Ascorbic Acid Inhibits Apoptosis Induced by X Irradiation in HL60 Myeloid Leukemia Cells

Abstract

Exposure of cells to ionizing radiation can cause apoptosis. Since antioxidants have been shown to protect against radiation-induced apoptosis, in this study we have evaluated the putative protective effect of ascorbate against radiation-induced apoptosis as well as the production of peroxides in the cells. HL60 cells transport the oxidized form of ascorbic acid, dehydroascorbic acid (DHA), and accumulate reduced ascorbate. Exposure of the cells to 5-40 Gy X radiation resulted in induction of apoptosis. Preincubation of the cells with DHA reduced the level of apoptosis after exposure to 5-20 Gy. Exposure of the cells to 5 or 20 Gy X radiation did not affect the intracellular concentration of peroxides, while phorbol myristate acetate (PMA), which is known to induce production of H(2)O(2) in cells (and served as a control), resulted in an increase in peroxides and a decrease in intracellular ascorbate. Irradiation of the cells with 1-3 Gy resulted in up-regulation of expression of BCL2 without affecting the level of apoptosis. At higher doses of radiation, enhanced BCL2 expression did not prevent radiation-induced apoptosis. Loading of the cells with ascorbate prior to their exposure to 1-3 Gy X radiation did not affect the enhanced BCL2 expression observed in the irradiated cells. At higher doses of radiation, ascorbate decreased apoptosis and restored the level of BCL2 in the cells. Exposure of the cells to 3-20 Gy X radiation enhanced the cell surface expression of TNFRSF6 (formerly known as Fas/APO-1) antigen and enhanced anti-TNFRSF6 antibody-induced apoptosis of the cells. Ascorbate loading did not affect expression of TNFRSF6 and did not overcome the anti-TNFRSF6 antibody-induced apoptosis. In conclusion, our data demonstrate that exposure of HL60 cells to radiation enhanced BCL2 and TNFRSF6 expression. Ascorbate did not affect BCL2 or TNFRSF6 expression. We therefore conclude that it protects HL60 cells against radiation-induced apoptosis, although the mechanisms of protection must still be elucidated.

Mechanism of Vitamin C Inhibition of Cell Death Induced by Oxidative Stress in Glutathione-depleted HL-60 Cells

“…HL-60 cells accumulate millimolar concentrations of vitamin C, and their resistance to H 2 O 2 increases in proportion to the intracellular concentration of ascorbic acid in a dose-dependent manner. Similar results have been observed in other cell lines where preloading with ascorbic acid was shown to protect from H 2 O 2 generated by xanthine oxidase (35) and by direct treatment with H 2 O 2 or radiation (7,8,36).…”

“…Oxidative damage has been linked to many disease states (3,4) and to the development of cancer via the oxidation of DNA bases (5,6). The role of antioxidant defenses in tumor cells themselves, however, has been poorly studied, and the role of antioxidants in neoplastic cell responses to radiation and chemotherapy is not understood completely (7)(8)(9)(10).…”

Vitamin C protects HL60 and U266 cells from arsenic toxicity

“…37,63,64 To circumvent the in vitro pro-oxidant effects of AA, we loaded HL60 and U266 cells (which do not transport AA directly) with vitamin C by incubating them with DHA. [68][69][70] Cells loaded with vitamin C were significantly protected from As 2 O 3 toxicity. Increasing concentrations of intracellular vitamin C in HL60, U266, and RPMI-8226 cells provided increasing protection from As 2 O 3 cytotoxicity with maximum protection achieved at 2 to 8 mM intracellular AA.…”

Regulation of Apoptosis by Vitamin C

“…These events culminate in activation of the effector caspases, including caspase-3 that cleaves a number of structural and regulatory proteins (49,50). Oxidant stress can initiate apoptosis, and this can be partly prevented by cellular anti-oxidants, including ascorbate (10,11). In our current experiments, exposure of HUVEC to NH 2 Cl or glycine-NHCl for 60 min resulted in an increase in apoptosis that was not greatly affected by ascorbate.…”