Uncoupling Of Mitochondrial Oxidative Phosphorylation Research Articles

P-171 Mitochondrial complex I, a key component of the mitochondrial respiratory chain, is necessary for maintaining chromosomal stability during the final stages of oocyte maturation

Abstract Study question What is the role of mitochondrial complex I during the final stages of human oocyte maturation? Summary answer Mitochondrial complex I is necessary for the cytoplasmic maturation of germinal vesicle (GV) oocytes and the subsequent organization of the meiotic spindle. What is known already Mitochondria play a pivotal role in oogenesis. Primordial human oocytes are characterized by an abundance of immature mitochondria, which exhibit low activity and a diminished capacity to produce adenosine triphosphate (ATP). During oocyte recruitment, mitochondrial fusion and fission play an essential role, contributing to oocyte maturation and oocyte developmental competence. Furthermore, an increase in ATP levels during polar body extrusion has been associated with higher fertilization rates, suggesting a reliance on mitochondria as ATP suppliers during oocyte maturation. However, the precise role of mitochondria during the final stages of human oocyte maturation is yet to be understood. Study design, size, duration At total of 99 immature oocytes, including 56 GVs and 40 metaphase I (MI) oocytes, were included in the study. These were obtained from 40 oocyte donation cycles, performed at a single IVF centre between August 2023 and September 2023. Participants/materials, setting, methods Immature denuded oocytes were cultured in G2TM PLUS medium to reach nuclear maturation (rescue in vitro maturation; rIVM). rIVM was performed in the absence (0.1% DMSO, control) or presence of 0.5 µM Rotenone (an inhibitor of mitochondrial complex I) or 10 µM FCCP (an uncoupler of mitochondrial oxidative phosphorylation). To assess mitochondrial and nuclear status, we performed immunofluorescence analyses for TOMM20 and the meiotic spindle (DNA and TUBA). Main results and the role of chance After 40 hours of culture, 64.3% of control GV oocytes progressed to the MII stage, 7.1% reached MI, while 21.4% arrested, and 7.1% degenerated. Control MI oocytes showed a 90% rIVM rate after 24 hours, with none arresting and 10% degenerating. Different mitochondrial inhibitors had varied effects on GV and MI oocytes. FCCP resulted in the degeneration of all treated oocytes. When treated with rotenone, 87.5% of GVs advanced to the MI stage, after which they arrested, but no signs of degeneration were observed. Notably, 100% of MIs and MIIs derived from GVs subjected to rotenone treatment presented with misaligned chromosomes, compared to 33.3% in the control group. When starting from MIs, the rIVM rate decreased to 41.7% following rotenone treatment, with no signs of degeneration. Here, misalignments were observed in 40% of the rIVM-MII oocytes and 43% of those that arrested, compared to 33.3% in controls. These results suggest that both GV and MI oocytes have adequate complex I activity, essential for proper nuclear maturation at the MII stage. We further highlight the importance of complex I activity, especially in GVs, for processes such as polar body extrusion during oocyte maturation. Limitations, reasons for caution Rescue in vitro maturation poses a challenge due to its limited effectiveness in achieving sufficient MII developmental competence. Consequently, mitochondrial inhibitors may have unforeseen effects on mitochondria and spindle formation. The specific role of complex I should be confirmed by employing other complex I inhibitors not related to rotenone. Wider implications of the findings Our results suggest that complex I activity, a key component of the mitochondrial respiratory chain, plays a crucial role in spindle organization during oocyte maturation. This relationship between mitochondrial function and chromosomal stability in oocytes suggests that interventions targeting the mitochondrial respiratory chain may be promising for enhancing oocyte quality. Trial registration number Not applicable

Low-Dose Exposure of WS2 Nanosheets Induces Differential Apoptosis in Lung Epithelial Cells.

