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NATURAL COMPOUNDSIN CANCER PREVENTION AND TREATMENT
BOOK OF ABSTRACTS
OCTOBER 13-15, 2009, SMOLENICE CASTLE, SLOVAKIA
CANCER RESEARCH INSTITUTE,SLOVAK ACADEMY OF SCIENCES
ANDSLOVAK CANCER RESEARCH FOUNDATION
INTRODUCTION
The health-beneficial effects of plants or their extracts and chemical structure diversityrepresentthefountofinspirationfornewwaysincancerchemopreventionandtreatment.Theconferenceisdedicatedtocommemoratethe50thanniversaryofpioneeringworkofprof.Drobnica’steaminthefieldofcancerostaticeffectsofnaturalandsyntheticisothiocyanates,theresultsofwhichwerepublishedinNeoplasmajournal.
ThegoaloftheproposedconferenceistobringtogetherEuropeanfoodengineers,epidemiologists,cancerresearchersandclinicianstoexplorenewutilizationofplantagentsincancerpreventionandtheirpossibleinvolvementinfuturetreatmentprotocols.
SCIENTIFIC AND ORGANIZING COMMITTEE
SedlakJan,SlovakRepublicMithenRichard,UnitedKingdomHunakovaLuba,SlovakRepublicHalkierBarbaraAnn,DenmarkGerhaeuserClarissa,GermanyDurajJozef,SlovakRepublicCollinsAndrew,Norway
SCHEDULE
TUESDAY 13 OCT 2009
10:00-12:00 Walking City Tour through Old Town of Bratislava DepartingfromHotelCarlton-Hviezdoslavovonám.3,81102Bratislava.
12:00-14:00 Bus transfer to conference venue (Smolenice Castle) 12:00 departingfromhotelCarlton-Hviezdoslavovonám.3,81102Bratislava 12:20 BustransferstopsatMainTrainStationofBratislava-Predstaničné námestie1,81101Bratislava 12:40 BustransferstopsatMainBusStationMlynskéNivy-MlynskéNivy31, 82109Bratislava 13:00 BustransfertoSmoleniceCastlestopsatBratislavaInternationalAirport- LetiskoM.R.Štefánika,82311Bratislava22 14:00 ArrivaltoSmoleniceCastle
14:00-16:00 Lunch, registration, check in
16:00-17:30 Opening remarks & The ITC research in Slovakia • Coevals of prof. Drobnica,TheHistoryofITCResearchinSlovakia:TheFiftiesofthe LastCentury. • Jan Sedlak, director of Cancer Research Institute SAS,TheLast10yearsofITC researchinSlovakia. • Jana Jakubikova,Dana-FarberCancerInstitute,Boston,USA,CancerResearchInstitute SAS,Bratislava,Slovakia:IsothiocyanatesExertAnti-myelomaActivityandEnhance TherapeuticCytotoxicity.
17:30-18:00 Coffee break
18:00-19:40 Section V Intervention Studies and Functional Food
• 18:00-18:45 RichardMithen,IFRNorwich,UK:HumanInterventionStudieswith BroccoliandtheModeofActionofSulforaphaneinVivo. • 18:45-19:00 DanaCholujova,CancerResearchInstituteSAS,Bratislava,Slovakia: BioBranModulatestheInnateImmuneSystemFunctions:Multiple MyelomaPatients’Study. • 19:00-19:15 EmilTomasik,INFREDPharm,Slovakia:ThePerspectiveUseofthe PreparationMellozaninOncology. • 19:15-19:30 KeanAshurst,Sulforaphane(ITC’s)ApplicationsforImprovedHealth- Yesterday!–Today!–Tomorrow!
20:00- Welcome party Smolenice Castle
WEDNESDAY 14 OCT 2009
8:00-9:00 Breakfast
9:00-10:45 Section I Chemoprevention, chemoprotection
• 9:00-9:45 ClarissaGerhauser,GermanCancerResearchCenter,Heidelberg,Germany: ProstateCancerPreventivePotentialofCruciferousVegetables. • 9:45-10:00 YongpingBao,UniversityofEastAnglia,Norwich,UK:NovelTargetsof DietaryIsothiocyanatesinCancerPrevention. • 10:00-10:15 KatarzynaSkupinska,NationalMedicinesInstitute,Warsaw,Poland: IsothiocyanatesasChemopreventiveAgentsAgainstPolycyclicAromatic Hydrocarbons. • 10:15-10:30MonikaKassayova,MelatonininthePreventionofExperimentalMammary Carcinogenesis. • 10:30-10:45RudolfŠtětina,TheInfluenceofAntioxidants(EllagicAcid,Epigallocatechin Gallate)ontheRemovalofDNADamageInducedWithDifferentChemical Mutagens.
9:45-11:15 Coffee break
11:15-12:45 Section III Biomarkers and Epidemiology
• 11:15-12:00AndrewCollins,DepartmentofNutrition,UniversityofOslo,Norway: Non-antioxidantEffectsofAntioxidants. • 12:00-12:15Jian-MinYuan,UniversityofMinnesota,MN,USA:Isothiocyanatesand CancerPrevention–arewereadytomoveforwardfromobservational studiestolargeclinicaltrials? • 12:15-12:30KristinMoy,UniversityofMinnesota,MN,USA:AProspectiveStudyof UrinaryBiomarkerforIsothiocyanatesandRiskofGastricCancerin Shanghai,China. • 12:30-12:45KatarinaVolkovova,OxidativeDamageandAntioxidantProtectionin RelationtoAgeing.
13:00-14:00 Lunch
14:00-15:30 Section IV Plant Biochemistry/Biotechnology and Organic Chemistry
• 14:00-14:45MichaelDalgaardMikkelsen,FacultyofLifeSciences,Universityof Copenhagen,Frederiksberg,Denmark:EngineeringBenzylglucosinolateinto Tobacco. • 14:45-15:00EvaCellarova,FacultyofScience,UPJS,Kosice,Slovakia:Biotechnological AlternativeinProductionofBioactiveSubstancesintheGenusHypericum.
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• 15:00-15:15JanImrich,InstituteofChemistry,FacultyofScience,UPJS,Kosice,Slovakia: SynthesisandAntitumorActivityofCompoundsBasedon 9-Isothiocyanatoacridine. • 15:15-15:30VladimirFrecer,CancerResearchInstituteSAS,Bratislava,Slovakia: Computer-assistedCombinatorialDrugDesign.
15:45 Departure of sightseeing trip to Červený Kameň Castle (Red Stone Castle)
16:30-18:00 Visit of the Červený Kameň Castle
18:00 Departing from Červený Kameň Castle to Fugger Manor in Častá Village
18:30-19:00 Wine tasting in vine cellars of Fugger Manor
19:00-21:00 Farewell dinner in vine cellars of Fugger Manor House
21:00 Departure to Smolenice Castle
21:30 Poster session
THURSDAY 15 OCT 2009
8:00-9:00 Breakfast, check out
9:00-10:00 Section II Treatment and Signaling Pathways
• 9:00-9:30 Jan Sedlak,CancerResearchInstituteSAS,Bratislava,Slovakia:Exploiting ITC-modulatedSignalingPathwaysinCancerTherapy. • 9:30-9:45 PeterFerenc,InstituteofBiologyandEcology,FacultyofScience,UPJS, Kosice,Slovakia:Down-regulationofBCL-2andAKTInducedby CombinationofPhotoactivatedHypericinandGenisteininHumanBreast CancerCells. • 9:45-10:00JozefDuraj,CancerResearchInstituteSAS,Bratislava,Slovakia: AdministrationofIsothiocyanate(E-4IB)andCisplatinLeadstoAltered SignallingandLysosomalExportinHumanOvarianCarcinomaSensitiveand Cisplatin-ResistantCells.
10:00-10:30 Coffee break
10:30-12:00 Section II Treatment and Signaling Pathways (Cont.)
• 10:30-10:45AntoniettaMelchini,UniversityofMessina,Italy:Selective AntiproliferativeActivityinVitroofStructurallyRelatedIsothiocyanates, ErucinandSulforaphane,onHumanProstateCells. • 10:45-11:00EvaHorvathova,LaboratoryofMutagenesisandCarcinogenesis,Cancer ResearchInstituteSAS,Bratislava,Slovakia:EffectsofCarvacroland RosemaryOilSupplementationonOxidativeDNADamageInducedin PrimaryRatHepatocytesby2,3-dimethoxy-1,4-naphthoquinone(DMNQ). • 11:00-11:15VeronikaSackova,InstituteofBiologyandEcology,FacultyofScience, UPJS,Kosice,Slovakia:ManumycinAasAgentSensitizingAdenocarcinoma CelltoPhotodynamicTreatment. • 11:15-11:30PeterSolar,InstituteofBiologyandEcology,FacultyofScience,UPJS, Kosice,Slovakia:HSP90ProteinasaTargetforImprovingPhotodynamic Therapy. • 11:30-11:45JaromirMikes,InstituteofBiologyandEcology,FacultyofScience,UPJS, Kosice,Slovakia:TheRoleofProteinP53inPhotodynamicTherapywith Hypericin. • 11:45-12:00MartinKello,InstituteofBiologyandEcology,FacultyofScience,UPJS, Kosice,Slovakia:PotentiationofPhotodynamicTherapywithHypericinby ExogenousPolyunsaturatedFattyAcids.
12:00 Closing remarks
13:00-14:00 Lunch
14:00-15:50 Bus transfer departure from Smolenice Castle to Bratislava
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LECTURES
• L1 JanaJakubikova,Dana-FarberCancerInstitute,Boston,USA,CancerResearch InstituteSAS,Bratislava,Slovakia:IsothiocyanatesExertAnti-myelomaActivityand EnhanceTherapeuticCytotoxicity.• L2 RichardMithen,IFRNorwich,UK:HumanInterventionStudieswithBroccoli andtheModeofActionOfSulforaphaneinVivo.• L3 DanaCholujova,CancerResearchInstituteSAS,Bratislava,Slovakia:BioBran ModulatestheInnateImmuneSystemFunctions:MultipleMyelomaPatients’Study.• L4 EmilTomasik,INFREDPharm,Slovakia:ThePerspectiveUseofthePreparation MellozaninOncology.• L5 KeanAshurst,Sulforaphane(ITC’s)ApplicationsforImprovedHealth- Yesterday!–Today!–Tomorrow!• L6 ClarissaGerhauser,GermanCancerResearchCenter,Heidelberg,Germany: ProstateCancerPreventivePotentialofCruciferousVegetables• L7 YongpingBao,UniversityofEastAnglia,Norwich,UK:NovelTargetsOf DietaryIsothiocyanatesinCancerPrevention• L8 KatarzynaSkupinska,NationalMedicinesInstitute,Warsaw,Poland: IsothiocyanatesasChemopreventiveAgentsAgainstPolycyclicAromatic Hydrocarbons.• L9 MonikaKassayova,MelatonininthePreventionofExperimentalMammary Carcinogenesis.• L10RudolfŠtětina,TheInfluenceofAntioxidants(EllagicAcid,Epigallocatechin Gallate)ontheRemovalofDNADamageInducedWithDifferentChemical Mutagens. • L11AndrewCollins,DepartmentofNutrition,UniversityofOslo,Norway: Non-antioxidantEffectsofAntioxidants.• L12Jian-MinYuan,UniversityofMinnesota,MN,USA:IsothiocyanatesandCancer Prevention–AreWeReadytoMoveForwardfromObservationalStudiestoLarge ClinicalTrials?• L13KristinMoy,UniversityofMinnesota,MN,USA:AProspectiveStudyof UrinaryBiomarkerForIsothiocyanatesandRiskofGastricCancerinShanghai, China.• L14KatarinaVolkovova,OxidativeDamageandAntioxidantProtectionin RelationtoAgeing.• L15MichaelDalgaardMikkelsen,FacultyofLifeSciences,Universityof Copenhagen,Frederiksberg,Denmark:EngineeringBenzylglucosinolateintotobacco.• L16EvaCellarova,FacultyofScience,UPJS,Kosice,Slovakia:Biotechnological AlternativeinProductionofBioactiveSubstancesintheGenusHypericum.• L17JanImrich,InstituteofChemistry,FacultyofScience,UPJS,Kosice,Slovakia: SynthesisandAntitumorActivityofCompoundsBasedon9-Isothiocyanatoacridine.• L18VladimirFrecer,CancerResearchInstituteSAS,Bratislava,Slovakia: Computer-assistedCombinatorialDrugDesign.
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• L19JanSedlak,CancerResearchInstituteSAS,Bratislava,Slovakia:Exploiting ITC-modulatedSignalingPathwaysinCancerTherapy.• L20PeterFerenc,InstituteofBiologyandEcology,FacultyofScience,UPJS, Kosice,Slovakia:Down-regulationofBCL-2andAKTInducedbyCombinationof PhotoactivatedHypericinandGenisteininHumanBreastCancerCells.• L21JozefDuraj,CancerResearchInstituteSAS,Bratislava,Slovakia: AdministrationofIsothiocyanate(E-4IB)andCisplatinLeadstoAlteredSignalling AndLysosomalExportInHumanOvarianCarcinomaSensitiveandCisplatin- ResistantCells.• L22AntoniettaMelchini,UniversityofMessina,Italy:SelectiveAntiproliferative ActivityinVitroofStructurallyRelatedIsothiocyanates,ErucinandSulforaphane,on HumanProstateCells.• L23EvaHorvathova,LaboratoryofMutagenesisandCarcinogenesis,Cancer ResearchInstituteSAS,Bratislava,Slovakia:EffectsofCarvacrolandRosemaryoil SupplementationonOxidativeDNADamageInducedinPrimaryRatHepatocytesBy 2,3-dimethoxy-1,4-naphthoquinone(DMNQ).• L24VeronikaSackova,InstituteofBiologyandEcology,FacultyofScience,UPJS, Kosice,Slovakia:ManumycinAasAgentSensitizingAdenocarcinomaCellto PhotodynamicTreatment.• L25PeterSolar,InstituteofBiologyandEcology,FacultyofScience,UPJS,Kosice, Slovakia:HSP90ProteinasaTargetforImprovingPhotodynamicTherapy.• L26JaromirMikes,InstituteofBiologyandEcology,FacultyofScience,UPJS, Kosice,Slovakia:TheRoleofProteinP53inPhotodynamicTherapywithHypericin.• L27MartinKello,InstituteofBiologyandEcology,FacultyofScience,UPJS, Kosice,Slovakia:PotentiationofPhotodynamicTherapywithHypericinby ExogenousPolyunsaturatedFattyAcids.
