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Commitment to apoptosis by ceramides depends on mitochondrial respiratory function, cytochrome C release and caspase-3 activation in HEP-G2 cells

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Abstract

Apoptosis and necrosis are distinct forms of cell death that occur in response to various agents. We studied the action of N-Acetyl-D-sphingosine (C2-ceramide) or N-hexanoyl-D-sphyngosine (C6-ceramide) in human hepatoma HepG2 cell line. The cells were treated in vitro for 1–24 h. Cell toxicity was evaluated by MTT assay. DNA content was estimated by gel electrophoresis and flow cytometry. Measurement of mitochondrial respiration, analysis of cytochrome c release and caspase-3 activation were assessed in order to determine if either of these events in the induction of apoptosis and/or necrosis was predominant. We have demonstrated that C2 and C6-ceramide were cytotoxic in a time and dose-dependent manner. After 24 h of treatment with 100 μM of C2 and C6 the morphology (May-Giemsa staining) of treated cells displayed an apoptotic phenotype in C6-treated cells, confirmed by a high (sub-G1 peak > 20%) proportion by flow cytometry while a necrotic morphology was observed after C2-ceramide treatment, confirmed by DNA smearing in DNA electrophoresis. After C6-ceramide incubation, the respiratory chain was functional only slightly inhibited (20%), there was production of ATP, cytochrome c release without ROS production, activation of caspase-3 and induction of apoptosis. On the contrary, C2-ceramide inhibit the respiratory chain more intensely (80%) increased significantly ROS production, which resulted in an arrest of ATP production, no cytochrome c release and absence of caspase-3 activation. Finally after complete exhaustion of intracellular ATP, mitochondrial explosion induce necrotic cell death. In conclusion, evidence suggest that mitochondrial respiratory chain function is essential for controlling the decision of the cell to enter a apoptotic or necrosis process.

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References

  1. Hannun YA: The sphyngomyelin cycle and second messenger function of ceramide. J Biol Chem 269: 3125-3128, 1994

    Google Scholar 

  2. Jarvis WD, Grant S, Kolesnick RN: Ceramide and the induction of apoptosis. Clin Cancer Res 2: 1-6, 1996

    Google Scholar 

  3. Bose R, Verheij M, Haimovitz-Friedman A, Scotto K, Fuks Z, Kolesnick R: Ceramide synthetase mediates daunorubicin-induced apoptosis: An alternative mechanism for generating death signals. Cell 82: 405-441, 1995

    Google Scholar 

  4. Gudz TI, Tserng K-Y, Hoppel CL: Direct inhibition of mitochondrial respiratory chain complex III by cell-permeable ceramide. J Biol Chem 272: 24154-24158, 1997

    Google Scholar 

  5. Garcia-Ruiz C, Colell A, Mari M, Morales A, Fernadez-Checa J: Direct interaction of ceramide on the mitochondrial electron transport chain leads to generation of reactive oxygen species. J Biol Chem 272: 11369-11377, 1997

    Google Scholar 

  6. Quillet-Mary A, Jaffrézou JP, Mansat V, Bordier C, Naval J, Laurent G: Implication of mitochondrial hydrogen peroxide generation in ceramide-induced apotosis. J Biol Chem 272: 21388-21395, 1997

    Google Scholar 

  7. Arora AS, Jones BJ, Patel TC, Bronk SF, Gores GJ: Ceramide induces hepatocytes cell death through disruption of mitochondrial function in the rat. Hepatology 25: 957-963, 1997

    Google Scholar 

  8. Darzynkiewicz Z, Juan G, Li X, Gorczyca W, Murakami T, Traganos F: Cytometry in cell necrobiology: Analysis of apoptosis and accidental cell death (necrosis). Cytometry 27: 1-20, 1997

    Google Scholar 

  9. Kroemer G, Reed J: Mitochondrial control of cell death. Nature Med 6: 513-519, 2000

    Google Scholar 

  10. Eguchi Y, Shimizu S, Tsujimoto Y: Intracellular ATP levels determine cell death fate by apoptosis or necrosis. Cancer Res 57: 1835-1840, 1997

    Google Scholar 

  11. Hirch T, Marchetti P, Suzin SA, Dallaporta B, Zamzami N, Marzo I, Geuskens M, Kroemer G: The apoptosis-necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death. Oncogene 15: 1573-1582, 1997

    Google Scholar 

  12. Goldstein JC, Waterhouse NJ, Juin P, Evan GI, Green DR: The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant. Nature Cell Biol 2: 156-162, 2000

    Google Scholar 

  13. Cai J, Yang J, Jones DP: Mitochondrial control of apoptosis: The role of cytochrome c. Biochem Biophys Acta 1366: 139-149, 1998

    Google Scholar 

  14. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X: Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates and apoptotic protease cascade. Cell 91: 479-489, 1997

