S1626 A Novel Mechanism of Bile Acid Induced Damage: Relevance to Esophageal Carcinogenesis

on autophagy in BE cells. Methods: Barrett's esophagus CP-A cells were treated for 10 minutes with medium acidified to pH 4 and/or 0.2mM hydrophobic bile acid cocktail following 170 minutes incubation in normal medium. As a positive control for autophagy CP-A cells were also exposed to rapamycin or amino acid starvation. Autophagy was assessed by evaluating the LC3II/LC3I ratio, by quantification of cells showing the punctuate pattern of LC3-GFP and by transmission electronmicroscopy (TEM). In addition, we also determined the localization and expression of proautophagy protein Beclin1 and Lamp1, a specific marker of autophagic vacuole maturation, in the cells exposed to different treatments by confocal microsopy and western blot analysis. Results: Our results indicate that acidified medium in combination with bile acids induces autophagy in BE cells as demonstrated by the increased conversion of LC3-I (the cytosolic form) into LC3-II (membrane bound), the increased expression of Beclin 1 and the formation of typical autophagosomes by TEM. Furthermore, 53.5±4.0% of cells transfected with GFP-LC3 showed a punctuated pattern after exposure to acidified medium and bile acids, while only 17.4±2.5% of untreated cells showed this pattern. Supporting this, we observed an apparent expansion of the lysosomal compartment in cells treated with acidified medium and bile acids compared with untreated cells as visualized with the lysosomal-specific marker, Lamp1. No significant increase in these markers was detected in the cells exposed to 0.2mM bile acid cocktail or to medium acidified to pH 4 alone. Rapamycin and amino acid starvation induced similar changes in these autophagic markers as acid in combination with bile acids. Conclusion: The autophagic pathway is activated in response to stressful condition. Here we report that acute exposure of BE cells to hydrophobic bile acids in combination with acid induce autophagy, which may play a key role in the survival of BE cells exposed to refluxate and progression to cancer.