Escalating the production and application of tungsten disulfide (WS2) nanosheets inevitably increases environmental human exposure and warrants the necessity of studies to elucidate their biological impacts. Herein, we assessed the toxicity of WS2 nanosheets and focused on the impacts of low doses (≤10 μg/mL) on normal (BEAS-2B) and tumorigenic (A549) lung epithelial cells. The low doses, which approximate real-world exposures, were found to induce cell apoptosis, while doses ≥ 50 μg/mL cause necrosis. Focused studies on low-dose exposure to WS2 nanosheets revealed more details of the impacts on both cell lines, including reduction of cell metabolic activity, induction of lipid peroxidation in cell membranes, and uncoupling of mitochondrial oxidative phosphorylation that led to the loss of ATP production. These phenomena, along with the expression situations of a few key proteins involved in apoptosis, point toward the occurrence of mitochondria-dependent apoptotic signaling in exposed cells. Substantial differences in responses to WS2 exposure between normal and tumorigenic lung epithelial cells were noticed as well. Specifically, BEAS-2B cells experienced more adverse effects and took up more nanosheets than A549 cells. Our results highlight the importance of dose and cell model selection in the assessment of nanotoxicity. By using doses consistent with real-world exposures and comparing normal and diseased cells, we can gain knowledge to guide the development of safety precautions for mitigating the adverse impacts of nanomaterial exposure on human health.

Metabolic reprogramming through mitochondrial biogenesis drives adenosine anti-inflammatory effects: new mechanism controlling gingival fibroblast hyper-inflammatory state.

Fibroblasts are the dominant stromal cells in the gingival lamina propria with a well-established relevance in regulation of inflammation, and in innate immunity. This is exemplified by their hypersecretion of CXCL8, enhancing leukocyte infiltration in chronic and sustained inflammatory conditions. We have previously shown adenosine to be a key metabolic nucleoside that regulates stromal inflammation, but the underlying mechanisms linking adenosine to the metabolic status of fibroblasts and to the resultant inflammatory response are unclear. This study examined, by seahorse real-time cell metabolic analysis, the bioenergetics of the stromal fibroblast response to extracellular adenosine and IL-1β, focusing on CXCL8 secretion by primary human gingival fibroblasts (HGF). Markers of the glycolytic pathway and mitochondrial biogenesis were tracked through immunoblot. Further, the influence of adenosine on mitochondrial accumulation was measured by uptake of MitoTracker Red fluorescent probe and assessment of the role of FCCP (a mitochondrial uncoupler) in CXCL8 secretion and mitochondrial accumulation. Our results show that the anti-inflammatory response of HGF to extracellular adenosine, typified by reduced CXCL8 secretion, is mediated by mitochondrial oxidative phosphorylation, reflected in higher oxygen consumption rate (OCR). In the presence of IL-1β, adenosine-treated cells induced higher ATP production, basal respiration and proton leak compared to IL-1β without adenosine. Surprisingly, adenosine had no additional effect on the IL-1β-induced higher glycolysis rate demonstrated by the extracellular acidification rate (ECAR). In addition, the higher OCR in adenosine-stimulated cells was not due to the mitochondrial fuel dependency or capacity, but due to an increase in mitochondrial biogenesis and accumulation in the cells with concomitant decrease in mitophagy-required p-PINK1 marker. We detected the accumulation of functional mitochondria with increased activation of the AMPK/SIRT1/PGC-1α pathway. The adenosine-induced uptake of MitoTracker was abrogated by PGC-1α inhibition with SR-12898. In addition, the adenosine effects on reduced CXCL8 were ablated by treatment with FCCP, a potent uncoupler of mitochondrial oxidative phosphorylation. Our findings reveal a key role for mitochondrial bioenergetics in regulation of CXCL8-mediated inflammation by HGF through the adenosine/AMPK/SIRT1/PGC-1α axis. Therapeutically targeting this pathway in gingival fibroblasts might be a promising future strategy to modulate stromal-mediated sustained hyper-inflammatory responses.

Mitochondrial Dysfunction Links to Impaired Hippocampal Serotonin Release in a Mouse Model of Alzheimer's Disease.

