title: TNF-related apoptosis-inducing ligand-induced apoptosis of melanoma is associated with changes in mitochondrial membrane potential and perinuclear clustering of mitochondria.
abstract: Past studies have shown that TNF-related apoptosis-inducing ligand (TRAIL) induced apoptosis in a high proportion of cultured melanoma by caspase-dependent mechanisms. In the present studies we have examined whether TRAIL-induced apoptosis of melanoma was mediated by direct activation of effector caspases or whether apoptosis was dependent on changes in mitochondrial membrane potential (MMP) and mitochondrial-dependent pathways of apoptosis. Changes in MMP were measured by fluorescent emission from rhodamine 123 in mitochondria. TRAIL, but not TNF-alpha or Fas ligand, was shown to induce marked changes in MMP in melanoma, which showed a high correlation with TRAIL-induced apoptosis. This was associated with activation of proapoptotic protein Bid and release of cytochrome c into the cytosol. Overexpression of B cell lymphoma gene 2 (Bcl-2) inhibited TRAIL-induced release of cytochrome c, changes in MMP, and apoptosis. The pan caspase inhibitor z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) and the inhibitor of caspase-8 (z-Ile-Glu-Thr-Asp-fluoromethylketone; zIETD-fmk) blocked changes in MMP and apoptosis, suggesting that the changes in MMP were dependent on activation of caspase-8. Activation of caspase-9 also appeared necessary for TRAIL-induced apoptosis of melanoma. In addition, TRAIL, but not TNF-alpha or Fas ligand, was shown to induce clustering of mitochondria around the nucleus. This process was not essential for apoptosis but appeared to increase the rate of apoptosis. Taken together, these results suggest that TRAIL induces apoptosis of melanoma cells by recruitment of mitochondrial pathways to apoptosis that are dependent on activation of caspase-8. Therefore, factors that regulate the mitochondrial pathway may be important determinants of TRAIL-induced apoptosis of melanoma.
authors.affiliation: Department of Oncology and Immunology, Newcastle, New South Wales, Australia.
authors.forename: W D
authors.initials: WD
authors.lastname: Thomas
authors.forename: X D
authors.initials: XD
authors.lastname: Zhang
authors.forename: A V
authors.initials: AV
authors.lastname: Franco
authors.forename: T
authors.initials: T
authors.lastname: Nguyen
authors.forename: P
authors.initials: P
authors.lastname: Hersey
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000276:immunology*; D051017:Apoptosis Regulatory Proteins; D051023:BH3 Interacting Domain Death Agonist Protein; D002352:Carrier Proteins / Q000378:metabolism; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007113:Immunity, Innate; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D008024:Ligands; D008545:Melanoma / Q000201:enzymology / Q000276:immunology / Q000378:metabolism* / Q000473:pathology*; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects / Q000276:immunology; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism / Q000502:physiology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000235:genetics; D053221:TNF-Related Apoptosis-Inducing Ligand; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000502:physiology*
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D051017:Apoptosis Regulatory Proteins; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D002352:Carrier Proteins; D061945:Caspase Inhibitors; D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D008024:Ligands; D008562:Membrane Glycoproteins; D019253:Proto-Oncogene Proteins c-bcl-2; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases