Antiproliferative effects of isoflavonoids on prostate cancer cells and antitumorigenic effects in xenograft nude mice

181 Pages·2001·6.3 MB·English

by  Samedi, Von G.

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