Opening of voltage dependent anion channels promotes reactive oxygen species generation, mitochondrial dysfunction and cell death in cancer cells.

Enhancement of aerobic glycolysis and suppression of mitochondrial metabolism characterize the pro-proliferative Warburg phenotype of cancer cells. High free tubulin in cancer cells closes voltage dependent anion channels (VDAC) to decrease mitochondrial membrane potential (ΔΨ), an effect antagonized by erastin, the canonical promotor of ferroptosis. Previously, we identified six compounds (X1-X6) that also block tubulin-dependent mitochondrial depolarization. Here, we hypothesized that VDAC opening after erastin and X1-X6 increases mitochondrial metabolism and reactive oxygen species (ROS) formation, leading to ROS-dependent mitochondrial dysfunction, bioenergetic failure and cell death. Accordingly, we characterized erastin and the two most potent structurally unrelated lead compounds, X1 and X4, on ROS formation, mitochondrial function and cell viability. Erastin, X1 and X4 increased ΔΨ followed closely by an increase in mitochondrial ROS generation within 30-60 min. Subsequently, mitochondria began to depolarize after an hour or longer indicative of mitochondrial dysfunction. N-acetylcysteine (NAC, glutathione precursor and ROS scavenger) and MitoQ (mitochondrially targeted antioxidant) blocked increased ROS formation after X1 and prevented mitochondrial dysfunction. Erastin, X1 and X4 selectively promoted cell killing in HepG2 and Huh7 human hepatocarcinoma cells compared to primary rat hepatocytes. X1 and X4-dependent cell death was blocked by NAC. These results suggest that ferroptosis induced by erastin and our erastin-like lead compounds was caused by VDAC opening, leading to increased ΔΨ, mitochondrial ROS generation and oxidative stress-induced cell death.

Erastin-Like Anti-Warburg Agents Prevent Mitochondrial Depolarization Induced by Free Tubulin and Decrease Lactate Formation in Cancer Cells.

In Warburg metabolism, suppression of mitochondrial metabolism contributes to a low cytosolic ATP/ADP ratio favoring enhanced aerobic glycolysis. Flux of metabolites across the mitochondrial outer membrane occurs through voltage-dependent anion channels (VDAC). In cancer cells, free dimeric tubulin induces VDAC closure and dynamically regulates mitochondrial membrane potential (ΔΨ). Erastin, a small molecule that binds to VDAC, antagonizes the inhibitory effect of tubulin on VDAC and hyperpolarizes mitochondria in intact cells. Here, our aim was to identify novel compounds from the ChemBridge DIVERSet library that block the inhibitory effect of tubulin on ΔΨ using cell-based screening. HCC4006 cells were treated with nocodazole (NCZ) to increase free tubulin and decrease ΔΨ in the presence or absence of library compounds. Tetramethylrhodamine methylester (TMRM) fluorescence was assessed by high-content imaging to determine changes in ΔΨ. Compounds were considered positive if ΔΨ increased in the presence of NCZ. Using confocal microscopy, we identified and validated six lead molecules that antagonized the depolarizing effect of NCZ. Lead compounds and erastin did not promote microtubule stabilization, so changes in ΔΨ were independent of tubulin dynamics. The most potent lead compound also decreased lactate formation. These novel small molecules represent a potential new class of anti-Warburg drugs.

ATP/ADP Turnover and Import of Glycolytic ATP into Mitochondria in Cancer Cells Is Independent of the Adenine Nucleotide Translocator.

Non-proliferating cells oxidize respiratory substrates in mitochondria to generate a protonmotive force (Δp) that drives ATP synthesis. The mitochondrial membrane potential (ΔΨ), a component of Δp, drives release of mitochondrial ATP(4-) in exchange for cytosolic ADP(3-) via the electrogenic adenine nucleotide translocator (ANT) located in the mitochondrial inner membrane, which leads to a high cytosolic ATP/ADP ratio up to >100-fold greater than matrix ATP/ADP. In rat hepatocytes, ANT inhibitors, bongkrekic acid (BA), and carboxyatractyloside (CAT), and the F1FO-ATP synthase inhibitor, oligomycin (OLIG), inhibited ureagenesis-induced respiration. However, in several cancer cell lines, OLIG but not BA and CAT inhibited respiration. In hepatocytes, respiratory inhibition did not collapse ΔΨ until OLIG, BA, or CAT was added. Similarly, in cancer cells OLIG and 2-deoxyglucose, a glycolytic inhibitor, depolarized mitochondria after respiratory inhibition, which showed that mitochondrial hydrolysis of glycolytic ATP maintained ΔΨ in the absence of respiration in all cell types studied. However in cancer cells, BA, CAT, and knockdown of the major ANT isoforms, ANT2 and ANT3, did not collapse ΔΨ after respiratory inhibition. These findings indicated that ANT was not mediating mitochondrial ATP/ADP exchange in cancer cells [corrected]. We propose that suppression of ANT contributes to low cytosolic ATP/ADP, activation of glycolysis, and a Warburg metabolic phenotype in proliferating cells.

Voltage-dependent anion channels modulate mitochondrial metabolism in cancer cells: regulation by free tubulin and erastin.

Respiratory substrates and adenine nucleotides cross the mitochondrial outer membrane through the voltage-dependent anion channel (VDAC), comprising three isoforms--VDAC1, 2, and 3. We characterized the role of individual isoforms in mitochondrial metabolism by HepG2 human hepatoma cells using siRNA. With VDAC3 to the greatest extent, all VDAC isoforms contributed to the maintenance of mitochondrial membrane potential, but only VDAC3 knockdown decreased ATP, ADP, NAD(P)H, and mitochondrial redox state. Cells expressing predominantly VDAC3 were least sensitive to depolarization induced by increased free tubulin. In planar lipid bilayers, free tubulin inhibited VDAC1 and VDAC2 but not VDAC3. Erastin, a compound that interacts with VDAC, blocked and reversed mitochondrial depolarization after microtubule destabilizers in intact cells and antagonized tubulin-induced VDAC blockage in planar bilayers. In conclusion, free tubulin inhibits VDAC1/2 and limits mitochondrial metabolism in HepG2 cells, contributing to the Warburg phenomenon. Reversal of tubulin-VDAC interaction by erastin antagonizes Warburg metabolism and restores oxidative mitochondrial metabolism.