TGR5-Mediated NADPH Oxidase NOX5-S Expression Contributes to Bile Acid-Induced DNA Damage in Barrett's Esophageal Adenocarcinoma Cells
Dan Li, Murray Resnick, Jack Wands, Weibiao Cao. Rhode Island Hospital and the 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). There is considerable data suggesting that bile acids may contribute to the pathogenesis of progression from BE to dysplasia and adenocarcinoma. However, the mechanisms whereby bile acid reflux may accelerate this progression are not fully understood. We have previously shown that bile acid-induced NOX5-S expression depends on activation of bile acid receptor TGR5. In this study we examined whether TGR5-mediated NOX5-S expression is involved in bile acid-induced DNA damage in a Barrett's EA cell line FLO.
Design: DNA damage was measured by using a Comet assay which is based on the ability of denatured cleaved DNA fragments to migrate out of the cell under the influence of an electric potential. It is quantitated by measuring the tail length, tail area and tail moment. The phosphorylation of histone H2AX was examined by Western blot analysis.
Results: Bile acid taurodeoxycholic acid (TDCA, 10-11M, 24h) significantly increased tail length from 0.3±0.1 to 8.2±0.5 pixels, tail area from 15.5±5.3 to 272.4±16.6 pixels, and tail moment from 0.9±0.3 to 3.1±0.3, suggesting that TDCA increases DNA damage. To further confirm this result, the phosphorylation of histone H2AX, an indicator of double strand DNA break, was examined. TDCA markedly increased histone H2AX phosphorylation. TDCA-induced increase in tail length, tail area, tail moment and histone H2AX phosphorylation was significantly decreased by knockdown of NOX5-S with NOX5 siRNA. Conversely, overexpression of NOX5-S significantly increased tail length, tail area, tail moment and histone H2AX phosphorylation. Pre-treatment with diphenylene iodonium, an inhibitor of NOX, prevented the bile acid-induced increase in tail length, tail area and tail moment. In addition, TDCA-induced increase in tail length, tail area and tail moment was significantly decreased by knockdown of TGR5 with TGR5 siRNA, whereas overexpression of TGR5 with TGR5 plasmid significantly increased DNA damage.
Conclusions: Bile acid treatment causes DNA damage via activation of TGR5 and NOX5-S. It is possible that in Barrett's esophagus bile acid activates TGR5 and NOX5-S, and increases ROS production, which causes DNA damage, thereby contributing to the progression from BE to EA.
Supported by NIH NIDDK R01 DK080703.
Monday, March 4, 2013 11:30 AM
Proffered Papers: Section G2, Monday Morning