oligomycins and Glioma

Oxidative phosphorylation and glycolysis are important features, by which cells could bypass oxidative stress. The level of oxidative stress, and the ability of cells to promote oxidative phosphorylation or glycolysis, significantly determined proliferation or cell demise. In the present work, we have employed selective mitochondrial probe MitoTracker™ Orange CMTM/Ros (MTO) to estimate the level of oxidative stress in cancer cells at different stressed conditions. MTO is partially sensitive to decrease of mitochondrial membrane potential and to reactive oxygen species (ROS) generated in mitochondria. We have demonstrated, that fluorescence lifetime of MTO is much more sensitive to oxidative stress than intensity-based approaches. This method was validated in different cancer cell lines. Our approach revealed, at relatively low ROS levels, that Gö 6976, a protein kinase C (PKC) α inhibitor, and rottlerin, an indirect PKCδ inhibitor, increased mitochondrial ROS level in glioma cell. Their involvement in oxidative phosphorylation and apoptosis was investigated with oxygen consumption rate estimation, western blot and flow-cytometric analysis. Our study brings new insight to identify feeble differences in ROS production in living cells.

Topics: Acetophenones; Antimycin A; Apoptosis; Benzopyrans; Carbazoles; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Line, Tumor; Flow Cytometry; Glioma; Glutathione; Humans; Kinetics; Microscopy, Fluorescence; Mitochondria; Molecular Imaging; Oligomycins; Oxidative Stress; Oxygen Consumption; Rotenone; Superoxides; Time Factors

Gliomas contain a small number of treatment-resistant glioma stem cells (GSCs), and it is thought that tumor regrowth originates from GSCs, thus rendering GSCs an attractive target for novel treatment approaches. Cancer cells rely more on glycolysis than on oxidative phosphorylation for glucose metabolism, a phenomenon used in 2-[(18)F]fluoro-2-deoxy-D-glucose positron emission tomography imaging of solid cancers, and targeting metabolic pathways in cancer cells has become a topic of considerable interest. However, if GSCs are indeed important for tumor control, knowledge of the metabolic state of GSCs is needed. We hypothesized that the metabolism of GSCs differs from that of their progeny. Using a unique imaging system for GSCs, we assessed the oxygen consumption rate, extracellular acidification rate, intracellular ATP levels, glucose uptake, lactate production, PKM1 and PKM2 expression, radiation sensitivity, and cell cycle duration of GSCs and their progeny in a panel of glioma cell lines. We found GSCs and progenitor cells to be less glycolytic than differentiated glioma cells. GSCs consumed less glucose and produced less lactate while maintaining higher ATP levels than their differentiated progeny. Compared with differentiated cells, GSCs were radioresistant, and this correlated with a higher mitochondrial reserve capacity. Glioma cells expressed both isoforms of pyruvate kinase, and inhibition of either glycolysis or oxidative phosphorylation had minimal effect on energy production in GSCs and progenitor cells. We conclude that GSCs rely mainly on oxidative phosphorylation. However, if challenged, they can use additional metabolic pathways. Therefore, targeting glycolysis in glioma may spare GSCs.

Topics: Adenosine Triphosphate; Blotting, Western; Cell Line, Tumor; Clone Cells; Deoxyglucose; Energy Metabolism; Glioma; Glucose; Glycolysis; Humans; Immunohistochemistry; Lactates; Neoplastic Stem Cells; Oligomycins; Oxygen Consumption; Positron-Emission Tomography; Proteasome Endopeptidase Complex; Reactive Oxygen Species; Stem Cells; Tissue Array Analysis; Uncoupling Agents

Topics: Adenosine Triphosphate; Amino Acid Sequence; Bepridil; Biological Transport; Brain Ischemia; Calcium; Deoxyglucose; Glioma; Glycolysis; Glycosylation; Humans; Hypoxia, Brain; L-Lactate Dehydrogenase; Models, Neurological; Molecular Sequence Data; Neuroprotective Agents; Oligomycins; Peptides; Phosphorylation; Sodium; Sodium-Calcium Exchanger; Tumor Cells, Cultured

The intracellular nonmitochondrial calcium pools of saponin-permeabilized NG108-15 cells were characterized using inositol 1,4,5-trisphosphate (IP3) and GTP. IP3 or GTP alone induced release of 47 and 68%, respectively, of the calcium that was releasable by A23187. GTP induced release of a further 24% of the calcium after IP3 treatment, whereas IP3 induced release of a further 11% of the calcium after GTP treatment. Guanosine 5'-O-(3-thio)triphosphate had little effect on IP3-induced calcium release but completely inhibited GTP-induced calcium release. In contrast, heparin inhibited the action of IP3 but not that of GTP. The results imply the existence of at least three nonmitochondrial pools: (a) 31% is releasable by IP3 and GTP, (b) 11% is releasable by IP3 alone, and (c) 24% is releasable by GTP alone. GTP enhanced calcium uptake in the presence of oxalate with an EC50 of 0.6 microM and stimulated calcium release in the absence of oxalate with an EC50 of 0.32 microM. The similar EC50 values for these dual effects of GTP on calcium movement suggest that GTP exerts its dual action by the same mechanism.

Topics: Adenosine Triphosphate; Animals; Biological Transport, Active; Calcimycin; Calcium; Calcium-Transporting ATPases; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Line; Cell Membrane Permeability; Glioma; Guanosine Triphosphate; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Kinetics; Mice; Neuroblastoma; Oligomycins; Rats

Rat glioma cells grown in culture secrete cyclic adenosine 3':5'-monophosphate (cyclic AMP) into the culture medium following stimulation by beta-agonistic catecholamines. Agents which reduced cellular ATP levels such as valinomycin, oligomycin, and uncouplers of oxidative phosphorylation, inhibited cyclic AMP efflux. Secretion of cyclic AMP was also prevented by prostaglandin A-1 and pharmacological agents including probenecid and papaverine. Of the latter agents, only papaverine reduced ATP levels. These results suggest that the transport of cyclic AMP across animal cell membranes is energy-dependent and subject to regulation.

Topics: Animals; Biological Transport; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Membrane; Cells, Cultured; Clone Cells; Cyclic AMP; Glioma; Neoplasms, Experimental; Oligomycins; Papaverine; Potassium Chloride; Probenecid; Propranolol; Prostaglandins A; Prostaglandins E; Prostaglandins F; Rats; Time Factors; Valinomycin