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Abundant epidemiological evidence links acid reflux to adenocarcinoma in Barrett's esophagus, but few studies have examined the cellular mechanisms by which acid promotes this neoplastic progression. We hypothesized that extracellular acid exposure causes intracellular acidification that triggers MAPK signaling and proliferation in Barrett's epithelial cells. We tested that hypothesis in a Barrett's-derived esophageal adenocarcinoma cell line (SEG-1). SEG-1 cells were exposed to varying concentrations of acid, and intracellular pH (pH(i)) was measured by 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein microfluorimetry. After acid exposure, ERK and p38 MAPK activation were measured by Western blot analysis and an immune complex kinase assay. Proliferation was measured by Coulter counter cell counts and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide incorporation assay. Exposure of SEG-1 cells to solutions with a pH between 3 and 6.5 caused a rapid, reversible decrease in pH(i) to a level approximately equal to extracellular pH. Acid exposure caused a rapid activation of both ERK and p38 MAPKs and also resulted in pH-dependent increases in cell number, with a maximum increase of 41% observed at pH 6.0. The MAPK activation and proliferation in SEG-1 cells induced by acid exposure could be blocked by pretreatment with disodium 4,4'-diisothiocyanatostilbine-2,2'-disulfonate (DIDS), which prevents intracellular acidification by inhibiting the Cl(-)/HCO(3)(-) exchanger. In conclusion, in SEG-1 cells, extracellular acid exposure causes intracellular acidification, which activates MAPK and causes proliferation. The magnitude of these effects is pH dependent, and the effects can be inhibited by preventing intracellular acidification with DIDS.

Background: The mechanisms by which gastroesophageal reflux promotes malignant progression in Barrett's esophagus are poorly understood. The phosphatidylinositol-3-kinase (PI3 kinase)/Akt pathway regulates proliferation and apoptosis. We hypothesized that the PI3 kinase/Akt pathway mediates the pro-proliferative and antiapoptotic effects of bile., Methods: The Barrett's adenocarcinoma cell line, SEG-1, was exposed to the conjugated bile salt, glycochenodeoxycholic acid (GCDA). Cell number was measured by the MTT incorporation assay and by Coulter counter. PI3 kinase/Akt activity was inferred from Western blots of phosphorylated and total Akt. Proliferation and apoptosis were determined by BrdU incorporation and cell death ELISA., Results: A dose-dependent cell number increase was seen with a 20-minute exposure to GCDA. On Western blot, 200 micromol/L GCDA caused a 3-fold increase in Akt phosphorylation within 20 minutes, which was inhibited by 90% with the addition of PI3 kinase inhibitor, LY294002. LY294002 produced dose-dependent inhibition of GCDA-induced cell number increases. 200 micromol/L GCDA decreased apoptosis by 25%. Addition of LY294002 did not completely inhibit the antiapoptotic effect of bile., Conclusions: Bile salts activate the PI3 kinase/Akt signaling pathway and stimulate cell growth in SEG-1. The majority of this PI3 kinase-mediated effect is secondary to increases in proliferation rather than to decreases in apoptosis., (Copyright 2004 Elsevier Inc.)

Bile reflux has been implicated in the neoplastic progression of Barrett's esophagus (BE). Bile salts increase proliferation in a Barrett's-associated adenocarcinoma cell line (SEG-1 cells) by activating ERK and p38 MAPK pathways. However, it is not clear that these findings in cancer cells are applicable to non-neoplastic cells of benign BE. We examined the effect of bile salts on three human cell lines: normal esophageal squamous (NES) cells, non-neoplastic Barrett's cells (BAR cells), and SEG-1 cells. We hypothesized that bile salt exposure activates pro- proliferative and antiapoptotic pathways to promote increased growth in BE. NES, BAR, and SEG-1 cells were exposed to glycochenodeoxycholic acid (GCDA) at a neutral pH for 5 mm. Proliferation was measured by Coulter counter cell counts and a 5-bromo-2′-deoxyuridine (BrdU) incorporation assay. GCDA-induced MAPK activation was examined by Western blot analysis for phosphorylated ERK and p38. Apoptosis was measured by TdT-mediated dUTP nick-end labeling and annexin V staining after GCDA and UV-B exposure. Statistical significance was determined by ANOVA. NES cells exposed to 5 mm of GCDA did not increase cell number. In BAR cells, GCDA exposure increased cell number by 31%, increased phosphorylated p38 and ERK levels by two- to three-fold, increased BrdU incorporation by 30%, and decreased UV-induced apoptosis by 15-20%. In conclusion, in a non-neoplastic Barrett's cell line, GCDA exposure induces proliferation by activation of both ERK and p138 MAPK pathways. These findings suggest a potential mechanism whereby bile reflux may facilitate the neoplastic progression of BE. [ABSTRACT FROM AUTHOR]

Abundant epidemiological evidence links acid reflux to adenocarcinoma in Barrett's esophagus, but few studies have examined the cellular mechanisms by which acid promotes this neoplastic progression. We hypothesized that extracellular acid exposure causes intracellular acidification that triggers MAPK signaling and proliferation in Barrett's epithelial cells. We tested that hypothesis in a Barrett's-derived esophageal adenocarcinoma cell line (SEG-1). SEG-1 cells were exposed to varying concentrations of acid, and intracellular pH (pHi) was measured by 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein microfluorimetry. After acid exposure, ERK and p38 MAPK activation were measured by Western blot analysis and an immune complex kinase assay. Proliferation was measured by Coulter counter cell counts and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide incorporation assay. Exposure of SEG-1 cells to solutions with a pH between 3 and 6.5 caused a rapid, reversible decrease in pHi to a level approximately equal to extracellular pH. Acid exposure caused a rapid activation of both ERK and p38 MAPKs and also resulted in pH-dependent increases in cell number, with a maximum increase of 41% observed at pH 6.0. The MAPK activation and proliferation in SEG-1 cells induced by acid exposure could be blocked by pretreatment with disodium 4,4′-diisothiocyanatostilbine-2,2′-disulfonate (DIDS), which prevents intracellular acidification by inhibiting the Cl-/HCO3- exchanger. In conclusion, in SEG-1 cells, extracellular acid exposure causes intracellular acidification, which activates MAPK and causes proliferation. The magnitude of these effects is pH dependent, and the effects can be inhibited by preventing intracellular acidification with DIDS. [ABSTRACT FROM AUTHOR]