Gao Chun, Fang Long, Zhang Hui, Zhang Wei-Shuo, Li Xiao-Ou, Du Shi-Yu
Department of Gastroenterology, China-Japan Friendship Hospital, Beijing 100029, People's Republic of China.
Department of Gastroenterology, Beijing Tiantan Hospital, Capital Medical University, Beijing 100070, People's Republic of China.
Cancer Manag Res. 2020 Jul 14;12:5803-5811. doi: 10.2147/CMAR.S257966. eCollection 2020.
Metformin may exert the anticancer effect on multiple types of cancers and some potential mechanisms have been suggested. Our study was designed to determine the effect of metformin on the cell autophagy and autophagic flux via the AMPK-mTOR signaling pathway in human hepatocellular carcinoma (HCC) cells.
MHCC97H and HepG2 cell lines were cultured and treated without and with metformin at various concentrations (2, 5, 10 and 20 mM) for 48 h. Then, 10 mM was determined as the optimal concentration and the HCC cells were treated with metformin for 12, 24, 48, and 72 h. MTT assay was used to assess the cell viability and Western blotting was used to determine the expression of proteins (LC3-II, p62, phospho-AMPKα, phospho-mTOR, mTOR, phospho-p70 S6 Kinase, p70 S6 Kinase, PARP1, Caspase-9 and Caspase-3). Autophagy inhibitor 3-methyladenine, EGFP-LC3 and mCherry-GFP-LC3B plasmid transfection were used for further study.
Metformin inhibited significantly the viability of MHCC97H and HepG2 cells in a dose- and time-dependent manner. For the apoptotic properties, activation of Caspase-9 and Caspase-3 and PARP cleavage were not observed after treatment with metformin. MHCC97H cells were transfected with a EGFP-LC3 plasmid and treatment with metformin could lead to the increased level of LC3-II and decreased level of p62. In metformin-induced autophagy, AMPK expression was activated, and the phosphorylation levels of mTOR and p70 S6 Kinase were inhibited. Metformin treatment and mCherry-GFP-LC3B plasmid transfection showed that metformin could induce the autophagic flux. 3-Methyladenine (3-MA) partly abolished this effect.
Metformin could induce the autophagy, autophagic flux, and activate the AMPK-mTOR signaling pathway in human HCC cells.
二甲双胍可能对多种癌症发挥抗癌作用，并且已经提出了一些潜在机制。我们的研究旨在确定二甲双胍通过AMPK-mTOR信号通路对人肝癌（HCC）细胞的细胞自噬和自噬通量的影响。
培养MHCC97H和HepG2细胞系，分别用不同浓度（2、5、10和20 mM）的二甲双胍处理48小时，其中不添加二甲双胍的为对照组。然后，确定10 mM为最佳浓度，并将HCC细胞用二甲双胍处理12、24、48和72小时。采用MTT法评估细胞活力，并用蛋白质印迹法测定蛋白质（LC3-II、p62、磷酸化AMPKα、磷酸化mTOR、mTOR、磷酸化p70 S6激酶、p70 S6激酶、PARP1、Caspase-9和Caspase-3）的表达。使用自噬抑制剂3-甲基腺嘌呤、EGFP-LC3和mCherry-GFP-LC3B质粒转染进行进一步研究。
二甲双胍以剂量和时间依赖性方式显著抑制MHCC97H和HepG2细胞的活力。对于凋亡特性，用二甲双胍处理后未观察到Caspase-9和Caspase-3的激活以及PARP的切割。用EGFP-LC3质粒转染MHCC97H细胞，并用二甲双胍处理可导致LC3-II水平升高和p62水平降低。在二甲双胍诱导的自噬中，AMPK表达被激活，mTOR和p70 S6激酶的磷酸化水平被抑制。二甲双胍处理和mCherry-GFP-LC3B质粒转染表明二甲双胍可诱导自噬通量。3-甲基腺嘌呤（3-MA）部分消除了这种作用。
二甲双胍可诱导人HCC细胞的自噬、自噬通量，并激活AMPK-mTOR信号通路。
