Inhibition by ascorbic acid of apoptosis induced by oxidative stress in HL-60 myeloid leukemia cells

Abstract The human myeloid leukemia cell line HL-60 transports the oxidized form of ascorbic acid, dehydroascorbic acid (DHA), and accumulates reduced ascorbic acid. We studied the effect of ascorbic acid loading on apoptosis induced by serum- and glucose-free culture and by oxidative stress induced by H 2 O 2 . Uptake accumulation studies indicated that incubation of HL-60 cells with DHA resulted in the accumulation of intracellular ascorbic acid which decreased with time when cells were incubated in DHA-free medium. Exposure of HL-60 cells to increasing concentrations of H 2 O 2 resulted in dose-dependent intracellular accumulation of peroxides, as determined by the use of the oxidation-sensitive fluorescent probe 2′,7′-dichlorofluorescin-diacetate (DCFH-DA), which was accompanied by a decrease in intracellular ascorbic acid and an increase in apoptosis. A dramatic decrease in intracellular ascorbic acid was noted when preloaded HL-60 cells were exposed to 150 μM H 2 O 2 (the concentration dropped from 5.2 ± 0.6 mM to 3.6 ± 0.1 mM in cells preincubated with 150 μM DHA). A dose-dependent protective effect of DHA was observed. Ascorbic acid loading also provided strong protection from apoptosis associated with serum- and glucose-free culture. Flow cytometry studies showed that exposure of HL-60 cells to 150 μM H 2 O 2 resulted in decreased Bcl-2 expression that was associated with enhanced apoptosis (up to 33.6 ± 2.6%). No significant variation of Bcl-2 expression was measured following exposure of HL-60 cells, loaded with ascorbic acid, to 150 μM H 2 O 2 and only a slight increase (up to 10.1 ± 3.1%) in apoptosis. These findings indicate that ascorbic acid can inhibit apoptosis induced by oxidative stress in HL-60 cells.