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

Akihiro Ito, Takashi Uehara, Ai Tokumitsu, Yasunobu Okuma, Yasuyuki Nomura

Research output: Contribution to journalArticlepeer-review

63 Citations (Scopus)

Abstract

Ceramide is characterized as a second messenger of apoptosis induced by various agents such as tumor necrosis factor (TNF-α), 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-CH2-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.

Original languageEnglish
Pages (from-to)263-274
Number of pages12
JournalBiochimica et Biophysica Acta - Molecular Cell Research
Volume1452
Issue number3
DOIs
Publication statusPublished - Dec 9 1999

Keywords

  • Apoptosis
  • Caspase
  • Ceramide
  • Cytochrome c
  • Neuron

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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