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Diagnostic, Predictive and Experimental
ONCOLOGY Days ABSTRACT BOOK December 02 - 03, 2014 hotel NH Collection Olomouc Congress Legionarska 21, 779 00 Olomouc, Czech Republic
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Diagnostic, Predictive and Experimental
ONCOLOGY Days
Pořadatel Ústav molekulární a translační medicíny LF UP a FN Olomouc Ústav klinické a molekulární patologie LF UP a FN Olomouc MedChemBio - zájmové sdružení právnických osob
Odborná garance Sekce diagnostické a prediktivní onkologie České onkologické společnosti ČLS JEP Komplexní onkologické centrum Olomouc
Prezident akce doc.MUDr. Marián Hajdúch, Ph.D.
Organizační výbor doc. Mgr. Jiří Drábek, Ph.D., RNDr. Radek Trojanec Ph.D., MUDr. Kateřina Bouchalová, Ph.D., MUDr. Petr Džubák, Ph.D., MUDr. Josef Srovnal, Ph.D., doc. RNDr. Ondřej Slabý, Ph.D., doc. MUDr. Marek Svoboda, Ph.D.
Organizátor Ústav molekulární a translační medicíny Lékařské fakulty Univerzity Palackého v Olomouci Hněvotínská 5, 779 00, Olomouc Kontaktní osoba: Mgr. Peter Vanek mob.: +420 775 050 355, email: peter.vanek@upol.cz
PROGRAM / PROGRAM
Program satelitního workshopu X. dnů diagnostické, prediktivní a experimentální onkologie Prediktivní diagnostika nádorů 1. prosince, 2014 Část 1
(moderuje: Libor Staněk, Jiří Drábek)
10:30 – 10:50 Prediktivní genetické biomarkery na signální dráze Ras u karcinomu kolorekta Jiří Drábek 10:50 – 11:10 Prediktivní genetické biomarkery u GIST Alena Kalfusová
11:10 – 11:30 Biomarker HER2 u karcinomu prsu a žaludku Magdalena Uvírová
11:30 – 12:30 Přestávka na oběd 12:30 – 12:50 Biomarker EGFR u NSCLC Libor Staněk 12:50 – 13:10 Vyšetření prediktivních markerů u maligního melanomu Hana Vošmiková 13:10 – 13:30 Možnosti tekuté biopsie u solidních nádorů Libor Staněk
13:30 – 13:50 Komplexní genetické biomarkery v predikci u karcinomu prsu Marek Svoboda 13:50 – 14:25 Přestávka na kávu a exkurzi Část 2
14:25 – 14:55 Souhrn stávajících prediktivních biomarkerů a výhled na rok 2015 Marián Hajdúch Část 3
(moderuje: Libor Staněk, Jiří Drábek)
14:55 – 15:55 Uzavřená sekce pro pracovníky referenčních laboratoří Libor Staněk, Jana Stránská a další
Powered by: Ústav molekulární a translační medicíny, Lékařská fakulta, Univerzita Palackého Olomouc Hněvotínská 5, 779 00 Olomouc, Česká republika
tašky hl. papír
IMTM
PROGRAM / PROGRAM X. DNY DIAGNOSTICKÉ, PREDIKTÍVNÍ A EXPERIMENTÁLNÍ ONKOLOGIE / X. DIAGNOSTIC, PREDICTIVE AND EXPERIMENTAL ONCOLOGY DAYS ÚTERÝ / TUESDAY - 2. prosince 2014 / December 2, 2014 9.45
Zahájení X. Dnů diagnostické, prediktivní a experimentální onkologie / Opening ceremony
10:00 – 12:00 Biomarkery nádorových onemocnění I / Cancer biomarkers I Předsedajíci / Chairs: Marek Svoboda, Ondřej Slabý 10.00 – 10.30
Luminální/Her-2 negativní karcinom prsu a možnosti využití komplexních prognostických systémů založených na analýze genetických molekulárních biomarkerů.
Marek Svoboda 10:30 – 11:00 11:00 – 11:15 11:15 – 11:30 11:30 – 11:45
11:45 – 12:00
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Plasma proteomic biomarkers for early detection and characterization of colorectal cancer
Marián Hajdúch Circulating tumor cells in pancreatic cancer Josef Srovnal Circulating tumor cells - a tool to monitor cancer treatment Anna Jakabová MiR-31-3p a miR-31-5p predikují čas do progrese u pacientů s metastatickým kolorektálním karcinomem s nemutovaným onkogenem KRAS léčených cetuximabem Jitka Mlčochová MikroRNA u solidních nádorů: Od biomarkerů po terapeutické cíle Ondřej Slabý OBĚD / LUNCH
13:00 – 14:30 Nové modely a technologie / New models and technologies Předsedajíci / Chairs: Viswanath Das, Karel Koberna 13:00 – 13:15 Mouse transgenic models to study tumor initiation and progression in the gut Michaela Krausová 13:15 – 13:30 Technology of programmable nucleases and reporter mouse lines engineering for cancer research Dominika Fričová 13:30 – 13:45 Genome editing and gene modification technologies: an overview and update Sylvia Petrezselyová 13:45 – 14:00 Understanding biological heterogeneity with the CyTOF 2 Mass Cytometer Mark D. Lynch 14:00 – 14:15 Three-dimensional cultures: physiologically-relevant in vitro tumor models Viswanath Das 14:15 – 14:30 Advantages of high-throughput qPCR for gene expression profiling. Analysis and applications Vlasta Korenková 14:30 – 15:00
PŘESTÁVKA / BREAK
15:00 – 17:00 Molekulární cíle a protinádorová léčiva I / Molecular targets and anticancer drugs I Předsedajíci / Chairs: Marián Hajdúch, Tomáš Eckschlager 15:00 – 15:30 Lactate transport in hypoxic cancer cells is facilitated by non-catalytic function of carbonic anhydrase IX Holger Becker 15:30 – 15:45 Carborane - Based Carbonic Anhydrase IX Inhibitors Jana Štěpánková 15:45 – 16:00 5-Azacytidine Nucleosides and their Derivatives: Molecular hallmarks of Drug Resistance & Alternative Therapeutic Regimen Kushboo Agrawal 16:00 – 16.15 The activity of disulfiram towards breast cancer Zdeněk Škrott
PROGRAM / PROGRAM 16:15 – 16:30 16:30 – 16:45 16:45 – 17:00
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Valproic Acid Increases CD133 Positive Cells that Show Stem Cell Characters in Neuroblastoma Mohamed Khalil Distinctive activity of ginger phenylpropanoids against lymphoblastic leukemia cells Ermin Schadich Tumor microenvironment: mesenchymal stromal cell contribution Lucia Kučerová
SPOLEČENSKÝ VEČER / CONFERENCE DINNER
STŘEDA / WEDNESDAY, 3. prosince 2014 / December 3, 2014 8:00 – 9:00 Poškození a reparace DNA / DNA damage and repair Předsedajíci / Chairs: Kateřina Bouchalová, Martin Mistrík 8:00 – 8:15 Function of cyclin-dependent kinase 12 in DNA repair pathway Jiří Kohoutek 8:15 – 8:30 New approach to quantitative image analysis of laser induced DNA damage in live cells Eva Veselá 8:30 – 8:45 BRCA-mutation status combined with BCL2 protein in prediction of relapse in triple-negative breast cancer (TNBC) treated with adjuvant anthracycline-based hemotherapy. Kateřina Bouchalová 8:45 – 9:00 2‘-deoxy-5-ethynyluridine (EdU): a thymidylate synthase inhibitor with DNA damaging activity Anna Ligasová 9:00 – 9:30
PŘESTÁVKA / BREAK
9:30 – 11:30 Biomarkery nádorových onemocnění II / Cancer biomarkers II Předsedajíci / Chairs: Jitka Berkovcová, Pavel Dundr 9:30 – 9:45 Možnosti predikce léčebné odpovědi na cílenou anti-EGFR a anti-VEGF terapii metastatického kolorektálního karcinomu (mCRC) Radim Němeček 9:45 – 10:00 Management and prognosis of childhood acute lymphoblastic leukemia on protocols ALL-BFM 90, 95 and ALL IC-BFM 2002: a retrospective single-center study Jana Volejníková 10:00 – 10:15 Využití metody sekvenování nové generace v intratumorové heterogenitě kolorektálního karcinomu Jitka Berkovcová 10:15 – 10:30 Introduction to RAS pyrosequencing of FFPE samples Gabriela Gabčová 10:30 – 10:45 MiR-215 as an important tumor suppressor in colorectal cancer patients Petra Vychytilová-Faltejsková 10:45 – 11:00 Overexpression of Filamin-A protein is associated with aggressive clinicopathological features and poor survival outcome in NSCLC patients treated with platinum-based combination chemotherapy Mariam Gachechiladze 11:00 – 11:15 Genetic changes in recurrent Glioblastoma - Is there any significance? Zuzana Crlíková 11:15 – 11:30 Assay destinguishing integrated and episomal form of HPV16, 18, 31 and 58 Hana Ondryášová
PROGRAM / PROGRAM 11:30 – 12:40
OBĚD / LUNCH
12:40 – 14:10 Molekulární cíle a protinádorová léčiva II / Molecular targets and anticancer drugs II Předsedajíci / Chairs: Petr Džubák, Jan Hraběta 12:40 – 12:55 Use of the FUCCI probe in HTS/HCA screening campaigns. Petr Džubák 12:55 – 13:10 Validation of Cell Based Assays for High-Throughput Screening Soňa Gurská 13:10 – 13:25 Molecular mechanisms of cell death induction by novel taxanes effective in resistant breast cancer cells Michael Jelínek 13:25 – 13:40 Novel triterpenoid fluoroderivatives with anticancer activity Jiří Řehulka 13:40 – 13:55 Proteomic profiling of oxaliplatin treated cell line revealed nucleoli and ribosomal protein downregulation Tomáš Oždian 13:55 – 14:10 Apoferritin and its applications in nanomedicine Simona Dostálová 14:10 – 14:30
PŘESTÁVKA / BREAK
14:30 – 15:15 Biomarkery nádorových onemocnění III / Cancer biomarkers III Předsedajíci / Chairs: Radek Trojanec, Jiří Drábek 14:30 – 14:45 Identification of prognostic and predictive factors in non-small cell lung cancer patients treated by Adjuvant chemotherapy Jana Potočková 14:45 – 15:00 A workflow for the identification and validation for N-glycoprotein biomarkers in mouse xenograft serum Lakshman Varanasi 15:00 – 15:15 System for reliable identification of alterations using protein mass-spectrometry Miroslav Hruška 15:15 - 15:45
PŘESTÁVKA - POSTEROVÁ SEKCE / POSTER SESSION BREAK
15:45 – 16:05 Farmakologie protinádorových léčiv a in vivo imaging / Pharmacology of anticancer drugs and in vivo imaging Předsedajíci / Chairs: Miloš Petřík, Josef Srovnal 15:45 – 16:00 Assessing pharmacokinetic properties in drug discovery: screening versus in silico Alice Nová 16:15 – 16:30 68Ga labelled peptides for glioblastoma imaging Miloš Petřík 16:30 – 16:45 Imaging of glioblastoma multiforme in mice using microPET/CT systém Zbyněk Nový 16:45 – 17:00
UKONČENÍ KONFERENCE/CLOSING CEREMONY
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Exprese HNF-1β v karcinomech děložního čípku Kristyna Nemejcova Temperature uniformity of the thermocyclers tested using melting temperature of the same amplicon Eva Hruskova Inhibition of proliferation by inhibitors of histone deacetylases Tomas Groh Multi-walled carbon nanotubes as a promising doxorubicin nanocarrier and their effects on neuroblastoma cell lines Tereza Cerna Prognostický a prediktivní význam sérových onkomarkerů u pacientů s pokročilým neskvamózním NSCLC léčených pemetrexedem Ondrej Fiala Sérové onkomarkery jako prediktivní biomarker u pacientů s pokročilým NSCLC léčených EGFR-TKI Ondrej Fiala Influence of quercetin and taxifolin on microRNA 375 expression in HepG2 cell line and human hepatocytes. Zdenek Dostal PHAGE λ as a suitable doxorubicin nanocarrier Simona Dostalova Fluorescenční charakterizace zlatem modifikovaného liposomu s uzavřenými protinádorovými léčivy a s připojenou antisense N-myc DNA uchycenou k magnetickým částicím Sylvie Skalickova SLEDOVÁNÍ INTERAKCE DOXORUBICINU A ELIPTICINU S BIOLOGICKY ODBOURATELNOU SLOUČENINOU - ALBUMINEM Sylvie Sklickova STUDY OF AFFINITY OF COORDINATION COMPLEXES OF METAL IONS TO DNA BASED ON THE CHANGE OF FLUORESCENCE INTENSITY OF INTERCALATION LABELS (DOXORUBICIN AND ETHIDIUM BROMIDE) Sylvie Skalickova Nanotools in microRna isolation and detection Marketa Vaculovicova Charakterizace dlouhé nekódující RNA MALAT-1 u buněk karcinomu prsu Jaroslav Juracek DEREGULACE PIWIL PROTEINŮ A VYBRANÝCH PIRNA U RCC A CRC Robert Iliev
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Navýšení exprese miR-215 in vivo ovlivňuje velikost nádoru v animálním modelu kolorektálního karcinomu Jana Merhautova Molecular genetic analysis of pheochromocytomas and paragangliomas in Czech patients Musil Zdenek Skp2 associates with Slug and androgen receptor in patients with high Gleason score and lymph node metastasis of prostate cancer Gvantsa Kharaishvili Ubiquitin specific peptidase 7 (USP7) regulates DNA damage bypass pathway through stabilizing Cdc7 kinase and RAD18 ubiquitin ligase. Zsofia Turi Is Epithelial to Mesenchymal Transition Followed by Global DNA Methylation Changes? Bozena Smolkova The effect of acquired resistance to paclitaxel on expression of ABC transporters at breast cancer cells: role of ABCB1 Matej Behounek Vliv rentgenového záření na genovou expresi v lidských chlupových folikulech Hanus Slavik Sada 6-ti mikroRNA predikuje celkové přežívání u pacientů s multiformním glioblastomem Jiri Sana The comparison of 2-D and 3-D model of gene therapy mediated by genetically modified mesenchymal stem cells on human ovarian carcinoma cells SKOV-3. Lenka Toro MESENCHYMAL STROMAL CELLS PLAY AN IMPORTANT ROLE IN MIGRATION AND CHEMOSENSITIVITY OF BREAST CANCER CELL LINES Svetlana Skolekova A novel non-laborious and cost-effective approach for mammalian cell synchronization Martin Liptay MIF: A prognostic marker in Glioblastoma multiforme? Nato Narsia Ultra deep amplicon sequencing of RAS genes and its use for mCRC predictive diagnostics Jana Stranska Výhody vysokokapacitního qPCR pro genově expresní profilování Analýza a aplikace Vlasta Korenková
Abstract book
Biomarkery nádorových onemocnění I Cancer biomarkers I
Předsedajíci / Chairs: Marek Svoboda, Ondřej Slabý úterý / 2. prosince 2014 / Tuesday / December 2, 2014 / 10.00 - 12.00 hod. Luminální/Her-2 negativní karcinom prsu a možnosti využití komplexních prognostických systémů založených na analýze genetických molekulárních biomarkerů. úterý / 2. prosince 10.00 - 10.30 hod.
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Marek Svoboda Klinika komplexní onkologické péče Masarykův onkologický ústav, Žlutý kopec 7, 656 53 Brno Karcinom prsu tvoří skupinu jednotlivých podtypů s odlišným biologickým chováním, jejichž charakterizace je založena na histologických a molekulárních znacích, vycházejících z jejich rozdílného genetického profilu. V současnosti tak rozlišujeme 5 hlavních podtypů karcinomu prsu, a to: 1. luminální – typ „A“, 2. luminální – typ „B“, 3. Her-2 pozitivní/luminální, 4. Her-2 pozitivní/neluminální, 5. triple-negativní, jejichž stanovení je již rutinní součástí klinické praxe, neboť se sebou přináší prognostické a prediktivní informace nezbytné k určení správného algoritmu onkologické léčby. Nejvyšší prevalence mezi pacientkami s karcinomem prsu dosahují luminální (A i B)/Her-2 negativní karcinomy. V populaci českých pacientek tvoří 65 - 70 % (data MOÚ z let 2005 – 2009). Vyznačují se zejména expresí estrogenového receptoru alfa (ERalfa) a přítomností nemutované formy genu Her-2. Jsou sice obecně prognosticky příznivější, nicméně i mezi nimi existuje značná heterogenita ve vývoji onemocnění, a to i v těch případech, kdy jsou zachyceny v časném stádiu, tj. kdy je nádor omezen pouze na prsní žlázu.
Ve srovnání s ostatními podtypy u nich častěji dochází k pozdním relapsům. V dosavadní klinické praxi jsou pacientky s luminálním/Her-2 negativním typem karcinomu prsu členěny do skupin s odlišným rizikem relapsu na základě klasických a molekulárních prognostických biomarkerů, jako jsou: věk, velikost tumoru, postižení lymfatických uzlin, grading, zastoupení nádorových buněk exprimujících estrogenový receptor alfa a progesteronový receptor, markery proliferace (např. Ki-67) a případně další. Pacientky ve zvýšeném riziku relapsu pak podstupují intenzivnější adjuvantní systémovou léčbu, a to v podobě sekvenční a/nebo prodloužené hormonoterapie nebo aplikace chemoterapie před hormonální léčbou. V těchto případech však současně dochází k navyšování toxicity léčby, při podávání chemoterapie významnou měrou, a s tím souvisejících přímých i nepřímých nákladů, tj. spojených s vlastní protinádorovou léčbou nebo léčbou její toxicity. Z tohoto pohledu největší problém činí stanovení prognózy, a tedy i indikace intenzivnější adjuvantní systémové léčby, u pacientek s luminálním/Her-2 negativním typem karcinomu prsu, jejichž onemocnění: a) prokazatelně nedisseminovalo do regionálních lymfatických uzlin (N0), přitom jejich tumor přesahuje 2 cm, b) postihlo sentinelové a/ nebo maximálně 3 lymfatické uzliny, c) má přítomny jiné prognosticky nepříznivé parametry (např. vyšší grading, zvýšená proliferační aktivita, nižší – ale stále pozitivní exprese estrogenové receptoru). Přestože určitého konsensu v užívání a interpretaci výše uvedených biomarkerů bylo dosaženo prostřednictvím prognostických
schémat a systémů, které vychází z doporučení odborných panelů (např. závěry z konferencí v St. Gallen, NCCN a ASCO doporučení) nebo z klinických registrů (např. Adjuvant! Online), problém správného určení strategie léčby ve výše uvedených případech tímto způsobem zcela vyřešen není. Důvodů omezenosti uvedených prognostických schémat je několik, mezi hlavní patří i ta skutečnost, že stanovení gradingu, exprese estrogenového, progesteronového receptoru a Ki-67 podléhá do značné míry technické a interindividuální variabilitě, a dále, že výše uvedené biomarkery u takto vymezené skupiny pacientek (luminální/Her-2 negativní karcinom) již nejsou schopny blíže diferencovat biologickou povahu onemocnění. Zcela zásadní změnu v určení prognózy těchto pacientek přinesly prognostické systémy založené na metodicky jednotném, vysoce přesném stanovení a interpretaci několika biomarkerů pomocí metod molekulární genetiky. Několik těchto komplexních molekulárních prognostických systémů se již dostalo do užívání v klinické praxe a další se tomu blíží Jejich použití u indikované skupiny pacientek s luminálním/Her-2 negativním karcinomem prsu vede ke změně postupu léčby v rozmezí 27 – 74 % případů, a to v závislosti na použitém a srovnávavném prognostickém systému, vždy však s převahou případů, kdy jsou pacientky adjuvantní chemoterapie ušetřeny (20 – 40 % pacientek). Cílem naší přednášky bude shrnout stávající situaci v oblasti komplexních molekulárních prognostických systémů, se zaměřením se na jejich dostupnost, přednosti, případně slabá místa. Diskutovány budou testy Breast Cancer Index, Clarient Insight Dx, EndoPredict, eXagenBC, MammaPrint, Oncotype DX® Breast
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Cancer, Prosigna Breast cancer prognostic gene signature assay a další.
Plasma proteomic biomarkers for early detection and characterization of colorectal cancer úterý / 2. prosince 10.30 - 11.00 hod.
2014
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S.Surinova1*,M. Dziechciarková2,A. Sethi1,Ch.-Y.Chang3,T. Clough3,L.Radova2,M. Skrovina4, K. Vyslouzil5,M. Okoniewski6,H.Weisser1,E. Cattaneo7,O.Vittek3,G.Marra7, R.Aebersold1, M.Hajdúch2 Institute of Molecular Systems Biology, ETH Zurich, Switzerland; 2 Institute of Molecular and Translational Medicine, Palacký University and University Hospital in Olomouc, Czech Republic 3 Department of Statistics, Purdue University, West Lafayette IN, USA 4 J.G.Mendel Oncology Centre, Novy Jicin, Czech Republic 5 Department of Surgery, University Hospital in Olomouc, Czech Republic, 6 Functional Genomics Center, and 7 Institute of Molecular Cancer Research, University of Zurich, Switzerland 1
The current management of colorectal cancer (CRC) would greatly benefit from non-invasive prognostic and predictive biomarkers indicative of clinical and molecular tumor characteristics. Here we employed targeted proteomic profiling of 80 glycoprotein biomarker candidates across a well annotated patient cohort with comprehensive CRC characteristics. Clinical data included 8-year overall survival, tumor staging, histological grading, regional localization, and molecular pathway state of microsatellites and the KRAS gene. The obtained quantitative proteomic data set was subjected to the development of biomarker signatures predicting the respective clinical endpoints.
Protein candidates were selected into the signatures based on significance testing and a stepwise protein selection, each within 10-fold cross validation. A sixprotein biomarker signature of patient outcome could predict survival beyond clinical stage, and was able to stratify patients into groups of better and worse prognosis. Additional signatures predicting colon versus rectal tumor localization, mutation in the KRAS gene, and metastatic disease were also identified. The integration of rich clinical data, quantitative proteomic technologies, and tailored computational modeling facilitated the characterization of these signatures in patient circulation. These findings propose the promise of a simultaneous assessment of important prognostic and predictive disease characteristics within a single measurement.
Circulating tumor cells in pancreatic cancer úterý / 2. prosince 11.00- 11.15 hod.
2014
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Josef Srovnal1, Andrea Prokopova1, Martin Lovecek2, Roman Havlik2, Dusan Klos2, Jana Vrbkova1, Marian Hajduch1 Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic, 2 Department of Surgery, University Hospital Olomouc, Olomouc, Czech Republic Introduction The aim of this ongoing study was to evaluate the hypothesis that presence of circulating tumor cells (CTCs) is a negative prognostic factor in pancreatic cancer. Pancreatic cancer is one of the most aggressive malignancies with a poor prognosis. Assessment of CTCs could prevent aggressive surgery in patients with advanced systemic dissemination. Materials/methods 1
This was a prospective study to test the presence of CTCs in systemic blood, tumor draining blood, bone marrow and peritoneal lavage of 175 pancreatic carcinoma patients at the time of surgery using realtime RT-PCR for carcinoembryonic antigen (CEA), epidermal growht factor receptor 1 (EGFR1) and human telomerase (hTERT). Gene expression of tested markers was correlated with clinical/pathological characteristics and overall survival. Results and conclusions Overall, 151 of 175 (86.3%) pancreatic carcinoma patients died, 81 of 175 (46.3%) of patients underwent curative surgery (R0 surgery). A significant association between EGFR expression levels in the tumor draining blood and clinical stage was found, where patients with advanced disease have a higher expression of EGFR in the tumor draining blood, than patients with lower clinical stage (p<0.001). In addition, significant association between CEA expression levels in the peritoneal lavage and surgical radicality was found (p<0.01). The pancreatic cancer patients with the presence of occult tumor cells detected in the peritoneal lavage using CEA had significantly shorter overall survival (p<0.006). It enables identify patients with advance disease for whom radical surgery is of small benefit. However, we didn‘t find any significant correlation of CTC presence in blood and bone marrow and overall survival. It seems, that CTCs play minimal role in pancreatic cancer prognosis due to high agressivness of the disease. Acknowledgements: Supported by grants IGA UP LF_2014_019, CZ.1.07/2.3.00/30.0004, NPU LO1304 and TAČR TE02000058.
Circulating tumor cells - a tool to monitor cancer treatment úterý / 2. prosince 11.15 - 11.30 hod.
2014
Anna Jakabová1,2, Michael Pinkas1,2, Petra Eliášová3, Zuzana Ušiaková4, Robin Strnad5, Katarína
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Abstract book
Kološtová1,2, Robert Gürlich3, Vladimír Bobek1,2 Department of Tumor Biology, Third Faculty of Medicine, Charles University in Prague, Prague, Czech Republic, 2 Department of Laboratory Genetics, University Hospital Kralovske Vinohrady, Prague, Czech Republic, 3 Department of Surgery, University Hospital Kralovske vinohrady, Prague, Czech Republic, 4Oncology Clinic, General University Hospital Prague, Prague, Czech Republic, 5Department of Surgery, Thomayer’s Hospital, Prague, Czech Republic Introduction Introduction: Circulating tumor cells (CTCs) are shed from tumor to blood circulation of patients with cancer diseases and it is believed that they are responsible for the formation of metastasis. Monitoring of them can be useful for determination of disease progression and treatment response. CTCs isolation from peripheral blood is in comparison to tumor tissue biopsy less invasive and can be repeated. Circulating tumor cells (CTCs) are shed from tumor to blood circulation of patients with cancer diseases and it is believed that they are responsible for the formation of metastasis. Monitoring of them can be useful for determination of disease progression and treatment response. CTCs isolation from peripheral blood is in comparison to tumor tissue biopsy less invasive and can be repeated. Materials/methods Methodology: Patients with colorectal carcinoma (CRC) (n=84) and breast carcinoma (BC) (n=14) were included into the study report. CTCs were tested in a prospective manner and the peripheral blood was taken in parallel with clinical examination (every 3 months). All of the CRC patients were undergoing primary surgery, if possible blood from tumor draining vein has been withdrawn, too. Within the surgery peritoneal washings were taken and analyzed for disseminated 1
tumor cells (DTC) presence. In the group of BC patients CTC-presence was tested to monitor the effect of neoadjuvant therapy. CTCs were enriched from unclothed peripheral blood (4-8 ml) by sizebased separation (MetacellTM). CTC- presence is confirmed by cytomorphological evaluation in parallel with gene expression analysis of tumor associated genes. Fluorescent dyes for labelling both nucleus (NucBlue®) and cytoplasm (CellTrackerTM) in viable cells were used. Partly, CTCs were stained by May-Grünwald or Diff. Quick protocol. Gene expression of tumor associated genes (e.g. KRT7, KRT18, KRT19, KRT20, EpCAM, MUC1, EGFR, MIF...) was tested in an enriched fraction of CTCs immediately after separation and/ or after in vitro culture (3-5days). The gene expression profiles were compared to the profiles obtained in whole peripheral blood. CD45, CD68 and -actin were used as normalizers. If CTCs were present the gene expression analysis of chemoresistance-associated genes followed. Results were analyzed by GENEX software 6.0 (MultiD). Results and conclusions Results: Based on cytomorphological evaluation CTCs were found in 41% (24/59) of CRC patient samples of peripheral blood, and DTCs in 31% (14/46) of peritoneal washings. Interestingly, in 27% of the patients CTCs were found in peripheral blood and in tumor blood in parallel. Subsequently, gene expression profiles of CTCs and DTCs were compared to primary tumor tissue. The results have shown, that the gene expression profiles of CTCs are closely related to DTCs profile, additionally, DTCs expression profiles are related to primary tumor tissue. In the group of BC patients 78% (11/14) were CTC-positive before the therapy starts. CTCs gene expression profiles have changed during the therapy. The most interesting case studies of CRC and
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BC patients will be reported. Conclusion: CTCs analysis could be used within regular clinical examination to monitor possible recurrence and therapy developed resistance.
