Myeloperoxidase-dependent caspase-3 activation and apoptosis in HL-60 cells: protection by the antioxidants ascorbate and (dihydro)lipoic acid

Abstract

The heme enzyme myeloperoxidase (MPO) has recently been implicated in hydrogen peroxide H₂O₂-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 10⁶ cells/ml media) with H₂O₂ (0-200 μM) 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 μM H₂O₂. Pre-incubation of the cells with the MPO-specific inhibitor 4-aminobenzoic acid hydrazide (ABAH) and the heme enzyme inhibitor 3-aminotriazole (100 μM each) resulted in complete and partial inhibition, respectively, of intracellular MPO, caspase-3 activity, and apoptosis following addition of 50 μM H₂O₂. 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 H₂O₂. Addition of high concentrations of H₂O₂ (200 μM) to cells pre-incubated with lipoic acid, however, resulted in cytotoxicity. Overall, our data indicate that MPO-derived oxidants, rather than H₂O₂ 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.