Antiproliferative effects of isoflavonoids on prostate cancer cells and antitumorigenic effects in xenograft nude mice PDF
Preview Antiproliferative effects of isoflavonoids on prostate cancer cells and antitumorigenic effects in xenograft nude mice
ANTIPROLIFERATIVEEFFECTSOFISOFLAVONOIDS ONPROSTATECANCERCELLS ANDANTITUMORIGENICEFFECTSINXENOGRAFTNUDEMICE By VONG.SAMEDI ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT OFTHEREQUIREMENTSFORTHEDEGREEOF DOCTOROFPHILOSOPHY UNIVERSITYOFFLORIDA 2001 Thisdissertation isdedicatedto mycaringand supportiveparents,mymotherMarie ThereseSamedi,myfatherLucienJacquesLapierre,mylovinggrandmotherSceurette 'Marraine'Augustin,mysisterChantalandmybrotherBabal. ACKNOWLEDGEMENTS IwouldliketoexpressmysinceregratitudetoDr.KathleenShiverickforher mentorship, support, guidance, friendship and commitment to sharpen my scientific skills. Iwouldalsoliketothankherforgivingmetheopportunitytopursuethisexciting areaofresearch. Ithankthemembersofmycommittee,Drs.SusanPercival,TomRowe, andDietmarSiemann,fortheirencouragementandconstructivecriticism. Iwouldliketo thank,inparticular.Dr.LoriRiceforallherinsightfulsuggestionsandforallowingmeto be involved in collaborative projects between our laboratories. I also thank Carol SweeneyfromDr.Rice'slaboratoryforallherinput. IappreciateallthehelpfromDr. JaimeFurman,whomIthankverymuch. IthankMelissaChenforallherhelpwiththe flowcytometryanalysis. MyworkinDr. Shiverick'slaboratorywouldnothavebeen possiblewithouttheinstrumentalinputofTheresaMedrano,soIdeeplythankher. My experienceasagraduatestudenthascertainlybeenenhancedbythefriendshipofallthe membersofDr. Shiverick'slaboratory. 1alsoacknowledgeBarbara,Dorma,Judy,and Patsyinthepharmacologyofficefortheirsupportthroughoutmygraduatestudies. Iwant tothankLynnRaynorforallthecomputersupportshehasprovidedmeasagraduate assistant. Finally,IthanktheNationalCancerInstitute(NCI)forfinanciallysupporting mytrainingattheUniversityofFlorida. iii TABLEOFCONTENTS page ACKNOWLEDGEMENTS iii ABSTRACT vii ''-[ CHAPTERS \l 1 INTRODUCTION 1 StudyObjectives 1 ProstateCancer 2 IncidenceandMortalityRates 2 RiskFactors 3 MolecularProgression 5 NutritionandChemoprevention 7 Isoflavonoids 8 Source 8 StructureandMetabolism 9 AnticancerProperties 10 ProstateCancerCellLines 15 LNCaPCellLine 15 PC-3CellLine 16 StudyOutline 16 2 MATERIALSANDMETHODS 23 Materials 23 Methods 24 CellCultureandTreatments 24 CellViabilityAssay 25 ApoptosisAssay 25 [^HJThymidineIncorporationAssay 26 AnalysisofCellCyclePhaseDistribution 26 WesternBlotAnalysis 27 IsolationofRNA 28 MicroarrayDNAAnalysis 28 AnimalStudies 30 iv DataAnalysis 31 3 EFFECTSONCELLPROLIFERATION 33 Introduction 33 RegulationoftheCellCycle 34 Apoptosis 36 ACellCyclePhenomenon 36 MechanismofProgrammedCellDeath 38 Results 39 EffectsonCellViability 39 InductionofDNAFragmentation 40 EffectsonDNASynthesis 40 EffectonCellCycleProgression 41 EffectsonCyclins 42 EffectsonCycHn-DependentKinaseInhibitors 43 EffectsofTyrosineKinaseInhibitorTyrphostin25 44 Discussion 45 4 INVIVOEFFECTS 76 Introduction 76 HumanTumorXenograftModel 77 TheHostModel 77 Anchorage-MediatedTumorGrowth 78 GrowthCharacteristics 79 ExperimentalModel 80 ExperimentModelI 