Prostaglandin EP2 Receptor Expression is Increased in Barrett's Oesophagus and Oesophageal Adenocarcinoma

P. Jiménez; E. Piazuelo; C. Cebrian; J. Ortego,; M. Strunk; M. A. GarcíA-Gonzalez; S. Santander; J. Alcedo; A. Lanas


Aliment Pharmacol Ther. 2012;31(3):440-451. 

In This Article


The demographic characteristics of patients included in the study are summarized in Table 1. Use of aspirin/NSAIDs was reported in one patient of the control group and another in the oesophagitis group. None of the patients of the Barrett's oesophagus-intraepithelial neoplasia-adenocarcinoma groups was taking NSAIDs. Forty of 46 (86.9%) patients with Barrett's oesophagus and low-grade intraepithelial neoplasia and 6 of 18 (33.33%) patients of the cancer group were under proton pump inhibitor (PPI) therapy.

For the purpose of this study, patients in the cancer group included patients with high-grade intraepithelial neoplasia and patients with adenocarcinoma because the difference between these two conditions is narrow and in many cases, the presence of high-grade intraepithelial neoplasia co-exists with the presence of adenocarcinoma.

Immunohistochemical Staining for COX Isoenzymes and EP Receptors in Human Oesophageal Biopsies

Immunohistochemical analysis was performed to determine protein expression levels and the specific cellular localization of COX isoenzymes and EP receptors. EP2 protein was present in all biopsies analysed, including normal squamous epithelium. A statistically significant increase in staining was found in all stages of the Barrett's metaplasia-intraepithelial neoplasia-adenocarcinoma sequence when compared with control patients. Most patients had moderate staining (degree = 2); interestingly, 35.7% of patients with Barrett's metaplasia and 46.7% of patients with low-grade intraepithelial neoplasia showed strong staining (degree = 3) (Table 2a), whereas only 9.1% of patients with adenocarcinoma showed this grade of staining. The cellular localization of EP2 protein was cytoplasmic and preferentially perinuclear (Figure 2). No statistically significant differences in EP1 expression were found across the different lesions of the oesophageal adenocarcinogenetic sequence (Table 2a). EP1 protein was localized in both cytoplasm and nucleus.

Figure 2.

Immunohistochemical analysis of EP2 protein expression. (a) Normal oesophageal epithelium showed cytoplasmic staining for the EP2 protein, preferentially perinuclear and mostly localized in the basal layer. (b) Barrett's metaplasia showed moderate granular cytoplasmic staining. (c) Expression of EP2 receptor increased notably in Barrett's metaplasia with low-grade intraepithelial neoplasia. (d) Oesophageal adenocarcinoma: EP2 protein is expressed within tumour cells. (Magnification x100; the top picture shows the same fields at ×400 magnification).

Immunohistochemical staining showed that EP3 protein levels were markedly decreased along the Barrett's metaplasia-intraepithelial neoplasia-adenocarcinoma sequence. Thus, 66.7% of control patients showed a moderate-strong staining, whereas 100% of patients with Barrett's and low-grade intraepithelial neoplasia and 92.3% of patients with high-grade intraepithelial neoplasia and adenocarcinoma showed weak or no staining (Table 2a). EP3 protein was localized in the cytoplasm.

Immunohistochemical staining for EP4 receptor showed a predominantly weak or no staining in the groups studied, except in oesophageal adenocarcinoma samples, which showed a moderate intensity of staining in 46.2% of patients (Table 2a). Normal oesophageal epithelium showed cytoplasmic staining for EP4 preferentially at the basal layer. Cytoplasmic immunoreactivity for EP4 was also observed in tumour cells (Figure 3).

Figure 3.

Immunohistochemical analysis of EP4 protein expression. (a) Normal oesophageal epithelium showed cytoplasmic staining. (b) Barrett's metaplasia with weak cytoplasmic staining in the gland. (c) Barrett's metaplasia with low-grade intraepithelial neoplasia showing moderate granular cytoplasmic staining. (d) Oesophageal adenocarcinoma exhibited cytoplasmic immunoreactivity for EP4 in the tumour cells. (Magnification ×100; the top picture shows the same fields at ×400 magnification).