Deprivation of extracellular serotonin has been linked to cognitive decline and neuropsychiatric disturbances in Alzheimer's disease (AD). However, despite degeneration of serotonin-producing neurons, whether serotonin release is affected in AD-sensitive brain regions is unknown. This study investigated the impact of mitochondrial dysfunction in decreased hippocampal serotonin release in AD amyloidosis mouse model 5xFAD mice. Electrochemical assays were applied to examine hippocampal serotonin release. We also employed multidisciplinary techniques to determine the role of oligomeric amyloid-β (Aβ) in hippocampal mitochondrial deficits and serotonin release deficiency. 5xFAD mice exhibited serotonin release decrease and relatively moderate downregulation of serotonergic fiber density as well as serotonin content in the hippocampal region. Further experiments showed an inhibitory effect of oligomeric amyloid-β (Aβ) on hippocampal serotonin release without affecting the density of serotonergic fibers. Pharmaceutical uncoupling of mitochondrial oxidative phosphorylation (OXPHOS) disrupted hippocampal serotonin release in an ex vivo setting. This echoes the mitochondrial defects in serotonergic fibers in 5xFAD mice and oligomeric Aβ-challenged primary serotonergic neuron cultures and implicates a link between mitochondrial dysfunction and serotonin transmission defects in AD-relevant pathological settings. The most parsimonious interpretation of our findings is that mitochondrial dysfunction is a phenotypic change of serotonergic neurons, which potentially plays a role in the development of serotonergic failure in AD-related conditions.

Resistance to energy metabolism - targeted therapy of AML cells residual in the bone marrow microenvironment.

In response to the changing availability of nutrients and oxygen in the bone marrow microenvironment, acute myeloid leukemia (AML) cells continuously adjust their metabolic state. To meet the biochemical demands of their increased proliferation, AML cells strongly depend on mitochondrial oxidative phosphorylation (OXPHOS). Recent data indicate that a subset of AML cells remains quiescent and survives through metabolic activation of fatty acid oxidation (FAO), which causes uncoupling of mitochondrial OXPHOS and facilitates chemoresistance. For targeting these metabolic vulnerabilities of AML cells, inhibitors of OXPHOS and FAO have been developed and investigated for their therapeutic potential. Recent experimental and clinical evidence has revealed that drug-resistant AML cells and leukemic stem cells rewire metabolic pathways through interaction with BM stromal cells, enabling them to acquire resistance against OXPHOS and FAO inhibitors. These acquired resistance mechanisms compensate for the metabolic targeting by inhibitors. Several chemotherapy/targeted therapy regimens in combination with OXPHOS and FAO inhibitors are under development to target these compensatory pathways.

Regulation of autophagy in chick myotube cultures: Effect of uncoupling mitochondrial oxidative phosphorylation.

Abstracts: Skeletal muscles have a high demand for ATP, which is met largely through mitochondria oxidative phosphorylation. Autophagy is essential for the maintenance of skeletal muscle mass under catabolic conditions. This study investigated the effect of uncoupling mitochondrial oxidative phosphorylation on autophagy in chicken skeletal muscle. Chick myotubes were incubated with the mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP) at 25 μM for 3h. CCCP prevented the phosphorylation of p70 ribosomal S6 kinase 1 (Thr389), S6 ribosomal protein (Ser240/244), and eukaryotic translation initiation factor 4E-binding protein 1 (Thr37/46), which are the measures of the mechanistic target of rapamycin complex 1 (mTORC1) activity. CCCP significantly increased cytoplasmic and mitochondrial LC3-II content, which act as indices of index for autophagosome formation and mitophagy, respectively, but did not influence the expression of autophagy-related genes LC3B, GABARAPL1, and ATG12. Finally, surface sensing of translation method revealed that protein synthesis, a highly energy consuming process, was significantly decreased upon CCCP treatment. These results indicate that the uncoupling of mitochondrial oxidative phosphorylation stimulates autophagy and inhibits protein synthesis through mTORC1 signaling in chick myotube cultures.

Nab3 nuclear granule accumulation is driven by respiratory capacity.

