Characterization of squamous esophageal cells resistant to bile acids at acidic pH: implication for Barrett's esophagus pathogenesis

Abstract

Barrett's esophagus (BE) is a premalignant condition, where normal squamous epithelium is replaced by intestinal epithelium. BE is associated with an increased risk of developing esophageal adenocarcinoma (EAC). However, the BE cell of origin is not clear. We hypothesize that BE tissue originates from esophageal squamous cells, which can differentiate to columnar cells as a result of repeated exposure to gastric acid and bile acids, two components of refluxate implicated in BE pathology. To test this hypothesis, we repeatedly exposed squamous esophageal HET1A cells to 0.2 mM bile acid (BA) cocktail at pH 5.5 and developed an HET1AR-resistant cell line. These cells are able to survive and proliferate after repeated 2-h treatments with BA at pH 5.5. HET1AR cells are resistant to acidification and express markers of columnar differentiation, villin, CDX2, and cytokeratin 8/18. HET1AR cells have increased amounts of reactive oxygen species, concomitant with a decreased level and activity of manganese superoxide dismutase compared with parental cells. Furthermore, HET1AR cells express proteins and activate signaling pathways associated with inflammation, cell survival, and tumorigenesis that are thought to contribute to BE and EAC development. These include STAT3, NF-κB, epidermal growth factor receptor (EGFR), cyclooxygenase-2, interleukin-6, phosphorylated mammalian target of rapamycin (p-mTOR), and Mcl-1. The expression of prosurvival and inflammatory proteins and resistance to cell death could be partially modified by inhibition of STAT3 signaling. In summary, our study shows that long-term exposure of squamous cells to BA at acidic pH causes the cells to display the same characteristics and markers as BE.

The role of nitric oxide in the induction of caudal-type homeobox 2 through epidermal growth factor receptor in the development of Barrett's esophagus

“…Next, we found the role of EGFR in CDX2 induction not only by AG1478 but also by siRNA specific for human EGFR, since the treatment of AG1478, commonly used as an EGFR inhibitor by inhibiting EGFR tyrosine kinase, might have nonspecific effects on EGFR [32,36]. The roles of EGFR in CDX2 induction were supported by previous studies [5,9,38] about the induction of CDX2 through the activation of EGFR by bile acids via both ligand-dependent and ligand-independent mechanisms. Taken together, we demonstrated that the exposure to NO or peroxynitrite ligand-independently enhanced CDX2 expression though the activation of EGFR in KYSE30.…”

“…Concerning the role of NO in EGFR activation, we showed that the treatment of NOC7, peroxynitrite or SIN-1 induced EGFR phosphorylation in a ligandindependent manner and that the effects of treatment with NOC7 were significantly inhibited by Carboxy-PTIO in a dose-dependent manner. The mechanisms in this signaling were described in previous studies [34,35,38,44] demonstrating that exogenous NO might induce the trans-activation of tyrosine residue in cytoplasm and that exogenous peroxynitrite might activate extracellular signal-regulated kinases in the EGFR signal transduction pathway. Next, we found the role of EGFR in CDX2 induction not only by AG1478 but also by siRNA specific for human EGFR, since the treatment of AG1478, commonly used as an EGFR inhibitor by inhibiting EGFR tyrosine kinase, might have nonspecific effects on EGFR [32,36].…”

“…One of these plasma membranous receptors that can interact with NO is epithelial growth factor receptor (EGFR), a 170-kDa glycoprotein that is a member of the receptor tyrosine kinase superfamily [1,3,[34][35][36][37]. A recent study [38] showed that ROS generated by the exposure of bile acid might enhance the CDX2 expression through the activation of EGFR. Therefore, we aimed to elucidate the hypothesis that the stimulation of large amounts of NO could directly induce CDX2 expression through EGFR activation in the development of BE.…”

Gastroesophageal reflux activates the NF-κB pathway and impairs esophageal barrier function in mice

“…IL-8 and IL-1␤ have been shown to be upregulated, even in endoscopy-negative patients (11,14,15,20,26,28). In vitro experiments also showed that exposure of human esophageal epithelial cells to acid or bile acid activated the NF-B pathway and upregulated IL-6 and IL-8 (10,12,17,35) as a function of the degree of acidity and exposure time (13). In this study, activation of the NF-B pathway was observed in the esophageal epithelium with duodenal or mixed reflux (Table 1, Fig.…”

Role of epidermal growth factor receptor tyrosine kinase inhibitors in the treatment of esophageal carcinoma and the suggested mechanisms of action

“…EGFR plays a key role in the process of normal esophageal epithelial cell carcinogenesis. BE tissue may originate from esophageal squamous cells (12). Long-term exposure of squamous cells to bile and gastric Role of epidermal growth factor receptor tyrosine kinase inhibitors in the treatment of esophageal carcinoma and the suggested mechanisms of action (Review)…”