MiR-31-3p a miR-31-5p predikují čas do progrese u pacientů s metastatickým kolorektálním karcinomem s nemutovaným onkogenem KRAS léčených cetuximabem úterý / 2. prosince 11.30- 11.45 hod.
2014
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Jitka Mlcochova2, Petra Vychytilova-Faltejskova1,2, Hana Mlcochova1,2, Radim Nemecek1, Jana Nekvindova3, Lenka Radova2, Manuela Ferracin4, Barbara Zagatti4, Rostislav Vyzula1, Massimo Negrini4, Ondrej Slaby1,2 Klinika komplexní onkologické pece MOU, Brno, Czech Republic, 2 CEITEC, Masarykova Univerzita, Brno, Czech Republic, 3 Ústav klinické biochemie a diagnostiky, FN Hradec Králové, Hradec Králové, Czech Republic, 4 Laborator experimentální medicíny a diagnostiky, Ferrara, Italy Introduction Terapie metastatického kolorektálního karcinomu (mCRC) se rozšířila o anti-EGFR terapii (cetuximab, panitumumab), která cílí a blokuje receptor pro epidermální růstový faktor (EGFR). Úspěšnost léčby je podmíněná nemutovaným onkogenem KRAS (wt-KRAS). I přesto má klinický benefit z léčby jen u přibližně pětina pacientů s wt-KRAS. To je důvodem nutnosti nalézt nové biomarkery schopné predikovat odpověď k anti-EGFR terapii, která v případě neúčinnosti zbytečně oddaluje možnost podání jiné potenciálně efektivní terapie. Těmito biomarkery by mohly být miRNA zapojené do klíčových signálních drah a regulující také jednotlivé složky signální dráhy EGFR. 1
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Materials/methods Do studie bylo zařazeno 71 klinicky charakterizovaných pacientů s mCRC s wt-KRAS léčených cetuximabem a 25 léčených panitumumabem. Pacienti byli rozděleni na základě odpovědi na anti-EGFR terapii. Ze 41 FFPE vzorků odebraných před zahájením léčby byla vyizolována celková RNA obohacená o frakci krátkých RNA, která byla následně použita pro stanovení expresních profilů miRNA pomocí technologie Agilent miRNA MicroArrays. Následovala validace na nezávislé kohortě 30 pacientů léčených cetuximabem a 25 pacientů léčených panitumumabem pomocí qRT-PCR. Results and conclusions Výsledky: Po vyhodnocení profilů exprese 723 miRNA bylo identifikováno 9 miRNA s nejsignifikantnějším rozdílem v expresi mezi pacienty s dobrou a špatnou odpovědí na terapii cetuximabem (P ≤ 0,01). Těchto 9 miRNA bylo dále validováno a bylo zjištěno, že hladiny exprese miR-315p, miR-31-3p (P < 0,001) korelují s časem do progrese (TTP) u pacientů léčených cetuximabem (miR-31-3p – 43 vs. 14 týdnů, miR-31-5p, 44 vs. 14 týdnů), ale nikoliv pacientů léčených panitumumabem (miR-313p – 20 vs. 29 týdnů, miR-31-5p, 20 vs. 30 týdnů). Závěr: Na základě získaných výsledků se zdá, že miR-31-5p, miR-31-3p by mohly sloužit jako nové prediktivní biomarkery k predikci odpovědi na cetuximab u pacientů s mCRC s wt-KRAS, což by přispělo k vyšší individualizaci léčby. Následné studium miRNA a jejich zapojení do regulace signální
dráhy EGFR by mohlo poodhalit další z molekulárních mechanizmů rezistence k anti-EGFR terapii. Práce byla podpořena grantovým projektem IGA MZČR NT 138604/2012.
MikroRNA u solidních nádorů: Od biomarkerů po terapeutické cíle úterý / 2. prosince 11.45- 12.00 hod.
2014
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Ondřej Slabý1,2 Masarykův onkologický ústav, Klinika komplexní onkologické péče, Brno; 2 CEITEC – Středoevropský technologický institut, Masarykova univerzita, Brno; 1
MikroRNA (miRNA) jsou krátké nekódujcí RNA dlouhé 18-25 nukleotidů, které post-transkripčně regulují genovou expresi v průběhu rozličných buněčných procesů jako jsou apoptóza nebo diferenciace, ale maligní transformace. Změny v expresních profilech miRNA již byly pozorovány u většiny solidních nádorů. Mechanistické studie v nádorové buňce prokázaly schopnost jednotlivých miRNA vykazovat funkci nádorových supresorů a onkogenů. Nejnovější pozorování navíc popisují potenciál jedné miRNA vykazovat v závislosti na kontextu jak funkci nádorového supresoru tak onkogenu. Tato pozorování zásadním způsobem rozšířila koncept molekulární patogeneze nádorových onemocnění a naznačila potenciál miRNA nejen jako diagnostických biomarkerů, ale také jako potenciálních terapeutických cílů.
Sdělení zahrnuje novinky z oblasti biogeneze a funkce miRNA, izomiRs, koncept kompetujících endogenních RNA (ceRNAs), význam miRNA v nádorové biologii a jejich zapojení do hlavních znaků maligního nádoru, biologie nádorové kmenové buňky či autofagie. V kontextu našich výsledků bude diskutována schopnost vybraných miRNA složit jako tkáňové biomarkery (prognostické a prediktivní), sérové a močové biomarkery (diagnostické) a potenciální terapeutické cíle u kolorektálního karcinomu, renálního karcinomu a glioblastomu. Specifické expresní profily miRNA byly u pacientů se solidními nádory úspěšně využity ke stanovení prognózy, k predikci léčebné odpovědi na vybrané terapeutické režimy nebo upřesnění diagnostiky u metastáz neznámého původu. Přítomnost miRNA byla prokázána v krevním séru a plazmě, ale také moči nebo mozkomíšním moku, kde vykazovaly nejen vysokou míru stability, ale u vybraných solidních nádorů rovněž velice dobré analytické vlastnosti. V současné době je kromě možného diagnostického využití cirkulujících miRNA intenzivně studován jejich původ a příčiny jejich extrémně vysoké stability. MiRNA jsou také velice slibnými terapeutickými cíli, přičemž první protinádorová terapie na bázi miRNA vstupuje do klinického hodnocení začátkem příštího roku. Výzkum byl podpořen granty IGA MZ ČR NT13549-4/2012, NT13860 4/2012, NT-13547 -04/2012, NT13514-4/2012 a NT112144/2010.
obchodní a servisní zastoupení firem Abstract book A13 obchodní a servisní zastoupení firem THERMO Scientific – divize laboratorní techniky THERMO Scientific – divize laboratorní techniky BIOQUELL . ERLAB . HMC EUROPE . LANCER . RUSKINN BIOQUELL . ERLAB. SYNGENE . HMC EUROPE . LANCER . RUSKINN SYNBIOSYS . LABOGENE . TECNIPLAST SYNBIOSYS . SYNGENE . LABOGENE . TECNIPLAST
… řešení pro vaši laboratoř … řešení pro vaši laboratoř
biohazardy, laminární boxy, izolátory centrifugy - ultracentrifugy biohazardy, laminární boxy, izolátory centrifugy - ultracentrifugy biohazard boxy biohazard boxy laminární boxy laminární boxy boxy pro PCR boxy pro PCR biohazardy tř. III biohazardy tř. III bezodtahové digestoře bezodtahové digestoře boxy pro laboratorní zvířata boxy pro laboratorní zvířata peroxidová dekontaminace peroxidová dekontaminace
mikrocentrifugy a malé centrifugy mikrocentrifugy a malé centrifugy multifunkční centrifugy multifunkční centrifugy vysokootáčkové centrifugy vysokootáčkové centrifugy velkokapacitní centrifugy velkokapacitní centrifugy ultracentrifugy ultracentrifugy zkumavky pro centrifugaci zkumavky pro centrifugaci
gel—imaging a analýza inkubátory - termostaty gel—imaging a analýza inkubátory - termostaty
gelová dokumentace a analýza gelová dokumentace a analýza chemiluminiscence, fluorescence chemiluminiscence, fluorescence software pro 1D i 2D analýzu software pro 1D i 2D analýzu transiluminátory transiluminátory elektroforézy elektroforézy zdroje pro elfo zdroje pro elfo
CO2, CO2/O2 inkubátory CO2, CO2/O2 inkubátory termostaty, sterilizátory termostaty, sterilizátory chlazené termostaty chlazené termostaty třepací termostaty třepací termostaty anaerobní a mikroaerofilní boxy anaerobní a mikroaerofilní boxy klimatické boxy, růstové komory, systémy pro monitoring teploty klimatické boxy, růstové komory, systémy pro monitoring teploty
mrazicí a chladící boxy, kryoboxy mikrodestičková instrumentace mrazicí a chladící boxy, kryoboxy mikrodestičková instrumentace
spektrofotometry, fotometry spektrofotometry, fotometry fluorometry, luminometry fluorometry, luminometry dávkovače dávkovače promývačky promývačky třepací termostaty pro mikrodestičky třepací termostaty pro mikrodestičky KingFisher KingFisher
chladící boxy 0°C až 15°C chladící boxy 0°C až 15°C mrazící boxy - 5°C až - 40°C mrazící boxy - 5°C až - 40°C mrazící boxy do - 86°C a - 150°C mrazící boxy do - 86°C a - 150°C řízené zamrazování LN2 -180°C řízené zamrazování LN2 -180°C skladovací boxy LN2 - 180°C skladovací boxy LN2 - 180°C transportní boxy, výrobníky ledu, šokové zmrazovače transportní boxy, výrobníky ledu, šokové zmrazovače
myčky , autoklávy koncentrátory vzorků myčky , autoklávy koncentrátory vzorků
laboratorní myčky laboratorní myčky velkokapacitní myčky velkokapacitní myčky myčky pro chovné boxy myčky pro chovné boxy sušičky sušičky autoklávy 25 až 160 litrů autoklávy 25 až 160 litrů čidla pro validaci autoklávů čidla pro validaci autoklávů
vakuové centrifugační koncentrátory vakuové centrifugační koncentrátory velkoobjemové lyofilizátory velkoobjemové lyofilizátory stolní lyofilizátory stolní lyofilizátory vakuové sušárny vakuové sušárny
pipety a laboratorní plast chov laboratorních zvířat pipety a laboratorní plast chov laboratorních zvířat
IVC individuálně ventilované boxy IVC individuálně ventilované boxy chovné nádoby a příslušenství chovné nádoby a příslušenství izolátory izolátory ochranné a přestýlací boxy ochranné a přestýlací boxy metabolické klece metabolické klece komplexní projekty komplexní projekty
Pipety Finnpipette Pipety Finnpipette steppery a dávkovače steppery a dávkovače pipetovací nástavce pipetovací nástavce kompletní sortiment špiček kompletní sortiment špiček spotřební plastik Nalgene, Nunc spotřební plastik Nalgene, Nunc stripy a destičky stripy a destičky
drobné laboratorní přístroje příprava čisté vody drobné laboratorní přístroje příprava čisté vody
magnetické míchačky magnetické míchačky topné desky, blokové lázně topné desky, blokové lázně třepačky a vortexy třepačky a vortexy
reverzní osmóza reverzní osmóza deionizační systémy deionizační systémy kompaktní laboratorní systémy kompaktní laboratorní systémy
servis kontakt servis kontakt
validace přístrojů validace přístrojů záruční a pozáruční servis záruční a pozáruční servis akreditovaná kalibrační laboratoř akreditovaná kalibrační laboratoř
www.trigon-plus.cz www.trigon-plus.cz mail@trigon-plus.cz mail@trigon-plus.cz tel. +420 272 680 190, mob. +420 602 313 571 tel. +420 272 680 190, mob. +420 602 313 571
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Abstract book
Nové modely a technologie New models and technologies
Předsedajíci / Chairs: Viswanath Das, Karel Koberna úterý / 2. prosince 2014 / Tuesday / December 2, 2014 / 13.00 - 14.30 hod. Mouse transgenic models to study tumor initiation and progression in the gut
addition, currently utilized mouse models of intestinal cancer will be presented. úterý / 2. prosince 2014 / Results and conclusions 13.00 - 13.15 hod. The talk will illustrate how genetic Michaela Krausova, Bohumil manipulations in mouse deepened our knowledge about cellular Fafilek, Vladimir Korinek architecture of adult gut tissue. The Institute of Molecular Genetics AS contribution of various signaling CR, v.v.i., Prague, Czech Republic pathways to normal stem cell physiology and to tumor initiation and Introduction The epithelia of the gastrointestinal progression will also be discussed. tract represent one of the most Technology of programmable rapidly self-renewing tissues in the nucleases and reporter mouse adult mammalian body. The single- lines engineering for cancer layer epithelium of the small intestine research is formed by microscopic projections úterý / 2. prosince 2014 / into the intestinal lumen called villi, 13.15 - 13.30 hod. and invaginations into underlying mesenchyme, so-called crypts of Dominika Fricova, Maja Lieberkühn. Tissue homeostasis Sabol, Petr Kaspar, Petr is maintained by intestinal stem Kasparek, Trevor Epp, Inken cells residing in the bottom part of the crypts. The stem cells divide Beck, Radislav Sedlacek asymmetrically, giving rise to fast- Laboratory of Transgenic Models cycling transit amplifying cells of Diseases and Czech centre for located above the stem cell zone. Phenogenomics, BIOCEV, Institute At the crypt orifice these cells of Molecular Genetics of the ASCR, differentiate to generate specialized Prague, Czech Republic cell lineages of the gut. Differentiated Introduction cells continue their movement TALE (Transcription-activator like up to the top of the villus, where effector) and CRISPR (Clustered they are extruded to the intestinal Regularly Interspaced Short lumen. The process of epithelial Palindromic Repeats) associated self-renewal is completed in 3-5 nucleases are able to generate genetic days. The tract lining is concurrently modifications by introduction of exposed to chemical stress and double-stranded breaks. Subsequent mechanical tensions. Consequently, activation of repair pathways and the combination of high proliferation homologous recombination (HR) rate and environmental stress results represent molecular basis for knockin the accumulation of somatic in strategies. This approach allows mutations hitting tumor suppressor genomic insertions (1) within the genes or proto-oncogenes leading to locus of the gene of interest or (2) in the tumor formation. unrelated, well described genomic Materials/methods region as ROSA26, which provides The talk will be focused on recent ubiquitous transgene expression with methodologies employed for the no adverse consequences on mouse identification of specific markers phenotype. In the order to facilitate of the intestinal stem cells and for the analysis of the function(s) of genes cell tracking in transgenic mice. In involved in senescence (p16Ink4a
and p21) and DNA damage response (Fancd2 and 53BP1), we employed programmable nucleases mediated targeting systems to generate mouse lines expressing reporters as luciferase and/or fluorescent protein inserted in desired genomic region. Materials/methods Programmable nucleases including TALENs and CRISPR/Cas9 system enable generation of genetic modifications in cultured cells, as well as in whole animals. In order to create reporter mouse lines we used different strategies. We generated targeting constructs containing reporters and specific homology arms for targeting specific genomic region. In Fancd2 and 53BP1 targeting strategy, gene encoding for citrine was inserted behind the start codon using TALENs and CRISPRs in two different approaches. The donor vectors were assembled using ligation-independent cloning, and contained homology arms from region surrounding the ATG site. For p16Ink4a and p21 reporter models we prepared constructs containing genes encoding for luciferase and tdTomato fluorescent protein in tandem fusion under p16Ink4a or p21 promoter control. After in vitro confirmation of its specific activity in senescent cells, we cloned reporter construct into donor vector containing homology arms from first intron of the ROSA26 locus. TALENs or CRISPRs combined with the linearized donor construct were injected into mouse zygotes. Results and conclusions Reporter gene mouse lines represent crucial tool for in vivo studies in variety of scientific fields from functional genomics, proteomics, cell biology to cell-based drug screenings. In this study we designed and used both TALENs and CRISPR/Cas9 assisted targeting for creation of reporter mouse lines for genes involved in DNA
Abstract book
damage response (Fancd2, 53BP1) and senescence (p16Ink4a and p21). To prepare models expressing citrine-tagged versions of protein products of Fancd2 and 53BP1 we targeted directly genes loci of these genes. In p16Ink4a and p21 reporter mice strategy we inserted genes encoding for luciferase and tdTomato under p16Ink4a (or p21) promoter control into ROSA26 locus. Mouse embryonic fibroblasts were developed from created mouse lines and the activity of tagged proteins was examined. Our models could be used for deeper analysis of the role of senescence and DNA damage response in context of cancer development and its treatment.
Genome editing and gene modification technologies: an overview and update úterý / 2. prosince 13.30 - 13.45 hod.
2014
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Silvia Petrezselyova, Radislav Sedlacek Laboratory of Transgenic Models of Diseases and Czech centre for Phenogenomics, BIOCEV, Institute of Molecular Genetics of the ASCR, v.v.i., Prague, Czech Republic Introduction Functional gene studies require precise and efficient genome engineering technologies and gene targeting via homologous recombination (HR) has been extensively used to generate specific mutant species for decades. However, the use of this technique has been hampered because of several limitations: low HR rate in mammalian cells, labor-intensive and time-consuming selection/screening strategies and construction of large targeting vectors. In recent years, discovery of new genome-editing technologies, such as ZFN (zinc finger nucleases), TALENs (transcription activator-like effector nucleases) and CRISPR (the Clustered Regularly Interspaced Short Palindromic Repeats) associated with Cas9 (CRISPR-associated 9) protein, has revolutionized
generation of targeted mutations in many model organisms. All these technologies work on the same principle, i.e. sequence-guided DNA endonucleases induce DNA doublestrand breaks that subsequently stimulate either non-homologous end joining (NHEJ) recombination and/or homology-directed repair (HDR) system at targeted loci. The NHEJ recombination is an errorprone event forming small insertion or deletion (indels) and therefore results in a frame-shift mutation. Because the HDR system requires a DNA template to repair a DSB, it is used to introduce a desired point mutation or insert a missing gene/ reporter gene. Materials/methods In our laboratory, we employ both TALEN and CRISPR/Cas9 approaches to create site specific modifications in the mouse genome. Knock-out mice are prepared by targeting one site or two sites simultaneously that allows frame-shift and deletion mutations, respectively. Double-stranded constructs or single-stranded oligonucleotide are introduced via HR to generate point mutation changes, reporter genes or conditional alleles. TALEN and CRISPR/Cas9-related constructs are microinjected into mouse zygotes to generate gene knockout or knock-in mice or transfected into embryonic stem (ES) cells for the creation of recombinant ES cell clones. Results and conclusions Transgenic and knock-out mice are valuable models for understanding of physiological functions of proteins and their mechanisms. Our model mice will be applied in many human disease studies, including cancer. Several examples (single point mutation mice, simple/conditional knock-outs and reporter mice) out of about 39 finalized and 30 ongoing projects will be presented.
Understanding biological heterogeneity with the CyTOF 2 Mass Cytometer úterý
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13.45- 14.00 hod.
Mark D. Lynch Fluidigm Europe BV, Les Ulis, France Introduction Single-cell biology endeavors to unravel biological heterogeneity using multidimensional approaches. Cells use DNA, RNA, and proteins to power biological networks. Materials/methods The C1TM Single-Cell Auto Prep System delivers an easy, complete workflow that captures and prepares individual cells for gene expression and sequencing, while the CyTOF® 2 Mass Cytometer allows highspeed acquisition of multiparametric single-cell proteins. Results and conclusions With these innovative tools, multidimensional single-cell biology is providing profound insights into the dynamic elements of biological heterogeneity.
Three-dimensional cultures: physiologically-relevant in vitro tumor models úterý / 2. prosince 14.00 - 14.15 hod.
2014
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Viswanath Das, Marta Zbožínková, Marián Hajdúch Institute of Molecular and Translational Medicine, Olomouc, Czech Republic Introduction Three-dimensional (3D) cultures of cancer cells are increasingly being recognized as physiologicallyrelevant in vitro tumor models for effective selection of anticancer drugs in preclinical studies. Herein, we present a comparative study of the effect of a few selected anticancer agents that are clinically used in the treatment of various solid tumors in two-dimensional (2D) and 3D cultures of colorectal HCT116 and HT29 carcinoma cells. Materials/methods Methods Cytotoxic effects of anticancer agents in 2D and 3D cultures of
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HCT116 and HT29 cells were examined using MTT cell proliferation assay and bright-field imaging. Cell cycle alteration in 3D cultures was monitored by flow cytometry. Changes in the expression of proteins in 2D and 3D cultures were detected by Western blot analyses. Results and conclusions Anticancer drugs that are highly cytotoxic in 2D cultures show low cytotoxicity in 3D cultures of HCT116 and HT29 cells. 3D cultures of HCT116 and HT29 cells showed G0/G1 cell cycle arrest and an increase in the expression of carbonic anhydrase IX. Our results illustrate relative closeness of three-dimensional cultures to primary tumors and provide further support for the potential use of 3D cell cultures for the screening of promising anticancer agents in preclinical screens.
Výhody vysokokapacitního qPCR pro genově expresní profilování. Analýza a aplikace. (Advantages of highthroughput qPCR for gene expression profiling. Analysis and applications.) úterý / 2. prosince 14.15 - 14.30 hod.
2014
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Vlasta Korenková1, Lucie Langerová1, Vendula Novosadová1, Mikael Kubista1,2 Biotechnologický ústav AV CR, v.v.i., Praha, Czech Republic, 2 TATAA Biocenter, Goteborg, Sweden Introduction Expresní profilování představuje měření exprese více genů najednou za účelem vytvoření obrazu o buněčné funkci. Profily genové exprese mohou být použity například pro diagnostické monitorování odpovědi pacienta na léčbu a tím k optimalizaci individuální terapie. Materials/methods Vysokokapacitní zařízení BioMark (Fluidigm), které je umístěno 1
v qPCR servisním pracovišti Biotechnologického ústavu AV ČR, umožňuje současnou analýzu exprese až 96 genů v 96 vzorcích (9216 reakcí) v rámci jediného experimentu. Měření probíhá pomocí kvantitativní polymerázové řetězové reakce (qPCR), která je jednou z nejcitlivějších metod pro kvantifikaci mRNA či mikroRNA. Možnost analyzovat větší množství genů najednou vybízí k použití multivariantních statistických analýz. Results and conclusions Využití vysokokapacitního qPCR přístroje BioMark poskytuje množství výhod. 1. Efektivita: Výrazná úspora vzorku (5 µl pro kompletní analýzu), detekční chemie, mastermixu a spotřebního materiálu. 2. Rychlost: Celý vysokokapacitní experiment lze provést v rámci několika hodin, ne dnů či týdnů. 3. Automatizace a vysoká kapacita: Díky zautomatizovanému pracovnímu postupu je minimalizována možnost lidské chyby. Není potřeba mezidestičkové kalibrace. 4. Sensitivita a flexibilita qPCR systému. 5. Možnost dalších aplikací: vysokokapacitní genotypování a digitální PCR.
Abstract book
Molekulární cíle a protinádorová léčiva I Molecular targets and anticancers drugs I
Předsedajíci / Chairs: Marián Hajdúch, Tomáš Eckschlager úterý / 2. prosince 2014 / Tuesday / December 2, 2014 / 15.00 - 17.00 hod. Lactate transport in hypoxic cancer cells is facilitated by non-catalytic function of carbonic anhydrase IX úterý / 2. prosince 15.00- 15.30 hod.
2014
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Somayeh Jamali1, Michael Klier1,2, L. Felipe Barros3, Robert McKenna4, Joachim W. Deitmer2, Holger M. Becker1 Division of Zoology / Membrane Transport, University of Kaiserslautern, Kaiserslautern, Germany, 2 Division of General Zoology, University of Kaiserslautern, Kaiserslautern, Germany, 3 Centro de Estudios Científicos (CECS), Valdivia, Chile, 4 Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, USA Introduction The most aggressive and invasive tumor cells, which often reside in hypoxic environments, rely on extensive glycolysis to meet their large demand for energy and biosynthetic precursors. Thereby they release vast amounts of lactate and protons via monocarboxylate transporters (MCTs), which exacerbates extracellular acidification and supports the formation of a hostile environment. In the present study we investigated the mechanisms that regulate lactate transport in cancer cells during the switch from normoxia to hypoxia. Materials/methods Changes in intracellular pH and lactate concentration in single MCF7 breast cancer cells were monitored by pH imaging and with the lactatesensitive FRET nanosensor Laconic, respectively. Hypoxia-induced changes in the expression levels of various enzymes and transport 1
Translational Medicine, Olomouc, Czech Republic, 2 Institute of Organic Chemistry and Biochemistry ASCR, v.v.i., Structural Biology, Prague, Czech Republic, 3 Institute of Inorganic Chemistry of the AS CR, v.v.i., Department of Syntheses, Husinec Rez, Czech Republic Introduction Carbonic anhydrase IX (CA IX) is highly overexpressed in different solid tumors. It is a transmembrane isoform of carbonic anhydrase with an extracellular-facing catalytic site and therefore is well positioned to act in the control of tumor pH. Function of CA IX can be inhibited by CA IX-selective sulphonamides. Inhibition its function perturbs in vitro survival under hypoxia conditions. The aim of this study is to evaluate the effect of new carboranes with functional sulfonamide residues on CA IX function. Carboranes, icosahedral clusters containing boron, carbon and hydrogen replace various hydrophobic structures in biologically active molecules. Materials/methods The most interesting drugs were Carborane - Based Carbonic chosen based on enzymatic assay. Anhydrase IX Inhibitors úterý / 2. prosince 2014 / To characterize their CA IX inhibition mode in cellular level, several cell 15.30- 15.45 hod. biology methods were used. Drug Jana Stepankova1, Pavlina cytotoxicity and consequently extracellular pH of carboranes Rezacova2, Brynda Jiri2, treated cell lines under hypoxia Harvanova Monika1, Masek conditions was evaluated. Data Vlastimil1, Nova Alice1, shows reversed hypoxia-induced Schiller Michal1, Das decline of extracellular pH in treated Viswanath1, Dolezal Dalibor1, HT-29 and 4T1 cell lines. Moreover, we set up a new method of Raman Grüner Bohumir3, Sicha 3 1 spectroscopy locating carboranes Vaclav , Konecny Petr , Znojek Pawel1, Dzubak Petr1, distribution in cells. To extend this study, pharmacokinetics and Hajduch Marian1 pharmacology profile describe 1 Faculty of Medicine and Dentistry ADME methods and experiments on Palacky University Olomouc, animal model. Institute of Molecular and proteins were analyzed in MCF-7 cells by qRT-PCR and western blot. Functional interactions between MCTs and carbonic anhydrase CAIX were determined in Xenopus oocytes by single-site mutation and subsequent measurements of intracellular pH with ion-sensitive microelectrodes. Results and conclusions Under hypoxia, expression of MCT1 and MCT4 in MCF-7 breast cancer cells remained unchanged, while expression of carbonic anhydrase IX (CAIX) was greatly enhanced. Realtime measurements of intracellular pH and lactate concentration show that CAIX augments lactate flux via MCT1 by a non-catalytic interaction. Mutation studies in Xenopus oocytes indicate that CAIX, via its intramolecular H+-shuttle His200, functions as a „protoncollecting/distributing antenna“ to facilitate rapid lactate flux via MCT1. Knockdown of CAIX significantly reduced proliferation of MCF-7 cancer cells, suggesting that rapid efflux of lactate and H+, as enhanced by CAIX, contributes to cancer cell survival under hypoxic conditions.
