Ceramide increases oxidative damage due to inhibition of catalase by caspase-3-dependent proteolysis in HL-60 cell apoptosis

We investigated through which mechanisms ceramide increased oxidative damage to induce leukemia HL-60 cell apoptosis. When 5 muM N-acetylsphingosine (C-2-ceramide) or 20 muM H2O2 alone induced little increase of reactive oxygen species (ROS) generation as judged by the 2'-7'-dichlorofluorescin diacetate method, 20 muM H2O2 enhanced oxidative damage as judged by ROS accumulation, and thiobarbituric acid-reactive substance production after pretreatment with 5 muM 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 H2O2, and in contrast, purified catalase inhibited the enhancement of oxidative damage by H2O2 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-AspMet-Gln-Asp-aldehyde. Taken together, it is suggested that H2O2 enhances apoptosis in ceramide-pretreated cells, because ceramide increases oxidative damage by inhibition of ROS scavenging ability through caspase-3-dependent proteolysis of catalase.