膀胱癌细胞中舞茸Dfraction与干扰素的协同增效作用.docx
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膀胱癌细胞中舞茸Dfraction与干扰素的协同增效作用.docx
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膀胱癌细胞中舞茸Dfraction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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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-α合用时,是否能提高IFN-α的的抗癌活性。
材料与方法:
评估72小时后不同浓度IFN-α2b(0-50000IU/ml),PDF(0-700μg/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-α2b能显著降低细胞的生长(≈50%),当IFN-α2b达到50000IU/m时,生长下降达到66%。
PDF浓度小于200μg/ml时,对癌细胞生长没有影响,但当其浓度为400和700μg/ml时,其生长减少率分别达到≈20%和≈53%。
当不同浓度的IFN-α2b和PDF组合时,10000IU/ml的IFN-α2b和200μg/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-α2b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-α2b和200μg/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-α2b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,Pages1011-1015
SynergisticpotentiationofinterferonactivitywithmaitakemushroomD-fractiononbladdercancercells
BrandonLouie,SrinivasRajamahanty,JohnWon,MuhammadChoudhuryandSensukeKonno
DepartmentofUrology,NewYorkMedicalCollege,Valhalla,NY,USA
Acceptedforpublication9June2009
OBJECTIVE
Toexaminewhetherthecombinationofinterferon(IFN)-αandmaitakemushroomD-fraction(PDF),abioactivemushroomextract,mightpotentiatetheanticanceractivityofIFN-αinbladdercancerT24cellsinvitro.
MATERIALSANDMETHODS
EffectsofrecombinantIFN-α2b(0–50000IU/mL),PDF(0–700μg/mL),ortheircombinationswereassessedonT24cellgrowthat72h.Cellcycleanalysisandassaysfordouble-strandedDNA-dependentproteinkinase(DNA-PK)wereperformedtoexplorepossibleantiproliferativemechanismoftheseagents.
RESULTS
IFN-α2bwasabletoinduceasignificant(≈50%)growthreductionat20000IU/mL,whichfurtherdeclinedto≈66%at50000IU/mL.PDFhadnoeffectsupto200μg/mL,buttherewasan≈20%and≈53%growthreductionat400and700μg/mL,respectively.WhenthevaryingconcentrationsofIFN-α2bandPDFwerecombined,10000IU/mLofIFN-α2bcombinedwith200μg/mLofPDFresultedinan≈75%growthreduction.ThiswasaccompaniedbyaG1cellcyclearrest,shownbycellcycleanalysis.Concurrently,DNA-PKactivityinIFN-α2b/PDF-treated
cellswasalmostthree-foldhigherthancontrols.
CONCLUSIONS
ThecombinationofIFN-α2b(10000IU/mL)andPDF(200μg/mL)reducedgrowthby≈75%inT24cells.Thisappearstobeduetoasynergisticpotentiationofthesetwoagents,inducingaG1arrestwithDNA-PKactivation.Therefore,theIFN-α2b/PDFcombinationcouldtriggerDNA-PKactivationthatmayactonthecellcycletoceasecancercellgrowth.
KEYWORDS
interferon,D-fraction,combinedtherapy,synergism,bladdercancer
.
INTRODUCTION
Thebladderisthemostcommonsiteofcancerintheurinarysystemand≈90%ofbladdercancersareTCCs.OftheseTCCs,≈80%arediagnosedassuperficialbladdertumours[1].Transurethralresectionistheprimarymethodforremovalofthosesuperficialbladdertumours;however,nearly65%ofpatientswillhavetumourrecurrencein5yearswhile10–20%willhaveprogressiontomuscleinvasion[2,3].Itisthusconceivablethattheprimarytherapeuticaimistopreventmultiplerecurrencesandprogressiontoamoreadvanced,invasivedisease.
Severalcytotoxicandimmunemodifyingagentshavebeenusedintravesicallyfortherapeuticpurpose.Amongthem,intravesicaladministrationofBCGishighlyeffectiveinreducingtherecurrencerateandalteringtheprogressionrateofthediseasewithanincreasedsurvivalrate[4].Infact,adjuvantintravesicalBCGtherapyaftersurgicalresectionhasbecomeestablishedtherapyforsuperficialbladdercancers,resultingin≈40%reductionincancerrecurrence[4,5].However,itsbenefitsaresometimesoutweighedbyitssevereside-effects:
cystitisoccursin90%ofpatientsandotherpotentialadverseeffects(fever,allergicreactions,sepsis,etc.)cannotbeexcluded[6,7].Thesedrawbacksthuslimititsuseinclinicalpracticeandrequestasaferandeffectivetreatmentmethodwithfewside-effects,promotingtheuseofunconventionaltherapieswithothervariousimmunomodulators.