Numerous biological processes involve proteins capable of transiently assembling into subcellular compartments necessary for cellular functions. One process is the RNA polymerase II transcription cycle which involves initiation, elongation, co-transcriptional modification of nascent RNA, and termination. The essential yeast transcription termination factor Nab3 is required for termination of small non-coding RNAs and accumulates into a compact nuclear granule upon glucose removal. Nab3 nuclear granule accumulation varies in penetrance across yeast strains and a higher Nab3 granule accumulation phenotype is associated with petite strains, suggesting a possible ATP-dependent mechanism for granule disassembly. Here, we demonstrate the uncoupling of mitochondrial oxidative phosphorylation by drug treatment or deletions of nuclear-encoded ATP synthase subunit genes were sufficient to increase Nab3 granule accumulation and led to an inability to proliferate during prolonged glucose deprivation, which requires respiration. Additionally, by enriching for respiration competent cells from a petite-prone strain, we generated a low granule-accumulating strain from a relatively high one, providing another link between respiratory competency and Nab3 granules. Consistent with the resulting idea that ATP is involved in granule accumulation, the addition of extracellular ATP to semi-permeabilized cells was sufficient to reduce Nab3 granule accumulation. Deleting the SKY1 gene, which encodes a kinase that phosphorylates nuclear SR repeat-containing proteins and is involved in efficient stress granule disassembly, also resulted in increased granule accumulation. This observation implicates Sky1 in Nab3 granule biogenesis. Taken together, these findings suggest there is normally an equilibrium between termination factor granule assembly and disassembly mediated by ATP-requiring nuclear machinery.

Investigating mitochondria-immune responses in zebrafish, Danio rerio (Hamilton, 1822): A case study with the herbicide dinoseb

The dinitrophenol herbicide dinoseb is an uncoupler of mitochondrial oxidative phosphorylation (OXPHOS). Studies in fish demonstrate impaired OXPHOS is associated with altered immune system responses and locomotor activity in fish. The objective of this study was to determine the effect of dinoseb on zebrafish (Danio rerio) during early stages of development. We measured oxygen consumption rates of embryos, transcripts related to OXPHOS, growth, and the immune system (cytokines and immune-signaling transcripts), and locomotor activity. We hypothesized that OXPHOS of fish would be impaired in vivo, leading to altered basal immune system expression and locomotor activity. Oxidative respiration assessments in embryos revealed that dinoseb decreased both mean basal respiration and oligomycin-induced ATP-linked respiration. Expression levels of cytochrome c oxidase complex IV, 3-hydroxyacyl-COA dehydrogenase and superoxide dismutase 1 were decreased in larvae following exposure to dinoseb while succinate dehydrogenase complex flavoprotein subunit A, insulin growth factor 1 (igf1) and igf2a mRNA were increased in abundance. Immune-related transcripts chemokine (C-X-C motif) ligand 1 and matrix metallopeptidase 9 (MMP-9) were decreased in expression levels while toll-like receptor 5a and 5b were increased in expression. In addition, a visual motor response test was conducted on both 6 and 7 dpf larvae to determine if dinoseb impaired locomotor activity. Dinoseb decreased locomotor activity in 7 dpf larvae but not 6 dpf. This study improves knowledge of toxicity mechanisms for dinoseb in early stages of fish development and demonstrates that mitochondrial toxicants may disrupt immune signaling in zebrafish.

Niclosamide Is a Much More Potent Toxicant of Mitochondrial Respiration than TFM in the Invasive Sea Lamprey (Petromyzon marinus).

Invasive sea lampreys in the Laurentian Great Lakes are controlled by applying TFM (3-trifluoromethyl-4-nitrophenol) and niclosamide to streams infested with their larvae. Both agents uncouple oxidative phosphorylation in the mitochondria, but TFM specifically targets lampreys, which have a lower capacity to detoxify the lampricide. Niclosamide lacks specificity and is more potent than TFM. However, its greater potency is poorly understood. We tested the hypothesis that niclosamide is a stronger uncoupler of mitochondrial oxidative phosphorylation than TFM by measuring oxygen consumption rates in isolated liver mitochondria exposed to physiologically relevant concentrations of TFM, niclosamide, or their mixture (100 TFM:1 niclosamide) at environmentally relevant temperatures (7, 13, and 25 °C). Niclosamide increased State 4 respiration and decreased the respiratory control ratio (RCR) at much lower concentrations than TFM. Calculations of the relative EC50 values, the amount of TFM or niclosamide required to decrease the RCR by 50%, indicated that niclosamide was 40-60 times more potent than TFM. Warmer temperature did not appear to decrease the sensitivity of mitochondria to niclosamide or TFM, as observed in the intact sea lamprey exposed to TFM in warmer waters. We conclude that the extreme sensitivity of mitochondria to niclosamide contributes to its greater in vivo toxicity in the whole animal.

