Oxidative Stress and JNK Activation cause Mitochondrial Dysfunction and Cell Death in Hepatocarcinoma after VDAC-Tubulin Antagonists

Background: Flux of metabolites into mitochondria sustains membrane potential (ΔΨ) and reactive oxygen species (ROS) formation. Free α,β-tubulin dimers close VDAC in vitro and high free tubulin in cancer cells closes VDAC. The small molecule erastin antagonizes the inhibition of tubulin on VDAC. Here, we hypothesized that VDAC-tubulin antagonists (VTA) open VDAC, increase mitochondrial metabolism, decrease glycolysis and activate c-jun N-terminal kinase (JNK), culminating in mitochondrial dysfunction and death of hepatocarcinoma (HCC) cells. Our AIM was to evaluate the effects of VTAs on mitochondrial ΔΨ, NAD(P)H+ and ROS, lactate generation, JNK activation and cell killing in HepG2 and Huh7 HCC cells.Methods: Confocal/multiphoton fluorescence microscopy assessed ΔΨ (tetramethylrhodamine methylester), ROS (chloromethyldichlorofluorescein [cmDCF]; MitoSOX Red) and NAD(P)H (autofluorescence). JNK was assessed by Western blotting and cell killing by propidium iodide fluorometry.Results: Erastin and small molecules X1-2 identified in a high-throughput screen increased ΔΨ, NAD(P)H and cmDCF and MitoSOX fluorescence. The mitochondrial antioxidant MitoQ blocked ROS increases. Mitochondrial hyperpolarization was followed by depolarization. Additionally, erastin and X1 activated JNK. JNK inhibitors (JNK VIII and SP600125, 30 µM) blocked X1-induced hyperpolarization/depolarization and decreased ROS formation. Lactate generation after X1 decreased by 60%. Both X1 and X2 killed cancer cells (∼93% and ∼76% respectively), which the antioxidant N-acetylcysteine (100 µM) blocked and JNKVIII and SP600125 decreased. By contrast, X1-2 caused <25% cell death in primary rat hepatocytes. X1 (5 mg/kg, i.p. daily) decreased tumor growth in a Huh7 xenograft model.Conclusion: AVTs are anti-Warburg compounds that promote mitochondrial metabolism, inhibit glycolysis and cause mitochondrial generation of ROS, which in turn leads to JNK activation, mitochondrial dysfunction and selective death of cancer cells that is prevented by antioxidants and JNK inhibitors.