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Results and conclusions The results shows the ability of these compounds inhibit CA IX in enzymatic level and cellular level. Thus, novel carboranes with functional sulfonamide residues indicate new selective CA IX inhibitors as potential anticancer drugs. Acknowledgement: ProMedChem, reg. n.: CZ.1.07/2.3.00/30.0060, BIOMEDREG, reg. n.: CZ.1.05/2.1.00/01.0030
5-Azacytidine Nucleosides and their Derivatives: Molecular Hallmarks of Drug Resistance & Alternative Therapeutic Regimen úterý / 2. prosince 15.45- 16.00 hod.
2014
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Khushboo Agrawal1, Dušan Holub1, Petr Vojta1, Ivo Frydrych1, Zuzana Maceckova1, Miroslav Otmar2, Petr Džubák1, Marián Hajdúch1 Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic, 2 Institute of Organic Chemistry and Biochemistry, ASCR v.v.i., Prague, Czech Republic Introduction Aberrant DNA methylation turning ‘off’ the gene expression remains the consistent hallmark due to its frequent involvement in all types of cancer. The prototypal DNA methylation inhibitors, 5-azacytidine and 2 -deoxy-5-azacytidine, are currently one of the most effective epigenetic drugs, for the treatment of blood malignancies. However, the chemotherapeutic resistance to these medicines is the major obstacle, forefending the successful epigenetic therapy. Materials/methods We developed several HCT116 p53 wild-type cell clones, resistant towards 2‘-deoxy-5-azacytidine. 1
Principal methods used to study the molecular alterations during the development of resistance included, flow cytometry based analyses, high throughput RNA sequencing based transcriptomics, and mass spectrometry based proteomics, utilizing stable isotope labelling of amino acids in cell culture (SILAC). Further, we used MTT cytotoxicity assays to determine the crossresistance or sensitivity of the resistant clones towards other epigenetic inhibitors. Results and conclusions Flow cytometry based studies revealed significant up-regulation of DNA and RNA synthesis. Molecular profiling of resistant clones unveiled 8010 genes and 3352 proteins, which were differentially expressed (ANOVA p<0.05) compared to parental cell line. The major affected cellular pathways were (i) Cell cycle: nucleocytoplasmic transport of CDK/ cyclins, role of 14-3-3 proteins in cell cycle regulation, G1/S transition and initiation of mitosis (ii) Apoptosis and survival: granzyme A signaling, BAD phosphorylation, p53 dependent apoptosis (iii) Transcription: role of heterochromatin protein I family in transcriptional silencing (iv) DNA damage: role of SUMO in p53 regulation. During MTT cytotoxicity assays, resistant clones exhibited cross-resistance towards all the tested epigenetic inhibitors, however, significant sensitivity was exceptionally observed for bromodomain inhibitors, which was further validated by down-regulation of BET bromodomain, BRD4 gene, in all the resistant cell clones. Validation of relevant genes and/or proteins as biomarkers of drug resistance, and bromodomains as alternative therapeutic target, for re-sensitizing the cancer patients, resistant to DNA methylation inhibitors is currently ongoing. The present study will aid to the understanding of the molecular basis of acquired tumor resistance to 2‘-deoxy-5-azacytidine and help in predicting its clinical response, as well as in designing alternative
treatment regimens for overcoming resistance, hence furthering clinical development.
The activity of disulfiram towards breast cancer úterý / 2. prosince 16.00- 16.15 hod.
2014
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Zdenek Skrott1, Martin Mistrik1, Boris Cvek2, Pavla Pouckova3, Jiri Bartek1,4 Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic, 2 Department of Cell biology and Genetics, Faculty of Science, Palacky University, Olomouc, Czech Republic, 3 st 1 Medical Faculty, Charles University, Prague, Czech Republic, 4 Danish Cancer Society Research Canter, Copenhagen, Denmark Introduction Drug repurposing is a new viable approach of drug development. Disulfiram (Antabuse), as an old drug used for decades in alcohol aversion therapy, shows promising anticancer activity. The potent disulfiram´s antitumor effect is attributed to the complex formed from the reaction between disulfiram and copper in the human body. It is assumed that disulfiram-copper complex kills cancer cells by inhibition of ubiquitin-proteasome system, which is responsible for degradation of cellular proteins. Materials/methods The toxicity of the main metabolites of disulfiram was evaluated in vitro with various breast cancer cell lines (MDA MB231, MCF7, HS578T, T47D, Cal51) and in vivo using MDA MB231 xenografts. The cellular effect of disulfiram-copper complex was measured in selected breast cancer cell lines or Ub-(G67V)-GFP expressing Hela cell line by standard methods (Western blot, fluorescent microscopy). Results and conclusions 1
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Disulfiram complexed with copper supress cancer cell viability and ubiquitin-dependent protein degradation. In contrast to previous report, the complex does not exhibit any inhibition of 20S core particle of the proteasome. On the other hand, disulfiram-copper complex induces notable accumulation of ubiquitinated proteins, indicating that the degradation of proteins is impaired. Moreover, the complex stabilizes both, physiological proteasome substrate IκBα, and artificial Ub(G76V)-GFP reporter. Collectively, these data indicate that disulfiram´s anticancer activity is linked to the inhibition of the ubiquitin-proteasome system by a mechanism that is distinct to the conventional proteasome inhibitors.
Valproic Acid Increases CD133 Positive Cells that Show Stem Cell Characters in Neuroblastoma úterý / 2. prosince 16.15- 16.30 hod.
2014
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Mohamed Ashraf Khalil1, Jan Hraběta1, Tomáš Groh1,2, Pavel Procházka3, Helena Doktorová1, Šimon Cipro4, Tomáš Eckschlager1 Department of Pediatric Hematology and Oncology, 2nd Medical Faculty, Charles University and University Hospital Motol, Prague, Czech Republic, 2 Department of Biochemistry, Faculty of Science, Prague, Czech Republic, 3 Institute of Experimental Medicine, Academy of Sciences, Prague, Czech Republic, 4 Department of Pathology and Molecular Medicine, 2nd Medical Faculty, Charles University and University Hospital Motol, Prague, Czech Republic Introduction Recently, Cancer stem cell model proposes that tumor consists of variety of cells with different proliferative and tumorigenic capacities. According to this theory, a small population of cancer stem 1
cells (CSCs) was suggested to drive tumor growth and be responsible for its resistance, recurrence and metastasis. CSCs can be identified by the presence of a single or combination of markers. CD133 is one of the most important markers for CSCs in neuroblastoma (NB) and solid tumors. It is obvious that epigenetic modifications such as histone acetylation and promoter methylation regulate CD133 transcription. Valproic acid (VPA), a well known antiepileptic drug, is a histone deacetylase (HDAC) inhibitor that demonstrates antitumor activities. Hence, we evaluated the effect of VPA on expression of CD133 in NB cell lines. Materials/methods Cell lines: neuroblastoma UKF-NB3, IMR 32, SH-SY5Y and UKF-NB4. Western blot: detection of CD133/1 and acetylated histone H3 & H4. Flow Cytometry: detection of CD133/2, cell cycle and cleaved caspase 3. Colony formation assay. Bisulfite conversion. drugs used: Valproic acid , Valpromide (VPA analogue) and 5-aza-2’-deoxycytidine (demethylating agent). Results and conclusions Flow cytometric and western blot analysis showed increased CD133+ population to several folds as far as VPA is included in the culture of UKF-NB3 and SH-SY-5Y cells. The increase of CD133 was accompanied by increase of histones H3 and H4 acetylation level. CD133+ cells were mainly present in S and G2/M phases of cell cycle and showed less cleaved caspase-3 compared to CD133- cells when treated with VPA and cytostatics. VPA treated UKF-NB3 cells have acquired higher colony formation capacity, unlike IMR-32 and UKF-NB4 which were not expressing CD133 protein and had no increase in colony formations in VPA pretreated samples. CD133 has been reactivated in IMR-32 and UKF-NB4 after using demethylating agent 5-aza-2’-deoxycytidine.
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Reappearance of CD133 protein was in parallel with demethylation of CD133 promoter P1. In conclusion, we proved that VPA can induce CD133+ cells which show CSCs features in some NB cell lines. In addition, we confirm the epigenetic regulation of CD133 expression in NB. This work was support by GACR, grant No. 14-18344S.
Distinctive activity of ginger phenylpropanoids against lymphoblastic leukemia cells úterý / 2. prosince 16.30- 16.45 hod.
2014
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Ermin Schadich1, Marek Handl2, Soňa Křupková1, Ivo Ivo Frydrich1, Petr Džubák1, Marián Hajdúch1 Faculty of Medicine and Dentistry, Palacký University Olomouc, Olomouc, Czech Republic, 2 Faculty of Science, Palacký University, Olomouc, Czech Republic Introduction Plant phenylpropanoids are considered as useful source for development of novel drugs due to their anti-oxidant, anti-inflammatory and chemopreventive properties. Specifically, previous studies showed that some of them might also have the pro-oxidant effect associated with distinct cytotoxic activity only in the certain cancer lines. The aim of our studies was to assess the cytotoxic activity of phenylpropanoids from rhizome of ginger (Zingiber officinale) against three different cancer cell lines, hepatocellular carcinoma (HepG2) cells, glioblastoma (U-87 MG) and acute T lymphoblastic leukemia (CCRF-CEM (CRM-CCL-119)) cells, and fibroblasts (CRL-2522) , immortalized keratinocytes (HaCat) and human embryonic kidney (HEK293) cells. Materials/methods The phenylpropanoids from methanol extract of ginger rhizome were tested for components 1
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and cytotoxic activity against all seven cell lines using liquid chromatography (LC-MS) and MTT assay respectively. Their property to modulate the antioxidant and anti-inflammatory pathway was assessed using three different luciferase reporter cell lines, HepG2 and HaCat cell line with antioxidant response element reporter and rat glioma (CCL-107) cell line with NF-ÎşB (nuclear factor kappa-lightchain-enhancer of activated B cells) reporter. Results and conclusions The results LC-MS analysis showed that methanol extract of ginger rhizome contained both two major phenylpropanoids, 6-gingerol and 6-shogaol and their derivatives. These phenylpropanoids increased the signaling of all of the three reporter cells lines showing their antioxidant and anti-inflammatory property. Significantly, they had only the cytotoxic activity against CCRFCEM cells. Ginger phenylpropanoids of ginger rhizome have the distinct cytotoxic activity against CCRF-CEM cells indicating their potential for development of novel chemotherapeutic drugs against lymphoblastic leukemia.
Tumor microenvironment: mesenchymal stromal cell contribution ĂşterĂ˝ / 2. prosince 16.45- 17.00 hod.
2014
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Lucia Kucerova Molecular Oncology Department, Cancer Research Institute SAS, Bratislava, Slovakia Introduction Tumor microenvironment substantially affects tumor progression and tumor cell behavior. The extrinsic factors and signals coming from the surrounding stromal tissue alter numerous processes in the tumor cells. It represents a complex structure composed of various non-malignant cell types and secreted molecules including extracellular matrix, growth factors, chemokines and cytokines. Mesenchymal stromal cells (MSCs) significantly contribute to tumor microenvironment and a complex cross talk between heterogeneous malignant cells and the MSC together with the extracellular matrix determines also the tumor therapeutic responses. Materials/methods MSCs represent a population of cells with multilineage differentiation capability, which secrete wide array of chemokines and growth factors. The MSCs are defined by their ability to differentiate into osteoblasts, adipocytes and chondrocytes, but they can also differentiate
into other cell types; pericytes; cancer stem cell niche cells; and tumor associated fibroblasts in the tumor microenvironment.We have evaluated the interactions by live-cell kinetic imaging, viability and apoptotic assays, expression analysis and functional migration assays. Results and conclusions The MSCs can function in promoting or inhibiting tumor initiation and progression. The MSCs may directly promote tumor cell proliferation and inhibit cell death. The MSCs can exert their effect on tumor also indirectly by promoting angiogenesis or affecting some immune functions. Tumor metastasis might be supported at multiple steps (from initial dissemination to formation of novel niches in distal tissues) by the MSCs. They can also regulate cancer stem cell self-renewal and differentiation. The detailed understanding of the interactions in tumor microenvironment could unravel novel intervention points for anticancer therapies. Our recent data will demonstrate data multilevel nature of the interaction between MSC and breast cancer cells with the focus on the biological relevance. The MSCs represent a valuable cell source for regenerative therapies therefore it is of critical importance to examine the safety of MSCs clinical application in the patients with cancer predisposition or history.
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Poškození a reparace DNA DNA damage and repair
Předsedajíci / Chairs: Martin Mistrík, Kateřina Bouchalová středa / 3. prosince 2014 / Wednesday / December 3, 2014 / 8.00 - 9.00 hod. Function of cyclin-dependent kinase 12 in DNA repair pathway středa / 3. prosince 8.00 - 8.15 hod.
2014
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Hana Paculová1, Marta Dzimková1, David Vrábel1, Matija Peterlin2, Dalibor Blažek3, Jiří Kohoutek1 Veterinary Research Institute, Brno, Czech Republic, 2 School of Medicine, University of California San Francisco, San Francisco, USA, 3 Central European Institute of Technology, Masaryk University, Brno, Czech Republic Introduction In many aspects, genome stability is essential for protection from various diseases, including cancer. The DNAdamage-response (DDR) pathway is a cellular mechanism which has evolved to protect cellular integrity by detection and repair of DNA lesions. The DDR is orchestrated by diverse type of proteins and among them, transcriptional cyclin-dependent kinase (Cdk) complexes, and phosphorylation of the C-terminal domain of RNA polymerase II were identified to play a key role. Materials/methods We have demonstrated that the cyclindependent kinase 12 (CDK12), and its binding partner cyclin K, maintains genome stability via regulation of expression of DDR genes. Depletion of the CycK/CDK12 complex from cells led to aberrant expression of several critical regulators of genome stability, specifically, BRCA1, RAD51, ATR, FANCI and FANCD2 proteins. Importantly, down regulation of the CycK/CDK12 complex caused induction of the 53BP1 and γH2AX foci, markers of spontaneous DNA damage signaling followed by accumulation of cells in 1
Copenhagen, Denmark Introduction Many fluorescence microscopy techniques enable powerful combination of single cell analysis and high-content (HC) and highthroughput (HT) screening. With the use of reporter cell lines expressing fluorescently tagged proteins, live cell analysis can be implemented to reduce costs and obtain even more valuable data such as various dynamic studies. Here we focused on laser scanning microscopy (LSM) which usage for HC/HT screening is rather limited due to low speed of image acquisition. On the other hand, LSM mode offers, apart from simple visualization, several approaches for photo-manipulation techniques, which are only poorly exploited in HC/ HT protocols. We optimized dynamic readouts of protein translocation to sites of laser-induced DNA lesions and fluorescence recovery after photo-bleaching (FRAP). This novel approaches could be implemented into automated HC/HT mode for screening of compounds and/or factors interfering with important cellular processes involved in cancer New approach to quantitative biology. image analysis of laser induced Materials/methods DNA damage in live cells Two reporter cell lines with středa / 3. prosince 2014 / fluorescently tagged proteins, 8.15 - 8.30 hod. U2OS-MDC1-GFP and U2OS1 1 53BP1-GFP were used to address Eva Veselá , Martin Mistrík , two different DNA-damage Tomáš Fürst1,2, Hana repair pathways, homologous Hanzlíková3, Ivo Frydrych1, recombination and non-homologous Jirí Bártek1,4 end-joining. Cell visualization 1 and DNA damage induction was Institute of Molecular and performed on AxioObserver Z.1 Translational Medicine, Olomouc, inverted microscope combined with Czech Republic, 2 LSM780 confocal module (Zeiss). Department of Mathematical Quantitative image analysis was Analysis and Applications of done by specialized software which Mathematics, Olomouc, Czech was developed and optimized as a Republic, 3 part of this study. Institute of Molecular Genetics, ASCR, Prag, Czech Republic, 4 Danish Cancer Society, the G2-M phase of the cell cycle. Finally, the loss of the CycK/CDk12 complex rendered cell sensitivity to various DNA damaging agents, including camptothecin, etoposide and mitomycin. Of note, mutations in Cdk12 have been identified in several malignancies, among them ovarian cancer with defective homologous recombination in half of the tumors was characterized with recurrent mutations in kinase domain of CDK12 gene. Importantly, detail analyses of mRNA expression in tumors with mutated CDK12 revealed aberrant expression of several DDR genes and impaired DNA damage repair. Results and conclusions In summary, we believe that understanding the physiological role of CDK12 will help us in the future to dissect precise function of CDK12 in the pathophysiology of the tumor. We propose that CDK12 is a new tumor suppressor. The project is supported by the grant from the Ministry of Agriculture MZE002716202 and the Internal Grant Agency of the Ministry of Health, NT14599-3/2013.
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Rakovina věc veřejná N A D A C E
P R O
V Ý Z K U M
R A K O V I N Y
Počet nově diagnostikovaných nádorů roste v České republice každých deset let o čtvrtinu. I proto byla Proto byla v roce l997 založena nadace pro výzkum rakoviny Olomouc, jejímž cílem je podpora výzkumu a vývoje v oblasti nádorových onemocnění, v oblasti primární a sekundární prevence zhoubných novotvarů a jiných závažných civilizačních nemocí.
Nadace Rakovina věc veřejná založila v roce 2012 stejnojmennou obecně prospěšnou společnost, pomocí níž chce zvýšit povědomí o problematice infekce HPV virem (Human Papillomavirus) mezi ženami. Hodlá je také motivovat k molekulárnímu vyšetření HPV infekce, a to zejména infekce nejrizikovějšími genotypy tohoto viru, takzvanými hrHPV (high-risk HPV). V případě pozitivního výsledku na hrHPV infekci klientky vyhledají svého gynekologa, který na základě cytologických nebo histologických vyšetření doporučí další kroky.
- V rámci projektu „Pošli příběh“ se uskutečnilo divadelní představení s Vandou Hybnerovou ve studiu Alta nebo veřejné čtení veršů Tomáše Tajchnera, které četl Saša Rašilov - Výstava fotografií Pavly Hodkové za podpory Jana Saudka - Dražba darů sportovců, účastníků Olympijských her - Každoroční nadační kalendáře, v letošním roce se fotilo s dětmi, které prodělaly náročnou onkologickou léčbu - Tradiční benefice, spojené s módní přehlídkou v Praze a Olomouci
Děkujeme všem partnerům, známým osobnostem i široké veřejnosti, kteří se v roce 2014 2013 zapojili do benefičních projektů nadace Rakovina věc veřejná.
Přejeme hodně úspěchů v roce 2015 2014. Renata Hilšer Grmolenská ředitelka nadace
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Results and conclusions The presented method is based on atypical microscope settings and specialized image analysis software. It is possible to follow DNA damage recognition and repair in live cells in several time-points after localized UV DNA damage. Compared to commonly used handbased techniques which usually analyze about dozen of cells, this approach enables to evaluate tens to hundreds cells per sample in an automatic mode. The method was tested on two different LSM systems and several experimental settings (various concentrations of pre-sensitizer, compounds directly interfering with observed proteins). It may become a useful tool for characterization of chemicals and factors interfering with DNA damage response, which is a general principle of multiple existing or tested anticancer treatment strategies. The work was supported by Ministry of the interior of the Czech Republic, grant nr. VG20102014001 and GACR, grant no. P305/11/P683.
BRCA-mutation status combined with BCL2 protein in prediction of relapse in triple-negative breast cancer (TNBC) treated with adjuvant anthracycline-based chemotherapy. středa / 3. prosince 8.30 - 8.45 hod.
2014
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K. Bouchalova1,5, M. Svoboda2, G. Kharaishvil3, J. Vrbkova1, L. Radova1, J. Bouchal3, R. Trojanec1, V. Koudelakova1, K. Cwiertka4, M. Hajduch1, Z. Kolar3 Laboratory of Experimental Medicine, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Olomouc, Czech Republic, 2 Masaryk Memorial Cancer Institute, Brno, Czech Republic, 3 Laboratory of Molecular Pathology, Faculty of Medicine and Dentistry, Palacky University, Olomouc, 1
Czech Republic, Department of Oncology, Faculty of Medicine and Dentistry and Faculty Hospital, Olomouc, Czech Republic, 5 Department of Pediatrics, Faculty of Medicine and Dentistry and Faculty Hospital, Olomouc, Czech Republic Introduction TNBC is aggressive and the prognosis is poor. Patients cannot benefit from targeted treatment based on hormonal or HER2 receptors. For this reason, there is an urgent need for markers that will predict the outcome of chemotherapy (Bouchalova et al. Current Drug Targets 2014). The role of BRCA mutation status as a predictor in TNBC is unclear. Data show an association of high BCL2 expression and resistance to anthracyclines. BCL2, size and nodal status are independent predictors for both relaps and death in TNBC treated with adjuvant anthracyclines (Bouchalova et al. ASCO 2012). The objective of this study was to determine whether combination of BCL2 and BRCA1 status predicts outcome in TNBC patients treated with adjuvant anthracycline-based therapy. Materials/methods The study included 187 patients with TNBC, 178 of whom were treated with adjuvant chemotherapy (164 had anthracyclines). BCL2 analysis was performed using IHC. BRCA1 status was obtained from patients records: mutation (mut), wildtype (wt) and unknown status were present in 21.39, 19.25, and 59.36 %, respectively. The data were analysed with software Statistica and R. Results and conclusions Among six BCL2/BRCA1 TNBC subtypes, BCL2high/BRCA1wt predicts the worst, while BCL2low/ BRCA1mut the best RFS (logrank p<0.05). BCL2high protein expression predicts poor relapse free survival (RFS) in BRCA1wt TNBC patients treated 4
with adjuvant anthracycline-based regimens (logrank p=0.007, hazard ratio, HR 13.24, 95%CI 1.19147.93). Interestingly, there was no significant difference in RFS between BCL2low/BRCA1mut and BCL2high/ BRCA1mut, but between BCL2low/ BRCA1mut and BCL2high/BRCA1wt (logrank p=0.009) TNBC patients treated with adjuvant anthracyclinebased therapy. BCL2high/BRCA1wt predicts trend to the worst overall survival (OS) analyzed together with other subtypes treated with adjuvant anthracycline-based regimens (logrank p=0.081). Conclusions: Dividing TNBC into subtypes according BCL2 protein expression and BRCA1 mutation status predicts good, vs. poor outcome in patients treated with adjuvant anthracycline-based chemotherapy. BCL2 expression together with BRCA1 status could facilitate decision making on adjuvant therapy. Underlying mechanisms could be revealed by further research. In patients with BCL2high/BRCA1wt other types of adjuvant therapy should be considered. The study was presented at ASCO Annual Meeting 2014 [Bouchalova et al., J Clin Oncol 32, 2014 (suppl; abstr 1132)]. Grants: IGA NT14599-32013 and IGA NS10286-3. Infrastructural part of this project (Institute of Molecular and Translational Medicine) was supported from the Operational Programme Research and Development for Innovations (project CZ.1.05/2.1.00/01.0030), National Sustainability Programme (LO1304), and Czech Technology Agency (Center of competence for molecular diagnostics and personalized medicine,TE02000058).
2‘-deoxy-5-ethynyluridine (EdU): a thymidylate synthase inhibitor with DNA damaging activity středa / 3. prosince 8.45 - 9.00 hod.
2014
Anna Ligasová1, Dmytro Strunin2, David Friedecký1,
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Tomáš Adam1, Karel Koberna1 Institute of Molecular and Translational Medicine, Faculty of Medicine, Palacký University, Olomouc, Czech Republic, 2 Institute of Organic Chemistry and Biochemistry ASCR, Prague, Czech Republic Introduction The use of 2‘-deoxy-5-ethynyluridine (EdU) as an anti-viral substance was already studied in the nineteen seventies [1,2]. Although this analogue of 2‘-deoxyuridine evinced an anti-HSV-1 and HSV-2 effect and also an impact against the vaccinia virus, the effective concentration also inhibited the growth and metabolism of non-infectious cells [1]. Similar results were also obtained in 2007 in the case of cytomegalovirus [3]. EdU was also successfully tested as a possible inhibitor of the cell growth of human breast cancer cells [4]. The mechanism of the inhibition, however, remained unknown, although some of the data indicated that EdU can act as an inhibitor of thymidylate synthase [5]. The interest in EdU was greatly revived in 2008 when this nucleoside analogue was used as a marker of cellular replicational activity [6]. Due to its simple and fast visualization, EdU immediately became very strong competitor of the most frequently used marker to date nucleoside 5-bromo-2‘-deoxyuridine (BrdU). In contrast to BrdU detection based on the use of specific antibodies, the reaction between the azido group of the tag molecule and the ethynyl group of EdU is employed in EdU detection [6]. Due to the renewed interest in EdU and the high number of cell lines used in various studies, new findings about the impact of EdU on cell metabolism were obtained indicating that EdU incorporation can lead to DNA breaks followed by cell death, suppression of in vitro population expansion and in vivo tumour progression [7]. On the bases of immunolocalisation studies of the proteins γH2AX and p53BP1 it was suggested that EdU induces 1
double-stranded DNA breaks as well [8]. Although it is evident that EdU toxicity is highly dependent on the cell line used [3,4,8,9], the reason for the different effect of EdU in various cell lines remained unknown. In the study presented, we have focused on the possibility that the different cytotoxic effect of EdU could be related to the different rate of EdU incorporation in DNA. We also studied (i) the changes in the rate of DNA replication and cell cycle progression, (ii) the possibility that EdU can generate interstrand crosslinks and (iii) the role of the metabolism of 2‘-deoxythymidine (dT) in EdU-mediated toxicity. Materials/methods Cells lines used HeLa cells, 143B PML BK TK and HCT116 cells. MTT assay The MTT assay was performed according to [10]. Microscopic analysis The incorporated EdU was visualised by means of a click reaction, the nuclear DNA was stained by DAPI. Nucleotide pools analysis by HPLCMS The nucleotide pools were performed according to [11-13]. Doubling time and the relative length of S phase The doubling were calculated using wide-field microscopy. The relative length of the S phase was determined on the basis of the determination of the fraction of EdU-labelled cells after 1-hour labelling with EdU as compared to the overall number of the cells. Analysis of the cell cycle and BrdU detection The analysis of the cell cycle was performed using DAPI labelling followed by image cytometry. In the case of the analysis of the DNA replication, the cells were labelled with BrdU followed by its revelation using copper(I) ions [14] and indirect immunofluorescence. Reverse comet assay
The comet assays were performed according to [15] with the modification of Dr. Dušínska (http://www.cometassayindia.org/ Dusinska-protocol.PDF). Data analysis The data of particular experiments were analysed using following software: CellProfiller, Microsoft Excel, GraphPad Prism 6 and CometScore software. Results and conclusions a) EdU toxicity positively correlates with the efficiency of its incorporation and this efficiency is different in different cell lines. b) The incorporation of EdU is dependent on the intracellular concentrations of dT and 2‘-deoxythymidine 5‘-monophosphate (dTMP). c) EdU incorporation in DNA leads to the deceleration and deformation of the cell cycle including the slowdown of the S phase accompanied by a decrease in the DNA synthetic activity. d) Although the in vivo inhibitory effect of EdU on the activity of thymidylate synthase is substantially lower when compared to 5-fluoro2‘-deoxyuridine (FdU), this effect contributes to the high toxicity of EdU especially at higher EdU concentrations. It results in a lowering of the dTMP, dTDP and dTTP pools and subsequently in the higher incorporation of EdU in DNA. e) EdU induces interstrand crosslinks. f) The use of non-toxic concentrations of EdU (less than 1% cells die using a standard cytotoxicity test) for labelling replicated DNA results in a substantial decrease of the signal when compared to the maximal signal or does not allow any labelling at all. The non-toxic concentration is lower than 0.501 µM, 0.044 µM and 0.47 µM in HeLa, 143B PML BK and HCT116 cells, respectively. References 1. De Clercq E, et al (1979) PNAS 76: 2947-2951. 2. De Clercq E, et al. (1980) J Infect
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Dis 141: 563-574. 3. Cristofoli WA, et al. (2007) J Med Chem 50: 2851-2857. 4. Meneni S, et al. (2007) Bioorg Med Chem 15: 3082-3088. 5. De Clercq E, et al. (1978) Mol Pharmacol 14: 422-430. 6. Salic A, Mitchison TJ (2008) PNAS 105: 2415-2420. 7. Ross HH, et al. (2011) J Neurooncol 105: 485-498.
