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.