Abstract A88: Openers of voltage dependent anion channels are anti-Warburg agents that enhance mitochondrial metabolism, decrease glycolysis, activate JNK and induce ROS-dependent killing of cancer cells
Authors: DeHart David N. , Gooz Monika , Lemasters John J. , Lemasters John J. , Maldonado Eduardo N. 
Published: May 2016

Background: Warburg metabolism is characterized by enhanced aerobic glycolysis and suppressed mitochondrial metabolism. Voltage dependent anion channels (VDAC) located in the mitochondrial outer membrane control flux of metabolites into mitochondria. Free α,β-tubulin closes VDAC in planar lipid bilayers (Rostovtseva et al., PNAS 105:18746), and high free tubulin in cancer cells decreases mitochondrial membrane potential (ΔΨ) by limiting ingress of respiratory substrates and ATP that supportΔΨ formation (Maldonado et al., Cancer Res. 70:10192). The small molecule erastin opens VDAC by antagonizing the inhibitory effect of tubulin on VDAC (Maldonado et al., JBC 288:11920). Here, we hypothesized that erastin and erastin-like antagonists of VDAC-tubulin interaction increase mitochondrial redox state, decrease glycolysis, 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 erastin/erastin-like compounds on mitochondrialΔΨ, NAD(P)H+ and ROS, lactate generation, JNK activation and cell killing in HepG2 and Huh7 hepatocarcinoma cells.

Methods: Confocal/multiphoton fluorescence microscopy assessedΔΨ (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: In both HepG2 and Huh7, erastin and small molecules X1 and X2 identified in a high-throughput screen increasedΔΨ and NAD(P)H. The three compounds also prevented mitochondrial depolarization by cytosolic high free tubulin induced by nocodazole (microtubule depolymerizing agent). Initial increases ofΔΨ were followed by mitochondrial depolarization occurring 1-2 h after X1 and X2 and 3-4 h after erastin. Lactate generation tested after X1 decreased by 60%. cmDCF and MitoSOX fluorescence increased 30 min after X1 and 60 min after X2 and erastin. The mitochondrially targeted antioxidant MitoQ blocked this increase of ROS. Additionally, erastin caused JNK activation with maximal phosphorylation within 1 h. Both X1 and X2 caused killing of cancer cells (~93% and ~76% respectively), which the antioxidant N-acetylcysteine (100µM) blocked. By contrast, X1 and X2 caused &lt;25% cell death in primary rat hepatocytes.

Conclusion: Antagonists of VDAC-tubulin interaction promote mitochondrial metabolism and inhibit glycolysis. These anti-Warburg drugs also 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. Grants: T32DK083262 to DND; DK073336, DK037034 and 14.Z50.31.0028 to JJL; ACS 13-043-01 and COBRE Pilot Project GM103542 to ENM.

Citation Format: David N. DeHart, Monika Gooz, John J. Lemasters, John J. Lemasters, Eduardo N. Maldonado. Openers of voltage dependent anion channels are anti-Warburg agents that enhance mitochondrial metabolism, decrease glycolysis, activate JNK and induce ROS-dependent killing of cancer cells. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A88.