8. Zhao H, et al. (2013) Cytometry A 83: 979-988. 9. Diermeier-Daucher S, et al. (2009) Cytometry A 75: 535-546. 10. Freshney RI (2005) In: Freshney RI, editor. 5th. ed. New Jersey: John Wiley and Sons, Inc. pp. 365-369. 11. Bennett BD, et al. (2008) Nat Protoc 3: 1299-1311. 12. Ivanisevic J, et al. (2013) Anal Chem 85: 6876-6884. 13. Yuan M, et al. (2012) Nat Protoc
7: 872-881. 14. Ligasovรก A, et al. (2012) PLoS One 7: e52584. 15. Singh NP, et al. (1988) Exp Cell Res 175: 184-191. 16. This work was supported by the Technology Agency of the Czech Republic [TA03010598, TA03010719 and TE02000058] and the Ministry of Education, Youth and Sports [LO1304].
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Biomarkery nádorových onemocnění II Cancer biomarkers II
Předsedajíci / Chairs: Jitka Berkovcová, Pavel Dundr středa / 3. prosince 2014 / Wednesday / December 3, 2014 / 9.30 - 11.30 hod. Možnosti predikce léčebné odpovědi na cílenou antiEGFR a anti-VEGF terapii metastatického kolorektálního karcinomu (mCRC) středa / 3. prosince 9.30 - 9.45 hod.
2014
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Radim Nemecek1,2 Masarykuv onkologicky ustav, Brno, Czech Republic, 2 Lekarska fakulta MU, Brno, Czech Republic Introduction Systémová léčba mCRC je založena na použití chemoterapie v kombinaci s monoklonálními protilátkami proti VEGF a EGFR. Na tuto léčbu však pozitivně odpoví jen část pacientů, je proto vyvíjena intenzivní snaha o nalezení faktorů predikujících odpověď či rezistenci ještě před zahájením onkologické terapie. Zatímco v případě dráhy pro VEGF predikce dosud selhává, v případě receptoru EGFR je prediktorů známo hned několik a některé z nich se již používají v běžné klinické praxi. Materials/methods Mezi „pozitivní prediktory“ odpovědi na anti-EGFR terapii se řadí zvýšený počet kopií genu pro EGFR a hladina exprese mRNA dvou hlavních ligandů EGFR - epiregulinu a amphiregulinu. Z tzv. „klinických“ prediktorů je diskutována role akneiformního exantému, hypomagnesémie a tzv. „časné regrese nádoru“, resp. hloubky nádorové odpovědi. V klinické praxi se tyto prediktory dosud nepoužívají. „Negativních prediktorů“ je známo více. Vychází z jednotlivých článků signálních kaskád vedoucích od EGFR k jádru buňky (RAS-RAFMAP2K-ERK a PI3K-AKT-mTOR). Mutace v onkogenu RAS (mRAS) je nejrozšířenější a jako jediná se používá v rutinní klinické praxi. Nachází se u cca 50% pac. s CRC 1
a je zodpovědná za trvalou aktivaci kinázy RAS vysílající stimulační signály k jádru bez ohledu na stav EGFR. Nicméně i po vyloučení této mutace (a tedy potvrzení nemutovaného genu RAS - wt RAS) odpoví na anti-EGFR terapii jen cca polovina pacientů. Hledají se tedy další prediktory s cílem léčbu ještě více „personalizovat“. Mutace v onkogenu BRAF (V600E) se nachází u cca 5-15% pac. s CRC. Nevyskytuje se nikdy současně s mRAS. Kromě evidentně negativně prognostického vlivu byl prokázán i její vliv negativně prediktivní, obzvláště u předléčených pacientů. Mutace onkogenu PIK3CA se vyskytuje u 15-18% pacientů s mCRC. Byly identifikovány 2 varianty - mutace v exonu 9 (60-65% pac.) a v exonu 20 (20-25% pac.). Negativně prediktivní vliv byl prokázán pouze u druhé z nich. Dráha PI3K-AKT-mTOR je inhibována kinázou PTEN, jejíž mutace vedou k nedostatečnému efektu anti-EGFR terapie u mCRC. Exprese PTEN souvisí s mírou mikrosatelitové nestability a je rozdílná v primárním nádoru a metastázách. Existují rovněž data o prediktivním vlivu statutu HER2 a mutace p53. Results and conclusions Personalizovaná onkologická léčba založená na genovém profilování nádorů je považována za léčebnou strategii budoucnosti. Se současnou úrovní znalostí však stojíme na samém začátku této nesmírně zajímavé kapitoly.
Jana Volejníková1,2, Jana Vrbková2, Marián Hajdúch2, Vladimír Mihál1,2
Department of Pediatrics, Palacky University and University Hospital, Olomouc, Czech Republic, 2 Institute of Molecular and Translational Medicine, Olomouc, Czech Republic Introduction Great progress has been made in the diagnostics and treatment of childhood acute lymphoblastic leukemia (ALL) over the past decades. The vast majority of children are cured, but there is still room for improvement, especially in specific patient subgroups. In this study, we retrospectively evaluated treatment and prognosis of children with ALL enrolled in three consecutive treatment protocols ALL-BFM 90, 95 and ALL IC-BFM 2002 between years 1990 and 2007. Materials/methods We compared characteristics and survival data of 97 patients treated for ALL at University Hospital Olomouc with records of 924 children from 7 remaining centers in the Czech Republic. Results and conclusions Within the entire Czech Republic, successive improvement in both event-free (EFS) and overall survival (OS) among protocols were observed (RFS: 81.1±2.2%, 78.6±2.2% and 88.2±1.9%, p=0.01; EFS: 70.5±2.4%, 70.4±2.7% and 84.5±2.1%, p=8.4e-05; OS: 76.3±2.3%, Management and prognosis of 79.0±2.5% and 91.3±1.7%, p=6echildhood acute lymphoblastic 06, respectively). There was also a trend to better survival in a single leukemia on protocols ALLcenter on ALL-BFM 95 trial than in BFM 90, 95 and ALL IC-BFM the rest of the Czech Republic (EFS: 2002: a retrospective single83.3±6.2% vs. 70.4±2.7%, p=0.11; center study OS: 91.7±4.6% vs. 79.0±2.5%, středa / 3. prosince 2014 / p=0.07). This fact might be explained 9.45 - 10.00 hod. by a significantly lower proportion of 1
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boys and an inclusion of only one high risk patient in our center. However, multiple minor changes among protocols (regarding diagnostics, patient enrollment and risk group stratification, cranial irradiation and course of chemotherapy) might have influenced the mentioned results and will be discussed in detail. Cytogenetic examination in our center showed an exceptionally high yield rate, but its result was not decisive for the risk stratification. In conclusion, we demonstrate a gradual progress and a high quality of ALL diagnostics and treatment in the Czech Republic in general, which is possible to maintain in a single center setting. The given outcomes surpassed international results of the ALL IC-BFM 2002 trial (5-year EFS 74%, OS 82%) and were comparable with worldś leading childhood leukemia trials - e.g. a 7-year EFS 80.4% and OS 91.8% for AIEOP-BFM ALL 2000; 5-year OS 90.4% for the Childrenś Oncology Group. Due to advances in molecular diagnostics, excellent collaboration on both national and international level and high overall standards of care, Czech Republic has survival rates comparable with leading international study groups for childhood ALL. The future of ALL treatment lies in its individualization, including pharmacogenetics. One of the most important predictors of outcome and risc stratification criteria is the level of minimal residual disease (MRD), which quantification is highly specialized, laborious and expensive. Due to our collaborative effort, the Czech Republic has since 2010 joined the MRD-based international „state of the art“ AIEOP-BFM ALL 2009 protocol for childhood ALL. Supported by: NPU LO1304
Využití metody sekvenování nové generace v intratumorové heterogenitě kolorektálního karcinomu středa / 3. prosince 10.00 - 10.15 hod.
2014
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Jitka Berkovcová1, Lenka Radová2, Šárka Rathouzská1, Pavel Fabian1 Masarykuv onkologický ústav, odd. onkologické patologie, Brno, Czech Republic, 2 CEITEC, Výzkumná skupina Lékarská genetika, Brno, Czech Republic Introduction Nádorová heterogenita je dobře známý jev (Kinjn et al. 2011), stejně jako teorie multiklonáního modelu progrese nádorů v čase (Marusyk et al. 2010). Rozdíl ve stanovení mutačního stavu genu KRAS bývá popisován asi ve 4 % případů kolorektálních karcinomů. Metody sekvenování nové generace (NGS) díky svojí vysoké senzitivitě umožňují zachytit minoritní molekuly DNA v analyzovaném vzorku. Je to tedy vhodný nástroj ke studiu heterogenity nádorové populace. Cíle: Přesná mutační analýza genů s klinickým významem u pacientů s kolorektálním karcinomem. Porovnání vzdáleně oddálených odběrů rozsáhlých tumorů, případně morfologicky odlišných klonů u jednoho pacienta. Materials/methods Do souboru bylo zařazeno 22 pacientů s operovaným kolorektálním karcinomem v roce 2014. Jako hlavní kriterium byla zvolena velikost nádoru minimálně 40mm. Celkem byla izolována DNA z 51 vzorků s vysokým zastoupením nádorové populace (40-70 %). Příprava DNA knihovny byla provedena pomocí TruSight Tumor kitu (illumina). Tento kit je designován tak, že obsahuje exony 26 vybraných genů s „Hot Spots“ oblastmi (celkem 175 amplikonů). Základní analýza sekvencí byla provedena pomocí softwarů illumina dostupných v BaseSpace, detekce variant byla provedena pomocí softwaru VariantStudio. K následným datovým úpravám byly použity balíky R/Bioconductor. Results and conclusions Naše izlovaná DNA z tkáňových 1
bloků dosahuje velice dobré kvality a ze všech vzorků bylo možné připravit kvalitní DNA knihovny. Q Skore 30 dosahujeme u více jak 96 % sekvencí. Průměrná coverage dosahuje 4000 čtení. Sekvenace a analýza dat zatím proběhla u 12 pacientů. U 7 pacientů jsme detekovali mutaci v genu KRAS, u 1 mutaci v genu BRAF, u 7 pacientů mutaci v genu TP53, u 2 pacientů mutaci v genu APC, u 1 pacienta mutaci v genu PIK3CA a u 1 pacienta mutaci v genu FBXW7. U 1 pacienta byly detekovány rozdílné genetické klony v odlišných odběrech tumoru. Vedle toho zaznamenáváme řadu změn na úrovni subpopulací. Očekává se, že NGS napomůže zodpovědět řadu otázek souvisejících s heterogenitou nádorových buněk. Zároveň předpokládáme, že NGS povede k upřesnění predikce cílené protinádorové terapie. Podpořeno MZ ČR - RVO (MOÚ, 00209805)
Introduction to RAS pyrosequencing of FFPE samples středa / 3. prosince 10.15 - 10.30 hod.
2014
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Jana Stranska, Gabriela Gabcova, Rastislav Slavkovsky, Veronika Holinkova, Miroslava Rabcanova, Jiri Drabek Institute of Molecular and Translational Medicine, Palacky University, Olomouc, Czech Republic Introduction KRAS and NRAS (RAS) are components of the EGFR signaling network. EGFR regulates cancer-cell proliferation, apoptosis and tumorinduced angiogenesis. RAS links growth-promoting signals from the cell surface to the nucleus by acting as a molecular switch. The switch normally occurs transiently when growth factor receptors (such as EGFR) are activated. When specific mutations in codons 12, 13, 59, 61, 117 and 146 of RAS genes occur,
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the resulting KRAS/NRAS protein is constitutively active. Anti-EGFR monoclonal antibodies (cetuximab and panitumumab) treat only RAS wildtype patients with metastatic colorectal cancer (mCRC). The high number of tested mutations makes probe-based qPCR genotyping methods problematic and while sequencing techniques can identify all mutations. In this project, we test feasibility of pyrosequencing for RAS genotyping in mCRC. Materials/methods Primers for detection of mutations of KRAS and NRAS exons 2, 3 and 4 were designed according to the literature and PyroMark Assay Design Software 2.0. Three different PCR mixes (separate reagents (Thermo Scientific), HotStarTaq Plus Master Mix Kit (Qiagen), PyroMark PCR Kit (Qiagen)) were compared. Concentration of MgCl2 (1.5 - 3 mM) and PCR profile for all mutation mixes analyzed in one run were optimized. Volumes of 10 and 15 μl with or without purification of amplicons (QIAquick PCR Purification Kit, Qiagen) were tested. Pyrosequencing was performed with PyroMark® Q96 ID (Qiagen) according to the machine manual. Results and conclusions All primer sets provided amplicons of corresponding length. The best amplification and pyrosequencing results were obtained with HotStarTaq Plus Master Mix Kit, 3 mM MgCl2, 15 μl, without purification step. All read sequences corresponded to their design. Further experiments for sensitivity evaluations will continue. The work was supported by grants CZ.1.05/3.1.00/14.0307, CZ.1.07/2.3.00/30.0060 and CZ.1.07/2.3.00/30.0041.
MiR-215 as an important tumor suppressor in colorectal cancer patients středa / 3. prosince 10.30 - 10.45 hod.
Petra VychytilovaFaltejskova1,2, Jitka
2014
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Mlcochova2, Jana Merhautova3, Lenka Radova2, Marek Svoboda1,2, Rostislav Vyzula1, Ondrej Slaby1,2 Masaryk Memorial Cancer Institute, Brno, Czech Republic, 2 CEITEC, Masaryk University, Brno, Czech Republic, 3Department of Pharmacology, Faculty of Medicine, Masaryk University, Brno, Czech Republic Introduction Colorectal cancer (CRC) is one of the most common types of cancers worldwide with the high incidence and mortality. Therefore, several efforts have been made to find new diagnostic, prognostic and predictive biomarkers, but also therapeutic targets. One of the promising approaches is the characterization of the solid tumors using microRNAs (miRNAs). MiRNAs are small, non-coding RNAs that post-transcriptionally regulate gene expression. They regulate many biological processes such as cell cycle, apoptosis, proliferation or invazivity and they can also affect anti-cancer therapy efficiency, therefore, their deregulation leads to the origin and progression of many severe diseases including CRC. However, only little is known about miRNAs‘ target molecules and signaling pathways in which they are involved. Materials/methods We have analyzed expression profiles of 667 miRNAs in 8 patients diagnosed for CRC and 8 paired adjacent non-tumoral tissues using TaqMan Low Density miRNA arrays. We have found miR-215 to be one of the most deregulated miRNAs, therefore its expression was further validated on independent cohort of 250 paired samples and correlated with clinicopathological features of the patients. Afterwards, we investigated its involvement in CRC pathogenesis. We have overexpressed miR-215 in CRC stable cell lines (DLD-1, HCT 116, HT-29, SW620) and analyzed the effect on the viability (MTT test), proliferation (cell counting), cell cycle 1
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and apoptosis (flow cytometry), migration of the cells (scratch assay, transwell migration assay) and the ability to found new colonies (colony forming assay). Subsequently, several targets of miR-215 have been identified using quantitative real time PCR, and effects of this miRNA have been also analyzed in vivo on mouse model. Results and conclusions We have observed significantly decreased levels of miR-215 in primary CRC tissues and also in metastatic tissue. Moreover, we have found correlation between low expression of this miRNA, clinical stage, lymph node metastasis and disease free survival of the patients. In vitro analyses proved that higher levels of miR-215 lead to the arrest of cell cycle, increased apoptosis and reduced migration and proliferation of the cells. Using qRT-PCR we have identified several potential targets of miR-215 including XIAP, CD164 or HOXB9. The results of in vivo analyses will be the part of the message. The reduced expression of miR-215 has been observed in several tumor diseases, therefore we presume that this miRNA functions as an important tumor suppressor. The results of our study also indicated that miR-215 could serve as a new diagnostic and prognostic biomarker and also potential therapeutic target for the treatment of CRC patients. This work has been supported by IGA MZČR NT13549-4/2012 and NT13860-4/2012.
Overexpression of Filamin-A protein is associated with aggressive clinicopathological features and poor survival outcome in NSCLC patients treated with platinum-based combination chemotherapy středa / 3. prosince 10.45 - 11.00 hod.
2014
Mariam Gachechiladze1,2, Josef Škarda1,2, Maria Janikova1,2, Gvantsa Kharaishvili1,2, Vítězslav
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Kolek3, Jiří Klein4, Alžběta Poprachová5 Department of Clinical and Molecular Pathology, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic, 2 Institute of Molecular and Tranlational Medicine, Olomouc, Czech Republic, 3 Department of Tuberculosis and Respiratory Diseases, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic, 4 Laboratory of Experimental Medicine, Departments of Pediatrics and Oncology, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic, 5 Department of Normal Anatomy, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic Introduction An actin-binding protein Filamin A connects the actin filament network to cell membrane receptors, and acts as a scaffold for various signaling pathways related to cancer growth and progression. Recently, it has been reported that Filamin A is required for efficient regulation of early stages of DNA repair process. Moreover, some in vitro studies showed that the overexpression of Filamin A determines resistance to various cytotoxic drugs, including cisplatin. We aimed to analyse the expression of Filamin A protein in resected NSCLC specimens, to investigate the association of the level of Filamin A protein expression and other clinicopathological features, and possible relationship between the expression of Filamin A and survival outcome in NSCLC patients, treated with platinumbased combination chemotherapy. Materials/methods We performed FLNA protein immunohistochemistry on formalinfixed and parrafin-embedded tissue sections from 134 NSCLC patients, using EP2405Y antibody against C-terminus of FLNA (LSBio). 1
Cytoplasmic, membranous and nuclear staining were evaluated semiquantitatively, scored according to Histoscore method, and correlated with all available clinicopathological factors. Patients were divided in two groups for survival analysis (I group - patients with adjuvant platinum based chemotherapy, II group patients with surgical treatment only). Results and conclusions We found significant positive correlation between Filamin-A protein expression and NSCLC stage (r=.249; p<0,05), lymph node metastases (N) (r=0.205; p<0,05) and distant metastases (M) (r=0.332; p<0.01). The increased expression of Filamin A protein was also positively associated with primary tumor size (T), grade (G), and with lymphovascular invasion (LVI). Multivariate Cox proportional hazards regression analysis showed that Filamin A overexpression >90, represents a risk factor for disease relapse together with tumor stage, size and metastases (HR=1.723, 95%CI [1.021:2.909], p<0.05). Increased Filamin A protein expression has been significantly assosiated with poor survival outcomes in patients with adjuvant platinum-based chemotherapy: OS (HR=1.005, 95%CI[1.000;1.010], p=0.037), DFS (HR=1.004, 95%CI [1.001:1.008], p=0,017). Our study results suggest the important role of Filamin A protein in NSCLC porgression and it might represent not only a prognostic marker, but also one of the additional predictive markers for platinum-based treatment outcome in patients with NSCLC.
GENETIC CHANGES IN RECURRENT GLIOBLASTOMA-IS THERE ANY SIGNIFICANCE? středa / 3. prosince 11.00 - 11.15 hod.
2014
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Zuzana Crlikova1, Magdalena Megova1, Jana Vrbkova1, Radek Trojanec1, Lucie Tuckova2, Jana Potockova1,
Sona Mlcochova1, Marian Hajduch1 Laboratory of Experimental Medicine, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic, 2 Laboratory of Molecular Pathology, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic Introduction Glioblastoma multiforme belongs to the most aggressive tumors with very short-time survival. Treatment of this disease is very limited and only temozolomide and radiation is widely used in clinical practice. New biomarkers could allow deeper insight in gliomagenesis and also can help to facilitate possible targets in prognosis and prediction which may lead to development of new therapy regimes. Materials/methods In our study, status of genes and chromosomes (MDM2/chr.12, EGFR1/chr.7, BCR/chr.22, P53/ chr.17, RB1/chr.13, C-MET/chr.7, PTEN/10p, 19q/19p, 1p/1q, 9p/ chr.9) was investigated, using FISH assay. We examined 25 patients with recurrent glioblastoma. Control group comprising of 111 patients with glioblastoma was used for comparison (both groups with treatment regime 54Gy and 40 days of temozolomide). Results and conclusions Statistical analysis revealed significant difference in age (P=0.0002) and loss of p53 (P=0.093) between control group and recurrent glioblastoma group. Other significant relevances of this study are summarized and clinical relevance of our findings will be associated with intended additional study. Support: NT 13581; TE02000058; LF_2014_019; CZ.1.05/2.1.00/01.0030. 1
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Assay destinguishing integrated and episomal form of HPV16, 18, 31 and 58 stĹ&#x2122;eda / 3. prosince 2014 / 11.15 - 10.30 hod.
Hana Ondryasova, Vladimira Koudelakova, Rastislav Slavkovsky, Veronika Venskova, Marian Hajduch Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic Introduction Cervical cancer is the third most common malignancy in women. Early detection screening is in Czech Republic available and covered by insurance since 2008. This screening detect precancerous states caused by aggressive subtypes of human papillomavirus (mainly HPV16 and 18). The problem of the screening, which standardly uses cytology, is a high false negative rate, which is reported in up to 20% of cases. Only ambiguous cases with abnormal cytology (ASC-US, LSIL) are sent to test at level of DNA (HPV DNA). HPV
DNA diagnostics are much more sensitive than standard cytology which was demonstrated by large studies. HPV DNA diagnostics should be therefore a part of primary screening However, commercially available HPV DNA diagnostics can also mis HPV positive cases (paradoxically the serious ones). Low viral load below the detection limit of the method is the first reason. The fragmentation of the gene (which detection kit is based on) occurs during the integration of the virus into the human genome, may be another reason. The paradox is that just tumors with lower viral load and related degradation of some viral genome fragments are more aggressive with worse prognosis and response to treatment. For proper diagnosis of cervical cancer is therefore necessary to capture also tumors in whom conventional diagnostic kits fail. From this reason we developed method for semiquantification and differentiation of free-episomal from integrated HPV form in cervical cells. Materials/methods Cervical smears have been tested by Cobas 4800 system (Roche) for
presence of HPV16/18 and other hrHPV genotypes (12 hrHPV). All samples have been also analyzed by PapilloCheck HPV- Screening system (Greiner Bio-one) detecting 18 hrHPV and 6 lrHPV genotypes. Samples positive for HPV16, 18, 31 and 56 have been analyzed for presence of episomal, integrated or mixed form of HPV infection by multiplex real-time PCR assay. GAPDH has been used as internal control. Results and conclusions Our assay is able to distinguish integrated and episomal form of HPV16, 18, 31 and 58. Detection limit of this assay is 4 copies of HPV per reaction. We collect clinicopathological data from HPV positive patients in these days. We have analysed 116 HPV positive samples. It is necessary to analyze large set of HPV positive samples with known cytology/histology results to prove that this assay could help to recognize patients in real risk of cervical cancer. (Supported by: CZ.1.05/3.1.00/14.0307 , CZ.1.05/2.1.00/01.0030.)
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Molekulární cíle a protinádorová léčiva II Molecular targets and anticancer drugs II
Předsedajíci / Chairs: Petr Džubák, Jan Hraběta středa / 3. prosince 2014 / Wednesday / December 3, 2014 / 12.40 - 14.10 hod. Use of the FUCCI probe in HTS/HCA screening campaigns. středa / 3. prosince 12.40 - 12.55 hod.
2014
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Petr Dzubak, Pawel Znojek, Sona Gurska, Marian Hajduch Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University in Olomouc, Olomouc, Czech Republic Introduction High content screening (HCS) is a method that uses automatic microscopy and image analysis techniques to extract multiple physiologically relevant measurements at cellular level. Our goal was to use HCS to develop robust, homogeneous and inexpensive assay for cell cycle phase detection as alternative to FACS. Materials/methods In our endeavors we used HeLa cells stably expressing FUCCI (Fluorescent Ubiquitination-based Cell-Cycle Indicator) probe. The FUCCI probe was generated by fusing red and green fluorescent proteins with ubiquitination domains of Cdt1 and Geminin respectively. As a means of tracking cell cycle progression FUCCI exploits cyclical expression and degradation of the ubiquitination oscillators Cdt1 and Geminin to specifically mark cell cycle phases in living cells. Cell cycle perturbation can be evaluated by measuring mean fluorescence intensity of Cdt1-Red and GemininGreen in single cell and eventually based on fluorescence intensity range cells can be classified as one of G1, G1/S, S/G2/M and M cell cycle phase. The assay was designed for full automatization of
cell plating, addition of compounds, cytotoxicity measurement and high content microscopy on our triple robotic system. For setting up and validation of the assay, dose response curve was generated for 8 reference compounds with known activity on cell cycle to choose desired concentrations for which there is evident block of cell cycle progression in specific phase. Results and conclusions Chosen compounds were used to assess assay reproducibility over plates and experimental days by evaluation of Z‘ calculated for each phase of cell cycle. Overall we obtained good assay reproducibility and robustness with an overall Z‘ value of above 0.5 for G1, S/G2/M and M phases. Finally, we tested our approach against commercially available LOPAC library which includes 90 drug-like molecules used as model drugs in the fields of cell signaling and neuroscience. The results of cytotoxicity assay allowed to validate 26 compounds as active based on dose response curve fitting. High content analysis of FUCCI probe showed that several agents among these can be identified as possible cell cycle modulators. Results demonstrated here shows that this newly developed assay is simple to set up, robust, sensitive and inexpensive. Project was supported by the Ministry of Education of the Czech Republic (LO1304 and CZ.1.07/2.3.00/30.0041).
Validation of Cell Based Assays for High-Throughput Screening
Univesity, Olomouc, Czech Republic Introduction High-Throughput Screening (HTS) is an important step in the discovery of new drugs. It allows the assaying of a large number of potential biological modulators against a chosen set of defined targets. The chemical compounds are tested in the primary screen firstly. Only compounds with positive results (HITs) are investigated in the secondary screen and the IC50 values are calculated. This testing is performed by biological and biochemical tests. Materials/methods For validation of our HTS, the LOPAC Library (Library of Pharmacologically Active Compounds) was used. This library contains 90 compounds with known biological activity. Their cytotoxic effects were testing on the group of 10 cell lines by MTT and XTT methods. The IC50 value for each compound was calculated. Results and conclusions To quantify the suitability of the used cytotoxic assays in a HTS the Z-factor was determined for each plate. Obtained results and some troubles and errors will be presented and discussed.
Molecular mechanisms of cell death induction by novel taxanes effective in resistant breast cancer cells středa / 3. prosince 13.10 - 13.25 hod.
2014
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Michael Jelínek, Kamila Balušíková, Adéla Kábelová, středa / 3. prosince 2014 / Jan Kovár 12.55 - 13.10 hod.
