oligomycins and Necrosis

Acidic lysosomes are indispensable for cancer development and linked to chemotherapy resistance. Chloroquine (CQ) and functional analogues have been considered as a potential solution to overcome the cancer progression and chemoresistance by inhibiting the lysosome-mediated autophagy and multidrug exocytosis. However, their anti-cancer efficacy in most clinical trials demonstrated modest improvement. In this study, we investigated the detailed mechanisms underlying the acquired resistance of K562 leukemic cells to CQ treatment. In response to 5-80 μM CQ, the lumen pH of endosomal-lysosomal system immediately increased and gradually reached dynamic equilibrium within 24 h. Leukemic cells produced more acidic organelles to tolerate 5-10 μM CQ. CQ (20-80 μM) concentration-dependently triggered cytosolic pH (pHi) rise, G0/G1 arrest, mitochondrial depolarization/fragmentation, and necrotic/apoptotic cell death. Oxidant induction by CQ was responsible for the mitochondria-dependent cytotoxicity and partial pHi elevation. Cells that survived the CQ cytotoxicity were accompanied with increased mitochondria. Under the 80 μM CQ challenge, co-treatment with the inhibitor of F

Topics: Apoptosis; Autophagy; Cell Line, Tumor; Chloroquine; Humans; Hydrogen-Ion Concentration; Lysosomes; Mitochondria; Necrosis; Oligomycins

Cellular metabolism can impact cell life or death outcomes. While metabolic dysfunction has been linked to cell death, the mechanisms by which metabolic dysfunction regulates the cell death mode called necroptosis remain unclear. Our study demonstrates that mitochondrial oxidative phosphorylation (OXPHOS) activates programmed necrotic cell death (necroptosis) in human lung epithelial cells. Inhibition of mitochondrial respiration and ATP synthesis induced the phosphorylation of mixed lineage kinase domain-like protein (MLKL) and necroptotic cell death. Furthermore, we demonstrate that the activation of AMP-activated protein kinase (AMPK), resulting from impaired mitochondrial OXPHOS, regulates necroptotic cell death. These results suggest that impaired mitochondrial OXPHOS contributes to necroptosis in human lung epithelial cells.

Topics: Acrylamides; Adenosine Triphosphate; AMP-Activated Protein Kinases; Apoptosis; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Respiration; Cells, Cultured; Epithelial Cells; Humans; Lung; Mitochondria; Necrosis; Oligomycins; Oligopeptides; Oxidative Phosphorylation; Sulfonamides

The physical law of diffusion imposes O2 concentration gradients from the plasma membrane to the center of the cell. The present study was undertaken to determine how such intracellular radial gradients of O2 affect the fate of isolated single cardiomyocytes. In single rat cardiomyocytes, mitochondrial respiration was moderately elevated by an oxidative phosphorylation uncoupler to augment the intracellular O2 gradient. At physiological extracellular O2 levels (2-5%), decreases in myoglobin O2 saturation and increases in NADH fluorescence at the center of the cell were imaged (anoxic cell core) while the mitochondrial membrane potential (DeltaPsim) and ATP levels at the anoxic cell core were relatively sustained. In contrast, treatment with 0.5 mM iodoacetamide (IA) to inhibit creatine kinase (CK) resulted in depletion of both DeltaPsim and ATP at the anoxic cell core. Even at normal extracellular Po2, actively respiring cardiomyocytes developed rigor contracture followed by necrotic cell death. Furthermore, such rigor was remarkably accelerated by IA, whereas cell injury was perfectly rescued by mitochondrial F1Fo inhibition by oligomycin. These results suggest that increases in radial gradients of O2 potentially promote cell death through the reverse action of F1Fo in mitochondria located at the anoxic cell core. However, in the intact cardiomyocyte, the CK-mediated energy flux from the subsarcolemmal space may sustain DeltaPsim at the cell core, thus avoiding uncontrolled consumption of ATP that can lead to necrotic cell death. Mitochondria at the anoxic core can cause necrotic cell death in cardiomyocytes at physiological extracellular Po2.

