Role of Glycolysis and Fatty Acid Synthesis in the Activation and T Cell-Modulating Potential of Dendritic Cells Stimulated with a TLR5-Ligand Allergen Fusion Protein.

Trained immune responses, based on metabolic and epigenetic changes in innate immune cells, are de facto innate immune memory and, therefore, are of great interest in vaccine development. In previous studies, the recombinant fusion protein rFlaA:Betv1, combining the adjuvant and toll-like receptor (TLR)5-ligand flagellin (FlaA) and the major birch pollen allergen Bet v 1 into a single molecule, significantly suppressed allergic sensitization in vivo while also changing the metabolism of myeloid dendritic cells (mDCs). Within this study, the immune-metabolic effects of rFlaA:Betv1 during mDC activation were elucidated. In line with results for other well-characterized TLR-ligands, rFlaA:Betv1 increased glycolysis while suppressing oxidative phosphorylation to different extents, making rFlaA:Betv1 a suitable model to study the immune-metabolic effects of TLR-adjuvanted vaccines. In vitro pretreatment of mDCs with cerulenin (inhibitor of fatty acid biosynthesis) led to a decrease in both rFlaA:Betv1-induced anti-inflammatory cytokine Interleukin (IL) 10 and T helper cell type (TH) 1-related cytokine IL-12p70, while the pro-inflammatory cytokine IL 1ß was unaffected. Interestingly, pretreatment with the glutaminase inhibitor BPTES resulted in an increase in IL-1ß, but decreased IL-12p70 secretion while leaving IL-10 unchanged. Inhibition of the glycolytic enzyme hexokinase-2 by 2-deoxyglucose led to a decrease in all investigated cytokines (IL-10, IL-12p70, and IL-1ß). Inhibitors of mitochondrial respiration had no effect on rFlaA:Betv1-induced IL-10 level, but either enhanced the secretion of IL-1ß (oligomycin) or decreased IL-12p70 (antimycin A). In extracellular flux measurements, mDCs showed a strongly enhanced glycolysis after rFlaA:Betv1 stimulation, which was slightly increased after respiratory shutdown using antimycin A. rFlaA:Betv1-stimulated mDCs secreted directly antimicrobial substances in a mTOR- and fatty acid metabolism-dependent manner. In co-cultures of rFlaA:Betv1-stimulated mDCs with CD4+ T cells, the suppression of Bet v 1-specific TH2 responses was shown to depend on fatty acid synthesis. The effector function of rFlaA:Betv1-activated mDCs mainly relies on glycolysis, with fatty acid synthesis also significantly contributing to rFlaA:Betv1-mediated cytokine secretion, the production of antimicrobial molecules, and the modulation of T cell responses.

Reprint of: Ubisemiquinone Is the Electron Donor for Superoxide Formation by Complex III of Heart Mitochondria.

Much evidence indicates that superoxide is generated from O2 in a cyanide-sensitive reaction involving a reduced component of complex III of the mitochondrial respiratory chain, particularly when antimycin A is present. Although it is generally believed that ubisemiquinone is the electron donor to O2, little experimental evidence supporting this view has been reported. Experiments with succinate as electron donor in the presence of antimycin A in intact rat heart mitochondria, which contain much superoxide dismutase but little catalase, showed that myxothiazol, which inhibits reduction of the Rieske iron-sulfur center, prevented formation of hydrogen peroxide, determined spectrophotometrically as the H2O2-peroxidase complex. Similarly, depletion of the mitochondria of their cytochrome c also inhibited formation of H2O2, which was restored by addition of cytochrome c. These observations indicate that factors preventing the formation of ubisemiquinone also prevent H2O2 formation. They also exclude ubiquinol, which remains reduced under these conditions, as the reductant of O2. Since cytochrome b also remains fully reduced when myxothiazol is added to succinate- and antimycin A-supplemented mitochondria, reduced cytochrome b may also be excluded as the reductant of O2. These observations, which are consistent with the Q-cycle reactions, by exclusion of other possibilities leave ubisemiquinone as the only reduced electron carrier in complex III capable of reducing O2 to O2-.

