膀胱癌细胞中舞茸D-fraction与干扰素的协同增效作用

1/1膀胱癌细胞中舞茸D-fraction与干扰素的协同增效作用声明：

本文摘自BJUInternational（2010年4月，第105卷第7期），即BritishJournalofUrologyInternational《英国国际泌尿学杂志》。

是国际泌尿学领域的主要杂志，发表关于成人及小儿泌尿学的原创论文、综述、评论等，为泌尿科、肾病科、肿瘤科、放射科、妇科、男科、外科及儿科的临床医生提供最新的技术、治疗方法等信息。

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本文摘自舞茸D-fraction官方网站，转载请注明SynergisticpotentiationofinterferonactivitywithmaitakemushroomD-fractiononbladdercancercells膀胱癌细胞中舞茸D-fraction与干扰素的协同增效作用目的：

检验在膀胱癌T24细胞的体外试验中，一种具有生物活性的菇类提取物舞茸D-fraction(PDF)与IFN-合用时，是否能提高IFN-的的抗癌活性。

材料与方法：

评估72小时后不同浓度IFN-2b（0-50000IU/ml），PDF(0-700g/ml)，以及两者的组合对T24细胞的作用。

进行细胞周期分析以及双链DNA依赖蛋白激酶试验来发现这些药物可能的抗增殖机制。

结果：

浓度为20000IU/ml的IFN-2b能显著降低细胞的生长（50%），当IFN-2b达到50000IU/m时，生长下降达到66%。

PDF浓度小于200g/ml时，对癌细胞生长没有影响，但当其浓度为400和700g/ml时，其生长减少率分别达到20%和53%。

当不同浓度的IFN-2b和PDF组合时，10000IU/ml的IFN-2b和200g/ml的PDF联合使用时，细胞生长减少率约为75%。

细胞周期分析发现，与此同时出现了G1细胞周期停滞现象。

同时，经IFN-2b和PDF联合处理后的细胞内的DNA依赖蛋白激酶活性比对照组几乎高3倍。

结论：

T24细胞中10000IU/ml的IFN-2b和200g/ml的PDF联合使用能导致约75%的细胞生长减少。

这表明这两者具有协同作用，并能激活DNA依赖蛋白激酶活性并诱导G1细胞周期停滞。

所以，IFN-2b和PDF联合使用能触发DNA依赖蛋白激酶活性，它可能作用于细胞周期使癌细胞停止生长。

关键词：

干扰素，D-fraction，联合疗法，协同作用，膀胱癌BJUInternational,Volume105,Issue7,April2010,Pages1011-1015SynergisticpotentiationofinterferonactivitywithmaitakemushroomD-fractiononbladdercancercellsBrandonLouie,SrinivasRajamahanty,JohnWon,MuhammadChoudhuryandSensukeKonnoDepartmentofUrology,NewYorkMedicalCollege,Valhalla,NY,USAAcceptedforpublication9June2009OBJECTIVEToexaminewhetherthecombinationofinterferon(IFN)-andmaitakemushroomD-fraction(PDF),abioactivemushroomextract,mightpotentiatetheanticanceractivityofIFN-inbladdercancerT24cellsinvitro.MATERIALSANDMETHODSEffectsofrecombinantIFN-2b(050000IU/mL),PDF(0700g/mL),ortheircombinationswereassessedonT24cellgrowthat72h.Cellcycleanalysisandassaysfordouble-strandedDNA-dependentproteinkinase(DNA-PK)wereperformedtoexplorepossibleantiproliferativemechanismoftheseagents.RESULTSIFN-2bwasabletoinduceasignificant(50%)growthreductionat20000IU/mL,whichfurtherdeclinedto66%at50000IU/mL.PDFhadnoeffectsupto200g/mL,buttherewasan20%and53%growthreductionat400and700g/mL,respectively.WhenthevaryingconcentrationsofIFN-2bandPDFwerecombined,10000IU/mLofIFN-2bcombinedwith200g/mLofPDFresultedinan75%growthreduction.ThiswasaccompaniedbyaG1cellcyclearrest,shownbycellcycleanalysis.Concurrently,DNA-PKactivityinIFN-2b/PDF-treatedcellswasalmostthree-foldhigherthancontrols.CONCLUSIONSThecombinationofIFN-2b(10000IU/mL)andPDF(200g/mL)reducedgrowthby75%inT24cells.Thisappearstobeduetoasynergisticpotentiationofthesetwoagents,inducingaG1arrestwithDNA-PKactivation.Therefore,theIFN-2b/PDFcombinationcouldtriggerDNA-PKactivationthatmayactonthecellcycletoceasecancercellgrowth.KEYWORDSinterferon,D-fraction,combinedtherapy,synergism,bladdercancer.INTRODUCTIONThebladderisthemostcommonsiteofcancerintheurinarysystemand90%ofbladdercancersareTCCs.OftheseTCCs,80%arediagnosedassuperficialbladdertumours[1].Transurethralresectionistheprimarymethodforremovalofthosesuperficialbladdertumours;however,nearly65%ofpatientswillhavetumourrecurrencein5yearswhile1020%willhaveprogressiontomuscleinvasion[2,3].Itisthusconceivablethattheprimarytherapeuticaimistopreventmultiplerecurrencesandprogressiontoamoreadvanced,invasivedisease.Severalcytotoxicandimmunemodifyingagentshavebeenusedintravesicallyfortherapeuticpurpose.Amongthem,intravesicaladministrationofBCGishighlyeffectiveinreducingtherecurrencerateandalteringtheprogressionrateofthediseasewithanincreasedsurvivalrate[4].Infact,adjuvantintravesicalBCGtherapyaftersurgicalresectionhasbecomeestablishedtherapyforsuperficialbladdercancers,resultingin40%reductionincancerrecurrence[4,5].However,itsbenefitsaresometimesoutweighedbyitssevereside-effects:cystitisoccursin90%ofpatientsandotherpotentialadverseeffects(fever,allergicreactions,sepsis,etc.)cannotbeexcluded[6,7].Thesedrawbacksthuslimititsuseinclinicalpracticeandrequestasaferandeffectivetreatmentmethodwithfewside-effects,promotingtheuseofunconventionaltherapieswithothervariousimmunomodulators.Interferons(IFNs)havebeenwidelyusedasimmunotherapyforvarioushumanmalignanciesincludingprostate,bladder,andRCCs[810].Particularly,IFN-hasbeenusedasanintravesicalagentfortreatingsuperficialbladdercancer,resultinginan40%responserateinpatients[11].AlthoughthisresponserateislowerthanthatofBCGtherapy,IFN-causesonlyminimallocalandsystemictoxicity(comparedwithBCG)[11,12].ToimprovetheefficacyofIFN-therapy,combinedtherapy(e.g.IFN-/BCG)hasbeenproposedandisbeingassessedinpilotclinicaltrialsandanimalstudies,whichareshowingbetterandencouragingoutcomes[13,14].Thissuggeststhatfurtherexplorationofeffectivetreatmentmethodssuchasanalternativeand/orcombinedtherapyiswarranted.D-fraction(PDF)isabioactiveproteoglucanextractedfrommaitakemushrooms(Grifolafrondosa)[15].ThestandardizedPDFhasbeencommerciallyavailableformedicalandscientificresearch.SeveralpublishedandunpublishedstudieshavetodatesuggestedtheimmunomodulatoryandantitumouractivitiesofPDF[16,17].ItwasshowninananimalmodelthatPDFwascapableofactivatingimmune-competentcellssuchasnaturalkillercellsandcytotoxicT-cellswithaconcomitantincreaseininterleukin-1production[16,17],indicatingstimulationofimmuneresponses.Meanwhile,thesafetyofPDFissupportedbythefactthattheUSAFoodandDrugAdministration(FDA)hasexemptedaphaseIstudyoftoxicologytests.Additionally,theFDAhasapprovedPDFfortheInvestigationalNewDrugapplicationforaphaseIIpilotstudyonpatientswithadvancedbreastandprostatecancer[18].Accordingly,weinvestigatedwhetherIFN-,PDFortheircombinationmighthaveagrowthinhibitoryeffectonbladdercancerT24cellsinvitro,andtheunderlyingmechanismofsuchactivitywasalsoexplored.MATERIALSANDMETHODSThehumanbladdercancerT24cells,derivedfromapatientwithTCC,wereobtainedfromtheAmericanTypeCultureCollection(Rockville,MD,USA).CellsweremaintainedinMcCoys5amediumcontaining10%fetalbovineserum,penicillin(100U/mL),andstreptomycin(100g/mL).Routinely,culturemediumwaschangedevery34daysandthepassageofcellswasperformedweekly.Forexperiments,cellswereseededinT-75flasksorsix-wellcultureplatesattheinitialcelldensityof2105cells/mLandwereculturedwithrecombinantIFN-2b(ScheringCorp.,Kenilworth,NJ,USA),PDF(MaitakeProducts,Inc.,Paramus,NJ,USA)ortheircombinations.CellnumberswerethenassessedatspecifiedtimesusingtheTrypanblueexclusionmethod.CellcycleanalysiswasperformedusingaFACScanflowcytometer(Becton-Dickinson),equippedwithadoublediscriminationmodule.About1106cellswereresuspendedin500Lofpropidiumiodidesolution(20g/mLpropidiumiodide,0.2mg/mLRNase,0.2mg/mLEDTA,0.5%NP-40)andincubatedatroomtemperaturefor1h.Inall,10000nucleiwereanalysedforeachsample,andCellFitsoftwarewasusedtoquantifycellcyclecompartmentsandestimatecellcyclephasefractions.Invitrophosphorylationassaywasperformedaspreviouslydescribed[19].CelllysateswerefirstpreparedfromcontrolandIFN-2b/PDF-treatedcellsbythreecyclesoffreeze-thawinliquidnitrogen.A5galiquotofcelllysatepreparationwasaddedtothephosphorylationcocktailcontaining55.5kBqof[-32P]-ATP(specificactivity:166.5TBq/mmol)andincubatedat37Cfor15min.Toactivateendogenousdouble-strandedDNA-dependentproteinkinase(DNA-PK),2g/mLoffragmentedcalfthymusDNAwasalsoincludedinthereactionmixture.Phosphoproteinswerethenseparatedby10%SDSandanalysedbyautoradiography.IntensitiesofspecificDNA-PKbandswerethenquantifiedusingascandensitometer(SilkScientific,Oregon,UT,USA).Forstatisticalanalysis,alldatawerepresentedasthemean(SD),andstatisticaldifferencesbetweengroupswereassessedwiththeunpairedStudentst-test;P0.05wasconsideredtoindicatestatisticalsignificance.RESULTSEFFECTSOFIFN-2bANDPDFONT24CELLGROWTHToexaminepossibleeffectsofIFN-2bandPDFonT24cellproliferation,cellswereculturedwiththevaryingconcentrationsofIFN-2b(050000IU/mL)orPDF(0700g/mL)for72h.IFN-2bcausedan50%reductionincellnumberat20000IU/mL,whichfurtherdeclinedto66%at50000IU/mL(Fig.1A).PDFhadnoeffectsupto200g/mL,buttherewasan20%and53%growthreductionat400and700g/mL,respectively(Fig.1B).Thus,theseresultsshowthat20000IU/mLofIFN-2bor700g/mLofPDFisrequiredtosignificantlyinhibitthegrowthofT24cells.SYNERGISTICGROWTHINHIBITORYEFFECTSOFIFN-2bANDPDFWenextexaminedwhetherthecombinationofIFN-2bandPDFmighthaveabettergrowthinhibitoryeffect.CellswereculturedwithcombinationsofIFN-2bandPDFatthevaryingconcentrationsandcellgrowthwasassessedat72h.Thecombinationof10000IU/mLIFN-2band200g/mLPDFresultedinan75%reductionincellgrowth(Fig.2).ThissuggeststhattheIFN-2b/PDF-inducedgrowthreductionisduetoasynergisticeffect,becausetheeffectofthetwoagentswasgreaterthantheeffectofeachagentindividually(Fig.1A,B).FIG.1.EffectsofIFN-2borPDFonT24cellgrowth.T24cellswereculturedwithvaryingconcentrationsofeitherIFN-2b(050000IU/mL)orPDF(0700g/mL),andcellnumbersinIFN-2b-treated(A)orPDF-treated(B)weredeterminedat72h.AlldataarethemeanSDfromthreeseparateexperiments;*P0.03;**P0.05.FIG.2.EffectsofcombinationsofIFN-2bandPDFoncellgrowth.CellswereculturedwithvaryingconcentrationsofIFN-2b/PDFcombinationfor72h,andcellgrowthwasassessedbythepercentagerelativetothecellnumberincontrol(100%).Cellgrowthincontrol,IFN-2b(10000IU/mL)-treated,PDF(200g/mL)-treated,orIFN-2b(10000IU/mL)/PDF(200g/mL)-treatedcellsisshown.ThedataaremeanSDfromthreeindependentexperiments;*P0.02.EFFECTSOFCOMBINEDIFN-2bANDPDFONTHECELLCYCLEToexploretheunderlyingmechanismofsuchasynergisticgrowthinhibitioninducedbytheIFN-2b/PDFcombination,cellsweretreatedwithIFN-2b(10000IU/mL),PDF(200g/mL),ortheircombinationfor72handsubjectedtocellcycleanalysis.AsshowninTable1,IFN-2borPDFalonehadlittleeffectssimilartocellcyclephasedistributionincontrolcells;incontrast,theIFN-2b/PDFcombinationcausedan63%decreaseincellnumberintheSphasewithaconcomitant55%increaseintheG1-phasecellpopulation,comparedwiththoseincontrols.TheseresultsthusindicatethattheIFN-2b/PDFcombinationmaycauseablockageofcellsenteringfromtheG1totheSphase,knownasaG1cellcyclearrest.INVOLVEMENTOFDNA-DEPENDENTPROTEINKINASE(DNA-PK)INGROWTHINHIBITIONAsmanyproteinsandenzymesareknowntobemodulatedbyIFNs[20,21],itisfeasiblethatthegrowth-inhibitoryactivityofIFN-2bwithPDFmayinvolvespecificPK(s)actingonthesignaltransductionpathwayforcellproliferation.Particularly,weareinterestedinaPK,namelydouble-strandedDNA-PK,whoseactivityreliesessentiallyonsmalldouble-strandedDNA[22].Accordingly,cellextractsobtainedfromcontrolandIFN-2b(10000IU/mL)/PDF(200g/mL)-treatedcellsat72hweresubjectedtoinvitrophosphorylationassayinthepresenceorabsenceofexogenousDNA(servedasaDNA-PKactivator).ThebasalphosphorylationstateofDNA-PKinIFN-2b/PDF-treatedcellswasthree-foldhigherthancontrols(comparelanes1with3inFig.3),andsuchphosphorylationwasincreased(autophosphorylated)byadditionalthree-foldwithDNA(comparelanes3with4inFig.3).Therefore,thesestudiesshowthattheIFN-2b/PDF-inducedgrowthinhibitionisaccompaniedbyactivationofDNA-PK.FIG.3.Invitrophosphorylationassays.Celllysatesfromcontrol(lanes1and2)andIFN-2b/PDF-treatedcells(lanes3and4)for72hweresubjectedtoinvitrophosphorylationassayswith(lanes2and4)orwithout(lanes1and3)DNA,asdescribedintheMethods.AutophosphorylatedDNA-PKdetectedonautoradiogramisshown,andtherelativeintensitiesofDNA-PKbandswerequantified(byascandensitometer)andexpressedbyarbitraryvalues.DISCUSSIONInanattempttoestablishanimprovedmethodforbladdercancertherapy,pilotclinicaltrialsusingcombinationsofIFN-andBCGhasbeenconducted.ThesetrialsshowedthatsuchcombinedtherapycouldlowerBCGtoxicityagainsttumours[13].However,theexactmechanismbywhichIFN-potentiatesBCG-mediatedanti-bladdercancerimmunityhasnotbeenfullyunderstood.Inaddition,IFN-therapyhasseveraldrawbacks,suchashighcostandrepeatedadministration.AstandardintravesicalIFN-instillation(combinedwithBCG)iscarriedoutwith50100106IUofIFN-[23],althoughsuchahighdosageappearstobeinexcessoftheactualamountneededforaneffectiveantitumourimmunity.Moreover,evenahighdoseofIFN-maynotbesufficienttoinduceoptimalimmunitybecauseofitsshortretentiontimeinsidethebladder[23].Consequently,weexploredanalternativeapproachforbladdercancerimmunotherapybycombiningIFN-2bandPDF,abioactiveproteoglucanofmaitakemushroom.WefoundthatIFN-2batconcentrationsof20000IU/mLwereabletoinduceasignificant(50%)growthreductioninT24cells.PDFcouldbealsoeffectiveatarelativelyhighconcentrationof700g/mL,leadingtoan53%growthinhibition.ThisPDFconcentrationseemstobeyethigherthanaphysiologicallyachievableconcentration,althoughsuchaconcentrationhasnotbeenestablishedatthistime.Nevertheless,thepossibilitythatPDFmightbeabletoenhanceorpotentiateantiproliferativeactivityofIFN-2bwasthentested.Thecombinationof10000IU/mLIFN-2band200g/mLPDFwascapableofinducingan75%growthreduction(Fig.2).Thisenhancedgrowthinhibitionprobablyresultedfromasynergisticpotentiationofthetwoagents,becauseneitherIFN-2b(10000IU/mL)norPDF(200g/mL)alonehadsuchgrowth-inhibitoryactivity(Fig.1A,B).Italsoshowsthatarelativelylowconcentration(10000IU/mL)ofIFN-2bwasrequiredtoattainabettergrowthinhibitoryeffect(75%)whencombinedwithPDF(Fig.2),comparedwiththeinhibitoryeffect(66%)attainedby50000IU/mLIFN-2balone(Fig.1A).Inotherwords,comparedwiththeamountofIFN-2bneededtobeeffectiveasasoleagent,merelyafifthofthatIFN-2bdosewouldbeneededtoexhibitthebetter,enhancedgrowth-inhibitoryactivitywhencombinedwithPDF.Thus,itisplausiblethatPDFmaynotonlyhelppotentiateIFN-2bactivitybutmayalsohelptoreducethecostoftreatment.WenextexaminedtheeffectsofIFN-2b/PDFcombinationoncellcycleregulationtoexplorethegrowthinhibitorymechanism.Cellcycleanalysisshowedan63%decreaseintheS-phasecellnumberwithaconcomitant55%increaseintheG1-phasecellnumberaftertreatmentwiththeIFN-2b/PDFcombination(Table1).Thus,theseresultsconfirmthattheIFN-2b/PDF-inducedgrowthinhibitionwasmediatedthroughablockageoftheG1Sphasetransition(i.e.aG1arrest).ThisfurthersuggeststhatthegrowthinhibitoryactionoftheIFN-2b/PDFcombinationmaytargetprimarilytheG1Sphaseprogressioninthecellcycle.TodatethereisverylittleinformationrelevanttosuchIFN-combinedtherapy,butIFN-hasbeenshowntoblocktheSG2phasetransitioninsomecancersincludingbladdercancercells[24].Yet,thisdifferenceincellcyclearrest(fromthepresentstudy)ismostprobablyduetothedifferentbladdercancercellsthatwerestudied.Asmentionedearlier,IFNscanregulatecellproliferationviasignaltransductionpathwaysinvolvingspecificPKs[20,21].Also,ithasbeenreportedthatIFNscaninduceDNAfragmentation,leadingtoanaccumulationofsmallorlow-molecularweightDNA[25].Accordingly,wefocusedononeofsuchPKs,DNA-PK,whichrequiressmalldouble-strandedDNAforitsactivation[22]andplaysanimportantroleincellcycleregulation[26].ItshouldbementionedthatourpilotDNAanalysisverifiedDNAfragmentationdetectedinIFN-2b/PDF-treatedcells(datanotshown).ThepossibleinvolvementofDNA-PKinsuchanIFN-2b/PDF-inducedgrowthinhibitionwasthenexaminedusinginvitrophosphorylationassays.ThebasalphosphorylationstateofDNA-PKwassignificantly(aboutthree-fold)higherinIFN-2b/PDF-treatedcellsandwasfurtherincreasedinresponsetoexogenouslyaddedDNA.ThisDNA-PKautophosphorylation(self-phosphorylation)withDNAisindicativeofitsenzymaticactivation[26].Thus,theseresultsconfirmthatendogenousDNA-PKisindeedactivatedbytheIFN-2b/PDFcombinationinT24cells,althoughtheexactroleofDNA-PKinthisIFN-2b/PDF-inducedgrowthinhibitionyetremainstobefullydefined.Inconclusion,thepresentstudyshowsthatPDFsynergisticallypotentiatestheanticancer/antiproliferativeactivityofIFN-2bonbladdercancerT24cells.ThisenhancedgrowthinhibitionresultsfromaG1cellcyclearresttogetherwithactivationofDNA-PK.Itisconceivablethatsuchanantiproliferativemechanismisassociatedwithanaccumulationoflow-molecularweightDNA,triggeringDNA-PKactivationthatmightactprimarilyonthecellcycletoceasecancercellgrowth.Therefore,thelow-doseIFN-2b/PDFcombinationmayprovideanalternative,improvedimmunotherapyforsuperficialbladdercancerandthusclinicalstudies/trialsarewarranted.CONFLICTOFINTERESTNonedeclared.Sourceoffunding:departmental.REFERENCES1LeeR,DrollerMJ.Thenaturalhistoryofbladdercancer.Implicationsfortherapy.UrolClinNorthAm2000;27:1132WitjesJA,MuldersPF,DebruyneFM.Intravesicaltherapyinsuperficialbladdercancer.Urology1994;43(Suppl.):263LammDL.Long-termresultsofintravesicaltherapyforsuperficialbladdercancer.UrolClinNorthAm1992;19:573804KurthKH,BouffiouxC,SylvesterR,vanderMeijdenAP,OosterlinckW,BrausiM.Treatmentofsuperficialbladdertumors:achievementsandneeds.TheEORTCGenitourinaryGroup.EurUrol20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