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.

Cancer metabolism is emerging as a chemotherapeutic target. Enhanced glycolysis and suppression of mitochondrial metabolism characterize the Warburg phenotype in cancer cells. The flux of respiratory substrates, ADP, and Pi into mitochondria and the release of mitochondrial ATP to the cytosol occur through voltage-dependent anion channels (VDACs) located in the mitochondrial outer membrane. Catabolism of respiratory substrates in the Krebs cycle generates NADH and FADH2 that enter the electron transport chain (ETC) to generate a proton motive force that maintains mitochondrial membrane potential (ΔΨ) and is utilized to generate ATP. The ETC is also the major cellular source of mitochondrial reactive oxygen species (ROS). αβ-Tubulin heterodimers decrease VDAC conductance in lipid bilayers. High constitutive levels of cytosolic free tubulin in intact cancer cells close VDAC decreasing mitochondrial ΔΨ and mitochondrial metabolism. The VDAC–tubulin interaction regulates VDAC opening and globally controls mitochondrial metabolism, ROS formation, and the intracellular flow of energy. Erastin, a VDAC-binding molecule lethal to some cancer cell types, and erastin-like compounds identified in a high-throughput screening antagonize the inhibitory effect of tubulin on VDAC. Reversal of tubulin inhibition of VDAC increases VDAC conductance and the flux of metabolites into and out of mitochondria. VDAC opening promotes a higher mitochondrial ΔΨ and a global increase in mitochondrial metabolism leading to high cytosolic ATP/ADP ratios that inhibit glycolysis. VDAC opening also increases ROS production causing oxidative stress that, in turn, leads to mitochondrial dysfunction, bioenergetic failure, and cell death. In summary, antagonism of the VDAC–tubulin interaction promotes cell death by a “double-hit model” characterized by reversion of the proproliferative Warburg phenotype (anti-Warburg) and promotion of oxidative stress.

Opening of voltage dependent anion channels (VDAC) by the erastin-like compound X1 and the multikinase inhibitor sorafenib promotes oxidative stress and mitochondrial dysfunction in hepatocarcinoma cells. Here, we hypothesized that X1 and sorafenib induce mitochondrial dysfunction by increasing ROS formation and activating c-Jun N-terminal kinases (JNKs), leading to translocation of activated JNK to mitochondria. Both X1 and sorafenib increased production of reactive oxygen species (ROS) and activated JNK. X1 and sorafenib caused a drop in mitochondrial membrane potential (ΔΨ), a readout of mitochondrial metabolism, after 60 min. Mitochondrial depolarization after X1 and sorafenib occurred in parallel with JNK activation, increased superoxide (O 2•− ) production, decreased basal and oligomycin sensitive respiration, and decreased maximal respiratory capacity. Increased production of O 2 •− after X1 or sorafenib was abrogated by JNK inhibition and antioxidants. S3QEL specific inhibitor of site IIIQ o, at Complex III prevented depolarization induced by X1. JNK inhibition by JNK inhibitors VIII and SP600125 also prevented mitochondrial depolarization. After X1, activated JNK translocated to mitochondria as assessed by proximity ligation assays.

Azoles are antifungals that are widely utilized due to relatively low toxicity and cost of treatment. One of their drawbacks, however, is that azoles are primarily cytostatic, leaving fungal cells capable of developing drug resistance. The human pathogen Cryptococcus neoformans acquires resistance to the azole drug fluconazole (FLC) through the development of aneuploidy, leading to elevated expression of key resistance genes, a mechanism that is also common for Candida albicans (K. J. Kwon-Chung and Y. C. Chang, PLoS Pathog 8:e1003022, 2012, https://doi.org/10.1371/journal.ppat.1003022; J. Morschhäuser, J Microbiol 54:192–201, 2016, https://doi.org/10.1007/s12275-016-5628-4). However, the exact ways in which FLC contributes to increased resistance in either of these important fungal pathogens remain unclear. Here we found that FLC treatment leads to an increase in DNA content in C. neoformans through multiple mechanisms, potentially increasing the size of a pool of cells from which aneuploids with increased resistance are selected. This study demonstrated the importance of FLC’s inhibitory effects on growth and cytokinesis in the generation of cell populations with decreased sensitivity to the drug.

