 hours and then 0.1 mg III. Results diphenylterrazolium bromide (Sigma, St Louis, MO, USA) was RBCCD changes of venule and arteriole both added to the wells before another 4 hoursâ&#x20AC;&#x2122; culture. The occurred under the illumination of 75mW/cm2 and supernatant fluid was removed when the culture was completed 150mW/cm2 laser. Data collected from the image playback and 0.15 ml dimethylsufoxide was added. Culture plates were of experiment groups were summarized in Figure 1. First, oscillation slightly for 10 min and the optical density (OD) of all the data obtained from the observation of arteriole were the wells was measured by an Elisa (318-MC, Xinke Inc,

shown in Figure 1A, followed by the data obtained from

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Cancer Therapy Vol 4, page 273 the observation of arteriole shown in Figure1B. In Figure 1A, there was an indication that under the low fluence rate illumination there occurred less microvasculature damage than high fluence rate in the early period of PDT, while there was no significant difference between the two kinds of illumination near the end of the treatment. Arteriole RBCCDrel was obviously lower under 150mW/cm2 illumination than 75mW/cm2 when light dose is 36 J/cm2 (p=0.0031), 54 J/cm2 (p=0.0024) and 72 J/cm2 (p=0.036). The administration of light dose up to 90 J/cm2 and above seemed to make similar damages to the arteriole at both light fluence rate. For the light dose of 18 J/cm2 increase of RBCCDrel occurred under 150 mW/cm2 illumination. That might due to the physiological reaction to the high light fluence rate. The RBCCDrel of the venule decreased more quickly against the light dose than that of the arteriole as shown in Figure 1B, while no statistically significant difference in the RBCCDrel of the venule was observed under 150mW/cm2 and 75mW/cm2 illumination though one point at the light dose of 54 J/cm2 gave some peradventure. It was observed that under the illumination of 75mW/cm2 light the RBCCD of venules dropped slowly when the illuminated fluence was less than 54 J/cm2, but rapidly when between 54 J/cm2 and 72 J/cm2. Therefore we could assume that at 75 mW/cm2 the threshold energy

dose causing the vessels damaged severely was between 54 J/cm2 and 72 J/cm2. Similarly, at 150 mW/cm2 that threshold might be between 36 J/cm2 and 54 J/cm2. The difference of the two thresholds might do some contribution to the exception at 54 J/cm2. To determine the PDT induced direct MDAMB 543 cells killing at various fluence rates the survival rates were examined. The mean OD valuewas measured as the scale of the fraction surviving. For control group it was the average OD of 82 wells in the control plate. OD value of each well in the experiment plates was measured and normalized to the mean control value. For the 150mW/cm2 illuminated group the wells number of each plate was 36 and for OD 75 mW/cm2 illuminated group it was 38. The illuminating light dose of both groups was 1.5 J/cm2, 3 J/cm2, 4.5 J/cm2. Mean normalized OD value and SD of each plate were calculated and summarized in Figure 2. The surviving fraction was 0.624 versus 0.526(1.5 J/cm2), 0.516 versus 0.384(3 J/cm2), and 0.319 versus 0.219(4.5 J/cm2) under 150mW/cm2 versus 75mW/cm2 illuminating. It was recognized in a statistic way that the MDAMB 543 cells survival fractions under fluence rate of 75mW/cm2 was less than that under 150 mW/cm2, indicating more effective cell killing under lower fluence rate illumination in PDT.

Figure 1. RBCCD rel changes against the light dose in different fluence rates for arteriole (A) and venule (B). Values are meansÂąSD of 10 observation results in each experiment group. Those data points that show, based on the Wilcoxon signed-ranks test, a statistically significantly difference between the 75 mW/cm 2 group and 150 mW/cm 2 group are indicated with an asterisk (*). Figure 2. Cell fraction surviving after illuminating with light dose of 1.5 J/cm 2, 3 J/cm2, 4.5 J/cm 2 in PDT. Values were experimental OD normalized to the control group that was protected from light. Cell fraction surviving of the two experiment groups were significantly different (p=0.041, 0.033, 0.021).

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Xu and Li: How could the light fluence rate influence the cure effect of photodynamic therapy? The initial oxygen concentration at some points in a range of 100#m surrounding the vascular, including the point just at the outer wall Cout(0), could be obtained from the experimental study of Tsai et al, 1998. We expanded the range through fitting those points and got the initial oxygen distributing in a range of 1.5mm. Crank-Nicolson method was employed to solve equation (1) starting from those initial values. The variety of oxygen concentration against time t and space r was displayed by Figure 3A (fluence rate of 75mW cm-2) and Figure 3B (fluence rate of 150mW cm-2). The oxygen threshold in the photodynamic reaction was considered to be 3.54#M in Figure 3A and 5.2#M in Figure 3B. This result indicated a more powerful PDT impetus under low fluence rate than high one. It could be informed from Figure 3 that photodynamic reaction could not start in some area where the oxygen concentration was less than those thresholds. For 75mW/cm2 illuminating the inactive area was region of r>1.16mm and for 150mW/cm2 illuminating it is r>0.8mm. It is 40.2% and 71.6% of the model tumor volume respectively.

due to the extremely vulnerable venule circulation. In theory, less damage to microvasculature indicates timely oxygen supply to recover the photochemical consumption of oxygen in PDT. As tissue oxygen is one of the elementary factors of PDT, the timely supply of oxygen is fairly important for the destruction of tumors. Some clinical and experimental data have been reported recently pointing to a significant tumor cure effect (Robinson et al 1998; Langmack et al 2001). From the result of our cell killing experiment, we can conclude that lower fluence rate treatments may be more effective in increasing the killing rate of tumor cells, which directly indicates better local tumor control. For an experiment performed in vitro, the oxygen consumption of PDT and the photosensitizer concentration in the medium can be regarded as constant. Thus the cell killing effect may vary against the photosensitizer bleaching and singlet oxygen generating, which changes with the light fluence rate. While recently studies have reported that higher photobleaching seem to occur under illuminating of lower fluence rate both in vivo and vitro experiments (Coutier et al 2001; Finlay et al 2004). So we can only contribute the higher cell killing rate caused by lower fluence rate to higher singlet oxygen generating. In vitro experiment with sufficient oxygen supplying, sharply consumption of triplet oxygen caused by high fluence rate illuminating will increase the chance that excited singlet oxygen return to the ground state. As a result the possibility that singlet oxygen reaction with the tumor cell is decreased, means a low cell killing rate. In the PDT process, the decay rates of the photosensitizer triplet, kp, is decided by the concentration of triplet photosensitizer. Under a higher fluence rate illuminating, more photosensitizer molecules are excited to be singlet and triplet status. High irradiance energy causes high frequence of energy level transition. Consequently the tendency of energy transferring from triplet photosensitizer to triplet oxygen is wakened. So under higher fluence rate illuminating the bimolecular rate of triplet quenching by triplet oxygen, kot, takes a lower value.

IV. Discussion From this work it can be recognized that light fluence rate can improve the therapy effect through the blood oxygen supply, the cell killing rate and the reaction impetus. Low fluence rate treatments can be more effective in decreasing vascular lesions than high fluence rate treatments, if the same light fluence is applied during the PDT process. Other studies have also concluded lower fluence rate treatments can preserve the status of oxygen for a more effective PDT (Tromberg et al 1990; Foster et al 1991; Sitnik and Henderson 1998; Sitnik et al 1998), and it has been recognized that light fluence rates play a major role in the tumor oxygenation status during PDT exposure. Our work explains the mechanism of oxygen preservation under low fluence illumination in PDT to some extent. It can be concluded from Figure 1 that lower fluence brings less microvasculature damage during the PDT process, though there seems to be no difference in venules at the two different fluence rates, which may be

Figure 3. Oxygen concentration changes against time t and space r in 75 mW/cm2 (A) and 150 mW/cm 2 (B) illuminating PDT. Values are deduced step by step with Crank-Nicolson method from the Krogh model tumor. C(r,0) is obtained through fitting the dates in Tsaiâ&#x20AC;&#x2122;s paper, 1998.

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Cancer Therapy Vol 4, page 275 Krogh A (1919) The number and distribution of capillaries in muscles with calculations of the oxygen pressure head necessary for supplying the tissue. J Physiol 52, 409-415. Langmack K, Mehta R, Twyman P, Norris P (2001) Topical photodynamic therapy at low fluence rates-theory and practice. J Photochem Photobiol B. 60, 37-43. Mitra S, Foster TH (2000) Photochemical Oxygen Consumption Sensitized by a Porphyrin Phosphorescent Probe in Two Model Systems. Biophys J 78, 2597-2605. Nichols MG and Foster TH (1994) Oxygen diffusion and reaction kinetics in the photodynamic therapy of multicell tumour spheroids. Phys Med Biol 39, 2161-2181. Pogure BW, Oâ&#x20AC;&#x2122;Hara JA, Wilmot CM, Paulsen KD and Swartz HM (2001) Estimation of oxygen distribution in RIF-1 tumors by diffusion model-based interpretation of pimonidazole hypoxia and eppendorf measurements. Radiat Res 155, 15-25. Robinson DJ, de Bruijn HS, van der Veen N, Stringer MR, Brown SB, Star WM (1998) Fluorescence photobleaching of ALA-induced protoporphyrin IX during photodynamic therapy of normal hairless mouse skin: the effect of light dose and irradiance and the resulting biological effect. Photochem Photobiol 67, 140-146. Rosenthal DJ, Glatstein E (1994) Clinical applications of photodynamic therapy. Ann Med 26, 405-409. Schunck T, Poulet P (2000) Oxygen consumption through metabolism and photodynamic reactions in cell cultured on microbeads. Phys Med Biol 45, 103-119. Sitnik TM, Hampton JA, Henderson BW (1998) Reduction of tumour oxygenation during and after photodynamic therapy in vivo: effects of fluence rate. Br J Cancer 77, 1386-1394. Sitnik TM, Henderson BW (1998) The effect of fluence rate on tumor and normal tissue responses to photodynamic therapy. Photochem Photobiol 67, 462-466. Slonim NB and Hamilton LA (1981) In Respiratory Physiology, 4th edn. Mosby,St Louis. MO. Tang PS (1933) On the rate of oxygen consumption by tissues and lower organisms as a function of oxygen tension. Q Rev Biol 8, 260-274. Tromberg BJ, Orenstein A, Kimel S, Barker SJ, Hyat J, Nelson JS, Berns MW (1990) In vivo tumour oxygen tension measurements for the evaluation of the efficiency of photodynamic therapy. Photochem Photobiol 52, 375-385. Tsai AG, Friesenecker B, Mazzoni MC, Kerger H, Buerk DG, Johnson PC and Intaglietta M (1998) Microvascular and tissue oxygen gradients in the rat mesentery. Proc Natl Acad Sci U S A95,6590-6595. Veenhuizen RB, Stewart FA (1995) The importance of fluence rate photodynamic therapy: was there a parallel with ionizing radiation dose-rate effects? Radiother Oncol 37, 131-135. Xu T (2004) The theoretical and experimental studies on the tumor oxygen content in photodynamic therapy. PhD thesis, Tianjin University. Xu T, Wu X, Li YX (2005) Application of lower fluence rate for less microvasculature damage and more cell killing during photodynamic therapy. Lasers Med Sci 19, 257-262.

