表没食子儿茶素没食子酸酯对肝癌HepG2细胞自噬信号的影响及意义  

Effect of(-)-epigallocatechin-3-O-gallate on autophagic signaling in HepG2 cells

作　　者：陈丽[1] 叶会兰[2] 张国[2] 姚文敏[2] 陈兴洲[2] 梁钢[3] 

机构地区：[1]广西医科大学第一附属医院新药研发中心,广西壮族自治区南宁市530021 [2]广西壮族自治区人民医院消化内,广西壮族自治区南宁市530021 [3]广西医科大学药学院药理教研室,广西壮族自治区南宁市530021

出　　处：《世界华人消化杂志》2015年第19期3022-3028,共7页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;Nos.81160532;81172260;81360077;81102495~~

摘　　要：目的:观察表没食子儿茶素没食子酸酯[(-)-epigallocatechin-3-O-gallate,EGCG]作用下肝癌Hep G2细胞自噬改变,探讨其对肿瘤细胞增殖、死亡的影响及意义.方法:DMEM培养基常规培养Hep G2细胞,分组给予不同浓度EGCG作用.通过透射电镜观察细胞自噬泡形成、实时定量逆转录P C R及免疫印迹分析自噬相关基因及蛋白分子表达、MTT法测定细胞增殖率以及台盼蓝染色法检测细胞死亡数,进一步通过自噬干预实验综合判断细胞自噬变化是否参与EGCG的肝癌治疗作用.结果:EGCG明显抑制Hep G2细胞生长,药物浓度与细胞增殖率呈负相关(r=-0.9341,P<0.001).在有效抑制细胞增殖的EGCG作用下,Hep G2细胞自噬相关基因Beclin1和Atg5的mR NA及蛋白质表达下降,作用底物P62表达增加,细胞内自噬泡形成总体减少.使用Raypamycin上调自噬水平明显削弱EGCG对Hep G2细胞的杀伤作用(t=9.95,P<0.01),6-氨基-3-甲基嘌呤(3 methyladenine,3MA)抑制自噬可显著增强药物的杀癌效应(t=22.82,P<0.01).结论:EGCG可通过下调肝癌细胞自噬有效抑制癌细胞增殖、促进癌细胞死亡,是EGCG发挥治疗肝癌作用的重要药理机制.AIM：To investigate the changes of autophagy in hepatocellular carcinoma（HCC） Hep G2 cells in response to（-）-epigallocatechin-3-Ogallate（EGCG）,and to explore its impact on cell proliferation and death.METHODS：HepG 2 cells were routinely cultured and re-plated in Dulbecco＇s modified eagle＇s medium（DMEM） in the presence of EGCG of different concentrations.Transmission electron microscopic technique was used to record the formation of autophagosomes in Hep G2 cells.Real-time RT-PCR and Western blot were used to detect the mR NA and protein expression of autophagy-related genes,respectively.MTT and trypan blue assays were carried out to determine the cellular proliferation and death.Autophagic intervention experiment was performed to evaluate whether changes in autophagy are involved in the anti-cancer efficacy of EGCG in HCC.RESULTS：The proliferation of Hep G2 cells was significantly inhibited by EGCG and was negatively related to the concentrations of this compound（r =-0.9341,P 0.001）.Doses of EGCG that could effectively inhibit the proliferation of Hep G2 cells significantly decreased the mR NA and protein expression of Beclin1 and Atg5,with increments of P62 named autophagic substrate as well as substantiallyreduced numbers of autophagasomes found in these cells.Moreover,up-regulating autophagy with rapamycin was found to apparently impair the effect of EGCG in killing HepG 2 cells（t = 9.95,P 0.01）,while 3-MA,an autophagy inhibitor,dramatically exaggerated the anti-cancer effects of EGCG（t = 22.82,P 0.01）.CONCLUSION：EGCG substantially inhibits cell proliferation and promotes cell death in HCC cells via down-regulation of autophagy,which indicates a novel critical pharmacological mechanism of EGCG for hepatoma therapy.

关 键 词：表没食子儿茶素没食子酸酯 肝癌 自噬 细胞增殖 药理机制 

分 类 号：R735.7[医药卫生—肿瘤]