80 ExperimentModelII 80 DataAnalysis 81 Results 81 DatafromExperimentalDesignI 81 DatafromExperimentalDesignII 82 Discussion 83 5 EFFECTSONGENEEXPRESSION 97 Introduction 97 PrincipleofMethodsofDNAMicroarrayTechnology 98 HumanProstate-SpecificMicroarrayMembranes 100 MethodsforAnalyzingExpressionData 101 NormalizationandRelativeFold-Changes 101 DataMining 102 ClusterAnalysis 102 V Results 106 EliminatingError 106 IdentificationofGeneswithAlteredExpressionLevel 107 CellularFunctionsTargetedbyGenistein(37^iM)inLNCaP 108 CellularFunctionsTargetedbyBiochanin(37^M)inLNCaP 109 CellularFunctionsTargetedbyGenistein(111|^M)inPC-3 110 CellularFunctionsTargetedbyBiochanin(111[iM)inPC-3 Ill ClusteringofGenesandHybridizations 113 Discussion 113 6 CONCLUSIONSANDFUTUREDIRECTIONS 140 LISTOFREFERENCES 148 BIOGRAPHICALSKETCH 171 vi AbstractofDissertationPresentedtotheGraduateSchool oftheUniversityofFloridainPartialFulfillmentofthe RequirementsfortheDegreeofDoctorofPhilosophy ANTIPROLIFERATIVEEFFECTSOFISOFLAVONOIDS ONPROSTATECANCERCELLS ANDANTITUMORIGENICEFFECTSINXENOGRAFTNUDEMICE By VonG.Samedi August2001 Chair:KathleenT.Shiverick MajorDepartment:PharmacologyandTherapeutics Epidemiologicalstudieshavesuggestedthatthephytochemicalisoflavonoidsmay beinvolved inprotectiveeffects againstprostatecancer. Thisstudyinvestigatedthe anticancerpropertiesoftwobiologicallyactiveisoflavonoids,genisteinanditspreciu^sor biochaninA,intwohumanprostatecancercelllinesLNCaP(androgen-sensitive)and PC-3 (androgen-independent). LNCaPorPC-3cellsculturedinthepresenceofthese isoflavonoids for48hoursexhibitedadose-dependentdecreaseincellviability. The levelofapoptosisincreasedwithincreasingconcentrationoftheisoflavonesinLNCaP cells. In contrast, PC-3 cells were more resistant to induction ofapoptosis. The isoflavonoidsproducedadose-dependentinhibitionofDNAsynthesisinbothLNCaP andPC-3withcompleteinhibitionat37and111\iM,respectively. Theseconcentrations weresubsequentlyusedtostudymechanismsofgrowthinhibition. InLNCaP,genistein accumulatedcellsintheG2/Mphase,whereasbiochaninproducedaGl accumulation. vii InPC-3,bothisoflavonoidsinducedaG2/Marrest. CyclinBproteinlevelwasmarkedly decreasedbybothphytochemicalsinLNCaP,andbybiochanininPC-3. Thecellcycle inhibitoryproteinp21 expressionwasinducedbygenisteininbothcelllines,whereas biochanindecreaseditby40%inLNCaP. Theeffectsoftheseplantchemicalsongene expressionwere investigatedusingcDNAmicroarraytechnology. Inbothcell lines, genisteinandbiochaninalteredexpressionofsharedanduniquegeneswhichareinvolved inmultiplecellularfunctions,includingDNAsynthesis,transcription,translation,protein degradation, signaltransduction,cellproliferation,andcell adhesion. Lastly,athymic micewereimplantedwithLNCaPxenografts, himicetreatedwithbiochaninat400 p.g/dayfor10days,meantumorvolumewassignificantlysmallercomparedtocontrolsat 3and6weeks,andtumorincidencewas54%comparedto89%incontrolat3weeks. In mice with established tumors, genistein and biochanin treatment slowed the rate of growth compared to controls. In one oftwo experiments, biochanin significantly decreasedthemitoticindexinthetumors,whereasinanotheronegenisteinincreasedit. No changes in microvessel densitywere observed