Mitochondrial ΔΨ Research Articles

FDA-approved disulfiram induces ferroptosis via alteration of redox balance and lipid peroxidation and overcomes carfilzomib resistance in multiple myeloma

Multiple myeloma (MM), a malignant plasma cell disorder, remains incurable due to inevitable chemo-resistance development, including carfilzomib (CFZ) leading to relapse and poor patient outcomes. Therefore, new treatment strategies, including using FDA-approved alcohol deterrent disulfiram (DSF) are under investigation. The present study investigated the effect of DSF on ferroptosis and CFZ resistance in MM cells. Our findings indicate that DSF increases the production of cytosolic and mitochondrial reactive oxygen species, and causes a loss of mitochondrial Δψ and an elevation in lipid peroxidation in MM cells. DSF treatment in MM cell lines led to the significant downregulation of ferroptotic genes, including glutathione peroxidase 4. Moreover, ferroptosis inhibitor liproxstatin-1rescued DSF-induced ferroptosis by promoting glutathione peroxidase 4 upregulation. DSF alone overcomes CFZ resistance through lipid peroxidation elevation and acts synergistically with CFZ in CFZ-resistant MM cell lines. Our results suggest that DSF is a promising anti-myeloma agent for overcoming CFZ resistance in MM through ferroptosisinduction.

New advances in the protective mechanisms of acidic pH after ischemia: Participation of NO

BackgroundIt has been previously demonstrated that the maintenance of ischemic acidic pH or the delay of intracellular pH recovery at the onset of reperfusion decreases ischemic-induced cardiomyocyte death. ObjectiveTo examine the role played by nitric oxide synthase (NOS)/NO-dependent pathways in the effects of acidic reperfusion in a regional ischemia model. MethodsIsolated rat hearts perfused by Langendorff technique were submitted to 40 min of left coronary artery occlusion followed by 60 min of reperfusion (IC). A group of hearts received an acid solution (pH = 6.4) during the first 2 min of reperfusion (AR) in absence or in presence of l-NAME (NOS inhibitor). Infarct size (IS) and myocardial function were determined. In cardiac homogenates, the expression of P-Akt, P-endothelial and inducible isoforms of NOS (P-eNOS and iNOS) and the level of 3-nitrotyrosine were measured. In isolated cardiomyocytes, the intracellular NO production was assessed by confocal microscopy, under control and acidic conditions. Mitochondrial swelling after Ca2+ addition and mitochondrial membrane potential (Δψ) were also determined under control and acidosis. ResultsAR decreased IS, improved postischemic myocardial function recovery, increased P-Akt and P-eNOS, and decreased iNOS and 3-nitrotyrosine. NO production increased while mitochondrial swelling and Δψ decreased in acidic conditions. l-NAME prevented the beneficial effects of AR. ConclusionsOur data strongly supports that a brief acidic reperfusion protects the myocardium against the ischemia-reperfusion injury through eNOS/NO-dependent pathways.

Altered Hepatic Mitochondrial Bioenergetics and Oxidant Emission with Ischemia and Reperfusion