Sona Gurska, Pawel Znojek, Petr Džubák, Marián Hajdúch Institute of Molecular and Translational Medicine, Palacky
Department of Biochemistry, Cell and Molecular Biology, Division of Cell and Molecular Biology, Third Faculty of Medicine, Charles University, Prague, Czech Republic
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Introduction The classical taxanes paclitaxel (Taxol®) and docetaxel (Taxotere®) are routinely used in therapy of solid tumors. Unfortunately, cancer cells often become resistant to taxanes. Therefore, new taxanes were prepared and tested for using in the therapy of resistant cancer cells. As for mechanisms of cell death induction, taxanes are known to block depolymerization of tubulin and to induce programmed cell death. However, the exact mechanisms of cell death induction are still not understood. To help to elucidate this issue, we investigated breast cancer cells after application of classical and novel taxanes. Materials/methods We studied taxane-induced cell death in two breast cancer cell lines SK-BR-3 and MCF-7. We used paclitaxel and novel taxanes SB-T-1216 and SB-T-12854 (being more efficient against resistant cancer cells) at a death inducing concentration. We also employed western blot analysis, specific siRNAs and cell fractionation followed by western blot anylysis. For testing of mitochondria functions we made imunoflorescence and also FACS analysis. Results and conclusions Programmed cell death was observed in both tested cell lines after application of classical as well as novel taxanes. Next we studied the role of proteins of Bcl-2 family. We confirmed the importance of proteins Bad and Bim in cell death induction described previously and in addition we found that proteins Bik and Bok also seem to function in cell death induction. The translocation of cytochrome C and decrease of mitochondrial membrane potential were observed only in SK-BR-3 cells and not in MCF-7 cells, so the role of mitochondria is questionable. On the other hand, caspase -2, -8, -9, -7 and -3 (caspase-3 only in SK-BR-3 cells) were activated in SK-BR-3 as well as MCF-7 cells 36 h after application of taxanes. As previously reported, caspase-2 seems to be the most
apical caspase in both cell lines. Using of specific siRNA showed that also caspase-3 and caspase-7 had also important function in apoptotic cascade induced by novel taxanes. We found that classical and novel taxanes induced cell death very similarly in breast cancer cells. Proteins from Bcl-2 family, (Bad, Bim, Bik and Bok) and caspase-2, -3 and -7 probably play the most important role in cell death induction. In summary, new taxanes could be used in future anticancer therapy of resistant breast cancer cells
Novel triterpenoid fluoroderivatives with anticancer activity středa / 3. prosince 13.25 - 13.40 hod.
2014
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Jiri Rehulka1, Milan Urban1, Lucie Borkova2, Ivo Frydrych1, Jan Sarek2, Petr Dzubak1, Marian Hajduch1 Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital, Olomouc, Czech Republic, 2 Department of Organic Chemistry, Institute of Molecular and Translational Medicine, Faculty of Science, Palacky University, Olomouc, Czech Republic Introduction Pentacyclic triterpenes are secondary metabolites usually found in plants that have a variety of biological activities. Some of their semisynthetic derivatives are extensively studied for anti-cancer, anti-HIV, and various protective effects. The aim of this study was to evaluate the newly synthesised betulinic acid fluoroderivatives, which were designed to enhance cytotoxic effect towards cancer cell lines. Materials/methods MTT assay, Flow cytometric analyses Results and conclusions We prepared a set of fluorinated derivatives of betulinic acid and 1
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allobetulin in order to increase the cytotoxic activity of the parent compounds. Previously unknown 2,2-difluorodihydrobetulinic acid and its esters had significant cytotoxicity on several cancer cell lines with somewhat insufficient selectivity in comparisson with normal human fibroblasts. Derivatives of allobetulin were inactive. In the context of other triterpenoid derivatives prepared earlier, we may conclude that the introduction of an electron withdrawing substituent to the position 2 of betulinic acid enhanced its potency. In the case of fluorinated compounds, however, more studies need to be done in order to lower the toxicity on non-cancer cells. The biological part of this work (MTT tests, cell cycle analysis etc.) was supported by grant from the Ministry of Education, Youth and Sports of the Czech Republic (No. CZ.1.07/2.3.00/30.0041) and internal grant of Palacky University IGA_LF_2014_010. The infrastructural part (Institute of Molecular and Translational Medicine) was supported from the Operational Program Research and Development for Innovations (project CZ.1.05/2.1.00/01.0030) and NPUII project LO1304. The chemical part was supported by Technology Agency of the Czech Republic (TE01020028) and by the Ministry of Education, Youth and Sport of the Czech Republic and by the European Social Fund (CZ.1.07/2.3.00/30.0060).
Proteomic profiling of oxaliplatin treated cell line revealed nucleoli and ribosomal protein downregulation středa / 3. prosince 13.40 - 13.55 hod.
2014
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Tomáš Oždian, Dušan Holub, Gabriela Rylová, Petr Džubák, Marián Hajdúch Laboratory of Experimental Medicine, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký
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University in Olomouc,, Olomouc, Czech Republic Introduction Oxaliplatin is a third generation of platinum drug, which shows no cross resistance with cisplatin. Nowadays the oxaliplatin is used in the monotherapy of colorectal canceror in combination with 5-fluorouracil and leucovorin where all drugs shows synergic effect. Although oxaliplatin is a powerful drug, patients suffers from several toxicities, with most serious neuropathy. Its known mechanism of action is binding of reactive platinum species to DNA and forming intraand inter-strand crosslinks with guanines and adenines leading to the DNA damage. The aim of our study was to analyse response to oxaliplatin at complex protein level. Materials/methods This has been achieved by influencing CCRF-CEM cell line (1 mil. cells / ml) by 5xIC50 (29.3 μM) for time corresponding to half-time to apoptosis (4h). Treated cells were mixed with SILAC labeled control cells, lyzed, digested and analyzed on LC/MS-MS. Approximately 3200 proteins were identified in each of three biological replicates. By applying Max Planck Institute‘s significance B statistical test, 74 proteins changed significantly in at least two replicates were identified. We have identified 26 ribosomal proteins with average ratio (treated/ control) 0.85, 21 nucleolar and RNA processing associated proteins and 26 proteins with different localization and function. Nucleolar toxicity of oxaliplatin was confirmed by Smetana toluidine blue staining and western blotting. Results and conclusions Up to date, there are only few studies describing nucleolar toxicity and ribosomal stress as the response to oxaliplatin treatment. The novelty of our work is to show down regulation of ribosomal and nucleolar proteins in response to oxaliplatin. However the direct mechanism leading to nucleolar toxicity was not identified.
This work was supported by grants LF_2014_010, TE02000058, CZ.1.05/3.1.00/14.0307. Institutional part of the work was supported by an EU Operational Program Research and Development for Innovations infrastructure grant project CZ.1.05/2.1.00/01.0030
APOFERRITIN AND ITS APPLICATIONS IN NANOMEDICINE středa / 3. prosince 13.55 - 14.10 hod.
2014
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Zbynek Heger1,2, Simona Dostalova1,2, Ondrej Zitka1,2, Vojtech Adam1,2, Tomas Eckschlager3, Marie Stiborova4, Rene Kizek1,2 Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic, 2 Department of Chemistry and Biochemistry, Laboratory of Metallomics and Nanotechnologies, Mendel University in Brno, Brno, Czech Republic, 3 Department of Paediatric Haematology and Oncology, 2nd Faculty of Medicine, Charles University, and University Hospital Motol, Prague, Czech Republic, 4 Department of Biochemistry, Faculty of Science, Charles University, Pragu, Czech Republic Introduction Nanomedicine as a continuously evolving discipline is still looking for a structure with perfect properties usable as a multifunctional transporter. Ferritins as the proteins naturally occurring in most living organisms throughout evolution show that they are the possible choice. These proteins show perfect biodegradability, biocompatibility and non-toxicity highly important for nanotransporters [1-3]. The structure of apoferritin is remarkably stable and robust, and it is able to withstand biologically extreme temperatures (up to 70 °C) and wide pH range (pH 2.0 - 10.0) for an appreciable period of time without significant disruption of their quaternary structure. Twenty1
four ferritin subunits form a spherical cage-shaped protein shell folded in a bundle of four long parallel and antiparallel α-helices (A, B, C, and D) with a fifth shorter C-terminal helix E, inclined at 60° to the major helix bundle [4]. Apoferritin structure has six 2-fold symmetry axes, four 3-fold symmetry axes and three 4-fold symmetry axes. It is known that there are narrow hydrophilic channels along the 3-fold symmetry axes consisting of negatively charged amino acids (glutamic and aspartic acid) and hydrophobic channels along the 4-fold symmetry axes. The protein shell forms an inner cavity with inner and outer diameters of 7-8 and 12-13 nm, respectively [4]. The inner cavity with an 80 Å diameter is capable to store up to 4500 Fe(III) atoms. Protein cage may be reversibly disassociated in unfavourable environment and after changing of environment conditions may be reconstituted backwards holding therapeutic agent in its cavity, protecting it against unwanted interactions. Moreover, a surface of protein cage may be easily functionalized with antibodies, aptamers, etc. to achieve highly specific nanoparticles. Materials/methods APOFERRITINS IN NANOMEDICINE As a drugs or contrast agents carriers, apoferritins could protect its cargo against degradation and pre-releasing causing undesired side effects. The first mention of the ability of ferritins to encapsulate the anti-cancer therapeutics was published in 2005 by Simsek and Kilic in the paper entitled „Magic ferritin: A novel chemotherapeutic encapsulation bullet“ [5]. As time goes by after seven years in 2012, Kilic et al. formed the apoferritin complex with doxorubicin commonly used cytostatic drug. The doxorubicin encapsulation was carried out using direct and stepwise change of pH of the solution from 2.5 to 7.4. It was found that up to 28 molecules of doxorubicin can be capsulated per apoferritin protein and no significant drug
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leakage occurs during several daysâ&#x20AC;&#x2DC; long storage [6]. Although apoferritin exhibits great attributes to serve as a platform for nanomedicine, the possible undesired immune response of patients still exists. The ideal nanotransporter has to go through a body undetected by immune system. Despite evidence that excessive amount of apoferritin administered for long period can trigger immune complex glomerulonephritis in mice [7], there is still lack of evidence about immune response in human. Another way of apoferritin utilization for medical purposes is photodynamic therapy in cancer treatment. The photodynamic therapy (PDT) is s a new therapeutic modality that is emerging as a powerful tool against malignant tumors [8]. Apoferritin nanocage, used as a delivery system for photosensitizers to the intracellular space, provides unique transporter protecting loaded photosensitizers from reactive biomolecules in the cell membranes. This enables further targeting of singlet oxygen upon specific light irradiation to tumour cells only. The gene therapy includes the insertion, removal or modification of the defective gene(s) for the treatment of genetically inherited diseases. The commonly used transporters for gene delivery are viral vectors, liposomes, peptides and cationic polymers. The main requirements of the gene delivery vector are the protection of delivered nucleic acid against nucleases, targeting, and ability to disrupt the endosomal membrane, and thus the DNA is delivered to the nucleus [9, 10]. Among the main obstacles of gene delivery vectors,
aggregation, instability, toxicity and their propensity to be captured by the mononuclear phagocyte system (MPS) are the most significant. Although the usage of apoferritin as a gene vector has not been published yet, it exhibits few advantages, but it is necessary to study apoferritins due to their colloidal behaviour, charge, possessing an electrostatic repulsion as well as the stability of encapsulated DNA. Results and conclusions One of the main goals of nanomedicine is to create a nanocarrier that can efficiently and specifically deliver therapeutic agents to target sites in the body. Moreover, to enable efficient and specific delivery, a nanocarrier needs to have an ability to be easily modified. Replacement of synthetic materials such as porous hollow silica nanoparticles, single-wall nanotubes, fullerenes, etc. with natural materials that are more acceptable to many people has become an attractive way of this field of research. The authors gratefully acknowledge financial support from the Grant Agency of the Czech Republic (NANOCHEMO GA CR 14-18344S). REFERENCES 1. Shevchenko, E.V., et al., Structural diversity in binary nanoparticle superlattices. Nature, 2006. 439(7072): p. 55-59. 2. Roney, C., et al., Targeted nanoparticles for drug delivery through the blood-brain barrier for Alzheimerâ&#x20AC;&#x2DC;s disease. Journal of Controlled Release, 2005. 108(2-3): p. 193-214. 3. Agnihotri, S.A., N.N. Mallikarjuna, and T.M. Aminabhavi, Recent advances on chitosanbased micro- and nanoparticles in
drug delivery. Journal of Controlled Release, 2004. 100(1): p. 5-28. 4. Uto, K., et al., Characterization of stable, electroactive protein cage/ synthetic polymer multilayer thin films prepared by layer-by-layer assembly. J. Nanopart. Res., 2013. 15(4): p. 1-11. 5. Simsek, E. and M.A. Kilic, Magic ferritin: A novel chemotherapeutic encapsulation bullet. J. Magn. Magn. Mater., 2005. 293(1): p. 509-513. 6. Kilic, M.A., E. Ozlu, and S. Calis, A Novel Protein-Based Anticancer Drug Encapsulating Nanosphere: ApoferritinDoxorubicin Complex. Journal of Biomedical Nanotechnology, 2012. 8(3): p. 508-514. 7. Li, M., et al., CD100 enhances dendritic cell and CD4(+) cell activation leading to pathogenetic humoral responses and immune complex glomerulonephritis. Journal of Immunology, 2006. 177(5): p. 3406-3412. 8. Saboktakin, M.R. and R.M. Tabatabaee, The novel polymeric systems for photodynamic therapy technique. International Journal of Biological Macromolecules, 2014. 65: p. 398-414. 9. Mahato, R.I., Non-viral peptide-based approaches to gene delivery. J. Drug Target., 1999. 7(4): p. 249-268. 10. Tan, P.H., C.L.H. Chan, and A.J.T. George, Strategies to improve non-viral vectors potential applications in clinical transplantation. Expert Opinion on Biological Therapy, 2006. 6(6): p. 619-630.
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Biomarkery nádorových onemocnění III Cancer biomarkers III
Předsedajíci / Chairs: Radek Trojanec, Jiří Drábek středa / 3. prosince 2014 / Wednesday / December 3, 2014 / 14.30 - 15.15 hod. IDENTIFICATION OF PROGNOSTIC AND PREDICTIVE FACTORS IN NON-SMALL CELL LUNG CANCER PATIENTS TREATED BY ADJUVANT CHEMOTHERAPY středa / 3. prosince 14.30 - 14.45 hod.
2014
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Jana Potockova1, Radek Trojanec1, Jiri Drabek1, Jana Stranska1, Jana Vrbkova1, Ivona Grygarkova2, Vladimira Koudelakova1, Zuzana Crlikova1, Sona Mlcochova1, Marian Hajduch1 Laboratory of Experimental Medicine, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Hnevotinska 5, 779 00, Czech Republic, 2 Department of Pulmonary Diseases and Tuberculosis, University Hospital in Olomouc, Olomouc, I.P. Pavlova 6, 779 00, Czech Republic Introduction Lung carcinomas represent one of the most common types of all tumors and can be classified into several groups according to various criteria such as biological behavior, histological composition, or localization. In practice, however, it is most useful to divide them into small-cell lung carcinoma SCLC (20-25%) and non-small-cell lung carcinoma NSCLC (75-80%). In comparison, NSCLC usually exhibits slower growth, so that in practice surgical removal becomes a favorable possibility, assuming that the tumor has not metastasized. NSCLC also responds less favorably to anti-tumor cytotoxic therapy and radiotherapy than SCLC. It is therefore that NSCLC has become the subject of our work. 1
Materials/methods In our study, we investigated a group of 124 patients with NSCLC who were treated with an adjuvant regimen consisting of a combination of platinum derivatives and vinorelbine. For the material, formalin-fixed paraffin embedded (FFPE) tissue samples were used. Using the FISH assay, the status of EGFR, FGFR, C-MET, the presence of rearrangements in ALK and ROS1 genes, and the number of copies of chromosomes 7 and 8 were determined. Concurrently, the qPCR method was used to investigate the mutational status in the K-RAS and B-RAF genes. Results and conclusions Statistical analyses of the clinical and laboratory results were performed; the control group comprised NSCLC patients without adjuvant therapy. Some interesting correlations were revealed. For example, patients with the higher copy number of chromosomes 7 or 8 and higher copy number of the genes EGFR and MET (located on these chromosomes), exhibited shorter relapse free survival (RFS). Other correlations will be also presented. Acknowledgement: This work was supported by grant project IGA MZ ČR NT/13569 and BIOMEDREG CZ.1.05/2.1.00/ 01.0030
A workflow for the identification and validation for N-glycoprotein biomarkers in mouse xenograft serum středa / 3. prosince 14.45 - 15.00 hod.
2014
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Lakshman Varanasi1, Miroslav Hruska2, Dusan Holub1, Petr Dzubak1, Marian Hajduch1 1
Institute of Molecular and Translational Medicine, Palacky University, Olomouc, Czech
Republic, Department of Computer Science, Palacky University, Olomouc, Czech Republic Introduction Serum is one of several types of patient samples routinely analyzed in the clinic and is often preferred because it can be obtained noninvasively in sufficient quantities. Serum proteins that indicate a condition or disease, its severity, the patient‘s chances of recovery and of the patient‘s possible response to therapy are of clinical value. Glycosylation of cellular proteins post-translation has been reported as an indicator of altered cellular physiology. We describe here the optimization of a workflow for the identification of such N-glycosylated proteins using the HCT116 mouse xenograft model system. The premise for this approach is that human proteins observed in mouse serum originate in the tumor xenograft. Materials/methods Hydrazide slurry was purchased from BioRad. All other reagents were obtained from SigmaAldrich. Samples were processed by SPEG (Solid-phase extraction of glycoproteins). Tandem massspectra were acquired using an Orbitrap Elite mass-spectrometer (Thermo). Acquired tandem mass spectra were matched with corresponding peptides by Proteome Discoverer (Thermo) in a database of human and mouse peptides. The peptide spectral matches were classified as uniquely human, uniquely murine or as peptides common to the two species, using a program developed in-house. Results and conclusions Altogether 30 uniquely human peptides were identified in the discovery phase. These are
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candidates for the validation phase of the study viz quantification in a colorectal cancer patient serum sample cohort by the single-reaction monitoring (SRM) technique.
System for reliable identification of alterations using protein massspectrometry stĹ&#x2122;eda / 3. prosince 15.00 - 15.15 hod.
2014
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Miroslav Hruska1,2, Jiri Voller1, Lakshman Varanasi1, Petr Vojta1, Dusan Holub1, Khushboo Agrawal1, Petr Dzubak1, Marian Hajduch1 Institute of Molecular and Translational Medicine, Olomouc, Czech Republic, 2 Department of Computer Science, Olomouc, Czech Republic Introduction Typical cancer cells carry mutations in up to hundreds of genes. Knowledge of mutation profiles allows us to understand which processes are altered in the cancer cells of individual patients and select the therapy accordingly. Modern high-throughput methods like SNP arrays or next-generation sequencing (NGS) are used for mutation screening. However, the mass spectrometry of proteome, another method with a great potential in personalized medicine, is not used for this purpose yet. The limiting factor is insufficient coverage of mutant variants in proteome databases and incompleteness of fragmentation spectrum for peptide de novo reconstruction. Materials/methods To advance the current state, we have developed a system for the identification of mutant, polymorphic proteins on protein level. The core consists of dedicated mutation/ SNPs database with unique coverage (number of both mutation and integrated information sources). The system, Decryptor, enables users to search for alterations in their MS/MS spectra. The identification 1
process itself is performed using Dymka, in-house developed, multiengine, cluster-powered system built on top of open-source system OpenMS. Decryptor works both with peptide database search engines and tag-based de novo systems. Obtained results are subjected to careful artefact analysis to separate relevant findings from potential artefacts. For example, one class of artefacts emerges because of peptide homeometricity, i.e., existence of different peptides with highly similar fragmentation spectra. Final results are then annotated from multiple data sources and presented in a form amenable to further investigations. Results and conclusions Performance of Decryptor was evaluated on colorectal cancer line HCT116. The necessity of artefact analysis was shown to be especially important for reliable identification of rare sequence variants. In such case peptide homeometric to mutant peptide is often found among posttranslationally modified reference peptides.
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Farmakologie protinádorových léčiv a in vivo imaging Pharmacology of anticancer drugs and in vivo imaging
Předsedajíci / Chairs: Miloš Petřík, Josef Srovnal středa / 3. prosince 2014 / Wednesday / December 3, 2014 / 15.45 - 16.30 hod. Assessing pharmacokinetic properties in drug discovery: screening versus in silico středa / 3. prosince 15.45 - 16.00 hod.
2014
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Alice Nová, Martina Michalová, Monika Harvanová, Milan Urban Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacky University Olomouc, Olomouc, Czech Republic Introduction ADME (absorbtion, distribution, metabolism and excretion) in silico modeling can be a useful tool in drug design and development to help in finding the most promising compounds, preferably prior synthetizing them; however in silico prediction has its pitfalls. Materials/methods Here, we compare in silico ADME predictions gained from free ADME prediction softwares to in vitro experimental data obtained from testing three groups of compounds. We also compare the ability and accuracy of in silico ADME tools to predict ADME properties depending on the complexity of the molecule structure of tested compounds. In order to predict ADME properties we have chosen three online software tools: ACD/I-lab, admetSAR and online BBB (blood brain barrier) predictor. We have tested compounds from the groups of aminopyrimidines (anti-inflammatory compounds), triterpenes (potential antitumor agents) and carboranes (potential inhibitors of carbonic anhydrase IX, which is selectively expressed in hypoxic tumors). Results and conclusions Aminopyrimidines can be characterized as small and relatively
simple molecules. Both ACD/I-lab and admetSAR predicted accurately GIT permeability, however active efflux was not correctly predicted. All three used software tools predicted brain penetration as sufficient for CNS activity and these results were in accordance with the data obtained from the in vitro BBB model using MDR1-MDCK cell line. Only ACD/I-lab is able to predict plasma protein binding and all tested compounds were generally satisfactorily predicted. Compounds from the group of triterpens represent more complex structures and larger molecules compared to aminopyrimidines. In silico prediction of triterpene GIT permeability achieved 75% reliability against Caco-2 cell line testing, however both predictors failed in active efflux prediction. In case of prediction of the brain penetration, in silico tools have diverged among themselves in BBB prediction. In vitro MDR1-MDCK permeability testing of CNS permeability of triterpenes is currently in the process. Carborane-based compounds are examples of such complex structures, that they cannot be processed by previously selected in silico predictors. In conclusion, GIT permeability and brain penetration of relatively uncomplicated molecules are possible to predict in silico with sufficient reliability. Metabolism and plasma stability are not possible to reliably predict using free in silico modeling at all. Here we wanted to point out, that the ADME in silico modeling has its place in the drug development, but it is necessary to take into account that it can never outperform the experimental data.
Ga labelled peptides for glioblastoma imaging 68
středa / 3. prosince 16.00 - 16.15 hod.
2014
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Milos Petrik, Jana Stepankova, Zbynek Novy Institute of Molecular and Translational Medicine, Olomouc, Czech Republic Introduction Glioblastoma multiforme (GBM) is the most aggressive type of primary brain tumors in humans with more than 90% 5-year mortality. Early diagnosis may be the key factor to improving patient survival rates through the prevention of tumor growth. Positron emission tomography (PET) is a useful imaging tool allowing early stage tumors identification, monitoring of treatment efficacy and follow up for disease recurrence. 18F-fluorodeoxyglucose (18F-FDG) is the most common clinically utilized PET tracer for imaging of GBM, although it has several limitations based on high glucose uptake within the brain. Here we report on the preclinical evaluation of two 68Ga labelled peptides as potential agents for GBM imaging and their comparison with 18F-fluorothymidine (18F-FLT), another PET tracer widely used in clinical practice. Materials/methods 68 Ga labelling of studied peptides (NODAGA-conjugated RGD dimer and DOTA-conjugated Substance P) was optimized concerning temperature, peptide amount and reaction time. For in vitro characterization, partition coefficient, protein binding properties and stability values in different media were determined. Ex vivo biodistribution and animal imaging was studied in Balb/c mice.
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Results and conclusions Both 68Ga labelled peptides were prepared with high radiochemical purity (> 97%) under various reaction conditions. 68Ga-NODAGARGD dimer showed hydrophilic properties and was stable in human serum, iron and DTPA solutions as well as at pH = 7. In contrast to 68Ga labelled RGD peptide, 68Ga-DOTASubstance P displayed much lower stability, especially in iron and DTPA solutions. Protein-bound activity of 68Ga-NODAGA-RGD dimer after 120 min incubation (3.9% ± 1.9) was found to be 7-fold lower than for 68Ga-DOTA-Substance P. In mice, both 68Ga labelled tracers revealed mainly renal elimination with low blood values 90 min p.i., while 18F-FLT showed much slower kinetics and not only renal, but also gastrointestinal excretion. Studied peptides bound 68Ga with high affinity, and 68Ga-NODAGARGD dimer, especially, displayed high in vitro stability and excellent pharmacokinetics. Both 68Ga labelled tracers showed superior in vivo properties compared to clinically used 18F-FLT. 68Ga-NODAGA-RGD dimer, in particular, holds promise for PET imaging of glioblastoma, which needs to be confirmed in U87MG xenografts. Animal experiments in U87MG xenograft tumors are currently ongoing.
Imaging of glioblastoma multiforme in mice using microPET/CT system středa / 3. prosince 16.15 - 16.30 hod.
2014
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Zbynek Novy1, Milos Petrik1, Hana Adamkova2, Marian Hajduch1 Faculty of Medicine and Dentistry, Palacky University in Olomouc, Olomouc, Czech Republic, 2 Faculty of Science, Palacky Universtity in Olomouc, Olomouc, Czech Republic Introduction Glioblastoma multiforme is a primary brain tumor with very poor prognosis. Early diagnosis is essential key 1
point for successful therapy of this disease. Non-invasive imaging techniques play very important role in the diagnosis and staging of glioblastoma multiforme. To facilitate preclinical in vivo research in the field of glioblastoma multiforme we have compared the biodistribution and the imaging potential of three clinically used radiopharmaceuticals for positron emission tomography in a mouse tumor model. Materials/methods Tumor mouse model was established using SCID mouse strain and U-87 MG tumor cells xenografted subcutaneously. Visualizations of tumors were performed by employing three PET radiopharmaceuticals administered already routinely in human medicine - 18F-fluorodeoxyglucose (FDG), 18F-fluorothymidine (FLT) and 18F-fluorocholine (FCH). Healthy non-tumorous mice were used as a negative control. MicroSPECT/ PET/CT system (Albira, Carestream, USA) was used to acquire images of tested mice after the application of the tracers. The mice in general anesthesia were PET-scanned during 90 minutes after administration of the radiopharmaceuticals to reveal the tracer´s biodistribution and the best time point for visualization of the tumor. The acquired data were quantified in PMOD software. Results and conclusions The biodistribution of three mentioned tracers in SCID mouse was determined using output data from PMOD software. As well as the tumor to brain rations were calculated to find out the specificity of the tracers. The most appropriate biodistribution (fast renal excretion) and in the same time the highest tumor to brain ratio (4,4) was observed in case of 18F-fluorothymide. The other two tested radiopharmaceuticals (FDG and FCH) have not showed satisfactory results for imaging of glioblastoma multiforme in mice. In conclusion, the benefits of used radiopharmaceuticals for imaging the studied tumor
were evaluated and as the most appropriate radiopharmaceutical for this examination was determined 18F-flurothymidine.