Ecdysterone prevents negative effect of acute immobilization stress on energy metabolism of rat liver mitochondria

Ecdysterone is a naturally occurring steroid hormone, which presents in arthropods and in a number of plants as an insect defence tool. There are many studies showing that application of ecdysterone can alter mitochondrial functions of mammalian cells, however it is not clear whether its effects are direct or mediated by activation of other cellular processes. In our study, we have shown how ecdysterone acts at the mitochondrial level in normal conditions and in certain pathology. We have demonstrated that application of immobilization stress to male rats causes uncoupling of mitochondrial oxidative phosphorylation, the preliminary application of ecdysterone prevents negative effect of immobilization stress on mitochondria. In-vitro experiments with isolated mitochondria have shown that ecdysterone can increase mitochondrial coupling and hyperpolarise mitochondria but without a noticeable effect on ADP/O ratio. Molecular docking experiments revealed that ecdysterone has high binding energy with mitochondrial FOF1 ATP synthase, but further biochemical analysis have not revealed either stimulatory or inhibitory effect of ecdysterone on FOF1 ATPase activity of the enzyme. Thus, ecdysterone can directly affect mitochondrial bioenergetics, though we assume that its preventive effect on mitochondria during immobilization stress is also coupled with the activation of some other cellular processes.

Detecting Effects of Low Levels of FCCP on Stem Cell Micromotion and Wound-Healing Migration by Time-Series Capacitance Measurement.

Electric cell–substrate impedance sensing (ECIS) has been used as a real-time impedance-based method to quantify cell behavior in tissue culture. The method is capable of measuring both the resistance and capacitance of a cell-covered microelectrode at various AC frequencies. In this study, we demonstrate the application of high-frequency capacitance measurement (f = 40 or 64 kHz) for the sensitive detection of both the micromotion and wound-healing migration of human mesenchymal stem cells (hMSCs). Impedance measurements of cell-covered electrodes upon the challenge of various concentrations of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), from 0.1 to 30 μM, were conducted using ECIS. FCCP is an uncoupler of mitochondrial oxidative phosphorylation (OXPHOS), thereby reducing mitochondrial ATP production. By numerically analyzing the time-series capacitance data, a dose-dependent decrease in hMSC micromotion and wound-healing migration was observed, and the effect was significantly detected at levels as low as 0.1 μM. While most reported works with ECIS use the resistance/impedance time series, our results suggest the potential use of high-frequency capacitance time series for assessing migratory cell behavior such as micromotion and wound-healing migration.

Study of Stem Cells Influence on Cardiac Cells Cultured with a Cyanide-P-Trifluoromethoxyphenylhydrazone in Organ-on-a-Chip System.

Regenerative medicine and stem cells could prove to be an effective solution to the problem of treating heart failure caused by ischemic heart disease. However, further studies on the understanding of the processes which occur during the regeneration of damaged tissue are needed. Microfluidic systems, which provide conditions similar to in vivo, could be useful tools for the development of new therapies using stem cells. We investigated how mesenchymal stem cells (MSCs) affect the metabolic activity of cardiac cells (rat cardiomyoblasts and human cardiomyocytes) incubated with a potent uncoupler of mitochondrial oxidative phosphorylation under microfluidic conditions. A cyanide p-trifluoromethoxyphenylhydrazone (FCCP) was used to mimic disfunctions of mitochondria of cardiac cells. The study was performed in a microfluidic system integrated with nanofiber mats made of poly-l-lactid acid (PLLA) or polyurethane (PU). The microsystem geometry allows four different cell cultures to be conducted under different conditions (which we called: normal, abnormal—as both a mono- and co-culture). Metabolic activity of the cells, based on the bioluminescence assay, was assessed in the culture’s performed in the microsystem. It was proved that stem cells increased metabolic activity of cardiac cells maintained with FCCP.

