Bax cleavage implicates caspase-dependent H2O2-induced apoptosis of hepatocytes.
Oxidative stress plays an important role in the development of ischemia/reperfusion (I/R)-induced apoptosis of hepatocytes. We aimed to examine the involvement of caspases and calpains in H2O2-induced hepatic cell apoptosis. TUNEL-positive apoptotic cells appeared in parallel with poly(ADP-ribose) polymerase (PARP) cleavage and procaspase-3 proteolysis by H2O2 treatment in a dose-dependent manner (250-1,000 micro M). Bcl-xL and intact Bax expression levels decreased when H2O2 was >250 micro M. The cleaved form of Bax appeared prior to caspase-3 activation, increasing in a dose-dependent manner. A pan-caspase inhibitor, Z-VAD-fmk, completely blocked H2O2-induced procaspase-3 proteolysis and PARP cleavage without changing Bax cleavage, but partially attenuated H2O2-induced apoptosis. Calpeptin, a calpain inhibitor, did not inhibit caspase-3 activation, Bax cleavage or apoptosis. Our results indicate that Bax cleavage is upstream signal of caspase-dependent apoptosis in hepatocytes exposed to H2O2, but not independent upon calpain. Molecular targeting of Bax cleavage may allow the development of strategies to prevent hepatic I/R injury.
Tamura
Hiroaki
H
Department of Surgery II, Graduate School of Biochemical Sciences, Nagasaki University, Nagasaki 852-8501, Japan.
Ohtsuru
Akira
A
Kamohara
Yukio
Y
Fujioka
Hikaru
H
Yanaga
Katsuhiko
K
Kanematsu
Takashi
T
Yamashita
Shunichi
S
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000187:drug effects / Q000378:metabolism*; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D053938:DNA Fragmentation / Q000187:drug effects; D004151:Dipeptides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D016877:Oxidants / Q000494:pharmacology*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000187:drug effects / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000187:drug effects / Q000378:metabolism; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
D000590:Amino Acid Chloromethyl Ketones; C494826:BAX protein, human; C494811:BCL2L1 protein, human; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D004792:Enzyme Precursors; D016877:Oxidants; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C071482:calpeptin; D006861:Hydrogen Peroxide; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; D002154:Calpain; D053148:Caspase 3; D020169:Caspases