Downregulation of p63 upon exposure to bile salts and acid in normal and cancer esophageal cells in culture

p63 is a member of the p53 protein family that regulates differentiation and morphogenesis in epithelial tissues and is required for the formation of squamous epithelia. Barrett's mucosa is a glandular metaplasia of the squamous epithelium that develops in the lower esophagus in the context of chronic, gastroesophageal reflux and is considered as a precursor for adenocarcinoma. Normal or squamous cancer esophageal cells were exposed to deoxycholic acid (DCA, 50, 100, or 200 μM) and chenodeoxycholic and taurochenodeoxycholic acid at pH 5. p63 and cyclooxygenase-2 (COX-2) expressions were studied by Western blot and RT-PCR. DCA exposure at pH 5 led to a spectacular decrease in the levels of all isoforms of the p63 proteins. This decrease was observed within minutes of exposure, with a synergistic effect between DCA and acid. Within the same time frame, levels of p63 mRNA were relatively unaffected, whereas levels of COX-2, a marker of stress responses often induced in Barrett's mucosa, were increased. Similar results were obtained with chenodeoxycholic acid but not its taurine conjugate at pH 5. Proteasome inhibition by lactacystin or MG-132 partially blocked the decrease in p63, suggesting a posttranslational degradation mechanism. These results show that combined exposure to bile salt and acid downregulates a critical regulator of squamous differentiation, providing a mechanism to explain the replacement of squamous epithelium by a glandular metaplasia upon exposure of the lower esophagus to gastric reflux.

Expression of Nitric Oxide Synthase Type 3 in Reflux-Induced Esophageal Lesions

BACKGROUND: The expression of endothelial constitutive nitric oxide synthase (NOS3) by squamous dysplasia and carcinomas of the head and neck has previously been described. We sought to compare NOS3 expression in squamous mucosa, glandular metaplasia, and adenocarcinoma of the esophagus. METHODS: Forty paraffin-embedded specimens from 20 patients with adenocarcinoma were stained with anti-NOS3 monoclonal antibody. The percentage of cells stained and the intensity of staining were determined for squamous epithelium, metaplasia, and adenocarcinoma. Staining characteristics were statistically analyzed according to clinical variables. RESULTS: NOS3 expression was significantly higher in adenocarcinoma and squamous epithelium compared with glandular metaplasia. Among the carcinomas, larger tumor size (T3/4), nodal positivity, and advanced TNM stage (III/IV) significantly correlated with increased NOS3 expression. CONCLUSIONS: NOS3 is expressed in refluxinduced lesions of the esophagus. Glandular metaplasia shows basal levels of NOS3 that significantly increase with malignant transformation and tumor progression. The role of free radicals in carcinogenesis is being actively studied.