The Role of Vitamin C as a Competition Modulator in Barrett’s Esophagus Cell Culture Model

A136 Barrett’s esophagus (BE), a condition caused by gastro-esophageal reflux, is associated with an incidence of esophageal adenocarcinoma (EA) of 0.5-1% per year (Sharma P. et al. 2006). BE is defined by the displacement of normal squamous by specialized intestinal epithelium. Inflammation, common in BE, generates reactive oxygen species, and is thought to contribute to the development of a variety of cancers. The role of antioxidants in protection against BE and EA remains unclear. In general, patients with EA were found to have lower intakes of antioxidants from citrus fruits and raw vegetables compared to control populations (Terry P. et al 2001). Recent studies have shown that BE patients have significantly lower plasma levels of vitamin C, xanthophylls and other antioxidants (Clements D. et al. 2005). BE tissue was found to contain significantly lower levels of vitamin C compared with normal squamous mucosa (Fountoulakis et al. 2004). >The aim of our study was to determine if antioxidants (i.e. vitamin C) are capable of altering the microenvironment of Barrett’s cells, thereby biasing the competition between Barrett’s and squamous cells in favor of the squamous cells. Our model acts by competing normal esophageal squamous cells (EPC2) in co-culture with one of 4 different BE cell lines, each with a constitutively-expressed GFP or DsRed protein. These competitions were measured over 14 days by monitoring the proportion of the cell lines by flow cytometry. Based on epidemiological data of vitamin C plasma levels correlating with BE, we investigated the effect that vitamin C could have in competition between normal squamous and BE cell lines. >Results indicated that vitamin C (50-500µM) does act to modulate competition between squamous and CP-D BE cells, with squamous increasing their proportion in the competition by day 7 and continuing past day 14. Less of an effect was observed for the CP-A and CP-C cells and no effect was observed for CP-B. Proliferation levels, based on Ki67 expression, were not different for squamous grown with vitamin C, yet a marked decrease was observed for CP-C and CP-D with increasing vitamin C. Apoptosis and necrosis, measured by annexin V and 7AAD, were decreased in squamous cells, yet increased in CP-D cells with increasing vitamin C. Apoptosis in the other BE cell lines (CP-A, CP-B and CP-C) were not different from their controls. Cell cycle analysis indicated that there was no change observed for normal squamous cells, yet for CP-D, more cells accumulated in G1 and less in S-phase with increasing vitamin C. These data (proliferation, apoptosis, cell cycle) result from 7-day exposure to vitamin C in monoculture conditions. >Vitamin C appears to alter the competition between squamous and BE cells. This decrease in proliferation is more likely due to an increase in cells remaining in the G1 phase of the cell cycle. Also, CP-D cells showed higher apoptosis due to vitamin C exposure than squamous cells. These results suggest that vitamin C may be used to modulate competition between Barrett’s and squamous esophageal epithelium and therefore, may be an effective EA prevention agent. Recent epidemiological results support this conclusion and show that vitamin C supplementation is associated with a decrease in cancer risk in the Seattle Barrett’s Esophagus cohort (Dong et al. in press).