Topics: Adenosine Triphosphate; Animals; Cell Hypoxia; Cell Respiration; Cell Survival; Creatine Kinase; Diffusion; Enzyme Inhibitors; Iodoacetamide; Membrane Potential, Mitochondrial; Mitochondria, Heart; Myocytes, Cardiac; Myoglobin; NAD; Necrosis; Oligomycins; Oxidative Phosphorylation; Oxygen; Phosphocreatine; Rats; Rats, Sprague-Dawley; Time Factors; Uncoupling Agents

Detailed mechanisms of the switch of the cell death mode from apoptosis to necrosis remain to be solved, although the intracellular level of ATP and that of free radicals have been postulated to be the major factors involved in the mechanisms. In the present study menadione (MEN)-induced cell injury processes were studied using rho0 cells derived from human osteosarcoma 143B cells and parental rho+ cells co-treated with inhibitors of electron transfer chain of mitochondria or oligomycin, an inhibitor of ATP synthesis. Treatment of rho+ cells with 100 microM MEN induced apoptosis, which reached the maximum at 6 h, and was followed by an abrupt decrease thereafter, while necrotic cells (NC) increased continuously when they were judged by Annexin V and PI double staining. On the other hand, MEN induced apoptotic and necrotic changes much faster in rho0 cells compared to rho+ cells. The frequency to find apoptotic cells (AP) in the former cells was distinctly smaller than that to find NC judged by Annexin V and PI double staining. Electron microscopically, a major population of rho0 cells treated with MEN for 6 h consisted of intermediate cells, and a small number of AP co-existed. At 9 h of the treatment intermediate cells were exclusively seen, and AP were hardly detected. When parental rho+ cells were treated with MEN in the presence of oligomycin or oligomycin plus antimycin A both apoptotic and necrotic changes of the cells were distinctly accelerated. The intracellular level of superoxide in rho0 cells continuously increased after the MEN treatment, whereas that of ATP remained distinctly low before and after the MEN treatment compared to that in rho+ cells. These data suggest that the intracellular level of superoxide may be a key factor controlling the switch from apoptosis to necrosis.

Topics: Adenosine Triphosphate; Apoptosis; Cell Line, Tumor; Electron Transport; Flow Cytometry; Humans; Membrane Potentials; Microscopy, Electron, Transmission; Mitochondria; Necrosis; Oligomycins; Oxidative Phosphorylation; Protein Synthesis Inhibitors; Reactive Oxygen Species; Vitamin K 3

In pathological situations, different modes of cell death are observed, and information on the role and uptake of nonapoptotic corpses is scarce. Here, we modeled two distinct forms of death in human Jurkat T cells treated with staurosporine: classical apoptosis under normal culture conditions and programmed death with necrotic morphology under ATP-depleting conditions (necPCD). When offered to phagocytes, both types of cell corpses (but not heat-killed unscheduled necrotic cells) reduced the release of the proinflammatory cytokine TNF from the macrophages. The necPCD cells were efficiently engulfed by macrophages and microglia, and from mixtures of necPCD and apoptotic cells macrophages preferentially engulfed the necrotic cells. Using a newly developed assay, we demonstrated that phosphatidylserine is translocated to the surface of such necrotic cells. We demonstrate that this can occur independently of calcium signals, and that surface phosphatidylserine is essential for the uptake of necrotic cells by both human macrophages and murine microglia.

