SR4 Uncouples Mitochondrial Oxidative Phosphorylation,Modulates AMP-dependent Kinase (AMPK)-Mammalian Target of Rapamycin (mTOR) Signaling,and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells |
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| Authors: | James L. Figarola Jyotsana Singhal Joshua D. Tompkins George W. Rogers Charles Warden David Horne Arthur D. Riggs Sanjay Awasthi Sharad S. Singhal |
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| Affiliation: | From the Departments of ‡Diabetes and Metabolic Diseases Research.;‖Molecular Medicine, and ;**Medical Oncology, Beckman Research Institute of the City of Hope, Comprehensive Cancer Center, Duarte, California 91010.;§Seahorse Biosciences, North Billerica, Massachusetts 01862, and ;the ¶Bioinformatics Program, University of Michigan, Ann Arbor, Michigan 48109 |
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| 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. |
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