Rationale: Ischemia-reperfusion injury (IRI) is inevitable in liver transplantation (LT) which can be mitigated by normothermic machine perfusion (NMP) before LT. The current NMP liver viability criteria for LT mainly rely on bile production and perfusate lactate levels, factors that can also be affected by external variables such as NMP conditions and perfusate additives. Incorporating mitochondrial bioenergetic data could improve the predictive value of current NMP viability criteria for LT. Thus, the goal of this study was to evaluate mitochondrial oxygen consumption rate (OCR), membrane potential (Δψ), and H2O2 oxidant emission in control livers and in livers with IRI and to explore underlying mechanisms responsible for any observed differences. Methods: Healthy control livers and livers exposed to 60 minutes warm ischemia time (WIT) through in situ clamping of the portal vein and hepatic artery followed by 60 minutes reperfusion (IRI) were harvested from adult male Sprague-Dawley rats. Mitochondria from control and IRI livers were isolated using established protocols and assessed for their bioenergetics responses. Three different substrate combinations, namely, pyruvate+malate (PM), glutamate+malate (GM), and succinate were used, followed by addition(s) of ADP and the uncoupler FCCP. Two different ADP addition protocols were designed to determine how different substrates influence mitochondrial OCR, Δψ, and H2O2 emission during oxidative phosphorylation (OxPhos) between control and IRI conditions. In one protocol, a single saturated dose of ADP addition, and in the other, sequentially increasing doses of ADP additions were made following substrate addition. The OCR and Δψ were measured simultaneously using a dual chamber Oroboros Oxygraph-2k Instrument coupled to a fluorometer using the TMRM dye. The H2O2 emission was measured spectrofluorometrically using the amplex red and horseradish peroxidase assay. Results: The measured data show that the kinetics and effciency of OxPhos defining mitochondrial OCR and Δψ responses in the liver are negatively affected by IRI, characterized by enhanced H2O2 emission, in a substrate-dependent manner. Interestingly, the respiratory rates are higher when GM and succinate are utilized as substrates, whereas the use of PM shows comparatively lower respiration in both conditions. Control mitochondria exhibited higher respiratory rates than IRI mitochondria irrespective of the substrate utilized. The ADP-induced state 3 OCR in IRI mitochondria was 50% lower than that in control mitochondria resulting in doubling the duration of state 3 OCR. Similar differences were observed in Δψ for both ADP addition protocols. Compromised mitochondrial bioenergetics during IRI was observed using both single and sequentially increasing doses of ADP and was concomitant with a higher rate of H2O2 emission. Conclusion: This study provided novel quantitative data demonstrating the substrate specific changes in mitochondrial bioenergetics in hepatic IRI, which can be used to improve the NMP viability criteria for LT. The obtained bioenergetics and H2O2 emission data indicate that, during IRI, mitochondrial function is highly affected which may critically impact energy dependent metabolism and lactate/pyruvate ratio. NIH R01-HL151587. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sex-Specific Mitochondrial Bioenergetics Differences in Heart and Kidney Cortex

Rationale: Mitochondrial bioenergetics of different tissues have been studied widely, but their sex-specific differences have not been systematically studied. The goal of this study was therefore to investigate the sex-specific differences in mitochondrial respiration (oxygen consumption rate; OCR) and membrane potential (Δψ) in the heart and kidney cortex and to explore underlying mechanisms responsible for any observed differences. Methods: Mitochondria were isolated from adult male and female Sprague-Dawley rat heart and kidney cortex and were used to study their bioenergetics responses. Three different substrate combinations, namely, pyruvate+malate (PM), glutamate+malate (GM), and succinate in the presence of complex I inhibitor rotenone (SR) were used. This was followed by two different ADP addition protocols to determine how different substrates influence mitochondrial OCR and Δψ to ADP perturbations during oxidative phosphorylation (OxPhos) in male and female heart and kidney cortex. In one, a single saturated dose of ADP, while in the other, sequentially increasing doses of ADP were added to mitochondria in the presence of different substrates. Mitochondrial OCR was measured using a dual chamber Oroboros Oxygraph-2k Instrument and Δψ was measured using a spectrofluorometer and rhodamine-123 dye. From these measurements, OCR and Δψ alterations under different respiratory states and as functions of added ADP concentration were determined for different substrates and for both male and female rats. Results: Male and female mitochondria exhibited distinct respiratory patterns in the kidney cortex and heart with different substrates. In the kidney cortex, male mitochondria showed significantly higher OCR than female mitochondria when fueled with PM or SR. However, no significant sex differences in the OCR were observed when GM were used as substrates. In contrast, heart mitochondria exhibited negligible sex differences in the OCR for PM and SR, but significant differences emerged with GM, where female mitochondria displayed higher OCR than their male counterparts. Interestingly, sex-specific variations of Δψ in ADP-stimulated respiration were not apparent for either tissue or substrate when a single saturated dose of ADP was used. Instead, a trend similar to the OCR emerged when ADP was administered in gradually increasing concentrations. Conclusion: Heart and kidney cortex mitochondrial Δψ for males and females did not differ conclusively with the two ADP addition protocols. However, there were significant differences in the OCR of mitochondria from the heart and kidney cortex of males and females, depending on the respiratory substrate utilized. Males were found to have enhanced OCR in the kidney cortex, whereas females exhibited higher OCR in the heart. These observed sex-specific differences in the OCR could be attributed to differential regulation of mitochondrial metabolic pathways by respiratory substrates and sex hormones. NIH R01-HL151587. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Ameliorative Role of Mitochondrial Therapy in Cognitive Function of Vascular Dementia Mice.

