VDAC Opening Drugs to Induce Mitochondrial Dysfunction and Cell Death

Abstract

Background: Mitochondrial membrane potential (ΔΨ) and reactive oxygen species (ROS) formation depend on metabolite flux into mitochondria through voltage dependent anion channels (VDAC). Free tubulin closes VDAC, and high free tubulin levels decrease ΔΨ in cancer cells. Erastin opens VDAC by antagonizing the inhibitory effect of tubulin. Here, we hypothesized that erastin and erastin-like compounds open VDAC, increase mitochondrial metabolism and ROS formation, and activate c-jun N-terminal kinase (JNK), leading to mitochondrial dysfunction and cell killing. Our AIM was to evaluate the effects of erastin/erastin-like compounds on ΔΨ, ROS, NADH, JNK activation, cell killing and protection by antioxidants in HepG2 cells.Methods: Confocal/multiphoton fluorescence microscopy assessed ΔΨ (tetramethylrhodamine methylester), ROS (chloromethyldichlorofluorescein [cmDCF], MitoSOX Red) and NADH (autofluorescence). JNK was assessed by Western blotting and cell killing by propidium iodide assay.Results: Erastin increased ΔΨ by 46% and NADH by 30% and blocked the depolarizing effect of microtubule destabilizers in HepG2 human hepatoma cells. Increased ΔΨ after erastin/erastin-like compounds induced mitochondrial hyperpolarization that was followed by depolarization. Small molecules X1 and X2, identified in a high-throughput screening, caused a more rapid drop of ΔΨ (<1 h) compared to erastin (3-4 h). Erastin, X1 and X2 also maximally increased cmDCF and MitoSOX Red fluorescence after 1-2 h. Additionally, JNK activation peaked at 60 min. JNK activation and ROS formation preceded mitochondrial depolarization. Cell killing promoted by X1 (93%) and X2 (76%) after 12 h was blocked by the antioxidant N-acetyl cysteine (100 µM).Conclusion: Mitochondrial hyperpolarization caused by VDAC opening drugs causes oxidative stress, which in turn leads to JNK activation, mitochondrial dysfunction and cell death that is prevented by antioxidants. Grants DK073336, DK037034 and 14.Z50.31.0028 to JJL and ACS 13-043-01 to ENM.