For the promotion of photodynamic reaction is positive going with the product of kot and oxygen concentration, there need to be a high oxygen environment to ensure the proceeding of photodynamic reaction. Similar viewpoint are presented in appendix equation (A9) by Finlay et al, 2004. As a result PDT under a higher fluence rate will stop at a higher oxygen concentration threshold. Oxygen concentration ascending showed in Figure 3 is owned to the oxygen diffusion in the model tumor. Though there is no reaction occurring, the light illuminating aggravates the oxygen diffusion to the inactive area.

References Coutier S, Mitra S, Bezdetnaya LN, Parache RM, Georgakoudi I, Foster TH and Guillemin F (2001) Effects of fluence rate on cell survival and photobleaching in meta-tetra(hydroxyphenyl)chlorin-photosensitized colo 26 multicell tumor spheroids. Photochem Photobiol 73, 297-303. Dougherty TJ, Gomer CJ, Henderson BW, Jori G et al (1998) Photodynamic therapy. J Natl Cancer Inst 90,889-905. Fingar VH, Kik PK, Haydon PS, Cerrito PB, Tseng M, Abang E, Wieman TJ (1999) Analysis of acute vascular damage after photodynamic therapy using benzoporphyrin derivative (BPD). Br J Cancer 79, 1702-1708. Fingar VH, Wieman TJ, Wiehle SA, Cerrito PB (1992) The role of microvascular damage in photodynamic therapy: the effect of treatment on vessel constriction, permeability and leukocyte adhesion. Cancer Res 52, 4914-4921. Finlay JC, Soumya M, Patterson MS, Foster TH (2004) Photobleaching kinetics of photofrin in vivo and in multicell tumour spheroids indicate two simultaneous bleaching mechanisms. Phys Med Biol 49, 4837-4860. Foster TH (1990) Magnetic resonance, optical , and mathematical methods applied to studies in the biological physics of tissues. Ph.D theisis of the University of Rochester. Foster TH, Hartley DF, Nicholas MG, Halif R (1993) Fluence rate effects in photodynamic therapy of multicell tumour spheroids. Cancer Res 53, 1249-1254. Foster TH, Murant RS, Bryant RG, Knox RS, Gibson SL, Halif R (1991) Oxygen consumption and diffusion effects in photodynamic therapy. Radiat Res 126, 296-303. Geogakoudi I, Nichols MG and Foster TH (1997) The mechanism of photofrin photobleaching and its consequences for photodynamic dosimetry. Photochem Photobiol 65, 135144. Guyton AC (1981) Physiology: Textbook of Medical Physiology, 6th edn. Saunders, Philadelphia, PA. Henderson BW, Busch TM, Vaughan LA, Frawley NP, Babich D, Sosa TA, Zollo JD, Dee AS, Cooper MT, Bellnier DA, Greco WR, Oseroff AR (2000) Photofrin Photodynamic Therapy Can Significantly Deplete or Preserve Oxygenation in Human Basal Cell Carcinomas during Treatment, Depending on Fluence Rate. Cancer Res 60, 525-529.

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Surgical treatment of gastric cancer in South Western Greece Research Article

Konstantinos Vagenas1, Charalambos Spyropoulos1, Pantelis Tsamalos1, Panagiotis Papadopoulos2, George Skroubis1, John Spiliotis2 1 2

Department of Surgery, University of Patras, Medical School of Patras, Greece Department of Surgery, “Hatzikosta” General Hospital, Mesolongi, Greece

__________________________________________________________________________________ *Correspondence: Konstantinos Vagenas, Associate Professor of Surgery, 16, Aou Street, 26442, Patras (Exo Aghia), Greece; Tel. +32610-455-635; Fax +32610-993-984; E-mail: kvagenas@hotmail.com Key words: Gastric Cancer, Nodal Status, Gastrectomy, Lymphadenectomy, Survival Abbreviations: partial gastrectomy, (PG); total gastrectomy, (TG) Received: 3 May 2006; Revised: 20 November 2006 Accepted: 01 December 2006; electronically published: December 2006

Summary Curative resection is the treatment of choice for gastric cancer, but the role of the type of dissection, (D1) versus (D2) and the lymph node involvement in survival, remains unclear. The aim of this study is to evaluate the different surgical procedures in the 5-year survival rates.The files of 139 patients with gastric cancer, treated surgically, were evaluated retrospectively from 1996 to 2005. The study compared the effectiveness, estimated by 5-year survival rates, of two different surgical procedures, total gastrectomy (TG) versus partial gastrectomy (PG), two types of lymph node dissections according to the number of lymph nodes finally excised, A versus B and the nodal status, N0 versus N+. Total gastrectomy demonstrated statistically significant differences (p < 0.002) compared to partial gastrectomy. The number of lymph node dissection demonstrated a survival benefit for B resection while the 5-year survival rates for N0 status were superior to N positive status (50% versus 11% respectively). Our results support the data from Japanese and Western studies, proposing total gastrectomy with advanced lymph node dissection in patients with gastric carcinoma.

of lymph nodes finally excised, A versus B and the nodal status, N0 versus N+, in patients with gastric carcinoma, according to data retrieved from two hospitals in South Western Greece during the last decade (1996–2005).

I. Introduction Surgical approach remains the only hope for curative treatment of gastric carcinoma, mainly when this aggressive type of neoplasm is localized. The prognosis after surgical resection is strongly related to tumor stage and lymph node involvement (de Almeida et al, 1994). The value of different types of gastric resections or lymph node dissections remains controversial between Japanese surgeons who routinely perform extended lymph node dissection and they are so convinced that extended lymphadenectomy in positive nodes reduces the incidence of local recurrence (Katai et al, 1994) and improves survival (Sasako et al, 1995), that a randomized trial comparing D1 versus D2 resection in Japan would be regarded as ethically unacceptable (Roukos et al, 1998). The objective of this retrospective study was to compare the effectiveness, estimated by 5-year survival rates, of two different surgical procedures, total gastrectomy (TG) versus partial gastrectomy (PG), two types of lymph node dissections according to the number

II. Patients and methods The files of 139 patients (97 men and 42 women, age range 25–101 years, mean age 64.5 years) with gastric cancer were evaluated retrospectively. All clinical and pathological data were obtained form the surgical records and histopathological reports. Total gastrectomy was performed in 66 patients (47.5%) and partial gastrectomy in 73 patients (52.5%). The degree of lymph node dissection was determined according to the number of lymph nodes evaluated in the histopathological results. The operation with < 20 lymph nodes evaluated histopathologically was classified as A dissection while B dissection was characterized the operation with > 20 nodes evaluated.

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Vagenas et al: Surgical treatment of gastric cancer in south western Greece The nodal status was considered as N0 if the nodes were negative for metastatic disease and N positive (N+) if at least one node was found positive for metastatic disease. This type of surgery was performed with curative aim and this group is the object of the current report.

A lymph node dissection. Further analysis of lymph node involvement (N0 versus N+) showed statistical difference in outcome (p < 0.001), with 5-years survival rates reaching 50% and 11% respectively (Figure 3). Correlation analysis of nodal status and dissection type revealed that N0 disease survival rates are similar for A and B lymph node resection (p > 0.1), but statistical difference in N+ status with B resection demonstrates better prognosis than A lymph node resection (p < 0.001) (Figure 4). When correlation analysis was conducted for the type of surgical resection, the nodal status was found to be the most important factor for longer survival in the group of patients where total gastrectomy was performed. In this group, there was no survival benefit in N0 patients either if A or B lymph node dissection was performed (p > 0.2), while in N+ patients the benefit of B resection was clearly demonstrated (p < 0.002) (Figure 5). The same analysis in the group of patients with partial gastrectomy and N0 or N+ status, showed no influence on survival by A or B procedure respectively (Figure 6, Figure 7).

III. Results The analysis of postoperative morbidity and mortality showed no statistical differences between total gastrectomy and partial gastrectomy groups (x2 = 3.91, p = 0.048, Table 1). Survival estimates between total gastrectomy versus partial gastrectomy demonstrated statistically significant difference (p < 0.002) in favor of total gastrectomy group. The mean survival for total gastrectomy group was 59 months versus 25 months of mean survival after partial gastrectomy, while the 5-years survival rates were 39% and 21% respectively (Figure1). The number of lymph node dissection (A versus B) determined the survival curves presented in Figure2, also demonstrating statistically significant difference (p < 0.001) with a mean survival of 58 months for B dissection versus 29 months after Table 1. Mortality and morbidity results.

Death Pneumonia Intestinal Fistula Postoperative Bleeding Infectious Complications (Catheter related, Urine or Incisional abscess) Deep Vein Thrombosis

Total Gastrectomy 2/66 (3.03%) 4/66 (6.06%) 1/66 (1.51%) 1/66 (1.51%) 8/66 (12.12%)

Partial Gastrectomy 2/73 (2.74%) 3/73 (4.11%) 2/73 (2.74%) 3/73 (4.11%) 10/73 (13.69%)

1/66 (1.51%)

2/73 (2.74%)

Figure 1. Kaplanâ&#x20AC;&#x201C;Meier curve in patients with gastric carcinoma. The role of the procedure type.

Figure 2. Kaplanâ&#x20AC;&#x201C;Meier curve in patients with gastric carcinoma. The role of the type of lymph node dissection.

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Figure 3. Kaplanâ&#x20AC;&#x201C;Meier curve in patients with gastric carcinoma. The role of the nodal status, according to tumor invasion.

Figure 4. The role of A versus B lymph node dissection in N+ lymph nodes.

Figure 5. The role of type of lymph node dissection (A versus B) in patients with total gastrectomy and N+ nodal status.

Figure 6. The role of type of lymph node dissection (A versus B) in patients with partial gastrectomy and N0 nodal status.

Figure 7. The role of type of lymph node dissection (A versus B) in patients with partial gastrectomy and N+ nodal status.

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Vagenas et al: Surgical treatment of gastric cancer in south western Greece

these procedures was found in favor of total gastrectomy, especially in node positive patients, with similar mortality and morbidity rates. These results agree with different studies from western countries (Wanebo et al, 1996; Llanos et al, 1999; Hartgrink et al, 2004). Gastric carcinoma is one of the commonest causes of cancer death worldwide, characterized by a high frequency of regional disease with nodal metastases. The prognosis is strongly related to complete resection, to penetration of the serosa layer and the lymph node involvement. Our data demonstrated that the node positivity was directly related to survival and support the beneficial effect of total gastrectomy and more aggressive lymph node dissection for gastric carcinoma.