Interaction of picolinamide fungicide primary metabolites UK-2A and CAS-649 with the cytochrome bc1 complex Qi site: mutation effects and modelling in Saccharomyces cerevisiae.

BACKGROUND: Fenpicoxamid and florylpicoxamid are picolinamide fungicides targeting the Qi site of the cytochrome bc1 complex, via their primary metabolites UK-2A and CAS-649, respectively. We explore binding interactions and resistance mechanisms for picolinamides, antimycin A and ilicicolin H in yeast by testing effects of cytochrome b amino acid changes on fungicide sensitivity and interpreting results using molecular docking. RESULTS: Effects of amino acid changes on sensitivity to UK-2A and CAS-649 were similar, with highest resistance associated with exchanges involving G37 and substitutions N31K and L198F. These changes, as well as K228M, also affected antimycin A, while ilicicolin H was affected by changes at G37 and L198, as well as Q22E. N31 substitution patterns suggest that a lysine at position 31 introduces an electrostatic interaction with neighbouring D229, causing disruption of a key salt-bridge interaction with picolinamides. Changes involving G37 and L198 imply resistance primarily through steric interference. G37 changes also showed differences between CAS-649 and UK-2A or antimycin A with respect to branched versus unbranched amino acids. N31K and substitution of G37 by large amino acids reduced growth rate substantially while L198 substitutions showed little effect on growth. CONCLUSION: Binding of UK-2A and CAS-649 at the Qi site involves similar interactions such that general cross-resistance between fenpicoxamid and florylpicoxamid is anticipated in target pathogens. Some resistance mutations reduced growth rate and could carry a fitness penalty in pathogens. However, certain changes involving G37 and L198 carry little or no growth penalty and may pose the greatest risk for resistance development in the field. © 2022 Society of Chemical Industry.

Bioenergetic Profiling of Human Pluripotent Stem Cells.

Cellular metabolism contributes to cell fate decisions. Bioenergetic profiling can therefore provide considerable insights into cellular identity and specification. Given the current importance of human pluripotent stem cells (hPSCs) for biomedical applications, assessing the bioenergetic properties of hPSCs and derivatives can unveil relevant mechanisms in the context of development biology and molecular disease modeling. Here, we describe a method to facilitate bioenergetic profiling of hPSCs in a reproducible and scalable manner. After simultaneous assessment of mitochondrial respiration and glycolytic capacity using Seahorse XFe96 Analyzer, we measure lactate concentration in the cellular media. Finally, we normalize the values based on DNA amount. We describe the procedures with specific requirements related to hPSCs . However, the same protocol can be easily adapted to other cell types, including differentiated progenies from hPSCs .

Inhibition of mitochondrial complex III induces differentiation in acute myeloid leukemia.

Although acute myeloid leukemia (AML) is a highly heterogeneous disease with diverse genetic subsets, one hallmark of AML blasts is myeloid differentiation blockade. Extensive evidence has indicated that differentiation induction therapy represents a promising treatment strategy. Here, we identified that the pharmacological inhibition of the mitochondrial electron transport chain (ETC) complex III by antimycin A inhibits proliferation and promotes cellular differentiation of AML cells. Mechanistically, we showed that the inhibition of dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme in de novo pyrimidine biosynthesis, is involved in antimycin A-induced differentiation. The activity of antimycin A could be reversed by supplement of excessive amounts of exogenous uridine as well as orotic acid, the product of DHODH. Furthermore, we also found that complex III inhibition exerts a synergistic effect in differentiation induction combined with DHODH inhibitor brequinar as well as with the pyrimidine salvage pathway inhibitor dipyridamole. Collectively, our study uncovered the link between mitochondrial complex III and AML differentiation and may provide further insight into the potential application of mitochondrial complex III inhibitor as a mono or combination treatment in differentiation therapy of AML.

Antimycin A shows selective antiproliferation to oral cancer cells by oxidative stress-mediated apoptosis and DNA damage.

