oligomycins and Melanoma

Canine malignant melanoma (CMM) is a highly aggressive and fatal neoplasm. To identify potential therapeutic compounds and/or targets, 320 compounds were screened for their growth inhibitory activity in a CMM line (CMM-1) using a chemical library known to target specific signaling pathways/cell growth-related molecules. Among the compounds screened, the F1Fo ATPase inhibitor oligomycin showed potent growth inhibitory effects in CMM-1 cells, while exhibiting less toxic effects in a non-neoplastic control cell line (MDCK cells). The growth inhibitory effect of oligomycin A was then examined using six CMM lines and MDCK cells. Three CMM lines were highly sensitive to oligomycin A, with around 3000-20 000 times lower IC

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Dog Diseases; Dogs; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Madin Darby Canine Kidney Cells; Melanoma; Oligomycins; Proton-Translocating ATPases

Hydrogen peroxide (H2O2) production due to oxidative stress is associated with apoptosis and melanogenesis in melanocytes. Here, we analyzed the effects of H2O2 on melanogenesis by measuring the melanin content and analyzing the expression of melanogenesis-related proteins, including cAMP-responsive element binding protein (CREB), microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), and phenylalanine hydroxylase (PAH). Treatment with 1mM H2O2 increased the cellular melanin content; the expression of PAH, TYR, and MITF; and the phosphorylation of CREB in B16F10 and SK-Mel-2 cells. In addition, H2O2 increased the expression of ATP synthase β (ATP5B), a mitochondrial F1 complex, and increased intracellular ATP levels. Studies using the ATP5B inhibitor oligomycin (OM) showed that the induction of cAMP resulted from an increase in ATP caused by the induction of ATP5B. OM treatment increased H2O2-mediated apoptosis via accelerated ATP depletion and apoptosis-related gene expressions. In summary, H2O2 may induce melanogenesis via the upregulation of PAH and activation of cAMP/p-CREB/MITF signaling by increasing intracellular cAMP levels through the induction of ATP5B.

Topics: Adenosine Triphosphate; Adenylyl Cyclase Inhibitors; Animals; Apoptosis; Cell Line, Tumor; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Humans; Hydrogen Peroxide; Imines; Melanins; Melanocytes; Melanoma; Mice; Microphthalmia-Associated Transcription Factor; Mitochondria; Mitochondrial Proton-Translocating ATPases; Oligomycins; Oxidative Stress; Phenylalanine Hydroxylase; Signal Transduction

Despite success with BRAFV600E inhibitors, therapeutic responses in patients with metastatic melanoma are short-lived because of the acquisition of drug resistance. We identified a mechanism of intrinsic multidrug resistance based on the survival of a tumor cell subpopulation. Treatment with various drugs, including cisplatin and vemurafenib, uniformly leads to enrichment of slow-cycling, long-term tumor-maintaining melanoma cells expressing the H3K4-demethylase JARID1B/KDM5B/PLU-1. Proteome-profiling revealed an upregulation in enzymes of mitochondrial oxidative-ATP-synthesis (oxidative phosphorylation) in this subpopulation. Inhibition of mitochondrial respiration blocked the emergence of the JARID1B(high) subpopulation and sensitized melanoma cells to therapy, independent of their genotype. Our findings support a two-tiered approach combining anticancer agents that eliminate rapidly proliferating melanoma cells with inhibitors of the drug-resistant slow-cycling subpopulation.

Topics: Antineoplastic Agents; Cisplatin; Drug Resistance, Neoplasm; Electron Transport; Gene Knockdown Techniques; Humans; Indoles; Jumonji Domain-Containing Histone Demethylases; Melanoma; Nuclear Proteins; Oligomycins; Repressor Proteins; Sulfonamides; Vemurafenib

DC-81, an antitumor antibiotic produced by the Streptomyces species, belongs to pyrrolo[2,1-c] [1,4]benzodiazepine (PBD), which are potent inhibitors of nucleic acid synthesis. We previously reported an efficient synthesis of PBD hybrids linked with indole carboxylates. This is the first demonstration on the mechanism of the anticancer effect of PBD hybrid (IN6CPBD) agent on human melanoma A375 cells. IN6CPBD-treated cells exhibited higher cytotoxicity than DC-81 and displayed several features of apoptosis, including an increase in the sub-G1 population, a significantly increased annexin V binding, a degradation of caspase-3, and poly (ADP-ribose) polymerase (PARP) cleavage. Because degradative changes associated with apoptosis are often preceded by the disruption of mitochondrial function, the assessment of mitochondrial function in IN6CPBD-treated cells is worthy of investigation. Our data revealed that treatment of A375 cells with IN6CPBD resulted in the loss of mitochondrial membrane potential (DeltaPsimt), a decrease in intracellular pH (pHi), a reduction of ATP synthesis, increased reactive oxygen species (ROS) generation, and cytochrome c release. Collectively, our studies indicate that IN6CPBD induces apoptosis in A375 cells through a mitochondrial dysfunction pathway, leading to caspase-3 substrate PARP cleavage and subsequent apoptotic cell death.