IV. Discussion The prognosis of patients with gastric carcinoma is determined by a number of tumorâ&#x20AC;&#x2122;s and patientâ&#x20AC;&#x2122;s associated factors (Allgayer et al, 1997; Siewert et al, 1998). Of particular interest are prognostic factors that may be influenced by therapeutic measures, such as the extent of regimen resection and the lymphadenectomy. Complete tumor removal with adequate margins of clearance, a R0 resection according to the UICC classification, has been widely accepted as a major factor of survival improvement in patients with gastric carcinoma (Bonenkamp et al, 1999; Bozzetti et al, 1999; Huscher et al, 2004). Our data demonstrate that total and partial gastrectomies performed as elective procedures have similar postoperative complication rates and surgical outcome. A 39% 5-year probability after total gastrectomy versus 21% in partial gastrectomy was found in our results, which are worse than other western surgical series. This may be due to relatively higher incidence of patients with tumors of the upper third of the stomach, a group well known to have a worse prognosis (Marubini at al 1993; Finlayson et al, 2003; Kunisaki et al, 2005). Data supporting the value of lymph node dissection D1 versus D2 are lacking. In our study a survival benefit of B dissection has been demonstrated, with mean survival of 58 months in this group of patients versus 29 months in the patients where A dissection was performed. These results are almost comparing to the large Japanese studies and published reports from specialized centers where typical D2 dissections were performed (Marujama et al, 1987; Sasako et al, 1995; Pacelli et al, 1999). The data of the present study also suggest that B type of lymph node resection has a beneficial effect in patients with nodeâ&#x20AC;&#x201C;positive disease, supporting the data of the literature (Roukos et al, 1998). A 50% 5-year survival probability for node-negative group versus only 11% in node-positive patients (p < 0.001) was observed. A recent meta-analysis (McCulloch et al, 2005) demonstrated a possible 32% survival benefit of D2 resection for patients with serosa positive tumors, although mortality rates were higher when D2 operation was performed. The present study confirms data from the literature that lymph node involvement is the most reliable prognostic factor available for final prognosis (Roder et al, 1995; Bonenkamp et al, 1999), while other studies clarify not only the level but also the number of the metastatic nodes to be closely related to 5-year survival rates (Adachi et al, 1994). Lymph node dissection in gastric carcinoma of the upper third of the stomach is a promising approach; however there is no consensus of the optimal management of these tumors. Therefore, it is of great importance to clarify the therapeutic strategy of these tumors through evaluation of the pattern of lymph node metastases and the efficacy of lymph node dissection (Kunisaki et al, 2005). Another important factor in our analysis is the impact of total or partial gastrectomy on 5-year survival probability. A statistically significant difference between

References Adachi Y, Kamakura T, Mori M, Baba H, Maehara Y, Sugimachi K (1994) Prognostic significance of the number of positive lymph nodes in gastric carcinoma. Br J Surg 81, 414-416. Allgayer H, Heiss MM, Schildberg FW (1997) Prognostic factors in gastric cancer. Br J Surg 84, 1651-1664. Bonenkamp JJ, Hermans J, Sasako M, van de Velde CJ, Welvaart K, Songun I, Meyer S, Plukker JT, Van Elk P, Obertop H, Gouma DJ, van Lanschot JJ, Taat CW, de Graaf PW, von Meyenfeldt MF, Tilanus H (1999) Extended lymph node dissection for gastric cancer. N Engl J Med 340, 908914. Bozzetti F, Marubini E, Bonfanti G, Miceli R, Piano C, Gennari L (1999) Subtotal versus total gastrectomy for gastric cancer: five-year survival rates in a multicenter randomized Italian trial. Italian Gastrointestinal Tumor Study Group. Ann Surg 230, 170-178. de Almeida JC, Bettencourt A, Costa CS, de Almeida JM (1994) Curative surgery for gastric cancer: study of 166 consecutive patients. World J Surg 18, 889-895. Finlayson EV, Goodney PP, Birkmeyer JD (2003) Hospital volume and operative mortality in cancer surgery: a national study. Arch Surg 138, 721-725. Hartgrink HH, van de Velde CJ, Putter H, Bonenkamp JJ, Klein Kranenbarg E, Songun I, Welvaart K, van Krieken JH, Meijer S, Plukker JT, van Elk PJ, Obertop H, Gouma DJ, van Lanschot JJ, Taat CW, de Graaf PW, von Meyenfeldt MF, Tilanus H, Sasako M (2004) Extended lymph node dissection for gastric cancer: who may benefit? Final results of the randomized Dutch gastric cancer group trial. J Clin Oncol 22, 2069-2077. Huscher CG, Mingoli A, Sgarzini G, Sansonetti A, Lirici MM, Napolitano C, Piro F (2004) Videolaparoscopic total and subtotal gastrectomy with extended lymph node dissection for gastric cancer. Am J Surg 188, 728-735. Katai H, Maruyama K, Sasako M, Sano T, Okajima K, Kinoshita T (1994) Mode of recurrence after gastric cancer surgery. Dis Surg 11, 99-103. Kunisaki C, Shimada H, Nomura M, Matsuda G, Otsuka Y, Ono H, Akiyama H (2005) Surgical outcome in patients with gastric adenocarcinoma in the upper third of the stomach. Surgery 137, 165-171. Llanos O, Guzman S, Pimentel F, Ibanez L, Duarte I (1999) Results of surgical treatment of gastric cancer. Dig Surg 16, 385-388. Marubini E, Bonfanti G, Bozzetti F, Boracchi P, Amadori D, Folli S, Nanni O, Gennari L (1993) A prognostic score for

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Cancer Therapy Vol 4, page 281 patients resected for gastric cancer. Eur J Cancer 29, 845850. Maruyama K, Okabayashi K, Kinoshita T (1987) Progress in gastric cancer surgery in Japan and its limits of radicality. World J Surg 11, 418-425. McCulloch P, Niita ME, Kazi H, Gama-Rodrigues JJ (2005) Gastrectomy with extended lymphadenectomy for primary treatment of gastric cancer. Br J Surg 92, 5-13. Pacelli F, Sgadari A, Doglietto GB (1999) Surgery for gastric cancer. N Engl J Med 341, 538-539. Roder JD, Bonenkamp JJ, Craven J, van de Velde CJ, Sasako M, Bottcher K, Stein HJ (1995) Lymphadenectomy for gastric cancer in clinical trials: update. World J Surg 19, 546-553. Roukos DH, Lorenz M, Encke A (1998) Evidence of survival benefit of extended (D2) lymphadenectomy in western patients with gastric cancer based on a new concept: a prospective long-term follow-up study. Surgery 123, 573578. Sasako M, McCulloch P, Kinoshita T, Maruyama K (1995) New method to evaluate the therapeutic value of lymph node dissection for gastric cancer. Br J Surg 82, 346-351. Siewert JR, Bottcher K, Stein HJ, Roder JD (1998) Relevant prognostic factors in gastric cancer: ten-year results of the German Gastric Cancer Study. Ann Surg 228, 449-461.

Wanebo HJ, Kennedy BJ, Winchester DP, Fremgen A, Stewart AK (1996) Gastric carcinoma: does lymph node dissection alter survival? J Am Coll Surg 183, 616-624.

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Cancer Therapy Vol 4, page 283 Cancer Therapy Vol 4, 283-288, 2006

Prevention of radiation enteritis Review Article

Mert Saynak1,*, Vuslat Yurut-Caloglu1, Gulden Bayir-Angin1, Murat Caloglu1, Rusen Cosar-Alas1, Seden Ozbilen-Kucucuk2, Cem Uzal1, Isik Aslay2 1 2

Radiotherapy Department, Trakya University, Faculty of Medicine, Edirne, Turkey Radiotherapy Department, Istanbul University Oncology Institute, Istanbul, Turkey

__________________________________________________________________________________ *Correspondence: Dr. Mert Saynak, Fatih Mahallesi, Alparslan Yalkin Caddesi, Caglayan Apartmani B-Blok Daire 6, Edirne, Turkey; Tel: +90 537 3563500; Fax: +90 2361074; E-mail: mertsaynak@yahoo.com Key words: Radiation enteritis, Treatment planning, Treatment delivery, Radioprotectans Abbreviations: computerized tomography, (CT); intensity modulated radiation therapy, (IMRT); small bowel displacement device, (SBDD); Three-dimensional conformal radiotherapy, (3-DCRT); up down table, (UDT) Received: 07 June 2006; Revised: 27 November 2006 Accepted: 4 December 2006; electronically published: December 2006

Summary In pelvic cancers, small bowel tolerance is a highly significant dose-limiting factor, because of early and late adverse effects. Acute reactions are experienced in most of the patients and many of these effects are self-limiting and rarely require active intervention. However, late intestinal toxicity is difficult to manage and adversely impacts the quality of life of long-term cancer survivors. Many surgical techniques have been used to exclude the small bowel from the pelvis. However these approaches may cause delay radiotherapy and add unnecessary morbidity such as an intense pelvic reaction. Including techniques to reduce the amount of exposed small intestine in the radiation field represent a critical strategy for prevention of this toxicity. In addition to modern techniques include 3-dimensional planning, conformal radiotherapy techniques, IMRT (Intensity modulated radiotherapy), the use of position techniques may useful to reduce intestinal toxicity. Clinicians must consider that the prevention of enteric toxicity is a part of the treatment of the pelvic tumors.

intestinal morbidity include variations in treatment planning or treatment delivery (Table 1). Aim of this manuscript is to review various prevention strategies for severe enteric radiation injury. We reviewed relevant clinical papers published in this field.

