title: zVAD-fmk upregulates caspase-9 cleavage and activity in etoposide-induced cell death of mouse embryonic fibroblasts.
abstract: Caspases are key effectors of programmed cell death. Down- and up-regulation of their activity are involved in different pathologies. In most cells, zVAD-fmk prevents apoptosis. However, unexpected effects of zVAD-fmk have been characterized in different laboratories, cell models and cell death processes. We have previously shown that zVAD-fmk accelerates p53-dependent apoptosis in rat embryonic fibroblasts. In this study, we pursued our investigations on zVAD-fmk effects and focused our study at the mitochondrial level in mouse embryonic fibroblasts (MEFs). In both primary and immortalized (by AgT or 3T9 protocol) MEFs, zVAD-fmk increased etoposide-induced loss of ΔΨm. This increase correlated with an increase of the number of apoptotic cells in primary and 3T9 MEFs, but did not in AgT MEFs. In both types of immortalized MEFs, zVAD-fmk regulated neither p53 levels nor transcriptional activities, suggesting that zVAD-fmk acts downstream of p53. In MEFs, zVAD-fmk increased p53-dependent loss of ΔΨm, cytochrome c release and caspase-9 activity. Indeed, zVAD-fmk inhibited effector caspases (caspases-3, -6, -7) as expected but increased caspase-9 cleavage and activity in etoposide-treated MEFs. Q-VD-OPh, another caspase inhibitor, also increased both loss of ΔΨm and caspase-9 cleavage in etoposide-treated MEFs. Invalidation of bax and bak suppressed p53-dependent cell death and zVAD-fmk regulation of this process. Invalidation of caspase-9 did not inhibit mitochondrial membrane depolarization but suppressed zVAD-fmk amplification of this process. Altogether, our data suggest that caspase-9 activity is up-regulated by zVAD-fmk and is involved in an amplification loop of etoposide-induced cell death at the mitochondrial level in MEFs.
authors.affiliation: Universite de Versailles-Saint-Quentin-en-Yvelines, Ecole Pratique des Hautes Etudes, Versailles, France.
authors.forename: Aida
authors.initials: A
authors.lastname: Rodríguez-Enfedaque
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Delmas
authors.forename: Arnaud
authors.initials: A
authors.lastname: Guillaume
authors.forename: Sébastien
authors.initials: S
authors.lastname: Gaumer
authors.forename: Bernard
authors.initials: B
authors.lastname: Mignotte
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Vayssière
authors.forename: Flore
authors.initials: F
authors.lastname: Renaud
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology*; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D053178:Caspase 6 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism*; D061945:Caspase Inhibitors / Q000494:pharmacology*; D002478:Cells, Cultured; D045304:Cytochromes c / Q000378:metabolism; D004357:Drug Synergism; D004622:Embryo, Mammalian; D004789:Enzyme Activation / Q000187:drug effects; D005047:Etoposide / Q000494:pharmacology*; D005347:Fibroblasts; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D015854:Up-Regulation; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D000972:Antineoplastic Agents, Phytogenic; C494773:Bak1 protein, mouse; C494827:Bax protein, mouse; D061945:Caspase Inhibitors; D016159:Tumor Suppressor Protein p53; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D005047:Etoposide; D045304:Cytochromes c; C505409:Casp3 protein, mouse; C505516:Casp6 protein, mouse; C506434:Casp9 protein, mouse; D053148:Caspase 3; D053178:Caspase 6; D053453:Caspase 9