Involvement of BH3-only proapoptotic proteins in mitochondrial-dependent Phenoxodiol-induced apoptosis of human melanoma cells.
Phenoxodiol is a chemically modified analogue of the plant hormone isoflavone with antitumour activities. In the present study, we have examined its ability to induce apoptosis in human melanoma cells and the mechanisms involved. Apoptosis was observed in Phenoxodiol-treated cells by using annexin V/propidium iodide staining and determining mitochondrial membrane potential. To determine which caspase pathways were involved in Phenoxodiol-induced apoptosis, studies were performed using specific caspase inhibitors. Western studies were performed to ascertain which proteins of the apoptosis cascade were affected to cause Phenoxodiol-induced apoptosis. We found that induction of apoptosis by Phenoxodiol was maximal at 48 h with a range of apoptosis of 12+/-4 to 48+/-5% in different melanoma lines. This apoptosis was mainly dependent on activation of caspase-3 and caspase-9. Apoptosis was associated with induction of changes in mitochondrial membrane potential and was inhibited by over-expression of Bcl-2. Variation in sensitivity to Phenoxodiol appeared related to events upstream of the mitochondria and the degree of conformational change in Bax. The p53-regulated BH3-only proteins (Bad, PUMA and Noxa) were increased in the sensitive, but not in the resistant lines, whereas Bim was increased in all the lines tested. Bim appeared, however, to be partially involved because reduction of Bim by RNA interference resulted in decreased levels of apoptosis. Together, these studies suggest that Phenoxodiol induces apoptosis of melanoma cells by induction of p53-dependent BH3 proteins (Bad, PUMA and Noxa) and the p53-independent Bim protein, resulting in activation of Bax and its downstream events.
Yu
Fu
F
Oncology and Immunology Unit, Newcastle Mater Hospital, New South Wales, Australia.
Watts
Ralph N
RN
Zhang
Xu Dong
XD
Borrow
Jodie M
JM
Hersey
Peter
P
D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism / Q000502:physiology*; D000072224:Bcl-2-Like Protein 11; D020169:Caspases / Q000502:physiology; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D006801:Humans; D007529:Isoflavones / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D008545:Melanoma / Q000473:pathology*; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology; D008928:Mitochondria / Q000187:drug effects*; D011487:Protein Conformation / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000378:metabolism / Q000502:physiology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000737:chemistry / Q000187:drug effects
D051017:Apoptosis Regulatory Proteins; C494826:BAX protein, human; C438378:BBC3 protein, human; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; D007529:Isoflavones; D008565:Membrane Proteins; C498702:PMAIP1 protein, human; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; C471183:phenoxodiol; D020169:Caspases