TRAIL联合蛋白酶抑制剂MG132诱导肺癌A549细胞凋亡的实验研究

摘要

目的 观察TRAIL对肺癌A549细胞的体外生长抑制及诱导凋亡作用,并探讨TRAIL与蛋白酶抑制剂MG132联合用药诱导细胞凋亡的作用及其机制.方法 采用MTT法检测不同浓度TRAIL对肺癌A549细胞增殖的影响;应用流式细胞仪技术检测TRAIL、蛋白酶抑制剂MG132及其两药联合干预24h对肺癌A549细胞凋亡率的影响;采用Western blotting检测药物干预前后细胞凋亡相关蛋白Caspase-8、Caspase-9以及核转录因子NF-κB的表达情况.结果 MTT结果显示不同浓度TRAIL对肺癌A549细胞的体外抑制作用与剂量效应正相关,24h IC50为96.322μg/ml,48h IC50为43.59 μg/ml;流式细胞术显示TRAIL与MG132联合用药后肺癌A549细胞凋亡率明显增高(平均67.65%),与TRAIL(平均23.55%)、MG132(平均25.91%)单药组比较,有显著性差异(P=0.016);Western blotting结果显示,与TRAIL单药组比较,两药联合后细胞凋亡相关蛋白Caspase-8、Caspase-9表达明显增强(P=0.006),抑制细胞凋亡因子NF-κB表达则明显减弱(P=0.04).结论 TRAIL可抑制A549细胞的增殖,呈浓度依赖关系;TRAIL与蛋白酶抑制剂MG132联合应用可以明显提高细胞凋亡率,可能与蛋白酶抑制剂MG132降低NF-κB的活性,增强细胞对TRAIL诱导凋亡的敏感性,促进凋亡相关蛋白Caspase-8及Caspase-9的表达有关.%Objective To investigate the cytotoxicity of TRAIL in lung adenocarcinoma cell line 3549,its synergetic effect combined with proteasome inhibitor MGI32 ,and the possible mechanism.Methods Survival fraction of the lung adenocarcinoma cell line A549 intervened by TRAIL combined with MGI32 at different conditions of concentrations were tested with MTT The apoptosis rate of the lung adenocarcinoma cell line A549 intervened by TRAIL or MGI32 and by TRAIL combined with MGI32 were detected by flow cytometry.The change of the expression of the apoptosis-related factors in cell line A549 interfered by TRAIL combined with protease inhibitor were measured by Western blotting analysis.Results TRAIL could induce apoptosis in the lung adenocarcinoma cell line A549,presenting obvious dose-dependent cytotoxicity through MTI, and ICso of 24h was 96.322μg/ml, while IC50 of 48h was 43.59 μg/ml.The apoptosis rate of the lung adenocarcinoma cell line A549 was obviously increased by intervention of TRAIL combined with MGI32 according to the flow cytometry(67.65％ vs.23.55％ ,P =0.0lb).The expression level of the apoptosis-related factors caspase-8, caspase-9 was upregulated in group of TRAIL combined with MGI32( P =0.006) as well as the expression level of NF-kB was increased in group of TRAIL( P = 0.04 ).Conclusion TRAIL combined with MG132 can apparently increase the apoptosis rate of the lung adenocarcinoma cell line A549.The expression level of the apoptosis-related factors caspase-8 and caspase-9 protein may be involved in the mechanism increased apoptosis.Moreover, the lung adenocarcinoma cell line A549 showing more sensitivity to TRAIL may be due to decreased activity of NF-κB inhibited by proteasome inhibitor MGI32.