POSTERS
• P1 RebekaTomasin,LaboratoryofNutritionandCancer,InstituteofBiology,State UniversityofCampinas,UNICAMP,Campinas,Brazil:AloeVeraandHoneyReduce TumourGrowthbyDecreasingitsCellProliferativeCapacityinTumour-bearingRats.• P2 KatarzynaLubelska,NationalMedicinesInstitute,Warsaw,Poland: SulforaphaneandAlyssinasChemopreventiveAgents.• P3 LubaHunakova,CancerResearchInstituteSAS,Bratislava,Slovakia: ModulationofMarkersAssociatedwithAggressivePhenotypeinMDA-MB-231 BreastCarcinomaCellsbySulforaphane.• P4 PeterKutschy,InstituteofChemicalSciences,FacultyofScience,UPJS,Kosice, Slovakia:1-MethoxyindolePhytoalexins:SynthesisandAnticancerActivity.• P5 MoniaLenzi,DepartmentofPharmacology,UniversityofBologna,Italy: CombinationofDoxorubicinandSulforaphaneforReversingDoxorubicin-resistant PhenotypeinMiceFibroblastswithp53(Ser220)Mutation.• P6 MagdalenaMilczarek,NationalMedicinesInstitute,Warsaw,Poland:Analysis ofInteractionofIsothiocyanateswith5-FluorouracilinChineseHamsterLung FibroblastCellLine.• P7 PetrBenes,DepartmentofExperimentalBiology,FacultyofScience,Masaryk University,Brno,CzechRepublic:WedelolactoneReducesGrowthandInduces DifferentiationandApoptosisofBreastCancerCells. • P8 AlexandraHudecova,CometAssay:AUsefulMethodForStudyingPapaver RhoeasandGentianaAsclepiadeaFlowerExtracts.• P9 AnnaSmiechowska,TheImpactofCultivationConditionsonPhytocomlex CompositionandChemopreventivePropertiesofWhiteCabbage.• P10PatriciaMaimone,AntiproliferativeEffectsofJuniperusCommunisl.Var. CommunisBerryExtractintheHumanHepatocellularCarcinomaCellLin.• P11 JungHyunKim,IncreasedHistoneAcetylationbyKaleSproutIntervention inaHumanProstateCancerXenograftMode.• P12MichalPastorek,RegulationOfHypoxicPathwayByNaturalIsothiocyanate SulforaphaneInDrug-resistantOvarianCarcinomaCellLines.
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L1
ISOTHIOCYANATES EXERT ANTI-MYELOMA ACTIVITY AND ENHANCE THERAPEUTIC CYTOTOXICITY
Jakubikova,J.1,2,3,Cervi,D.1,2,Ooi,M.1,2,Cholujova,D.3,Daley,J.F.1,2,Klippel,S.1,2,Leiba,M.1,2,Blotta,S.1,2,Richardson,P.G.1,2,Mitsiades,C.S.1,2,Sedlak,J.3
andAnderson,K.C.1,2
1Dana Farber Cancer Institute, Department of Medical Oncology, Boston MA 02115, USA2Department of Medicine, Harvard Medical School, Boston MA 02115, USA
3Cancer Research Institute, Department of Tumor Immunology, Vlarska 7, 83391 Bratislava, Slovak Republic
Multiplemyeloma (MM), a plasma cellmalignancy, remains largely incurablewith 3 to 5 years survival, despite the use of conventional and novel chemotherapies. Isothiocyanates (ITCs), a family of phytochemicals found in cruciferous vegetables,and provide chemopreventive effects associatedwith a reduced risk of several types of cancer.IthasalsobeenreportedthatITCsenhancethechemosensitivityofdiversetypesoftumorcells.Wethereforeevaluatedtheanti-myelomaactivityoftheITCs,sulforaphane(SFN) and phenylethyl isothiocyanate (PEITC), on a panel of human MM cell lines, sensitiveandresistanttoconventionalandnovelanti-myelomadrugs.ITCsinducedapop-toticdeathofMMcells;depletionofmitochondrialmembranepotential;cleavageofPARPandcaspases-3and -9;anddown-regulationofMcl-1,X-IAP,c-IAPandsurvivin. ITCsalso inducedG2/Mcell cycle arrest andmitoticphosphorylationofhistoneH3, accom-panied by decreased protein expression of cyclinB1 and p-cdc2.Multiplex analysis ofphoshorylationofdiversecomponentsofsignalingcascadesrevealedchanges inMAPK activation;increasedphosphorylationofc-junandHSP27;anddecreasedphoshorylationofAkt,GSK3α/βandp53. In addition to the cytotoxic effect of ITCs as single agents, synergy with established anti-MM agents has been studied. Our study revealed that SFN exhibited synergistic effects with established anti-MM drugs such as bortezomib, dexametha-sone,doxorubicin,andmelphalan;whereasPEITChadsynergisticeffectcombinedwith thalidomide derivative lenalidomide, bortezomib and melphalan. Bone marrow micro- environmenthasbeenessentialfortumorcellgrowthandsurvivalinvivoandcriticalformaintenanceofdisease.Importantly,ITCstreatment,bothaloneandincombinationwiththe aforementioned agents, also significantly suppressed proliferation ofMM cell lines co-culturedwiththehumanbonestromalcelllineHS-5.TheseresultsindicatethatSFNandPEITCcansuppressgrowthandpromotedeathofMM,bothaloneandincombinationwithcurrentlyestablishedanti-MMdrugs,suggestingtheirtherapeuticpotentialinMM.
L2
HUMAN INTERVENTION STUDIES WITH BROCCOLI AND THE MODE OF ACTION OF SULFORAPHANE IN VIVO
Mithen,R.,andTraka,M.
Institute of Food Research, Colney Lane, Norwich UK
Epidemiological studies provide evidence that consuming a moderate amountof broccoli per week can reduce the risk of incidence and progression of prostate cancer,andofcanceratseveralothersites.Isothiocyanates,derivedfromglucosinolatesthat accumulate in broccoli are widely considered to be likely candidates that mediate the protectiveeffectofbroccoli.Cellandanimalmodelshavebeenextensivelyusedtoexplorethepotentialbiologicalactivityofisothiocyanates,butthemajorityofthesestudieshaveused concentrations of ITCs that are far in excess of that obtained through the diet. Inthispaper,wesummarizeresultsfromaseriesofacuteandlongtermhumaninterventionstudiesandfromcellandanimalmodelsthatsuggestthatsulforaphane–themajorITCsobtainedfrombroccoliconsumption-actsinvivotodownregulatePI3K/AKTmediatedcellsignalingprobably,probablyduetoitsactivityasanonspecificreceptortyrosinekinaseinhibitor.
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L3
BIOBRAN MODULATES THE INNATE IMMUNE SYSTEM FUNCTIONS: MULTIPLE MYELOMA PATIENTS` STUDY
Cholujova,D.,Jakubikova,J.,Sulikova,G.,Chovancova,J.,Hunakova,L.,Duraj,J.,andSedlak,J.
Cancer Research Institute SAS, Laboratory of Tumor Immunology
Multiplemyeloma(MM)isincurablemalignancyofplasmacellsthataccumulateatmultiplesitesinthebonemarrow.PatientswithMMsufferfromhumoralandcellularimmunodeficienciesthatcanleadtolife-threateninginfections. BioBranisafoodsupplementderivedfromricebranhemicellulosehydrolyzedbyShiitakemushroomenzymeswithmodulatoryeffectsoninnateimmunecells,aswellasonadaptivelymphocytes. The impactofBioBranconsumptiononinnate immunesystemofMMpatientswasevaluatedintheplacebo-controlledstudy.Wemonitoredchangesinnaturalkiller(NK)cellactivitybyperformingflow-cytometriccytotoxicityassayandtheproportionofNKanddendriticcell(DC)subpopulationsinperipheralbloodbyimmunophenotypicanalysis.Usingmultiplexbeadarrayimmunoassayweanalyzedapanelof27cytokinesinpatients’plasma. BioBran showed modulatory effects on DC and NK cells, as we observed significant augmentation ofNK activity, aswell as increased percentage of overallDCpopulation andmyeloidDC subpopulation in comparison to placebo group.Therewas ashiftofTh1/Th2cytokinebalancetomoreactiveTh2immunityinMMpatientswhencomparedtohealthyblooddonors.Thelevelsof10cytokinesweresignificantlyincreasedafterBioBrantreatment. The results of the present study indicate that BioBran consumption could be beneficialasasupplementformaintenanceofmyeloidDCsubpopulationandNKactivity.
L4
THE PERSPECTIVE USE OF THE PREPARATION MELLOZAN IN ONCOLOGY
TomasikE.,MansurovaF.,.ZikriyahodzhaevD.Z.,andLaninaN.
INFREDpharm, s.r.o. (Slovakia), Research Institute of Gastroenterology of the Academy of Sciences of Tajikistan Republic, the Republican Clinical Centre of the Ministry of
Health of Tajikistan Republic
PreparationМellozan®produced by INFREDpharm, s.r.o. (Slovakia) is a sterile 0.4% solution for injections in ampoules of 1 and 3 ml. The pharmaceutical substanceis used in manufacturing process. The active substance is a complicated complex of compounds obtained by biotechnological processing of natural raw material – honey,whichisecologicallypure(itisconfirmedbyCertificateofBioproduct).Syntheticorother additives are not present. The preparation is registered officially in the Republic of Tajikistan under the trade name MELLINOL®, and in the Republics of Moldova and KyrgyzstanunderthetradenameMELLOZAN®.Theaprovedindicationoftheregisteredpreparationisthetreatmentofliverdiseasesofdifferentetiology.Theresultsofthecurrent research determined that preparation Mellozan® has the following pharmacodynamic effects:immunomodulative,cytoprotective,regenerative. NonclinicalstudiesshowedthatMellozanhasadose-dependentimmunoregulatoryactivity.This is demonstrated by its ability to induce production of different types of cy-tokines:proinflammatory (IL-1, IL-6, IL-8), anti-inflammatory (IL-10), immunoregulatory(IFN-γ,IL-4). ClinicalstudiesprovedthatthepreparationincreasesthenumberofT-lymphocytesandNK-cells,whichcorrespondswiththeenhancementofprotectiveimmunereactionsinpatientswiththechronicviralhepatitis. Ontheotherside,MellozandecreasestheimmunopathologicalactivityofB-lym-phocytes with its hyperproduction of antibodies of different isotypes. The administrationof thedrug topatientswith thedysfunctionof the immune systemcaused thenormaliza-tion of theT-lymphocytes number, normalized immunoregulatory index (ratioT-helpers/-cytotoxiclymphocytes),andincreasedthecytotoxicactivityNKcells.Italsoincreasesthesynthesisof the immunoglobulinesbytheplasmaticcells, increases thefunctionalactivityofmononuclear phagocytes, and decreases the level of the spontaneous apoptosis of the Th-lymphocytes,whichimprovestheantiviralimmunity.Widespectrumofthepharmacodynamiceffects,inparticularhepatoprotectiveandimmuno-modulativeallowedustoanticipateitspossibleefficacyinoncologicaltherapy,astheadjuvantpreparationforabettertoleranceofthechemo-andradio-therapy,decreaseofthesideeffectsbythecytostatictherapyandnormalisationoftheimmuneandhaematologicpatterns.
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The clinical studies of Mellozan® in the completion therapy of 82 patients with the different types of malignity, were performed in the Republic Clinical Oncology CentreandTajikStateMedicalUniversity(Dushanbe,Tajikistan), in the2005-2006.Thepatients underwentthetherapybyMellozan–10injectionsof3mleveryotherdayintramusculary(monotherapy)andincombinationwithchemotherapy,eitherbeforeoraftertheradiotherapy.Themodeofthetreatmentwasdeterminedaccordingtotheactivityoftumorousprocess,thepatientstatusandprevioustherapy.Morethan70%ofthepatientswerepatientswithprimarydiseasewithoutthepreviousantineoplastictherapy.Properantineoplasticeffectofthepreparationwasassessedonlyin7patientswithbreastcan-cerwhorefusedthechemotherapy.Uneasinessfeelings,depressions,generalweaknessandothergeneralsymptomswerethemostoccurredsymptomsnoticedbythepatientsbeforethetreatmentinthehospital.ThesepatientswentthroughthecourseofMellozantherapybeforethesurgical intervention.Thesomaticstatusofall thesepatientsshowedtheimprovementafterthistherapy.Itwaspossibletoseeescapingofthedepression,improvementoftheapetiteandincreaseoftheactivity.In6caseswasdeterminedthediminutionofthetumorsizeupto40-44% Theresultsof theclinicalstudiesshowedthat theuseof thecomplextherapybyMellozanincreasedtheefficacyofthecytostaticdrugs,activityofimmunesystem(92%pa-tients) independently from thedisseminationof theprocess.Mellozanshowed the regula-tiveimpactontheprocessesofthelymphocytesdifferentiationandactivatedantitumorous immune reactions. The increase of the terminal differentiated lymphocytes number (expressing Fas-receptor for induction of the apoptosis), expressing of the receptors totheIL-2andtransferrin-whichis thesignof theactiveproliferationof lymphocyteswas possibletoseeafterthefirstphaseofthetherapy. The results of the clinical studies confirmed that use of Mellozan increasesthe resistance to the chemo- and radio-therapy. The hepatotoxic effect of the cytostaticdrugs decreased.Themain indicators of the liver function restored to the normal values. AdministrationofMellozancorrectedthehaemopoiesisintheredandwhitecomponent,im-provedtheindicatorsoftheimmunesystemdestroyedbythechemo-andradio-therapy.AllpatientstreatedbycomplextherapywithMellozanwereablegothroughfullschemeofantitumortherapywithoutanecessaryinterruptionswhatimprovedtheresultsofthetreat-ment. TheachievedresultsoftheclinicalstudiesshowedthatMellozanisaperspectivedrugintheoncologicaltherapy.Mellozanisabletoincreasetheefficacyofthetherapy,thetolerancetothesideeffectsoftheradioandchemotherapyandimprovesthequalityoflife.
