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BMC Cancer Nov 2023Chronic myeloid leukemia (CML) is effectively treated with tyrosine kinase inhibitors (TKIs), targeting the BCR::ABL1 oncoprotein. Still, resistance to therapy, relapse...
Synergistic lethality in chronic myeloid leukemia - targeting oxidative phosphorylation and unfolded protein response effectively complements tyrosine kinase inhibitor treatment.
Chronic myeloid leukemia (CML) is effectively treated with tyrosine kinase inhibitors (TKIs), targeting the BCR::ABL1 oncoprotein. Still, resistance to therapy, relapse after treatment discontinuation, and side effects remain significant issues of long-term TKI treatment. Preliminary studies have shown that targeting oxidative phosphorylation (oxPhos) and the unfolded protein response (UPR) are promising therapeutic approaches to complement CML treatment. Here, we tested the efficacy of different TKIs, combined with the ATP synthase inhibitor oligomycin and the ER stress inducer thapsigargin in the CML cell lines K562, BV173, and KU812 and found a significant increase in cell death. Both, oligomycin and thapsigargin, triggered the upregulation of the UPR proteins ATF4 and CHOP, which was inhibited by imatinib. We observed comparable effects on cell death when combining TKIs with the ATP synthase inhibitor 8-chloroadenosine (8-Cl-Ado) as a potentially clinically applicable therapeutic agent. Stress-related apoptosis was triggered via a caspase cascade including the cleavage of caspase 3 and the inactivation of poly ADP ribose polymerase 1 (PARP1). The inhibition of PARP by olaparib also increased CML death in combination with TKIs. Our findings suggest a rationale for combining TKIs with 8-Cl-Ado or olaparib for future clinical studies in CML.
Topics: Humans; Tyrosine Kinase Inhibitors; Fusion Proteins, bcr-abl; Oxidative Phosphorylation; Thapsigargin; Drug Resistance, Neoplasm; Protein Kinase Inhibitors; Enzyme Inhibitors; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Oligomycins; Adenosine Triphosphate; Apoptosis
PubMed: 38012567
DOI: 10.1186/s12885-023-11623-6 -
Journal of Molecular and Cellular... Jun 2010Screening for cell surface proteins up-regulated under stress conditions may lead to the identification of new therapeutic targets. To search for genes whose expression...
Screening for cell surface proteins up-regulated under stress conditions may lead to the identification of new therapeutic targets. To search for genes whose expression was enhanced by treatment with oligomycin, a mitochondrial-F(0)F(1) ATP synthase inhibitor, signal sequence trapping was performed in H9C2 rat cardiac myoblasts. One of the genes identified was that for neural cell adhesion molecule (NCAM, CD56), a major regulator of development, cell survival, migration, and neurite outgrowth in the nervous system. Immunohistochemical analyses in a mouse myocardial infarction model revealed that NCAM was strongly expressed in residual cardiac myocytes in the infarcted region. Increased expression of NCAM was also found during the remodeling period in a rat model of hypertension-induced heart failure. Lentivirus-mediated knockdown of NCAM decreased the cell growth and survival following oligomycin treatment in H9C2 cells. In primary rat neonatal cardiac myocytes, NCAM was also found to be up-regulated and played a protective role following oligomycin treatment. Analyses of downstream signaling revealed that knockdown of NCAM significantly decreased the basal AKT phosphorylation level. In contrast, NCAM mimetic peptide P2d activated AKT and significantly reduced oligomycin-induced cardiomyocyte death, which was abolished by treatment with the PI3K inhibitor LY-294002 as well as overexpression of the dominant-negative AKT mutant. These findings demonstrate that NCAM is a cardioprotective factor up-regulated under metabolic stress in cardiomyocytes and augmentation of this signal improved survival.
