Smac调节奥沙利铂诱导大肠癌耐药细胞凋亡的研究
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摘要
第一章Smac,Survivin在大肠癌中的表达及其临床意义
目的：检测线粒体促凋亡因子Smac和凋亡抑制因子Survivin在正常大肠组织大肠肿瘤组织中的表达,并探讨其临床意义。
方法：RT-PCR方法及蛋白质免疫印迹技术(immunohistochem-istry)分别检测正常肠组织50例、肠癌组织50例中的Smac及Survivin的表达。应用SPSS统计学软件,采用非参数Mann-Whitney和Wilcoxon检验分析Smac及Survivin mRNA的表达与肠癌的组织类型及EOC细胞分极、临床分期的关系。
结果：1.Smac mRNA的表达与大肠正常组织和肠肿瘤组织中的表达：结果表明,Smac mRNA的表达在正常大肠组织>肠肿瘤组织(P<0.05),呈递减趋势。
2.Survivin mRNA在正常大肠组织和肠肿瘤组织中的表达：Survivin mRNA在正常大肠组织中有2例表达缺失,在恶性肠肿瘤组织中均有表达,呈递增趋势：正常大肠组织<肠癌组织(P<0.05)>。
3.Smac及Survivin蛋白的表达：Smac及Survivin蛋白在正常大肠组织及肠肿瘤组织中的表达与mRNA的表达趋势一致。
结论：Smac mRNA及蛋白在大肠癌中表达降低和Survivin mRNA及蛋白在肠癌中表达升高与大肠癌的发生及发展密切相关；Smac及Survivin mRNA及蛋白在大肠癌中的表达水平高低可作为判断打肠癌患者病情进展的指标。
第二章Smac对大肠癌细胞株SW480生长及对奥沙利铂敏感性影响的研究
目的：探讨外源性Smac基因过表达对大肠癌细胞株SW480生物学特性及对奥沙利铂敏感性的影响。
方法:1.脂质体2000介导Smac基因转染SW480细胞,G418筛选阳性克隆,RT-PCR,western blot鉴定稳定转染细胞中Smac的表达,倒置相差显策镜观察转染前后细胞形态,细胞计数法绘制细胞生长曲线。
2.采用四甲基偶氮唑盐(4,5-dimethylt hiazoleor 2,5-dipheny-ltetrazoliumb romide,MTT)法,检测不同浓度奥沙利铂作用不同时间对转染空载体或目的基因的SW480细胞的生长抑制率(growth inhibitory rate,GIR)。
3.流式细胞仪检测各转染细胞的细胞周期分布及10ug/ml奥沙利铂作用24h后,转染空载体或目的基因的SW480细胞的凋亡率；Hochst33342染色法检测10g/ml奥沙利铂作用24h后凋亡细胞形态。
结果：1.经过4G418筛选及RT-PCR和Western blotting鉴定,获得稳定表在Smac的SW480细胞。
2.转染Smac基因的SW480细胞形态及生长检测：转染Smac的SW480细胞胞体和细胞核增大,轻度肿胀,细胞变不短梭型；流式细胞仪检测显示细胞周期G0／G1期阻滞,S期比例下降,细胞生长变缓。
3.奥沙铂对SW480/neo,SW480/Smac细胞的生长抑制作用：奥沙利铂浓度为10,50,100,500ug/ml,作用24h对转染空载体的SW480细胞(SW480/neo)及SW480/Smac细胞的生长抑制率分别为9.55%、12.7%、18.53%、24.6%及33.11%、42.67%、57.63%、68.6%,68.6%.A2780／Smac细胞对奥沙利铂敏感性显著升高,具有时间及浓度依赖性(P<0.05)。
4.奥沙利铂通过诱导细胞凋亡发挥抗肿瘤作用：10ug／ml奥沙利铂作用24h后,SW480／neo及SW480／Smac细胞的凋亡率分别为9.20%和30.3%,细胞均呈现典型的凋亡形态。
结论：
1.外源性Smac基因转染可影响大肠癌细胞SW480的生长。
2.Smac SW480细胞对奥沙利铂具有敏感性,并呈时间及浓度的依赖性。
3.Smac增强奥沙利铂诱导大肠癌细胞凋亡作用的机制与细胞内促凋亡蛋白Smac抑制凋亡蛋白Survivin的活性有关。
第三章奥沙利铂耐药的大肠癌癌细胞株SW480／L-OHP的建立及Smac对其影响的研究
目的：建立人大肠癌SW480耐药株,并检测该耐药株的生物学特性,为应用基因技术逆转肿瘤多药耐药性提供实验模型。通过转染S m a c基因并联合奥沙利铂作用于人大肠癌耐药细胞株SW480／L-OHP研究二者联合作用对大肠癌细胞耐药性的影响。
方法：通过奥沙利铂(L-OHP)长期筛选建立人大肠癌耐药细胞株SW480／L-OHP.应用四甲基偶氮唑(MTT)法及流式细胞仪(FCM)对该细胞株的相关特性(药物敏感性、倍增时间、P-糖蛋白功能、细胞周期)进行系统研究。将克隆Smac的pCDA3-Smac质粒,通过脂质体2000介导转染人大肠癌耐药细胞株SW480／L-OHP,同时,在培养液中加入L-OHP,用MTT法分析细胞的生长情况；荧光显微镜观察两种细胞的形态改变；流式细胞仪对它们的细胞周期和凋亡情况进行分析。最后用罗丹明123蓄积实验检验联合作用对耐药株P-gp活性的影响。
