Therapeutic Concentrations of Statins Hyperpolarize Mitochondria and Inhibit Cell Proliferation Without Promoting Cell Death in Hepatocarcinoma Cells

Abstract

Statins, widely used inhibitors of HMG-CoA reductase to decrease cholesterol, may also decrease the risk of developing liver, breast, and lung cancer. Concentrations of statins (10-100 µM) typically used in vitro are 1,000- to 10,000-fold higher than plasmatic therapeutic concentrations (10-100 nM). In preliminary work, we showed that simvastatin (SIM) and lovastatin at 10 µM increased mitochondrial membrane potential (ΔΨ) and decreased respiration and ATP production in HepG2, Huh7 and HCC4006 cells. We also showed that the bcl-XL inhibitor ABT-737 prevents mitochondrial hyperpolarization induced by the ATP synthase inhibitor, oligomycin. Here, we hypothesize that SIM at nanomolar concentrations increases ΔΨ contributing to decreased cell proliferation and that both mitochondrial hyperpolarization and inhibition of cell proliferation are influenced by bcl-XL. Confocal fluorescence microscopy of tetramethylrhodamine methyl ester assessed ΔΨ. Cholesterol levels were determined using Amplex Red after lipid extraction from whole HepG2 cells and isolated mitochondria. Respiration was assessed using a Seahorse XFe96 Analyzer and cell proliferation by direct counting of live/dead cells. SIM (20 nM to 100 nM) increased mitochondrial ΔΨ in a dose-dependent fashion in HepG2 cells at 24 h. SIM (100 nM for 24 h and 10 µM for 6 h) increased ΔΨ by ∼120 % and 160 % respectively. High ΔΨ promoted by SIM (100 nM and 10 µM) was sustained for at least 72 h. ABT-737 (1 µM) blocked mitochondrial hyperpolarization induced by SIM. SIM at 100 nM and 10 µM decreased cell proliferation by ∼49% and 82% respectively without promoting cell killing. The inhibition of cell proliferation was blocked by pretreatment with ABT-737. Therapeutic concentrations of statins decreased cell proliferation without promoting cell killing possibly by modulation of mitochondrial metabolism mediated by bcl-XL.