Topics: Animals; Cardiotonic Agents; Cell Membrane; Cell Proliferation; Cell Survival; Enzyme Inhibitors; Male; Mice; Mice, Inbred C57BL; Models, Biological; Myocytes, Cardiac; Neural Cell Adhesion Molecules; Oligomycins; Proton-Translocating ATPases; Rats; Up-Regulation
PubMed: 19853610
DOI: 10.1016/j.yjmcc.2009.10.014 -
International Journal of Molecular... Feb 2021The conserved Blm10/PA200 proteins are proteasome activators. Previously, we identified PA200-enriched regions in the genome of SH-SY5Y neuroblastoma cells by chromatin...
The conserved Blm10/PA200 proteins are proteasome activators. Previously, we identified PA200-enriched regions in the genome of SH-SY5Y neuroblastoma cells by chromatin immunoprecipitation (ChIP) and ChIP-seq analysis. We also found that selective mitochondrial inhibitors induced PA200 redistribution in the genome. Collectively, our data indicated that PA200 regulates cellular homeostasis at the transcriptional level. In the present study, our aim is to investigate the impact of stable PA200 depletion (shPA200) on the overall transcriptome of SH-SY5Y cells. RNA-seq data analysis reveals that the genetic ablation of PA200 leads to overall changes in the transcriptional landscape of SH-SY5Y neuroblastoma cells. PA200 activates and represses genes regulating metabolic processes, such as the glycolysis and mitochondrial function. Using metabolic assays in live cells, we showed that stable knockdown of PA200 does not change basal respiration. Spare respiratory capacity and proton leak however are slightly, yet significantly, reduced in PA200-deficient cells by 99.834% and 84.147%, respectively, compared to control. Glycolysis and glycolytic capacity show a 42.186% and 26.104% increase in shPA200 cells, respectively, compared to control. These data suggest a shift from oxidative phosphorylation to glycolysis especially when cells are exposed to oligomycin-induced stress. Furthermore, we observed a preserved long and compact tubular mitochondrial morphology after inhibition of ATP synthase by oligomycin, which might be associated with the glycolytic change of shPA200 cells. The present study also demonstrates that the proteolytic cleavage of Opa1 is affected, and that the level of OMA1 is significantly reduced in shPA200 cells upon oligomycin-induced mitochondrial insult. Together, these findings suggest a role for PA200 in the regulation of metabolic changes in response to selective inhibition of ATP synthase in an in vitro cellular model.
Topics: Cell Line, Tumor; Chromatin Immunoprecipitation; GTP Phosphohydrolases; Gene Deletion; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Glycolysis; Humans; Mitochondria; Neuroblastoma; Nuclear Proteins; Oligomycins; Oxidative Phosphorylation; RNA, Small Interfering; Sequence Analysis, RNA
PubMed: 33562813
DOI: 10.3390/ijms22041629 -
American Journal of Physiology. Heart... Jul 2006Diabetic hearts are known to be more susceptible to ischemic disease. Biguanides, like metformin, are known antidiabetic drugs that lower blood glucose concentrations by...
Diabetic hearts are known to be more susceptible to ischemic disease. Biguanides, like metformin, are known antidiabetic drugs that lower blood glucose concentrations by decreasing hepatic glucose production and increasing glucose disposal in muscle. Part of these metabolic effects is thought to be mediated by the activation of AMP-activated protein kinase (AMPK). In this work, we studied the relationship between AMPK activation and glucose uptake stimulation by biguanides and oligomycin, another AMPK activator, in both insulin-sensitive and insulin-resistant cardiomyocytes. In insulin-sensitive cardiomyocytes, insulin, biguanides and oligomycin were able to stimulate glucose uptake with the same efficiency. Stimulation of glucose uptake by insulin or biguanides was correlated to protein kinase B (PKB) or AMPK activation, respectively, and were additive. In insulin-resistant cardiomyocytes, where insulin stimulation of glucose uptake was greatly reduced, biguanides or oligomycin, in the absence of insulin, induced a higher stimulation of glucose uptake than that obtained in insulin-sensitive cells. This stimulation was correlated with the activation of both AMPK and PKB and was sensitive to the phosphatidylinositol-3-kinase/PKB pathway inhibitors. Finally, an adenoviral-mediated expression of a constitutively active form of AMPK increased both PKB phosphorylation and glucose uptake in insulin-resistant cardiomyocytes. We concluded that AMPK activators, like biguanides and oligomycin, are able to restore glucose uptake stimulation, in the absence of insulin, in insulin-resistant cardiomyocytes via the additive activation of AMPK and PKB. Our results suggest that AMPK activation could restore normal glucose metabolism in diabetic hearts and could be a potential therapeutic approach to treat insulin resistance.
