Myeloperoxidase-dependent caspase-3 activation and apoptosis in HL-60 cells: protection by the antioxidants ascorbate and (dihydro)lipoic acid.
The heme enzyme myeloperoxidase (MPO) has recently been implicated in hydrogen peroxide H(2)O(2)-induced apoptosis of HL-60 human leukemia cells. The purpose of this study was to investigate the molecular mechanism(s) of MPO-mediated apoptosis, in particular caspase-3 activation, and to determine the effects of the antioxidants ascorbate and (dihydro)lipoic acid. Incubation of HL-60 cells (1 x 10(6) cells/ml media) with H(2)O(2) (0-200 microM) resulted in dose-dependent stimulation of caspase-3 activity, DNA fragmentation, and morphological changes associated with apoptosis. Caspase-3 activity, DNA fragmentation and apoptosis were maximal at approximately 50 microM H(2)O(2). Pre-incubation of the cells with the MPO-specific inhibitor 4-aminobenzoic acid hydrazide (ABAH) and the heme enzyme inhibitor 3-aminotriazole (100 microM each) resulted in complete and partial inhibition, respectively, of intracellular MPO, caspase-3 activity, and apoptosis following addition of 50 microM H(2)O(2). Enhancement of cellular antioxidant status by pre-incubation of the cells with dehydro-ascorbic acid and lipoic acid, which are reduced intracellularly to ascorbate and dihydrolipoic acid, respectively, afforded protection against caspase-3 activation and apoptosis following addition of H(2)O(2). Addition of high concentrations of H(2)O(2) (200 microM) to cells pre-incubated with lipoic acid, however, resulted in cytotoxicity. Overall, our data indicate that MPO-derived oxidants, rather than H(2)O(2) itself, are involved in caspase-3 activation and apoptosis in HL-60 cells, and the antioxidants ascorbate and (dihydro)lipoic acid inhibit caspase-3 activation and apoptosis in these cells, likely via scavenging the MPO-derived oxidants.
Myzak
Melinda C
MC
Linus Pauling Institute, Oregon State University, Corvallis, Oregon 97331-6512, USA.
Carr
Anitra C
AC
D000975:Antioxidants / Q000494:pharmacology*; D017209:Apoptosis; D001205:Ascorbic Acid / Q000494:pharmacology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D018922:HL-60 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism; D009195:Peroxidase / Q000378:metabolism*; D008063:Thioctic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology*
D000975:Antioxidants; D008063:Thioctic Acid; C015144:dihydrolipoic acid; D006861:Hydrogen Peroxide; D009195:Peroxidase; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D001205:Ascorbic Acid; D010100:Oxygen