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Activation of NADPH oxidases leads to DNA damage in esophageal cells.

ABSTRACT: Gastroesophageal reflux disease (GERD) is the strongest known risk factor for esophageal adenocarcinoma. In the center of tumorigenic events caused by GERD is repeated damage of esophageal tissues by the refluxate. In this study, we focused on a genotoxic aspect of exposure of esophageal cells to acidic bile reflux (BA/A). Analyzing cells generated from patients with Barrett's esophagus and human esophageal specimens, we found that BA/A cause significant DNA damage that is mediated by reactive-oxygen species. ROS originate from mitochondria and NADPH oxidases. We specifically identified NOX1 and NOX2 enzymes to be responsible for ROS generation. Inhibition of NOX2 and NOX1 with siRNA or chemical inhibitors significantly suppresses ROS production and DNA damage induced by BA/A. Mechanistically, our data showed that exposure of esophageal cells to acidic bile salts induces phosphorylation of the p47^phox subunit of NOX2 and its translocation to the cellular membrane. This process is mediated by protein kinase C, which is activated by BA/A. Taken together, our studies suggest that inhibition of ROS induced by reflux can be a useful strategy for preventing DNA damage and decreasing the risk of tumorigenic transformation caused by GERD.

SUBMITTER: Bhardwaj V

PROVIDER: S-EPMC5577233 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Activation of NADPH oxidases leads to DNA damage in esophageal cells.

Bhardwaj Vikas V Gokulan Ravindran Caspa RC Horvat Andela A Yermalitskaya Liudmila L Korolkova Olga O Washington Kay M KM El-Rifai Wael W Dikalov Sergey I SI Zaika Alexander I AI

Scientific reports 20170830 1

Gastroesophageal reflux disease (GERD) is the strongest known risk factor for esophageal adenocarcinoma. In the center of tumorigenic events caused by GERD is repeated damage of esophageal tissues by the refluxate. In this study, we focused on a genotoxic aspect of exposure of esophageal cells to acidic bile reflux (BA/A). Analyzing cells generated from patients with Barrett's esophagus and human esophageal specimens, we found that BA/A cause significant DNA damage that is mediated by reactive-o ...[more]

PMID: 28855537

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Project description:BackgroundThe incidence of esophageal adenocarcinoma (EAC) is rising rapidly in the US and Western countries. The development of Barrett's esophagus (BE) and its progression to EAC have been linked to chronic gastroesophageal reflux disease (GERD). Exposure of BE and EAC cells to acidic bile salts (ABS) in GERD conditions induces high levels of oxidative stress and DNA damage. In this study, we investigated the role of insulin-like growth factor binding protein 2 (IGFBP2) in regulating ABS-induced DNA double-strand breaks.MethodsReal-time RT-PCR, western blot, immunohistochemistry, immunofluorescence, co-immunoprecipitation, flow cytometry, and cycloheximide (CHX) chase assays were used in this study. To mimic GERD conditions, a cocktail of acidic bile salts (pH?4) was used in 2D and 3D organotypic culture models. Overexpression and knockdown of IGFBP2 in EAC cells were established to examine the functional and mechanistic roles of IGFBP2 in ABS-induced DNA damage.ResultsOur results demonstrated high levels of IGFBP2 mRNA and protein in EAC cell lines as compared to precancerous Barrett's cell lines, and IGFBP2 is frequently overexpressed in EACs (31/57). Treatment of EAC cells with ABS, to mimic GERD conditions, induced high levels of IGFBP2 expression. Knocking down endogenous IGFBP2 in FLO1 cells (with constitutive high levels of IGFBP2) led to a significant increase in DNA double-strand breaks and apoptosis, following transient exposure to ABS. On the other hand, overexpression of exogenous IGFBP2 in OE33 cells (with low endogenous levels of IGFBP2) had a protective effect against ABS-induced double-strand breaks and apoptosis. We found that IGFBP2 is required for ABS-induced nuclear accumulation and phosphorylation of EGFR and DNA-PKcs, which are necessary for DNA damage repair activity. Using co-immunoprecipitation assay, we detected co-localization of IGFBP2 with EGFR and DNA-PKcs, following acidic bile salts treatment. We further demonstrated, using cycloheximide chase assay, that IGFBP2 promotes EGFR protein stability in response to ABS exposure.ConclusionsIGFBP2 protects EAC cells against ABS-induced DNA damage and apoptosis through stabilization and activation of EGFR - DNA-PKcs signaling axis.

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Activation of NADPH oxidases leads to DNA damage in esophageal cells.

Publications

Activation of NADPH oxidases leads to DNA damage in esophageal cells.

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