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Posterová sekce / Poster section vyskytuje u většiny adenokarcinomů LightCycler 480 II, Roche). Exprese HNF-1β v karcinomech děložního čípku děložního hrdla, oproti tomu u Materials/methods
Kristyna Nemejcova, Pavel Dundr, Libor Stanek, Ctibor Povysil General Facuty Hospital, Prague, Czech Republic Introduction HNF-1β je maker běžně využívaný v diferenciální diagnostice světlobuněčných karcinomů ovária a endometria. Recentní studie prokázaly expresi HNF-1β v menším rozsahu i u dalších ovariálních a endometriálních tumorů. Ohledně děložního čípku byla exprese HNF-1β pouze okrajově zmíněna u některých typů adenokarcinomů. Nicméně systematická analýza exprese HNF1β u cervikálních karcinomů dosud nebyla provedena. Materials/methods Cílem studie bylo stanovit expresi HNF-1β v různých typech invazivních karcinomů čípku a zhodnotit přínos imunohistochemické analýzy exprese tohoto proteinu pro diferenciální diagnostiku jednotlivých histologických typů nádorů. Studie zahrnovala 155 případů invazivních karcinomů děložního čípku (86 dlaždicobuněčných (SCC), 56 adenokarcinomů (ACAs), 13 nediferencovaných karcinomů. Exprese HNF-1β byla určována imunohistochemicky a výsledky byly korelovány s expresí ER, PR, CEA, p16, p63, p40 a D2-40. Results and conclusions HNF-1β byl exprimován v 42/56 ACAs (75 %). Ve skupině SCC, byly pozitivní pouze 2/86 (2,32%) případy, které exprimovaly HNF1β v 70 % a ve 30 % nádorových buněk. Nediferencované karcinomy vykazovaly expresi HNF-1β ve 2/13 případů. Závěr ukázal, že exprese HNF-1β se
dlaždicobuněčných karcinomů jsme ji zastihli pouze raritně. Tento marker by tedy mohl být potenciálně využitelný jako pomocný marker v diferenciální diagnostice málo diferencovaných karcinomů děložního hrdla, jejichž odlišení je na morfologické úrovni někdy obtížné až nemožné. Samotnou expresi tohoto markeru však nelze nadřadit nad jiné rutinně používané markery jako je např. p63, p40 a D2-40. Acknowledgement: Práce byla podporována IGA MZ CR NT140013/2013
In the present study, we investigated if there is a possibility to check the performance of thermocycler regarding temperature uniformity over the heating block using PCR reagents readily available in the laboratory. We tried to find correlations between melting temperature, Tm, and location of the well by comparing melting temperature of the same amplified DNA sequence across the thermal block. We supposed that differences in Tm across the block are caused not only by small temperature Temperature uniformity of the heterogeneity of the thermal block but also by other factors (i.e. thermocyclers tested using imprecission of pipetting). melting temperature of the Results and conclusions same amplicon We tested several possibilities to Eva Hruskova, Jiri Drabek alleviate the sources of unwanted Institute of Molecular and variability with the aim to increase the Translational Medicine of the reproducibility of Tm measurements. Faculty of Medicine and Dentistry In this contribution, we present of Palacký University, Olomouc, our findings and suggestion for Czech Republic practical use of Tm for thermal block temperature uniformity testing. Introduction Supported Real-time PCR is one of the most Acknowledgement: widely used method in clinical by grants LO1304, NT13569 and diagnostic laboratories. Because CZ.1.05/3.1.00/14.0307. even a small change of the Inhibition of proliferation temperature can lead to insufficient denaturation or hybridization by inhibitors of histone resulting in loss of specificity, deacetylases sensitivity and reproducibility, the Tomas Groh1, Mohamed temperature needs to be precise Asraf Khalil2, Jan Hrabeta2, and uniform. To confirm the proper Blanka Jedlickova3, Tomas operation of thermocyclers and 2 fulfill ISO17025 and ISO15189 Eckschlager , Marie accreditation requirements, regular Stiborova1 calibration of thermal cyclers is 1 Department of Biochemistry, needed. Calibration is usually Faculty of Science, Charles performed by accredited calibration University, Prague, Czech Republic, laboratory using metrologically 2 Department of Pediatric traceable temperature probes. Hematology and Oncology, 2nd However, sometimes such calibration Medical School, Charles University is not possible because the cycler is and University Hospital Motol, inaccessible for thermoprobes (i. e. Prague, Czech Republic,
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Department of Chemistry and Clinical Biochemistry, 2nd Medical School, Charles University and University Hospital Motol, Prague, Czech Republic Introduction N-myc protein is a member of Myc family transcription factors. Different alterations in gene or protein function of Myc family members are connected to variety of human cancers. Myc family members are responsible for fast proliferation of cells, chemoresistance and metabolic adaptation of cancer cells. N-myc has been studied intensively since it was discovered as the most important prognostic factor in neuroblastoma (NB). NB is a most common extracranial solid tumor in infancy and some its forms has a very bad curability. N-myc amplification is related to a worse prognosis and poor survival of patients suffering from NB. Inhibitors of histone deacetylases (HDAC) are promising drugs for cancer treatment. Some of them are in clinical testing, for their ability to supress cancer cells growth, to stop cell cycle, differentiate cancer cells and ability to induce programmed cell death. Inhibitors of HDAC act as epigenetic modifiers of histone tails or change acetylation status of different proteins. Materials/methods Western blot was used for detection of N-myc and HIF-1a transcription factors. Flow cytometry was used to measure cell cycle distribution. Results and conclusions We studied mechanism of inhibition of cell growth of NB cells induced by inhibitor of HDAC valproate (VPA). We tested three different NB cell lines that amplify N-myc gene (IMR32, UKF-NB-3, UKF-NB-4). N-myc protein expression decreased after 24h and 48h treatment with nontoxic dose of VPA (1mM). That decrease was even under hypoxic conditions (1% O2). Cells stopped proliferate mostly under hypoxic conditions after treatment with 1mM VPA, that correlate with decrease 3
of both N-myc trascription factor and hypoxia inducible factor 1 alpha (HIF-1a). As we observed earlier, inhibition of HDAC by VPA increased acetylation of histone H3 and histone H4. Simultaneously VPA induced cell cycle arrest and increased doubling time of cells. We hypothesize that, decrease of N-myc transcription factor induced by VPA plays an important role in inhibition of proliferation NB cells. The study was supported by the Charles University in Prague, project GA UK No. 635712 and No. 620612.
Multi-walled carbon nanotubes as a promising doxorubicin nanocarrier and their effects on neuroblastoma cell lines
Tereza Cerná1, Jan Hrabeta2, Tomáš Groh1, Hoai Viet Nguyen3, Amitava Moulick3, Tomáš Eckschlager2, Marie Stiborová1 Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic, 2 Department of Pediatric Hematology and Oncology,2nd Medical Faculty, Charles University and University Hospital Motol, Prague, Czech Republic, 3 Laboratory metallomics and nanotechnology, Mendel University in Brno and Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic Introduction Neuroblastoma is the most common extracranial solid tumor of childhood. Despite advances in cancer diagnosis and therapy, gradually developing tumor cell chemoresistance is one of the major causes of treatment failure of high grade neuroblastoma. Currently nanocarriers are promising agents to improve drug therapeutic index, divert ABC-transporter mediated drug efflux mechanism and selectively target tumor cells. Selective nanocarrier targeting to tumor overcomes dose-limiting 1
side effects, lack of selectivity, tissue toxicity and limited drug access to tumor tissues, moreover it allows application of higher doses of cytostatics. Carbon nanotubes (CNTs) are emerging as a family of nanovectors for drug delivery into biological systems. Materials/methods To evaluate potential application of this technology for neuroblastoma therapy, the aim of this pilot study was to compare the cytotoxic effects of doxorubicin loaded multi-walled CNTs (MWCNTs) and free doxorubicin on sensitive and resistant neuroblastoma cell lines UKF-NB-4 in vitro in normoxic and hypoxic conditions. Cell viability was assessed using the MTS cytotoxicity assay and DNA doublestrand breaks were detected by flow cytometry as phosphorylation of histone H2A variant. Results and conclusions We show here that effect of doxorubicin loaded MWCNTs on human neuroblastoma cell lines is very similar to the effect of free doxorubicin. There were also detected hypoxia-induced resistance to doxorubicin both free and in MWCNTs . These results seem promising to begin animal experiments, where it is hoped that in vivo studies will demonstrate higher effectivity of MWCNTs. This work was supported by GACR, grant No. 14-18344S.
Prognostický a prediktivní význam sérových onkomarkerů u pacientů s pokročilým neskvamózním NSCLC léčených pemetrexedem
Ondřej Fiala1, Miloš Pešek2, Jindřich Fínek1, Vít Martin Matějka1, Luboš Holubec1, Zbyněk Bortlíček3, Ondřej Topolčan4 Onkologická a radioterapeutická klinika LF UK a FN Plzeň, Plzen, Czech Republic, 2 Klinika pneumologie a ftizeologie 1
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LF UK a FN Plzeň, Plzen, Czech Republic, 3 Institut biostatistiky a analýz, LF MU, Brno, Brno, Czech Republic, 4 Oddělení nukleární medicíny – imunoanalytická laboratoř LF UK a FN Plzeň, Plzen, Czech Republic Introduction Pemetrexed je antifolátové cytostatikum nové generace, jehož účinnost i bezpečnost byla prokázána v několika randomizovaných studiích fáze III. V současné době standardně užíván pro léčbu pacientů s pokročilým stádiem NSCLC (st. IIIB nebo IV), neskvamózního histologického typu, v kombinaci s platinovým derivátem v první linii a v monoterapii v linii druhé. Recentní studie rovněž pokázaly efektivitu udržovací léčby pemetrexedem. Cílem této práce bylo zjištění prediktivního a prognostického významu hladin sérových onkomarkerů karcinoembryonálního antigenu (CEA), fragmentu cytokeratinu 19 (CYFRA 21-1), Monototalu, chromograninu A, thymidin kinázy (TK) a antigenu skvamózních karcinomů (SCCA) u pacientů s pokročilým stadiem NSCLC, kteří byli léčeni pemetrexedem. Materials/methods Soubor pacientů čítá celkem 114 pacientů s pokročilým stadiem NSCLC (IIIB, IV) neskvamózního histologického typu, kteří byli léčeni pemetrexedem v kombinaci s platinovým derivátem nebo v monoterapii. U všech pacientů byla před zahájením léčby stanovena sérová hladina zkoumaných onkomarkerů. Přežití pacientů bylo hodnoceno metodikou podle Kaplana-Meiera, srovnání bylo provedeno log-rank testem. Prognostický a prediktivní význam onkomarkerů byl dále verifikován pomocí Coxova vícerozměrného modelu. Results and conclusions Mediány přežití bez známek progrese (PFS) a celkového přežití (OS) u pacientů s vysokou hladinou CEA činily 2,4 a 12,8 vs. 3,1 a
11,5 měsíce u pacientů s nízkou hladinou CEA (p=0,137 a p=0,866). Mediány PFS a OS u pacientů s vysokou hladinou CYFRA 21-1 činily 2,4 a 10,3 vs. 2,7 a 23,4 měsíce u pacientů s nízkou hladinou CYFRA 21-1 (p=0,899 a p<0,001). Mediány PFS a OS u pacientů s vysokou hladinou Monototalu činily 3,1 a 12.4 vs. 2,9 a 22,3 měsíce u pacientů s nízkou hladinou Monototalu (p=0,543 a p=0,451). Mediány PFS a OS u pacientů s vysokou hladinou chromograninu A činily 1,4 a 7,9 vs. 2,4 a 13,5 měsíce u pacientů s nízkou hladinou chromograninu A (p=0,196 a p<0,224). Mediány PFS a OS u pacientů s vysokou hladinou TK činily 2,2 a 11,3 vs. 2,8 a 23,4 měsíce u pacientů s nízkou hladinou TK (p=0,163 a p=0,003). Mediány PFS a OS u pacientů s vysokou hladinou SCCA činily 2,7 a 8,7 vs. 2,9 a 16,6 měsíce u pacientů s nízkou hladinou SCCA (p=0,557 a p=0,510). Coxův vícerozměrný model prokázal, že sérové hladiny CYFRA 21-1 (HR=2,26; p=0,001) a TK (HR=2,09; p=0,003) představují významný, nezávislý faktor predikující OS. Výsledky naší studie prokázaly, že vysoké předléčebné hladiny sérových onkomarkerů CYFRA 21-1 a TK představují významný, nezávislý faktor predikující krátké OS u pacientů s pokročilým NSCLC. Žádný ze zkoumaných onkomarkerů se neukázal jako významný prediktivní biomarker pro odhad efektu léčby pemetrexedem. Podpořeno MZ ČR - FNPl, 00669806.
Sérové onkomarkery jako prediktivní biomarker u pacientů s pokročilým NSCLC léčených EGFR-TKI Ondřej Fiala1, Miloš Pešek2, Jindřich Fínek1, Marek Minárik3, Lucie Benešová3, Zbyněk Bortlíček4, Ondřej Topolčan5 1 Onkologická a radioterapeutická klinika LF UK a FN Plzeň, Plzeň, Czech Republic, 2 Klinika pneumologie a ftizeologie LF UK a FN Plzeň, Plzeň, Czech Republic,
Centrum aplikované genomiky solidních nádorů (CEGES), Genomac výzkumný ústav, Praha, Praha, Czech Republic, 4 Institut biostatistiky a analýz, LF MU, Brno, Brno, Czech Republic, 5 Oddělení nukleární medicíny – imunoanalytická laboratoř LF UK a FN Plzeň, Plzeň, Czech Republic Introduction Zavedení inhibitorů tyrozinkináz receptoru pro epidermální růstový faktor (EGFR-TKI) do léčby nemalobuněčného plicního karcinomu (NSCLC) přispělo k prodloužení přežití pacientů s pokročilým stádiem tohoto onemocnění. Aktivační mutace genu EGFR představují spolehlivý prediktor dobrého efektu léčby EGFR-TKI u pacientů s pokročilým NSCLC. Výskyt těchto mutací v evropské populaci je nízký (cca 10-15%), navíc je stále mnoho pacientů, u kterých vyšetření na přítomnost aktivační mutace genu EGFR není možné provést z různých důvodů. Z tohoto důvodu je hledání dalších prediktivních biomarkerů nutné. Cílem této práce bylo zjištění významu sérových onkomarkerů CEA, CYFRA 21-1, NSE a TK pro predikci efektu léčby EGFR-TKI. Materials/methods Soubor pacientů, u kterých jsme hodnotili význam onkomarkerů CEA a CYFRA 21-1 čítá 144 pacientů a soubor pacientů, u kterých jsme hodnotili význam onkomarkerů NSE a TK čítá 163 pacientů s pokročilým stadiem NSCLC (IIIB, IV), kteří byli léčeni EGFR-TKI (erlotinib, gefitinib). U všech pacientů byla před zahájením léčby stanovena sérová hladina zkoumaných onkomarkerů. Přežití pacientů bylo hodnoceno metodikou podle Kaplana-Meiera, srovnání bylo provedeno log-rank testem. Prognostický a prediktivní význam onkomarkerů byl dále verifikován pomocí Coxova vícerozměrného modelu. Results and conclusions Mediány přežití bez známek progrese (PFS) a celkového přežití (OS) u pacientů s vysokou hladinou 3
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CEA činily 1,9 a 8,6 vs. 2,9 a 16,1 měsíce u pacientů s nízkou hladinou CEA (p=0,046 a p=0,116). Mediány PFS a OS u pacientů s vysokou hladinou CYFRA 21-1 činily 1,9 a 6,1 vs. 3,4 a 23,8 měsíce u pacientů s nízkou hladinou CYFRA 21-1 (p<0.001 a p<0.001). Mediány PFS a OS u pacientů s vysokou hladinou NSE činily 1,1 a 3,7 vs. 2,6 a 11,6 měsíce u pacientů s nízkou hladinou NSE (p=0,002 a p=0,003). Mediány PFS a OS u pacientů s vysokou hladinou TK činily 2,1 a 8,5 vs. 2,9 a 17,4 měsíce u pacientů s nízkou hladinou TK (p=0.026 a p=0.020). Coxův vícerozměrný model prokázal, že sérové hladiny CEA (HR=1,72; p=0,007), CYFRA 21-1 (HR=2,17; p<0,001) a NSE (HR=2,36; p=0,003) představují významný, nezávislý faktor predikující PFS a hladina CYFRA 21-1 (HR=2,74; p<0,001) představuje významný, nezávislý faktor predikující OS. Výsledky naší studie prokázaly, že vysoké předléčebné hladiny sérových onkomarkerů CEA, CYFRA 21-1 a NSE představují významný, nezávislý faktor predikující nízký efekt léčby EGFR-TKI a dále potvrdily negativní prognostický význam onkomarkeru CYFRA 21-1. Podpořeno MZ ČR - FNPl, 00669806.
Influence of quercetin and taxifolin on microRNA 375 expression in HepG2 cell line and human hepatocytes.
Zdenek Dostal1, Martin Sebera2, Josef Srovnal3, Michaela Sedlackova3, Lenka Radova4, Katerina Staffova3, Martin Modriansky1, Jitka Ulrichova1 Department of Medical Chemistry and Biochemistry, Faculty of Medicine, Palacky University, Olomouc, Czech Republic, 2 Department of Kinesiology, Faculty of sport studies, Masaryk University, Brno, Czech Republic, 3 Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky 1
University and University Hospital Olomouc, Olomouc, Czech Republic, 4 Central European Institute of Technology, Research Group Medical Genomics, Masaryk University, Brno, Czech Republic Introduction Taxifolin and quercetin belong to a group of polyphenols that are natural compounds present in fruits, vegetables, beverages such as tea, and in several other foods and natural products. In general these substances display positive biological effects and due to a relatively high consumption are they considered beneficial. According to many studies the daily dose of these substances may reach more than 1 g/day. One possible mode of action is an influence of these compounds on microRNA expression and the associated change in amount of target proteins. The change may reflect in cell phenotype and this affects its behavior. Materials/methods Quercetin and taxifolin were tested for their ability to influence the expression of microRNA 375 and its potential impact on the expression of one potential target gene, procaspase 3. The influence of both substances was estimated from chip array and verified by RT-PCR method. The impact on procaspase 3 expression was studied by western blot. Because liver is the first organ possibly affected by both substances, we selected HepG2 cell line as it is derived from human hepatoma and primary human hepatocytes. The dose of both substances was 1 mM based on toxicity studies. The treatment time period was established as 24 hours. Total RNA was isolated by phenolchloroform method. Results and conclusions Firstly, we compared the data obtained from microRNA chips with those obtained by RT-PCR. Our data show slight but stable increase in microRNA 375 expression by taxifolin in HepG2 cell line. On the
other hand, expression of microRNA 375 in human hepatocytes differed significantly among six individual samples. Quercetin causes an increase in expression of the microRNA of interest in both cell types. As both tested substances are perceived as cytoprotective, we selected to monitor the expression of the protein procaspase 3, which is one of several possible target genes of microRNA 375. However, western blot experiments showed no significant influence on procaspase 3 expression even though a trend toward attenuation of its expression in dose dependent manner was observed. Our data suggest that beneficial biological activity of taxifolin and quercetin may proceed through microRNA modulation. Acknowledgements : This work was supported by grants IGA_ LF_2014_14 and LO1304
PHAGE λ as a suitable doxorubicin nanocarrier
Simona Dostalova1,2, Dita Munzova2, Marketa Vaculovicova1,2, Tomas Eckschlager3, Marie Stiborova4, Vojtech Adam1,2, Rene Kizek1,2 Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic, 2 Department of Chemistry and Biochemistry, Laboratory of Metallomics and Nanotechnologies, Mendel University in Brno, Brno, Czech Republic, 3 Department of Paediatric Haematology and Oncology, 2nd Faculty of Medicine, Charles University, and University Hospital Motol, Prague, Czech Republic, 4 Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic Introduction One of frequently used cytotoxic drug for cancer treatment is doxorubicin. It is an anthracycline antibiotic drug. Doxorubicin can block enzyme topoisomerase II, resulting in 1
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disruption of newly replicated DNA. It also intercalates in DNA, which leads to DNA breaks and defects during replication and transcription; and forms free radicals. It is used in treatment of breast, lung, ovarian, bladder, thyroid gland, testicle or head and neck carcinomas, as well as neuroblastomas, leukaemia, lymphomas and many others. However, the administration of doxorubicin during the treatment leads to many severe side effects for healthy cells, lowering the wellbeing of patients. The side effects include nausea and vomiting, sores in the mouth and on the lips, hair loss, diarrhea, darkening of the soles, palms or nails, blood in the urine, unusual bleeding and bruising or swelling of feet. The most severe is its cardiotoxicity which can lead to patient‘s death. Encapsulation of doxorubicin in suitable nanocarrier can eliminate the negative side effects. Nanocarriers can be composed of many different materials, either inorganic or organic. Protein-based nanocarriers, including viral capsids, are more suitable for administration in patients in comparison with inorganic materials. Materials/methods Cultivation and purification of phage λ Phage λ-producing strain of Escherichia coli was cultivated in Luria-Bertani broth (1 % tryptone, 0.05 % yeast extract and 1 % sodium chloride) with 0.2 % maltose for 24 h at 37 °C and 600 rpm. After cultivation, the culture was mixed with chloroform at 6:1 ratio and incubated for 1 h at 25 °C to kill the producing E. coli. The samples were centrifuged at 5200 g and 4 °C for 10 min to remove E. coli and then at 10000 g and 4 °C for 6 min to remove remaining contaminants. Next the supernatant containing phage was ultracentrifuged at 130000 g and 4°C for 3 h. The pellet containg phage was resuspended in PBS at protein concentration of 15 μg/ml and stored at 4 °C.
Encapsulation of doxorubicin in phage λ The doxorubicin was encapsulated in purified phage by infusion method. 80 μl of phage was mixed with 80 μl of doxorubicin at different concentrations (200; 100; 50; 25; 12.5 and 0 μg/ml). Incubation was conducted for 2 h at 25 °C in dark. Free doxorubicin was subsequently dialyzed using Amicon 3K (MerckMillipore, MA, USA) for 15 min at 7000 g and 20 °C and phage was twice rinsed with water. The volume was then filled to original volume. Encapsulation of doxorubicin in apoferritin The doxorubicin was encapsulated in apoferritin by opening and closing of apoferritin in various pH. 80 μl of apoferritin (15 μg/ml) was mixed with 80 μl of doxorubicin at different concentrations (200; 100; 50; 25; 12.5 and 0 μg/ml). 1 μl of 1M hydrochloric acid was added to sample to lower the pH to 2 and open the apoferritin. The samples were mixed for 15 min at 25 °C and 600 rpm. 1 μl of 1M sodium hydroxide was added to higher the pH to 7 and encapsulate doxorubicin in apoferritin. Free doxorubicin was subsequently dialyzed using Amicon 3K (Merck-Millipore, MA, USA) for 15 min at 7000 g and 20 °C and phage was twice rinsed with water. The volume was then filled to original volume. Verification of doxorubicin encapsulation in nanocarriers Absorption spectra of 50 μl of nanocarriers with encapsulated doxorubicin were measured from 200 to 800 nm. Emission spectra of samples were measured with excitation at 480 nm (absorption maximum of doxorubicin) and emission from 515 to 815 nm. Results and conclusions In this work, well-studied coliphage λ was used as a nanocarrier for doxorubicin, using the infusion method of encapsulation. The successful encapsulation was proven by absorbance and fluorescence measurement of the
whole phage. The absorbance of phage λ at 480 nm increased from 0.06 to 0.22 after encapsulation of 200 μg/ml doxorubicin. The fluorescence of phage λ at 600 nm (the emission maximum of doxorubicin) increased from 1000 to 22000 after encapsulation of 200 μg/ ml doxorubicin. Encapsulation by phage λ was compared with well-studied nanocarrier apoferritin. At the same concentration, apoferritin was able to encapsulate 4 times lower amount of doxorubicin than phage. Undesired release of doxorubicin from apoferritin was 10 times higher in comparison with phage. It can be concluded, that phage λ can serve as a suitable nanocarrier for anticancer drug doxorubicin. The authors gratefully acknowledge financial support from the Grant Agency of the Czech Republic (NANOCHEMO GA CR 14-18344S).
Fluorescenční charakterizace zlatem modifikovaného liposomu s uzavřenými protinádorovými léčivy a s připojenou antisense N-myc DNA uchycenou k magnetickým částicím
Lukas Nejdl1,2, Sylvie Skalickova1,2, Jiri Kudr1,2, Ana Maria Jimenez Jimenez1, Branislav Ruttkay-Nedecky1,2, Tomas Eckschlager3, Marie Stiborova4, Vojtech Adam1,2, Rene Kizek1 Mendelova univerzita v Brne;, Agronomicka fakulta, Ustav chemie a biochemie, Brno, Czech Republic, 2 Stredoevropsky technologicky institut CEITEC VUT v Brne, Brno, Czech Republic, 3 Ustav pediatricke hematologie a onkologie, 2. lekarska fakulta Karlova univerzity, a univerzitni nemocnice Motol, Praha, Czech Republic, 4 Ustav biochemie, Prirodovedecka fakulta, Karlova univerzita, Praha, Czech Republic 1
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Introduction Onkogen N-myc hraje důležitou roli během vývoje a diferenciace neuroektodermu. Jeho nadměrná exprese výrazně přispívá k malignímu potenciálu buňky. Nadbytečná exprese tohoto genu je spojena se vznikem několika nádorů, převážně s neuroblastomem. Neuroblastom je maligní embryonální nádor dětského věku odvozený z nezralých a nediferencovaných buněk neurální lišty. V současné době mají v léčbě rakoviny velký potenciál liposomy pro cílený transport léčiv a genovou terapii. Cílem této studie bylo vyvinout nanokonstrukt skládající se z oligonukleotidu (AAA-ODNSH) značeného na jedné straně adeninovými repeticemi pro vazbu na tyminem modifikovanou magnetickou částici a na straně druhé thiolovou skupinou pro vazbu zlatem modifikovaného liposomu s uzavřenými léčivy (etoposidem, elipticinem a doxorubicinem). Námi navržený nanokonstrukt nesl specifickou sekvenci anti-sense oligonukleotidu (ODN sonda), který je schopen blokovat expresi N-myc genu. Doxorubicin, elipticin a etoposid a jejich enkapsulované varianty byly charakterizovány pomocí fluorescenční spektroskopie. Materials/methods Fluorescenční spektra byly získány pomocí automatického fluorimetru Tecan Infinite 200 PRO (TECAN, Švýcarsko). Excitační vlnové délky pro elipticin, doxorubicin a etoposid byly 420, 480 and 250 nm. Fluorescenční sken pro sledované látky byl v rozmezí 450 - 850 nm pro elipticin, 510 - 850 pro doxorubicin a 280 - 850 nm pro etoposid. Citlivost detektoru byla nastavena na 100%. Absorbance ssDNA byla měřena v rozmezí vlnových délek 260 - 280 nm. Vzorky objemu 2 µl byly dávkovány na 16 jamkovou nanodestičku Tecan NanoQuant plate (TECAN, Švýcarsko). Všechna měření byla provedena při 30 °C.
Results and conclusions Účinnost uzavření léčiv do liposomu byla sledována fluorescenční analýzou léčiv a léčiv uzavřených do liposomu. Z rozdílů fluorescence byla vypočítaná účinnost enkapsulace 50 %. Dále byl sledován výtěžek hybridizace nanokonstruktu, ukotveného na magnetické částici, stanovením koncentrace ssDNA. Po každém hybridizačním kroku byly nenavázané oligonukleotidy odstraněny promýváním ukotveného nanokonstruktu na magnetické částici. Pro oligonukleotid navázaný na magnetickou částici a liposom (AAA-ODN-SH) byl výtěžek 8 %, pro ODN sondu s cílovou sekvencí pro N-myc gen byl 6 % a konečný výtěžek hybridizace N-myc genu k nanokonstruktu byl 4%. Koncentrace enkapsulovaných léčiv v nanokonstruktu byla 0,8 µg/ml pro doxorubicin, 10 µg/ml pro etoposid a 60 µg/ml pro elipticin. V naší studii jsme uvedli možný způsob využití nanokonstruktu pro genovou terapii, tak jako pro značení cílových neuroblastomových buněk. Autoři děkují za finanční podporu grantové agentuře ČR (NANOCHEMO GA CR 14-18344S).
