Expression of bile acid transporting proteins in Barrett's esophagus and esophageal adenocarcinoma

Abstract

Objectives: Barrett's esophagus (BE) is a metaplastic lesion characterized by replacement of the normal squamous epithelium by columnar intestinal epithelium containing goblet cells. It is speculated that this process is an adaptation to protect cells from components of refluxate, such as gastric acid and bile acids. In contrast to the normal squamous epithelium, enterocytes of the distal ileum are adapted to transport bile acids from the intestinal lumen. Several bile acid transporters are utilized for effective removal of bile acids, including the apical sodium-dependent bile acid transporter (ASBT), the ileal bile acid-binding protein (IBABP), and the multidrug-resistant protein 3 (MRP3). We hypothesized that one of the possible functions of newly arising metaplastic epithelium, in the esophagus, is to transport bile acids. Our major goal was to evaluate the expression of bile acid transporters in normal squamous epithelium, BE with different grades of dysplasia, and esophageal adenocarcinoma (EAC).

Methods: A total of 101 patients were included in this study. Immunohistochemistry (IHC) and reverse transcriptase (RT)-PCR were used to detect the expression of these transporters at the mRNA and protein levels.

Results: Our immunohistochemical studies showed that all three bile acid transporters are expressed in BE glands, but not in squamous epithelium. ASBT was found in the apical border in BE biopsies. The highest frequency of ASBT expression was in patients with nondysplastic BE (9 of 15, 60%), and a progressive loss of ASBT was observed through the stages of dysplasia. ASBT was not detected in EAC (0 of 15). IBABP staining was observed in the cytoplasm of BE epithelial surface cells. Expression of IBABP was found in 100% of nondysplastic BE (14 of 14), in 93% of low-grade dysplasia (LGD, 15 of 16), in 73% of high-grade dysplasia (HGD, 10 of 14), and in 33% of EAC (5 of 15). MRP3 was expressed in the basolateral membrane in 93% of nondysplastic BE (13 of 14), in 60% of LGD (10 of 16), and in 86% of HGD (11 of 13). Only weak MRP3 staining was detected in EAC biopsies (5 of 15, 33%). In addition, RT-PCR studies showed increased expression of mRNA coding for ASBT (6.1x), IBABP (9.1x), and MRP3 (2.4x) in BE (N=13) compared with normal squamous epithelium (N=15). Significantly increased mRNA levels of IBABP (10.1x) and MRP3 (2.5x) were also detected in EAC (N=21) compared with normal squamous epithelium.

Conclusions: We found that bile acid transporters expression is increased in BE tissue at the mRNA and protein levels and that expression of bile acid transporter proteins decreased with progression to cancer.

Figure 1

Evaluation of bile acid transporter proteins, ASBT, IBABP and MRP3, in Barrett’s esophagus (BE), esophageal adenocarcinoma (EAC), and squamous epithelium (SQ) by immunohistochemistry. Matched BE esophagus negative controls (BE/NC) are also shown, in which rabbit or mouse IgG was added instead of primary antibody. The magnification used was ×1,000 for top three images and ×400 for all other images.

Figure 2

Evaluation of ASBT, IBABP, and MRP3 mRNA expression in squamous epithelium (SQ, N = 15), Barrett’s esophagus (BE, N = 13), and esophageal adenocarcinoma (EAC, N = 21) by real-time quantitative RT–PCR. Boxplots show median (white band), mean (red diamond), 25th and 75th percentiles (bounded by gray box), 1.5 times the interquartile range (whiskers), outliers within 3 times the first of third interquartile range (asterisk), and extreme outliers lying more than 3 times the first of third interquartile range (o). Significant differences are shown by horizontal bars with the respective P values shown.