Hydrogen sulfide induces Ca2+ release from intracellular Ca2+ stores and stimulates lactate production in spinal cord astrocytes

Hydrogen sulfide (H2S) is a well-known inhibitor of the mitochondrial electron transport chain (ETC). H2S also increases intracellular Ca2+ levels in astrocytes, which are glial cells and that supply lactate as an energy substrate to neurons. Here, we examined the relationship between H2S-induced metabolic changes and Ca2+ responses in spinal cord astrocytes. Na2S (150 μM), an H2S donor, increased the intracellular Ca2+ concentration, which was inhibited by an ETC inhibitor and an uncoupler of mitochondrial oxidative phosphorylation. Na2S also increased the accumulation of extracellular lactate. Na2S alone did not change intracellular ATP content, but decreased it when glycolysis was inhibited. The Na2S-induced Ca2+ increase and accumulation of extracellular lactate were inhibited by emetine, an inhibitor of translocon complex, which mediates Ca2+ leak from the endoplasmic reticulum (ER). Furthermore, an inhibitor of the Ca2+-sensitive NADH shuttle decreased Na2S-mediated accumulation of lactate. We conclude that inhibition of the mitochondrial ETC by H2S induces Ca2+ release from mitochondria and the ER in spinal cord astrocytes, which increases lactate production. H2S may promote glycolysis by activating the Ca2+-sensitive NADH shuttle and facilitating the supply of lactate from astrocytes to neurons.

Selective cytotoxicity of marine-derived fungal metabolite (3S,6S)-3,6-dibenzylpiperazine-2,5-dione against cancer cells adapted to nutrient starvation.

The cancer cells that are adapted to the hypoxic and nutrient-starved conditions of the tumor microenvironment have become a key target for anticancer therapies. In the course of search for selective cytotoxic substances against cancer cells adapted to nutrient starvation, (3S,6S)-3,6-dibenzylpiperazine-2,5-dione (1) was isolated from culture extract of marine-derived Paecilomyces formous 17D47-2. Compound 1 showed cytotoxic activity on the human pancreatic carcinoma PANC-1 cells adapted to glucose-starved conditions with IC50 value of 28 µM, whereas no effect was observed against PANC-1 cells under general culture conditions up to 1000 µM. Further studies on the mechanism of the selective cytotoxicity of 1 against the glucose-starved PANC-1 cells suggest that it may function via uncoupling of mitochondrial oxidative phosphorylation.

Fatty Acid Metabolism, Bone Marrow Adipocytes, and AML.

Acute myeloid leukemia (AML) cells modulate their metabolic state continuously as a result of bone marrow (BM) microenvironment stimuli and/or nutrient availability. Adipocytes are prevalent in the BM stroma and increase in number with age. AML in elderly patients induces remodeling and lipolysis of BM adipocytes, which may promote AML cell survival through metabolic activation of fatty acid oxidation (FAO). FAO reactions generate acetyl-CoA from fatty acids under aerobic conditions and, under certain conditions, it can cause uncoupling of mitochondrial oxidative phosphorylation. Recent experimental evidence indicates that FAO is associated with quiescence and drug-resistance in leukemia stem cells. In this review, we highlight recent progress in our understanding of fatty acid metabolism in AML cells in the adipocyte-rich BM microenvironment, and discuss the therapeutic potential of combinatorial regimens with various FAO inhibitors, which target metabolic vulnerabilities of BM-resident, chemoresistant leukemia cells.

Mitochondrial metabolic study guided by proteomics analysis in hepatocellular carcinoma cells surviving long-term incubation with the highest dose of sorafenib.

