雷帕霉素联合缺氧诱导肺腺癌A549细胞凋亡及其分子机制  

Rapamycin induces apoptosis of lung adenocarcinoma cell A549 under hypoxia and its molecular mechanism

作　　者：陈斌[1] 顾蔚卿[1] 倪健[1] 

机构地区：[1]同济大学附属上海市肺科医院肿瘤科,上海200433

出　　处：《肿瘤》2010年第4期293-297,共5页Tumor

摘　　要：目的:探讨哺乳动物雷帕霉素靶向(mammalian target of rapamycin,mTOR)通路抑制剂雷帕霉素联合缺氧诱导人肺腺癌细胞株A549细胞凋亡的分子机制。方法:应用雷帕霉素联合缺氧处理人肺腺癌细胞株A549后,Hoechst33258染色法观察细胞凋亡情况,实时定量PCR和Western印迹法分别检测A549细胞中survivin mRNA和蛋白的表达变化,Western印迹法检测缺氧诱导因子1α(hypoxia-inducible factor1α,HIF1α)表达的改变。结合染色质免疫共沉淀技术,探讨雷帕霉素通过诱导细胞凋亡而发挥抗肺癌效应的分子机制。结果:雷帕霉素联合缺氧可在体外诱导A549细胞凋亡,雷帕霉素联合缺氧可诱导A549细胞中survivin mRNA(P<0.01)和蛋白的表达水平明显下调。雷帕霉素通过抑制缺氧条件下诱导的HIF-1α表达增加,进而抑制survivin的表达。结论:雷帕霉素通过抑制缺氧条件下A549细胞中HIF-1α的表达,降低HIF-1α与survivin核心启动子的结合,下调缺氧引发的凋亡抑制基因survivin表达,进而发挥抗肿瘤的生物学效应。Objective：To investigate the molecular mechanism underlying apoptosis of lung adenocarcinoma cell line A549 induced by rapamycin,a type of mammalian target of rapamycin（mTOR） signal pathway inhibitor,combined with hypoxia treatment.Methods：Rapamycin was applied to treat A549 cell line under hypoxia for 16 h.Then the discrepancy of cell apoptosis between treated and untreated control was compared by Hochest 33258 staining.After being treated with rapamycin combined with hypoxia,the mRNA and protein expressions of survivin,a kind of anti-apoptotic molecule,were tested by real-time PCR and Western blotting.Western blotting was employed to test the difference of hypoxia-inducible factor（HIF）-1α expression level before and after rapamycin was added.In additon,chromatin immunoprecipitation（ChIP） assay was further applied to explore the mechanism underlying the effects of rapamycin under hypoxia in inducing apoptosis of A549 cells.Results：Rapamycin induced apoptosis of A549 cell line under hypxia in vitro.In A549 cell line,the expressions of survivin at mRNA（P0.01） and protein levels,were both decreased by rapamycin combined with hypoxia treatment.Besides,ChIP assay revealed that rapamycin inhibited the expression of survivin at the transcriptional level through decreasing the hypoxia-induced up-regulation of HIF-1α and its binding with the promoter of survivin gene.Conclusion：Rapamycin exerted anti-tumor effects by inhibiting the expression of HIF-1α and decreasing the binding of HIF-1α with the promoter of survivin gene,thereby down-regulating the increase in the expression of apoptosis inhibiting gene survivin.

关 键 词：癌 非小细胞肺 腺癌 西罗莫司 缺氧诱导因子1 细胞 A549 

分 类 号：R734.2[医药卫生—肿瘤]