The antibiotic antimycin A (AMA) is commonly used as an inhibitor for the electron transport chain but its application in anticancer studies is rare. Recently, the repurposing use of AMA in antiproliferation of several cancer cell types has been reported. However, it is rarely investigated in oral cancer cells. The purpose of this study is to investigate the selective antiproliferation ability of AMA treatment on oral cancer cells. Cell viability, flow cytometry, and western blotting were applied to explore its possible anticancer mechanism in terms of both concentration- and exposure time-effects. AMA shows the higher antiproliferation to two oral cancer CAL 27 and Ca9-22 cell lines than normal oral HGF-1 cell lines. Moreover, AMA induces the production of higher reactive oxygen species (ROS) levels and pan-caspase activation in oral cancer CAL 27 and Ca9-22 cells than in normal oral HGF-1 cells, providing the possible mechanism for its selective antiproliferation effect of AMA. In addition to ROS, AMA induces mitochondrial superoxide (MitoSOX) generation and depletes mitochondrial membrane potential (MitoMP). This further supports the AMA-induced oxidative stress changes in oral cancer CAL 27 and Ca9-22 cells. AMA also shows high expressions of annexin V in CAL 27 and Ca9-22 cells and cleaved forms of poly (ADP-ribose) polymerase (PARP), caspase 9, and caspase 3 in CAL 27 cells, supporting the apoptosis-inducing ability of AMA. Furthermore, AMA induces DNA damage (γH2AX and 8-oxo-2'-deoxyguanosine [8-oxodG]) in CAL 27 and Ca9-22 cells. Notably, the AMA-induced selective antiproliferation, oxidative stress, and DNA damage were partly prevented from N-acetylcysteine (NAC) pretreatments. Taken together, AMA selectively kills oral cancer cells in an oxidative stress-dependent mechanism involving apoptosis and DNA damage.

S-methyl-L-cysteine Protects against Antimycin A-induced Mitochondrial Dysfunction in Neural Cells via Mimicking Endogenous Methionine-centered Redox Cycle.

Mitochondrial superoxide overproduction is believed to be responsible for the neurotoxicity associated with neurodegeneration. Mitochondria-targeted antioxidants, such as MitoQ, have emerged as potentially effective antioxidant therapies. Methionine sulfoxide reductase A (MsrA) is a key mitochondrial-localized endogenous antioxidative enzyme and it can scavenge oxidizing species by catalyzing the methionine (Met)-centered redox cycle (MCRC). In this study, we observed that the natural L-Met acted as a good scavenger for antimycin A-induced mitochondrial superoxide overproduction in PC12 cells. This antioxidation was largely dependent on the Met oxidase activity of MsrA. S-methyl-L-cysteine (SMLC), a natural analogue of Met that is abundantly found in garlic and cabbage, could activate the Met oxidase activity of MsrA to scavenge free radicals. Furthermore, SMLC protected against antimycin A-induced mitochondrial membrane depolarization and alleviated 1-methyl-4-phenylpyridinium (MPP+)-induced neurotoxicity. Thus, our data highlighted the possibility for SMLC supplement in the detoxication of mitochondrial damage by activating the Met oxidase activity of MsrA.

Kitamycin C, a new antimycin-type antibiotic from Streptomyces antibioticus strain 200-09.

An actinomycete strain 200-09, isolated from a soil sample collected from the coast of Hawaii, USA, was identified as Streptomyces antibioticus on the basis of its morphological, physiological and biochemical characteristics as well as 16S rDNA analysis. A new antimycin-type antibiotic, kitamycin C (1), together with kitamycin A (2), kitamycin B (3), urauchmycin B (4), deisovaleryblastomycin (5) was isolated from a cultured broth of strain 200-09. The structure of the new compound was determined by spectroscopic data, including HR-ESI-MS and NMR. All the compounds exhibited antifungal activities against Candida albicans with MIC of about 25.0 µg mL-1.

Genetic Evidence for Cytochrome b Qi Site Inhibition by 4(1H)-Quinolone-3-Diarylethers and Antimycin in Toxoplasma gondii.