I. Introduction Pelvic radiation therapy is often indicated in the treatment of patients with pelvic malignity. Although the goal of the radiotherapy is tumor control, this must be done with the minimum amount of toxicity to prevent worsening of the quality of life. The risk of small bowel toxicity is strongly correlated with some patient characteristics and treatment factors. Also the severity of problems are related to the total dose and dose per fraction, irradiated volume of intestine, the use of single field treatment daily, use of concomitant chemotherapy, co morbidities, previous abdominal surgery and observation time (Letschert et al, 1995). Pelvic radiotherapy includes a considerable volume of the small bowel. The intestinal complications of radiation therapy are often progressive, and may be lethal. A number of surgical and non-surgical techniques have been investigated to minimize the amount of small bowel within the radiotherapy portals with some success. Radiotherapy modifications intended to minimize

II. Radiation enteritis Intestinal complications of radiation therapy are classified as either acute or chronic. Following radiotherapy for pelvic carcinoma, the acute effects are caused by ionizing radiation on epithelium of the intestine. The small intestine is particularly sensitive to radiotherapy, since the intestinal epithelium has a rapid turnover. Intestinal epithelium is composed of differentiated cells involved in the transport of intestinal nutrients, water and electrolyte movements, mucus secretion, immune defense or the modulation of intestinal complication. Early gastrointestinal symptoms are expressed during or immediately after therapy and include nausea, vomiting, abdominal pain, bacterial infection, and diarrhea. These effects to the intestines occur at

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Saynak et al: Prevention of radiation enteritis approximately 1000 cGy. Symptoms of acute radiation enteritis can generally be treated with medical and nutritional therapy, with the expectation of complete symptom resolution within 6 weeks to 6 months following the end of therapy (Chemberline et al, 1999). The incident of late small bowel damage is one of the most important dose-limiting factors in radiation treatment of the pelvis. The chronic effects of radiotherapy are resulted from the induction of vasculitis and fibrosis, and they are more serious than the acute effects. Most of chronic injuries occur between 12 and 24 months after radiation (Touboul, 1996). Late small bowel complications are generally irreversible that are an indirect result of progressive scarring and blood vessel injury. Prominent structural features include mucosal atrophy, intestinal fibrosis and vascular sclerosis. For pelvic malignancies following pelvic radiotherapy, incidence of late chronic radiation injury of the small intestine varies between 0.5 and 25%. However, Yoeh and colleagues stated that it was under estimated and supported the hypothesis that most patients with this condition do not seek medical help until a severe complication occurs (Yoeh et al, 1993). In some patients, acute reactions fail to heal completely and persist into the late period. These effects, termed consequential late effects, add to the overall damage. Consequential late effects are increasingly being observed as the introduction of new aggressive treatment regimens with combined modalities, such as chemoradiotherapy protocols (Stone et al, 2003). Prevention of intestinal complications can be achieved by limiting the small bowel volume with in the radiation field. Numerous surgical techniques have been used to reduce the small bowel volume. These include absorbable polyglycolic acid mesh sling, tissue expanders and pelvic displacement prothesis (Rodier et al, 1991; Herbert et al, 1992; Sezeur et al, 1999). However, these procedures may bring about abdominal discomfort and the morbidity related to the surgical procedures. Fortunately, the small bowel is mobile structure and segments of small bowel can move in and out of the irradiated volume.

Repositioning of normal tissues can be accomplished by mechanical rather than invasive surgical techniques. For this reason, the position techniques such as the use of a belly board, an open table top and an up down table have been described (Shanahan et al, 1990; Huh et al, 1998; Capirci et al, 2001). In addition to modern techniques such as computerized tomography (CT) planning and intensity modulated radiation therapy (IMRT), the use of these techniques may be useful to reduce intestinal toxicity.

III. Treatment planning The volume of the irradiated small bowel in the radiation portals for pelvic carcinoma is considered to be an important factor with regard to the severity of acute and chronic morbidity. Letschert found in 1994 an incidence of chronic diarrhea and malabsorption of 22 vs. 39% with irradiated small bowel volumes below 178 cm3vs. over 178 cm3for patients with rectal carcinoma and pelvic postoperative radiotherapy to 50 Gy (Letschert et al, 1994). Several methods may be used to prevent intestinal toxicity. The benefits of multiple fields are well known. Instead of shooting the beam once from front to back, 1/4 of the dose may be given from the front, 1/4 from the back and 1/4 from each of the lateral sides. Four-field radiotherapy has been utilized to reduce the complications resulting from two field pelvic irradiation. Yamazaki and colleagues performed in 2000 a trial to compare acute enteric toxicity between two patient groups that using the irregular shaped four field technique or twofield technique for postoperative radiotherapy. They reported that the incidence of grade 2-3 intestinal complications in the irregularly shaped four field technique group was significantly lower than that in the two field technique group (p<0.05). There was no statistical difference in survival, relapse free survival and pelvic control rates between two groups (Yamazaki et al, 2000).

Table 1. Prevention methods of radiation enteritis Surgical Techniques

Surgical fixation of the intestine with the other organs Absorbable mesh Non-absorbable mesh Tissue expanders Pelvic displacement prosthesis

Treatment Planning

Small bowel contrast studies Normal tissue blocks Multiple field 3-D planning Conformal therapy Bladder distention Positioning techniques Belly board Abdominal-wall compression Up down table device

Treatment Delivery

Radioprotectans

Amifostine (WR-2721) 284

Cancer Therapy Vol 4, page 285 The use of computerized radiation dosimetry to design the best treatment plan and the use high-energy treatment machines, such as linear accelerators, that deliver a high dose to tumor volume while sparing the normal structures (Minsky, 1988). The use of three-dimensional conformal radiotherapy (3-DCRT) in the treatment of pelvic cancer was proven to be superior to conventional pelvic radiotherapy in reducing the irradiated volume of normal tissues. This way the maximal effect is in the area where the beams cross which is targeted on the tumor, and the normal tissues get less radiation (Gerstner et al, 1999; Boyer et al, 2001). Several studies have reported the superiority of IMRT, over the conventional technique, in reducing the small bowel dose. Roeske et al. showed in 10 gynecologic cancer patients that IMRT decreased irradiated small bowel volume at doses 30 Gy compared with standard four-field whole pelvic RT in supine position. They reported that the small bowel volume was reduced from 33.8% to 17.4% (p= 0.0005), inside the 90% isodose (Roeske et al, 2000). Portelance et al. reported statistically significant decreases in irradiated small bowel volume with IMRT compared with conventional RT in cervical cancer patients. They assessed whether IMRT could achieve adequate dose coverage for different lymph node regions in the pelvic and paraaortic areas, although sparing the small bowel, rectum, and bladder more effectively than conventional four-field whole pelvic radiation. Consequently, the volume of the small bowel irradiated with the prescribed dose was reduced by 50% by the IMRT technique (Portelance et al, 2001). Mundt and colleagues also demonstrated in 2002 that IMRT planning results in excellent planning target volume (PTV) coverage, with considerable sparing of the surrounding normal tissues in women with gynecologic malignancies. They noted grade 2 acute gastrointestinal toxicity was less common in the IMRT group than in the conventional RT group (60% vs 91%). The chronic GI toxicity was found to be lower in the IMRT treated patient group (11.1 vs. 50.0%, P = 0.02) (Mundt et al, 2002).

pelvic adhesions. So in the pelvic radiotherapy field too much intestine may be included (Figure 1). Most radiotherapists, therefore, seriously worried about enhanced small bowel complications with post-surgery radiotherapy. Green and colleagues observed in 1983 small bowel fixation in over 60% of patients who had pelvic surgery. Some investigators have treated patients in the prone position to displace small bowel loops out of the pelvic fields. Caspars and Hop used small bowel contrast studies to evaluate prospectively the impact of positioning in small-bowel displacement from the pelvis. The volumes have been calculated for the supine and the prone position. In comparison, they showed the prone position to be superior to the supine position in 78 % of patients (Caspars et al, 1983). In 1986, Gallagher and colleauges described the “Grid Method” for the measurement of the irradiated small bowel volume for standardization. They used barium contrasted bowel loops on simulation radiographs and divided the bowel region into a grid of 1 cm x 1 cm squares. This method allowed a quantitative comparison of the efficacy of the different technical innovation. Consequently, they reported that a compression pillow with bladder distention in the prone position provided maximum sparing of small bowel radiation field and found a profound effect of the volume of irradiated small bowel on late toxicity (Gallagher et al, 1986). Particularly for rectal cancer, prone positioning on a belly board has been among the most common strategies to displace the small bowel from the pelvic radiation fields and has been highly effective in pelvic RT. With patients in the prone position, the opening in the table top allows the abdomen to fall below the level of the table. Thus, gravity is used to help displace the small bowel from the pelvis. Shanahan and colleagues prospectively evaluated in 1990 the benefit of the “belly board” in 30 consecutive patients undergoing pelvic radiotherapy. Contrastenhanced CT scanning and custom-designed computer software was used to determine the volume of small bowel in the treatment portals. The mean small bowel volume was reduced by 66% (299 versus 102cm3) in patients simulated in the prone position with the “belly board” compared with the supine position. In 13 patients without prior pelvic surgery, the small bowel volume reduction was a more dramatic 74% (334 cm3 to 88 cm3) (Shanahan et al, 1990). According to results of the bellyboard trials, the small bowel volume can be significantly reduced with a mean of 50 to 66%, with the belly-board device compares with supine positioning (Shanahan et al, 1990; Fu et al, 1995; Das et al, 1997). Holst and colleagues described in 1995 a small bowel displacement device (SBDD) that is fixed to the treatment table. According to them, placement of the SBDD allowed a mean reduction of the small bowel within the field up to 57% compared to the quantity of small bowel visualized in the treatment field with prone positioning alone (Holst et al, 1995). Nevertheless a disadvantage of this method is difficult verification of patient positioning. Because of this, in 1998, Huh et al used customized small bowel displacement system the most caudal small bowel by using

IV. Treatment delivery In 1975, Green and colleagues have suggested that oral contrast is a useful adjunct in treatment planning to localize the dose limiting small intestines. Authors studied 54 patients with small bowel radiographs taken with contrast with medium in the prone, supine and 45 degree Trendelenburg position. In prone position, 50% of the patients showed small bowel displacement in the cephalad direction. They proposed that this information could be used to modify the radiation regimen and minimize small bowel complications (Green, 1975). Treating a patient with a full bladder may be helped passing out of the pelvis of small bowel when pelvic radiotherapy is given. Green noted that in many patients distention of the bladder might help displacing small bowel from the pelvis (Green, 1983). The incidence and severity of intestinal radiation toxicity also depends on previous pelvic surgery. Patients with previous abdominal surgery are more likely to have 285

Saynak et al: Prevention of radiation enteritis Figure 1. PA simulation film in prone position of a patient postoperatively treated for endometrial cancer. All opacified small bowel within the radiotherapy field.

