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

Biochim Biophys Acta. 1999 Dec 9;1452(3):263-74. doi: 10.1016/s0167-4889(99)00131-7.


Ceramide is characterized as a second messenger of apoptosis induced by various agents such as tumor necrosis factor (TNF-alpha), Fas ligand, hydrogen peroxide, heat shock and ionizing radiation. In this study, we investigated the mechanism of ceramide-induced apoptosis using a human neuroblastoma cell line, SK-N-MC. N-Acetyl-sphingosine (C2-ceramide), a cell-permeable ceramide analogue, was able to induce apoptosis in SK-N-MC cells as estimated by DNA fragmentation and chromatin condensation. C2-ceramide-induced DNA fragmentation was blocked by caspase inhibitor (Z-Asp-CH(2)-DCB). An increase in caspase-3 (CPP32)-like protease activity was evident during C2-ceramide-induced 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-ceramide. To elucidate which types of caspase are activated in C2-ceramide-treated cells, we performed Western blot analysis using antibodies against each 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 activate caspases (caspase-9, -3, and -2) during C2-ceramide-induced apoptosis of SK-N-MC cells.

MeSH terms

  • Apoptosis*
  • Caspase Inhibitors
  • Caspases / genetics
  • Caspases / metabolism*
  • Cell-Free System
  • Cytochrome c Group / antagonists & inhibitors
  • Cytochrome c Group / metabolism*
  • Cytosol / enzymology
  • DNA Fragmentation
  • Enzyme Activation
  • Enzyme Inhibitors / pharmacology
  • Humans
  • L-Lactate Dehydrogenase / analysis
  • Neuroblastoma
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sphingosine / analogs & derivatives*
  • Sphingosine / pharmacology
  • Time Factors
  • Tumor Cells, Cultured


  • Caspase Inhibitors
  • Cytochrome c Group
  • Enzyme Inhibitors
  • N-acetylsphingosine
  • L-Lactate Dehydrogenase
  • Caspases
  • Sphingosine