Abstract
Metformin (MetF) is widely used worldwide as a first-line therapy for type 2 diabetes. Recently, there has been increasing interest in the pleiotropic effects of MetF, such as its anti-cancer and anti-aging properties. However, the molecular target of the MetF and the detailed mechanism underlying its cell growth inhibitory effects through autophagy induction remain incompletely elucidated. In this study, using an innovative label-free DARTS/LC-MS/MS methods, we discovered that the mitochondrial voltage-dependent anion channel 1 (VDAC1) is a novel binding protein in the autophagy-related cell death activity of high-dose MetF in hepatocellular carcinoma (HCC). Computational alanine scanning mutagenesis revealed that MetF and VDAC1 (Asp9, Glu203) interact electrostatically. MetF disrupts the IP3R-GRP75-VDAC1 complex, which plays a key role in stabilizing mitochondria-associated ER membranes (MAMs) by binding to VDAC1. This disruption leads to increased cytosolic calcium levels, which contributes to autophagy induction. MetF also decreased the AMP/ATP ratio and activated the AMPK pathway. Cells genetically knocked down for VDAC1 mimicked the activity of MetF. In conclusion, this study provides new insights into the involvement of MetF in ionic interactions with VDAC1, contributing to its anticancer effects in HCC. These findings help to elucidate the diverse biological and pharmacological effects of MetF, particularly its influence on autophagy, as well as the potential of MetF as a therapeutic agent for diseases in which VDAC1 is overexpressed.