the SBDS and the (SBDS) made for each patient individually. They reported that the mean small-bowel volume exposed was reduced by 59% on postero-anterior and 51% on lateral-simulation films. Ninety three percent of the patients showed upward displacement of the most caudal small bowel by using the SBDS. Furthermore, in patients treated with the SBDS, a significantly lower incidence of diarrhea requiring medication was observed. They noted that the average daily random setup variations were small (1-3 mm) and compliance with setup was excellent in their study. However, the use of this technique has been criticized for time consuming (Huh et al, 1998). Kim and colleagues demonstrated in 2005 that the combination of the belly board device and the distended bladder exhibited an additive effect, and constituted the most effective method for the reduction of irradiated small bowel volume in the preoperative radiotherapy of rectal cancer patients (Kim et al, 2005). For the same purpose, Capirci and colleagues described in 2001 the “up down table” (UDT) device”. They evaluated 277 patients with uterine cervical cancer who were treated with using UDT device for comparing their historic series. They reported that the average volume of small bowel within the planning target volume was 100 cm3 (median 49 ± 114) in the series treated with standard box technique and 23 cm3 (median 0 ± 64) in the series treated with the UDT (p < 0.001). The average volume of small bowel included in any isodose (any-dose volume) was 505 cm3 (median 447 ± 338) and 158 cm3 (median 69 ± 207), respectively (p< 0.001). The incidence of G1, G2, and G3 acute enteric toxicity in the UDT series was 16%, 15%, and 1.5%; in the standard box technique, it was 28%, 25%, and 3%, respectively (p < 0.05). They showed the

incidence of acute enteric toxicity directly correlated with the irradiated small bowel volume (Capirci et al, 2001). It is known that 3-D CRT in the treatment of gynecological cancer is superior to conventional pelvic RT in reducing the irradiated volume of normal tissues. Several studies were made to evaluate benefit of using 3-D CRT and positions techniques together. Huh et al. have been used a customized SBDS in the 3-D CRT to displace the small bowel maximally out of the pelvic radiation fields (Huh et al, 2004). In their series 10 consecutive patients were referred for pelvic radiotherapy for uterine cervical cancer. They showed that the median small bowel volume with SBBS was reduced by 56.4 % compared with small bowel volume in prone position alone. At the prescription dose, the median volume of the irradiated small bowel was significantly reduced by use of the SBDS (9.8% vs. 1.2%). An important issue in the SBDS-assisted IMRT is the setup accuracy. Kim et al. reported that the custom-made abdominal board improves the reproducibility of a given pelvic field, with minimal set-up error. They investigated the inter-fractional setup accuracy of the customized SBDS and showed that the mean inter-fractional deviation of the isocenter, along the right–left, craniocaudal, and posterior–anterior directions were 1.2 ± 1.6, 1.0 ± 3.0, and 0.9 ± 4.4 mm, respectively (Kim et al, 2000). Historically, women with locally advanced cervical cancer were treated with radiotherapy alone. However, concurrent chemoradiotherapy is obligatory in the management of locally advanced cervical cancer at present. This current trend is a poorly understood biological variable. Administration of chemotherapy concurrently with pelvic radiation therapy theoretically increases toxic effects on small bowel. That’s why, bowel-

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Cancer Therapy Vol 4, page 287 â&#x20AC;&#x153;Up down tableâ&#x20AC;? device. Int J Radiat Oncol Biol Phys 51, 465-473. Caspers RJL, HopWCJ (1983) Irradiation of true pelvis for bladder and prostatic carcinoma in supine, prone or Trendelenburg position. Int J Radiat Oncol Biol Phys 9, 589-593. Chemberlein RS, Jacobs TS, Orkin BA (1999) Radiation enteritis: A primer on bowel injury due to radiation treatments for cancer. Ostomy Quarterly 36, 36-39. Das IJ, Lanciano RM, Movsas B, Kagawa K, Barnes SJ (1997) Efficacy of a belly board device with CT-simulation in reducing small bowel volume within pelvic irradiation fields, Int J Radiat Oncol Biol Phys 39, 67-76. Denham JW, Hauer-Jensen M, Kron T, Langberg CW (2000) Treatment time dependence models of early and delayed radiation injury in rat small intestine. Int J Radiat Oncol Biol Phys 48, 871-887. Fu YT, Lam JC, Tze JM. (1995) Measurement of irradiated small bowel volume in pelvic irradiation and the effect of a bellyboard, Clin Oncol 7, 188-192. Gallagher MJ, Brereton HD, Rostock RA, Zero J M, Zekoski D A, Poyss L F, Richter M P, Kligerman M M (1986) A prospective study of treatment techniques to minimize the volume of pelvic small bowel with reduction of acute and late effects associated with pelvic irradiation. Int J Radiat Oncol Biol Phys12, 1565-1573. Gerstner N, Wachter S, Knocke TH, Fellner C, Wambersie A, Potter R (1999) The benefit of Beam's eye view based 3D treatment planning for cervical cancer. Radiother Oncol 51, 71-78. Green N (1983) The avoidance of small intestine injury in gynecologic cancer. Int J Radiat Oncol Biol Phys 9, 13851390. Green N, Iba G, Smith WR (1975) Measures to minimize small intestine injury in the irradiated pelvis. Cancer 35, 16331640. Herbert SH, Solin LJ, Hoffman JP, Hanks GE (1992) Volumetric analysis of small bowel displacement from radiation portals with the use of a pelvic tissue expander. Int J Radiat Oncol Biol Phys 25, 885-893. Holst R, La Couture T, Koprowski C, Goldschmidt E (1995) A simple manual method of repositioning small bowel during pelvic irradiation. Med Dosim 20, 123-129. Huh SJ, Lim DH, Ahn YC, Kim DY, Kim MK, Wu HG, Choi DR (1998), Effect of customized small bowel displacement system in pelvic irradiation, Int J Radiat Oncol Biol Phys 40, 623-627. Huh SJ, Park W, Ju SG, Lee JE, Han Y (2004) Small-bowel displacement system for the sparing of Small bowel in threedimensional conformal radiotherapy for cervical cancer. Clinical Oncology 16, 467-473. Kim MK, Kim DY, Ahn YC, Huh SJ, Lim DH, Shin KH (2000) Analysis of the inter-and intra-treatment isocenter deviations in pelvic radiotherapy with small bowel displacement system. J Korean Soc Ther Radiol Oncol 18, 114-119. Kim TH, Chie EK, Kim DY, Park SY, Cho KH, Jung KH, Kim YH, Sohn DK, Jeong SY, Park JG (2005) Comparison of the belly board device method and the distended bladder method for reducing irradiated small bowel volumes in preoperative radiotherapy for rectal cancer patients. Int J Radiat Oncol Biol Phys 62, 769-75. Letschert JG, Lebesque JV, Aleman BM, Bosset JF, Horiot JC, Bartelink H, Cionini L, Hamers JP, Leer JW, van Glabbeke M (1994) The volume effect in radiation-related late small bowel complications: results of a clinical study of the EORTC Radiotherapy Cooperative Group in patients treated for rectal carcinoma. Radiother Oncol 32, 116-123.

exclusion techniques must be considered especially important at the present time.

V. Radioprotectans Various agents that confer protection against radiation have been developed, of which the most promising is amifostine (WR-2721). Amifostine is a prodrug that is dephosphorylated by alkaline phosphatase to the active metabolite WR-1065, which appears selective in its entry in nonmalignant cells. After the demonstration of the efficacy of amifostine as a radioprotector in reducing the severity of acute and late xerostomia without compromising the efficacy of radiotherapy in patients with head and neck cancer several randomized trials tested the role of amifostine in reducing radiation esophagitis and radiation-related pneumonitis (Sauer et al, 1999). The few clinical trial data available on the use of amifostine in pelvic cancer patients suggest benefit in reducing intestinal toxicity (Athanassiou et al, 2003). However, role of amifostine is not clear for enteric toxicity and larger, randomized trials are still needed.

VI. Conclusion As a conclusion, radiation enteritis continues to be a limiting factor in the application of pelvic radiotherapy and may even be life threatening. Particularly, after having late toxicity on small bowel, treatment is very difficult. Therefore, to prevent intestinal toxicity must be attached importance. In the literature, successful measures to prevent radiation-induced small bowel injury have been described. The use of small bowel contrast during RT simulation is one technique used to localize the small bowel. Also, bladder-filling techniques may be helpful to prevent intestinal toxicity. Many surgical interventions have been proposed to reduce the volume of small bowel. Fortunately, small bowel is a mobile structure that can be maneuvered variety techniques. Therefore, instead of invasive surgical techniques various radiotherapy techniques can be used for repositioning of normal tissues. These techniques can be used without causing serious setup errors. Also advances in the techniques of delivery of radiotherapy to pelvic organs may decrease the incidence of intestinal complications. Clinicians must consider the prevention of enteric toxicity is a part of the treatment of the pelvic tumors.

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Touboul E, Balosso J, Schlienger M, Laugier A (1996) Radiation injury of the small intestine. Radiobiological, radiopathological aspects; risk factors and prevention. Ann Chir 50, 58-71. Yamazaki A, Shirato H, Nishioka T, Hashimoto S, Kitahara T, Kagei K, Miyasaka K (2000) Reduction of late complications after irregularly shaped four-field whole pelvic radiotherapy using computed tomographic simulation compared with parallel-opposed whole pelvic radiotherapy. Jpn J Clin Oncol 30, 180-184. Yeoh E, Horowitz M, Russo A, Muecke T, Robb T, Maddox A, Chatterton B (1993) Effect of pelvic irradiation on gastrointestinal function: a prospective longitudinal study. Am J Med 95, 397-406.

Mert Saynak

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Suppression of tumor metastasis by adenoviralmediated gene transfer of Motility Related Protein â&#x20AC;&#x201C; 1 (MRP-1/CD9) Research Article

Jun Zhang1,2,5, Bogdan Ceacareanu3, Haruhito Azuma4, Aviv Hassid3, Lisa K. Jennings1 and Yi Lu1,2,5,* 1

Department of Pathology and Laboratory Medicine, 2Department of Medicine, Department of Physiology 5 University of Tennessee Cancer Institute, University of Tennessee Health Science Center, Memphis, Tennessee, USA 4 Department of Urology, Osaka Medical College, Osaka, Japan 3

__________________________________________________________________________________ *Correspondence: Yi Lu, Ph.D., Departments of Pathology and Medicine, University of Tennessee Cancer Institute, University of Tennessee Health Science Center, 956 Court Avenue, H300, Memphis, TN 38163, USA; Tel: (901) 448-5436; Fax: (901) 448-5496; Email: ylu@utmem.edu Key words: CD9, breast cancer, tumor metastasis, motility Abbreviations: Crk associated substrate, (p130Cas); extracellular matrix, (ECM); fluorescein isothiocyanate, (FITC); human immunodeficiciency virus, (HIV); matrix metalloproteinase, (MMP); multiplicity of infection, (moi); phosphoinositide 3-kinase, (PI 3kinase); polyacrylamide gel electrophoresis, (PAGE); Wiskott-Aldrich syndrome WASP family verproline-homologous protein 2, (WAVE2) Received: 11 September 2006; Revised: 16 November 2006 Accepted: 15 December 2006; electronically published: December 2006

Summary Metastasis, the major cause of deaths of cancer patients, is a complex multi-step process in which cell motility plays an important role. The motility related protein-1, MRP-1/CD9 (or CD9), is a tetraspanin membrane glycoprotein and it has been implicated in playing roles in cell adhesion, spreading, and motility. In this study, a replicationdeficient recombinant adenovirus expressing CD9 (Ad-CD9) was generated. By using a highly metastatic breast cancer cell line JygMC(A) as the model, we evaluated the effects of adenoviral-mediated CD9 expression on suppression of tumor metastasis, and related biological and biochemical parameters. By comparing with untreated or control virus-treated cells, Ad-CD9 transduced JygMC(A) cells had an increased cell motility and increased adhesion to laminin and fibronectin, but adhesion to the matrigel did not change. Overexpression of CD9 in JygMC(A) cells suppressed cell growth and in vitro invasion ability to penetrate matrigel. Moreover, Ad-CD9 transduced JygMC(A) cells formed less metastasis in the lungs and livers in nude mice than control groups in a spontaneous metastasis animal model. These results suggest that CD9 may act as an anti-metastasis gene, and suppress breast cancer growth and metastases. Adenoviral-mediated CD9 gene transfer may have the potential for breast cancer gene therapy.