Mitochondrial dysfunction plays a vital role in the progression of vascular dementia (VaD). We hypothesized that transfer of exogenous mitochondria might be a beneficial strategy for VaD treatment. The study was aimed to investigate the role of mitochondrial therapy in cognitive function of VaD. The activity and integrity of isolated mitochondria were detected using MitoTracker and Janus Green B staining assays. After VaD mice were intravenously injected with exogenous mitochondria, Morris water maze and passive avoidance tests were used to detect cognitive function of VaD mice. Haematoxylin and eosin, Nissl, TUNEL, and Golgi staining assays were utilized to measure neuronal and synaptic injury in the hippocampus of VaD mice. Detection kits were performed to detect mitochondrial membrane potential (ΔΨ), SOD activity and the levels of ATP, ROS, and MDA in the brains of VaD mice. The results showed that isolated mitochondria were intact and active. Mitochondrial therapy could ameliorate cognitive performance of VaD mice. Additionally, mitochondrial administration could attenuate hippocampal neuronal and synaptic injury, improve mitochondrial ΔΨ, ATP level and SOD activity, and reduce ROS and MDA levels in the brains of VaD mice. The study reports profitable effect of mitochondrial therapy against cognitive impairment of VaD, making mitochondrial treatment become a promising therapeutic strategy for VaD.

Effect of Fusidic Acid and Some Nitrogen-Containing Derivatives on Liposomal and Mitochondrial Membranes.

The paper assesses the membranotropic action of the natural antibiotic fusidic acid (FA) and its derivatives. It was found that a FA analogue with ethylenediamine moiety (derivative 2), in contrast to native FA and 3,11-dioxime analogue (derivative 1), is able to increase the mobility of the lipid bilayer in the zone of lipid headgroups, as well as to induce permeabilization of lecithin liposome membranes. A similar effect of derivative 2 is also observed in the case of rat liver mitochondrial membranes. We noted a decrease in the microviscosity of the mitochondrial membrane and nonspecific permeabilization of organelle membranes in the presence of this agent, which was accompanied by a decrease in mitochondrial Δψ and OXPHOS efficiency. This led to a reduction in mitochondrial calcium retention capacity. The derivatives also reduced the production of H2O2 by mitochondria. The paper considers the relationship between the structure of the tested compounds and the observed effects.

Fatty acid oxidation protects cancer cells from apoptosis by increasing mitochondrial membrane lipids.

Overcoming resistance to chemotherapies remains a major unmet need for cancers, such as triple-negative breast cancer (TNBC). Therefore, mechanistic studies to provide insight for drug development are urgently needed to overcome TNBC therapy resistance. Recently, an important role of fatty acid β-oxidation (FAO) in chemoresistance has been shown. But how FAO might mitigate tumor cell apoptosis by chemotherapy is unclear. Here, we show that elevated FAO activates STAT3 by acetylation via elevated acetyl-coenzyme A (CoA). Acetylated STAT3 upregulates expression of long-chain acyl-CoA synthetase 4 (ACSL4), resulting in increased phospholipid synthesis. Elevating phospholipids in mitochondrial membranes leads to heightened mitochondrial integrity, which in turn overcomes chemotherapy-induced tumor cell apoptosis. Conversely, in both cultured tumor cells and xenograft tumors, enhanced cancer cell apoptosis by inhibiting ASCL4 or specifically targeting acetylated-STAT3 is associated with a reduction in phospholipids within mitochondrial membranes. This study demonstrates a critical mechanism underlying tumor cell chemoresistance.

Pharmaceutical Drug Metformin and MCL1 Inhibitor S63845 Exhibit Anticancer Activity in Myeloid Leukemia Cells via Redox Remodeling.