Pathogenic basidiomycetous yeast, Cryptococcus neoformans, causes fatal meningitis in immunocompromised individuals. Fluconazole (FLC) is a fungistatic drug commonly administered to treat cryptococcosis. Unfortunately, FLC-resistant strains characterized by various degree of chromosomal instability were isolated from clinical patients. Importantly, the underlying mechanisms that lead to chromosomal instability in FLC-treated C. neoformans remain elusive. Previous studies in fungal and mammalian cells link chromosomal instability to the reactive oxygen species (ROS). This study provides the evidence that exposure of C. neoformans to FLC induces accumulation of intracellular ROS, which correlates with plasma membrane damage. FLC caused transcription changes of oxidative stress related genes encoding superoxide dismutase (SOD1), catalase (CAT3), and thioredoxin reductase (TRR1). Strikingly, FLC contributed to an increase of the DNA damage in vitro, when complexed with iron or copper in the presence of hydrogen peroxide. Strains with isogenic deletion of copper response protein metallothionein were more susceptible to FLC. Addition of ascorbic acid (AA), an anti-oxidant at 10 mM, reduced the inhibitory effects of FLC. Consistent with potential effects of FLC on DNA integrity and chromosomal segregation, FLC treatment led to elevated transcription of RAD54 and repression of cohesin-encoding gene SCC1. We propose that FLC forms complexes with metals and contributes to elevated ROS, which may lead to chromosomal instability in C. neoformans.

Statins, widely used to treat hypercholesterolemia, inhibit the 3‐hydroxy‐3‐methylglutary1‐coenzyme A reductase, the rate‐limiting enzyme of de novo cholesterol (Chol) synthesis. Statins have been also reported to slow tumor progression. In cancer cells, ATP is generated both by glycolysis and oxidative phosphorylation. Mitochondrial membrane potential (ΔΨ), a readout of mitochondrial metabolism, is sustained by the oxidation of respiratory substrates in the Krebs cycle to generate NADH and flavin adenine dinucleotide, which are further oxidized by the respiratory chain. Here, we studied the short‐term effects of statins (3–24 h) on mitochondrial metabolism on cancer cells. Lovastatin (LOV) and simvastatin (SIM) increased in HepG2 and Huh7 human hepatocarcinoma cells and HCC4006 human lung adenocarcinoma cells. Mitochondrial hyperpolarization after LOV and SIM was dose and time dependent. Maximal increase in ΔΨ occurred at 10 µM and 24 h for both statins. The structurally unrelated atorvastatin also hyperpolarized mitochondria in HepG2 cells. Cellular and mitochondrial Chol remained unchanged after SIM. Both LOV and SIM decreased basal respiration, ATP‐linked respiration, and ATP production. LOV and SIM did not change the rate of lactic acid production. In summary, statins modulate mitochondrial metabolism in cancer cells independently of the Chol content in cellular membranes without affecting glycolysis.—Christie, C. F., Fang, D., Hunt, E. G., Morris, M. E., Rovini, A., Heslop, K. A., Beeson, G. C., Beeson, C. C., Maldonado, E. N. Statin‐dependent modulation of mitochondrial metabolism in cancer cells is independent of cholesterol content. FASEB J. 33, 8186–8201 (2019). http://www.fasebj.org

Mitochondrial membrane potential (ΔΨm) is a global indicator of mitochondrial function. Previous reports on heterogeneity of ΔΨm were qualitative or semiquantitative. Here, we quantified intercellular differences in ΔΨm in unsynchronized human cancer cells, cells synchronized in G1, S, and G2, and human fibroblasts. We assessed ΔΨm using a two-pronged microscopy approach to measure relative fluorescence of tetramethylrhodamine methyl ester (TMRM) and absolute values of ΔΨm. We showed that ΔΨm is more heterogeneous in cancer cells compared to fibroblasts, and it is maintained throughout the cell cycle. The effect of chemical inhibition of the respiratory chain and ATP synthesis differed between basal, low and high ΔΨm cells. Overall, our results showed that intercellular heterogeneity of ΔΨm is mainly modulated by intramitochondrial factors, it is independent of the ΔΨm indicator and it is not correlated with intercellular heterogeneity of plasma membrane potential or the phases of the cell cycle.

The concept of osmotic stability during renal replacement therapy has received limited attention thus far. We report an illustrative case of a previously healthy 22 year old male presenting after prolonged ventricular fibrillation with 75 minutes of resuscitative efforts before regaining spontaneous perfusing rhythm. Central nervous system protecting hypothermia protocol and veno-arterious (VA) extracorporeal membrane oxygenator (ECMO) therapy were initiated at hospital admission due to refractory hypoxemia. Cardiovascular imaging procedures described global hypokinesis. Due to the combination of anuria, mixed acidosis and hemodynamic instability, we started continuous renal replacement therapy (CRRT) in continuous veno-venous hemodiafiltration functionality with added hypertonic saline solution (HTS) protocol, calculated to stabilize his serum sodium between 148 and 150 mEq/L. Serum osmolality also ranged between 321 and 317 mOsm/kg thereafter. Course was complicated by an acute right leg ischemia distal to VA ECMO cannula placement, requiring salvage therapy with cryoamputation. Vasoactive medication requirement and hemodynamics improved after the addition of intravenous (IV) hydrocortisone. Brain magnetic resonance imaging (MRI) 22 days post-arrest showed signals of limited hypoxic injury. He left the hospital in stable condition with limited neurologic sequelae. Therefore, the use of HTS during CRRT is a viable way to address potential or manifest cerebral edema and reduce the degree of cerebral injury.