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 journalArticle

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
Externally publishedYes

Fingerprint

Ceramides
Caspases
Cytochromes c
Apoptosis
Caspase 2
Caspase 3
Caspase 9
DNA Fragmentation
Cytosol
Sphingosine
Fas Ligand Protein
Caspase Inhibitors
Cell-Free System
N-acetylsphingosine
Second Messenger Systems
Ionizing Radiation
Neuroblastoma
Hydrogen Peroxide
Chromatin
Shock

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 journalArticle

@article{6b5ee4b1d1fb4c5ea5cb70696bc3b968,
title = "Possible involvement of cytochrome c release and sequential activation of caspases in ceramide-induced apoptosis in SK-N-MC cells",
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.",
keywords = "Apoptosis, Caspase, Ceramide, Cytochrome c, Neuron",
author = "Akihiro Ito and Takashi Uehara and Ai Tokumitsu and Yasunobu Okuma and Yasuyuki Nomura",
year = "1999",
month = "12",
day = "9",
doi = "10.1016/S0167-4889(99)00131-7",
language = "English",
volume = "1452",
pages = "263--274",
journal = "Biochimica et Biophysica Acta - Molecular Cell Research",
issn = "0167-4889",
publisher = "Elsevier",
number = "3",

}

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 -