Increase of ceramides and its inhibition by catalase during chemically induced apoptosis of HL-60 cells determined by electrospray ionization tandem mass spectrometry.

A change in all ceramide species during chemically induced apoptosis of HL-60 cells was determined using electrospray tandem mass spectrometry. Ceramides of C16:0, C24:1 and C26:1 increased significantly 4 h after the addition of actinomycin D, when the activation of caspase-3 was maximal. Addition of catalase, which inhibited apoptosis, the activation of caspase-3-like protease, and the release of cytochrome c from mitochondria to cytosol caused by actinomycin D or daunorubicin, significantly inhibited the increase of these ceramides at all time points. Ceramides of C16:0, C24:1, C18:0, C22:1 and C26:1 increased significantly 4 h after the addition of daunorubicin to HL-60 cells. Catalase also significantly inhibited the increase of these ceramides induced by daunorubicin. Based on time courses of events and inhibition studies, it is concluded that the increase of ceramides is downstream from both generation of hydrogen peroxide and cytochrome c release from mitochondria and takes place almost simultaneously with the activation of caspase-3.