L6
PROSTATE CANCER PREVENTIVE POTENTIAL OF CRUCIFEROUSVEGETABLES
Kim,J.H.1,Bähr,M.1,Wagner,J.2,DeNicola,G3,Bub,A4,Rüfer,C4,Jung,M2,Iori,R3,Gerhäuser,C.1
1German Cancer Research Center Heidelberg, Germany 2Universtiy of Freiburg, Germany
3Research Center for Industrial Crops, Agriculture Research Council, Bologna, Italy 4Max Rubner-Institute, Karlsruhe, Germany
Cruciferous vegetables are rich in glucosinolates, which are converted to isothiocyanates (ITCs) and indole-based compounds by the enzymatic activity of myrosinase.Highintakeofcruciferousvegetableshasbeenassociatedwithalowincidenceof prostate cancer in various case control studies. However,many other epidemiologicstudiesreportednoornegativecorrelationbetweenriskofprostatecancerandcruciferousvegetableconsumption.Mechanismsofchemopreventiveactivitiesofselectedcompoundsderived from cruciferous vegetables, such as the isothiocyanates sulforaphane and the indolebasedcompoundsindole-3-carbinoland3,3’-diindolylmethane,includemodulationof detoxifying enzymes, anti-inflammatory activity (NF-κB inhibition), anti-angiogenic properties, induction of cell cycle arrest, apoptosis and autophagy, and modulation of signaltransductionpathways.Tumor-growthinhibitoryefficacyinanimalmodelshasbeendemonstratedatvariousorgansitesincludingtheprostate.However,excepttheinduction of detoxifying enzymes, relatively little is known on mechanisms affected by dietary interventionwithcruciferousvegetables,whichrepresentamixtureofvariousconstituentspotentiallyenhancingorblockingtheiractivities(Pappaetal.,Carcinogenesis2007). Inthepresentstudy,wetestedthetumorgrowthinhibitorypotentialofkalesproutsin a human prostate cancer xenograftmodel. LNCaP human prostate cancer cellswere injectedtomalenudeBalb/cmice.Micewerefedwitheitherregularrodentchoworchowsupplementedwith 20%kale sprouts (containing about 60µmol glucosinolates per 5 gchowconsumeddaily,togetherwithactivemyrosinase)fromaweekbeforecellinjectionuntilsacrificeafter7.5weeksofintervention. Comparedwithnormaldiet,kalesproutinterventiondidnotsignificantlyinhibit tumor growth. Secreted prostate specific antigen (PSA), which is used as a biomarkerfor prostate cancer, was highly correlated with tumor size in both intervention groups. Interestinglyhowever,PSAplasma levels in thekale sproutgroupwere2.5-foldhigher(p<0.05)thaninthecontrolgroup.mRNAexpressionofhormone-regulatedgenessuchasandrogenreceptor,insulinlikegrowthfactor-1anditsreceptor,andPSAweresignificantlyincreased.AlthoughtherewasnodifferenceintheexpressionofPCNAasaproliferation
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marker,phosphorylatedhistoneH3stainingasamarkerofmitoticarrestwasenhanceinthekalesproutgroup.Also,weobservedanincreaseinhistoneH4acetylation,whichwecouldnotlinktoaninhibitionofHDACactivity,butwhichmightbeinvolvedinup-regulationofp21mRNAandproteinexpression.Interestingly,hemoglobinlevelsintumorcelllysateandvesselsintumorsectionsweresignificantlyreducedbyconsumingkalesproutpowder, indicating anti-angiogenic potential. This was associated with down-regulated VEGF-c expression.
In conclusion, our study indicate that the combination of components in kalesproutsaffectbothpro-proliferative(hormonesignaling)andanti-proliferativemechanisms(histone H4 acetylation, cell cycle arrest, anti-angiogenesis), whichmay abrogate their effectontumorgrowth.Theseresultsmaycontributetoabetterunderstandingoftheeffectsofcruciferousvegetableconsumptionincancerprevention.
Pappa et al., Quantitative combination effects between sulforaphane and 3,3’-diindolylmethane on proliferation of human colon cancer cells in vitro. Carcinogenesis 28, 1471-1477 (2007)
L7
NOVEL TARGETS OF DIETARY ISOTHIOCYANATES IN CANCER PREVENTION
Bao,Y.
School of Medicine, Health Policy and Practice,University of East Anglia, UK
Epidemiological studies suggest that a diet rich in cruciferous vegetables is associatedwith a decreased risk ofmany common cancers.Their protective effects areattributed,atleastinpart, toisothiocyaniates,thebreakdownproductsofglucosinolates.Many studies have demonstrated that isothiocyanates can modulate multiple cellular targetsincludingphaseIcarcinogen-activatingenzymes,phaseIIdetoxificationenzymes,cellcyclearrestandapoptosis. Recent studies suggest that sulforaphane canupregulate sequestosomep62 andinduceautophagy(cellularself-eating).p62isacommoncomponentofproteinaggregatesassociatedwithproteinmisfoldingdiseasesandplayanimportantroleinthelifeanddeathdecisionsofthecell.Interestingly,wehaveshownthatsulforaphanecaninduceNrf2trans-location into nucleus and increase p62 expression in humanhepatocytes.Therefore,wespeculatethatthisstimulationoftheNrf2pathwaybysulforaphaneandconsequentincreaseinp62couldfacilitatetargetingofdamagedcellularcomponentstoautophagosomesandplayaroleincancerprevention. Furthermore, we have shown that sulforaphane can down-regulate COX-2 expression in colon and bladder cancer cells. Moreover, we have established that synergistic interactions occur between isothiocyanates and mineral selenium in the regulation of antioxidant gene expression including thioredoxin reductase (TR1) and gastrointestinalglutathioneperoxidase(GI-GPx). Very recently,we have found that sulforaphane down-regulated the expressionof serotonin receptors and serotonin reuptake transporter (SERT), promoting signals for serotoninreleasebymediatingG-proteinsandactivatingneurotransmitterreceptors,whichareimportantintheproliferationofcoloncancercells. In summary, the effect of sulforaphane on the expression of p62, COX-2 and neurotransmitterreceptorsmayprovidenewinsightsintothedevelopmentofstrategiesthatutilizedietaryisothiocyanatesforcancerprevention.
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L8
ISOTHIOCYANATES AS CHEMOPREVENTIVE AGENTS AGAINST POLYCYCLIC AROMATIC HYDROCARBONS
Skupińska, K., Lubelska, K., Misiewicz, I., Kasprzycka-Guttman, T.
National Medicines Institute, 00-725 Warsaw, Poland, Chełmska 30/34
Plantsareagoodsourceofchemopreventivecompounds.Itwasshowninepide-miologicstudiesthatdietrichinBrassicavegetablesdecreaseriskofcancerdevelopment.Isothiocyanates (ITC), present in these vegetables are responsible for these properties. Awell-knownisothiocyanatepresentmainlyinbroccolisproutsissulforaphane(SFN),whichinhibitsthegrowthofcancercells,inducesapoptosisandactivityofII-phaseenzymesandalsoinhibitsI-phaseenzymesactivity.TheliteraturedataindicatethatmodificationofITCstructurecanalteritschemopreventiveproperties.Hence,twosulforaphaneanaloguesweresynthesized:isothiocyanate-2-oxohexylwithexchangedsulfuratomintocarbon,andalyssinwithprolongedaliphaticchain.Polycyclic aromatic hydrocarbons (PAH) are environmental pollutant.After entering thebody they aremetabolized byCYP1A1 andCYP1A2 belonging to I-phase enzymes to oxy-derivativeswhichareabletobindtoDNA,whatcaninitiatecarcinogenesis.PAHsinduceCYP1A1andCYP1A2activityviaArylHydrocarbonReceptor(AhR)-dependentpathwayincreasingtherateof itsownmetabolismtocarcinogenicmetabolites.Thus, inhibitionofCYP1A1andCYP1A2enzymesactivityisconsideredasachemopreventionstrategy. TheaimofthestudywastodetermineifsulforaphaneanditsanaloguescaninhibittheCYP1A1andCYP1A2activityinducedbypolycyclicaromatichydrocarbonsdifferinginthestructureandcarcinogenicity.ThisstudyaimedalsotocheckifsynthesizedanaloguespossessbetterchemopreventivepropertiesthanSFN.ThemechanismofITCactionwasalsostudied. Theresearchwasconductedontwocelllines:HepG2–ahumanlivercancercelllineandMcf7–humanbreastcancercell line.TheabilityofITCtoinhibit theCYP1A1andCYP1A2 activitywas assessedwith EROD andMROD assay. Investigation of ITC actionmechanismincluded:ITCabilitytodirectlyinhibitcatalyticactivityofCYP1A1andCYP1A2enzymes,ITCimpactontheenzymaticproteinlevel,andITCabilitytoblockthetranslocationofAhreceptortothenucleusinducedbyPAHs.TostudytheproteinlevelandAhRlocalizationimmunocytochemistryandconfocalmicroscopywereapplied. TheresultsrevealedthatonlyinMcf7cellsthesecompoundsactedasinhibitors.Thestrongestinhibitorypropertieswereshownbyalyssin.ThestudyrevealedalsothatCYP1A1wasinhibitedmainlybythedirectinhibitionofcatalyticactivity,whileCYP1A2isinhibitedbydecreasingtheenzymeproteinlevel.Aboveresultsindicatethatstudiedcompoundscanbeconsideredaschemopreventiveagents,especiallyincaseofestrogen-dependingcancers.
L9
MELATONIN IN THE PREVENTION OF EXPERIMENTAL MAMMARY CARCINOGENESIS
KassayováM.,AhlersI.,OrendášP.,BojkováB.,KubatkaP.,AhlersováE.,KiskováT.,GarajováM.
Department of Animal Physiology, Institute of Biology and Ecology, Faculty of Science, P.J. Šafárik University, Moyzesova 11, 041 54 Košice, Slovak Republic
Melatonin(N-acetyl-5-methoxytryptamine,MEL)isaphylogeneticallyveryold,conservativemoleculewith pleiotropic effects detected in awide spectrum of bacteria,algae, plants, fungi andvarious taxa of invertebrates andvertebrates.Well-known is itsregulatoryfunctionofsleep-wakecycle,circadianandcircaannualrhythms.Ithasalsoanimmunomodulatory,antioxidativeandoncostaticproperties. This contribution summarizes the experimental results of our working group duringthelast10yearswhenwehaveinvestigatedthechemopreventiveandantineoplasticpropertiesofMEL(aloneorthecombinationwiththeotherchemopreventivesubstances)inthemodelofchemicallyinducedmammarycarcinogenesisinfemaleSprague-Dawleyrats. Mammarycarcinogenesiswas inducedbytwochemocarcinogens:a)N-methyl-N-nitrosourea (NMU)administered in two intraperitoneal doses, eachof 50mg/kgb.w.or b) 7,12-dimethylbenz(a)anthracene (DMBA) administered intragastrically in a singledoseof20mg/animalorinathreedosesof10mg/animal.Chemocarcinogensweregiven duringthesensitiveperiodofmammaryglanddevelopment–between40.-55.Postnataldays.Melatoninwasadministeredasasolutionintapwaterataconcentrationof20μg/mland4μg/ml,respectively.Theanimalsweredrinkingmelatonindailybetween3p.m.and8a.m.(from8a.m.to3p.m.theyaredrinkingtapwater).ChemopreventionwithMELbegan5-15daysbeforecarcinogenadministrationandlasteduntiltheendofexperiment(20-26weeks). Basictumorgrowthparameters- tumorincidence,frequencypergroupandperanimal,tumorvolumeandlatencywereevaluated.MELeffectwasnotmarkedlydependentonthetypeofchemocarcinogenusedorontheMELconcentration.Sporadicdecreaseoftumorincidence,frequencypergroupandtumorvolumewasobservedinthegroupstreatedwithMELalone.MEL increased thechemopreventive influenceof theother substancesusedinourexperiments:retinylacetate,raloxifen,indomethacinandcelecoxib.Ourdatasuggest rather irregular actionof isolatedMEL,but the combinationwithother chemo- preventive agents displayed an enhanced effect in mammary carcinogenesis inhibition.ProbablyahigherdoseofMELwillbeneededinourfutureresearch.
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L10
THE INFLUENCE OF ANTIOXIDANTS (ELLAGIC ACID, EPIGALLOCATECHIN GALLATE) ON THE REMOVAL OF DNA DAMAGE
INDUCED WITH DIFFERENT CHEMICAL MUTAGENS
Štětina,R.1,Polívková,Z.2,Šmerák,P.2,Strejčková,L.3,Flutková,K3,Tlučhořová,D.3,ČernohorskáH.4,andBrabníková,Z.4
1Faculty of Military Health Sciences, Hradec Králové2Institute of the General Biology and Genetics; 3rd Medical Faculty, Prague
3Pharmaceutical Faculty, Charles University, Hradec králové4University of Pardubice, Faculty of Health Studies
[email protected]; [email protected]
Wehavefollowedtheinfluenceofseveralantioxidants(ellagicacid-EA,genisteinand epigallocatechin gallate -EGCG)on themutagenic effect ofN-methyl-N-nitrosourea(MNU),aflatoxinB1and2-amino-3-metylimidazo(4,5-f)quinoline(IQ).Allthreeantimuta-gensinhibitedtheinductionofmutationsinSalmonellatyphimuriumassayandtheinductionofchromosomeaberrationsinbonemarrowmironucleiassayinmice.TheyalsoinhibitedtheinductionofDNAbreaksinCHOandHepG2cellstreatedwithMNUorH2O2invitro,aswefollowedusingthecometassay.WithrespecttotheircapacitytoinhibittheinductionofDNAdamagebydifferenttypesofmutagens,theireffectcanbehardlyexplainedonlyonthebasisofantioxidantandscavengingproperties.Therefore,wetestedalsotheirpossibleeffectontheDNArepair.TheremovalofDNAbreaksinducedinHepG2orAA8withMNUorH2O2wasfaster,whencellswereafterthetreatmentwithmutagenincubatedinthepresenceofEA,orEGCG. TheeffectofEGCGontherepairofoxidisedbasesinducedbyH2O2wasstudiedinmoredetail.WhenwefollowedthekineticsofrepairofDNAdamageinducedwithH2O2wefound,thatsitescleavablewithFPGenzyme(cleavespredominantly8-oxo-guaniesandaba-sicsitesintheDNA)areremovedfromtheDNAsignificantlyslowercomparedtositessensi-tivetoendonucleaseIII(cleavesoxidisedpyrimidinesandabasicsites),ortrueDNAbreaks.ThiskineticswassimilarbothinnormalAA8cellsandUV-20mutant,whichisdeficientinthestepofincisionofthenucleotideexcisionrepair.Wefound,thatinthepresenceofEGCGtherepairofFPGsitesissloweddown,whiletherapidityoftherepairofDNAstrandbreaksandoxidisedpyrimidinesisnormal.Resultsshow,thatEGCGmayaffectsomeofpathwaysofbaseexcisionrepair,whichmayhaveanimpacttomutageniceffectofparticularmutagen.However,effectsoftestedantioxidantsarequitecomplicatedandmuchmoreexperimentalworkisnecessarytoelucidatethenatureoftheirbioprotectiveproperties.
This study was supported by the Grant of Czech Ministry of Health - NR. 8985-3 and by the grant of Czech ministry of Defence no: OVUOFVZ200810
L11
NON-ANTIOXIDANT EFFECTS OF ANTIOXIDANTS
Collins,A.R.1,Azqueta,A1,Brevik,A2,Lorenzo,Y1,3
1 University of Oslo, Department of Nutrition, Oslo, Norway2 Norwegian Institute of Public Health, Division of Environmental Medicine,
Oslo, Norway3 Faculty of Medicine, Universidad Autónoma de Madrid, Spain
Endogenous antioxidant defences, notably the antioxidant enzymes superoxide dismutases and catalase, sulphydryl-rich glutathione and its associated enzymes (peroxidases and transferases), are essential for survival. They maintain the balance betweenessentialoxidativeprocessesandreactiveoxygen-mediatedcellregulationontheonehand,andanexcessiveproductionoffreeradicals,i.e.oxidativestress,ontheotherhand.Sodoweneedantioxidantsfromthedietaswell?Inexcess,couldtheyevenupsetthisbalance?Theevidencefromlarge-scaleinterventionstudiesisthatpureantioxidantsathighdosesmaydomoreharmthangood. Epidemiological evidence still indicates that plant-derived foods can protectagainstcancerandcardiovasculardisease,butthismaynotbeonaccountoftheirantioxidant content,ashasbeenwidelyassumed.Thereare,ofcourse,manyotherwell-characterised effects of phytochemicals, such as inhibition or activation of phase I or phase II metabolisingenzymes,variouseffectsoncellsignallingpathways,and–recently–DNArepair. Usingthecometassay,wehavestudiedDNArepairintwoways:followingthetimecourseofremovaloflesionsinspecificallydamagedcells,andmeasuringtherepairactivityofacellextractinvitroonasubstrateofDNAcontainingdefinedlesions.Supple- menting the diet with kiwifruit, or eating a high fruit and vegetable diet, enhances lymphocyteBERintheinvitroassay;intriguingly,wefoundadecreaseinNERafterthehighfruitandvegetablediet.Incellculture,thecarotenoidβ-cryptoxanthinacceleratedtherepairofstrandbreaksandoxidisedbases. Itseemsthattheemphasisondietaryantioxidantsismisplaced;phytochemicalshaveotherimportantprotectiverolestoplay.