Topics: Animals; Annexin A5; Antibodies, Monoclonal; Apoptosis; Calcium; CD36 Antigens; Cell Line; Cell Membrane; Cells, Cultured; Escherichia coli; Formaldehyde; Humans; Inflammation; Ionomycin; Jumonji Domain-Containing Histone Demethylases; Jurkat Cells; Lipopolysaccharide Receptors; Liposomes; Macrophages; Membrane Lipids; Mice; Microglia; Microscopy, Confocal; Microscopy, Fluorescence; Necrosis; Oligomycins; Oligopeptides; Phagocytosis; Phosphatidylserines; Polymers; Receptors, Cell Surface; Staurosporine; Tumor Necrosis Factor-alpha

In order to investigate the relationship between nitric oxide-mediated regulation of mitochondrial function and excitotoxicity, the role of mitochondrial ATP synthesis and intracellular redox status on the mode of neuronal cell death was studied. Brief (5 min) glutamate (100 microM) receptor stimulation in primary cortical neurons collapsed the mitochondrial membrane potential (psi(m)) and transiently (30 min) inhibited mitochondrial ATP synthesis, causing early (1 h) necrosis or delayed (24 h) apoptosis. The transient inhibition of ATP synthesis was paralleled to a loss of NADH, which was fully recovered shortly after the insult. In contrast, NADPH and the GSH/GSSG ratio were maintained, but progressively decreased thereafter. Twenty-four hours after glutamate treatment, ATP was depleted, a phenomenon associated with a persistent inhibition of mitochondrial succinate-cytochrome c reductase activity and delayed necrosis. Blockade of either nitric oxide synthase (NOS) activity or the mitochondrial permeability transition (MPT) pore prevented psi(m) collapse, the transient inhibition of mitochondrial ATP synthesis, early necrosis and delayed apoptosis. However, blockade of NOS activity, but not the MPT pore, prevented the inhibition of succinate-cytochrome c reductase activity and delayed ATP depletion and necrosis. From these results, we suggest that glutamate receptor-mediated NOS activation would trigger MPT pore opening and transient inhibition of ATP synthesis leading to apoptosis in a neuronal subpopulation, whereas other groups of neurons would undergo oxidative stress and persistent inhibition of ATP synthesis leading to necrosis.

Topics: 2-Amino-5-phosphonovalerate; Adenosine Triphosphate; Animals; Apoptosis; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cells, Cultured; Cerebral Cortex; Cyclosporine; Electron Transport; Electron Transport Complex I; Energy Metabolism; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Glutamic Acid; Isoenzymes; Membrane Potentials; Mitochondria; Models, Biological; NAD; NADH, NADPH Oxidoreductases; Necrosis; Nerve Tissue Proteins; Neurons; Neurotoxins; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Oligomycins; omega-N-Methylarginine; Permeability; Rats; Rats, Wistar; Receptors, Glutamate; Rotenone; Single-Blind Method; Succinate Cytochrome c Oxidoreductase

Cell death due to necrosis results in acute inflammation, while death by apoptosis generally does not. The effect of adenosine triphosphate (ATP) on the pattern of cell death induced by oxidants was examined in bovine endothelial cells. ATP levels were altered by hydrogen peroxide (H2O2), glutamine (Gln), and metabolic inhibition (MI), to determine if necrosis can be shifted to apoptosis during oxidant injury. The form of cell death was determined by fluorescence microscopic techniques and the pattern of DNA degradation on agarose gels. ATP levels were measured using the luciferase-luciferin assay. Apoptosis occurred with 100 microM H2O2 without an alteration in ATP levels. ATP was significantly lowered with 5 mM H2O2, and necrosis occurred. MI, in combination with 100 microM H2O2, decreased ATP and resulted in necrosis. MI alone, however, did not cause cell death. Gln partially restored ATP levels in cells injured with 5 mM H2O2 and resulted in a significant increase in apoptosis. DNA laddering on agarose gels confirmed the apoptotic changes seen by fluorescence microscopy. In summary, a threshold level of ATP 25% of basal levels is required for apoptosis to proceed after oxidant stress, otherwise necrosis occurs. Agents like glutamine that enhance ATP levels in oxidant-stressed cells may be potent means of shifting cell death during inflammation to the noninflammatory form of death--apoptosis.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Cattle; Cell Survival; DNA Fragmentation; Endothelium, Vascular; Glutamine; Hydrogen Peroxide; Microscopy, Fluorescence; Necrosis; Oligomycins; Oxidative Stress; Pulmonary Artery; Time Factors