Metabolic landscape and sensitivity to apoptosis induction play a crucial role in acute myeloid leukemia (AML) resistance. Therefore, we investigated the effect of metformin, a medication that also acts as an inhibitor of oxidative phosphorylation (OXPHOS), and MCL-1 inhibitor S63845 in AML cell lines NB4, KG1 and chemoresistant KG1A cells. The impact of compounds was evaluated using fluorescence-based metabolic flux analysis, assessment of mitochondrial Δψ and cellular ROS, trypan blue exclusion, Annexin V-PI and XTT tests for cell death and cytotoxicity estimations, also RT-qPCR and Western blot for gene and protein expression. Treatment with metformin resulted in significant downregulation of OXPHOS; however, increase in glycolysis was observed in NB4 and KG1A cells. In contrast, treatment with S63845 slightly increased the rate of OXPHOS in KG1 and KG1A cells, although it profoundly diminished the rate of glycolysis. Generally, combined treatment had stronger inhibitory effects on cellular metabolism and ATP levels. Furthermore, results revealed that treatment with metformin, S63845 and their combinations induced apoptosis in AML cells. In addition, level of apoptotic cell death correlated with cellular ROS induction, as well as with downregulation of tumor suppressor protein MYC. In summary, we show that modulation of redox-stress could have a potential anticancer activity in AML cells.

Recent studies on NCLX in health and diseases

The mitochondria is a major hub for cellular Ca 2+ signaling. The identification of MCU, the mitochondrial Ca 2+ influx mediator, and the mitochondrial Ca 2+ extruder NCLX, were major breakthroughs in this field. Their identification provided novel molecular tools and animal models to interrogate their physiological function and mode of regulation. Here we will focus on the mitochondrial Na + / Ca 2+ exchanger NCLX that plays a dual role in mitochondrial Na + and Ca 2+ signaling. We will discuss recent advances in NCLX mods of regulation by kinases and mitochondrial ΔΨ. We will also focus on the heterogeneity of its expression in distinct mitochondrial populations and the pathophysiological implication of its excessive degradation. We will describe the ongoing debate on the stoichiometry of Na + to Ca 2+ transport, mediated by NCLX, and its physiological implication. We will focus on the major effects of mitochondrial Na + signaling by NCLX on mitochondrial metabolism in health; and finally, we will discuss the role NCLX plays in a wide range of health disorders, from heart failure and cancer to Parkinson and Alzheimer disease, making it a prime candidate for therapeutic targeting.

Vitamin C activates pyruvate dehydrogenase (PDH) targeting the mitochondrial tricarboxylic acid (TCA) cycle in hypoxic KRAS mutant colon cancer.

Background: In hypoxic tumors, positive feedback between oncogenic KRAS and HIF-1α involves impressive metabolic changes correlating with drug resistance and poor prognosis in colorectal cancer. Up to date, designed KRAS-targeting molecules do not show clear benefits in patient overall survival (POS) so pharmacological modulation of aberrant tricarboxylic acid (TCA) cycle in hypoxic cancer has been proposed as a metabolic vulnerability of KRAS-driven tumors.Methods: Annexin V-FITC and cell viability assays were carried out in order to verify vitamin C citotoxicity in KRAS mutant SW480 and DLD1 as well as in Immortalized Human Colonic Epithelial Cells (HCEC). HIF1a expression and activity were determined by western blot and functional analysis assays. HIF1a direct targets GLUT1 and PDK1 expression was checked using western blot and qRT-PCR. Inmunohistochemical assays were perfomed in tumors derived from murine xenografts in order to validate previous observations in vivo. Vitamin C dependent PDH expression and activity modulation were detected by western blot and colorimetric activity assays. Acetyl-Coa levels and citrate synthase activity were assessed using colorimetric/fluorometric activity assays. Mitochondrial membrane potential (Δψ) and cell ATP levels were assayed using fluorometric and luminescent test.Results: PDK-1 in KRAS mutant CRC cells and murine xenografts was downregulated using pharmacological doses of vitamin C through the proline hydroxylation (Pro402) of the Hypoxia inducible factor-1(HIF-1)α, correlating with decreased expression of the glucose transporter 1 (GLUT-1) in both models. Vitamin C induced remarkable ATP depletion, rapid mitochondrial Δψ dissipation and diminished pyruvate dehydrogenase E1-α phosphorylation at Serine 293, then boosting PDH and citrate synthase activity.Conclusion: We report a striking and previously non reported role of vitamin C in the regulation of the pyruvate dehydrogenase (PDH) activity, then modulating the TCA cycle and mitochondrial metabolism in KRAS mutant colon cancer. Potential impact of vitamin C in the clinical management of anti-EGFR chemoresistant colorectal neoplasias should be further considered.

