title: Caspase 3 is activated through caspase 8 instead of caspase 9 during H2O2-induced apoptosis in HeLa cells.
abstract: Oxidative stress is known to be involved in a variety of pathological processes including atherosclerosis, diabetes, and neurodegenerative diseases. Understanding how intracellular signaling pathways respond to oxidative stress will have a significant implication in the therapy of these diseases. In this study, we applied hydrogen peroxide (H(2)O(2)) to trigger apoptosis and investigated the dynamic activation of various caspases using a FRET technique. We measured the activation dynamics of caspase 3 and caspase 9 based on two reporter systems, SCAT 3 and SCAT 9. We found that caspase 3 activation was earlier than that of caspase 9 following H(2)O(2) treatment. Caspase 3 was activated rapidly, reaching a maximum in 12±3 min, while the average duration of caspase 9 activation was 21±3 min. When cells were pretreated with Z-LEHD-fmk, a caspase 9 specific inhibitor, caspase 3 activation and apoptosis by H(2)O(2) treatment were little affected, although the caspase 9 activation was completely inhibited. When cells were pretreated with Z-DEVD-fmk, a caspase 3 specific inhibitor, the activation of both caspase 3 and caspase 9, as well as apoptosis, were inhibited. When cells were pretreated with Z-IETD-fmk, a caspase 8 specific inhibitor, the activation of caspase 3 and caspase 9 were significantly delayed. Finally, we found that Bax did not translocate from the cytosol to the mitochondrial membrane during H(2)O(2)-induced apoptosis. Our results suggest that, during H H(2)O(2)-induced apoptosis, caspase 3 is activated directly through caspase 8 and is not through the mitochondria-dependent caspase 9 activation.
authors.affiliation: School of Medicine, Department of Biological Sciences and Biotechnology, State Key Laboratory of Biomembrane and Membrane Biotechnology, Tsinghua University, Beijing, China.
authors.forename: Yinyuan
authors.initials: Y
authors.lastname: Wu
authors.forename: Dianjun
authors.initials: D
authors.lastname: Wang
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Wang
authors.forename: Yinyin
authors.initials: Y
authors.lastname: Wang
authors.forename: Fangli
authors.initials: F
authors.lastname: Ren
authors.forename: Donald
authors.initials: D
authors.lastname: Chang
authors.forename: Zhijie
authors.initials: Z
authors.lastname: Chang
authors.forename: Baoqing
authors.initials: B
authors.lastname: Jia
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism*; D053181:Caspase 8 / Q000235:genetics / Q000378:metabolism*; D053453:Caspase 9 / Q000235:genetics / Q000378:metabolism; D061945:Caspase Inhibitors; D045304:Cytochromes c / Q000032:analysis; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology; D018384:Oxidative Stress / Q000187:drug effects*; D010957:Plasmids; D021381:Protein Transport / Q000187:drug effects; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D051028:bcl-2-Associated X Protein; D045304:Cytochromes c; D006861:Hydrogen Peroxide; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9