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L12
ISOTHIOCYANATES AND CANCER PREVENTION – ARE WE READY TO MOVE FORWARD FROM OBSERVATIONAL STUDIES TO LARGE
CLINICAL TRIALS
Yuan,J.M.
Masonic Cancer Center and Division of Epidemiology and Community Health,School of Public Health, University of Minnesota
Humansareexposedtoisothiocyanates(ITC)throughconsumptionofcruciferous vegetables (Brassica). ITC is biologically active against chemical carcinogenesis dueto its ability to induce phase II conjugating enzymes. ITC also is believed to confer protection against the development of cancer by stimulating apoptosis. Experimental studies have shown that ITC, in particular phenethyl isothiocyanate (PEITC), inhibitsthe tobaccocarcinogen inducedlungcancer inanimals. Indole-3-carbinol(I3C),another compound present in cruciferous vegetables, is also shown to be chemopreventive in experimental animals. Epidemiological studies have consistently showed that subjectswith high consumption of cruciferous vegetables or ITCs are associated with reducedriskoflung,colorectalandgastriccancers.Theprotectionisprimarilyseeninindividuals genetically deficient in phase II conjugating enzymes that metabolize ITC. There are limitedstudiesonI3Cinrelationtotheriskofdevelopingcancerinhumans.GiventhehighcorrelationbetweendietaryITCandI3C,observationalstudieswouldhavelimitedpowertoseparatethechemoprotectiveeffectofI3CfromITC.Randomized,placebo-controlledphase II clinical trials are required to separately examine the potential chemoprotective effect and to shed light on the biological mechanism(s) of these compounds in cancer protection.The phase II trialswill provide clinical data formaking the evidence-based decisiononlaunchinglargerandomizedphaseIIIclinicaltrials.
L13
A PROSPECTIVE STUDY OF URINARY BIOMARKER FOR ISOTHIOCYA-NATES AND RISK OF GASTRIC CANCER IN SHANGHAI, CHINA
Moy,K.A.,andYuan,J.M.
The Masonic Cancer Center and Division of Epidemiology and Community Health, School of Public Health, University of Minnesota, Minneapolis
Epidemiologic data suggests an inverse association between consumption of cruciferousvegetablesandriskofhumancancers.However,dataonspecificcompoundssuchasisothiocyanate(ITC)presentincruciferousvegetablesandcancerriskaresparse. Weconductedanestedcase-controlstudyofgastriccancerwithintheShanghaiCohort Study, a prospective cohort of 18,244middle-agedmen in Shanghai, China, to examine the associations between urinary total ITC and the risk of developing gastric cancer.Thestudyincluded307incidentgastriccancercases,andmorethan2000cancer-free individuallymatchedcontrol subjects. Inaddition toquantificationofurinary totalITC, information on lifestyle, dietary, and other environmental exposureswas collectedthroughin-personinterviews.Overall,highurinarytotalITCwasassociatedwithreducedriskofgastriccancer.Comparedwiththelowestquartile,thehighestquartileofITCwasassociatedwithastatisticallysignificantapproximate40%reducedriskofgastriccancer.
The risk reductionwasmore apparent in individuals carrying one or both nullgenotypes of glutathioneS-transferase (GST)M1 andGSTT1. The inverse associationbetweenurinarytotalITCandcancerriskbecamestrongerwithlongerdurationfromthecollectionofbaselineurine for ITCmeasurement to thediagnosisof cancer.This study provided direct evidence in support of natural compound ITC present in cruciferous vegetablesforprotectionagainstthedevelopmentofgastriccancerinhumans.
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L14
OXIADATIVE DAMAGE AND ANTIOXIDANT PROTECTION IN RELATION TO AGEING
Volkovová,K.,andDušinská,M.
Slovak Medical University in Bratislava
katarina.volkovova @szu.sk
Endogenous oxidative damage to proteins, lipids and DNA is thought to be important etiologic factor in ageingand thedevelopmentof chronicdiseases in elderly. Epidemiologicalevidencesuggeststhatincreasedconsumptionoffruitandvegetablecansubstantially enhance the protection againstmany common types of cancer in humans.HigherlevelsofvitaminsEandCinserumhavebeenshowntocorrelatewithlowerlevelsoflipidperoxides. Inasmallmolecularepidemiologicalstudyweinvestigatedaltogether291healthysubjects,151young(78menand73women,20to25yearsold)and140elderly(39menand101women,65to70yearsold). WemeasuredlevelsofvitaminC,α-tocopherol,γ-tocopherol,β-carotene,retinol, lycopene and xantophyl in plasma by HPLC. Lipid peroxidation was determined byHPLCaslevelsofmalondialdehyde(MDA)inplasma.OxidativeDNAdamageandDNA repairweremeasuredinisolatedlymphocytesbythecometassay.Chromosomalaberra-tions(ABC)andnumberofmicronuclei(NMN)weremeasuredinstimulatedlymphocytes.Totalantioxidantcapacityofplasma(FRAP)wasmeasuredspectrophotometrically. Comparison of biomarkers of oxidative damage and antioxidant protection betweenyoungandoldgroups indicated several significantdifferences.OxidativeDNAdamage (net FPG)was significantly higher in lymphocytes of young people comparedto elderly (p=0.011), as well as higher in young women compared to elderly women(p=0.005). WomenhadsignificantlyhigherrepairrateofoxidativeDNAdamagecomparedto men (p=0.001).We found significantly more micronuclei in women’s lymphocytes(p=0.001)comparedtomen.Elderlypeople(p=0.001)hadmoremicronucleithanyoung.Similarly,wedetectedmorechromosomalaberrationsinelderlygroupcomparedtoyoung(p=0.001). Thereweresignificantdifferencesinthetotalantioxidantcapacityofplasma.Allelderlyhadhigherlevelsthanyoung(p=0.001).Thesamedifferencesappearedinelderlywomenvs.youngwomen(p=0.001),aswellaselderlymenvs.youngmen(p=0.001).Wefoundsignificantdifferencesinplasmaconcentrationsofvitaminsbetweengroups. Vitamin C levels inversely correlated with DNA damage (netAlka) in allgroup (p=0.002), and also in the young (p=0.01) and elderly people (p=0.03). Retinol inverselycorrelatedwithnetFPGintheallpersons(p=0.03),howeverb-carotenepositively
correlatedwithnetFPGinallgroup(p=0.05)andintheyoung(p=0.001).Repairrateof oxidativeDNAdamagepositivelycorrelatedwithlevelsoflycopene(p=0.013)inallgroupsandnegativelywithlevelsofg-tocopherolinallgroups(p=0.03)andinelderly(p=0.05). OurresultssuggestthathigherlevelsofantioxidantvitaminsinplasmaandtotalantioxidantprotectionoforganismmighthaveprotectiveeffectonoxidativeDNAdamage,thusbeingbeneficialincancerprevention.However,itisnecessarytoconfirmthisresultinotherstudies.
This project was supported by the Slovak grant agency for Science APVT - 21 013202, and by the EU project GOCE 037019.
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L15
ENGINEERING BENZYLGLUCOSINOLATE INTO TOBACCO
Mikkelsen,M.D.,Geu-Flores,F.,Nielsen,M.T.,Nafisi,M.,Møldrup,M.E.,Olsen,C.E.,Motawia,M.S.,andHalkier,B.A.
Department of Plant Biology and Biotechnology, Faculty of Life Sciences,University of Copenhagen
Thelinkbetweenregularconsumptionofcruciferousvegetablesandareducedriskofdevelopingcanceriswellestablished.Thiscancer-preventiveactivityhasbeenattributedtoglucosinolates,whicharedefencecompoundscharacteristicofcruciferousplants.We report engineering the production of the bioactive benzylglucosinolate in the non-cruciferous plant Nicotiana benthamiana. The study includes identification of abiosynthetic enzyme, γ-glutamyl peptidase 1 (GGP1), which substantially increases benzylglucosinolateproduction.GGP1wasshowntometabolizeaglutathioneconjugatethat otherwise accumulates to large extents. Its discovery elucidates an uncharacterizedstepinthebiosynthesisofglucosinolatesandexpandsthereactionrepertoireofenzymescontainingglutamineamidotransferasedomains.Theabilitytoengineerbenzylglucosino-lateprovidesthebasisforheterologousproductionofglucosinolatesaimedatutilizationincancer-prevention.Theengineeringstrategydevelopedhasgreatpotentialasagenerallyapplicabletechnologicalplatformforgenediscoveryandproof-of-conceptengineeringofcomplexbiosyntheticpathwaysleadingtovaluablephytochemicals.
L16
BIOTECHNOLOGICAL ALTERNATIVE IN PRODUCTION OF BIOACTIVE SUBSTANCES IN THE GENUS HYPERICUM
Čellarova,E.
P. J. Šafárik University in Košice, Faculty of Science,Institute of Biology and Ecology/Genetics
MedicinalplantsfromthegenusHypericum,especiallyH.perforatumthatisthemoststudiedonefromabout450species,areattractingconsiderableinterestforcenturies.Modernpharmacologicalstudiesdemonstratehighefficacyofsomemetabolites,namelynaphthodianthronesandacylphloroglucinolswithawiderangeofactivitiesandsometimeswith insufficiently defined pharmaceutical function.Knowledge on biosynthesis of bio-activesubstances in therepresentativesof thegenusHypericumandgenesencodingforkeyenzymes in theirbiosyntheticpathways isvery limited.Thisallsuggests forfurtherdetailbiologicalandgeneticstudiesofthegenus/species.Theinconsistentchemicalprofilesarearesultofgeneticsrelatedtosecondarymetabolitebiosynthesis,physiologyandplant-environmentalinteractions. Inearlyninetiesofthelastcenturywhennewactivitiesofadianthronehypericin,suchasanticancerandantiviralhavebeenreported,H.perforatumL.asanaturalsourceofhypericinhasdevelopedintoamodelspeciesforbiotechnologicalandgeneticstudiesinourlaboratory. The biotechnological approach in study/production of desirable secondary metabolitesbytherepresentativesofthegenusHypericumcomprises: i) developmentofaneffectiveregenerationsysteminvitrobyeitherdirectorgano genesisorsomaticembryogenesisforhigh-producinggenotypes/species; ii) wideningofgeneticvariationviaindirectregenerationfromcallus; iii) developmentofaneffectivetransformationsystemforstudyingthegenefunction and/orforproductionpurposes; iv) studyingthecandidategenesinbiosynthesisofbioactivecompoundsandaffect- ingtheirexpression; v) designofsmall-scalecultivationofexplantsinbioreactorundercontrolledcondi- tions; vi) developmentofaneffectivecryopreservationprotocolsforestablishmentofgene bank.
This project was supported by the Slovak Research and Development Agency under the contract No.VVCE-0001-07 “CeXignal
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L17
SYNTHESIS AND ANTITUMOR ACTIVITY OF COMPOUNDS BASEDON 9-ISOTHIOCYANATOACRIDINE
Imrich,J.,Balentová,E.,Géci,I.,Vilková,M.,Tomaščiková,J.,Ungvarský,J.,Drajna,L.,andKristian,P.
P. J. Šafárik University, Faculty of Science, Institute of Chemistry,Moyzesova 11, 04167 Košice, Slovak Republic
Anoriginalsynthon,9-isothiocyanatoacridine,preparedbyKristianin1961,hasbeenbroadlyutilizedinourlaboratoryfornumeroussynthesesofvariousclassesofnewacyclicandheterocycliccompoundspossessingmoderateantitumoractivityinpastfifteenyears. The sets of newly prepared compounds comprised acridinyl and 9,10-dihydro-acridinyl thioureas, ureas, thiocarbamates, dithiocarbamates, thiosemicarbazides and hydrazones as intermediates and spirodihydroacridines and acridinyl thiazolidines, imidazolines,thiazines,isooxazolines,andoxadiazolesasfinalheterocyclicstructures. Thissyntheticvariabilityinspiredoursearchfornewsyntheticanalogues,wherestructures based on 3,6-disothiocyanatoacridine, 9-isothiocyanato-1,2,3,4-tetrahydro- acridine and sugar isothiocyanates proved to be promising synthons, fluorogens, and/orbiomarkers. Due to thepresenceof aplanar acridine skeleton, intercalatingpropertieshavebeenstudiedbyspectralmethodsandantitumoractivityhasbeenprovedformanytargetcompoundsincollaborationswithseverallaboratoriesinSlovakiaandabroad.
L18
COMPUTER-ASSISTED COMBINATORIAL DRUG DESIGN
Frecer,V.
Cancer Research Institute, Slovak Academy of Sciences, SK-83391 Bratislava, Slovakia
The last decadeshaswitnessed a technological revolution inmoleculardesign,material science and nanotechnology. Combinatorial chemistry, parallel synthesis and high-throughputscreeningaswellascomputationaldesigntechniqueshavebeenembracedbynumerousindustrysectorsasthemeanstoacceleratethediscoveryofnewmoleculesandmaterialswiththedesiredproperties.Thecentralaimofthecomputer-assistedmolecu-larandmaterialdesigntechniquesistohelpthescientiststomakethediscoveryprocessfaster,cheaperandsaferandconsequentlytofostertheindustrialresearchandinnovation. Wenowhavethecompletegenomesequencesofmorethan100organismsandcan employ the tools of bioinformatics to identify genes and proteins as potential drugtargets.Thebreadthofinnovativetechniquesrangingfromfragment-based,analog-based,structure-basedandcombinatoriallibrarydesignallowsustodesign,synthesize,orscreenformoleculesactingonthechosendrugtargetsmoreefficiently. Principlesand selectedexamplesof theuseofcomputer-assistedcombinatorialdesigntechniquesinthepharmaceuticalindustrywillbepresentedanddiscussed.Virtuallibrarydesign,focusing,virtualscreeningaswellaspharmacokineticpropertiesprediction,willbeillustratedonanexampleofanalog-baseddesignofantiviralcompounds.