(Marshall, 1993). The prevention of tumor metastasis is one of the most important challenges in the design of therapies for patients with malignancies. The metastatic spread of a primary tumor to distant organs involves a series of predictable processes that include tumor cell detachment from the primary tumor, migration and invasion through the basement membranes of blood and lymph vessels, embolization, arrest and binding to vascular endothelium at secondary organs, extravasation, and invasion of the secondary organ. In these processes,

I. Introduction Breast cancer is the most frequently diagnosed cancer and the second leading cause of cancer deaths in American women today. It is estimated that there will be 212,920 new cases of breast cancer and 48,970 breast cancer deaths in American women in the year 2006 (Jemal et al, 2006). Metastasis, the spread of tumor cells from the primary site to a distant organ to form a secondary tumor, is a major cause of deaths of breast cancer patients

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Zhang et al: CD9 suppresses breast cancer metastasis cell motility is one of the essential cellular functions that plays an important role in tumor metastasis (Miyake and Hakomori, 1991). Motility related protein-1 (MRP-1) was originally recognized, and thus isolated by a monoclonal antibody that inhibits cell motility and tumor cell metastasis (Miyake and Hakomori, 1991; Miyake et al, 1991). The sequence of MRP-1 protein and cDNA revealed that it is an identical gene to CD9, a cell surface antigen initially discovered on B lineage leukemic cells and cloned by two different groups independently (Boucheix et al, 1991; Lanza et al, 1991). The MRP-1/CD9 (also named as CD9 or p24/CD9) is a tetraspanin transmembrane glycoprotein (Boucheix et al, 1991; Lanza et al, 1991) that is located at the cell surface in a specific membrane microdomain called a lipid raft (Ishii et al, 2006). CD9 has been implicated in playing roles in cell adhesion, spreading (Cook et al, 1999), and motility (Ikeyama et al, 1993; Garcia-Lopez et al, 2005). CD9 is highly expressed in macrophages (Kaji et al, 2001) and forms a complex with integrin !IIb"3 and CD63, another member of the tetraspanin superfamily, in activated platelets (Israels et al, 2001). In addition, CD9 has been shown to play critical roles in sperm and egg fusion (Komorowski et al, 2006; Rubinstein et al, 2006). Recent data also suggest that extracellular domains of tetraspanin CD9 protein can inhibit infection of macrophage by HIV virus, probably by blocking viral entry via modulation of the activity of viral receptors that form complexes with endogenous tetraspanins (Ho et al, 2006). Among all these CD9-associated phenotypes and functions, cumulative data have demonstrated one prominent CD9 feature, i.e., suppression of tumor progression and metastasis. Downregulation of CD9 expression appears to be an acquired event in the development of malignant tumors. There is an inverse relationship between CD9 expression and metastatic potential in several cancers. CD9 protein levels in the metastatic lymph nodes were found to be lower than those in the respective primary breast cancers in half of the cases analyzed (Miyake et al, 1995). A similar inverse relationship was also observed in colon cancer (Mori et al, 1998), oesophageal cancer (Uchida et al, 1999), endometrical cancer (Miyamoto et al, 2001), oral cancer (Kusukawa et al, 2001), and melanoma (Si and Hersey 1993). By using differential display cloning technique in matched primary and metastatic derived human colon carcinoma cell lines, Cajot and colleagues confirmed in 1997 that CD9 gene was more highly expressed in the primary tumor as compared to its metastatic counterpart. Moreover, reduced CD9 expression was found to be associated with a poor prognosis in the patients with breast cancer (Seymour et al, 1990; Miyake et al, 1996), nonsmall cell lung cancer (Higashiyama et al, 1995), and pancreatic cancer (Sho et al, 1998). Transfection of human MRP-1/CD9 cDNA revealed that cell motility was altered in the MRP-1/CD9-expressing cells (Ikeyama et al, 1993), suggesting that MRP-1/CD9 regulates cell motility. Restored expression of CD9 in highly metastatic human small-cell lung cancer cells reduced liver metastasis in SCID mice. Moreover, no detectable levels of CD9 were

expressed in metastatic tumor cells in mice bearing CD9transfected SCLC cells (Zheng et al, 2005). Taken together, these data suggest that CD9 may be a tumormetastasis suppressor gene which may reduce tumor metastasis via the inhibition of cell proliferation and motility, and that decreased expression of CD9 may contribute to the malignant progression of tumors. Gene therapy has been demonstrated to be a novel and promising modality to combat malignant diseases (Hanania et al, 1995; Marchisone et al, 2000). Adenovirus vectors have multiple advantages over other viral vectors to serve as a gene delivery vehicle. Adenovirus is able to infect a wide variety of cell types, and is able to infect non-dividing cells, it has a high transduction efficiency and a high level of expression of transgene. It can accommodate a large piece of foreign DNA and can be concentrated at high titers (Lu, 2001). In this report, we generated a recombinant adenovirus expressing human wild-type CD9 (Ad-CD9), and analyzed the potential therapeutic effects of CD9 on suppression of breast tumor metastasis. In addition, we also used Ad-CD9 as a gene transfer vehicle to analyze the mechanism of CD9mediated biological modulation.

II. Materials and Methods A. Cell culture and medium 293 cell line was purchased from American Type Culture Collection (ATCC) (Manassas, VA) and was grown in Dulbecco’s modified Eagle medium (DMEM) 10% fetal bovine serum (FBS). Mouse breast cancer cell line JygMC(A) (Azuma et al, 2002) was grown in DMEM medium with 10% FBS. All cell lines were grown in medium containing 100 units/ml penicillin, 100 µg/ml streptomycin at 370C in 5% CO 2.

B. Generation of recombinant adenovirus Ad-CD9 Ad-CD9, the replication-deficient recombinant adenovirus, containing human MRP-1/CD9 cDNA under the control of a CMV promoter, was generated as following: a 846-bp fragment containing the full-length human CD9 cDNA gene was released from plasmid pRc/CMVp24/CD9 (Cook et al, 1999) by EcoR I and Xba I double digestion. After EcoR I and Xba I digestion of an E1-deleted adenoviral shuttle vector, pacAd5CMVKNpA (University of Iowa Research Foundation, Iowa City, IA), it was ligated to the above-mentioned 846-bp CD9 cDNA to generate the resultant adenoviral shuttle vector pacAd5-CD9. The pacAd5-CD9 and pacAd5 9.2-100, an adenoviral type 5 genome backbone plasmid (Anderson et al, 2000), were cotransfected into 293 cells by FuGENE 6 reagent (Roche, Indianapolis, IN) according to the manufacturer’s protocol. The individual plaques were screened by the direct plaque screening PCR method (Lu et al, 1998; Steiner et al, 2000) using a pair of primers recognizing the adenoviral genome and CD9 cDNA respectively.

C. Adenoviral vector preparation, titration and transduction Individual clones of Ad-CD9 were obtained by plaque purification. The construction of control adenovirus Ad-lacZ (Lu et al, 1999) was described previously. Individual clones of AdCD9 and Ad-lacZ were propagated in 293 cells. The culture medium of the 293 cells showing the complete cytopathic effect was collected, and adenovirus was purified by BD Adeno-X Virus Purification Kits (BD Biosciences, Palo Alto, CA). The viral titration and transduction were performed as previously

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Cancer Therapy Vol 4, page 291 described (Graham and Prevec, 1991).

medium with 1% BSA per well) were then placed into the upper compartment of the chamber and serum-free medium with 1% BSA was placed into the lower chamber. Chambers were incubated at 370C with 5%CO2 for 3 h. The filters were then removed and nonmigrating cells remaining on the upper side of the filter were scraped off. The cells that had migrated to the lower side of the filter were fixed in Giemsa staining solution (Sigma, St. Louis, MO). After extensive washing with water, the migrated cells were counted in five different fields under a microscope at x200 magnification. Migratory activity was expressed as the mean number of cells that migrated to the lower side of the filter, and results were represented as sum of total cell numbers in five randomly selected fields of view. Untreated or Ad-lacZ transduced cells were used as controls.

D. Western blot Cells were extracted and processed for gel electrophoresis as previously described (Lu et al, 1998; 2000). Cell lysates (100 µg) were loaded on polyacrylamide gels and subjected to sodium dodecylsulfate (SDS) gel electrophoresis, then transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). The membrane was treated with blocking solution (15% nonfat milk, 0.02% sodium azide in phosphate-buffered saline) overnight at 40C. The membrane was incubated for 1 hr at room temperature with the first antibody (anti-CD9). The membrane was then incubated for 1 hr at room temperature with the second antibody coupled to peroxidase (ECL Kit, Amersham, Buckinghamshire, England), and enhanced chemiluminescent (ECL) staining was performed according to the manufacturer's protocol. The antibody against CD9, mAb7, a mouse anti-human CD9 monoclonal antibody, was purified from this laboratory (Lanza et al, 1991; Cook et al, 2002).

I. Adhesion assay To examine the CD9 effects on ability of cells to adhere extracellular matrix (ECM) components including laminin, fibronectin, and matrigel, we transduced cells with Ad-CD9 at moi of 200 for 48 h. The cells were harvested and replated at 4x105 per well in the 24-well culture plate precoated with fibronectin (10 µg/ml) (GibcoBRL, Gaithersburg, MD), laminin (10 µg/ml) (GibcoBRL), or matrigel (Collaborative Biomedical Inc., Bedford, MA). After incubation for 4 h at 370C, the nonadherent cells were removed by washing the plate with PBS. The adherent cells were fixed with 4% of paraformaldehyde solution for 10 min at room temperature. The cells were stained with 1% toluidine blue for 5 min and rinsed with water. Cells were then solubilized by adding 1% SDS and quantified using a microtiter plate reader at 595 nm. Untreated or Ad-lacZ transduced cells were used as controls.

E. Immunofluorescence staining Cells were grown on coverslips and either untreated or transduced with virus (Ad-CD9 or Ad-lacZ) at moi=200 for 48 h. The cells were fixed with 3.5% formaldehyde in PBS for 30 min at room temperature. After washing 3 times with PBS, the cells were processed to immunofluorescence staining under nonpermeabilization conditions as described previously (Lu et al, 2000). The cells were incubated with 10% normal goat serum (NGS) in PBS for 20 min for blocking, then with 4 µg/ml antiCD9 antibody mAb7 in 10% NGS in PBS for 1 h at room temperature, followed by incubation with 5 µg/ml FITC-labeled goat anti-mouse IgG for 1 h at room temperature. After washing and fixing, the coverslips were mounted on the slides and photographed under fluorescence microscopy.

