A proteomic approach to the identification of early molecular targets changed by L-ascorbic acid in NB4 human leukemia cells

The pro-oxidant effect Of L-ascorbic acid (LAA) is toxic to leukemia cel Is. LAA induces the oxidation of glutathione to its oxidized form (GSSG) and this is followed by a con centrati on-dependent H202 accumulation, which occurs in parallel to the induction of apoptosis. To identify early protein targets of LAA in leukemia cells, we used a differential proteomics approach in NB4 human leukemia cellstreated with 0.5 mM of LAA for 30 min. This exposure was determined to efficiently block cellular proliferation and to activate oxidative stress-inducible apoptosis. We identified nine proteins that sensitively reacted to LAA treatment by using two-dimensional (20 gel electrophoresis and matrixassisted laser desorption ionization time-of-flight-MS. A subunit of protein-disulficle isomerase (a thiol/disulficle exchange catalyst) and immunoglobulin-heavy-chain binding protein (BiP, identical to Hsp70 chaperone) showed quantitative expression profile differences. A myeloid leukemia associated antigen protein (a tropomyosin isoform) showed changes in p1 as a result of phosphorylation. Our studies demonstrate for the first time that the addition of LAA to cells results in an immediate change in the intracellular thiol/disulfide condition and that this includes an increase inthe GSH oxidation with changes in the superfamily of thiol/disulficle exchange catalysts. These results suggest that LAA oxidizes intracellular reduced glutathione and modulates disulfide bond formation in proteins.