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L19
EXPLOITING ITC-MODULATED SIGNALING PATHWAYS IN CANCER THERAPY
Sedlak,J.1,2,Jakubikova,J.2,3,Bodo,J.2,Sedlakova,O.4,Cholujova,D.2,Pastorek,M.2,Balaz,P.5,Hunakova,L.1,2,andDuraj,J.1,2
1Cancer Research Institute SAS, Centre of Excellence for Signalosome Research, Bratislava, Slovakia
2Cancer Research Institute SAS, Laboratory of Tumor Immunology,Bratislava, Slovak Republic
3Dana-Farber Cancer Institute, Department of Medical Oncology, Boston MA, USA4Institute of Virology SAS, Bratislava, Slovak Republic5Institute of Geotechnics SAS, Kosice, Slovak Republic
Glucosinolates(beta-thioglucoside-N-hydroxysulfates),theprecursorsofisothio-cyanates(ITCs)havebeenfoundinfamiliesofangiosperms,manyofwhichareedibleandcouldprovidesubstantialamountofchemopreventive,dietaryorpharmacologicalagents.SincethemiddleofthelastcenturythesystematicinvestigationofITCsrevealedanumberofmechanismsthatmaybeinvolvedintheirchemopreventive/anticancereffects.Initially demonstrated as potent inducers of phase II enzymes activation and effective indirect antioxidants,ITCsareknowntoaffectmanyotherssignaling/regulatorypathways.Theinvitromodelsofprimaryorpermanentcelllineshelptodissectthemolecularmechanismsinfluenced by ITC treatmentmany ofwhichwere confirmed in animalmodels such as activation of apoptosis, modulation of intracellular redox state or inhibition of HDAC activity.Anti-inflammatory, -angiogenic, -neoplasticactivitiesofITCsin invivomodelsmay stem from the fact that theymodulate epithelial-mesenchymal transitionand targetpopulation of tumor-initiating cells as well as phoshorylation signaling pathways and epigeneticregulation.ThepromisingperspectivearisesfromobservationsthatITCsbehave synergistically with chemotherapeutic drugs to achieve cytotoxic effect. These small,multi-target reactive molecules represent the plentiful source of interesting compounds modulatingtheessentialcellularpathways.
This project was supported by the Slovak Research and Development Agency under the contract No.VVCE-0001-07 “CeXignal
L20
DOWN-REGULATION OF BCL-2 AND AKT INDUCED BY COMBINATION OF PHOTOACTIVATED HYPERICIN AND GENISTEIN IN HUMAN BREAST
CANCER CELLS
Ferenc,P.,Solár,P.,Kleban,J.,Mikeš,J.,andFedoročko,P.
Institute of Biology and Ecology, Faculty of Science, P.J. Šafárik University
Thecombinedefectofphotodynamictherapy(PDT)andgenistein(G)onMCF-7andMDA-MB-231humanbreastcancercelllineswasstudied.WeinvestigatedtheeffectofGontheresponseofestrogen-responsiveMCF-7andestrogen-unresponsiveMDA-MB-231cells toPDT,both identified inour conditions asPDT-hypericin (PDT-H) resistant.For combined therapy, low toxic concentrations of both agentswere selected based onMTTassay.Theapoptoticprocesswasmonitoredusingcellcycle,nuclearmorphologyand Western blot analyses supported by caspase-7 activity assay. PDT-H combined with G revealedsignificantincreaseinthepercentageofapoptoticcellsandPARPcleavage,whichcorrelatedwithdepletionofBcl-2andincreaseofBaxprotein,aswellaswithaccumula-tionofMCF-7andMDA-MB-231cellsintheG2phaseofthecellcyclespecificallyforthecombinationofPDT-H+G.Furthermore,GeliminatedPDT-HinducedactivationofAktandErk1/2phosphorylationstatusinMCF-7cells.TheonlydeclineinErk1/2phosphoryla-tionwasobservedinMDA-MB-231cellsaftercombinedtherapy.OurresultsindicatethatcombinedtreatmentwithPDT-HandtyrosinekinaseinhibitorGmaysignificantlyimprovetheeffectivenessofPDTtherapybyincreasingthesensitivityofMCF-7aswellasMDA-MB-231cellstoPDT-H.
This project was supported by the Slovak Research and Development Agency under the contract No.VVCE-0001-07 “CeXignal
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L21
ADMINISTRATION OF ISOTHIOCYANATE (E-4IB) AND CISPLATIN LEADS TO ALTERED SIGNALLING AND LYSOSOMAL EXPORT IN HUMAN
OVARIAN CARCINOMA SENSITIVE AND CISPLATIN-RESISTANT CELLS
Duraj,J.,Gronesova,P.,Hunakova,L.,Cholujova,D.,Bodo,J.,andSedlak,J.
Cancer Research Institute, Slovak Academy of Sciences,SK-83391 Bratislava, Slovak Republic
Theaimofthisstudywastocomparetheeffectofanewsyntheticisothiocyanate derivative, ethyl 4-isothiocyanatobutanoate E-4IB and cisplatin (CDDP) in CDDP- sensitivehumanovariancarcinomacellline(A2780)anditsresistantsubline(A2780/CP). In parental cells, in comparison to untreated cells, sequential administration of both compounds led to higher exosomal dye (LysoTrackerGreenDND-26) retention and to alterations ofmitogen-activated protein kinases (MAPKs), JNK,ERK and p38, orAkt, accompanied by changes in several anti- and pro-apoptotic molecules and lysosomal protein LAMP-1, as detected byWestern blotting. On the contrary, variantA2780/CPcellswereresistanttoCDDP-ortocombinedsensitizer(E-4IB)/inducer(CDDP)-related apoptosisinductionandexertedminorchangesinthelevelsofthesemolecules.
This project was supported by the Slovak Research and Development Agency under the contract No.VVCE-0001-07 “CeXignal
L22
SELECTIVE ANTIPROLIFERATIVE ACTIVITY IN VITRO OF STRUCTURALLY RELATED ISOTHIOCYANATES, ERUCIN AND SULFORAPHANE,
ON HUMAN PROSTATE CELLS
Melchini,A.1,2,Costa,Ch.3,Catania,S.3,Taviano,M.F.4,Miceli,N.4,Mithen,R.2,Traka,M.2
1Foundation “Prof. Antonio Imbesi”, University of Messina, Italy2Institute of Food Research, Norwich Research Park, Norwich, United Kingdom
3Interdepartmental Centre of Experimental, Environmental and Occupational Toxicology, University of Messina, Italy
4Pharmaco-Biological Department, School of Pharmacy, University of Messina, Italy
Cruciferousvegetablesarearichsourceofisothiocyanates(ITCs)thatarerecentlyidentifiedaspromisingdietaryconstituentsabletoprotectagainstthemostcommoncancertypes,suchas lung,prostate,breastandcoloncancers.Aprimarygoal in theanticancerdrugsresearchisthediscoveryanddevelopmentoffunctionalmoleculescharacterizedbyselectivecytotoxicityagainstcancercellsandlowtoxicityforhealthycells.LiteraturedatashowtherelevantantiproliferativeactivityofITCsinvariouscancercelllines.However,theireffectinnormalcellshasnotbeenfullyinvestigated.Previousstudieshaveprovided evidence that the 4- (methylthio) butyl isothiocyanate (erucin, ER) is selective in its effects,inducingastrongantiproliferativeeffectonhumanleukemiacells,butnotinnon-transformedhumanperipheralTlymphocytes. Recently,theselectivityofβ-phenylethylisothiocyanate(PEITC)againstonco-genicallytransformedovarianepithelialcells,includingcisplatin-resistantcells,hasbeen reported. The aim of this study was to evaluate the differential sensitivity between normal,hyperplasicandcancercellsfromhumanprostatetoERanditsoxidizedanalog,the4-(methylsulfinyl)butylisothiocyanate(sulforaphane,SF). Theeffectof increasingconcentrationsofSFandERon threedifferenthuman prostatic cell lines, normal prostate epithelium (PNT1A), benign prostatic hyperplasia(BPH-1)andprostateadenocarcinomacells(PC3)wasdeterminateusinginvitroprolifera-tionassays. Dataobtainedshowedacomparabledose-dependentdecreaseinPC3cellviabilityafterERandSFtreatment.BothITCswereveryeffectiveininhibitingproliferationofPC3cellsalreadyatconcentrationsthatmaybeachievableinhumans(1-2µM)afterconsump-tionofcruciferousvegetables.Cellviabilitywasreducedbyaround90%after75µMERand50µMSFtreatment. Interestingly,PNT1AandBPH-1cellswerelesssensitivetobothITCs,andthe
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trendforreductionincellviabilitywasdifferentcomparedtoPC3cells.Inthesecelllines,ITCs started exhibiting significant cytotoxicity at higher concentrations (≥50 µM ER,SF)andreducedcellviabilitybymore than70%after125µMERand75µMSF.Our preliminary data demonstrate that these structurally related ITCs exhibit a stronger cytotoxicityagainstcancercellscomparedtononmalignantcells.Thus,understandingthespecificactionofITCsononcogenicsignallingpathwaysmaybenecessarytomakethepotentialselectivityofdietaryITCsasignificanttherapeuticapplication.
L23
EFFECTS OF CARVACROL AND ROSEMARY OIL SUPPLEMENTATION ON OXIDATIVE DNA DAMAGE INDUCED IN PRIMARY RAT HEPATOCYTES BY
2,3-DIMETHOXY-1,4-NAPHTHOQUINONE (DMNQ)
Horváthová,E.,andSlameňová,D.
Cancer Research Institute, Slovak Academy of Sciences, Laboratory of Mutagenesisand Carcinogenesis, Vlárska 7, 833 91 Bratislava, e-mail: [email protected]
Freeradicalscanalterthestructureofbiomacromoleculesandinducecanceraswellasheart,eyeandneurologicaldiseases.Thedetrimentaleffectsofoxidationcausedbyradi-calscanbediminishedbysyntheticornaturalcompounds thatcanbind these free radicals. However, the use of synthetic preparations has caused some problems due to their highlyvolatilenature, instabilityathightemperaturesandstrict lawrestrictions.Consequently, it isnecessary tousesaferandmore reliablechemicals topreventoxidation reactions.Recently,therehasbeenagrowing interest inplantsasnaturalsourcesofcompoundsappropriate forthe prevention of oxidation, controlling pathogens and/or toxin-producing microorganisms in food, and for the treatment of several diseases. Although plant volatiles are common ingredients of the diet and are known especially for their positive action, the increased humanexposurerequiresacarefulre-assessmentoftheirpositiveandnegativecharacteristics indifferentexperimentalsystems. Theaimofthisstudywastodetermineinexvivoexperimentsthepotentialprotectiveeffectsofcarvacrol,acomponent,-orrosemaryoil,awholeplantessentialcompound,-supple-mentationonthelevelofoxidativeDNAdamageinducedinprimaryrathepatocytesby2,3-dimethoxy-1,4-naphthoquinone(DMNQ). Hepatocyteswereisolatedeitherfromcontrolratsorratstowhichcarvacrolintwoconcentration(30or60mg/kg/day)for7daysorrosemaryoilinthreeconcentrations(0.125;0.25or0.5‰)for14daysweregivenasadrinkingcomponent.PrimaryrathepatocyteswerethenexposedtoDMNQ.ThelevelsofoxidativeDNAlesionsincontrolandtreatedcellsweremeasuredbythemodifiedsinglecellgelelectrophoresis,whichincludesdigestionwithspecificrepairenzymemixtureofendonucleaseIII(EndoIII)andformamidopyrimidine-DNAglycosy-lase(Fpg). Our results showed that carvacrol- or rosemary oil-supplementation for 7 or 14days, respectively, didnot induce anynegative effect on thehealth conditionof animalsoron the levelofDNAdamage in isolatedhepatocytes.On theotherhand,wefoundout that supplementationofratswithcarvacrolorrosemaryoilefficientlydecreasedthelevelofDMNQ-inducedoxidativeDNAlesionsinprimaryrathepatocytes.
This study was supported by the Slovak Research and Development Agency grant APVV 51-015404 and by Scientific Grant Agency of the Ministry of Education of Slovak Republic and the Academy of Sciences grant VEGA 2/0072/09.
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L24
MANUMYCIN A AS AGENT SENZITIZING ADENOCARCINOMA CELLS TO PHOTODYNAMIC TREATMENT
Sačková,V.,Mikeš,J.,Jendželovský,R.,andFedoročko,P.
Institute of Biology and Ecology, Department of Cellular Biology, Faculty of Science, Pavol Jozef Šafárik University, Moyzesova 11, 040 01 Košice, Slovak Republic
Severalphotosensitizersandfarnesyltransferaseinhibitorsareextensivelystudied as promising anticancer drugs. In this study, hypericin, a photoactive component of Hypericum perforatum, was used as photosensitive drug and manumycinA, a naturalproduct of Streptomyces parvulus, was used as selective farnesyltransferase inhibitor.Our experimentsweredesigned to investigate the apoptoticmachinery and thepossible participation of caspase-independent pathway activated by hypericinmediated photody-namic therapy, alone and in combination with manumycin, on HT-29 adenocarcinomacells.Viability, survival,clonogenicandfluorogeniccaspaseactivityassaysandwesternblotanalysisof selectedproteins inwholeand fractionatedcell lysateswereperformed.Photoactivated hypericin and manumycin, used separately, reduced cell viability and proliferation, causedactivationofcaspasesandalteredexpressionofapoptoticproteins.Pre-treatmentofcellswithnon-cytotoxicconcentrationsofmanumycinpriortohypericin light activation, sensitized cells to effect of photoactivated hypericin. This combined therapy increased inhibition of clonogenic tumor growth and resulted in an enhanced apoptotic response of HT-29 adenocarcinoma cells (elevated caspase activity, massivePARPcleavage,completeBaxcleavage,Aktcleavage)comparedwiththeeffectsoncellstreatedwithhypericinandmanumycinalone.Basedontheseresults,theeffectiveanticancer activity of photosensitive drug and farnesyltransferase inhibitor combination could representanewmodalityapproachfortreatmentofthecancer. This project was supported by the Slovak Research and Development Agency under the contract No.VVCE-0001-07 “CeXignal
L25
HSP90 PROTEIN AS A TARGET FOR IMPROVING PHOTODYNAMIC THERAPY
Solár,P.,Chytilová,M.,Ferenc,P.,Solárová,Z.*,andFedoročko,P.