J. In vitro invasion assay The in vitro cell invasion assay was performed with 6-wellplate Biocoat matrigel invasion chambers (Becton Dickinson Labware, Bedford, MA) according to the manufacturer’s procedure. Briefly, the chamber was first rehydrated with serumfree media (DMEM) for 2 hr at 370C. After rehydration, the chambers were placed in the lower compartment previously loaded with DMEM media containing 5% FBS. Meanwhile, the JygMC(A) mouse breast cancer cells of untreated control, as well as control virus (Ad-lacZ) treated and Ad-CD9 transduced cells (moi=200 for 48 hr) were harvested. The cell suspensions were adjusted to 2.5x105 cells/ml with serum-free medium. The cell suspension (2ml per well) was immediately added to the upper compartment of the chamber. The cells were then allowed to invade through the matrigel for 22 hrs at 370C, and the noninvading cells were removed by scrubbing the upper surface with a wet cotton swab. The filters were stained with Diff-Quick stain kit (Dade Behring Inc., Newark, DE), drained and counted.

F. Flow cytometry assay The assay followed the procedure described previously (Cook et al, 1999). Briefly, untreated or virus transduced cells were harvested, the cells were suspended in DMEM with 5% NGS for 30 min for blocking, then 5x105 cells were labeled with 4 µg/ml anti-CD9 antibody mAb7 for 1 h on ice, followed by incubation with 5 µg/ml FITC-labeled goat anti-mouse IgG for 1 h on ice. After washing and centrifugation, the cells were resuspended in PBS and subjected to antibody binding assay using a FACS Calibur flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA).

G. Growth inhibition assay The growth inhibition assay followed the protocol as previously described (Steiner et al, 2000). Briefly, three groups of cells were used for each breast cancer cell line: (a) control untreated, (b) control virus Ad-lacZ treated, and (c) Ad-CD9 treated. Cells were transduced at multiplicity of infection (moi) of 200. Cellular proliferation was measured by cell counting of the attached cells at day 5 post viral transduction. Student’s t-test was used for statistical analysis throughout the entire project.

J. Spontaneous metastasis assay Highly metastatic breast cancer JygMC(A) cells were either untreated, or transduced with Ad-lacZ or Ad-CD9 (both at moi of 200). Forty-eight hours after viral infection, the cells were harvested and the viable cell numbers were counted in a hemocytometer using trypan blue exclusion. Cells (1x107 cells per mouse) were injected subcutaneously into the flanks of 6week-old female nude mice (athymic nude mice, Harlan, Indianapolis, IN). Three groups of mice, with five mice in each group, were formed corresponding to the three groups of cells mentioned above. Size of primary tumors were measured every three days with calipers, and tumor volumes were calculated using the standard formula: width2 x length x 0.5 (Steiner et al, 2000). All the nude mice were sacrificed at day 34 post inoculation when some of them became moribund. At the time of sacrifice, the

H. Cell motility assay The cell migration/motility was measured by a modified Boyden’s chamber method using a Transwell plate containing polycarbonate filters with a pore size of 8.0 µm (Nunc, Roskilde, Denmark). The filters were precoated on the undersurface (between upper and lower chambers) with 10 µg/ml fibronectin at 370C for 3 h. Cells were transduced with Ad-CD9 at moi of 200 for 48 h, the cells were harvested and counted. Cell suspensions (2 ml of 2.5x104 cells/ml in serum-free DMEM

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Zhang et al: CD9 suppresses breast cancer metastasis allograft primary tumors were collected and weighed. To examine metastasis, lung, liver, spleen, and kidney were removed and metastases were counted under the dissection stereoscope after fixation in Bouin's solution (Sigma) (Lu et al, 1994). Other organs, including heart, brain, pancreas, lymph nodes, bone, and lumbar spinal muscle were also carefully checked under microscopy to detect potential metastasis.

transduced cells.

B. CD9 inhibits breast cancer cell growth in vitro To determine the effects of p16 on breast cancer cell growth, JygMC(A) cells were treated with Ad-CD9, AdlacZ (both at moi=200), or no virus in vitro. As shown in Figure 4, Ad-CD9 inhibited the growth of JygMC(A) cells with a 45.26% inhibition, compared with untreated control cells, whereas no significant inhibition was observed from the control virus-treated group.

III. Results A. Ad-CD9 expresses high levels of p16 protein in breast cancer cells To confirm successful CD9 expression at the protein level by Ad-CD9 transduction, JygMC(A) cells were transduced by Ad-CD9 and subjected to Western blot analysis. As shown in Figure 1, indeed an overexpression of exogenous CD9 in Ad-CD9 transduced were observed. To determine whether the Ad-CD9 mediated expression of CD9 was at the cell-surface, where the majority of native CD9 protein is presumably located, immunofluorescent staining was performed under nonpermeable conditions. We observed a clearly cell-surface labeled staining for CD9 expression in Ad-CD9 transduced neonatal rat smooth muscle cells (SMC) (Figure 2), but not in Ad-lacZ transduced SMC cells. In addition, expression of CD9 at the cell-surface was also confirmed in Ad-CD9 transduced JygMC(A) cells by flow cytometry (Figure 3). Taken together, these data demonstrated that we had generated a recombinant adenovirus, Ad-CD9, which effectively expresses functional CD9 protein at the cell surface in

C. Motility assay Cell migration is an important aspect of the tumor metastatic process. To analyze the effect of CD9 on breast cancer cell migration, JygMC(A) cells treated with AdCD9 at moi of 20 for 48 h were harvested and plated in the upper compartment of the chamber which has been precoated with fibronectin on the undersurface of the chamber/filter. By comparison with the untreated control cells, Ad-CD9 transduced cells exhibited an almost twofold increase on cell motility, as evaluated by counting the cells that were on the undersurface of the filter. The control virus (Ad-lacZ) transduced cells did not cause a significant change on the cell motility compared with untreated control cells (Figure 5). This result suggested that CD9 promoted breast cancer cell migration towards a fibronectin-enriched environment, that is, a haptotactic cell motility to fibronectin.

Figure 1. Expression of CD9 protein in Ad-CD9 transduced cells. JygMC(A) cells transduced by Ad-CD9 at various moi as indicated were harvested 72 h post transduction. Protein extracts (100 Âľg/well) were loaded on a 12% SDS-PAGE gel under non-reducing conditions. Mouse anti-human CD9 monoclonal antibody mAb7 (Lanza et al, 1991; Cook et al, 2002) were used as the primary antibody. Goat anti-mouse IgG coupled with peroxidase was used as the secondary antibody. The size of the CD9 protein was shown at 25-kDa as expected. The same blot was also immunoblotted with anti-actin antibody as an internal control for protein loading.

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Figure 2. Immunofluorescence staining of CD9 protein expression in Ad-CD9 transduced cells. Baby rat smooth muscle cells (SMC) were transduced with Ad-CD9 (A and C) or Ad-lacZ (B and D) at moi=100 for 48 h. Under non-permeable conditions, cells were incubated with primary antibody against CD9 (mAb7), followed by secondary antibody conjugated with FITC. The images A and B were taken under a fluorescence microscope, and images C and D were taken under a light contrast microscope.

Figure 3. Flow cytometry analysis of cell-surface CD9 protein expression. JygMC(A) cells were transduced with Ad-CD9 at moi=200 for 48 h and processed to flow cytometry analysis for cell-surface CD9 expression by using mAb7 as the primary antibody (Fig. B). Shown are Ad-CD9 transduced (open area in Fig. A and B) and control virus Ad-lacZ transduced (filled area in Fig. A and B) cells. As negative controls, the same cells were also labeled by using mouse IgG as the primary antibody (Fig. A).

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Zhang et al: CD9 suppresses breast cancer metastasis Figure 4. Effect of Ad-CD9 on growth of breast cancer cells. JygMC(A) cells were untreated or transduced with either control virus Ad-lacZ or Ad-CD9 (moi=200). Cell numbers were counted using a Coulter cell counter at day 5 post viral transduction. The results represent data from at least two independent experiments with duplicate tests.

Figure 5. CD9 expression increased cell motility in JygMC(A) cells. JygMC(A) cells were untreated or transduced with either control virus Ad-lacZ or Ad-CD9 (moi=200) for 48 h. The cells were then harvested and used for cell motility assay by modified Boydenâ&#x20AC;&#x2122;s chamber method as described in Materials and Methods section. The sum of total cell numbers in five randomly selected fields was presented. The results represent data from at least two independent experiments with duplicate tests.

or control virus treated groups (Figure 6B).

D. Adhesion assay Effects of CD9 on cell adhesion were likewise analyzed both on fibronectin, laminin and matrigel. Compared with the untreated control group, Ad-CD9 transduced JygMC(A) cells had a significantly increased adhesion on fibronectin (66.2% increase) and laminin (132.2% increase), respectively; whereas the control virus transduced group showed no significant effects by these treatments (Figure 6A). These data indicate that overexpression of CD9 protein altered adhesion phenotype of breast tumor cells to the extracellular matrix proteins. Interestingly, CD9 caused a less dramatic effect of breast tumor cell adhesion towards matrigel, with only a moderate increase (10% increase) compared to untreated

E. In vitro invasion assay To study CD9 effects on the invasive ability of breast cancer cells, Ad-CD9 transduced JygMC(A) cells were analyzed by the in vitro invasion assay using Biocoated matrigel invasion chambers. After 22 h incubation, cells which penetrated through the matrigel were counted and represented as the in vitro invasiveness of the tumor cells. As shown in Figure 7, by comparing to the untreated control group, Ad-CD9 transduced cells penetrated through matrigel at a significantly reduced number, i.e., an 40.2% inhibition, whereas the control virus Ad-LacZ transduced group had only a minor reduction (5.1%

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Cancer Therapy Vol 4, page 295 inhibition). These results indicate that CD9 reduced the invasive ability of breast cancer cells.

sites. This suggests that CD9 expression did not affect tumorigenicity of JygMC(A) cells. The majority of metastasis of malignant JygMC(A) cells after subcutaneous injection in nude mice was found in the lungs (Figure 8) and livers, with a minor portion of metastases found in kidneys and spleens. No metastases were found in any other organs. Ad-CD9 treated mice had significantly less metastasis with a 36.7% average reduction of total metastases (sum of all metastases in lung, liver, kidney and spleen) compared to untreated mice, whereas the Ad-lacZ treated group had a moderate but nonsignificant reduction of metastasis (Figure 9).

F. CD9 inhibited tumor metastasis in a spontaneous metastasis animal model JygMC(A) cells are highly metastatic breast cancer cells that are able to form metastasis in nude mice in a spontaneous metastasis model (Azuma et al, 2002). JygMC(A) cells were either untreated, or transduced with control virus or Ad-CD9 at moi=200, and were harvested 48 hr later, then 1x107 cells per mouse were injected subcutaneously into the flank of female nude mice. All mice formed allograft primary tumors on the injection

Figure 6. CD9 expression increased a significant adhesion of JygMC(A) cells to fibronectin and laminin, and a moderate adhesion to matrigel. JygMC(A) cells were untreated or transduced with either control virus Ad-lacZ or Ad-CD9 (moi=200) for 48 h. The cells were then replated on a 24-well plate precoated with 10 Âľg/ml fibronectin, 10 Âľg/ml laminin (A); or matrigel (B) and incubated for 4 h. After washing, the adherent cells were solubilized and quantified by OD reading at 595 nm. The results represent data from at least two independent experiments with duplicate tests. Some error bars are too small to show in this scale.