Toxoplasma gondii is an apicomplexan parasite that causes fatal and debilitating brain and eye disease. Endochinlike quinolones (ELQs) are preclinical compounds that are efficacious against apicomplexan-caused diseases, including toxoplasmosis, malaria, and babesiosis. Of the ELQs, ELQ-316 has demonstrated the greatest efficacy against acute and chronic experimental toxoplasmosis. Although genetic analyses in other organisms have highlighted the importance of the cytochrome bc1 complex Qi site for ELQ sensitivity, the mechanism of action of ELQs against T. gondii and the specific mechanism of ELQ-316 remain unknown. Here, we describe the selection and genetic characterization of T. gondii clones resistant to ELQ-316. A T. gondii strain selected under ELQ-316 drug pressure was found to possess a Thr222-Pro amino acid substitution that confers 49-fold resistance to ELQ-316 and 19-fold resistance to antimycin, a well-characterized Qi site inhibitor. These findings provide further evidence for ELQ Qi site inhibition in T. gondii and greater insight into the interactions of Qi site inhibitors with the apicomplexan cytochrome bc1 complex.

Biochemical effects of the flavanol-rich lychee fruit extract on the melanin biosynthesis and reactive oxygen species.

An ingredient of fruit polyphenol, resveratrol, is known to have an inhibitory effect on melanogenesis. In order to examine the functional differences between resveratrol and other fruit polyphenols, we compared biochemical effects of a resveratrol-free polyphenol, flavanol-rich lychee fruit extract (FRLFE), with other phenolic compounds including resveratrol. FRLFE as well as hydroquinone and resveratrol suppressed growth of B16F1 melanoma cells more significantly than rhododendrol or arbutin. Resveratrol suppressed mushroom tyrosinase at the lowest concentration (23.0 µmol/L) among the compounds tested. FRLFE and hydroquinone suppressed tyrosinase at almost the same concentration (half maximal inhibitory concentration [IC50 ], 83.5 and 94.6 µmol/L, respectively), which was higher than rhododendrol, ascorbic acid and arbutin (IC50 , 245, 345 and 421 µmol/L, respectively). Western blot analysis revealed that although resveratrol decreased expressions of tyrosinase and tyrosinase-related protein 1, FRLFE did not affect their expressions. Both FRLFE and resveratrol suppressed antimycin A-mediated reactive oxygen species (ROS) production in melanocytic cells. Resveratrol-mediated ROS suppression was inhibited by nicotinamide, a SIRT1 inhibitor. However, FRLFE-mediated suppression was not affected by nicotinamide. Moreover, FRLFE directly decreased superoxide in vitro, as detected by superoxide dismutase-like scavenging activity assay. These results suggest that FRLFE can protect melanocytes from cytotoxicity caused by an excess amount of melanin and ROS in a different manner from resveratrol.

Unsaturated fatty acids-dependent linkage between respiration and fermentation revealed by deletion of hypoxic regulatory KlMGA2 gene in the facultative anaerobe-respiratory yeast Kluyveromyces lactis.

In the yeast Kluyveromyces lactis, the inactivation of structural or regulatory glycolytic and fermentative genes generates obligate respiratory mutants which can be characterized by sensitivity to the mitochondrial drug antimycin A on glucose medium (Rag(-) phenotype). Rag(-) mutations can occasionally be generated by the inactivation of genes not evidently related to glycolysis or fermentation. One such gene is the hypoxic regulatory gene KlMGA2. In this work, we report a study of the many defects, in addition to the Rag(-) phenotype, generated by KlMGA2 deletion. We analyzed the fermentative and respiratory metabolism, mitochondrial functioning and morphology in the Klmga2Δ strain. We also examined alterations in the regulation of the expression of lipid biosynthetic genes, in particular fatty acids, ergosterol and cardiolipin, under hypoxic and cold stress and the phenotypic suppression by unsaturated fatty acids of the deleted strain. Results indicate that, despite the fact that the deleted mutant strain had a typical glycolytic/fermentative phenotype and KlMGA2 is a hypoxic regulatory gene, the deletion of this gene generated defects linked to mitochondrial functions suggesting new roles of this protein in the general regulation and cellular fitness of K. lactis. Supplementation of unsaturated fatty acids suppressed or modified these defects suggesting that KlMga2 modulates membrane functioning or membrane-associated functions, both cytoplasmic and mitochondrial.

Inhibition of Lon blocks cell proliferation, enhances chemosensitivity by promoting apoptosis and decreases cellular bioenergetics of bladder cancer: potential roles of Lon as a prognostic marker and therapeutic target in baldder cancer.