with biochanin treatment. These resultsindicatethatgenisteinandbiochaninhaveantiproliferativeeffectsbothinvitro andinvivo,characterizedbysharedanddistinctmechanisms. Vlll CHAPTER 1 INTRODUCTION StudyObjectives Canceroftheprostate(Figure1-1)isthesecondleadingcauseofcancerdeathsin Americanmales. Intheyear2000,anestimated 180,400newcasesand31,900deaths werereportedintheUnitedStates(Woolam,2000). Thehighestratesofprostatecancer areobservedinpopulationswithWesternlifestylesthatincluderelativelyhighfat,meat- based,lowfiberdiets. Incontrast,thelowestratesaretypicallyseeninpopulationswith Eastern lifestyles that include plant-based diets with a high content ofsoyproducts (Adlercreutz, 1990;ParkinandMuir, 1992;Roseetal, 1986). Thereportedincidence variesfrom5to70-foldbetweenpopulations. StudiesonAsianimmigrantsintheUS indicate that this striking geographical difference in incidence rate is evidence that environmentalfactorsaresignificant(Koloneletal,1985;Koloneletal,1988). Asian men who migrate to America tend to adopt the prostate cancer incidence ofthe indigenous population within one or two generations. These epidemiological data supporttheconceptthatenvironmentalfactors,includingdiets,mayinhibitthepromotion andprogressionofprostatecancerinAsianmen. Thusithasbeensuggestedthatadiet richinplantsincludingsoyfoods,whicharewidelyusedinAsiancultures,mayconfer somelevelofprotectionagainstprostatecancer(AdlercreutzandMazur,1997;Barneset al, 1995;Dunn, 1975). Someofthemostbioactivecompoundsfoundinsoyarethe isoflavonoids. 1 2 Thisresearchwasundertakentoinvestigatetheanticancerpropertiesandmodes ofactionofthesephytochemicals,specificallygenisteinandbiochaninA. Asianmen havehighurinaryandplasmalevelsoftheseisoflavonoidscomparedtoAmericanand Western European men with low levels (Adlercreutz etal, 1993). The compounds genisteinandbiochaninAhavebeenreportedtoinhibittheproliferationofmanycancer celllinesthroughmultiplemechanisms(Bergamaschietal,1993;PetersonandBarnes, 1991, 1993; Shaoetal, 1998; Spinozzi etal, 1994; Yanagiharaetal, 1993). The hypothesis ofthis studyisthatthe isoflavones genistein andbiochanin exhibit their antiproliferativeeffectsinprostatecancercellsthroughcellcyclearrestbymodifyingthe expressionofcellcycleregulators. Thefirstobjectivewastodeterminetheeffectsofthe isoflavones on cell proliferation, cell cycle phase distribution and onkeycell cycle regulatory proteins in two established prostate cancer cell lines LNCaP (androgen- responsive)(Horoszewiczetal, 1980)andPC3(androgen-independent)(Kaighnetal, 1979). A second objecfive was to evaluate in vivo the chemoprevenfive and anfiproliferative effectsoftheseplantcompoundsinxenograftnudemice. Finally, a thirdobjecfivewastoobtainaprofileoftheeffectsofgenisteinandbiochaninAongene expressionintheprostatecancercelllines. Suchanapproachprovidesaglobalviewof theneteffectsofthesecompoundsinthecancercells. ProstateCancer IncidenceandMortalityRates Prostatecancerincidencerates varywidelyfi-omonegeographical locationto anotherwidelybyalmost70-foldaroundtheworld(ParkinandMuir,1992). Menliving intheFarEastandontheIndiansubcontinenthavethelowestrecordedrates,whereas
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