title: Reversibility of caspase activation and its role during glycochenodeoxycholate-induced hepatocyte apoptosis.
abstract: The accumulation of glycochenodeoxycholate (GCDC) induced hepatocyte apoptosis in cholestasis. However, many hepatocytes still survived GCDC-induced apoptosis. The molecular mechanism for the survival of hepatocytes remains unclear. In the present study, isolated rat hepatocytes were cultured in William's E medium and treated with 50 microM GCDC. DNA, RNA, cell lysate, and nuclear proteins were collected at different intervals for DNA fragmentation assay, reverse transcription PCR, Western blotting, and gel mobility shift assay, respectively. GCDC-induced active caspases were detected as early as 2 h by Western blotting and kinetic caspase assay, whereas hepatocyte apoptosis was found at 4 h by DNA fragmentation and terminal deoxynucleotidyl transferase-mediated dUPT nick-end labeling assay. When GCDC was removed, the increased caspases as well as NF-kappaB could be restored to control level. A1/Bfl-1 and inducible nitric oxide synthase (iNOS) were up-regulated in 2 h of GCDC stimulation. After GCDC was removed, hepatocytes decreased expression of A1/Bfl-1, but not iNOS, to the control level. NF-kappaB activation coincided with the change of A1/Bfl-1. Survivin, cIAP1, cIAP2, XIAP, and A1/Bfl-1, but not iNOS, were down-regulated by pan-caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone. In addition, benzyloxycarbonyl-VAD-fluoromethyl ketone inhibited release of cytochrome c and suppressed NF-kappaB activation. Our data suggested that caspase pathway is an important regulatory factor during hepatocyte apoptosis. GCDC-induced caspase response is reversible, which may activate anti-apoptotic genes to protect hepatocytes from apoptosis.
authors.affiliation: Department of Surgery, Loyola University Medical Center, Maywood, Illinois 60153, USA. kwang1@lumc.edu
authors.forename: Kewei
authors.initials: K
authors.lastname: Wang
authors.forename: John J
authors.initials: JJ
authors.lastname: Brems
authors.forename: Richard L
authors.initials: RL
authors.lastname: Gamelli
authors.forename: Jinwen
authors.initials: J
authors.lastname: Ding
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D001483:Base Sequence; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D017931:DNA Primers; D004789:Enzyme Activation; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects*; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D017931:DNA Primers; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D020169:Caspases