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

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 H2O2. Uptake accumulation studies indicated that incubation of HL-60 cells with DHA resulted in the accumulation of intracellular ascorbic acid which decre ased with time when cells were incubated in DHA-free medium. Exposure of HL -60 cells to increasing concentrations of H2O2 resulted in dose dependent i ntracellular accumulation of peroxides, as determined by the use of the oxi dation-sensitive fluorescent probe 2',7'-dichlorofluorescin-diacetate (DCFH -DA), which was accompanied by a decrease in intracellular ascorbic acid an d an increase in apoptosis. A dramatic decrease in intracellular ascorbic a cid was noted when preloaded HL-60 cells were exposed to 150 mu M H2O2 (the concentration dropped from 5.2 +/- 0.6 mM to 3.6 +/- 0.1 mM in cells prein cubated with 150 mu M DHA). A dose-dependent protective effect of DHA was o bserved. Ascorbic acid loading also provided strong protection from apoptos is associated with serum- and glucose-free culture. Flow cytometry studies showed that exposure of HL-60 cells to 150 mu M H2O2 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 followin g exposure of HL-60 cells, loaded with ascorbic acid, to 150 mu M H2O2 and only a slight increase (up to 10.1 +/- 3.1%) in apoptosis. These findings i ndicate that ascorbic acid can inhibit apoptosis induced by oxidative stres s in HL-60 cells. (C) 1999 Elsevier Science Inc.