NADPH Oxidase NOX5-S Mediates Acid-Induced Increase in H2O2 Production and Cell Proliferation in Barrett's Esophageal Adenocarcinoma Cells
J Hong, J Behar, M Resnick, LJ Wang, RA DeLellis, J Wands, W Cao. Rhode Island Hospital and Warren Alpert Medical School of Brown University, Providence, RI
Background: Gastro-esophageal reflux disease complicated by Barrett's esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). The mechanisms whereby acid reflux may accelerate the progression from BE to EA are not fully understood. We therefore investigated the role of NADPH oxidases in acid-induced changes in Barrett's EA cell line FLO.
Design: FLO cells or BE mucosal biopsies were exposed to pH 4.0 for 1hr, then washed and cultured at pH 7.2 for 24hrs. The mRNA levels of NOX5-S were measured by real time PCR. Cell proliferation was determined by measuring thymidine incorporation. Rho kinase activity was determined by a Cyclex ROCK assay kit.
Results: RT-PCR and 5'-RACE showed that NOX5-S was the major isoform of NADPH oxidase in FLO cells. NOX5-S mRNA expression was significantly greater in EA cell lines (FLO, and OE33) than in esophageal squamous epithelial cell line HET-1A. The levels of NOX5 mRNA were also markedly increased in BE mucosal biopsies with moderate dysplasia and in EA tissues, when compared with normal esophageal mucosa or BE mucosa. Immunohistochemical studies showed that NOX5-S was present in the cytosol of FLO cells. In FLO-EA cells, knockdown of NOX5-S with NOX5 siRNA significantly decreased cell proliferation at basal condition and inhibited acid-induced increase in cell proliferation. Acid treatment significantly increased H2O2 production in both FLO cells and BE mucosa. Acid-induced H2O2 production was blocked by the NADPH oxidase inhibitor apocynin. Acid increased mRNA expression of NOX5-S, and knockdown of NOX5-S abolished acid-induced H2O2 production in FLO cells. Acid-induced NOX5-S expression and H2O2 production were significantly decreased by MAP kinase kinase inhibitor PD98059 and Rho kinase inhibitor Y27632. In addition, acid treatment significantly increased the activity of Rho kinase and the phosphorylation of ERK2 MAP kinase. Acid-induced increase in phosphorylation of ERK2 MAP kinase was abolished by Y27632.
Conclusions: In EA cells acid induces H2O2 production by activation of NOX5-S and causes upregulation of NOX5-S expression through sequential activation of Rho kinase and ERK2 MAP kinase. In these cells NOX5-S contributes to increased cell proliferation. Supported by NIH NIDDK R21 DK073327-01.
Tuesday, March 10, 2009 8:30 AM
Platform Session: Section G 1, Tuesday Morning