索拉非尼诱导肝癌HepG2细胞自噬的机制及意义  

Mechanism and significance of sorafenib-induced autophagy in HepG2 cells

作　　者：秦利燕[1,2] 王滨[3] 倪振洪[3] 连继勤[3] 宋方洲[1,2] 袁成福[1,2] 刘革力[1,2] 

机构地区：[1]重庆医科大学基础医学部生物化学与分子生物学教研室,重庆400016 [2]重庆医科大学分子医学与肿瘤研究中心,重庆400016 [3]第三军医大学基础医学部生物化学与分子生物学教研室,重庆400038

出　　处：《第三军医大学学报》2013年第22期2435-2438,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(30800410)~~

摘　　要：目的探讨索拉非尼诱导肝癌HepG2细胞自噬的机制及意义。方法用GFP-LC3质粒转染HepG2细胞后观察索拉非尼对自噬小体形成的影响;免疫共沉淀法检测索拉非尼对Mcl-1与Beclin1之间相互作用的影响;使用siRNA沉默自噬相关基因Atg5或用氯喹(CQ)抑制自噬后观察索拉非尼抗肝癌细胞的效应;Western blot检测自噬与凋亡相关蛋白Atg5、Beclin1、LC3、Mcl-1与PARP表达的变化。结果索拉非尼处理HepG2细胞后LC3-Ⅱ蛋白表达量呈剂量依赖性增强,索拉非尼处理后转染GFP-LC3的HepG2细胞中出现明显的自噬小体样点状荧光,Western blot结果显示索拉非尼处理后HepG2细胞中Mcl-1表达下调,Beclin1无明显变化,Co-IP证实在HepG2细胞中Mcl-1和Beclin1间存在蛋白相互作用,索拉非尼能减弱Mcl-1和Beclin1的结合。抑制自噬后能明显增强索拉非尼的杀细胞效应。结论索拉非尼可能通过下调Mcl-1来诱导肝癌HepG2细胞自噬,抑制自噬后可显著增强索拉非尼的杀细胞效应。Objective To investigate the mechanism and significance of sorafenib-induced autophagy in HepG2 cell line. Methods HepG2 cells were transfected with GFP-LC3 plasmid to observe the effect of sorafenib on autophagosome formation. The interaction of Mcl-1 and Beclin1 was tested by co-immunoprecipitation （Co-IP）. The anti-tumor effect of sorafenib was detected after autophagy inhibition with chloroquine（CQ） or knock down of autophagy-related gene Atg5 by siRNA. Changes of autophagic and apoptotic proteins Atg5, Beclin1, LC3, Mcl-1 and PARP were detected by Western blotting. Results In HepG2 cells, sorafenib upregulated LC3-Ⅱ protein expression in a dose-dependent manner. HepG2 cells transfected with GFP-LC3 plasmid showed autophogosome-like dot fluorescence when treated with sorafenib. Mcl-1 protein level was downregulated while Beclin1 remained unchanged when treated with sorafenib, as shown by Western blotting. The interaction between MCL-1 and Beclin1 were observed and could be reduced by sorafenib treatment, as shown by Co-IP. The anti-tumor effect of sorafenib was enhanced when autophagy was inhibited. Conclusion Sorafenib may induce autophagy by down-regulating Mcl-1 expression. The anti-tumor effect of sorafenib is enhanced when autophagy is inhibited.

关 键 词：索拉非尼 自噬 MCL-1 凋亡 

分 类 号：R735.7[医药卫生—肿瘤] R966[医药卫生—临床医学]