Antagonists of the Inhibitory Effect of Free Tubulin on VDAC Induce Oxidative Stress and Mitochondrial Dysfunction

Abstract

Bakground: Mitochondrial membrane potential (ΔΨ) and generation of reactive oxygen species (ROS) requires a respiratory chain fueled by the flux of metabolites into mitochondria through voltage dependent anion channels (VDAC). Free tubulin induces reversible blockage of VDAC both in vitro and in cells. Erastin, a small molecule lethal to cancer cells, antagonizes the inhibitory effect of free tubulin on VDAC and upregulates mitochondrial metabolism. Here, we hypothesized that erastin and erastin-like compounds open VDAC, increase mitochondrial metabolism and ROS formation, and activate JNK, which in turn cause mitochondrial dysfunction. Our AIM was to evaluate the effects of erastin/erastin-like compounds on ΔΨ, NADH, ROS and JNK in intact cells. METHODS: ΔΨ was assessed by confocal microscopy of tetramethylrhodamine methylester (TMRM) fluorescence and ROS by chloromethyldichlorofluorescein (cmDCF) and MitoSOX Red fluorescence. Mitochondrial NADH autofluorescence was assessed by multiphoton microscopy. Total and phosphorylated JNK was determined by Western blotting. RESULTS: In lipid bilayers, erastin antagonized tubulin inhibition on VDAC. In HepG2 human hepatoma cells, erastin increased ΔΨ by 46% and NADH by 30%. Mitochondrial hyperpolarization plateaued after 2 h. Subsequently depolarization occurred (3-4 h), suggesting mitochondrial dysfunction. Erastin-like compounds X1 and X2, identified in a high-throughput screening, similarly caused mitochondrial hyperpolarization/depolarization. Erastin also caused increases of DCF and Mitosox Red fluorescence beginning after ∼30 min and reaching a maximum after 2 h. Additionally, erastin activated JNK (maximum pJNK at 60 min). JNK activation and ROS formation both preceded mitochondrial depolarization. Conclusion: Erastin relieves tubulin-dependent inhibition of VDAC conductance, leading to mitochondrial hyperpolarization that increases ROS production with concomitant activation of the stress kinase JNK. These events, in turn, may induce the mitochondrial permeability transition, mitochondrial dysfunction and ultimately cell death.