结果：L-OHP浓度梯度递增法连续作用SW480细胞3个月,得到大肠癌耐药细胞株SW480/L-OHP。经MTT法检测耐药株IC50是敏感株的36.2倍,耐药细胞的倍增时间延长了20.21h,罗丹明123蓄积实验证实耐药株的P-糖蛋白活性高于敏感株,细胞周期分析发现其S期与G0／G1期细胞减少、G2／M期细胞增多。Smac转染SW480/L-OHP后,细胞的生长受到抑制：在转染第4天,SW480/L-OHP的生长抑制率达49.62%,L-OHP作用组的生长抑制率达57.68%,联合作用组细胞的生长抑制最明显,为78.62%。奥沙利铂(32.18yg/ml)作用24h后,SW480/neo及SW480/L-OHP细胞的凋亡率分别为9.23%和33.82%。
结论：
1、成功建立了人大肠癌SW480耐药细胞株,该细胞株的生物学特性明显不同于敏感细胞株；
2、Smac可增强SW480细胞对奥沙利铂的敏感性,并具有时间及浓度依赖性。
3 Smac增强奥沙利铂诱导大肠癌细胞凋亡作用的机制与细胞内促凋亡蛋白Smac,抑制凋亡蛋白Survivin的活性有关.
PART I The EXPRESSION OF SMAC AND SURVIVIN IN COLORECTAL CANCER AND THEIR CLINICAL SIGNIFICANCE
Obective: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 APOPTOSIS
Objective: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.
PARTⅢEstablishment 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-OHP
Obective: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).
Results
SW480/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.
Conclusion
1 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.
目的：检测线粒体促凋亡因子Smac和凋亡抑制因子Survivin在正常大肠组织大肠肿瘤组织中的表达,并探讨其临床意义。
方法：RT-PCR方法及蛋白质免疫印迹技术(immunohistochem-istry)分别检测正常肠组织50例、肠癌组织50例中的Smac及Survivin的表达。应用SPSS统计学软件,采用非参数Mann-Whitney和Wilcoxon检验分析Smac及Survivin mRNA的表达与肠癌的组织类型及EOC细胞分极、临床分期的关系。
结果：1.Smac mRNA的表达与大肠正常组织和肠肿瘤组织中的表达：结果表明,Smac mRNA的表达在正常大肠组织>肠肿瘤组织(P<0.05),呈递减趋势。
2.Survivin mRNA在正常大肠组织和肠肿瘤组织中的表达：Survivin mRNA在正常大肠组织中有2例表达缺失,在恶性肠肿瘤组织中均有表达,呈递增趋势：正常大肠组织<肠癌组织(P<0.05)>。
3.Smac及Survivin蛋白的表达：Smac及Survivin蛋白在正常大肠组织及肠肿瘤组织中的表达与mRNA的表达趋势一致。
结论：Smac mRNA及蛋白在大肠癌中表达降低和Survivin mRNA及蛋白在肠癌中表达升高与大肠癌的发生及发展密切相关；Smac及Survivin mRNA及蛋白在大肠癌中的表达水平高低可作为判断打肠癌患者病情进展的指标。
第二章Smac对大肠癌细胞株SW480生长及对奥沙利铂敏感性影响的研究
目的：探讨外源性Smac基因过表达对大肠癌细胞株SW480生物学特性及对奥沙利铂敏感性的影响。
方法:1.脂质体2000介导Smac基因转染SW480细胞,G418筛选阳性克隆,RT-PCR,western blot鉴定稳定转染细胞中Smac的表达,倒置相差显策镜观察转染前后细胞形态,细胞计数法绘制细胞生长曲线。
2.采用四甲基偶氮唑盐(4,5-dimethylt hiazoleor 2,5-dipheny-ltetrazoliumb romide,MTT)法,检测不同浓度奥沙利铂作用不同时间对转染空载体或目的基因的SW480细胞的生长抑制率(growth inhibitory rate,GIR)。
3.流式细胞仪检测各转染细胞的细胞周期分布及10ug/ml奥沙利铂作用24h后,转染空载体或目的基因的SW480细胞的凋亡率；Hochst33342染色法检测10g/ml奥沙利铂作用24h后凋亡细胞形态。
