二甲双胍体外诱导人肝癌Huh-7细胞凋亡及其作用机制  

Metformin induces apoptosis in hepatocellular carcinoma Huh-7 cells in vitro and its mechanism

作　　者：林芬[1] 严威[1] 文霆[1] 吴国洋[1] 

机构地区：[1]厦门大学附属中山医院普通外科,361004

出　　处：《中华肿瘤杂志》2013年第10期742-746,共5页Chinese Journal of Oncology

基　　金：国家自然科学基金（81172285）

摘　　要：目的 探讨二甲双胍对人肝癌Huh-7细胞增殖、凋亡的影响及其可能的作用机制.方法 二甲双胍干预Huh-7细胞后,四甲基偶氮唑蓝（MTT）法检测其对细胞活力及生长抑制的影响；Western blot检测凋亡相关蛋白及CD133蛋白的变化；流式细胞术检测细胞凋亡及肝癌干细胞相关标志物CD133的表达；无血清悬浮培养观察二甲双胍对肿瘤干细胞球形成的影响；逆转录聚合酶链反应（RT-PCR）检测其对肿瘤干细胞相关基因CD133、β-catenin、ABCG2 mRNA表达的影响.以加入与实验组相同的培养液但未给予药物处理的组为对照组.结果 MTT检测结果显示,与对照组比较,随着二甲双胍浓度的增高,肝癌Huh-7细胞的增殖率下降,差异均有统计学意义（均P＜0.05）.10 mmol/L二甲双胍干预48 h后肝癌Huh-7细胞的早期凋亡率为（22.29±0.80）％,对照组的早期凋亡率为（6.70±0.50）％,差异有统计学意义（P ＜0.05）；10 mmol/L二甲双胍干预48 h后肝癌Huh-7细胞的晚期凋亡率为（13.87±1.20）％,对照组的晚期凋亡率为（1.10±0.02）％,差异有统计学意义（P＜0.05）.25 mmol/L二甲双胍干预48 h后细胞的早期凋亡率为（15.28±2.10）％,晚期凋亡率为（25.89±2.30）％,均高于对照组（均P＜0.05）.Western blot检测结果显示,25 mmol/L二甲双胍干预Huh-7细胞48 h后,p-Akt、Bcl-2/Bax比值表达下调,PTEN表达上调.随着二甲双胍浓度的增加,CD133蛋白的表达下调.流式细胞仪检测结果显示,25 mmol/L二甲双胍干预Huh-7细胞48 h前后,Huh-7细胞中CD133＋细胞的表达率分别为（40.7±2.1）％和（14.6±1.85）％ （P ＜0.05）.在低黏附培养皿、无血清培养液的生长环境中,对照组细胞球体积较大,中心厚重,周围清亮,折光度强；10mmol/L二甲双胍组细胞球体积较小,球体折光度较弱,细胞球数量少于对照组.RT-PCR检测结果显示,肿瘤干细胞相关基因CD133、β-catenin、ABCG2 mRNA表达水平�Objective to investigate the effects of antidiabetic drug metformin on proliferation and apoptosis in human hepatocellular carcinoma cell line Huh-7 ceils. Methods Huh-7 cells were treated with metformin at different concentrations. Cell viability was determined by MTT assay. Cell apoptosis and CD133＋ expression rate were detected by flow cytometery （FCM）. Expressions of PTEN, Akt, p-Akt, Bcl- 2, Bax proteins in the ceils were measured by Western blot. The effect of metformin on the hepatosphere formation was observed in the serum-free suspension culture. Reverse transcription-polymerase chain reaction （RT-PCR） was used to validate the expression levels of sternness marker genes CD133, β-catenin, and ABCG2 mRNA in the hepatospheres. Results The proliferation of Huh-7 cells was inhibited by mefformin in a dose- and time-dependent manner. The early and late cell apoptosis rates induced by metformin at dose of 10 mmol/L for 48 hrs were （ 22.29 ±0. 8 ） % and （ 13.87 ±1.2 ） % , respectively, and 25 mmoL/L for 48 hrs （ 15.28 ± 2. 1 ） % and （ 25.89 ± 2.3 ） %, respectively. Western blotting results revealed that theexpression of CD133, phosphorylated Akt and the Bcl-2/Bax ratio were downregulated, and PTEN was upregulated in the Huh-7 cells after treated with 25 mmol/L mefformin for 48 hrs. Mefformin inhibited the formation of hepatospheres. Metformin also downregulated the expression of several cancer stem cells （CSCs）-related genes which are involved in the signaling pathways governing the self-renewal, proliferation and differentiation of CSCs in the hepatospheres. Conclusion.s Metformin inhibits the proliferation of human hepatocellular carcinoma Huh-7 cells and enhances their apoptosis in vitro. It may be related to the downregulation of PI3K/Akt signal pathway and selectively targeting CD133 ＋cells.

关 键 词：二甲双胍 肝肿瘤 细胞凋亡 细胞增殖 PI3K Akt CD133 

分 类 号：R735.7[医药卫生—肿瘤]