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.