Cancer Management and Research (Jul 2020)
Metformin Induces Autophagy via the AMPK-mTOR Signaling Pathway in Human Hepatocellular Carcinoma Cells
Abstract
Chun Gao,1 Long Fang,1 Hui Zhang,2 Wei-Shuo Zhang,1 Xiao-Ou Li,1 Shi-Yu Du1 1Department of Gastroenterology, China-Japan Friendship Hospital, Beijing 100029, People’s Republic of China; 2Department of Gastroenterology, Beijing Tiantan Hospital, Capital Medical University, Beijing 100070, People’s Republic of ChinaCorrespondence: Shi-Yu DuDepartment of Gastroenterology, China-Japan Friendship Hospital, Ministry of Health, No. 2 Yinghua East Road, Beijing 100029, People’s Republic of ChinaTel/ Fax +86-10-84205503Email [email protected]: 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.Methods: 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.Results: 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.Conclusion: Metformin could induce the autophagy, autophagic flux, and activate the AMPK-mTOR signaling pathway in human HCC cells.Keywords: metformin, hepatocellular carcinoma, autophagy, autophagic flux, AMPK-mTOR signaling pathway