Interferons(IFNs)havebeenwidelyusedasimmunotherapyforvarioushumanmalignanciesincludingprostate,bladder,andRCCs[8–10].Particularly,IFN-αhasbeenusedasanintravesicalagentfortreatingsuperficialbladdercancer,resultinginan≈40%responserateinpatients[11].AlthoughthisresponserateislowerthanthatofBCGtherapy,IFN-αcausesonlyminimallocalandsystemictoxicity(comparedwithBCG)[11,12].ToimprovetheefficacyofIFN-αtherapy,combinedtherapy(e.g.IFN-α/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-α,PDFortheircombinationmighthaveagrowthinhibitoryeffectonbladdercancerT24cellsinvitro,andtheunderlyingmechanismofsuchactivitywasalsoexplored.
MATERIALSANDMETHODS
ThehumanbladdercancerT24cells,derivedfromapatientwithTCC,wereobtainedfromtheAmericanTypeCultureCollection(Rockville,MD,USA).CellsweremaintainedinMcCoy’s5amediumcontaining10%fetalbovineserum,penicillin(100U/mL),andstreptomycin(100μg/mL).Routinely,culturemediumwaschangedevery3–4daysandthepassageofcellswasperformedweekly.Forexperiments,cellswereseededinT-75flasksorsix-wellcultureplatesattheinitialcelldensityof2×105cells/mLandwereculturedwithrecombinantIFN-α2b(ScheringCorp.,Kenilworth,NJ,USA),PDF(MaitakeProducts,Inc.,Paramus,NJ,USA)ortheircombinations.CellnumberswerethenassessedatspecifiedtimesusingtheTrypanblueexclusionmethod.
CellcycleanalysiswasperformedusingaFACScanflowcytometer(Becton-Dickinson),equippedwithadoublediscriminationmodule.About1×106cellswereresuspendedin500μLofpropidiumiodidesolution(20μg/mLpropidiumiodide,0.2mg/mLRNase,0.2mg/mLEDTA,0.5%NP-40)andincubatedatroomtemperaturefor1h.Inall,10000nucleiwereanalysedforeachsample,andCellFitsoftwarewasusedtoquantifycellcyclecompartmentsandestimatecellcyclephasefractions.
Invitrophosphorylationassaywasperformedaspreviouslydescribed[19].CelllysateswerefirstpreparedfromcontrolandIFN-α2b/PDF-treatedcellsbythreecyclesoffreeze-thawinliquidnitrogen.A5μgaliquotofcelllysatepreparationwasaddedtothephosphorylationcocktailcontaining55.5kBqof[γ-32P]-ATP(specificactivity:
166.5TBq/mmol)andincubatedat37°Cfor15min.Toactivateendogenousdouble-strandedDNA-dependentproteinkinase(DNA-PK),2μg/mLoffragmentedcalfthymusDNAwasalsoincludedinthereactionmixture.Phosphoproteinswerethenseparatedby10%SDS-PAGEandanalysedbyautoradiography.IntensitiesofspecificDNA-PKbandswerethenquantifiedusingascandensitometer(SilkScientific,Oregon,UT,USA).
Forstatisticalanalysis,alldatawerepresentedasthemean(SD),andstatisticaldifferencesbetweengroupswereassessedwiththeunpairedStudent’st-test;P<0.05wasconsideredtoindicatestatisticalsignificance.
RESULTS
EFFECTSOFIFN-α2bANDPDFONT24CELLGROWTH
ToexaminepossibleeffectsofIFN-α2bandPDFonT24cellproliferation,cellswereculturedwiththevaryingconcentrationsofIFN-α2b(0–50000IU/mL)orPDF(0–700μg/mL)for72h.IFN-α2bcausedan≈50%reductionincellnumberat20000IU/mL,whichfurtherdeclinedto≈66%at50000IU/mL(Fig.1A).PDFhadnoeffectsupto200μg/mL,buttherewasan≈20%and≈53%growthreductionat400and700μg/mL,respectively(Fig.1B).Thus,theseresultsshowthat20000IU/mLofIFN-α2bor700μg/mLofPDFisrequiredtosignificantlyinhibitthegrowthofT24cells.
SYNERGISTICGROWTHINHIBITORYEFFECTSOFIFN-α2bANDPDF
WenextexaminedwhetherthecombinationofIFN-α2bandPDFmighthaveabettergrowthinhibitoryeffect.CellswereculturedwithcombinationsofIFN-α2bandPDFatthevaryingconcentrationsandcellgrowthwasassessedat72h.Thecombinationof10000IU/mLIFN-α2band200μg/mLPDFresultedinan≈75%reductionincellgrowth(Fig.2).ThissuggeststhattheIFN-α2b/PDF-inducedgrowthreductionisduetoasynergisticeffect,becausetheeffectofthetwoagentswasgreaterthantheeffectofeachagentindividual
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