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

• Published in: Journal of cellular biochemistry 2006-12, Vol.99 (6), p.1628-1641
• Main Authors: Park, Seyeon, Lee, Joomin, Yeom, Chang-Hwan
• Format: Article
• Language: English
• Subjects: Apoptosis; Ascorbic Acid - pharmacology; Cell Line, Tumor; Cell Proliferation; Humans; L-ascorbic acid; Leukemia - pathology; NB4; Oxidation-Reduction; Protein Disulfide-Isomerases - metabolism; Proteome; proteomic approach; Proteomics; thiol/disulfide condition; tropomyosin; Tropomyosin - pharmacology; tropomyosin, thiol/disulfide condition
• ISSN: 0730-2312; 1097-4644
• DOI: 10.1002/jcb.20971

The pro‐oxidant effect of L‐ascorbic acid (LAA) is toxic to leukemia cells. LAA induces the oxidation of glutathione to its oxidized form (GSSG) and this is followed by a concentration‐dependent H2O2 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 cells treated 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 (2‐D) gel electrophoresis and matrix‐assisted laser desorption ionization time‐of‐flight‐MS. A subunit of protein‐disulfide isomerase (a thiol/disulfide 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 pI 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 in the GSH oxidation with changes in the superfamily of thiol/disulfide exchange catalysts. These results suggest that LAA oxidizes intracellular reduced glutathione and modulates disulfide bond formation in proteins. J. Cell. Biochem. 99: 1628–1641, 2006. © 2006 Wiley‐Liss, Inc.