ATP-dependent Lon protease within mitochondrial matrix contributes to the degradation of abnormal proteins. The oxidative or hypoxic stress which represents the stress phenotype of cancer leads to up-regulation of Lon. However, the role of Lon in bladder cancer remains undefined. Here, we found that Lon expression in bladder cancer tissues was significantly higher than those in noncancerous tissues; down-regulation of Lon in bladder cancer cells significantly blocked cancer cell proliferation via suppression c-Jun N-terminal kinase (JNK) phosphorylation due to decreased reactive oxygen species (ROS) production and enhanced the sensitivity of bladder cancer cells to chemotherapeutic agents by promoting apoptosis. We further found that Lon down-regulation in bladder cancer cells decreased cellular bioenergetics as determined by measuring aerobic respiration and glycolysis using extracellular flux analyzer. The tissue microarray (TMA) results showed that high expression of Lon was related to the T and TNM stage, as well as histological grade of bladder cancer patients. We also demonstrated that Lon was an independent prognostic factor for overall survival of bladder cancer. Taken together, our data suggest that Lon could serve as a potential diagnostic biomarker and therapeutic target for treatment of bladder cancer, as well as for prediction of the effectiveness of chemotherapy.

Isoflurane modulates cardiac mitochondrial bioenergetics by selectively attenuating respiratory complexes.

Mitochondrial dysfunction contributes to cardiac ischemia-reperfusion (IR) injury but volatile anesthetics (VA) may alter mitochondrial function to trigger cardioprotection. We hypothesized that the VA isoflurane (ISO) mediates cardioprotection in part by altering the function of several respiratory and transport proteins involved in oxidative phosphorylation (OxPhos). To test this we used fluorescence spectrophotometry to measure the effects of ISO (0, 0.5, 1, 2mM) on the time-course of interlinked mitochondrial bioenergetic variables during states 2, 3 and 4 respiration in the presence of either complex I substrate K(+)-pyruvate/malate (PM) or complex II substrate K(+)-succinate (SUC) at physiological levels of extra-matrix free Ca(2+) (~200nM) and Na(+) (10mM). To mimic ISO effects on mitochondrial functions and to clearly delineate the possible ISO targets, the observed actions of ISO were interpreted by comparing effects of ISO to those elicited by low concentrations of inhibitors that act at each respiratory complex, e.g. rotenone (ROT) at complex I or antimycin A (AA) at complex III. Our conclusions are based primarily on the similar responses of ISO and titrated concentrations of ETC. inhibitors during state 3. We found that with the substrate PM, ISO and ROT similarly decreased the magnitude of state 3 NADH oxidation and increased the duration of state 3 NADH oxidation, ΔΨm depolarization, and respiration in a concentration-dependent manner, whereas with substrate SUC, ISO and ROT decreased the duration of state 3 NADH oxidation, ΔΨm depolarization and respiration. Unlike AA, ISO reduced the magnitude of state 3 NADH oxidation with PM or SUC as substrate. With substrate SUC, after complete block of complex I with ROT, ISO and AA similarly increased the duration of state 3 ΔΨm depolarization and respiration. This study provides a mechanistic understanding in how ISO alters mitochondrial function in a way that may lead to cardioprotection.

Decreased NAA in gray matter is correlated with decreased availability of acetate in white matter in postmortem multiple sclerosis cortex.

Multiple sclerosis (MS) is an inflammatory neurodegenerative disease of the central nervous system (CNS) which leads to progressive neurological disability. Our previous studies have demonstrated mitochondrial involvement in MS cortical pathology and others have documented decreased levels of the neuronal mitochondrial metabolite N-acetyl aspartate (NAA) in the MS brain. While NAA is synthesized in neurons, it is broken down in oligodendrocytes into aspartate and acetate. The resulting acetate is incorporated into myelin lipids, linking neuronal mitochondrial function to oligodendrocyte-mediated elaboration of myelin lipids in the CNS. In the present study we show that treating human SH-SY5Y neuroblastoma cells with the electron transport chain inhibitor antimycin A decreased levels of NAA as measured by HPLC. To better understand the significance of the relationship between mitochondrial function and levels of NAA and its breakdown product acetate on MS pathology we then quantitated the levels of NAA and acetate in MS and control postmortem tissue blocks. Regardless of lesion status, we observed that levels of NAA were decreased 25 and 32 % in gray matter from parietal and motor cortex in MS, respectively, compared to controls. Acetate levels in adjacent white matter mirrored these decreases as evidenced by the 36 and 45 % reduction in acetate obtained from parietal and motor cortices. These data suggest a novel mechanism whereby mitochondrial dysfunction and reduced NAA levels in neurons may result in compromised myelination by oligodendrocytes due to decreased availability of acetate necessary for the synthesis of myelin lipids.

