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SurKexPharmacodynamicstudyofanewanti-lungcanceroligoneuleotidedrugSurKex20052iSurKexiiSurKex80SurvivinSurvivinSurvivinDNAsurvivinSurKex1SurKex2NCI-H460PCRSurKex1SurKex2SurvivinmRNAiiiSurKexRT-PCRsurvivinNCI-H460SurKexNCI-H460RNAPCRSurKexNCI-H460survivinmRNASurKexsurvivinmRNA5.0µMSurKex1SurKex2survivinmRNASurKex1SurKex2SurKexNCI-H460SurKexNCI-H460SurvivinSurKexSurvivin5.0µMSurKex1SurKex2SurvivinNCI-H460SurKex18mg/kgSurKex1NCI-H4606mg/kgSurKex11.5mg/kgivSurvivinDNASurKexNCI-H460survivinsurvivinsurvivinNCI-H460vSurKexPharmacodynamicstudyofanewanti-lungcanceroligoneuleotidedrugSurKexABSTRACTLungcancerisamongthedeadliestcancers,andtakestheleadingpositioninoccurrenceandcancerdeathintheworld.ItisalsothemostcommontumorinChina.Lungcancercanbedividedintonon-smallcellandsmallcellforms,nearly80%ofwhichisnon-smallcell.Withahighlevelofunmetdemand,innovativeanti-cancerdrugsareexpectedtoachievehighclinicaluptake.AsanIAPfamilymember,Survivinistheapoptosisinhibitoraswellasthemitoticregulator.Survivinisabsolutelyundetectableinnormaltissues,butover-expressedinallthemostcommonhumancancers.Also,itisrequiredformaintainingcancer-cellviability.Thus,Survivinisconsideredasatherapeutictargetincancer.Genesilencingcanbeinducedbymethylationinthepromotorregion.Thismechanismmightbeusedtocurediseasesbyinhibitingtheexpressionofacertaingeneharmfultohealth.viWedesigneddrugsSurKex1andSurKex2toinhibittheanti-tumortargetsurvivinexpressionusingourmethylationtechniqueinducinggenesilencing.WeexaminedtheexpressionofSurvivinmRNAandproteinonhumannon-smallcelllungcancercelllineNCI-H460byQRT-PCRandWesternblotting.ThemoreeffectiveSurKexdrugwaschoosedtotakeananimaltestexaminingitsanti-tumoreffectinvivo.First,wevalidatedtheover-expressionofsurvivininNCI-H460celllinebyRT-PCR.Then,NCI-H460cellsweretreatedwithSurKex,RNAwasextractedandsurvivinmRNAabundancewasquantitatedbyRTandQRT-PCR.TheresultshowsthatSurKexhasanobviouseffectonsurvivinmRNAexpression.Underthedoseof5.0µM,SurKex1andSurKex2almostinhibittheexpressionofsurvivinmRNA.Thisinhibitionisdose-dependentandSurKex1hasthebettereffectthanSurKex2underthesamedose.Also,NCI-H460cellsweretreatedwithSurKex,proteinwasextractedandSurvivinproteinabundancewasexaminedbyimmunoprecipitationandWesternblotting.TheresultshowsthatSurKexhasanobviouseffectonSurvivinproteinexpression.Underthedoseof5.0µM,SurKex1andSurKex2almostinhibittheexpressionofSurvivinprotein.viiSurKexFinally,themodeloflungcanceronnudemicewereproducedandtreatedwithdrugsthroughIPinjection.TumorgrowthwasexaminedtostudytheeffectofSurKexanditscombinedusewithcisplatin,acommonchemotherapydrug.TheresultshowsthatUnderthedoseof18mg/Kg,SurKex1absolutelyinhibitthevolumeandweightofthetumor.Thecombinationof6mg/kgSurKex1and1.5mg/kgCisplatinhasdistinctsynergismandreducedtoxicity.ConsideringSurvivinasthetherapeutictargetinlungcancer,thisresearchprojectmakesuseofoligonucleotidedrugSurKextospecificallyinhibitsurvivinexpressioninNCI-H460cellsandtumorgrowthinnudemice.Hopefully,SurKexmightbedevelopedasanewdrugagainstlungcanceroranassistanttochemotherapytoprovidepatientswitheffectivetherapeuticmethods.Survivinisover-expressedinalmostalltumorcellsandisrequiredfortumorsurvival,thisresearchprojectcanbereferencedintherapiesofvariouscancers.KEYWORDSlungcancer,survivin,genesilencing,NCI-H460viii1..............................................................................................................11.1........................................................................................................11.2Survivin...........................................................................32.1.1Survivin.................................................................................32.1.2Survivin.......................................................................................................42.1.3Survivin.......................................................................................................62.1.4Survivin..........................................................................................71.3................................................................................................................91.3.1..................................................................................................91.3.2...........................................................................................101.3.3.......................................................................................111.3.4DNA......................................................................................111.4........................................................................................131.4.1..................................................................................................................131.4.2..................................................................................................................162SurvivinNCI-H460................................................172.1..........................................................................