Commitment to apoptosis by ceramides depends on mitochondrial respiratory function, cytochrome c release and caspase-3 activation in Hep-G2 cells.
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-sphingosine (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 microM 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 inhibited 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 induced 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.
Gentil
Benoit
B
Laboratoire de Bioénergétique Fondamentale et Appliquée, INSERM 02-21, Université Joseph Fourier, Grenoble cedex 9, France.
Grimot
Fabrice
F
Riva
Catherine
C
D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D002518:Ceramides / Q000378:metabolism*; D004396:Coloring Agents / Q000494:pharmacology; D045304:Cytochromes c / Q000378:metabolism*; D004247:DNA / Q000737:chemistry; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D004579:Electron Transport; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005434:Flow Cytometry; D006801:Humans; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism / Q000473:pathology; D017382:Reactive Oxygen Species; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D013997:Time Factors
D002518:Ceramides; D004396:Coloring Agents; C064769:N-acetylsphingosine; D017382:Reactive Oxygen Species; D013778:Tetrazolium Salts; D013844:Thiazoles; D045304:Cytochromes c; D004247:DNA; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; C022616:thiazolyl blue; D013110:Sphingosine