Topics: AMP-Activated Protein Kinases; Animals; Biguanides; Cells, Cultured; Dose-Response Relationship, Drug; Drug Combinations; Enzyme Activation; Glucose; Insulin; Insulin Resistance; Male; Multienzyme Complexes; Myocytes, Cardiac; Oligomycins; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar
PubMed: 16489105
DOI: 10.1152/ajpheart.01269.2005 -
The Journal of Physiology Jun 19671. Slices of brain cortex from rabbits were incubated in Ringer solution and in Ringer modified by the removal of calcium and sodium, and the addition of ouabain,...
1. Slices of brain cortex from rabbits were incubated in Ringer solution and in Ringer modified by the removal of calcium and sodium, and the addition of ouabain, oligomycin or extra potassium. The potassium content of the tissue, the oxygen consumption and the lactate production from glucose were measured and found to be interrelated.2. Incubation in high-K Ringer caused an increase in oxygen consumption that was prevented by ouabain, oligomycin and deprivation of sodium. Lactate production was also raised, but this increase was unaffected by ouabain and raised further by oligomycin.3. Calcium omission raised metabolism; the tissue K content was unaffected. Oligomycin always decreased oxygen consumption and raised lactate production further. The metabolic responses to calcium, potassium, ouabain and oligomycin depended on sodium.4. After anaerobic incubation, the tissue potassium concentration was still 5 times higher than that in Ringer. It was unaffected by oligomycin but lowered markedly by ouabain.5. The synergistic effects of sodium with potassium, oligomycin, calcium, and calcium plus ouabain suggest that the metabolic responses of brain cortex slices to a high-K Ringer depend on the operation of the sodium pump.
Topics: Animals; Biological Transport, Active; Calcium; Cell Membrane Permeability; Cerebral Cortex; Drug Synergism; In Vitro Techniques; Lactates; Oligomycins; Ouabain; Oxygen Consumption; Potassium; Rabbits; Sodium
PubMed: 6051789
DOI: 10.1113/jphysiol.1967.sp008230 -
Cells Feb 2023Understanding the role of astrocytes in the development of the nervous system and neurodegenerative disorders implies a necessary knowledge of the oxidative metabolism...
Understanding the role of astrocytes in the development of the nervous system and neurodegenerative disorders implies a necessary knowledge of the oxidative metabolism of proliferating astrocytes. The electron flux through mitochondrial respiratory complexes and oxidative phosphorylation may impact the growth and viability of these astrocytes. Here, we aimed at assessing to which extent mitochondrial oxidative metabolism is required for astrocyte survival and proliferation. Primary astrocytes from the neonatal mouse cortex were cultured in a physiologically relevant medium with the addition of piericidin A or oligomycin at concentrations that fully inhibit complex I-linked respiration and ATP synthase, respectively. The presence of these mitochondrial inhibitors for up to 6 days in a culture medium elicited only minor effects on astrocyte growth. Moreover, neither the morphology nor the proportion of glial fibrillary acidic protein-positive astrocytes in culture was affected by piericidin A or oligomycin. Metabolic characterization of the astrocytes showed a relevant glycolytic metabolism under basal conditions, despite functional oxidative phosphorylation and large spare respiratory capacity. Our data suggest that astrocytes in primary culture can sustainably proliferate when their energy metabolism relies only on aerobic glycolysis since their growth and survival do not require electron flux through respiratory complex I or oxidative phosphorylation.
