SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMP-dependent Kinase (AMPK)-Mammalian Target of Rapamycin (mTOR) Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells

Abstract

Mitochondrial oxidative phosphorylation produces most of the energy in aerobic cells by coupling respiration to the production of ATP. Mitochondrial uncouplers, which reduce the proton gradient across the mitochondrial inner membrane, create a futile cycle of nutrient oxidation without generating ATP. Regulation of mitochondrial dysfunction and associated cellular bioenergetics has been recently identified as a promising target for anticancer therapy. Here, we show that SR4 is a novel mitochondrial uncoupler that causes dose-dependent increase in mitochondrial respiration and dissipation of mitochondrial membrane potential in HepG2 hepatocarcinoma cells. These effects were reversed by the recoupling agent 6-ketocholestanol but not cyclosporin A and were nonexistent in mitochondrial DNA-depleted HepG2 cells. In isolated mouse liver mitochondria, SR4 similarly increased oxygen consumption independent of adenine nucleotide translocase and uncoupling proteins, decreased mitochondrial membrane potential, and promoted swelling of valinomycin-treated mitochondria in potassium acetate medium. Mitochondrial uncoupling in HepG2 cells by SR4 results in the reduction of cellular ATP production, increased ROS production, activation of the energy-sensing enzyme AMPK, and inhibition of acetyl-CoA carboxylase and mammalian target of rapamycin signaling pathways, leading to cell cycle arrest and apoptosis. Global analysis of SR4-associated differential gene expression confirms these observations, including significant induction of apoptotic genes and down-regulation of cell cycle, mitochondrial, and oxidative phosphorylation pathway transcripts at 24 h post-treatment. Collectively, our studies demonstrate that the previously reported indirect activation of AMPK and in vitro anticancer properties of SR4 as well as its beneficial effects in both animal xenograft and obese mice models could be a direct consequence of its mitochondrial uncoupling activity. Hepatocellular carcinoma (HCC)2 is the most common and severe form of liver cancer, accounting for about 80 -90% of primary liver cancers and 5% of all human cancers. More than 600,000 deaths are attributed to HCC every year with 2:1 ratio for men versus women (1, 2). HCC is a primary cancer of hepatocytes that most typically occurs in the setting of known risk factors, including cirrhosis and chronic hepatitis B virus or hepatitis C virus infections (2, 3), although recently, several lines of evidence suggest that type 2 diabetes is also an independent risk factor for HCC development (4). HCC is an aggressive tumor and represents a major health problem as its incidence is increasing. Systemic chemotherapies have proven ineffective against advanced HCC, so it typically leads to death within 6 -20 months (5). Hepatocarcinogenesis is a multistep process involving inflammation, hyperplasia, and dysplasia that finally leads to malignant transformation. In recent years, mitochondria have been found to provide a novel targeting site for new anticancer drugs (known as "mitocans") that ca...

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