Topics: Adenosine Triphosphate; Antineoplastic Agents; Apoptosis; Azepines; Benzodiazepines; Blotting, Western; Caspase 3; Catalase; Cell Line, Tumor; Cell Survival; Collagen Type XI; Cytochromes c; Dioxins; Dose-Response Relationship, Drug; Flow Cytometry; G1 Phase; Humans; Hydrogen-Ion Concentration; Indoles; Melanoma; Membrane Potential, Mitochondrial; Mitochondria; Nucleic Acids; Oligomycins; Organophosphorus Compounds; Reactive Oxygen Species; Superoxide Dismutase

In this paper the specific mitochondrial respiratory chain inhibitors rotenone and antimycin A and the highly specific mitochondrial ATP-synthase inhibitor oligomycin are shown to induce an apoptotic suicide response in cultured human lymphoblastoid and other mammalian cells within 12-18 h. The mitochondrial inhibitors do not induce apoptosis in cells depleted of mitochondrial DNA and thus lacking an intact mitochondrial respiratory chain. Apoptosis induced by respiratory chain inhibitors is not inhibited by the presence of Bcl-2. We discuss the possible role of mitochondrial induced apoptosis in the ageing process and age-associated diseases.

Topics: Animals; Antimycin A; Apoptosis; Cell Nucleus; Culture Media; DNA; Energy Metabolism; Humans; Leukemia; Melanoma; Mice; Oligomycins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rotenone; Tumor Cells, Cultured

The energy requirements via glycolytic pathways were directly measured in migrating tumor cells. Motility in the metastatic human melanoma cell line A2058, stimulated by insulinlike growth factor I (IGF-I), depends on glycolysis in the presence of glucose as its principal source of energy. Motility in glucose-free medium was 75% reduced and utilized mitochondrial respiration (inhibited by oligomycin). With increasing (physiologic) glucose concentrations, there was a dramatic shift to anaerobic glycolysis as the energy source and 93% elimination of the oligomycin inhibition of motility. Oxamate, an inhibitor of glycolysis, inhibited motility at all glucose concentrations. CO2 production from glycolysis and from the hexose monophosphate shunt was measured in migrating tumor cells. The time course and glucose-dose dependence of glycolytic CO2 production correlated directly with motility. In contrast, mitochondrial CO2 production was inversely related to glucose concentration. A monoclonal antibody for the IGF-I receptor inhibited both motility and glycolytic CO2 production, indicating that both processes are receptor mediated.

Topics: Antibodies, Monoclonal; Chemotaxis; Cycloheximide; Energy Metabolism; Glucose; Glycolysis; Humans; In Vitro Techniques; Insulin-Like Growth Factor I; Melanoma; Neoplasm Metastasis; Oligomycins; Oxamic Acid; Receptors, Cell Surface; Receptors, Somatomedin; Time Factors; Tumor Cells, Cultured

The action of lonidamine, 1,(2,4 dichlorobenzyl)-1H-indazol-3-carboxylic acid, on protein synthesis of neoplastic cells growing both in vivo and in vitro has been investigated. Lonidamine decreases amino acid incorporation in all cells tested, although the inhibition is partially relieved by glucose. The inhibition of labeled precursors into acid-insoluble material cannot be ascribed to an impairment of amino acid uptake which, on the contrary, is enhanced by the drug. Tests on cell-free systems showed that lonidamine does not inhibit the tobacco mosaic virus (TMV)-mRNA-directed in vitro protein synthesis, thus indicating that protein synthetic machinery per se is not affected. The inhibition of the rate of protein synthesis achieved by lonidamine must be ascribed to an effect on energy-yielding processes with a mechanism similar to that observed in other metabolic inhibitors. Lonidamine, however, because of its capacity to inhibit both respiration and glycolysis in neoplastic cells, is effective at 10 to 20 times lower concentrations. DNP and oligomycin potentiate the inhibitory effect of lonidamine on the rate of protein synthesis. This finding substantiates the idea that neoplastic cells, including those growing in ascitic form, utilize mitochondrial oxidative phosphorylation as the main source of ATP for their biosynthetic processes.

Topics: Amino Acids; Animals; Antineoplastic Agents; Carcinoma, Ehrlich Tumor; Cell Line; Cell-Free System; Dinitrobenzenes; Glycolysis; Humans; Indazoles; Kinetics; Lactates; Leukemia, Experimental; Male; Melanoma; Mice; Neoplasm Proteins; Oligomycins; Pyrazoles; Rabbits; Sarcoma, Experimental

Topics: Adenosine Diphosphate; Amobarbital; Animals; Antimycin A; Citric Acid Cycle; Dinitrophenols; Melanoma; Mice; Mitochondria; Neoplasm Transplantation; Neoplasms, Experimental; Oligomycins; Oxidative Phosphorylation; Oxygen Consumption; Rotenone; Transplantation, Homologous