Therapeutic Concentrations of Statins Hyperpolarize Mitochondria and Inhibit Cell Proliferation Without Promoting Cell Death in Hepatocarcinoma Cells

Statins, widely used inhibitors of HMG-CoA reductase to decrease cholesterol, may also decrease the risk of developing liver, breast, and lung cancer. Concentrations of statins (10-100 µM) typically used in vitro are 1,000- to 10,000-fold higher than plasmatic therapeutic concentrations (10-100 nM). In preliminary work, we showed that simvastatin (SIM) and lovastatin at 10 µM increased mitochondrial membrane potential (ΔΨ) and decreased respiration and ATP production in HepG2, Huh7 and HCC4006 cells. We also showed that the bcl-XL inhibitor ABT-737 prevents mitochondrial hyperpolarization induced by the ATP synthase inhibitor, oligomycin. Here, we hypothesize that SIM at nanomolar concentrations increases ΔΨ contributing to decreased cell proliferation and that both mitochondrial hyperpolarization and inhibition of cell proliferation are influenced by bcl-XL. Confocal fluorescence microscopy of tetramethylrhodamine methyl ester assessed ΔΨ. Cholesterol levels were determined using Amplex Red after lipid extraction from whole HepG2 cells and isolated mitochondria. Respiration was assessed using a Seahorse XFe96 Analyzer and cell proliferation by direct counting of live/dead cells. SIM (20 nM to 100 nM) increased mitochondrial ΔΨ in a dose-dependent fashion in HepG2 cells at 24 h. SIM (100 nM for 24 h and 10 µM for 6 h) increased ΔΨ by ∼120 % and 160 % respectively. High ΔΨ promoted by SIM (100 nM and 10 µM) was sustained for at least 72 h. ABT-737 (1 µM) blocked mitochondrial hyperpolarization induced by SIM. SIM at 100 nM and 10 µM decreased cell proliferation by ∼49% and 82% respectively without promoting cell killing. The inhibition of cell proliferation was blocked by pretreatment with ABT-737. Therapeutic concentrations of statins decreased cell proliferation without promoting cell killing possibly by modulation of mitochondrial metabolism mediated by bcl-XL.

Mitochondrial Subtype Identification and Characterization.

Healthy, functional mitochondria are central to many cellular and physiological phenomena, including aging, metabolism, and stress resistance. A key feature of healthy mitochondria is a high membrane potential (Δψ) or charge differential (i.e., proton gradient) between the matrix and inner mitochondrial membrane. Mitochondrial Δψ has been extensively characterized via flow cytometry of intact cells, which measures the average membrane potential within a cell. However, the characteristics of individual mitochondria differ dramatically even within a single cell, and thus interrogation of mitochondrial features at the organelle level is necessary to better understand and accurately measure heterogeneity. Here we describe a new flow cytometric methodology that enables the quantification and classification of mitochondrial subtypes (via their Δψ, size, and substructure) using the small animal model C. elegans. Future application of this methodology should allow research to discern the bioenergetic and mitochondrial component in a number of human disease and aging models, including, C. elegans, cultured cells, small animal models, and human biopsy samples. © 2018 by John Wiley & Sons, Inc.

Multidimensional mitochondrial energetics: Application to the study of electron leak and hydrogen peroxide metabolism.

Oxygen consumption is a valuable tool to link with measurements of mitochondrial electron leakage to form reactive oxygen species (ROS), which in mitochondria is predominantly superoxide and H2O2. However, oxygen consumption may respond differently to changes in conditions than superoxide/H2O2 production does, complicating the use of respiration as a sole indicator of mitochondrial energetics. The same equipment that is valuable for fluorescent monitoring of H2O2 efflux provides a straightforward means of estimating membrane potential (ΔΨ), thereby an alternative metric of mitochondrial energetics is readily added to complement studies on the link between mitochondrial energetics and electron leak. By combining multiple aspects of mitochondrial energetics a far more detailed picture emerges on why changes in superoxide/H2O2 formation arise with reduced dependence on assumptions. Here we illustrate integration of experimental methods via demonstration of linkages between mitochondrial ΔΨ, oxygen consumption and superoxide/H2O2 formation (the latter estimated by H2O2 efflux). In doing so we also expand on some pitfalls and cautions for these experimental manipulations of isolated mitochondria and, using these techniques, we raise the possibility that the oxygen affinity for respiration may be higher than the affinity for some sites of electron leak.

