Oxidative Stress Induced by Vdac Opening in Cancer Cells Depends on Cytosolic Free Tubulin and is Blocked by ROS Scavenging and Suppression of Superoxide Formation by Complex III

Background: Voltage dependent anion channels (VDAC) control flux of metabolites into mitochondria. Free α,β-tubulin decreases VDAC conductance and high free tubulin in cancer cells decreases mitochondrial membrane potential (ΔΨ). Erastin antagonizes the inhibitory effect of tubulin on VDAC. Here, we hypothesized that erastin and erastin-like compounds (X1 and X2) discovered during high-throughput screening of a small molecule library, increase mitochondrial formation of reactive oxygen species (ROS) and activate c-jun N-terminal kinase (JNK), culminating in mitochondrial dysfunction and death of cancer cells. Our AIM was to evaluate the effects of N-acetyl-L-cysteine (NAC), S3QEL-3 and paclitaxel (PTX) on mitochondrial dysfunction induced by erastin/erastin-like compounds. Methods: Confocal fluorescence microscopy assessed ΔΨ (TMRM) and ROS (cmDCF; MitoSOX Red). JNK was assessed by Western blotting and cell killing by propidium iodide fluorometry. Results: In HepG2 and Huh 7 hepatocellular carcinoma cells, erastin and X1 increased ΔΨ and NAD(P)H and activated JNK with maximal phosphorylation within 1-2 h. Initial increases of ΔΨ were followed by mitochondrial depolarization occurring 1-2 h after X1 and X2 and 3-4 h after erastin. X1 did not alter free/polymerized tubulin. The microtubule stabilizer PTX (10 µM) depleted cytosolic free tubulin and prevented depolarization induced by X1. The antioxidant NAC (100 µM) blocked mitochondrial dysfunction and cell death induced by X1. S3QEL-3 (100 µM), a suppressor of superoxide production at site IIIQo and JNK inhibitor SP600125 prevented mitochondrial superoxide formation and mitochondrial dysfunction. Conclusion: Antagonists of VDAC-tubulin interaction promote mitochondrial metabolism and increase mitochondrial ROS formation, which in turn leads to JNK activation, mitochondrial dysfunction and selective death of cancer cells that is prevented by antioxidants and JNK inhibition.