title: Ceramide generation occurring during 7beta-hydroxycholesterol- and 7-ketocholesterol-induced apoptosis is caspase independent and is not required to trigger cell death.
abstract: Biological activities of oxysterols seem tightly regulated. Therefore, the ability to induce cell death of structurally related oxysterols, such as those oxidized at C7(7alpha-, 7beta-hydroxycholesterol, and 7-ketocholesterol), was investigated on U937 cells at different times of treatment in a concentration range of 5-80 microg/ml. Whereas all oxysterols accumulate inside the cells, strong inhibition of cell growth and increased permeability to propidium iodide were observed only with 7beta-hydroxycholesterol and 7-ketocholesterol, which trigger an apoptotic process characterized by the occurrence of cells with fragmented and/or condensed nuclei, and by various cellular dysfunctions: loss of mitochondrial transmembrane potential, cytosolic release of cytochrome c, activation of caspase-9 and -3 with subsequent enhanced activity of caspase-3, degradation of poly(ADP-ribose) polymerase, and increased accumulation of cellular C16 : 0 and C24 : 1 ceramide species. This ceramide generation is not attributed to caspase activation since inhibition of 7beta-hydroxycholesterol- and 7-ketocholesterol-induced apoptosis by Z-VAD-fmk (100 microM), a broad spectrum caspase inhibitor, did not reduce C16 : 0 and C24 : 1 ceramide species accumulation. Conversely, when U937 cells were treated with 7beta-hydroxycholesterol and 7-ketocholesterol in the presence of fumonisin B1 (100 microM), a specific inhibitor of ceramide synthase, C16 : 0 and C24 : 1 ceramide species production was completely abrogated whereas apoptosis was not prevented. Noteworthy, 7alpha-hydroxycholesterol induced only a slight inhibition of cell growth. Collectively, these results are consistent with the notion that the alpha or beta hydroxyl radical position of oxysterols oxidized at C7 plays a key role in the induction of the apoptotic process. In addition, our findings demonstrate that 7beta-hydroxycholesterol- and 7-ketocholesterol-induced apoptosis involve the mitochondrial signal transduction pathway and they suggest that C16 : 0 and C24 : 1 ceramide species generated through ceramide synthase play a minor role in the commitment of 7beta-hydroxycholesterol- and 7-ketocholesterol-induced cell death.
authors: CHU/Hôpital du Bocage, Laboratoire de Biochimie Médicale, Inserm U 498, BP 1542, 21034 Dijon Cedex, France.
authors: C
authors: C
authors: Miguet
authors: S
authors: S
authors: Monier
authors: A
authors: A
authors: Bettaieb
authors: A
authors: A
authors: Athias
authors: G
authors: G
authors: Besséde
authors: A
authors: A
authors: Laubriet
authors: S
authors: S
authors: Lemaire
authors: D
authors: D
authors: Néel
authors: P
authors: P
authors: Gambert
authors: G
authors: G
authors: Lizard
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D002264:Carboxylic Acids / Q000494:pharmacology; D053148:Caspase 3; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000187:drug effects; D002518:Ceramides / Q000096:biosynthesis*; D003574:Cytochrome c Group / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D037341:Fumonisins; D006801:Humans; D006888:Hydroxycholesterols / Q000493:pharmacokinetics / Q000494:pharmacology*; D007653:Ketocholesterols / Q000493:pharmacokinetics / Q000494:pharmacology*; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011419:Propidium / Q000493:pharmacokinetics; D020298:U937 Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D002264:Carboxylic Acids; D061945:Caspase Inhibitors; D002518:Ceramides; D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D037341:Fumonisins; D006888:Hydroxycholesterols; D007653:Ketocholesterols; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D011419:Propidium; C056933:fumonisin B1; C011724:cholest-5-en-3 beta,7 alpha-diol; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases; C003001:7-ketocholesterol