Sorafenib is the standard first-line systemic therapy for hepatocellular carcinoma (HCC). However, the low objective response rates in clinical studies suggest the existence of certain HCC cells that are inherently insensitive to sorafenib. To understand the molecular basis of insensitivity of HCC cells to sorafenib, this study developed 3 kinds of insensitive HCC cells through exposure to various concentrations of sorafenib and performed a quantitative proteome analysis of the surviving HepG2 cells. 520 unique proteins were concentration-dependently upregulated by sorafenib. Bioinformatics-assisted analysis of 520 proteins revealed that the metabolic pathways involved in central carbon metabolism were significantly enriched, and 102 mitochondrial proteins, especially components of the electron transport chain (ETC), were incrementally upregulated in the 3 kinds of insensitive cells. Conversely, we identified a rapid holistic inhibitory effect of sorafenib on mitochondrial function by the direct targeting of the complex I-linked electron transport and the uncoupling of mitochondrial oxidative phosphorylation (OXHPOS) in HCC cells. Core metabolic reprogramming involved in a compensatory upregulation of OXHPOS combined with elevated glycolysis supports the survival of HCC cells under the highest dose of sorafenib treatment. Altogether, our work thus elaborates an ETC inhibitor and unveils the proteomic landscape of metabolic reprogramming in drug insensitivity.

The Anticancer Agent Elesclomol Has Direct Effects on Mitochondrial Bioenergetic Function in Isolated Mammalian Mitochondria

Elesclomol ((N-malonyl-bis(N′-methyl-N′-thiobenzoylhydrazide)); formerly STA-4783) is a mitochondria-targeted chemotherapeutic agent that has demonstrated efficacy in selective cancer cell killing in pre-clinical and clinical testing. The biologically active form of elesclomol is a deprotonated copper chelate (elesclomol:copper; E:C), which has been shown to enhance reactive oxygen species (ROS) production and induce a transcriptional gene profile characteristic of an oxidative stress response in vitro. Previous studies suggest that E:C interacts with the electron transport chain (ETC) to generate high levels of ROS within the organelle and ultimately induce cell death. The purpose of this study was to further explore the mechanism of cellular and mitochondrial toxicity of E:C by examining its direct effect on mitochondrial bioenergetic function. The results obtained indicate that E:C treatment in whole cells of non-tumorigenic origin at high concentrations (40 μM and higher) induces a rapid and substantial increase in mitochondrial superoxide levels and dissipation of mitochondrial membrane potential. Furthermore, similar higher concentrations of E:C act as a direct uncoupler of oxidative phosphorylation and generalized inhibitor of electron transport activity in isolated, intact mitochondria, and induce a dose-dependent inhibition of mitochondrial NADH-ubiquinone oxidoreductase activity in freeze-thawed mitochondrial preparations. The results of this study are important in that they are the first to demonstrate a direct effect of the E:C chelate on bioenergetic function in isolated mammalian mitochondria, and suggest the possibility that the increase in ROS production and cytotoxicity induced by E:C may in part be due to uncoupling of mitochondrial oxidative phosphorylation and/or inhibition of electron transport activity. These results also provide important information about the mechanisms of mitochondrial and cellular toxicity induced by E:C and will ultimately contribute to a better understanding of the therapeutic potential of elesclomol as an anticancer compound.

Application of ECIS to Assess FCCP-Induced Changes of MSC Micromotion and Wound Healing Migration.

Electric cell-substrate impedance sensing (ECIS) is an emerging technique for sensitively monitoring morphological changes of adherent cells in tissue culture. In this study, human mesenchymal stem cells (hMSCs) were exposed to different concentrations of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) for 20 h and their subsequent concentration-dependent responses in micromotion and wound healing migration were measured by ECIS. FCCP disrupts ATP synthesis and results in a decrease in cell migration rates. To detect the change of cell micromotion in response to FCCP challenge, time-series resistances of cell-covered electrodes were monitored and the values of variance were calculated to verify the difference. While Seahorse XF-24 extracellular flux analyzer can detect the effect of FCCP at 3 μM concentration, the variance calculation of the time-series resistances measured at 4 kHz can detect the effect of FCCP at concentrations as low as 1 μM. For wound healing migration, the recovery resistance curves were fitted by sigmoid curve and the hill slope showed a concentration-dependent decline from 0.3 μM to 3 μM, indicating a decrease in cell migration rate. Moreover, dose dependent incline of the inflection points from 0.3 μM to 3 μM FCCP implied the increase of the half time for wound recovery migration. Together, our results demonstrate that partial uncoupling of mitochondrial oxidative phosphorylation reduces micromotion and wound healing migration of hMSCs. The ECIS method used in this study offers a simple and sensitive approach to investigate stem cell migration and its regulation by mitochondrial dynamics.

