sodium-taurodeoxycholate and Esophageal-Neoplasms

Mechanisms of the progression from Barrett's oesophagus to oesophageal adenocarcinoma (OA) are not fully understood. Bile acids may have an important role in this progression. This study aimed at examining the role of NADPH oxidase NOX5-S and a novel bile acid receptor TGR5 in taurodeoxycholic acid (TDCA)-induced increase in cell proliferation.. Human Barrett's cell line BAR-T and OA cell line FLO were transfected by the Lipofectamine 2000 or Amaxa-Nucleofector-System. mRNAs were measured by real-time PCR. H(2)O(2) was measured by a fluorescent assay. Cell proliferation was determined by measurement of thymidine incorporation.. NOX5-S was present in FLO cells. TDCA significantly increased NOX5-S expression, H(2)O(2) production and thymidine incorporation in FLO and BAR-T cells. This increase in thymidine incorporation was significantly reduced by knockdown of NOX5-S. TGR5 mRNA and protein levels were significantly higher in OA tissues than in normal oesophageal mucosa or Barrett's mucosa. Knockdown of TGR5 markedly inhibited TDCA-induced increase in NOX5-S expression, H(2)O(2) production and thymidine incorporation in FLO and BAR-T cells. Overexpression of TGR5 significantly enhanced the effects of TDCA in FLO cells. TGR5 receptors were coupled with Galphaq and Galphai3 proteins, but only Galphaq mediated TDCA-induced increase in NOX5-S expression, H(2)O(2) production and thymidine incorporation in FLO cells.. TDCA-induced increase in cell proliferation depends on upregulation of NOX5-S expression in BAR-T and FLO cells. TDCA-induced NOX5-S expression may be mediated by activation of the TGR5 receptor and Galphaq protein. These data may provide potential targets to prevent and/or treat Barrett's OA.

Topics: Adenocarcinoma; Cholagogues and Choleretics; Esophageal Neoplasms; Gene Expression Regulation; Gene Knockdown Techniques; GTP-Binding Protein alpha Subunits, Gq-G11; Humans; Membrane Proteins; NADPH Oxidase 5; NADPH Oxidases; Neoplasm Proteins; Reactive Oxygen Species; Receptors, G-Protein-Coupled; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Taurodeoxycholic Acid; Tumor Cells, Cultured

Gastroesophageal reflux disease complicated by Barrett's esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). However, the mechanisms of the progression from BE to EA are not fully understood. Besides acid reflux, bile acid reflux may also play an important role in the progression from BE to EA. In this study, we examined the role of phosphatidylinositol-specific phospholipase C (PI-PLC) and a novel NADPH oxidase NOX5-S in bile acid-induced increase in cell proliferation. We found that taurodeoxycholic acid (TDCA) significantly increased NOX5-S expression, hydrogen peroxide (H(2)O(2)) production, and cell proliferation in EA cells. The TDCA-induced increase in cell proliferation was significantly reduced by U73122, an inhibitor of PI-PLC. PI-PLCbeta1, PI-PLCbeta3, PI-PLCbeta4, PI-PLCgamma1, and PI-PLCgamma2, but not PI-PLCbeta2 and PI-PLCdelta1, were detectable in FLO cells by Western blot analysis. Knockdown of PI-PLCgamma2 or extracellular signal-regulated kinase (ERK) 2 mitogen-activated protein (MAP) kinase with small interfering RNAs (siRNA) significantly decreased TDCA-induced NOX5-S expression, H(2)O(2) production, and cell proliferation. In contrast, knockdown of PI-PLCbeta1, PI-PLCbeta3, PI-PLCbeta4, PI-PLCgamma1, or ERK1 MAP kinase had no significant effect. TDCA significantly increased ERK2 phosphorylation, an increase that was reduced by U73122 or PI-PLCgamma2 siRNA. We conclude that TDCA-induced increase in NOX5-S expression and cell proliferation may depend on sequential activation of PI-PLCgamma2 and ERK2 MAP kinase in EA cells. It is possible that bile acid reflux present in patients with BE may increase reactive oxygen species production and cell proliferation via activation of PI-PLCgamma2, ERK2 MAP kinase, and NADPH oxidase NOX5-S, thereby contributing to the development of EA.

Topics: Adenocarcinoma; Bile Acids and Salts; Bile Reflux; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cholagogues and Choleretics; Enzyme Activation; Esophageal Neoplasms; Estrenes; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Hydrogen Peroxide; Isoenzymes; Membrane Proteins; Mitogen-Activated Protein Kinase 1; NADPH Oxidase 5; NADPH Oxidases; Phosphodiesterase Inhibitors; Phospholipase C gamma; Pyrrolidinones; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Taurodeoxycholic Acid

MUC4 (mucin 4) is a membrane-bound mucin overexpressed in the early steps of oesophageal carcinogenesis and implicated in tumour progression. We previously showed that bile acids, main components of gastro-oesophageal reflux and tumour promoters, up-regulate MUC4 expression [Mariette, Perrais, Leteurtre, Jonckheere, Hemon, Pigny, Batra, Aubert, Triboulet and Van Seuningen (2004) Biochem. J. 377, 701-708]. HNF (hepatocyte nuclear factor) 1alpha and HNF4alpha transcription factors are known to mediate bile acid effects, and we previously identified cis-elements for these factors in MUC4 distal promoter. Our aim was to demonstrate that these two transcription factors were directly involved in MUC4 activation by bile acids. MUC4, HNF1alpha and HNF4alpha expressions were evaluated by immunohistochemistry in human oesophageal tissues. Our results indicate that MUC4, HNF1alpha and HNF4alpha were co-expressed in oesophageal metaplastic and adenocarcinomatous tissues. Studies at the mRNA, promoter and protein levels indicated that HNF1alpha regulates endogenous MUC4 expression by binding to two cognate cis-elements respectively located at -3332/-3327 and -3040/-3028 in the distal promoter. We also showed by siRNA (small interfering RNA) approach, co-transfection and site-directed mutagenesis that HNF1alpha mediates taurodeoxycholic and taurochenodeoxycholic bile acid activation of endogenous MUC4 expression and transcription in a dose-dependent manner. In conclusion, these results describe a new mechanism of regulation of MUC4 expression by bile acids, in which HNF1alpha is a key mediator. These results bring new insights into MUC4 up-regulation in oesophageal carcinoma associated with bile reflux.

Topics: Bile Reflux; Cell Line, Tumor; Esophageal Neoplasms; Gene Expression Regulation, Neoplastic; Hepatocyte Nuclear Factor 1-alpha; Hepatocyte Nuclear Factor 4; Humans; Immunohistochemistry; Mucin-4; Mucins; Promoter Regions, Genetic; RNA, Small Interfering; Taurochenodeoxycholic Acid; Taurodeoxycholic Acid; Transcription, Genetic; Transfection; Up-Regulation