The Bax/Bcl-2 ratio determines the susceptibility of human melanoma cells to CD95/Fas-mediated apoptosis

Defective cytochrome c release and the resulting loss of caspase-3 activati on was recently shown to be essential for the susceptibility of human melan oma cells to CD95/Fas-induced apoptosis. Cytochrome c release from mitochon dria is regulated by the relative amounts of apoptosis-promoting and apopto sis-inhibiting Bcl-2 proteins in the outer membrane of these organelles. Th e assignment of Bax/Bcl-2 ratios by quantitative Western blotting in 11 mel anoma cell populations revealed a relation to the susceptibility to CD95-me diated apoptosis. We could show that a low Bax/Bcl-2 ratio was characterist ic for resistant cells and a high Bax/Bcl-2 ratio was characteristic for se nsitive cells. Low Bax expression was not a consequence of mutations in the p53 coding sequence. The Bax/Bcl-2 ratio was also in clear correlation wit h sensitivity to another cell death inducer, N-acetylsphingosine. Furthermo re, Bcl-2 overexpression abolished apoptosis triggered by both apoptotic st imuli, confirming the critical role of the Bax/Bcl-2 ratio as a rheostat th at determines the susceptibility to apoptosis in melanoma cells by regulati ng mitochondrial function. Interestingly, some chemotherapeutics lead to th e activation of death pathways by CD95L upregulation, ceramide generation, direct activation of upstream caspases, or upregulation of proapoptotic gen es. Taken together, these signals enter the apoptotic pathway upstream of m itochondria, resulting in activation of this central checkpoint. We therefo re assumed that apoptosis deficiency of malignant melanoma can be circumven ted by drugs directly influencing mitochondrial functions. For this purpose we used betulinic acid, a cytotoxic agent selective for melanoma, straight ly perturbing mitochondrial functions. In fact, betulinic acid induced mito chondrial cytochrome c release and DNA fragmentation in both CD95-resistant and CD95-sensitive melanoma cell populations, independent of the Bax/Bcl-2 ratio.