    Google Scholar 

  15. Appaix F, Minatchy MD, Riva-Lavieille C, Olivares O, Antonsson B, Saks V: Rapid spectrophotometic method for quantitation of cytochrome c release from isolated mitochondria or permeabilized cells revisited. Biochem Biophys Acta 1457: 175-181, 2000

    Google Scholar 

  16. Carmichael J, Mitchell JB, DeGraff WG, Gamson J, Gazdar AF, Johnson BE, Glatstein E, Minna JD: Chemosensitivity testing of human lung cancer cell lines using the MTT assay. Br J Cancer 57: 540-547, 1988

    Google Scholar 

  17. Searle J, Kerr JFR, Bishop CJ: Necrosis and apoptosis: Distinct modes of cell death with fundamentally different significance. Pathol Ann 17: 229-259, 1982

    Google Scholar 

  18. Hart LT, Morris NG, Bessin R, Leprince DJ, Todd WJ, Enright FM, Luther DG: Single-step technique for staining anaplasma marginale in bovine blood smears. Am J Vet Res 53: 1732-1733, 1992

    Google Scholar 

  19. Huang X, Frenkel K, Klein C, Costa M: Nickel induced oxidants in intact cultured mammalian cells as detected by dichlorofluorescein fluorescence. Pharmacol Toxicol 120: 29-36, 1993

    Google Scholar 

  20. Vento R, Giuliano M, Lauricella M, Carbillo M, Di Liberto D, Tesoriere G: Induction of programmed cell death in human retinoblastoma Y79 cells by C2-ceramide. Mol Cell Biochem 185: 7-15, 1998

    Google Scholar 

  21. Hung W-C, Chang H-C, Chuang L-Y: Activation of caspase-3-like proteases in apoptosis induced by sphyngosine and other long-chain bases in Hep3B hepatoma cells. Biochem J 338: 161-166, 1999

    Google Scholar 

  22. Singh NR: A simple method for accurate estimation of apoptotic cells. Exp Cell Res 256: 328-337, 2000

    Google Scholar 

  23. Puisieux A, Galvin K, Troalen F, Bressac B, Marcais C, Galun E, Ponchel F, Yakicier C, Ji J, Ozturk M: Retinoblastoma and p53 tumor suppressor genes in human hepatoma cell lines. Faseb J 7: 1407-1413, 1993

    Google Scholar 

  24. Ghafourifar P, Klein SD, Schucht O, Schenk U, Pruschy M, Rocha S, Richter C: Ceramide induces cytochrome c release from isolated mitochondria: importance of mitochondrial redox state. J Biol Chem 274: 6080-6084, 1999

    Google Scholar 

  25. Bernardi P, Scorrano L, Colonna R, Petronelli V, Di Lisa F: Mitochondria and cell death: Mechanistic aspect and methological issues. Eur J Biochem 264: 687-701, 1999

    Google Scholar 

  26. Venkataraman K, Futerman AH: Ceramide as a second messenger: Sticky solutions to sticky problems. Trends Cell Biol 10: 408-411, 2000

    Google Scholar 

  27. Verheij M, Bose R, Lin XH, Yao B, Jarvis WD, Grant S, Birrer MJ, Szabo E, Zon LI, Kyriakis JM, Haimovitz-Friedman A, Fuks Z, Kolesnick RN: Requirement for ceramide-initiated SAPK/JNK signaling in stress-induced apoptosis. Nature (Lond) 380: 75-79, 1996

    Google Scholar 

  28. Khaebanda S, Saxena S, Yoshida K, Pandey P, Kaneki M, Wang Q, Cheng K, Chen Y, Campbell A, Sudha T, Yuan Z, Narula J, Weichselbaum R, Nalin C, Kufe D: Translocation of SAPK/JNK to mitochondria and interaction with bcl-XL in response to DNA damage. J Biol Chem 275: 322-327, 2000

    Google Scholar 

  29. Corda S, Laplace C, Vicaut E, Duranteau J: Rapid reactive oxygen species production by mitochondria in endothelial cells exposed to tumor necrosis factor-alpha is mediated by ceramide. Am J Resp Cell Mol Biol 24: 762-768, 2001

    Google Scholar 

  30. Marchenko ND, Zaika A, Moll UM: Death signal-induced localization of p53 protein to mitochondria. J Biol Chem 275: 16202-16212, 2000

    Google Scholar 

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Authors and Affiliations

  1. Laboratoire de Bioénergétique Fondamentale et Appliquée, INSERM 02-21, Université Joseph Fourier, 38041, Grenoble cedex 9, France

    Benoit Gentil, Fabrice Grimot & Catherine Riva

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  1. Benoit Gentil
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  2. Fabrice Grimot
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  3. Catherine Riva
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Gentil, B., Grimot, F. & Riva, C. Commitment to apoptosis by ceramides depends on mitochondrial respiratory function, cytochrome C release and caspase-3 activation in HEP-G2 cells. Mol Cell Biochem 254, 203–210 (2003). https://doi.org/10.1023/A:1027359832177

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