SLEDOVÁNÍ INTERAKCE DOXORUBICINU A ELIPTICINU S BIOLOGICKY ODBOURATELNOU SLOUČENINOU - ALBUMINEM
Sylvie Sklickova1,2, Lukas Nejdl1,2, Martina Omastova1, Branislav Ruttkay-Nedecky1,2, Tomas Eckschlager3, Marie Stiborova4, Vojtech Adam1,2, Rene Kizek1,2 Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, Czech Republic, 2 Central European Institute of Technology - Brno University of Technology, Brno, Czech Republic, 3 Department of Paediatric Haematology and Oncology, 2nd Faculty of Medicine, Charles 1
University, and University Hospital Motol, Prague, Czech Republic, 4 Department of Biochemistry, Faculty of Science, Charles University, Pregue, Czech Republic Introduction Doxorubicin (hydroxyldaunorubicin) je antracyklinové cytostatikum s vysokou účinností. Jde o kardiotoxickou látku, proto jej lze podávat jen v omezených dávkách. Elipticin a jeho deriváty jsou účinné protinádorové látky, které fungují prostřednictvím různých mechanismů účastnících se zastavení buněčného cyklu a zahájení apoptózy. Interakcí s DNA však dochází k jejímu poškození. Bovinní sérový albumin (BSA) obsahuje 2 tryptofany (Trp-134, Trp212). Lidský sérový albumin (HSA) má naproti tomu jen 1 tryptofan (Trp-214). Tryptofan je velmi citlivý na změny okolního prostředí. Při jeho zkoumání se proto využívá vlastní fluorescence. Princip zhášení fluorescence je charakterizován bimolekulárními deaktivačními mechanismy, které zahrnují přenos energie z jedné molekuly na druhou. Jde o proces, který snižuje kvantový výtěžek fluorescence, vyvolaný molekulárními interakcemi se zhášecí molekulou. Snímání zhášení fluorescence tryptofanu BSA a HSA lze tedy využít pro sledování interakce doxorubicinu a elipticinu se sérovými proteiny. Materials/methods Fluorescenční spektra byla naměřena pomocí fluorimetru Tecan Infinite 200 PRO (TECAN, Švýcarsko). Excitační vlnová délka pro albumin byla 280 nm. Fluorescenční sken byl v rozsahu 280 - 430 nm. Citlivost detektoru byla nastavena na 100%. Vzorky byly měřeny 2x na 96 jamkové mikrotitrační destičce Costar (Fisher Scientific, USA). Všechna měření probíhala při 30 °C . Results and conclusions Interakci sérových proteinů s doxorubicinem a elipticinem lze sledovat prostřednictvím zhášení
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fluorescence tryptofanu bovinního sérového albuminu (BSA, 2 tryptofany) a lidského sérového albuminu (HSA, 1 tryptofan). Tímto zhášením fluorescence se snižuje kvantový výtěžek fluorescence, který je vyvolaný molekulárními interakcemi se zhášecí molekulou. Cílem práce byla charakterizace interakce albuminu s doxorubicinem a elipticinem v různém pufračním prostředí pomocí UV/VIS spektrofotometrie. Jako prostředí pro interakci byl zvolen fyziologický roztok (0,9 % NaCl). Pro interakci BSA a HSA s doxorubicinem byly stanoveny rovnice regrese y = -49,41x + 5033,9 a y = -30,459x + 2638,1 s R² = 0.9594 a 0.8956. Pro interakci BSA a HSA s elipticinem byly stanoveny rovnice regrese y = -622.61x + 103876 a y = -314.38x + 47559 s R² = 0.9203 a 0.8925. Z výsledků vyplývá, že se stoupajícími koncentracemi doxorubicinu a elipticinu se lineárně snižuje emitované záření albuminu. Strmější pokles intenzity fluorescence byl pak zjištěn u vzorků, které neprošly hodinovou inkubací. Autoři děkují za finanční podporu grantové agentuře ČR (GA CR CYTORES P301/10/0356).
STUDY OF AFFINITY OF COORDINATION COMPLEXES OF METAL IONS TO DNA BASED ON THE CHANGE OF FLUORESCENCE INTENSITY OF INTERCALATION LABELS (DOXORUBICIN AND ETHIDIUM BROMIDE)
Lukas Nejdl1,2, Jiri Kudr1,2, Sylvie Skalickova1,2, Branislav Ruttkay-Nedecky1,2, Tomas Eckschlager3, Marie Stiborova4, Vojtech Adam1,2, Rene Kizek1,2 Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic, 2 Department of Chemistry and Biochemistry, Laboratory of Metallomics and Nanotechnologies, Mendel 1
University in Brno, Brno, Czech Republic, 3 Department of Paediatric Haematology and Oncology, 2nd Faculty of Medicine, Charles University, and University Hospital Motol, Prague, Czech Republic, 4 Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic Introduction Metals are ubiquitous and essential for cellular processes in all living organisms [1, 2]. Metal ions coordination to the nucleic acids is critical for their structural properties and proper function [3]. Given that the cytostatic platinum-based drugs are successful in a number of cancer diseases, enormous research developing metal-based anticancer agents and to elucidating the mechanisms involved in the action of these compounds. The success of platinum complexes in cancer therapy is due to their ability to bind with coordination bond to the DNA bases [4, 5]. The formation of these bonds results in influencing the secondary structure of DNA and thereby blocking of important cellular processes such as replication and transcription [6]. Platinum compounds are still the most effective cytostatic drugs, although a lot of Ru(II), Os(II) and/or Ir(II) compounds have a quite similar properties [7]. In this work affinity of cobalt [Co3(tet)3(m-btc)](ClO4)3 and nickel [Ni2(tet)2(m-tda)](ClO4)2 coordination complexes, cisplatin and Hg(II) ions with neuroblastoma cell line SIMA was studied. Materials/methods All chemicals cisplatin, Hg(NO3)2 and ACS water were supplied from Sigma Aldrich. Preparation of coordination complexes [Co3(tet)3(m-btc)](ClO4)3 Triethylenetetramine hydrate (tet) (0.2 g, 1 mmol) was added to a stirred solution of Co(ClO4)2.6H2O (0.36 g, 1 mmol) in water (50 mL). Solution of 1,3,5-benzenetricarboxylic acid (btcH3) (0.07 g, 0.33 mmol)
neutralized with NaOH (0.04 g, 1 mmol) in water (5 mL) was added. Finally, pH was adjusted to 7 with addition of perchloric acid and solution was diluted with water to final volume of 100 mL. [Ni2(tet)2(m-tda)](ClO4)2 Tet (0.2 g, 1 mmol) was added to a stirred solution of Ni(ClO4)2.6H2O (0.36 g, 1 mmol) in water (50 mL). Solution of thiodiacetic acid (tdaH2) (0.075 g, 0.5 mmol) neutralized with NaOH (0.04 g, 1 mmol) in water (5 mL) was added. Finally, pH was adjusted to 7 with addition of perchloric acid and solution was diluted with water to final volume of 100 mL. UV / Vis spectrophotometry and fluorescence analysis Fluorescence and absorption spectra were acquired by a multifunctional microplate reader Tecan Infinite 200 PRO (TECAN, Switzerland). Wavelength of 545 nm was used as an excitation wavelength and the fluorescence scan was measured within the range from 575 to 800 nm per 2-nm steps. The detector gain was set to 100. 50 μl of sample was placed to each well. All measurements were performed at 30°C controlled by the Tecan Infinite 200 PRO (TECAN, Switzerland). Cyclic voltammetry Cyclic voltammetry was measured in the range of potentials -1 to 1 V. Screen-printed electrodes was used [8]. Changes in electrochemical signals were recorded with a potentiostat PGSTAT 101 (Metrohm, Herisau, Switzerland) and the results were evaluated by the Software NOVA 1.8 (Metrohm, Herisau, Switzerland). Results and conclusions Synthesis, electrochemistry and spectroscopic techniques are described for cobalt [Co3(tet)3(mbtc)](ClO4)3 and nickel [Ni2(tet)2(mtda)](ClO4)2 coordination complexes, cisplatin and Hg(II) ions. Their stability in water have been studied by electrochemical methods (cyclic voltammetry) using the screen printed electrode. It was electrochemically demonstrated that
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the studied complexes and Hg(II) ions in comparison with cisplatin are more stable in water, because cisplatin showed after 48 hours a significant change in reduction signal at potential -0.13 V. The affinity of these substances with DNA isolated from the neuroblastoma cell line SIMA was also studied and assessed by fluorescence spectroscopy. The change in fluorescence intensity of doxorubicin and ethidium bromide (EtBr) was monitored. EtBr is a typical mutagen and intercalating dye for DNA and it has been studied for many years [9]. This method was investigated by Zhu et al. when studying interactions of DNA with 5-hydroxymethyl-2-furfural [10]. In contrast with doxorubicin can be observed decrease in fluorescence intensity upon intercalation into DNA. For all the studied substances higher affinity to DNA as to cisplatin was demonstrated. Acknowledgements The authors gratefully acknowledge financial support from the Grant Agency of the Czech Republic (GA CR CYTORES P301/10/0356). References [1] Gitlin J, Lill R (2006) Biochim. Biophys. Acta-Mol. Cell Res. 1763:577-577. DOI 10.1016/j. bbamcr.2006.06.011 [2] Florea AM, Büsselberg D (2011) Cancers 3:1351-1371. [3] Park KS, Park HG (2014) Curr. Opin. Biotechnol. 28:17-24. DOI 10.1016/j.copbio.2013.10.013 [4] Chang CL, Lando DY, Fridman AS, Hu CK (2012) Biopolymers 97:807-817. DOI 10.1002/bip.22077 [5] Brabec V, Kasparkova J (2005) Drug Resist. Update 8:131-146. DOI 10.1016/j. drup.2005.04.006 [6] Theile D, Detering JC, Herold-Mende C, Dyckhoff G, Haefeli WE, Weiss J, Burhenne J (2012) J. Pharmacol. Exp. Ther. 341:51-58. DOI 10.1124/ jpet.111.189621 [7] Dhahagani K, Mathan KS, Chakkaravarthi G, Anitha K, Rajesh J, Ramu A, Rajagopal G
(2014) Spectroc. Acta Pt. A-Molec. Biomolec. Spectr. 117:87-94. DOI 10.1016/j.saa.2013.07.101 [8] Prasek J, Trnkova L, Gablech I, Businova P, Drbohlavova J, Chomoucka J, Adam V, Kizek R, Hubalek J (2012) Int. J. Electrochem. Sci. 7:1785-1801. [9] Bi SY, Zhang HQ, Qiao CY, Sun Y, Liu CM (2008) Spectroc. Acta Pt. A-Molec. Biomolec. Spectr. 69:123-129. DOI 10.1016/j. saa.2007.03.017 [10] Zhu J, Chen L, Dong Y, Li J, Liu X (2014) Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy 124:78-83. DOI 10.1016/j.saa.2013.12.091
Nanotools in microRna isolation and detection
Kristyna Smerkova1,2, Marcela Vlcnovska2, Veronika Vlahova2, Marketa Vaculovicova1,2, Rene Kizek1,2 Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic, 2 Department of Chemistry and Biochemistry, Laboratory of Metallomics and Nanotechnologies, Mendel University in Brno, Brno, Czech Republic Introduction The microRNA (miRNA) molecules belong to small, non-coding RNAs that regulate gene expression. The miRNAs significantly affect different biological processes including pathological processes such as the cancer origin and development. The aberrant miRNA expressions were found at different tumor types and moreover the various tumors have specific expression profiles. Because of their importance as diagnostic and prognostic cancer biomarkers the rapid, low-cost and sensitive detection technique is required. The magnetic particles ensure specific miRNA isolation and in combination with electrochemistry detection it is possible to achieve nanomolar detection limit. The other nanoparticles used for miRNA determination in this work were 1
quantum dots (QDs), providing specific labeling. Materials/methods Magnetic particles-based miRNA isolation The magnetic microparticles (MPs) Dynabeads M-270 Streptavidin (Life Technologies) were used for miRNA isolation. After washing of MPs the biotinylated complementaryDNA probe immobilization on MPs surface was done using 500 µg of MPs and 3 µl of 100 µM biotinylated complementary-DNA probe. The mixture was incubated for 10 minutes. Further, the hybridization with the target miRNA was performed according to Huska et al. (Talanta, 2009). Subsequently, the MPs were resuspended in 50 µl of the elution solution (0.2 M NaCl, 0.1 M Na2HPO4 and 0.1 M NaH2PO4). During the elution, the sample was heated to 70°C for 5 minutes and this caused double-stranded RNA (dsRNA) denaturation and the miRNA was released from MPs surface. Conjugation of quantum dots with streptavidin, biotinylated probe and microRNA CdTe QDs capped with mercaptopropionic acid precipitated by isopropanol (0.1 mg/ml in water) were mixed with 10 µl of carbodiimidazole (10 mM in 100 mM PBS pH=7.4) and the solution was kept at 25°C for 30 min. The mixture was mixed with streptavidin solution (final concentration of streptavidin was 0.1 mg/ml) and the reaction took place at 25°C for 2 hours. Subsequently, streptavidincoated QDs were conjugated with biotinylated complementaryDNA probe (25°C for 1 hour). QDs modified by probe were hybridized with target miRNA at 25°C for 1 hour. Capillary electrophoresis with laserinduced fluorescence detection (CELIF) CE-LIF analysis was carried out using PACE/MDQ instrument (Beckman Coulter). Argon ion laser with wavelength 488 nm was used as an excitation light source and emission was measured at 510 nm.
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20 mM sodium borate buffer was used as separation electrolyte and separation voltage was 10 kV. The sample was injected by pressure 5 psi for 5 seconds into the capillary with 75 μm internal diameter, 63.5 cm total length and 53.5 cm effective length. Results and conclusions Isolation based on streptavidincoated MPs is thanks to their surface modification by biotinylated probe highly specific. Optimal amount of biotinylated complementary-DNA probe was found to be 1.02 µg per 500 µg of MPs to cover to MPs surface. The magnetic separation selectivity was verified using non-complementary oligonucleotides. The noncomplementary oligonucleotides signal was approximately 15 times lower in comparison with target complementary miRNA. This significantly higher isolation yield of target miRNA confirms the optimized magnetic separation specificity. The coupling of QDs with microRNA caused the fluorescence signal change of both the migration time and peak shape. The electrophoretic mobilities for QDs modified with streptavidin, QDs-streptavidin modified with probe and QDsstreptavidin-probe with hybridized miRNA were -16.84×10-9 m2/V/s, -20.83×10-9 m2/V/s and -21.66×109 m2/V/s, respectively. The authors gratefully acknowledge financial support from the Grant Agency of the Czech Republic (NanoBioTECell P102/11/1068).
Charakterizace dlouhé nekódující RNA MALAT-1 u buněk karcinomu prsu
Jaroslav Juracek1, Alexandra Kondelova2, Marek Svoboda1,2, Ondrej Slaby1,2 Masarykuv onkologický ustav, Klinika komplexni onkologicke pece, Brno, Czech Republic, 2 CEITEC, Masarykova univerzita, Brno, Czech Republic Introduction Přesto že strukturní geny představují 1
u člověka méně než 2 % lidského genomu, celogenomové studie odhalily, že až 90 % z celkového počtu genů je aktivně transkribováno. Tyto RNA, kterým však není přisuzována žádná protein-kódující kapacita, byly původně považovány za odpadní molekuly nahromaděné během evoluce. Nedávný výzkum však poukazuje na jejich důležitost ve fyziologických i patologických buněčných procesech. Na základě délky transkriptu jsou nekódující RNA děleny na krátké a dlouhé. Zatímco krátké nekódující RNA, mezi které se řadí např. mikroRNA, jsou dnes již velmi dobře charakterizovány, dlouhé nekódující RNA (lncRNA) byly dosud značně přehlíženy. Do této obsáhlé skupiny přitom spadá velké množství 200-10000 bp dlouhých RNA, které hrají klíčové role jak v transkripční, tak v post-transkripční regulaci genové exprese. Kromě toho byla u mnoha onemocnění včetně nádorových pozorována deregulovaná hladina lncRNA a proto lze předpokládat jejich zapojení do procesu maligní transformace. Většina lncRNA vystupuje jako onkogeny, mezi něž patří také MALAT-1, jejíž zvýšená exprese byla potvrzena i u karcinomu prsu. Právě u tohoto onemocnění je i přes snižující se mortalitu nutné hledat nové terapeutické cíle, zejména z důvodu častého rozvoje rezistence na stávající léčbu. Studium dlouhých nekódujících RNA v tomto kontextu představuje významný zdroj potenciálních cílových molekul. Materials/methods Z důvodu bližší identifikace působení dlouhé nekódující RNA MALAT-1 na buňky karcinomu prsu byly pomocí qRT-PCR stanoveny hladiny exprese této lncRNA u pěti stabilních buněčných liniích karcinomu prsu. Následná in vitro charakterizace byla provedena na dvou buněčných liniích, u kterých byl pozorován vliv utlumení MALAT-1 pomocí sekvenčně homologní siRNA na proliferaci, apoptózu, migraci a klonogenicitu. Results and conclusions Po umělém snížení hladiny MALAT-1
pomocí transfekce siRNA nebyl pozorován významný vliv této onkogenní lncRNA na proliferaci, apoptózu ani migraci. Snížení exprese MALAT-1 však mělo významný inhibiční účinek na tvorbu kolonií nádorových buněk u obou testovaných linií, což by mohlo naznačovat zapojení MALAT-1 do rozvoje metastáz.
DEREGULACE PIWIL PROTEINŮ A VYBRANÝCH PIRNA U RCC A CRC
Robert Iliev1, Petra Vychytilova1, Michal Stanik3, Jan Dolezel3, Michal Fedorko4, Dalibor Pacik4, Marek Svoboda2, Ondrej Slaby1,2 CEITEC, Laborator molekularni onkologie II - solidni nadory, Brno, Czech Republic, 2 MOU, Klinika komplexni onkologicke pece, Brno, Czech Republic, 3 MOU, Oddeleni urologicke onkologie, Brno, Czech Republic, 4 FN Brno, Urologicka klinika, Brno, Czech Republic Introduction RNA interagující s PIWIL proteiny jsou genetické a epigenetické buněčné regulační faktory, které udržují genomovou stabilitu a jsou začleněny v umlčování a regulaci genové exprese. PiRNA jsou krátké jednořetězcové RNA dlouhé 26 až 31 nukleotidů vázající se vážou na PIWIL proteiny. Jedná se podrodinu proteinů typu Argonaut. U lidí se rodina Piwi proteinů skládá z proteinů PIWIL1, PIWIL2, PIWIL3 a PIWIL4. Fyziologická exprese PIWIL proteiů byla nejdříve pozorována hlavně v zárodečných a kmenových buňkách. Současné studie ukazují, že deregulovaná exprese PIWIL proteinů je společná mnoha typům nádorů. Je prokázáno, že exprese PIWIL proteinů koreluje s klinickými parametry a s horší prognózou u pacientů s nádorem prsu, cervixu, vaječníku, střev a dalších. V prvních studiích bylo pozorováno, že se podílejí na umlčování 1
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transponovatelných elementů. Změněná exprese piRNA byla však v poslední době zjištěna i u nádorů prsu a žaludku. Materials/methods Pro náši pilotní studii byly k analýzám vybrány kromě PIWIL proteinů i piR-X a piR-Y, jejichž deregulovaná exprese byla zjištěna u různých typů nádorů. V experimentu jsme použili nádorovou a příslušnou nenádorovou tkáň 56 pacientů s renálním karcinomem (RCC). Z tkání jsme izolovali celkovou RNA a pomocí metody RT-qPCR byla stanovena exprese genů PIWIL, piR-X a piR-Y. Pro analýzu sérových piRNA jsem byl použit soubor 125 pacientů s RCC, 100 pacientů s kolorektálním karcinomem (CRC) a 75 kontrolních sér od zdravých jedinců. Results and conclusions Po porovnání hladin RNA jsme objevili významný rozdíl v expresních hladinách piR-X, piR-Y a PIWIL1 u párových vzorků mezi nenádorovou a nádorovou tkání. U hladin exprese PIWIL2, PIWIL3 a PIWIL4 jsme neprokázali statisticky významné rozdíly. Hladiny PIWIL2 a PIWIL4 u pacientů s RCC významně korelovaly se stagem a gradem. Identifikovali jsme také korelaci mezi vyšší hladinou PIWIL1, piR-X a piR-Y a delším celkovým přežitím. U piR-X byla zjištěna signifikantně snížená hladina v krevním séru pacientů s RCC. Po ROC analýze jsme odlišili pacienty a kontrolní jedince se sensitivitou 77 % a specificitou 72 %. U analyzovaných hladin sérové piR-Y v séru pacientů s RCC a CRC jsme zjistili signifikantní rozdíly v expresi oproti nenádorovým kontrolním vzorkům, přičemž pacienti s RCC a CRC měli sníženou hladinu exprese (RCC - p<0.0001, CRC – p<0.0001). Pacienty s RCC jsme po ROC analýze dokázali odlišit od kontrolních jedinců s 97% senzitivitou a 99% specificitou. Skupinu pacientů s CRC jsme od kontrolních jedinců dokázali odlišit s 94% senzitivitou a 91% specificitou. Po dalším detailním výzkumu role piR-Y v patogenezi RCC a CRC by
mohla sloužit nejen jako diagnostický prostředek, ale také jako potenciální terapeutický cíl.
Navýšení exprese miR-215 in vivo ovlivňuje velikost nádoru v animálním modelu kolorektálního karcinomu
Jana Merhautová1,2, Petra Vychytilová2, Regina Demlová1, Ondřej Slabý2 Farmakologický ústav, Lékařská fakulta, Masarykova univerzita, Brno, Czech Republic, 2 Skupina Molekulární medicína II – solidní nádory, CEITEC, Masarykova univerzita, Brno, Czech Republic Introduction MikroRNA jsou krátké nekódující RNA, které působí jako regulátory exprese většiny genů. Byla publikována řada prací prokazující dysregulaci mikroRNA u nádorových onemocnění, mimo jiné také u kolorektálního karcinomu (CRC), v jehož incidenci zaujímá ČR celosvětově přední příčky. V přecházející sérii experimentů jsme identifikovali miR-215 jako specifickou pro tkáň CRC. Tato mikroRNA v in vitro testech prokázala tumorově supresorové vlastnosti. Prezentované in vivo experimenty ověřují její působení v kontextu celého organismu. Posloužily také k zavedení a optimalizaci techniky subkutánní xenotransplantace na našem pracovišti. Materials/methods a) Transfekce a selekce klonů: Buňky linie HCT-116+/+ byly lipofekcí transfekovány CMV vektorem s klonovanou sekvencí pre-miR-215 a kontrolním mock-vektorem (OriGene Technologies, Rockville, MD, USA). Stabilně transfekované buňky byly selektovány pomocí G-418 a za fluorescenční kontroly exprese GFP. Po limitním naředění bylo provedeno namnožení 14 jednotlivých klonů buněk a jejich charakterizace (morfologie, rychlost proliferace, exprese miR-215 a genů XIAP, EREG, TYMS, HOXB9 a CD164). Vybrán byl klon s nejpříznivějším 1
expresním profilem a nejnižší rychlostí proliferace. b) Subkutánní xenotransplantace: 1. pilotní experiment - 3 samicím NSG myši bylo na dorzální stranu těla vpravo subkutánně aplikováno 5×106 buněk klonu 33 miR-215HCT-116+/+. Jako kontrola byl aplikován vlevo stejný počet buněk mock-HCT-116+/+. 27. den po aplikaci byl pokus ukončen, nádory byly změřeny a zváženy a byl proveden odběr vzorku nádorové tkáně do RNAlateru a neutrálního formalínu. 2. pilotní pokus - 5 samcům NSG myši bylo analogicky aplikováno 2,5×106 buněk klonu 33 miR-215-HCT-116+/+. Jako kontrola byl aplikován vlevo stejný počet buněk mock-HCT-116+/+. 24. den po aplikaci byl pokus ukončen, nádory byly změřeny a zváženy a byl proveden odběr vzorku nádorové tkáně do RNAlateru a neutrálního formalínu. Results and conclusions V předkládaném experimentu byly úspěšně připraveny stabilně transfekované buňky linie odvozené od CRC s navýšenou experesí miR215. Selekcí klonů s následnou charakterizací byl vybrán a namnožen klon s optimálními vlastnostmi (změna exprese miR215, genů XIAP, EREG a dalších). Namnožený klon byl subkutánně aplikován NSG myším. Po 1012 dnech byly v podkoží hmatné tumory, experimenty byly ukončeny 27. resp. 24. den po aplikaci. Odebraná nádorová tkáň byla histologicky zhodnocena, nádorové buňky byly nízce diferencované a vysoce mitoticky aktivní. Makroskopicky i mikroskopicky vykazovaly kontrolní tumory vyšší míru nekróz než tumory vzniklé z buněk s navýšenou expresí miR-215. Velikost nádorů byla signifikantně menší (p < 0,05) u tumorů vzniklých z buněk s navýšenou expresí miR215 než u kontrolních tumorů. Nebyly pozorovány mezipohlavní rozdíly v růstu nádorů u zvířat. MiR215 ovlivňuje proliferaci nádorů – v závislosti na stavu p53 indukuje zástavu buněčného cyklu a zvyšuje míru apoptózy nádorových buněk.
