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 language | English |
|---|---|
| Pages (from-to) | 263-274 |
| Number of pages | 12 |
| Journal | Biochimica et Biophysica Acta - Molecular Cell Research |
| Volume | 1452 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - Dec 9 1999 |
| Externally published | Yes |
Fingerprint
Keywords
- Apoptosis
- Caspase
- Ceramide
- Cytochrome c
- Neuron
ASJC Scopus subject areas
- Cell Biology
- Molecular Biology
- Biophysics
Cite this
Possible involvement of cytochrome c release and sequential activation of caspases in ceramide-induced apoptosis in SK-N-MC cells. / Ito, Akihiro; Uehara, Takashi; Tokumitsu, Ai; Okuma, Yasunobu; Nomura, Yasuyuki.
In: Biochimica et Biophysica Acta - Molecular Cell Research, Vol. 1452, No. 3, 09.12.1999, p. 263-274.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Possible involvement of cytochrome c release and sequential activation of caspases in ceramide-induced apoptosis in SK-N-MC cells
AU - Ito, Akihiro
AU - Uehara, Takashi
AU - Tokumitsu, Ai
AU - Okuma, Yasunobu
AU - Nomura, Yasuyuki
PY - 1999/12/9
Y1 - 1999/12/9
N2 - 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.
AB - 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.
KW - Apoptosis
KW - Caspase
KW - Ceramide
KW - Cytochrome c
KW - Neuron
UR - http://www.scopus.com/inward/record.url?scp=0032719378&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0032719378&partnerID=8YFLogxK
U2 - 10.1016/S0167-4889(99)00131-7
DO - 10.1016/S0167-4889(99)00131-7
M3 - Article
C2 - 10590315
AN - SCOPUS:0032719378
VL - 1452
SP - 263
EP - 274
JO - Biochimica et Biophysica Acta - Molecular Cell Research
JF - Biochimica et Biophysica Acta - Molecular Cell Research
SN - 0167-4889
IS - 3
ER -