Laboratory of Cell Biology, Institute of Biology and Ecology,Faculty of Sciences, P. J. Šafárik University, Košice, Slovak Republic
*Geriatric Nursing Clinic, Faculty of Medicine, P. J. Šafárik University, Košice, Slovak Republic
Ourresultsshowthatphotodynamictherapy(PDT)ofhypericininducesthephos-phorylationofErk1,2proteinsandincreasesthelevelofsurvivin,whichistheinhibitorofapoptosisprotein.Survivinisaclientproteinforthe90kDaheatshockprotein(Hsp90),andthebindingofsurvivintoHsp90assistsinthematuration,properfolding,assembly,andtrans-portofthisprotein.17-(dimethylaminoethylamino)-17-demethoxygeldanamycin(17-DMAG)isabenzoquinoneansamycinantibioticwithdestabilizingeffectondifferentHsp90clientproteins,suchasAkt,HER-2,Rafkinase,survivinandothers.Incontrasttoothergeldanamycin(GA)analogues,17-DMAGhasseveralpotentialadvantages,e.g.watersolubility,higheroralbioavailabilityand/orlesstoxicmetabolites. In our studywewere focusing on the determination of the 17-DMAGeffect onSKBR3breastcancercells(overexpressingHER-2oncogene)aswellasontheresponseofSKBR3cellsonPDTwithhypericinwhenmodulatedby17-DMAGadministration.Lowcon-centrationof17-DMAGwithouttheeffectonsurvivalofSKBR3cellssignificantlyreducedmetabolicactivity(MTTassay),viabilityandcellularityofSKBR3cellsinduced24and48hafterPDTbyhypericin.Moreover,weobservedasignificantdecreaseofSKBR3cellsinSphaseaswellasanincreaseinG1phaseofthecellcycleafter24htreatmentwithIC10concentrationof17-DMAGandanincreaseinG2phaseofSKBR3cellswhen17-DMAGwascombinedwithPDT.Furthermore,IC50concentrationof17-DMAGinduceddecreaseinHER-2,Akt,phosporylatedErk1,2andsurvivinproteinlevelinSKBR3cellsshorttimeafteritsapplication.Inthisregard,thelevelofphosphorylatedErk1,2andsurvivininduced24afterPDTstayeddecreasedwhenSKBR3cellwerepretreatedwith17-DMAG.Interestingly,IC10concentrationof17-DMAGledtototaldepletionofAkt,P-Erk1,2proteinsandtodecreaseofsurvivinlevelinanalyzedcellsafter48h.Ontheotherhand,17-DMAGdidnotchangeHER-2relativeexpression(mRNAlevel)inSKBR3cells,butcausedasignificantdecreaseofHER-2mRNAinMCF.7cellscharacterizedwithlowerHER-2expression. TheseresultsshowthattargetingHsp90clientproteinsincreasestheefficiencyofantineoplasticeffectofPDTinvitro.This project was supported by the Slovak Research and Development Agency under the contract No.VVCE-0001-07 “CeXignal
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L26
ROLE OF PROTEIN p53 IN PHOTODYNAMIC THERAPY WITH HYPERICIN
Mikeš,J,Jedležovský,R.,Kovaľ,J.,Kello,M.,Sačková,V.,Kulíková,L.,Šemerláková,M.,andFedoročko,P.
Laboratory of Cell Biology, Institute of Biology and Ecology, Faculty of Sciences,P. J. Šafárik University, Košice, Slovak Republic
*Geriatric Nursing Clinic, Faculty of Medicine, P. J. Šafárik University, Košice, Slovak Republic
Photodynamictherapywithhypericin(HY-PDT)knownasanefficientmodality for treatment of various cancerous and non-cancerous diseases is based on administra-tionofanon-orweakly-toxicphotosensitizeranditsactivationwithlightofappropriate wavelength.Although the roleofp53protein incelldeathsignaling iswellestablished,relativelylittleisknownofitsimpactontheefficiencyofHY-PDT. Inourexperimentswefoundprevalenceofnecrosisaccompaniedbysuppressionbycaspase-3activationastheprincipalresponsetoHY-PDTdespitedifferentPDTdosesandtheabsenceofanti-apoptoticBcl-2expressioninHT-29cells;evenifthesamecondi-tioninducedcaspase-3activityatsimilartoxicityinHeLacells.IntroductionofBcl-2intoHT-29cellsinvokedcaspase-3activation,changedBcl-XLexpressionpattern,increasedtheapoptosisratiowithnoeffectonoveralltoxicity,andsupportedarrestintheG2/M-phaseofcellcycle.SinceitisknownthatBcl-2suppressioninHT-29isreversibleandlinkedtotheover-expressionofmutatedp53andalsoconsideringourdata,wesuggestedthatthemuta-tioninp53andeventslinkedtothisfeaturemightplayroleincelldeathsignalingincoloncancer cells.To prove the concept, comparison of sensitivity and long-term survival ofp53-nullversuswt-p53expressingHCT-116cellswasaccomplishedinaddition.Wefoundthatthelackofp53functiondidnotaffectcellproliferationorattenuatetheinitialphases of programmed cell death. However, analyses of apoptosis in the final stages revealed suppression of its incidence and delayed activation of caspase-3 in p53-null cells. Long-termsurvivalregardedasclonogenicabilitywassignificantlyenhancedinp53-nullcells and especially in hypoxia. Bringing the evidence together, we prove that despite insignificantimpactofoveralltoxicity,expressionandstatusofp53affectsfinalizationof apoptoticprocessandthelong-thermsurvivaloftumorcells.SincedestructionoftumortissueanditsvascularsystembyPDTtendstoleadtohypoxia,superiorsurvivalofp53-defficient cells under given conditionsmight result in recurrence of cncer diseases andthereforep53-statusmightbeconsideredasabadprognosisfactorinp53-mutatedtumors.
This work was supported by the Slovak Research and Development Agency under the contract No. VVCE-0001-07, by the Science and Technology Assistance Agency under contract No. APVV 0321-07, by the Scientific Grant Agency of the Ministry of Education of the Slovac Republic No. VEGA 1/0240/08 and by Internal grant system of P.J.Šafárik University in Košice under contract No. VVGS 26/09-10.
L27
POTENTIATION OF PHOTODYNAMIC THERAPY WITH HYPERICIN BY EXOGENOUS POLYUNSATURATED FATTY ACIDS
Kello,M.,Mikeš,J.,Jendželovský,R.,Kovaľ,J.,andFedoročko,P.
Institute of Biology and Ecology, Faculty of Science,PJ. Šafárik University in Košice, Moyzesova 11, 040 01 Košice, Slovak Republic
Extensiveresearchoverthepastseveraldecadeshasidentifiednumerousdietaryand botanical natural compounds that have beneficial effect in prevention and cure ofoncologicalpatients.Exploitationof lipidemulsions innutritiondiethasbecomeanew promisingmodality in cancer therapy.Lipid emulsions containing polyunsaturated fatty acids (PUFAs) as essential components should been considered not only as a source ofenergybutalsoasa toolforspecificpharmacological targeting.PUFAsareimportant inseveralphysiologicalcellfunctions,suchassignal transduction,cellgrowth,differentia-tionandviability.Ascomponentsofmembranephospholipids,PUFAschangemembrane fluidity and remodel membrane structure, which influence other membrane-mediated cellular functions.Dietary therapyofoncologicalpatientsemployingspecificfattyacids(n-3,n-6)decreasestumourgrowthasasingletherapyandincreasestherapeuticresponseoftumourtobroadspectrumofchemotherapeutics. Hypericin,asphotochemicalextractfromSt.JohnsWort(Hypericumperforatum)andrelatedspecies,hasbeenshowntohavepotent,broadspectrumofactivityincludingproapoptotic.Photodynamictherapy(PDT)basedonhypericiniscurrentlybeingusedasanalternativetreatmentmodalityforsometypesofcancers.Combinationofhypericin-basedPDTwithnutrition therapybyPUFAscouldbringconsiderableasset in tumour therapy.Thisparticularapproachintumourtherapyisnotexaminedproperlythesedays.
This work was supported by the Slovak Research and Development Agency under the contract No. VVCE-0001-07, by the Science and Technology Assistance Agency under contract No. APVV-0321-07, by the Scientific Grant Agency of the Ministry of Education of the Slovak Republic No. VEGA 1/0240/08.
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P1
ALOE VERA AND HONEY REDUCE TUMOR GROWTH BY DECREASING ITS CELL PROLIFERATIVE CAPACITY IN TUMOUR-BEARING RATS
Tomasin,R.,Cintra,M.C.andGomesMarcondes,G.
Laboratory of Nutrition and Cancer, Department of Physiology and Biophysics,Institute of Biology, State University of Campinas, UNICAMP, Campinas, Brazil
Cancer is responsible for around8millionofdeaths and11millionnewcasesare diagnosed every year. Researches show the importance of co-adjuvant therapies incancercontrol.Aloeveracan reduce tumoralmassandmetastasis,whilehoney inhibitstumour growth.This research verified the influence ofAloe vera and honey on tumourgrowth, accessing tumoral size and cell proliferative rate after 7, 14 and 20 days of subcutaneousWalker256carcinomaimplantationinadultrats.WAgroupreceivedgavageofAloeveraandhoneysolutiondaily,whiletheCWgroupwasgavagedwith0.9%NaClsolution.TheeffectofAloeveraandhoneyagainsttumourgrowthcouldbeseencomparingCWandWAtumourgrowthevolution,(relativeweight(%)),sincethedifferencebetweenCWandWAincreasedespeciallyafter20thdayoftumourevolution(CW7d=0.79±0.32;WA 7d=0.68±0.43; CW 14d=4.14±2.08;WA 14d=3.17±1.38; CW 20d=7.57±2.98;WA20d=5.16±2.46). In order to justify the less tumoral growth evolution, the tumour cell proliferativerateswereobtainedbyimunohistochemestryassayforKi-67(anuclearproteincurrentduringG-1,S,G-2andmitosisphases).Thenumberofstainednuclei(%)inWA tumoursevidentlydecreasedinallaccesseddays(CW7d=71.0±10.9;WA7d=51.4±18.1;CW14d=69.6±13.5;WA14d=37.2±16.4;CW20d=59.1±22.7;WA20d=32.0±3.3).Thesedata suggest thatAloe vera and honey canmodulate tumour growth (reducing the cell proliferation), helping the less tumor evolution (tumour weight reduction). Certainly alargeandcomplexnumberofmechanismsare involved in thisprocesswhichAloeveraandhoneycanberelated, including themainpoint tomanageandmodulate the tumour proliferativecapacity.
P2
SULFORAPHANE AND ALYSSIN AS CHEMOPREVENTIVE AGENTS
Lubelska,K.,Skupińska,K.,Misiewicz,I.,andKasprzycka-Guttman,T.
National Medicines Institute, 00-725 Warsaw, Poland, Chełmska 30/34
Isothiocyanates(ITC)are thegroupofchemicalcompoundscontainingchemo-preventiveagents.TheyarefoundinvegetablesfromthefamilyCruciferous.Mainlytheyoccurinvegetablessuchasbroccoli,cabbage,brusselssprouts,caulifloweretc.TheresultsofepidemiologicalstudiesindicateacorrelationbetweenhighconsumptionofBrassicascontainingITCandlowerriskofhumancancers.ITCarestronginducersofphase2detoxi-ficationenzymeslikeglutathioneS-transferases,NADPH:quinineoxidoreductase1(QR),etc.Thisgens-encodingdetoxificationandantioxidantproteinsareregulatedbynuclearfactor-erythroid2p45-relatedfactor2(Nrf2).Underhomeostaticconditions,Nrf2ismainlysequesteredinthecytoplasmbyprotein–Keap1.OxidantsandelectrophilescausedthatNrf2isreleasedfromKeap1protectionandtranslocatestothenucleus.IthasbeenshownthatNrf2nullmicearehighlysensitivetocarcinogenandoxidativestress. InthisstudyweevaluatedchemopreventiveactivityofITC:sulforaphane(SFN)and alyssin, which was synthesized in order to search most efficient chemopreventiveagents.EachofthemarenaturallypresentinfamilyBrassicaceae.ThestudywascarriedoutinhumanintestinalCaco-2cells.InordertoevaluatechemopreventiveactivityofITCwestudieddoseandtime–dependentchangesinviabilityandQRactivity.WeperformedaMTT - cytotoxicity test, which is a quantitative colorimetricmethod formammaliancellsurvivalandcellproliferation.Theassaymeasurestheamountofformazonproducedwitchproportionaltothenumberoflivingcellspresentintheculture.Theabsorbancewas measuredwithShimadzuUVmini1240spectrophotometerat570nm.TheQRactivitywasdeterminedbymeasuring theNADPH-dependentmenadiol-mediated reductionofMTT.Theabsorptionwassetat thelevelof11300M-1cm-1atλ=610nm.TheQRactivitywas normalized permg of total protein and expressed inmU/mg protein.Total proteincontentwasassessedbytheBradfordassay.InordertostudymechanismofQRinductionwealsoexaminedthetime-dependentchangesinthesubcellularlocalizationofNrf-2by immunostainingwithanti-Nrf2antibodyanddetectiontheresultswitchhelpofconfocalmicroscope(OlympusFluoView500systemequippedwithOlympusIX70microscope). Our results have shown dose–dependent changes in cell viability and time– dependentchangesinQR,whichindicatesthatITCareeffectivechemopreventiveagentsforCaco-2cells.
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P3
MODULATION OF MARKERS ASSOCIATED WITH AGGRESSIVE PHENOTYPE IN MDA-MB-231 BREAST CARCINOMA CELLS BY
SULFORAPHANE
L.Hunakova,L.1,Sedlakova,O.2,Cholujova,D.1,Gronesova,P.1,Duraj,J.1andSedlak,J.1
1Tumor Immunology Laboratory, Cancer Research Institute,Vlarska 7, Bratislava 833 91, Slovak Republic
2Institute of Virology, Slovak Academy of Sciences,Dubravska cesta 9, 845 05 Bratislava, Slovak Republic
Becauseof themultiplicityof oncogenicpathways, some tumoursmaybehave extremely aggressively. This aggressivity is dependent on tumor microenvironment, feasibility of neoangiogenesis, degradation of extracellular matrix, disruption of epithelial integrity and release of cells from intercellular interactions, and acquisition of EMT. Sulforaphane (SFN) is a naturally occurring cancer chemopreventive isothiocyanatefoundincruciferousvegetables,consumptionofwhichhasbeenassociatedwith reducedriskofcancer. In this study,wedescribeeffectofSFNonvariousaspects determininginvasivebehaviorofMDA-MB-231humanbreastcarcinomacells. Westudiedmodulationofmoleculesassociatedwithepithelial tomesenchymal transition (EMT), hypoxic marker CA IX and mitochondrially located peripheral benzodiazepinereceptor(PBR)usingflowcytometry,geneexpressionofMMP1,3,7,9,14,POU5F1andTwist1mRNAbyRTPCR,andcytokineproductionbymultiplexbead assay.SFNdownregulatedPBRandvimentinexpressioninadosedependentmanner,butdid not significantly affect CA IX protein expression.Among studiedMMP’s,MMP14mRNA was downregulated and MMP7 significantly decreased while MMP1, 3 and 9 remained unchanged. We observed marked down regulation of Twist1 and POU5F1, transcriptionfactorsthatmediateEMTandtheself-renewalofundifferentiatedembryonicstemcells.SFNreducedalso theproductionofpro-inflammatorycytokines IL-1b, IL-6,TNF-alpha,IFN-gamma,immunomodulatingcytokineIL-4andgrowthfactorsinvolvedinangiogenesisPDGFandVEGF. Our results indicate thatSFNefficacy isassociatedwith the reversalofseveralbiologicalcharacteristicsconnectedwithEMTorimplicatedinthematrixdegradationandextracellularproteolysis,aswellaswithreducedproductionofpro-inflammatorycytokinesandpro-angiogenicgrowthfactors,butnotwithCAIXexpressioninMDA-MB-231cells.
This project was supported by the Slovak Research and Development Agency under the contract No.VVCE-0001-07 “CeXignal and Vega grant 02/0119/08.
