Roles of acid-induced NF-kB p65 in esophageal neoplasia

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Esophageal adenocarcinoma (EAC) is one of the most rapidly increasing incident cancers in the USA. Barrett’s esophagus (BE), caused by gastroesophageal reflux disease (GERD), is the obligate precursor lesion of this cancer. Previous studies have shown the involvement of gastric acid in the development of BE and EAC. Defective repair of mucosal lesions in the inflammatory milieu created by GERD may make esophageal epithelium prone to metaplasia as a primary adaptive mechanism, and later to neoplasia. Cancer develops either via enhanced cellular proliferation or by decreased cell death. Apoptosis is an established mechanism that mediates cell death and represents the principal means for organisms to maintain proper cell numbers. Cells gaining the potential to resist apoptosis may be more prone to develop into tumors. Tumor necrosis alpha (TNFα) is a potent apoptosis-inducing cytokine. Increased TNFα expression has also been reported during progression from BE to EAC. Moreover, acid induces esophageal epithelial inflammation, and previous studies have shown that low pH induces esophageal epithelial cell proliferation. NF-kB p65 is a member of the NF-kB family, comprising five transcription factors, with a demonstrated role in cellular proliferation; it is induced in other inflammatory conditions and has been detected in the progression of BE to EAC.

For these reasons, we performed 3-minute stimulations of SEG1 EAC cells with serum-free pH3 medium, then returned the cells to normal pH medium, mirroring in vivo physiologic GERD conditions. Acid exposure resulted in increased cellular proliferation, as determined by WST-1 assay. Interestingly, exposure of cells to pH3 serum-free medium for 3 minutes increased NF-kB p65 activity 4-fold at 20 minutes and 8-fold at 40 minutes post-exposure, compared with unstimulated cells.

Our results also showed that TNFα stimulation of SEG1 cells for 20h was a strong inducer of apoptosis, reflected by a 7-fold increase in caspase3 activity vs. unstimulated cells. Acid exposure alone did not modify caspase3 activity. Notably, acid exposure prior to TNFα stimulation reduced caspase3 activation by one-third, providing a stimulus that partially rescued the cells from apoptosis.

Our data regarding the effects of acid exposure suggest that NF-kB p65 is involved in both cellular proliferation and apoptosis. We are currently employing a specific NF-kB p65 inhibitor (JSH-23, Calbiochem, San Diego, CA) to clarify the role of NF-kB p65 signaling induced by acid stimulation of cells with regard to enhanced cell proliferation and decreased apoptosis. At the time of the submission of this abstract, our results now show that JSH-23 successfully inhibits NF-kB p65 activation in acid-stimulated SEG1 cells. If NF-kB is involved in the development of esophageal neoplasia, inhibition of this transcription factor may have application as a means of preventing the progression of BE to EAC.