吲哚美辛诱导食管癌EC109细胞凋亡的Smac依赖性机制

The study of Smac-dependent apoptosis induced by indomethacin in EC109 esophageal cancer cell line

目的:研究Smac表达下调对吲哚美辛诱导的食管癌EC109细胞凋亡的影响,并探索其内在分子机制。方法:使用Smac-siRNA脂质体法转染食管癌EC109细胞,将细胞分为空白对照组,siRNA-control阴性对照组和siRNA-Smac组。使用不同浓度的吲哚美辛处理各组细胞,MTT检测细胞活力变化,流式细胞术检测其对细胞凋亡的影响,Western blot法检测Caspase-9、3以及XIAP、survivin表达情况。结果:采用不同浓度吲哚美辛处理后,细胞活力明显受到抑制;Smac siRNA可明显降低Smac在RNA水平和蛋白水平的表达;吲哚美辛可引起各组凋亡率明显增加,单纯导入siRNA-Smac片段并不能引起细胞凋亡变化,siRNASmac联合药物能明显降低EC109细胞凋亡率(P<0.05)。在蛋白水平,siRNA-Smac组的survivin表达无明显变化,XIAP表达明显增强;Caspase-9及Caspase-3的活性片段明显表达减弱(P<0.05)。结论:NSAIDs药物吲哚美辛可诱导食管癌EC109细胞凋亡,这种作用一定程度上依赖于Smac的正常表达;Smac表达降低后其对XIAP的抑制减弱,Caspase-9和Caspase-3的活化受到抑制,而survivin在其中并不起决定性作用。

Objective：To study the effect and underlying mechanism of Smac knockdown on NSAIDs drug indometacin induced apoptosis in EC109 cell.Methods：We transfected with Smac-siRNA into EC109 cells by LipofectamineTM2000,and cells were divided into three groups：control group,siRNA-control group,and siRNA-Smac group.After treating with indomethacin,cell viability was detected by MTT,cell apoptosis was determined by FACS,and the expression of Caspase-9/3、XIAP and survivin were identified by Western blot.Results：After treating with different concentrations of indomethacin,the cell viability got significant changes compared with control group （P ＜ 0.05）.After transfecting with Smac-siRNA sequence into EC109 cells by LipofectamineTM 2000,the expression of Smac were decreased both in RNA level and protein level （P ＜ 0.05）.Smac knowdown alone could not induce apoptosis,whereas treating combined with indomethacin could increase apoptosis significantly （P ＜ 0.05）.The apoptosis of siRNA-Smac ＋ indomethacin group was significantly lower than the normal control ＋ indomethacin group （P ＜ 0.05）.In protein level,the expression of survivin had not significantly changed （P ＜ 0.05）,whereas XIAP was significantly increased （P ＜ 0.05）,and active fragments of Caspase-9 and Caspase-3 were significantly decreased （P ＜ 0.05）.Conclusion：NSAIDs drug indomethacin can induce the apoptosis of EC109 cells,and much of that depends on the expression of Smac.Smac-knockdown weakened the inhibition of XIAP,further the activation of Caspase-9 and Caspase-3 were inhibited.However,survivin did not play a critical role in the process.