Ascorbate modulation of H2O2 and camptothecin-induced cell death in Jurkat cells

The effect of ascorbate on cell death was examined in Jurkat cells (human T-cell leukemia) by incubation with dehydroascorbate (DHA), which is rapidly taken up by cells and efficiently reduced to ascorbate. Apoptosis was evaluated by caspase-3 activity in cell extracts and flow cytometry of annexin V-labeled cells. In parallel, necrosis was estimated by the release of lactate dehydrogenase. Minor effects on cell death were observed when Jurkat cells were incubated with either DHA alone (100–1,000 M) or a single dose of 10 M H₂O₂. However, pre-incubation with DHA followed by exposure to H₂O₂ clearly stimulated both apoptosis and necrosis. In complete contrast, pre-incubation of cells with DHA significantly inhibited apoptosis, but did not affect necrosis, induced by the topoisomerase I inhibitor camptothecin. Our results indicate that intracellular ascorbate can modulate cell death in a manner which depends upon the nature of the apoptotic stimulus, which in turn has critical implications regarding the mechanism and potential application of ascorbate in cancer therapy.Abbreviations CPT 20-S-camptothecin lactone - DHA Dehydroascorbate - LDH Lactate dehydrogenase - Ac-DEVD-AMC Ac-Asp-Glu-Val-Asp-amino-4-methylcoumarin - PS Phosphatidylserine - GSH Glutathione - DTT Dithiothreitol