Possible involvement of cytochrome c release and sequential activation of caspases in ceramide-induced apoptosis in SK-N-MC cells

Ceramide is characterized as a second messenger of apoptosis induced by var ious agents such as tumor necrosis factor (TNF-alpha), Fas ligand, hydrogen peroxide, heat shock and ionizing radiation. In this study, we investigate d the mechanism of ceramide-induced apoptosis using a human neuroblastoma c ell line, SK-N-MC. N-Acetyl-sphingosine (C2-ceramide), a cell-permeable cer amide analogue, was able to induce apoptosis in SK-N-MC cells as estimated by DNA fragmentation and chromatin condensation. C2-ceramide-induced DNA fr agmentation was blocked by caspase inhibitor (Z-Asp-CH2-DCB). An increase i n caspase-3 (CPP32)-like protease activity was evident during C2-ceramide-i nduced apoptosis, suggesting that caspases are involved in this apoptosis. Moreover, enzymatic cleavage of VDVAD-AFC and LEHD-AFC (specific substrates for caspase-2 and -9, respectively) was increased by treatment with C2-cer amide. To elucidate which types of caspase are activated in C2-ceramide-tre ated cells, we performed Western blot analysis using antibodies against eac h isoform. Both proforms of caspase-2 and -3 were decreased in response to C2-ceramide in a time-dependent manner. Mitochondrial cytochrome c is also time-dependently released into the cytosol in response to treatment with C2 -ceramide. Addition of cytochrome c into the S-100 fractions prepared from SK-N-MC cells could activate caspase-2 in cell-free systems. These results- suggest the possibility that cytochrome c released to the cytosol can activ ate caspases (caspase-9, -3, and -2) during C2-ceramide-induced apoptosis o f SK-N-MC cells. (C) 1999 Elsevier Science B.V. All rights reserved.