Acid increases MAPK-mediated proliferation in Barret's esophageal adenocarcinoma cells via intracellular acidification through a Cl-/HCO3- exchanger

Dept. of Surgery, Medical University of Texas Southwestern Medical Center, Dallas, TX 75216, USA.
AJP Gastrointestinal and Liver Physiology (Impact Factor: 3.8). 01/2006; 289(6):G991-7. DOI: 10.1152/ajpgi.00215.2005
Source: PubMed


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.

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    • "Clinical studies have identified esophageal acid reflux as a risk factor for Barrett's esophagus and adenocarcinoma[3]. In vitro experiments using adenocarcionoma and non-neoplastic Barrett's esophageal cell lines established that short-term exposure to an acidic environment results in increase cell proliferation [5-7]. In addition, activation of signal transduction pathways associated with cell proliferation is observed with acid exposure. "
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    ABSTRACT: Esophageal reflux and Barrett's esophagus represent two major risk factors for the development of esophageal adenocarcinoma. Previous studies have shown that brief exposure of the Barrett's-associated adenocarcinoma cell line, SEG-1, or primary cultures of Barrett's esophageal tissues to acid or bile results in changes consistent with cell proliferation. In this study, we determined whether similar exposure to acid or bile salts results in gene expression changes that provide insights into malignant transformation. Using previously published methods, Barrett's-associated esophageal adenocarcinoma cell lines and primary cultures of Barrett's esophageal tissue were exposed to short pulses of acid or bile salts followed by incubation in culture media at pH 7.4. A genome-wide assessment of gene expression was then determined for the samples using cDNA microarrays. Subsequent analysis evaluated for statistical differences in gene expression with and without treatment. The SEG-1 cell line showed changes in gene expression that was dependent on the length of exposure to pH 3.5. Further analysis using the Gene Ontology, however, showed that representation by genes associated with cell proliferation is not enhanced by acid exposure. The changes in gene expression also did not involve genes known to be differentially expressed in esophageal adenocarcinoma. Similar experiments using short-term primary cultures of Barrett's esophagus also did not result in detectable changes in gene expression with either acid or bile salt exposure. Short-term exposure of esophageal adenocarcinoma SEG-1 cells or primary cultures of Barrett's esophagus does not result in gene expression changes that are consistent with enhanced cell proliferation. Thus other model systems are needed that may reflect the impact of acid and bile salt exposure on the esophagus in vivo.
    BMC Gastroenterology 02/2007; 7(1):24. DOI:10.1186/1471-230X-7-24 · 2.37 Impact Factor
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    • "The two main risk factors for OAC are oesophageal acid reflux and obesity [16-19]. Previous studies have shown that intermittent acid exposure stimulated proliferation and inhibited apoptosis in BO and OAC cell lines, although the involvement of Akt has not been examined [20-23]. Serum levels of the peptide hormone leptin are increased in obesity in proportion to body fact mass [24], recent data suggest that leptin receptors are expressed by BO and OAC [25,26] and studies have also shown that leptin can stimulate proliferation of OAC cells in culture [26,27]. "
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    ABSTRACT: The incidence of oesophageal adenocarcinoma is increasing rapidly in the developed world. The serine-threonine protein kinase and proto-oncogene Akt has been reported to regulate proliferation and apoptosis in several tissues but there are no data on the involvement of Akt in oesophageal carcinogenesis. Therefore we have examined the activation of Akt in Barrett's oesophagus and oesophageal adenocarcinoma and the functional effects of Akt activation in vitro. Expression of total and active (phosphorylated) Akt were determined in endoscopic biopsies and surgical resection specimens using immunohistochemistry. The functional effects of Akt were examined using Barrett's adenocarcinoma cells in culture. In normal squamous oesophagus, erosive oesophagitis and non-dysplastic Barrett's oesophagus, phospho-Akt was limited to the basal 1/3 of the mucosa. Image analysis confirmed that Akt activation was significantly increased in non-dysplastic Barrett's oesophagus compared to squamous epithelium and further significantly increased in high-grade dysplasia and adenocarcinoma. In all cases of high grade dysplasia and adenocarcinoma Akt was activated in the luminal 1/3 of the epithelium. Transient acid exposure and the obesity hormone leptin activated Akt, stimulated proliferation and inhibited apoptosis: the combination of acid and leptin was synergistic. Inhibition of Akt phosphorylation with LY294002 increased apoptosis and blocked the effects of acid and leptin both alone and in combination. Activation of Akt was associated with downstream phosphorylation and deactivation of the pro-apoptotic protein Bad and phosphorylation of the Forkhead family transcription factor FOXO1. Akt is abnormally activated in Barrett's oesophagus, high grade dysplasia and adenocarcinoma. Akt activation promotes proliferation and inhibits apoptosis in Barrett's adenocarcinoma cells and both transient acid exposure and leptin stimulate Akt phosphorylation. Downstream targets of Akt include Bad and Forkhead transcription factors. Activation of Akt in obesity and by reflux of gastric acid may be important in the pathogenesis of Barrett's adenocarcinoma.
    BMC Cancer 02/2007; 7(1):97. DOI:10.1186/1471-2407-7-97 · 3.36 Impact Factor

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