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Zhang et al: CD9 suppresses breast cancer metastasis Figure 7. CD9 expression decreased JygMC(A) cell ability to penetrate matrigel in the in vitro invasion assay. JygMC(A) cells were untreated or transduced with either control virus Ad-lacZ or Ad-CD9 (moi=200) for 48 h. The cells were then harvested and used for in vitro invasion assay on a 6well plate Biocoat matrigel invasion chamber (Becton Dickinson) according to the manufacturerâ&#x20AC;&#x2122;s instructions. The cells were allowed to invade through the matrigel for 22 h at 370C. The results represent data from at least two independent experiments with duplicate tests.

Figure 8. Representative lung metastasis in Ad-CD9 treated mice and control groups of mice are presented. JygMC(A) cells were untreated or transduced with either control virus Ad-lacZ or Ad-CD9 (moi=200) for 48 h. The cells were then harvested and 1x107 cells per mouse were injected subcutaneously into the flanks of female nude mice. The mice were sacrificed at day 34 after tumor inoculation for examining metastases. Shown are the representative lung metastases from mouse groups injected with untreated JygMC(A) cells (control), or JygMC(A) cells treated with control virus (Ad-lacZ), or JygMC(A) cells treated with Ad-CD9 (Ad-CD9).

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Cancer Therapy Vol 4, page 297 Figure 9. Total metastases in each mouse group. JygMC(A) cells, either untreated or transduced with control virus Ad-lacZ or Ad-CD9, were injected into mice as described above in the Fig. 8 legend. Various organs were examined at necropsy for metastases. The total number of metastases in each mouse (open triangle) and the average number of metastases in each group (red circle) are shown.

mechanism of CD9-mediated suppression of breast tumor metastasis, we analyzed protein expression levels of several tumor metastasis-associated proteinases, the enzymes which breakdown the extracellular matrix barrier to facilitate tumor cell spreading, in our model. Our Western blot analysis showed that MMP-2 and MMP-9 expression, at least at the protein level, were not altered by overexpression of CD9 in JygMC(A) tumors (data not shown), suggesting that CD9-mediated suppression of tumor metastasis was not due to the suppression of tumorassociated proteinases. In our study, CD9 enhances cell mobility and adhesion, whereas it inhibits invasion into matrigel. These two aspects are not contradictory if we take a detailed look for how the experiments have been done. Both cell mobility and cell adhesion assays were designed by measuring CD9’s attractiveness towards its receptor fibronectin, a component of ECM, precoated on the undersurface of the filter (mobility assay) or directly on plates (adhesion assay) within 3-4 h. Because fibronectin is suggested to be a receptor for CD9 (Cook et al., 2002), it is not surprising that CD9-overexpressing cells would have enhanced ability to bind to (adhesion) or migrate towards to (cell mobility) fibronectin-coated surface. On the other hand, in vitro invasion assay was done in 22 h by measuring cells’ ability to penetrate the matrigel, which is composed of ECM including fibronectin. Thus, CD9overexpressing cells would have more interaction (“ligand-receptor” binding) with ECM proteins in the matrigel, thus they are inclined to be “trapped” or retarded inside matrigel more easily than the control cells, reflecting as a reduced cell ability to penetrate the entire layer of the matrigel in the in vitro invasion assay. This reduced invasion ability, together with CD9-induced apoptosis and growth inhibition, contribute to reduced distant metastasis of CD9-overexpressing tumor cells. Expression of CD9 is significantly inversely associated with the stage of disease in human breast cancer (Miyake et al, 1995; Cajot et al, 1997). cDNA microarray results showed that CD9 was downregulated in metastatic

IV. Discussion In summary, in this study we showed that adenoviralmediated CD9 expression increased cell migration of the breast cancer line JygMC(A) cells in a fibronectin-coated transwell; CD9 significantly increased JygMC(A) cell adhesion to laminin and fibronectin with a minor increase in adhesion to matrigel; CD9 also suppressed JygMC(A) cell growth and in vitro invasion ability in terms of penetrating the matrigel. Moreover, Ad-CD9 mediated expression significantly decreased tumor metastasis of JygMC(A) cells in a spontaneous metastasis animal model. These results suggest that CD9 may have a therapeutic potential for clinical applications in suppressing malignant progression and metastasis of cancer. One of the practical examples of clinical application of Ad-CD9 is the adjuvant gene therapy for cancer patients: After surgical resection of the primary tumor, Ad-CD9 can be injected into the surgical wound sites to prevent the recurrence of tumor at the primary site and metastasis to other organs. Of course, the optimal goal of long-term is to develop a therapeutic gene therapy virus targeting metastatic cancer cells that can be delivered to the patients via systemic administration. In that case, either a regulatory promoter or cancer (or cell-type)specific promoter should be employed to ensure the therapeutic gene expression can be regulated or the transgene expression is within the desired target cells---the distant metastatic lesions (Lu, 2001). Our data showed that CD9 expression reduced invasion of JygMC(A) cells to penetrate matrigel (Figure 7). Consistently, CD9’s anti-invasion ability was shown in the in vitro mouse embryo implantation: blocking CD9 function in the embryo by either monoclonal antibody or antisense oligonucleotide against CD9 led to significantly enhanced embryo-outgrowth ability on the monolayer of uterus epithelial cells and stimulated matrix metalloproteinase 2 (MMP-2) production, suggesting that CD9 was able to impair embryo invasion and inhibit production of MMP-2 (Liu et al, 2006). To explore the

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Zhang et al: CD9 suppresses breast cancer metastasis breast carcinoma cells compared to primary breast carcinoma cells. Moreover, the relapse free survival in 5 years is significantly higher in CD9 positive cases than in negative cases (Mimori et al, 2005). CD9 downregulation has also been reported to be associated with tumor progression, metastasis and clinical outcome in various other solid tumors (Seymour et al, 1990a, b; Si and Hersey 1993; Higashiyama et al, 1995; Cajot et al, 1997; Mori et al, 1998; Sho et al, 1998; Uchida et al, 1999; Kusukawa et al, 2001; Miyamoto et al, 2001; Sauer et al, 2003; Mimori et al, 2005). However, one study reported that CD9 is not a prognostic factor in human osteosarcoma (Kubista et al, 2004). These data, taken together, suggest that CD9 may be best used as a prognostic factor for solid tumors. While several studies, including this report, have shown that expression of CD9 appears to suppress malignancy of tumor cells (Ikeyama et al, 1993; Miyake et al, 2000), one study showed, however, that CD9 overexpression did not affect in vivo tumorigenic or metastatic properties of human prostate cancer cells (Zvieriev et al, 2005). One explanation is that other proteins, such as CD9 partners, are needed for CD9 full anti-tumorigenic action in a particular cell type environment. While the exact mechanism of how CD9 functions to suppress tumor metastasis remains unclear, some reports demonstrate that CD9 cDNA transfection altered cell motility (Lanza et al, 1991; Ono et al, 1999) and induced apoptosis (Ono et al, 1999). One possibility is that CD9 may exert a malignant-suppressing effect via a combination of cell motility alteration and apoptotic induction. CD9’s anti-metastasis function was also demonstrated in a CD9-knockdown system by others: ovarian carcinoma cell line transfected with small interfering RNA against CD9, showed a CD9-negative phenotype and reduced adhesion to extracellular matrix and increased peritoneal dissemination (Furuya et al, 2005). These results suggest that downregulation of CD9 may be an acquired event in the process of tumor dissemination. Consistently, our results indicate that acquisition of CD9 expression in JygMC(A) breast cancer cells increased adhesion to extracellular matrix components (Figure 6). Cumulative data have suggested that CD9 and other tetraspanin family members, including KAI-1/CD82 (Ono et al, 1999) and CD63 (Israels et al, 2001), are involved in metastasis suppression, and this effect may be related to their association with integrins (Israels et al, 2001; Miyamoto et al, 2001). During tumor progression, a reduction of CD9 gene expression results in tumor cells with high metastatic potential. However, the mechanism of action of CD9 remains unclear. Microarray and real-time PCR were used to analyze the changes of gene expression in tumor cell lines and their CD9-transfectants. It has revealed that the Wnt signaling pathway may be the downstream of the CD9 signal (Huang et al, 2004). A recent study showed that CD9 regulates the actin cytoskeleton by downregulating the WAVE2, through the Wnt-independent signaling pathway (Huang et al, 2006). The mechanistic aspects of function of CD9 are currently under investigation in our lab. Our ongoing studies have

indicated that cells overexpressing CD9 have a significantly higher level of phosphorylated Akt than control transfected cells when plated on fibronectin, and inhibitors of the PI 3-kinase pathway inhibited CD9promoted haptotactic motility (data not shown), suggesting that the PI 3-kinase pathway may be involved in CD9mediated migration. In addition, our microarray results indicated that genes responsible for deacetylation of histones were increased in CD9 overexpressing cells (our unpublished data). We are in the process to examine whether trichostatin A (TSA), a histone deacetylase inhibitor, blocks the CD9-mediated actions. Moreover, our recent studies showed that CD9 appeared to modulate p130Cas (data not shown), an adaptor protein that is thought to be part of a “molecular switch” for induction of cell motility (Klemke et al, 1998). Therefore, CD9mediated motility and anti-tumor function may be regulated in a multiple-platform manner, and may involve many components in a complex network. Tetraspanins, including CD9, can form protein complexes with integrins. To investigate whether alteration of CD9 expression may change contents of intergrins, thus modulating integrinderived signalling pathway to regulate the cancer cell ability to adhere and invade, expression of several integrins at cell-surface (the functional integrins) of JygMC(A) cells were examined for potential alteration by the CD9 overexpression. We found that Ad-CD9 had no significant effect on cell-surface expression of integrin ß1; but Ad-CD9 caused a significant reduction of !5 integrin on cell-surface (mean fluorescent intensity of 28.2 in untransduced cells versus 5.11 in Ad-CD9 transduced cells). However, this alteration is probably not due to the CD9 expression, rather, it is due to the adenoviral protein(s), as the cells transduced by control adenovirus Ad-lacZ also showed a significant reduction of ß1 integrin on cell-surface (mean fluorescent intensity of 8.28). So we conclude that CD9 does not appear to affect integrins !5 and ß1 expression at cell-surface. Whether CD9 alters other integrins’ expression at cell-surface needs to be further investigated.

Acknowledgments This work was supported in part by National Institutes of Health grants DK65962 (Y.L.) and CA107162 (Y.L.), as well as by the Elsa U. Pardee Foundation (Y.L.), University of Tennessee Vascular Biology Center of Excellence Pilot and Feasibility grant (Y.L.) and the Cancer Research and Prevention Foundation (Y.L.). We thank Dr. Syamal Bhattacharya and Ms. Patti Johnson for reviewing this manuscript. We also thank Dr. Qiusha Guo for assembling the flow cytometry plots.

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