Peroxynitrite formation in nitric oxide-exposed submitochondrial particles: detection, oxidative damage and catalytic removal by Mn-porphyrins.

Peroxynitrite (ONOO(-)) formation in mitochondria may be favored due to the constant supply of superoxide radical (O(2)(∙-)) by the electron transport chain plus the facile diffusion of nitric oxide ((∙)NO) to this organelle. Herein, a model system of submitochondrial particles (SMP) in the presence of succinate plus the respiratory inhibitor antimycin A (to increase O(2)(∙-) rates) and the (∙)NO-donor NOC-7 was studied to directly establish and quantitate peroxynitrite by a multiplicity of methods including chemiluminescence, fluorescence and immunochemical analysis. While all the tested probes revealed peroxynitrite at near stoichiometric levels with respect to its precursor radicals, coumarin boronic acid (a probe that directly reacts with peroxynitrite) had the more straightforward oxidation profile from O(2)(∙-)-forming SMP as a function of the (∙)NO flux. Interestingly, immunospintrapping studies verified protein radical generation in SMP by peroxynitrite. Substrate-supplemented SMP also reduced Mn(III)porphyrins (MnP) to Mn(II)P under physiologically-relevant oxygen levels (3-30 µM); then, Mn(II)P were capable to reduce peroxynitrite and protect SMP from the inhibition of complex I-dependent oxygen consumption and protein radical formation and nitration of membranes. The data directly support the formation of peroxynitrite in mitochondria and demonstrate that MnP can undergo a catalytic redox cycle to neutralize peroxynitrite-dependent mitochondrial oxidative damage.

Deoxyactein stimulates osteoblast function and inhibits bone-resorbing mediators in MC3T3-E1 cells.

Osteoporosis is a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture. In order to improve the treatment of osteoporosis, identification of anabolic agents with minimal side effects is highly desirable. Cimicifuga racemosa has a long and diverse history of medicinal use and deoxyactein isolated from this species is one of the major constituents. In the present study, the effect of deoxyactein on the function of osteoblastic MC3T3-E1 cells was studied. Deoxyactein caused a significant elevation of cell growth, alkaline phosphatase activity, collagen content, and mineralization in the cells (P < 0.05). Moreover, deoxyactein significantly (P < 0.05) decreased the production of reactive oxygen species (ROS) and osteoclast differentiation-inducing factors such as TNF-α, IL-6 and receptor activator of nuclear factor-κB ligand in the presence of antimycin A, which inhibits mitochondrial electron transport and has been used as an ROS generator. These results demonstrate that deoxyactein may have positive effects on skeletal structure.

Honokiol isolated from Magnolia officinalis stimulates osteoblast function and inhibits the release of bone-resorbing mediators.

There has been a strong interest in searching for natural therapies for osteoporosis. Honokiol is a phenolic compound isolated from the bark of Magnolia officinalis, a plant widely used in traditional medicine. In the present study, the effects of honokiol on the function of osteoblastic MC3T3-E1 cells were studied. Honokiol caused a significant elevation of cell growth, alkaline phosphatase activity, collagen synthesis, mineralization, glutathione content, and osteoprotegerin release in the cells (P<0.05). Moreover, honokiol significantly (P<0.05) decreased the production of osteoclast differentiation inducing factors such as TNF-α, IL-6, and receptor activator of nuclear factor-kB ligand (RANKL) in the presence of antimycin A, which inhibits mitochondrial electron transport and has been used as a ROS generator. These results demonstrate that honokiol may have positive effects on skeletal structure.