结果：1.经过4G418筛选及RT-PCR和Western blotting鉴定,获得稳定表在Smac的SW480细胞。
2.转染Smac基因的SW480细胞形态及生长检测：转染Smac的SW480细胞胞体和细胞核增大,轻度肿胀,细胞变不短梭型；流式细胞仪检测显示细胞周期G0／G1期阻滞,S期比例下降,细胞生长变缓。
3.奥沙铂对SW480/neo,SW480/Smac细胞的生长抑制作用：奥沙利铂浓度为10,50,100,500ug/ml,作用24h对转染空载体的SW480细胞(SW480/neo)及SW480/Smac细胞的生长抑制率分别为9.55%、12.7%、18.53%、24.6%及33.11%、42.67%、57.63%、68.6%,68.6%.A2780／Smac细胞对奥沙利铂敏感性显著升高,具有时间及浓度依赖性(P<0.05)。
4.奥沙利铂通过诱导细胞凋亡发挥抗肿瘤作用：10ug／ml奥沙利铂作用24h后,SW480／neo及SW480／Smac细胞的凋亡率分别为9.20%和30.3%,细胞均呈现典型的凋亡形态。
结论：
1.外源性Smac基因转染可影响大肠癌细胞SW480的生长。
2.Smac SW480细胞对奥沙利铂具有敏感性,并呈时间及浓度的依赖性。
3.Smac增强奥沙利铂诱导大肠癌细胞凋亡作用的机制与细胞内促凋亡蛋白Smac抑制凋亡蛋白Survivin的活性有关。
第三章奥沙利铂耐药的大肠癌癌细胞株SW480／L-OHP的建立及Smac对其影响的研究
目的：建立人大肠癌SW480耐药株,并检测该耐药株的生物学特性,为应用基因技术逆转肿瘤多药耐药性提供实验模型。通过转染S m a c基因并联合奥沙利铂作用于人大肠癌耐药细胞株SW480／L-OHP研究二者联合作用对大肠癌细胞耐药性的影响。
方法：通过奥沙利铂(L-OHP)长期筛选建立人大肠癌耐药细胞株SW480／L-OHP.应用四甲基偶氮唑(MTT)法及流式细胞仪(FCM)对该细胞株的相关特性(药物敏感性、倍增时间、P-糖蛋白功能、细胞周期)进行系统研究。将克隆Smac的pCDA3-Smac质粒,通过脂质体2000介导转染人大肠癌耐药细胞株SW480／L-OHP,同时,在培养液中加入L-OHP,用MTT法分析细胞的生长情况；荧光显微镜观察两种细胞的形态改变；流式细胞仪对它们的细胞周期和凋亡情况进行分析。最后用罗丹明123蓄积实验检验联合作用对耐药株P-gp活性的影响。
结果：L-OHP浓度梯度递增法连续作用SW480细胞3个月,得到大肠癌耐药细胞株SW480/L-OHP。经MTT法检测耐药株IC50是敏感株的36.2倍,耐药细胞的倍增时间延长了20.21h,罗丹明123蓄积实验证实耐药株的P-糖蛋白活性高于敏感株,细胞周期分析发现其S期与G0／G1期细胞减少、G2／M期细胞增多。Smac转染SW480/L-OHP后,细胞的生长受到抑制：在转染第4天,SW480/L-OHP的生长抑制率达49.62%,L-OHP作用组的生长抑制率达57.68%,联合作用组细胞的生长抑制最明显,为78.62%。奥沙利铂(32.18yg/ml)作用24h后,SW480/neo及SW480/L-OHP细胞的凋亡率分别为9.23%和33.82%。
结论：
1、成功建立了人大肠癌SW480耐药细胞株,该细胞株的生物学特性明显不同于敏感细胞株；
2、Smac可增强SW480细胞对奥沙利铂的敏感性,并具有时间及浓度依赖性。
3 Smac增强奥沙利铂诱导大肠癌细胞凋亡作用的机制与细胞内促凋亡蛋白Smac,抑制凋亡蛋白Survivin的活性有关.
PART I The EXPRESSION OF SMAC AND SURVIVIN IN COLORECTAL CANCER AND THEIR CLINICAL SIGNIFICANCE
Obective: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 APOPTOSIS
Objective: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.
PARTⅢEstablishment 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-OHP
Obective: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).
Results
SW480/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.
Conclusion
1 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.
引文
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