title: Ceramide increases oxidative damage due to inhibition of catalase by caspase-3-dependent proteolysis in HL-60 cell apoptosis.
abstract: We investigated through which mechanisms ceramide increased oxidative damage to induce leukemia HL-60 cell apoptosis. When 5 microm N-acetylsphingosine (C(2)-ceramide) or 20 microm H(2)O(2) alone induced little increase of reactive oxygen species (ROS) generation as judged by the 2'-7'-dichlorofluorescin diacetate method, 20 microm H(2)O(2) enhanced oxidative damage as judged by ROS accumulation, and thiobarbituric acid-reactive substance production after pretreatment with 5 microm C(2)-ceramide at least for 12 h. The treatment with a catalase inhibitor, 3-amino-1h-1,2,4-triazole, increased oxidative damage and apoptosis induced by H(2)O(2), and in contrast, purified catalase inhibited the enhancement of oxidative damage by H(2)O(2) in ceramide-pretreated cells, suggesting that the oxidative effect of ceramide is involved in catalase regulation. Indeed, C(2)-ceramide inhibited the activity of immunoprecipitated catalase and decreased the levels of catalase protein in a time-dependent manner. Moreover, acetyl-Asp-Met-Gln-Asp-aldehyde, which dominantly inhibited caspase-3 and blocked the increase of oxidative damage and apoptosis due to C(2)-ceramide-induced catalase depletion at protein and activity levels. In vitro, active and purified caspase-3, but not caspase-6, -8, and -9, inhibited catalase activity and induced the proteolysis of catalase protein whereas these in vitro effects of caspase-3 were blocked by acetyl-Asp-Met-Gln-Asp-aldehyde. Taken together, it is suggested that H(2)O(2) enhances apoptosis in ceramide-pretreated cells, because ceramide increases oxidative damage by inhibition of ROS scavenging ability through caspase-3-dependent proteolysis of catalase.
authors.affiliation: Department of Hematology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Iwai
authors.forename: Tadakazu
authors.initials: T
authors.lastname: Kondo
authors.forename: Mitsumasa
authors.initials: M
authors.lastname: Watanabe
authors.forename: Takeshi
authors.initials: T
authors.lastname: Yabu
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Kitano
authors.forename: Yoshimitu
authors.initials: Y
authors.lastname: Taguchi
authors.forename: Hisanori
authors.initials: H
authors.lastname: Umehara
authors.forename: Atsushi
authors.initials: A
authors.lastname: Takahashi
authors.forename: Takashi
authors.initials: T
authors.lastname: Uchiyama
authors.forename: Toshiro
authors.initials: T
authors.lastname: Okazaki
mesh_terms: D017209:Apoptosis; D015152:Blotting, Northern; D015153:Blotting, Western; D053148:Caspase 3; D053178:Caspase 6; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002374:Catalase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002518:Ceramides / Q000378:metabolism* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D017353:Gene Deletion; D018922:HL-60 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism; D009195:Peroxidase / Q000378:metabolism; D011233:Precipitin Tests; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism; D017392:Thiobarbituric Acid Reactive Substances / Q000494:pharmacology; D013997:Time Factors
chemical_list: D002518:Ceramides; D004791:Enzyme Inhibitors; C064769:N-acetylsphingosine; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D017392:Thiobarbituric Acid Reactive Substances; D012313:RNA; D006861:Hydrogen Peroxide; D002374:Catalase; D009195:Peroxidase; C505407:CASP3 protein, human; C505514:CASP6 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053178:Caspase 6; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D013110:Sphingosine; D010100:Oxygen