The Effect Of Smac On Oxaliplatin Induced Apoptosis In Chemotherapy-resistant Colorectal Cancer

PART I The EXPRESSION OF SMAC AND SURVIVIN IN COLORECTAL CANCER AND THEIR CLINICAL SIGNIFICANCEObective:To study the expression of Smac and Survivin in normal colorectal tissues and colorectal tumor tissues and to Explore their clinical significant.Methods:Colorectal tissues were obtained from primary colorectal cancers(n=50),normal colorectal tissues(n=50).Reverse-transcription PCR and West blot were used to examine the mRNA and protein expressions of Smac and Survivin.The relationship between mRNA Expression of Smac, Survivin and colorectal tissue type as well as the tumor grade and clinicalst age of colorectal cancers patient was evaluated by nonparametric Mann-Whitney and Wilcoxon tests using SPSS software.Results:1.The mRNA expression of Smac in normal Colorectal issues and Smac tumor tissue:Signfcant decreased Smac expressionw as observed with a rank order:normal>malignanto colorectal cancers (p<0.05, respectively).2.The mRNA expression of Survivin in Colorectal tissues and Colorectal tumor tissue:Survivin mRNA was misse expressed in two cases of normal Colorectal tissues/while in Colorectal tumor tissue was all detectable.Significantly increased Survivin expression was observed ordered by normal and malignant Colorectal tissues(p<0.05,re spectively).3 The protein expression of Smac and Survivin:The protein expressionlevels of Smac and Survivin in normal Colorectal tissues and Colorectal tumor tissues were coincident with their mRNA expression levels.Conclusions:The depressed mRNA and protein expression of Smac and increased mRNA and protein expression of Survivin in Colorectal cancer play important roles in the carcinogenesis and progression of Colorectal cancer; The mRNA and protein expression levels of Smac and surviving maybe indicators of the progression of EOC patients. PART II EFFECT OF SMAC ON TUMOR GROWTH OF Colorectal CANCER CELL SW480 AND THE SENSITIZATION TO OXALIPLATIN INDUCED APOPTOSISObjective:To explore the effect of ectopic overexpression of Smac geneOn the proliferation of Colorectal cancer cell line SW480,and the sensitization to OXALIPLATIN induced apoptosis.Methods:1.Smac genes were introduced into ASW480 cells by liposome 2000, respectively. Subclone cells were obtained by persistent G418 selection. Cellular Smac in the transfected cells was assessd by RT-PCR and western blot. Cell morphous and cell growth curve were measured by phase-contrast inverted microscope and cell counting.2.MTT(4,5-dimethylth iazoleo r2,5-diphenylte trazoilumb romide, MTT) was adopted to detect the sensitivity of SW480 cells transfected with blank plasmid or targeting gene to OXALIPLATIN.3.FCM (flow cytometry) was used to detect the cell cycled is tribution and the rate of apoptosis upon 10ug/ml treatment OXALIPLATIN fo r24 h ours.Heochst33342 staining was used to detect apoptosis morphous of cells after treated with lOug/ml OXALIPLATIN for 24 hours.Results:1.Stable Smac transfected SW480 cell line were obtain after selected with G418 for 4 weeks and were conformed by RT-PCR and western blot.2.The observation of cell morphous and cell growth of Smac transfected SW480 cells.the cell body and nucleus of Smac transfected SW480 cell be came larger,swelled lightly and the cells s owed shorts pindle appearance;etopic expression of Smac SW480 cells resulted in a s lower cell proliferation rate accompanied by G0/G1 arrest and decreased proportion of S stage than vector controls.3 The cell growth inhibition effect of OXALIPLATIN on SW480/neo,or SW480/Smac cells:The cell gorwth inhibition rates of blank plasmid transfected SW480(SW480/neo) and SW480/Smac cells were 9.55%,12.7%,18.53%,24.6% and 33.11%,42.670/o,57.63%,68.6% respectively after treated with OXALIPLATINL at concentrations of 10, 50,100,500ng/mlfor24h.SW480/Smac showed higher sensitivity to time and concentration dependence (p<0.05).4.OXALIPLATINL inducedced cell death via apoptosisin ducing effect.The apoptosis rates of blank plasmid transfected SW480 and Smac transfected SW480 cells after treated with lOug/ml OXALIPLATINL for 24 hours were 9.20% and 39.49%.COLORECTAL cancer cells presented typical apoptosis morphous after treated with OXALI-PLATINL.Conclusions:1.Ectopic expression of Smac could affect cell growth of SW480 cells.2.Ectopic expression of Smac could sensitize SW480 cells to OXALIPLATINL induced apoptosis, with time and concentration dependence.PARTâ…¢Establishment of Human COLORECTAL cancer Multidrug Resistant Cell Line SW480/L-OHP and Effects of Combination of Smac and L-OHP on Growing of SW480/L-OHPObective:To establish multidrug resistant strain of human COLORECTAL cancer SW480 cell lines and to study its biological characteristics.To investigate the effects of the combination of transfected Smac gene and OXALIPLATIN (L-OHP) on drug resistance in human COLORECTAL cancer cell line.Methods:The cell line SW480 was induced to form a multidrug resistant cell subline(SW480/L-OHP) by exposing it to gradually increase concentration of L-OHP.MTT assay was used to evaluate drug sensitivity and to measure cell growth. Flow cytometric assay was employed to examine cell cycle and to determine drug influx and efflux.The cells were divided into four groups:the control group,the Smac group(the Smac transfected group),the L-OHP group(the L-OHP treatment group),the associated group(Smac transfected with L-OHP treatment group).The MTT assay was used to analyse the growth of SW480/L-OHP cells.The apoptosis of these cells were evaluated by flowcytometry(FCM).ResultsSW480/L-OHP cell line was established after 3 moths with stable resistance to L-OHP and resistance index 36.2;SW480/L-OHP cells exhibited cross resistance to many other chemotherapeutic agents.As compared with parental cells,the morphological and chromatosome number of SW480/L-OHP changed;its doubling time prolonged;and the number of cells in S phase and G0/G1 phase decreased while in G2/M phase increased by cell cycle analysis.The expression of Smac protein increased and Survivin protein was decreased in the SW480 cell line. SW480/L-OHP cells were inhibited 49.62%,At the same time,in the L-OHP treatment group and the associated group,SW480/L-OHP were inhibited 57.68% and 78.62% respectively.The apoptosis rates of SW480/L-OHP/pCDN3 and SW480/L OHP/Smac after treated for L-OHP 24h were 9.23%and33.82% respectively.Conclusion1 SW480/L-OHP cell line showed the typical multidrug resistant phenotype and might to serve as an ideal model for studying the mechanisms of resistance to L-OHP and filtrating reversing drug.SW480/L-OHP cell line possessed the basic characteristics of resistant cells.2 Ectopic expression of SmaC could sensitize SW480 cells to L-OHP induced apoptosis,with time and concentration dependence.3 The mechanism of apoptosis sensitization effect by SmaC was associated with significant up-regulation of Smac and down-regulation of Survivin.