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

Abstract

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.

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

“…In a previous high content small molecule screening, X1 and X4 (Fig. 2) were identified as the two most potent structurally unrelated lead compounds that antagonized mitochondrial depolarization caused by high cellular free tubulin in HCC4006 lung carcinoma cells [26]. In HepG2 cells loaded with TMRM, both 10 µM X1 (Fig.…”

“…Recently, we showed that erastin antagonizes the inhibitory effects of tubulin on VDAC. After identifying erastin as the first known pharmacological antagonist of the inhibitory effect of free tubulin on VDAC, we identified by high content cell-based screening several erastin-like small molecules that also appear to prevent VDAC closure by high free cytosolic tubulin [26]. Here, we assess the hypothesis that increased mitochondrial metabolism after VDAC opening leads to enhanced mitochondrial generation of reactive oxygen species (ROS), mitochondrial dysfunction, bioenergetic failure and cell death.…”

VDAC Regulation: A Mitochondrial Target to Stop Cell Proliferation

“…The six lead compounds hyperpolarized mitochondria without causing changes in tubulin polymerization in a dose-dependent fashion. Erastin and the most potent X1 not only increased mitochondrial metabolism but had an anti-Warburg effect evidenced by the decreased lactate release in HepG2 and Huh7 hepatocarcinoma cells and HCC4006 lung carcinoma cell lines (DeHart, Lemasters, & Maldonado, 2017). …”

“…The erastin-like anti-Warburg compound X1 also decreases glycolysis as evidenced by a decrease in lactate release (DeHart et al, 2017). The combination of reversal of Warburg metabolism and oxidative stress by the lead compound caused cell death to human hepatocarcinoma cell lines in culture and to xenografted Huh7 hepatocarcinoma cells (Fig.…”

Voltage-Dependent Anion Channels Influence Cytotoxicity of ME-344, a Therapeutic Isoflavone

“…It has been recently proposed that VDAC operates as a switch for global control of mitochondrial metabolism in cancer cells. Moreover, small molecules that cause VDAC opening increase mitochondrial metabolism and generation of ROS and decrease levels of enhanced glycolysis, acting as anti-Warburg compounds ( Maldonado, 2017 )( DeHart, 2017 )( Fang, 2018 )( Heslop, 2020 )(Maldonado, 2017; DeHart et al, 2018; Maldonado and Fang, 2018; Heslop et al, 2020).…”