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Molecular genetic analysis of pheochromocytomas and paragangliomas in Czech patients
Musil Zdenek1,2, Stanek Libor3, Vícha Aleš2, Vosecka Tatiana2,4, Krepelova Anna4, Zelinka Tomas5, Hamplova Barbora5, Widimsky Jiri5, Vocka Michal6, Turkova Hana6, Puchmajerova Alena4, Simandlova Martina4, Frysak Zdenek7, Vaclavik Jan7, Kohoutova Milada1 Institute of Biology and Medical Genetics of the 1st Faculty of Medicine and General Teaching Hospital, Charles University, Prague, Czech Republic, 2 Department of Paediatric Haematology and Oncology, University hospital Motol and 2nd Faculty of Medicine, Charles University, Prague, Czech Republic, 3 Institute of Pathology of the 1st Faculty of Medicine and General Teaching Hospital, Charles University, Prague, Czech Republic, 4 Department of Biology and Medical Genetics, University hospital Motol and 2nd Faculty of Medicine, Charles University, Prague, Czech Republic, 5 rd 3 Medical Department - Clinical Department of Endocrinology and Metabolism of the 1st Faculty of Medicine, Charles University, Prague, Czech Republic, 6 Department of Oncology of the 1st Faculty of Medicine and General Teaching Hospital, Charles University, Prague, Czech Republic, 7 rd 3 Clinical Department of Faculty of Medicine, Olomouc, Czech Republic Introduction Pheochromocytomas are tumors arising from adrenal gland, whereas paragangliomas are tumors arising from extra-adrenal chromaffin tissue. Functional paragangliomas 1
are secreting tumors which can be found mainly in the abdomen region, pelvis and chest. Contrarily, paragangliomas located in the skull region and neck are non-functional in most cases. Most of these tumors occur as sporadic tumors but up to 24% of them can be familial. The prevalence of pheochromocytoma can be estimated to lie between 1:1450 and 1:1700, with the annual incidence of 3-8 cases out of a million inhabitants in general public. Up to 10% of PHEO/PARA is malignant (they are able to produce distant metastases or they are tumors that recur). Genetic factors have a significant role in the PHEO/ PARA development. Currently, 18 genes (SDHA, SDHB, SDHC, SDHD, VHL, RET, NF1, MAX, TMEM 127, SDHAF2, KIF 1B, HIF 2A, H-RAS, K-RAS, IDH 1, PHD 2/ EGLN 1, FH, BAP 1) participating in PHEO/ PARA development are described. Pheochromocytomas and paragangliomas are also associated with genetic syndromes (MEN2, VHL, PGL 1 - 4, Pacak-Zhuang). Many mutations can be found as somatic mutations in cancerous tissues. Materials/methods Genetic testing, which should be indicated in every patient, is guided by the clinical presentation as well as by the secretory phenotype and the immunohistochemical characterization of the tumours. Genetic examinations may also have prognostic significance, since for example SDHB gene mutations are connected with significantly higher probability of malignancy. Mutation detection can be helpful in diagnostics of other manifestations of life-endangering genetic syndromes (for example MEN2related medullary carcinoma, or VHL-related central nervous system or abdominal tumors). Results and conclusions PHEO/PARA can be divided into two groups according to the expression change in genes being present in various signaling pathways: the first group leeds to the activation of pseudohypoxic pathway (VHL,
SDHA, SDHB, SDHC, SDHD, probably also HIF2A and FH), the second group includes receptor signaling kinases and protein translation pathways (NF1, RET, KIF1B , TMEM127 and MAX). This work was supported by the research projects PRVOUK-P27/ LF1/1, SVV-260023/2014 and the research organisation 00064203
Skp2 associates with Slug and androgen receptor in patients with high Gleason score and lymph node metastasis of prostate cancer
Gvantsa Kharaishvili1, Jan Bouchal1, Milan Kral2 Laboratory of Molecular Pathology, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic, 2 Department of Urology, University Hospital Olomouc, Olomouc, Czech Republic Introduction The Skp2 F-box protein is the substrate recruiting component of the SCF (Skp1-Cullin 1-F-box) type of E3 ubiqutin-ligase complexes. Skp2 plays important role in prostate tumorigenesis which needs further elucidation. Materials/methods Prostate cancer patients cohort (N=101) was analyzed by immunohistochemistry for the following proteins (Skp2, AR, Ki67, Slug, E-cadherin, beta-catenin and PPAR gamma) and statistically evaluated with clinico-pathological parameters. Results and conclusions High Gleason score (>8, N=30) was significantly associated with higher nuclear Skp2 and lower E-cadherin expression (p<0.001 and 0.011, respectively) and with a trend for higher androgen receptor (p=0.062). Patients with metastasis in lymph nodes (N=29) had lower E-cadherin and cytoplasmic Skp2, (p<0.001 and 0.018, respectively), while nuclear 1
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Skp2 and PPAR gamma, Slug (both nuclear and cytoplasmic), Ki-67 and androgen receptor showed a trend (p=0.176, 0.179, 0.103, 0.032, 0.079 and 0.12, respectively) towards higher expression in the prostate cancer cells. Similar changes of the mentioned proteins were observed also in risk categorization (based on serum PSA, pT classification and Gleason score). Nuclear Skp2 slightly correlated with AR in the whole patients cohort (Rs 0.37). In patients with high Gleason score, nuclear Skp2 potently correlated with AR, nuclear Slug and cytoplasmic PPAR gamma (Rs 0.53, 0.56 and 0.37, respectively). In patients with metastasis into lymph nodes, nuclear Skp2 similarly correlated with nuclear Slug and AR (Rs 0.56 and 0.37, respectively) while androgen receptor further correlated with Ki-67 (Rs 0.50). In summary, Skp2 correlates with androgen receptor and Slug which might contribute to agressive prostate cancer. Further mechanistical elucidation and clinical validation is needed.
Ubiquitin specific peptidase 7 (USP7) regulates DNA damage bypass pathway through stabilizing Cdc7 kinase and RAD18 ubiquitin ligase.
Zsofia Turi1, Marketa Senkyrikova1, Jiri Bartek1,2, Masayuki Yamada1 Laboratory of Genome Integrity, Institute of Molecular and Translational Medicine, Palacky University, Olomouc, Czech Republic, 2 Danish Cancer Society Research Center, Copenhagen, Denmark Introduction Cdc7 kinase is essential to replicate genomic DNA. However, its role in DNA repair and recombination, as well as its interplay with ubiquitinproteasome system, are poorly understood. We recently described a novel pathway that stabilizes the human Cdc7-ASK (also called Dbf4), its regulation, and its function in 1
cellular responses to compromised DNA replication. Stalled DNA replication evoked stabilization of the Cdc7-ASK complex in a manner dependent on ATR-Chk1-mediated checkpoint signaling and its interplay with the anaphase-promoting complex/ cyclosome(Cdh1) (APC/CCdh1) ubiquitin ligase. Mechanistically, Chk1 kinase inactivates APC/CCdh1 through degradation of Cdh1 upon replication block, thereby stabilizing APC/CCdh1 substrates, including Cdc7-ASK. Furthermore, motif C of ASK interacts with RAD18, a ubiquitin ligase essential for DNA damage bypass pathway, and this interaction is required for chromatin binding of RAD18. Impaired interaction of ASK with RAD18 disables foci formation by RAD18 and hinders chromatin loading of translesion DNA polymerase , suggesting that Cdc7 kinase plays a pivotal role in DNA damage bypass pathway. In this workshop, we will present new data which strongly suggest that USP7 is involved in DNA damage bypass pathway through stabilizing both Cdc7-ASK and RAD18. Materials/methods The protein abundance of Cdc7ASK and RAD18 as well as monoubiquitylated PCNA, which is catalysed by RAD18, were examined in USP7-depleted U2OS cells under replication stress by Western blotting. HCT116-derived USP7 knockout cell line was also used for the same analysis. Moreover, foci formation of RAD18 in USP7 knockdown cells was examined by immuostaining and subsequent quantitative data analysis with ScanR software. Results and conclusions We found a significant decrease of the protein levels of Cdc7-ASK and RAD18 in USP7 knockdown cells and USP7 knockout cells. We also found that monoubiqutylation of PCNA under replication stress was impaired in these cells. In addition, foci formation of RAD18 caused by replication stress was disturbed by knockdown of USP7. These data
indicate that USP7 regulates protein stability of Cdc7-ASK and RAD18, therefore plays an important role in DNA damage bypass pathway. Our new findings will define a novel mechanism that orchestrates ubiquitin-proteasome machinery and DNA damage bypass pathway to guard against replication collapse under conditions of replication stress.
Is Epithelial to Mesenchymal Transition Followed by Global DNA Methylation Changes?
Svetlana Skolekova1, Naouale El Ymani2, Mรกria Dusinska2, Viera Kajabova1, Tatiana Sedlackova3, Iveta Zmetakova1, Tomas Krivulcik1, Ivana Fridrichova1, Miroslava Matuskova1, Bozena Smolkova1 Cancer Research Institute of Slovak Academy of Sciences, Bratislava, Slovakia, 2 Health Effects Laboratory MILK, NILU- Norwegian Institute for Air Research, Kjeller, Norway, 3 Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia Introduction Deregulation of the epigenetic mechanisms, which developmentally influence gene expression via modifications to DNA, histone proteins, and chromatin, have been hypothesized to play a key role in many human diseases including cancer. Tumour cells before intravasation can undergo partial or total epithelial mesenchymal transition (EMT). Recent investigation of the mechanisms controlling EMT revealed a significant epigenetic regulatory impact. It was shown, that soluble factors in the tumour microenvironment can orchestrate EMT and induce cancer stem cells (CSC) formation from more differentiated tumour cells. We hypothesize, that cells in their epigenetically plastic 1
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state could be programmed by the microenvironment, to acquire epigenetic changes associated with tumourigenesis and EMT induction. Materials/methods The aim of our study was to induce EMT in in vitro model system and simultaneously investigate changes in global DNA methylation. As EMT is one of the main mechanisms underlying development of cancer metastasis, which induces stem like properties and confers drug resistance, reversing of EMT process could be a unique therapeutic approach. Taking into account reversible character of epigenetic processes, epigenetic therapy offers a potential way to influence those pathways directly. Results and conclusions After induction of EMT by mesenchymal stromal cells in primary breast cancer cell culture we evaluated EMT-associated changes in global and gene specific DNA methylation (CDH1 and TWIST promoter methylation). Understanding of the epigenetic regulation of EMT processes can contribute to the new approaches in epigenetic drugs discovery. This work was supported by the European Commission FP7 projects NANoREG [NMP.2012.1.3-3], Contract no. 310584; QualityNano [INFRA-2010-1.131] Contract no: 214547-2, NILU-TAF-279; VEGA 2/0169/14 grant and RFL2009 program founded by the Slovak Cancer Research Foundation.
The effect of acquired resistance to paclitaxel on expression of ABC transporters at breast cancer cells: role of ABCB1
Dana Kopperová1, Matej Behounek1, Kamila Balušíková1, Vlasta Nemcová1, Veronika Brynychová2, Viktor Hlavác2, Pavel Soucek2, Jan Kovár1 1
Department of Biochemistry, Cell and Molecular Biology, Division
of Cell and Molecular Biology, Third Faculty of Medicine, Charles University in Prague, Prague, Czech Republic, 2 Department of toxicogenomic, National Institute of Public Health, Prague, Czech Republic, Prague, Czech Republic Introduction Acquired resistance of cancer cells to chemotherapeutics is very spread phenomenon that prevent successful therapy of patients. The increased expression of ABC transporters represents one of main suspect mechanisms of acquirement of cancer cell resistance. Materials/methods We studied molecular mechanism of resistance of cancer cells to taxanes in breast cancer cell lines SKBR-3 and MCF-7 and in paclitaxelresistant sublines (derivate from the original lines by cultivation in medium with increasing concentration of paclitaxel). We determined the expression of all 49 human ABC transporters at mRNA level by realtime PCR in paclitaxel-sensitive and resistant sublines in SK-BR-3 a MCF-7 cells. Moreover, the role of transporter ABCB1 in resistance to paclitaxel was tested at protein level by western blot and by silencing of its expression by specific siRNA. Results and conclusions We studied molecular mechanism of resistance of cancer cells to taxanes in breast cancer cell lines SKBR-3 and MCF-7 and in paclitaxelresistant sublines (derivate from the original lines by cultivation in medium with increasing concentration of paclitaxel). We determined the expression of all 49 human ABC transporters at mRNA level by realtime PCR in paclitaxel-sensitive and resistant sublines in SK-BR-3 a MCF-7 cells. Moreover, the role of transporter ABCB1 in resistance to paclitaxel was tested at protein level by western blot and by silencing of its expression by specific siRNA. Conclusions: Taken together, we suppose increased efflux of paclitaxel from cell in result from induction of
transporter ABCB1 expression. This is probably significant but not the only one mechanism responsible for acquired resistance of tested breast cancer cells cancer.
Vliv rentgenového záření na genovou expresi v lidských chlupových folikulech
Hanuš Slavík, Pavlína Dušková, Laura Ewerlingová, Karolína Burdová, Jiří Drábek, Martin Mistrík UMTM, Olomouc, Czech Republic Introduction Vlasové a chlupové folikuly jsou lehce dostupným zdrojem genomické DNA a RNA. Jejich odběr je méně invazivní než biopsie a venopunkce a má potenciál poskytovat diagnostické biomarkery vlivu rentgenového záření. Cílem našeho projektu je zavést metodiku, která by umožnila kvantifikaci exprese genů na dráze ATM/CHEK2/p53, modifikovaných vlivem záření. Materials/methods Chlupy byly odebrány pracovníkům laboratoře pomocí speciální vakuové pistole, která zamezuje kontaminaci a degradaci vzorku a standardizuje množství odebíraného materiálu. Vzorky byly izolovány a analyzovány ihned po odběru nebo po předchozí 3 h či 24 h inkubaci v buněčném médiu, kdy část z nich byla vystavena záření dávkou 6,32 Gy. Celková RNA byla izolována pomocí miRNase Mini Kit a eluována do 30 µl. Množství a kvalita RNA byly analýzovány na Agilent Pico RNA Chipu, popřípadě fluorometricky. Následovala dvou-kroková RTqPCR pro geny SESN1, CDKN1A a MDM2, s výsledky normalizovanými na expresi provozního genu HPRT1. Reakce probíhaly jednotlivě (ve čtyřech zkumavkách) s detekcí signálu z interkalovaného fluoroforu SYBR Green I. Results and conclusions Izolovali jsme dostatečné množství RNA v dobré kvalitě i po 24 h inkubaci ve vhodně zvoleném buněčném médiu. Optimalizovali
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jsme qPCR pro dané geny pro dosažení specifické amplifikace. Prezentujeme předběžné výsledky optimalizace metodiky. Práce na projektu byly prováděny s podporou grantů LO1304 a CZ.1.05/3.1.00/14.0307
Sada 6-ti mikroRNA predikuje celkové přežívání u pacientů s multiformním glioblastomem
Jiri Sana1,2, Lenka Radova1, Radek Lakomy2, Leos Kren4, Pavel Fadrus3, Jana Nekvindova5, Rostislav Vyzula2, Ondrej Slaby1,2 Stredoevropsky technologicky institut - CEITEC, Masarykova univerzita, Brno, Czech Republic, 2 Klinika komplexni onkologicke pece, Masarykuv onkologicky ustav, Brno, Czech Republic, 3 Oddeleni neurochirurgie, Fakultni nemocnice Brno, Brno, Czech Republic, 4 Oddeleni patologie, Fakultni nemocnice Brno, Brno, Czech Republic, 5 Ustav klinicke biochemie a diagnostiky, Fakultni nemocnice Hradec Kralove, Hradec Kralove, Czech Republic Introduction Multiformní glioblastom (GBM) je nejčastěji se vyskytující maligní nádor mozku astrocytálního původu s mediánem celkového přežívání přibližně 13 měsíců od stanovení diagnózy. Ačkoliv je prognóza jednotlivých pacientů značně rozdílná, histologické profily tohoto nádorového onemocnění jsou si navzájem velmi podobné. Proto je velmi důležité najít takové molekulární markery, které by onkologům v klinické praxi umožnily s co největší přesností stanovit prognózu pacienta a predikovat odpověď na standardně podávanou adjuvantní konkomitantní chemoradioterapii s temozolomidem (RT/TMZ) a případně se tak rozhodnout pro intenzivnější či alternativní způsoby léčby, jež se v současné době začínají v léčbě GBM testovat. Z tohoto pohledu jsou nyní velmi diskutované mikroRNA 1
(miRNA), krátké nekódující RNA, které posttranskripčně regulují genovou expresi a jejich deregulace tak výrazně ovlivňuje biologii mnoha nádorových onemocnění, nevyjímaje GBM. Materials/methods Pomocí technologie TaqMan Low Density Array byla provedena globální expresní analýza miRNA u 58 vzorků FFPE tkáně GBM a 10 FFPE vzorků nenádorové mozkové tkáně získané z temporálních laloků resekovaných u pacientů trpících epilepsií. Results and conclusions Porovnáním exprese miRNA v nádorové a nenádorové tkáni bylo identifikováno 28 významně deregulovaných miRNA, které byly zpětně schopny všechny zkoumané vzorky správně klasifikovat. Navíc korelace s klinickými daty pacientů s GBM identifikovala sadu 6-ti miRNA (miR-31, miR-224, miR-432*, miR-454, miR-672 a miR-885-5p), která byla významně asociována jak s celkovým přežíváním, tak s přežíváním bez progrese onemocnění pacientů s GBM. Na základě dosažených výsledků se tedy domníváme, že zmíněné miRNA by mohly být slibnými diagnostickými a prognostickými markery u GBM. Následné in-vitro funkční analýzy navíc ukázaly, že zvýšená exprese miR-31 vedla k zástavě buněčného cyklu, což se projevilo na snížené proliferaci u GBM buněčných linií. Současně došlo u těchto buněk k významnému poklesu schopnosti migrovat. Práce byla podpořena grantovým projektem NT13514-4/2012 MZČR a projektem „CEITEC Středoevropský technologický institut” (CZ.1.05/1.1.00/02.0068).
The comparison of 2-D and 3-D model of gene therapy mediated by genetically modified mesenchymal stem cells on human ovarian carcinoma cells SKOV-3.
Lenka Toro, Lucia Kucerova Cancer Research Institute of
Slovak Academy of Sciences, Bratislava, Slovakia Introduction Adipose tissue-derived mesenchymal stem cells (AT-MSC) may serve as vehicles carrying genes capable of conversion of non-toxic substances into their toxic derivates to destroy the tumor. We engineered AT-MSC to express bacterial cytosine deaminase (BCD) or yeast cytosine deaminase::uracil phosphoribosyltransferase (CD::UPRT), capable of converting the prodrug 5-fluorocytosine (5-FC) into highly toxic 5-fluorouracil (5FU). Synthetised toxic metabolites induce apoptosis and together with bystander effect increase the elimination of tumor cells. SKOV3 represents a human ovarian carcinoma cell line resistant to a variety of chemotherapeutics. Materials/methods 2-D model: Cell line SKOV-3/GFP was cultured in concentration gradient of 5-FU and the effect of chemotherapeutic was evaluated by using fluorimetric assay, chemoluminescent assay and cell kinetic imaging. Also we tested the chemosensitivity of SKOV-3 cells alone or co-cultured with AT-MSC, BCD-MSC or CD::UPRT-MSC in the presence of 5-FC. The cytotoxic effect of therapeutical AT-MSC was evaluated using fluorimetric assay. 3-D model: SKOV-3/GFP cells were seeding acording to CellPlayerTM 96-well Kinetic 3-D Spheroid Protocol (Essen BioScience) with different ratio of CD::UPRT-MSC in the absence/presence of 5-FC. Evaluation was performed by chemoluminescence assay and cell kinetic imaging. Results and conclusions SKOV-3 cells showed chemosensitivity upon exposure to 5-FU. The treatment using CD::UPRT-MSC is more efficient than bacterial BCD-MSC under the same conditions. Remarkably the ratio 1:160 of CD::UPRT-MSC to SKOV-3 cells was efficient enough to eliminate 60-80% of tumor cells.
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3-D cultivation of SKOV-3 cells with CD:UPRT-MSC in the presence of 5-FC showed decreased response to the treatment in comparison to 2-D culture. Nevertheless, the 40% elimination of SKOV-3 cells was obtained, when ratio of CD::UPRTMSC to SKOV-3 cells was 1:10 in the presence of 5-FC.
MESENCHYMAL STROMAL CELLS PLAY AN IMPORTANT ROLE IN MIGRATION AND CHEMOSENSITIVITY OF BREAST CANCER CELL LINES
Svetlana Skolekova, Lucia Kucerova Cancer Research Institute of Slovak Academy of Sciences, Bratislava, Slovakia Introduction Mesenchymal stromal cells (MSC) are an important component of tumor microenvironment and can directly interact with tumor cells or alter the behaviour of the tumor cells in paracrine manner. MSC are capable of altering the behavior of tumor cells on multiple levels. Their preference for injured, inflamed and tumor tissue is exploited in regenerative medicine and also as a promising therapeutic approach in gene-directed enzyme prodrug therapy. However, it is very important to understand the crosstalk between MSC and tumor cells for their safe use. Materials/methods The tumor cells were cultivated in conditioned medium from adipose tissue-derived mesenchymal stromal cells (CM-MSC) or in chemotherapy pretreated CM-MSC. We analysed chemosensitivity, migration and proliferation of breast cancer cell lines by fluorimetric, mammosphere culture assay and IncuCyte ZoomTM Kinetic Imaging System. The expression of specific genes was analysed by PCR. Results and conclusions AT-MSC-secreted factors are able to increase migration of Sk-Br-3 and MCF-7 breast cancer cell lines. We propose the role of SCF/cKit signaling
in MSC-mediated migration of tumor cells as the expression of both increased in tumor cells cultivated in CM-MSC. Chemotherapy pretreated AT-MSC secreted factors protecting tumor cells against apoptosis and increasing resistance of tumor cells to cisplatin and doxorubicin in 2D and also 3D conditions. We have shown that AT-MSC express a wide scale of cytokines, chemokines and growth factors, which influence tumor cells depending on the context and are able to confer increased metastatic potential and resistance of breast cancer cells. Thus, there is a need to consider intrinsic properties of MSCs during their application in cancerrelated diseases and potential interaction that might be important for the therapeutic efficiency.
A novel non-laborious and cost-effective approach for mammalian cell synchronization
Martin Liptay, Kamila Jahodíková, Zuzana Loubalová, Juraj Kramara, Iva Protivánková, Martin Mistrík, Jiří Bártek Institute of Molecular and Translational Medicine, Olomouc, Czech Republic Introduction In the study of molecular and biochemical events and their consequences during the cell division, it is crucial to use a population of cell cycle synchronized cells. Many methods have been established to synchronize mammalian cells at specific phases of the cell-cycle. Commonly used cell synchronization techniques include pharmacological inhibition of processes that are essential for the cell cycle progression (e.g. thymidine block, mitotic poisons), serum starvation, elutriation, sorting and mitotic shake-off. These approaches differ in many essential aspects like the yield of synchronized cells, toxicity, cost- and labor demands. Choosing the appropriate technique
is then always a trade-off among these parameters. Materials/methods The method combines the principle of mitotic shake-off with so-called anchorage dependency. Constant vibration causes the loosely attached mitotic cells to detach from the support and remain in suspension. Anchorage dependency prevents the detached cells from finishing cell division and arrests them in the post-mitotic, bi-nuclear stage. Results and conclusions We will introduce a cell synchronization method that overcomes some of the above mentioned limitations and provides a simple means of getting synchronized cells from adherently growing mammalian cultures in sufficient amount and purity.
MIF: A prognostic marker in Glioblastoma multiforme?
Nato Narsia, Mariam Gachechiladze, Gvanca Kharaishvili Department of Clinical and Molecular Pathology, Institute of Molecular and Tranlational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic Introduction Glioblastoma Multifome (GBM, WHO grade IV astrocytic tumour) is one of the most aggressive malignant tumors in humans. Macrophage migration inhibitory factor (MIF) is a pluripotent cytokine which plays an important role in inflammation, immune response and in cancer development. Role of MIF in tumor growth and malignant behavior appears to be complex. Contrasting reports on MIF functions were published where in some cancers MIF acts as a tumor suppressor through the induction of various cytokines from macrophages, and in other cancers it functions as a promoting factor when neutralized by anti-MIF antibody. These conflicting results suggest that MIF exerts dual functions with regard to tumor cell growth and behavior, possibly due
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to localization of the MIF protein. In the current study MIF protein expression as a prognostic factor in different grades of gliomas is elucidated. Materials/methods FFPE tissue of 87 gliomas were included in this study out of which 15 were diffuse astrocytomas, 14 were anaplastic astrocytomas and 58 were glioblastomas. MIF protein expression was detected by imunohistochemistry. Nuclear and cytoplasmic expression was assessed semiquantitaively by histoscore. Logistic regression analysis was done to evaluate prognostic importance. Results and conclusions MIF protein expression was present diffusely in the cytoplasm of tumor cells in all tumor specimens examined. An increase in MIF expression by every 10 units resulted in 5% decrease of risk of death in GBM group (n=58). A significant association between cancer related death and MIF cytoplasmic expression was observed (p=0.039), indicating MIF as a good prognostic factor in GBM. However, studies on larger patient cohorts is necessary.
Ultra deep amplicon sequencing of RAS genes and its use for mCRC predictive diagnostics
Rastislav Slavkovsky, Jana Stranska, Veronika Venskova, Veronika Holinkova, Miroslava Rabcanova, Jiri Drabek Introduction EGFR pathway regulates cancercell proliferation, apoptosis and tumor-induced angiogenesis. Tumor DNA testing of KRAS and NRAS (RAS) genes,from the EGFR signaling network, is a prerequisite for proper personalized biological treatment using anti EGFR drugs (panitumumab and cetuximab) in mCRC. The anti-EGFR treatment is prescribed only in wildtype RAS patients. Detection of increasing
number of possible mutations with probe-based qPCR is cumbersome while amplicon ultra deep nextgeneration sequencing (NGS) has a potential to be suitable method for simultaneous direct detection of all somatic mutation within tested regions and with a defined detection limit. Aim of the study is to optimize and verify KRAS and NRAS NGS sequencing of tumor DNA samples for detection of mutations at codon 12, 13, 59, 61, 117, and 146 of NRAS and KRAS genes. Materials/methods DNA samples of mCRC patients with sufficient quality of DNA were used for testing. Two methods of NGS RAS assays with longer (189 – 295 bp) or shorter (70 - 150 bp) amplicons were introduced into laboratory. Both RAS assays consist of qPCR with a control of amplicons by melting curves and purification of the samples. “Long RAS assay” continues with tagmentation and index amplification, otherwise “short RAS assay” procedures consist of end-repair, adapter ligation, purification, and index amplification. After size selection of the products and their purification, samples from both assays were normalized, pooled, and applied to platform Illumina MiSeq. Results with at least 5 % mutation frequency were concluded as “mutation detected”, according to consensus of the Czech Society of Pathology. Results and conclusions In every RAS run, KRAS G13D (46 1 %) or NRAS Q61L (47 3 %) mutation standards were correctly found, confirming reproducibility of the method. The first pilot study of 24 samples was in 100 % concordance with therascreen® KRAS RGQ PCR Kit (KRAS codons 12 and 13, CE-IVD, Qiagen), in 96 % in last 50 samples. Interlaboratory comparison of 12 samples with CGB Ostrava yielded one discrepant sample that will be retested in both laboratories. External quality assessments yielded one result differing from consensus out of ten. Upon investigating the
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cause of this discrepance with EQA organizers, we found that sample was artificially prepared cell line that did not cover sequence of our primers. During almost one year, more than 237 samples were tested. So far we have detected mutations in 11 out of 12 investigated codons.Thirty six out of 40 samples that were not possible to be analyzed by “long RAS assay” due to DNA quality, were succesfully analyzed by “short RAS assay”. 4 samples (10 %, 1.7 % from total number of tested samples) remained untestable. Taken together we show that it is crucial to understand the topology of DNA around the studied mutations. Especially in case of FFPE derived samples the shortening of the sequencing area could be very beneficial for improving the quality of the results. The work was supported by grants CZ.1.07/2.3.00/30.0060 and CZ.1.07/2.3.00/30.0041.
Výhody vysokokapacitního qPCR pro genově expresní profilování. Analýza a aplikace. (Advantages of highthroughput qPCR for gene expression profiling. Analysis and applications.) úterý / 2. prosince 14.15 - 14.30 hod.
2014
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Vlasta Korenková1, Lucie Langerová1, Vendula Novosadová1, Mikael Kubista1,2 Biotechnologický ústav AV CR, v.v.i., Praha, Czech Republic, 2 TATAA Biocenter, Goteborg, Sweden Introduction Expresní profilování představuje měření exprese více genů najednou za účelem vytvoření obrazu o buněčné funkci. Profily genové exprese mohou být použity například pro diagnostické monitorování odpovědi pacienta na léčbu a tím k optimalizaci individuální terapie. Materials/methods 1
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Vysokokapacitní zařízení BioMark (Fluidigm), které je umístěno v qPCR servisním pracovišti Biotechnologického ústavu AV ČR, umožňuje současnou analýzu exprese až 96 genů v 96 vzorcích (9216 reakcí) v rámci jediného experimentu. Měření probíhá pomocí kvantitativní polymerázové řetězové reakce (qPCR), která je jednou z nejcitlivějších metod pro kvantifikaci mRNA či mikroRNA. Možnost analyzovat větší množství genů najednou vybízí k použití multivariantních statistických analýz. Results and conclusions Využití vysokokapacitního qPCR přístroje BioMark poskytuje množství výhod. 1. Efektivita: Výrazná úspora vzorku (5 µl pro kompletní analýzu), detekční chemie, mastermixu a spotřebního materiálu. 2. Rychlost: Celý vysokokapacitní experiment lze provést v rámci několika hodin, ne dnů či týdnů. 3. Automatizace a vysoká kapacita: Díky zautomatizovanému pracovnímu postupu je minimalizována možnost lidské chyby. Není potřeba mezidestičkové kalibrace. 4. Sensitivita a flexibilita qPCR systému. 5. Možnost dalších aplikací: vysokokapacitní genotypování a digitální PCR.
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