A pairwise chemical genetic screen identifies new inhibitors of glucose transport.

Oxidative phosphorylation (OXPHOS) and glycolysis are the two main pathways that control energy metabolism of a cell. The Warburg effect, in which glycolysis remains active even under aerobic conditions, is considered a key driver for cancer cell proliferation, malignancy, metastasis, and therapeutic resistance. To target aerobic glycolysis, we exploited the complementary roles of OXPHOS and glycolysis in ATP synthesis as the basis for a chemical genetic screen, enabling rapid identification of novel small-molecule inhibitors of facilitative glucose transport. Blocking mitochondrial electron transport with antimycin A or leucascandrolide A had little effect on highly glycolytic A549 lung carcinoma cells, but adding known glycolytic inhibitors 2-deoxy-D-glucose, iodoacetate or cytochalasin B, rapidly depleted intracellular ATP, displaying chemical synthetic lethality. Based on this principle, we exposed antimycin A-treated A549 cells to a newly synthesized 955 member diverse scaffold small-molecule library, screening for compounds that rapidly depleted ATP levels. Two compounds potently suppressed ATP synthesis, induced G1 cell-cycle arrest and inhibited lactate production. Pathway analysis revealed that these novel probes inhibited GLUT family of facilitative transmembrane transporters but, unlike cytochalasin B, had no effect on the actin cytoskeleton. Our work illustrated the utility of a pairwise chemical genetic screen for discovery of novel chemical probes, which would be useful not only to study the system-level organization of energy metabolism but could also facilitate development of drugs targeting upregulation of aerobic glycolysis in cancer.

Antimycin A-induced mitochondrial apoptotic cascade is mitigated by phenolic constituents of Phyllanthus amarus aqueous extract in Hep3B cells.

Antimycin A (AMA) treatment of cells blocks mitochondrial electron transport chain, and leads to elevated ROS generation, thereby causing damage to mtDNA, proteins and lipids, along with mitochondrial membrane depolarization, release of pro-apoptotic proteins into the cytoplasm, and induction of apoptosis. Prevention of such oxidative cellular damage by the aqueous extract of Phyllanthus amarus has been investigated in this study. The extract demonstrated significant potential in mitigating H(2)O(2)-induced membrane damage along with considerable recession in AMA-governed mitochondrial protein and lipid degradation in Hep3B cells. 8-OHdG analysis of mtDNA damage revealed substantial protective potential of the extract against mtDNA damage. SQ-PCR of selected mtDNA sequences confirmed the potential of the extract to alleviate levels of mtDNA damage. FACS analysis with JC-1 fluorescent dye established significant escalation of mitochondrial membrane potential by the extract in AMA-treated cells. Extract treatment resulted in a distinct decline in the degrees of AMA-induced release of cytochrome c and AIF into the cytoplasm along with consequent pacification of apoptosis. All protective efficiencies of the extract reported in this study were found to hold strong and significant (P<0.05) positive correlation to its total phenolic contents, thereby proving that polyphenolic constituents of P. amarus aqueous extract mitigate oxidative stress-induced cellular degeneration and aging.

Different oxidants and PKC isozymes mediate the opposite effect of inhibition of Q(i) and Q(o) site of mitochondrial complex III on calcium currents in rat cortical neurons.

The inhibition of the complex III of the mitochondrial respiratory chain under hypoxia-ischemia has been observed. However, the downstream events of this inhibition remain to be studied. In this paper, we used the Q(i) site inhibitor antimycin A and the Q(o) site inhibitor myxothiazol to inhibit the Q(i) site and the Q(o) site of the complex III and studied the effect and mechanism of the inhibition of these sites on voltage-gated Ca(2+) currents (I(Ca)) in rat prefrontal neurons with whole cell patch-clamp method in slices. The results showed that antimycin A inhibited I(Ca), but myxothiazol increased it. Further mechanism study showed that antimycin A inhibited I(Ca) via the H(2)O(2)-hydroxyl radicals/cPKC (mainly PKCbetaI) pathway, whereas myxothiazol increased I(Ca) via the superoxide anion/nPKC (mainly the PKCdelta) pathway.