Minerval (2-hydroxyoleic acid) causes cancer cell selective toxicity by uncoupling oxidative phosphorylation and compromising bioenergetic compensation capacity.

This work tests bioenergetic and cell-biological implications of the synthetic fatty acid Minerval (2-hydroxyoleic acid), previously demonstrated to act by activation of sphingomyelin synthase in the plasma membrane (PM) and lowering of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) and their carcinogenic signaling. We show here that Minerval also acts, selectively in cancer cell lines, as an ATP depleting uncoupler of mitochondrial oxidative phosphorylation (OxPhos). As a function of its exposure time, Minerval compromised the capacity of glioblastoma U87-MG cells to compensate for aberrant respiration by up-modulation of glycolysis. This effect was not exposure time-dependent in the lung carcinoma A549 cell line, which was more sensitive to Minerval. Compared with OxPhos inhibitors FCCP (uncoupler), rotenone (electron transfer inhibitor), and oligomycin (F1F0-ATPase inhibitor), Minerval action was similar only to that of FCCP. This similarity was manifested by mitochondrial membrane potential (MMP) depolarization, facilitation of oxygen consumption rate (OCR), restriction of mitochondrial and cellular reactive oxygen species (ROS) generation and mitochondrial fragmentation. Additionally, compared with other OxPhos inhibitors, Minerval uniquely induced ER stress in cancer cell lines. These new modes of action for Minerval, capitalizing on the high fatty acid requirements of cancer cells, can potentially enhance its cancer-selective toxicity and improve its therapeutic capacity.

Molecular mechanistic insights into uncoupling of ion transport from ATP synthesis

A procedure is evolved to assess the maximum uncoupling activity of the classical unsubstituted phenolic uncouplers of mitochondrial oxidative phosphorylation (OX PHOS) 2,4-dinitrophenol and 2,6-dinitrophenol. The uncoupler concentrations, C, required for maximum uncoupling efficacy are found to be a strong function of the pH, and a linear relationship of pC with pH is obtained between pH 5 to pH 9. The slopes of the uncoupler concentrations in the aqueous and lipid phases as a function of pH have been estimated. It is shown that the experimental results can be derived from first principles by an enzyme kinetic model for uncoupling that is based on the same equations as formulated for the coupling of ion transport to ATP synthesis in a companion paper after imposition of the special conditions arising from the uncoupling process. The results reveal the catalysis of a reaction that involves both the anionic and protonated forms of the phenolic uncouplers in the vicinity of their binding sites in a non-aqueous region of the cristae membranes of mitochondria. The rate-limiting step in the overall process of uncoupling has been identified based on the uncoupling data. The data cannot be explained by a simple conduction of protons by uncouplers from one bulk aqueous phase to another as postulated by Mitchell's chemiosmotic theory. It is shown that Nath's two-ion theory of energy coupling/uncoupling in ATP synthase is consistent with the results. A molecular mechanism for uncoupling of ATP synthesis by the dinitrophenols is presented and the chief differences between coupling and uncoupling in ATP catalysis are summarized. The pharmacological consequences of our analysis of uncoupling are discussed, with particular reference to the mode of action of the anti-tuberculosis drug bedaquiline that specifically targets the c-subunit of the F1FO-ATP synthase and uncouples respiration from ATP synthesis in Mycobacterium tuberculosis. Hence the work is shown to be important both from the point of view of fundamental biology and is also pregnant with possibilities for practical pharmaceutical applications.