NADPH Oxidase NOX5-S Mediates Acid-Induced DNA Damage in Barrett\'s Esophageal Adenocarcinoma Cells

AGA Abstracts

study we examine the role of mPGES1 in the acid-induced increase in PGE2 production and cell proliferation, and studied the role of NADPH oxidase NOX5-S in acid-induced upregulation of mPGES1 in an EA cell line FLO. RT-PCR shows that mPGES1, mPGES2 and cytosolic PGES (cPGES) are present in FLO cells. Pulsed acid treatment increased mPGES1 mRNA by 110% and mPGES2 by 10%, but did not have any effect on cPGES mRNA. Acid treatment significantly increased mPGES1 protein expression. Knockdown of mPGES1 by mPGES1 siRNA blocked acid-induced increase in PGE2 production and thymidine incorporation (an indicator of cell proliferation rate). Knockdown of NOX5-S by NOX5 siRNA significantly inhibited acid-induced increase in mPGES1 expression, PGE2 production and thymidine incorporation. Overexpression of NOX5-S significantly increased the luciferase activity in FLO cells transfected with a NF-κB In Vivo activation reporter plasmid pNF-κBLuc. Knockdown of NF-κB1 p50 by p50 siRNA almost abolished acid-induced increase in mPGES1 expression, PGE2 production and thymidine incorporation. In a chromatin immunoprecipitation assay, the promoter region of mPGES1 DNA was detectable in the immunoprecipitated chromatin sample of FLO cell lysate with a p50 antibody, indicating that p50 binds to mPGES1 promoter. We conclude that mPGES1 mediates acid-induced increase in PGE2 production and cell proliferation. Acid-induced mPGES1 expression depends on activation of NOX5-S and NF-κB1 p50. Microsomal PGES1 may be a potential target to prevent or treat EA. Supported by NIH NIDDK R01 DK080703.

Sa1083 A Possible Role for Toll-Like Receptor 4 Activation in Barrett's Esophagus in the Development of Esophageal Adenocarcinoma Romy E. Verbeek, Peter D. Siersema, Fiebo J. ten Kate, Kees Fluiter, Frank P. Vleggaar, Jantine W. van Baal BACKGROUND: Barrett's esophagus (BE) is thought to develop and progress to esophageal adenocarcinoma (EAC) in the setting of chronic inflammation. However, the underlying signaling pathways are incompletely understood. We hypothesize that Toll-Like Receptor (TLR) 4 activation in BE may be involved in the development of EAC through induction of COX2 and resistance to apoptosis. AIM: To determine the expression and functional activity of TLR4 in the esophagus and changes in COX2 expression and susceptibility to apoptosis following TLR4 activation in BE. METHODS: Esophageal biopsies were obtained during endoscopy from normal squamous epithelium (SQ), BE and EAC of 14 patients with BE and 8 with BE associated EAC. Expression of TLR4 mRNA was assessed by in situ hybridization using antisense oligonucleotides containing locked nucleic acid and 2'-Omethyl-RNA moieties, and TLR4 protein by immunohistochemistry. The former was quantified in frozen esophageal biopsies using Q-RT-PCR. To assess functional activity of TLR4, BE biopsies from 5 patients were stimulated with lipopolysaccharide (LPS), the natural TLR4 agonist. After 3 hours, IL8 production was determined in the supernatant by ELISA. RNA was isolated for determination of relative changes in COX2 expression by Q-RT-PCR. Western blot for the anti-apoptotic factors XIAP and BCL2 was performed in esophageal biopsies to study the effect on apoptosis. RESULTS: TLR4 mRNA as well as protein were for a large part expressed on the epithelial surface of BE and EAC but confined to the epithelial base in SQ. Q-RT-PCR showed a 9.1-fold (p