P4
1-METHOXYINDOLE PHYTOALEXINS: SYNTHESIS AND ANTICANCER ACTIVITY
Kutschy,P.1,Čurillová,Z.1,Salayová,A.1,Repovská,M.1,Mezencev,R.2,Mojžiš,J.3,andPilátová,M.3
1P.J. Šafárik University, Faculty of Science, Institute of Chemical Sciences,Department of Organic Chemistry, Moyzesova 11, 040 01 Košice, Slovak Republic
2Georgia Institute of Technology, College of Science, School of Biology, 315 Ferst Drive, Atlanta, GA 30332, USA
3P.J. Šafárik University, Faculty of Medicine, Department of Pharmacology,SNP 1, 040 66 Košice, Slovak Republic
Plantsproducedefensivesecondarymetabolitesinresponsetovariousformsofstress, includingmicrobial infection, physical or chemical stress. Since the 1940,whenprofessorMűllerdefinedthesesubstancesasphytoalexins(plantsprotectingcompounds),manynatural productsof thisgrouphavebeen identified, isolated and studied from thepoint of view of their biological activities. Among the others, indole-derived sulfur- containing phytoalexins isolated from the plant family Brassicaceae (syn. Cruciferae)appeared to be attractive chemopreventive and potentially anticancer (antiproliferative)agents.Wehave found that suitablydesigned analogsof cruciferous1-methoxyindolinephytoalexins may exhibit a higher cytotoxic activity against human cancer cell lines, compared to natural leads. In this contribution the syntheses and in vitro anticancer activityofselectedcruciferousphytoalexinsandtheiranalogswillbepresented.Althoughmechanismofactionofthesecompoundsisnotyetelucidated,glutathionedepletionandtopoisomerase II inhibition have been suggested as a part of their anticancer mode of action. Remarkable possibilities of structural variations of the analogs of indole and indolinephytoalexinsarenotmuchexploredandnewanalogswithimprovedcytotoxicityandselectivitytowardcancercelllinescouldbedesignedandsynthesized.
This work was supported by the Slovak Research and Development Agency under the contract No. APVV-0514-06
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P5
COMBINATION OF DOXORUBICIN AND SULFORAPHANE FOR REVERS-ING DOXORUBICIN-RESISTANT PHENOTYPE IN MICE FIBROBLASTS
WITH p53Ser220 MUTATION
Lenzi,M.,Fimognari,C.,Cantelli-Forti,G.,andHrelia,P.
Department of Pharmacology, Alma Mater Studiorum - University of Bologna
Traditional cytotoxic chemotherapeutic approaches cannot curemost advanced solidmalignancies.Themajorfactorthatlimitstheeffectivenessofchemotherapyinpatientswithadvancedcanceristheacquisitionofresistance.Chemoresistanceisamultifactorialprocess,whichincludesalterationsindrugaccumulation,increasedactivityofgluthationeS-transferases,lossoffunctionandmutationsofp53,etc.Onestrategyforreversingdrugresistanceistheconcomitantuseofchemopreventiveagents(i.e.non-cytotoxicagentsabletoblocktheprogressiontoinvasivecancer)thatarebythemselvesnon-toxicbutthatcauseabetterresponseratesthaneitherreagentalone.Sulforaphaneisoneofthemostpromising chemopreventive agent. Sulforaphane inhibits cell-cycle progression and induces apoptosisindifferenttumorcell lines.Thepro-apoptoticpotentialofsulforaphanecouldbeeffectiveeitheraloneor incombinationwithother therapeuticstrategies in reversing chemoresistance. We firstly investigated the effects of sulforaphane on mouse fibroblasts bearingadifferentp53status(wild-type,knock-out,mutated)forunderstandingwhetherits activityispreventedbyamutatedp53status.p53-knock-outfibroblastsfromnewbornmicetransfectedwiththep53Ser220mutation,observedindifferenthumancancers,wereusedasamodelofmutatedp53status.Moreover,sincep53Ser220mutationfibroblastsshoweda doxorubicin-resistant phenotype,we secondly treated the cellswith a combination ofdoxorubicinplussulforaphane.Toclarifytheoptimalscheduleofcombination,westudied theeffectsofsimultaneousandsequentialexposuretodoxorubicinandsulforaphane.Takentogether, our results suggest that amutated p53 status did not prevent the induction of apoptosis by sulforaphane and that sulforaphane was able to reverse the resistance todoxorubicin when administered simultaneously or after doxorubicin. The association sulforaphane-doxorubicinmay therefore allow doxorubicin to be administered at lowerdoses,therebyreducingitspotentialtoxicity.
P6
ANALYSIS OF INTERACTION OF ISOTHIOCYANATES WITH 5-FLUOROURACIL IN CHINESE HAMSTER LUNG FIBROBLAST CELL LINE
Milczarek, M., Misiewicz, I., Skupińska, K, Lubelska, K., Kasprzycka-Guttman, T.
National Medicines Institute, 00-725 Warsaw, Poland, Chełmska 30/34
Sulforaphane is a naturally derived compound that is present in the tissues of cruciferaeplantsintheformofglucosinolates.ItactivatesphaseIIenzymesinmanytypesofcells:inhepaticcells,prostatecancercells,lymphocytes,lymphoblastoidcells.ItaltersalsophaseIIImetabolism,byincreasedconjugationwithGSHandenhancedexcretionofconjugatesfromthecellsandalsobyalterationofactivityofABC-transporters,likePgporMRP1proteins.Athigherdosesitexhibitscytostaticandcytotoxicactivitybyinfluenceoncellcycleandinductionofapoptosis,whatwasproveninmanytypesoftissues,mostlyincancercelllines. This multipathway activity of sulforaphane makes it one of the most potentchemopreventivecompoundsamongallnaturally-derivedcompounds.Theresultsforminvivostudiesalsoshowthatsulforaphaneinhibitschemically-inducedcarcinogenesisbythemechanismdescribedabove.Thisformulationiswidelyavailableintheformofthedietary supplement. Because of the influence on the enzymes that metabolize xenobiotics the question arises about possible interactions with other drug substances. Sulforaphanewas also shown to act synergistically with 5-fluorouracil (5-Fu) in human adenocystic carcinomaattwomalignancyItwasshownthatsulforaphanecanactsynergisticallywithdoxorubicinwhatcouldresultinthelowertoxicityofdoxorubicininnormalcells. We investigated: antipoliferative, cytotoxic and combined effect of isothiocya-nates(ITC):sulforaphane,alyssin,2-oxohexylITCand5-fluorouracilinChinesehamsterlungfibroblastcellline.TheMTTassaywasusedtotestcellviability.Theinteractionswereevaluated in the two schemesof administration: co-administrationof5-Fuwith isothio-cyanatesandpre-treatmentwithisothiocyanateswiththesubsequenttreatmentwith5-Fu.Theinteractionof testedagentsweredeterminedbyusingamedianeffectanalysiswith mutuallynonexclusivemodelasdescribed. The evaluation of interactions show antagonistic interaction between 5-Fuand sulforaphane and between 5-Fu and 2-oxohehsyl ITC in the two schemes. After co-administrationof5-Fuandalyssinwasdetectedantagonistceffectbothcompoundsbutatsecondschemeofadministrationwasobservedadditiveeffect.Wepresentthefirststepofourstudy..
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P7
WEDELOLACTONE REDUCES GROWTH AND INDUCES DIFFERENTIATION AND APOPTOSIS OF BREAST CANCER CELLS
Benes,P1,Pinheiro,D2,Nemajerova,A1,Knopfova,L.1,Smarda,J.1
1Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic
2 Faculty of Science, Oporto University, Porto, Portugal
Wedelolactone,thenaturallyoccurringcoumestanderivedfromEcliptaprostrate and Wedelia calendulacea, is commonly used in South American medicine as snake antivenom.IntraditionalChinesemedicine,coumestansareusedtotreatsepticshock,liverdiseases and viral infections.Moreover, coumestans reduce cancer risk, potentially duetotheirstructuralsimilaritytophytoestrogens.Recently,itwasshownthatwedelolactoneinhibits growth of prostate and pituitary cancer cells. In this studywe tested the effectofwedelolactoneonbreastcancercelllineMDA-MB-231.Wefoundthat10and20µMwedelolactoneinhibitsgrowthofMDA-MB-231cellsbyblockingcellcycleprogressionintheS-phase.Growthinhibitoryeffectofwedelolactonewasassociatedwithcelldifferentia-tionasindicatedbychangesincellmorphology,accumulationoflipiddropletsandover-expressionofmilkfatglobule-EGFfactor8.Theadditionof30and50µMwedelolactonereducesviabilityandinducesapoptosisofMDA-MB-231cellsdocumentedbychromatin condensation, nuclear fragmentation and poly (ADP ribose) polymerase cleavage. We concludethatwedelolactonepossessstronganti-cancerpropertiesandcouldbeconsideredaspotentialdrugintherapyofbreastcancer.
This work was funded by grants IAA501630801 of the Grant Agency of AS CR, MSM0021622415 of the Ministry of Education, Youth ans Sports of the Czech Republic and 204/08/H054 of the Grant Agency of the Czech Republic. Diana Pinheiro was supported by LPP/Erasmus program from the Socrates National Agency.
P8
COMET ASSAY: A USEFUL METHOD FOR STUDYING PAPAVER RHOEAS AND GENTIANA ASCLEPIADEA FLOWER EXTRACTS
Hudecová,A.1,Hašplová,K.1,Gálová,E.1,Miadoková,E1,Volkovová,K.2,Dušinská,M.2,3
1Department of Genetics, Faculty of Natural Sciences, Comenius University, Mlynská dolina B1, 842 15 Bratislava, Slovak Republic
2Department of Experimental and Applied Genetics, Slovak Medical University, Limbová 12, 833 03 Bratislava, Slovak Republic
3Health Effects Group, CEE, NILU, Kjeller, Norway
Plants are universally recognized as a vital part of theworld’s natural heritageandupto80%ofthepopulationsrelyonplantsfortheirprimaryhealthcare.Varietiesof medicinal plants are known as a source of natural phytochemicals such as antioxidantsthat can protect from oxidative stress and from various chronic diseases [5]. The useof traditionalmedicine iswidespreadandplants still represent a large sourceofnatural phytochemicalsthatmightserveasleadsforthedevelopmentofnoveldrugs. Inourstudies,methanolicextractsfromPapaverrhoeasandGentianaasclepiadeawereevaluatedforgenotoxiceffectsusingthecometassay.DNAdamagewasmeasuredinculturedhumanandmonkeycellsobtainedfromdifferenttissues.ThemethodcandetectvarioustypeofDNAdamageinducedbygenotoxicagents,includingradiation,chemicalsandagentsinducingoxidativestress[3]. Papaveraceaeisafamilyoffloweringplants.Theflowersofpoppyhavealonghistory ofmedicinal usage, especially for ailments in the elderly and children. Chiefly employedasamildpainrelieverandasa treatmentfor irritablecoughs, italsohelps to reduce nervous over-activity [2]. The flowers and petals are anodyne, emollient, expectorant, hypnotic, slightly narcotic and sedatíve. An infusion is taken internallyin the treatment of bronchial complaints and coughs, insomnia, poor digestion, nervous digestive disorders and minor painful conditions. The flowers are also used in the treatmentof jaundiceand–inverysmallquantities,andunderexpertsupervision–asasleep-inducingdrug[1,2]. PlantsbelongingtoGentianaceaearewidelyusedintraditionalmedicinesinmanycountries.SomeofthemareusedasingredientsinChineseherbalmedicinesforstimulationof appetite andgastric secretion, gastro-duodenal protection, liver protection, antifungaltreatment,andinsomecasesforwomen´sdiseases.ItisknownthatfamilyGentianaceaecontainssecoiridoidglucosides,flavoneC-glucosides,andxanthonesaswellastheirglu-cosides.Ithasbeenreportedthatgentiopicroside,acompoundpresent inmanygentianaspecies,hasfreeradicalscavengingactivity.
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Experimentswerecarriedoutoncelllines:HepG2(humanhepatocellularlivercarcinomacellline),Caco2(humanepithelialcolorectaladenocarcinomacellline),Cos1(monkeykidneycelllines)andTK6(lymphoblastcellline).Thecelltypeswereselectedasrepresentantsofliver,kidney,digestiveandlymphaticsystem(naturalfoodsupplementsareusuallytakenperorally). Cellsweretreatedfor24hourswith5differentconcentrationsofplantextract(2,5%;0,5%;0,25%;0,05%and0,025%).Resultsshowedthatthehighestconcentration(2,5%)oftheplantextractscausedDNAdamageinmostofthecelltypes.ThemostsensitivecelllineswereTK6.TheyshowedverystrongDNAdamageafter24hourtreatmentwith2,5%and0,5%ofplantextract.100cometsperslidewerescoredusingPerceptiveimageanalysissystemCometassayIV. Future research will focus on determining the optimal Papaver rhoeas and Gentiana asclepiadea flower extract concentrations to produce these protective effects, andontheirmechanismsofaction.
This work was supported by the grant UK /203/2008 and the grants VEGA: 2/7033/7 and1/0008/08, SAIA - travel grant, NILUEEA grants (PNRF122) and by COMICS, EC STREP: LSHB-CT-2006-037575.
P9
THE IMPACT OF CULTIVATION CONDITIONS ON PHYTOCOMPLEX COMPOSITION AND CHEMOPREVENTIVE PROPERTIES OF WHITE
CABBAGE
Śmiechowska,A.,Piasek,A.,Szumska,A.,Kawecka,A.,Kusznierewicz,B.,Zemło,D.,Szulfer,I.,Kielbratowska,M.,Konieczka,Namieśnik,J.,Bartoszek,A.
Chemical Faculty, Gdańsk University of Technology
The phytochemicals found in white cabbage (Brassica oleracea var. capitata) display an array of biological activities important in cancer chemoprevention. Health promoting properties of this vegetable are determined by the content of bioactive substances,theabundanceorevencompositionofwhichdependoncultivationconditions.In order to investigate these interrelationswe cultivated the seedlings ofwhite cabbagederivedfromthesamesourcein10differentlocationsinNorthernPolandfromMaytolateSeptember.Thecultivationsitesvariedasregardsecologicalsettings,environmentalcondi-tions,soilquality,insolationandpestabundance. Afterharvest,theplantmaterialwasusedtopreparelyophilisatesandjuices.Inthesesamples,thefollowingchemicalparametersweredetermined:heavymetalcontentinvegetables,GLSinextractsfromlyophilisatesandindoleconcentrationinjuices.Theanti-oxidativeactivitywasmeasuredbyDPPH,ABTSandFCRtestfortheextractsandjuices.Thejuiceswerealsousedtodeterminetheabilitytoinhibitthecellulargrowthandinduc-tionofIIphaseenzymes(DT-diaphorase)inhumancoloncancerHT29cells.Theanalyzedsamples turned out to display surprisingly high variability in the case of all parametersdetermined.This suggests that the cultivation conditionsmay have crucial influence onchemopreventivevalueofcabbage.