Topics: Mice; Animals; Oxidative Phosphorylation; Electron Transport Complex I; Astrocytes; Mitochondria; Oligomycins
PubMed: 36899819
DOI: 10.3390/cells12050683 -
The Journal of Clinical Investigation Dec 1987To investigate the mechanisms responsible for urinary acidification in the terminal nephron, primary cultures of cells isolated from the renal papilla were grown as...
To investigate the mechanisms responsible for urinary acidification in the terminal nephron, primary cultures of cells isolated from the renal papilla were grown as monolayers in a defined medium. Morphologically, cultured cells were epithelial in type, and similar to collecting duct principal cells. Cell pH measured fluorometrically in monolayers grown on glass slides showed recovery from acid loads in Na+-free media. Recovery was inhibited by cyanide, oligomycin A, and N-ethylmaleimide. Cyanide and oligomycin inhibited recovery less in the presence than in the absence of glucose. When cells were first acid loaded in a Na+-free medium and then exposed to external Na+, pH recovery also took place. This recovery exhibited first-order dependence on Na+ concentration and was inhibited by 5-(N-ethyl-N-isopropyl)amiloride. These studies demonstrate that in culture, collecting duct principal cells possess at least two mechanisms for acid extrusion: a proton ATP-ase and an Na+-H+ exchanger. The former may be responsible for some component of the urinary acidification observed in the papillary collecting duct in vivo; the role of the latter in acid-base transport remains uncertain.
Topics: Acid-Base Equilibrium; Animals; Cyanides; Ethylmaleimide; Hydrogen-Ion Concentration; Kidney Medulla; Kidney Tubules; Kidney Tubules, Collecting; Microscopy, Electron; Oligomycins; Rats
PubMed: 3680519
DOI: 10.1172/JCI113255 -
The Journal of Biological Chemistry Jan 1994It has long been known that pyruvate is essential for survival of prenatal neurons in culture. To understand the role of exogenous pyruvate in neuronal calcium...
It has long been known that pyruvate is essential for survival of prenatal neurons in culture. To understand the role of exogenous pyruvate in neuronal calcium homeostasis, we have investigated the effects of pyruvate (plus malate) addition to dissociated adult rat hippocampal and cerebral cortex cells and cultured CNS neurons having an unrestricted glucose supply. We found that pyruvate (plus malate) increased the respiration rate while ATP levels were unchanged. At the same time, cytosolic free calcium concentrations, [Ca2+]i, decreased while total 45Ca2+ and 40Ca2+ accumulation increased. The extra Ca2+ accumulated by the cells is attributable to an increase in the size of the intracellular calcium pools. Two such pools were identified on the basis of their sensitivity to specific drugs. The first pool was mobilized by thapsigargin plus tert-butyl hydroquinone and caffeine while the second pool was discharged by the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxphenylhydrazone (FCCP) (plus oligomycin). The two pools represented about 15-20% and 15-30%, respectively, of the rapidly exchangeable 45Ca2+ pools in cerebral cortex cells. In cultured hippocampal neurons, the collapse of the mitochondrial membrane potential (as induced by uncouplers (FCCP) or respiratory chain inhibitors (antimycin) caused a large increase in [Ca2+]i which varied in size and shape among cells and was reduced by external Ca2+ chelation. The latter condition also resulted in a partial discharge of FCCP-releasable 45Ca2+. The effects of FCCP did not result simply from ATP depletion since incubation in glucose-free medium and sequential additions of 2 mM deoxyglucose and 10 microM oligomycin, conditions that led to a dramatic reduction in cellular ATP levels, did not abolish the FCCP-induced [Ca2+]i rise. Taken together, the results indicate that mitochondria harbor a significant proportion of cellular Ca2+. The sensitivity of the mitochondrial pool size to pyruvate (plus malate) questions previous hypotheses concerning a kinetic limitation for Ca2+ accumulation in mitochondria in resting neurons.