SPARC overexpression suppresses radiation-induced HSP27 and induces the collapse of mitochondrial Δψ in neuroblastoma cells.

Neuroblastoma is the cause of >15% of cancer-associated mortality in children in the USA. Despite aggressive treatment regimens, the long-term survival rate for these children remains at <40%. The current study demonstrates that secreted protein acidic and rich in cysteine (SPARC) suppresses radiation-induced expression of heat shock protein 27 (HSP27) in vivo and suppresses mitochondrial membrane potential (Δψ) in neuroblastoma cells. In the present study, the overexpression of SPARC in SK-N-BE(2) and NB1691 neuroblastoma cell lines suppresses radiation-induced G2M cell cycle arrest, proliferation, HSP27 expression (in vitro and in vivo) and induces the collapse of the mitochondrial Δψ. Gene ontology analysis demonstrated that the overexpression of SPARC combined with irradiation, induces the expression of dissimilar molecular function genes in SK-N-BE(2) and NB1691 cells, providing evidence of a dissimilar response signaling pathway. These results demonstrate that overexpression of SPARC suppresses radiation-induced HSP27 expression in neuroblastoma cells and the combination of SPARC and radiation induces the expression of protein 21, but suppresses neuroblastoma tumor density in in vivo mouse models. SPARC also induces mitochondrial Δψ collapse in SK-N-BE(2) and NB1691 neuroblastoma cells.

VDAC-Tubulin, an Anti-Warburg Pro-Oxidant Switch.

Aerobic enhanced glycolysis characterizes the Warburg phenotype. In cancer cells, suppression of mitochondrial metabolism contributes to maintain a low ATP/ADP ratio that favors glycolysis. We propose that the voltage-dependent anion channel (VDAC) located in the mitochondrial outer membrane is a metabolic link between glycolysis and oxidative phosphorylation in the Warburg phenotype. Most metabolites including respiratory substrates, ADP, and Pi enter mitochondria only through VDAC. Oxidation of respiratory substrates in the Krebs cycle generates NADH that enters the electron transport chain (ETC) to generate a proton motive force utilized to generate ATP and to maintain mitochondrial membrane potential (ΔΨ). The ETC is also the major source of mitochondrial reactive oxygen species (ROS) formation. Dimeric α-β tubulin decreases conductance of VDAC inserted in lipid bilayers, and high free tubulin in cancer cells by closing VDAC, limits the ingress of respiratory substrates and ATP decreasing mitochondrial ΔΨ. VDAC opening regulated by free tubulin operates as a “master key” that “seal–unseal” mitochondria to modulate mitochondrial metabolism, ROS formation, and the intracellular flow of energy. Erastin, a small molecule that binds to VDAC and kills cancer cells, and erastin-like compounds antagonize the inhibitory effect of tubulin on VDAC. Blockage of the VDAC–tubulin switch increases mitochondrial metabolism leading to decreased glycolysis and oxidative stress that promotes mitochondrial dysfunction, bioenergetic failure, and cell death. In summary, VDAC opening-dependent cell death follows a “metabolic double-hit model” characterized by oxidative stress and reversion of the pro-proliferative Warburg phenotype.

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.

Moderate dependence of reactive oxygen species production on membrane potential in avian muscle mitochondria oxidizing glycerol 3-phosphate.

Mitochondria are a major source of reactive oxygen species production in cells, and the production level is sensitive to the magnitude of the membrane potential (ΔΨ). The present study investigated the level of superoxide production in mitochondria oxidizing glycerol 3-phosphate (GP) and its dependence on ΔΨ in isolated avian muscle mitochondria. The levels of superoxide produced in mitochondria oxidizing GP were lower than those obtained with succinate and were similar to those obtained with NADH-linked substrates (glutamate/malate/pyruvate). The dependence of superoxide production on ΔΨ in mitochondria oxidizing GP was lower than that of mitochondria oxidizing succinate, and a weak dependence of GP-supported superoxide production on ΔΨ was observed in the presence of NADH-linked substrates or succinate. These results suggest that the levels of superoxide generated in response to GP are quantitatively low, but they are unsusceptible to changes in ΔΨ in avian muscle mitochondria.

Effects of trichlorotelluro-dypnones on mitochondrial bioenergetics and their relationship to the reactivity with protein thiols.