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P10
ANTIPROLIFERATIVE EFFECTS OF JUNIPERUS COMMUNIS L. VAR. COMMUNIS BERRY EXTRACT IN THE HUMAN HEPATOCELLULAR
CARCINOMA CELL LINE
Maimone,P.1,Taviano,M.F.1,Melchini,A.1,2,Güvenç,A.3,DePasquale,R.1,Trovato,A.1,andMiceli.N.1
1Pharmaco-Biological Department, School of Pharmacy, University of Messina, Italy 2Foundation “Prof. Antonio Imbesi”, University of Messina, Italy
3 Department of Pharmaceutical Botany, Ankara University, Turkey
Plantsusedinfolkmedicinecontinuetobeanimportantsourceofdiscoveryanddevelopmentofnoveltherapeuticagents.JuniperuscommunisL.var.communis(Jcc)isanevergreenshrubortreediffusedinEurope,AsiaandNorthAmerica,whoseberriesareknownfortheirbiologicalproperties.LiteraturedatasuggestthatJuniperusL.specieshavesomeprotectiveeffectsagainstcancer.InvitroandinvivoassayssuggestthatJ.chinensisL.possessantitumoractivitieswithamarkedantiproliferativeactivityintwomammaliancancer cell lines (HeLa,HGC-27).Recent studies show the cytotoxic effects both of J. communis and J. taxifolia in liver and colon carcinoma and in human promyelocytic leukemia HL-60 cells, respectively. The aim of this study was to evaluate in vitro the cytotoxic effects of berriesmethanol extract of Jcc on human hepatocellular carcinoma(HepG2) cells. The cytotoxic activity of the extract was determined on HepG2 cells after24hexposure.Resultsobtainedfromtheproliferationassayshowedadose-dependent decrease in HepG2 cell viability after treatment with Jcc extract.At the concentrationof 40 µg/mL Jcc extract markedly inhibited the growth of HepG2 cells with a reduc-tionofcellviabilitymorethan80%comparedtothecontrolcells.Thecytotoxiceffects observedinhumanhepatocellularcarcinomacellsafterexposuretotheextractsuggestthat JuniperuscommunisL.var.communiscouldrepresentagoodsourceofnaturalcompounds potentiallyimportantforcancerpreventionandtreatment.
P11
INCREASED HISTONE ACETYLATION BY KALE SPROUT INTERVENTION IN A HHUMAN PROSTATE CANCER XENOGRAFT MODEL
Kim,J.H.1,Bähr,M.1,Wagner,J.2,deNicola,G3,Rüfer,C.4,Jung,M.2,Iori,R.3,andGerhäuser,C.1
1German Cancer Research Center Heidelberg, Germany2Universtiy of Freiburg, Germany
3Research Center for Industrial Crops, Agriculture Research Council, Bologna, Italy4Max Rubner-Institute, Karlsruhe, Germany
Introduction:Prostatecanceristhemostcommonlydiagnosedmalignancyinmeninindustrializedcountries.Internationalepidemiologicalstudieshaveindicatedaninversecorrelation between prostate cancer and consumption of cruciferous vegetables such asbroccoliandkale.Cruciferousvegetablesarerichinglucosinolates,whichareconvertedtoisothiocyanatesandindole-basedcompoundsbytheenzymaticactivityofmyrosinase.Bothgroupsofcompoundshavecancerpreventivepotentialthroughvariousmechanismsincluding anti-proliferative activities. Isothiocyanate metabolites have been recently identifiedasinhibitorsofhistonedeacetylates(HDAC)whichhavethepotentialtoturnontheepigeneticallysilencedgenes,leadingtocellcyclearrestandapoptosis.Purpose: In this studywe investigated theeffectofkale sprouts commerciallyavailableas a food supplement (BS1000) to inhibit the growth of humanLNCaPprostate cancercells in a xenograftmodel.Wewere interestedwhether kale sprout interventionwould affecthistoneacetylationandinstudyingunderliningmechanisms,e.g.HDACsandhistone acetyltransferases(HAT). Treatmentandmethods:2x106LNCaPcellswereinjectedto therightandleftflankof6-7oldBalb/cmalenudemice.Tenmicepergroupwerefedwitheitherregularrodentchoworchowsupplementedwith20%kalesprouts(containingabout60µmolglu-cosinolatesper5gchowconsumeddaily,togetherwithactivemyrosinase)fromaweekbeforecellinjectionuntilsacrifice.7.5weeksafterinjection,tumortissuewascollected.HDACexpressionandhistoneH3andH4acetylationweredetectedbyWesternblottingusingantibodiesagainstHDACs1-5andacetylatedhistonesH3andH4.HDACactivityinnuclearextractsoftumortissuewasdeterminedbyusingZMALsubstrate(Fenicetal.,J.Androl.2008).mRNAexpressionofselectedHATs(HAT,p300,CBP,p300-CBPassoci-atedfactor(PCAF))wasquantifiedintumortissuebyqRT-PCR. Results: Kale sprout intervention did not significantly inhibit tumor growth inthexenograftmodel.However,globalacetylationlevelsofhistoneH4wereincreasedinthekalesprout-treatedgroup,whereasacetylatedhistoneH3levelswerenotsignificantlychanged.TheincreaseofH4lysine5,8,12and16acetylationwasindividuallyconfirmed.
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Amongthem,acetylatedH4K12wasmostabundant.Unexpectedly,HDACactivitywasnotsignificantlyinhibitedintumortissuebykalesproutintervention.Also,theexpression of HDAC family members HDAC1-5 was not changed overall, although HDAC 5 expression was reduced while HDAC2 expression was increased. Instead, kale sprout treatment significantly increased mRNA levels of PCAF and CBP by 36% and 23%, respectively, in tumor tissue (p< 0.001), which may contribute to increased H4 lysine acetylation. Conclusion:Takentogether,ourresultsindicatethatdietaryconsumptionofkalesprouts increased global acetylation in histoneH4 in LNCaP prostate cancer xenograftmodel,possiblybyinductionofHATexpressionratherthaninhibitionofHDACactivity.
P12
REGULATION OF HYPOXIC PATHWAY BY NATURAL ISOTHIOCYANATE SULFORAPHANE IN DRUG-RESISTANT OVARIAN CARCINOMA
CELL LINES
Pastorek,M.1,Hunakova,L.1,Barathova,M.2,andSedlak,J1.
1Tumor Immunology Laboratory, Cancer Research Institute,Vlarska 7, Bratislava 833 91, Slovak Republic
2Institute of Virology, Dubravska cesta 9, Bratislava 842 45, Slovak Republic
Isothiocyanate sulforaphane is well known because of its ability to induce apoptosis,detoxificationenzymes,cellcyclearrest,andforitschemopreventiveproperty. Development of chemoresistance and hypoxia are often serious problems in modern therapy, thereforewedecided todetermine the invitroeffectofsulforaphane inovarian carcinoma cell line A2780 and its two chemoresistant sublines during hypoxic and normoxicconditions.Wehaveconfirmedtheresponseofstudiedcelllinesonhypoxiaandrevealedsulforaphane-induceddecreaseofpromoteractivityofCAIXinaconcentration-dependent manner. Therefore we have focused on main transcription factor regulatinghypoxia and found out that sulforaphane affects hypoxia induced pathways primary bydecreasing stability ofHIF-1α, although othermechanisms of its regulation can not beexcluded.
This project was supported by the Slovak Research and Development Agency under the contract No.VVCE-0001-07 “CeXignal”.
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LIST OF PARTICIPANTS
1 Ashurst,Kean CaudillSeedInc,Kentucky(Corporate),USA
2 Bao,Yongping SchoolofMedicine,HealthPolicyandPractice, UniversityofEastAnglia,UK [email protected]
3 Benes,Petr DepartmentofExperimentalBiology,Facultyof Science,MasarykUniversity,Brno,CzechRepublic [email protected]
4 Busiková,Zuzana CancerResearchInstitute,SlovakAcademy ofSciences,SlovakRepublic
5 Collins,Andrew UniversityofOslo,DepartmentofNutrition,Oslo, Norway [email protected]
6 Čellárová,Eva P.J.ŠafárikUniversityinKošice,FacultyofScience, InstituteofBiologyandEcology/Genetics [email protected]
7 Duraj,Jozef CancerResearchInstitute,SlovakAcademy ofSciences,SlovakRepublic [email protected]
8 Fedoročko,Peter P.J.ŠafárikUniversityinKošice/InstituteofBiology andEcology,SlovakRepublic [email protected]
9 Ferenc,Peter InstituteofBiologyandEcology,FacultyofScience, P.J.ŠafárikUniversity,Košice [email protected]
10 Frecer,Vladimír CancerResearchInstitute,SlovakAcademyof Sciences,SK-83391Bratislava,SlovakRepublic [email protected]
11 Frecerová,Jana AcademiaTours/Organizer,SlovakRepublic
12 Gerhaeuser,Clarissa GermanCancerResearchCenterHeidelberg, Germany [email protected]
13 Gronesová,Paulína CancerResearchInstitute,SlovakAcademy ofSciences,SlovakRepublic [email protected]
14 Halkier,BarbaraAnn DepartmentofPlantBiologyandBiotechnology, FacultyofLifeSciences,UniversityofCopenhagen [email protected]
15 Hašplová,Katarína ComeniusUniversity,FacultyofNaturalSciences, DepartmentofGenetics,SlovakRepublic
16 Horváthová,Eva CancerResearchInstitute,SlovakAcademyof Sciences,LaboratoryofMutagenesisand Carcinogenesis,Vlárska7,83391Bratislava, SlovakRepublic [email protected]
17 Hudecová,Alexandra DepartmentofGenetics,FacultyofNatural Sciences,ComeniusUniversity,MlynskádolinaB1, 84215Bratislava,SlovakRepublic [email protected]
18 Hunáková,Ľuba CancerResearchInstitute,SlovakAcademy ofSciences,Slovakia,TumorImmunology Laboratory,CancerResearchInstitute,Vlarska7, Bratislava,83391,SlovakRepublic [email protected]
19 Cholujová,Dana CancerResearchInstitute,SlovakAcademy ofSciences,LaboratoryofTumorImmunology, SlovakRepublic [email protected]
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20 Imrich,Ján P.J.ŠafárikUniversity,FacultyofScience, InstituteofChemistry,Moyzesova11,04167Košice, SlovakRepublic
21 Jakubiková,Jana DanaFarberCancerInstitute,DepartmentofMedical Oncology,BostonMA02115,USA [email protected]
22 Kassayová,Monika DepartmentofAnimalPhysiology,Instituteof BiologyandEcology,FacultyofScience, P.J.ŠafárikUniversity,Moyzesova11,04154Košice, SlovakRepublic [email protected]
23 Kello,Martin InstituteofBiologyandEcology,FacultyofScience, PJ.ŠafárikUniversityinKošice,Moyzesova11, 04001Košice,SlovakRepublic [email protected]
24 Kim,JungHyun GermanCancerResearchCenterHeidelberg, Germany [email protected]
25 Kutschy,Peter P.J.ŠafárikUniversity,FacultyofScience, InstituteofChemicalSciences,Departmentof OrganicChemistry,Moyzesova11,04001Košice, SlovakRepublic [email protected]
26 Lanina,Natalia INFREDpharm(Corporate),SlovakRepublic
27 Lewandowski,GrzegoryAccompanyingperson Poland
28 Lenzi,Monia DepartmentofPharmacology,AlmaMaterStudiorum UniversityofBologna [email protected]
29 Lubelska,Katarzyna NationalMedicinesInstitute,Chełmska30/34, 00-725Warsaw,Poland [email protected]
30 Maimone,Patrizia Pharmaco-BiologicalDepartment,Schoolof Pharmacy,UniversityofMessina,Italy [email protected]
31 Melchini,Antonietta Foundation“Prof.AntonioImbesi”,Universityof Messina,Italy [email protected]
32 Mikeš,Jaromír LaboratoryofCellBiology,P.J.ŠafarikUniversity, Košice,SlovakRepublic [email protected]
33 Mikkelsen,MichaelD. DepartmentofPlantBiologyandBiotechnology, FacultyofLifeSciences,UniversityofCopenhagen
34 Milczarek,Małgorzata NationalMedicinesInstitute,Chełmska30/34 00-725Warsaw,Poland [email protected]
35 Mithen,Richard InstituteofFoodResearch,ColneyLane, NorwichUK [email protected]
36 Moy,Kristin TheMasonicCancerCenterandDivisionof EpidemiologyandCommunityHealth,Schoolof PublicHealth,UniversityofMinnesota,Minneapolis [email protected]
37 Pastorek,Michal TumorImmunologyLaboratory,CancerResearch Institute,Vlarska7,Bratislava,83391, SlovakRepublic [email protected]
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38 Sačková,Veronika InstituteofBiologyandEcology,Department ofCellularBiology,FacultyofScience,PavolJozef ŠafárikUniversity,Moyzesova11,04001Kosice, SlovakRepublic [email protected]
39 Sedlák,Ján CancerResearchInstituteSAS,CentreofExcellence forSignalosomeResearch,Bratislava,SlovakRepublic [email protected]
40 Skupińska,Katarzyna NationalMedicinesInstitute,Chełmska30/34, 00-725Warsaw,Poland [email protected]
41 Smiechowska,Anna ChemicalFaculty,GdańskUniversityofTechnology [email protected]
42 Solar,Peter LaboratoryofCellBiology,InstituteofBiologyand Ecology,FacultyofSciences,P.J.ŠafárikUniversity, Košice,SlovakRepublic [email protected]
43 Štetina,Rudolf FacultyofMilitaryHealthSciences,HradecKrálové [email protected];[email protected]
44 Šulíková,Margita CancerResearchInstitute,SlovakAcademyof Sciences,SlovakRepublic
45 Teng,Jin-Hong Accompanyingperson,USA
46 Tomasik,Emil INFREDpharm(Corporate),SlovakRepublic
47 Tomasin,Rebeka LaboratoryofNutritionandCancer,Departmentof PhysiologyandBiophysics,InstituteofBiology, StateUniversityofCampinas,UNICAMP,Campinas, Brazil,[email protected]
48 Volkovová,Katarína SlovakMedicalUniversityinBratislava [email protected]
49 Wang,Wei Accompanyingperson,UK
50 Yuan,Jian-Min MasonicCancerCenterandDivisionof EpidemiologyandCommunityHealth,Schoolof PublicHealth,UniversityofMinnesota [email protected]
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KRD is a biotechnology distribution company founded in 1998 with idea to supply the our local research comunities with products for : 1.Genomicresearch2.ClinicalPCR,RTPCRandrealtimeapplications3.Stemcellresearch4.Proteinengineering5.Tissuebanking6.Educationincellandmolecularbiology
Companyhastwooffices(Prague-Czechrepublic,Bratislava-Slovakia).KRDltd. KRDltd.Pekařská12 Saratovská26CZ-15500Prague5 SK-84102BratislavaCzechrepublic SlovakiaPhone:00420-2-57013400 Phone:00421-2-64534041Fax:00420-2-57013405 Fax:00421-2-64534043E-mail:[email protected] E-mail:[email protected] www.krd.sk
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PUBLISHED BY: CANCER RESEARCH INSTITUTE, SLOVAK ACADEMY OF SCIENCES AND SLOVAK CANCER RESEARCH FOUNDATION
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