Topics: Acetylcholine; Adenosine Diphosphate; Adenosine Triphosphate; Animals; Calcium; Calcium Radioisotopes; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cells, Cultured; Cerebral Cortex; Cytosol; Deoxyglucose; Hippocampus; Homeostasis; Hydrogen-Ion Concentration; Kinetics; Malates; Neurons; Oligomycins; Oxygen Consumption; Pyruvates; Rats; Ruthenium Red
PubMed: 7507925
DOI: No ID Found -
Nature Communications Sep 2016In most eukaryotes, mitochondria are inherited maternally. The autophagy process is critical for paternal mitochondrial elimination (PME) in Caenorhabditis elegans, but...
In most eukaryotes, mitochondria are inherited maternally. The autophagy process is critical for paternal mitochondrial elimination (PME) in Caenorhabditis elegans, but how paternal mitochondria, but not maternal mitochondria, are selectively targeted for degradation is poorly understood. Here we report that mitochondrial dynamics have a profound effect on PME. A defect in fission of paternal mitochondria delays PME, whereas a defect in fusion of paternal mitochondria accelerates PME. Surprisingly, a defect in maternal mitochondrial fusion delays PME, which is reversed by a fission defect in maternal mitochondria or by increasing maternal mitochondrial membrane potential using oligomycin. Electron microscopy and tomography analyses reveal that a proportion of maternal mitochondria are compromised when they fail to fuse normally, leading to their competition for the autophagy machinery with damaged paternal mitochondria and delayed PME. Our study indicates that mitochondrial dynamics play a critical role in regulating both the kinetics and the specificity of PME.
Topics: Animals; Autophagy; Caenorhabditis elegans; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Dynamics; Oligomycins; Uncoupling Agents
PubMed: 27581092
DOI: 10.1038/ncomms12569 -
Autophagy Nov 2016Pathological mutations in the mitochondrial DNA (mtDNA) produce a diverse range of tissue-specific diseases and the proportion of mutant mitochondrial DNA can increase...
Pathological mutations in the mitochondrial DNA (mtDNA) produce a diverse range of tissue-specific diseases and the proportion of mutant mitochondrial DNA can increase or decrease with time via segregation, dependent on the cell or tissue type. Previously we found that adenocarcinoma (A549.B2) cells favored wild-type (WT) mtDNA, whereas rhabdomyosarcoma (RD.Myo) cells favored mutant (m3243G) mtDNA. Mitochondrial quality control (mtQC) can purge the cells of dysfunctional mitochondria via mitochondrial dynamics and mitophagy and appears to offer the perfect solution to the human diseases caused by mutant mtDNA. In A549.B2 and RD.Myo cybrids, with various mutant mtDNA levels, mtQC was explored together with macroautophagy/autophagy and bioenergetic profile. The 2 types of tumor-derived cell lines differed in bioenergetic profile and mitophagy, but not in autophagy. A549.B2 cybrids displayed upregulation of mitophagy, increased mtDNA removal, mitochondrial fragmentation and mitochondrial depolarization on incubation with oligomycin, parameters that correlated with mutant load. Conversely, heteroplasmic RD.Myo lines had lower mitophagic markers that negatively correlated with mutant load, combined with a fully polarized and highly fused mitochondrial network. These findings indicate that pathological mutant mitochondrial DNA can modulate mitochondrial dynamics and mitophagy in a cell-type dependent manner and thereby offer an explanation for the persistence and accumulation of deleterious variants.
Topics: A549 Cells; Autophagy; Cytoplasm; DNA, Mitochondrial; Energy Metabolism; Humans; Microtubule-Associated Proteins; Mitochondria; Mitophagy; Mutation; Oligomycins; Sequestosome-1 Protein
PubMed: 27627835
DOI: 10.1080/15548627.2016.1226734