The effect of four trichlorotelluro-dypnones, named compounds 1, 2, 3, and 4, on the bioenergetics of isolated rat liver mitochondria (RLM) and cells was investigated. In a dose-dependent manner, the studied organotelluranes promoted Ca(2+)-dependent mitochondrial swelling inhibited by cyclosporine A and were associated with a decrease of the total mitochondrial protein thiol content. These effects characterize the opening of the classical mitochondrial permeability transition pore. Despite the reactivity with mitochondrial protein thiol groups, these compounds did not promote significant glutathione depletion. In the absence of Ca(2+), the organotelluranes promoted mitochondrial loss of ΔΨ in RLM concomitant with respiratory control decrease due to an increase of the state 4 respiration rate. In these conditions, mitochondrial swelling was absent, and thiol content was higher than that in the presence of Ca(2+). The differentiated effects observed in the presence and absence of Ca(2+) are probably related to the effects of that ion on membrane structure, with repercussions for the exposure of specific reactive protein thiol groups. In smooth muscle cells, these compounds promoted the loss of mitochondrial ΔΨ and apoptosis. The loss of ΔΨ was not preceded by a decrease of cell viability that is consistent with mitochondria as the primary targets for the action of these organotelluranes.

Reactive oxygen species-provoked mitochondria-dependent cell death during ageing of elm (Ulmus pumila L.) seeds.

Previous studies have shown that controlled deterioration treatment (CDT) induces programmed cell death in elm (Ulmus pumila L.) seeds, which undergo certain fundamental processes that are comparable to apoptosis in animals. In this study, the essential characteristics of mitochondrial physiology in elm seeds during CDT were identified by cellular ultrastructural analysis, whole-body optical imaging, Western blotting and semi-quantitative RT-PCR. The alteration in mitochondrial morphology was an early event during CDT, as indicated by progressive dynamic mitochondrial changes and rupture of the mitochondrial outer membrane; loss of mitochondrial transmembrane potential (Δψ(m)) ensued, and mitochondrial ATP levels decreased. The mitochondrial permeability transition pore inhibitor cyclosporine A effectively suppressed these changes during ageing. The in situ localization of production of reactive oxygen species (ROS), and evaluation of the expression of voltage-dependent anion-selective channel and cyclophilin D indicated that the levels of mitochondrial permeability transition pore components were positively correlated with ROS production, leading to an imbalance of the cellular redox potential and ultimately to programmed cell death. Pre-incubation with ascorbic acid slowed loss of mitochondrial Δψ(m), and decreased the effect of CDT on seed viability. However, there were no significant changes in multiple antioxidant elements or chaperones in the mitochondria during early stages of ageing. Our results indicate that CDT induces dynamic changes in mitochondrial physiology via increased ROS production, ultimately resulting in an irreversible loss of seed viability.

ATP/ADP ratio, the missed connection between mitochondria and the Warburg effect

Non-proliferating cells generate the bulk of cellular ATP by fully oxidizing respiratory substrates in mitochondria. Respiratory substrates cross the mitochondrial outer membrane through only one channel, the voltage dependent anion channel (VDAC). Once in the matrix, respiratory substrates are oxidized in the tricarboxylic acid cycle to generate mostly NADH that is further oxidized in the respiratory chain to generate a proton motive force comprised mainly of membrane potential (ΔΨ) to synthesize ATP. Mitochondrial ΔΨ then drives the release of ATP4− from the matrix in exchange for ADP3− in the cytosol via the adenine nucleotide translocator (ANT) located in the mitochondrial inner membrane. Thus, mitochondrial function in non-proliferating cells drives a high cytosolic ATP/ADP ratio, essential to inhibit glycolysis. By contrast, the bioenergetics of the Warburg phenotype of proliferating cells is characterized by enhanced aerobic glycolysis and the suppression of mitochondrial metabolism. Suppressed mitochondrial function leads to lower production of mitochondrial ATP and hence lower cytosolic ATP/ADP ratios that favor enhanced glycolysis. Thus, the cytosolic ATP/ADP ratio is a key feature that determines if cell metabolism is predominantly oxidative or glycolytic. Here, we describe two novel mechanisms to explain the suppression of mitochondrial metabolism in cancer cells: the relative closure of VDAC by free tubulin and the inactivation of ANT. Both mechanisms contribute to low ATP/ADP ratios that activate glycolysis.