Finally, COX-2 protein expression was absent or weak in normal oesophageal epithelium and its expression increased according to the severity of the lesion, reaching the highest levels in samples with high-grade intraepithelial neoplasia and adenocarcinoma. All high-grade intraepithelial neoplasia and adenocarcinoma biopsies analysed showed COX-2 expression (Table 2b). COX-2 was localized in the cytoplasm and in the cell membrane and was also found in plasma cells.

No immunoreactivity for COX-1 was observed in oesophageal tissues with the two antibodies used, independent of the lesion analysed. Thus, the expression of COX-1 protein was further analysed by western blot techniques. Figure 4a shows an immunoblot of mucosal biopsy lysates (50 μg/lane) from three cases with Barrett's oesophagus and matched-normal squamous mucosa. Western blotting showed a band of 70 kD in all biopsies evaluated. Once proteins were normalized with actin, quantitative densitometric analysis showed no statistically significant differences (Figure 4b).

Figure 4.

(a) Western blot demonstrating COX-1 protein expression in three Barrett's oesophagus cases (BO) compared with matched-normal squamous mucosa (N). A band at 70 kD was observed in all biopsies evaluated. Proteins were normalized for loading against actin on the same blot. (b) Quantitative densitometric analysis showed no statistically significant differences.

Expression of COX-1, COX-2 mRNA and EP Receptor Subtypes mRNA in Human Oesophageal Biopsies

To complement the information obtained from the immunohistochemical analysis, the expression of COX-1, COX-2 and the four subtypes of PGE2 receptors was analysed and quantified at mRNA level. Thus, a real time quantitative RT-PCR was performed in normal oesophageal mucosa, normal duodenal mucosa and in two critical stages of the oesophageal adenocarcinoma sequence: Barrett's oesophagus and adenocarcinoma.

COX Isoenzymes. Expression of COX-1 and COX-2 mRNA was detected in normal oesophageal mucosa, although levels of COX-2 transcripts were very low. COX-2 expression increased in Barrett's oesophagus (P = 0.051), although its expression was significantly greater in oesophageal adenocarcinoma specimens, reaching media values of 23.42-fold increase compared to normal squamous oesophageal epithelium (P < 0.01). In contrast, COX-1 was downregulated in both Barrett's oesophagus and oesophageal adenocarcinoma cases (Table 3).

EP Receptors. RT-PCR analysis showed that all four EP receptors subtypes are expressed in human oesophageal tissues. Levels of EP2 and EP4 mRNA were significantly elevated in Barrett's oesophagus when compared with normal mucosa. Of particular interest, EP2 receptor expression was extremely high (56.88-fold increase) in Barrett's oesophagus tissues. In oesophageal adenocarcinoma, only the expression of EP2 RNA was significantly upregulated, although to a lesser extent than that observed in Barrett's oesophagus (20.39-fold increase) (Table 3). No differences in EP1 and EP3 receptor expression were found in the groups analysed. EP2 and EP4 mRNA levels were also increased in duodenal samples compared with normal oesophageal mucosa (data not shown).

Effect of Bile and Acid on EP Receptor Expression

In a previous study, we had shown that the human adenocarcinoma oesophageal cell line OE33 expressed mRNAs for COX-1, COX-2, EP1, EP2 and EP4, but transcripts of EP3 receptors were negligible.[25] Therefore, to study whether acid and bile affect EP receptor expression, OE33 cells were incubated with acidified culture medium or deoxycholate. When OE33 cells were exposed to different pHs (7, 6.5, 5 and 4) for 1 h no differences between control (pH 7.4) and acidified cells were found (Table 4). The incubation of cells at pH 4 resulted in a marked mortality, as determined by LDH activity released into the culture supernatants (data not shown). However, exposure of OE33 cells to 0.3 mM DC (at pH neutral) was followed by a significant induction of COX-2 (37.79-fold induction; 95% CI = 20.15–70.89; P < 0.001), EP1 (3.96-fold induction; 95% CI = 1.41–11.12; P < 0.05), EP2 (3.49-fold induction; 95% CI = 1.20–10.13; P < 0.05) and EP4 (5.38-fold induction; 95% CI = 3.69–7.84; P < 0.01), but not COX-1 expression (3.37-fold induction; 95% CI = 0.76–14.92; n = 3). When the pH of the medium with DC was diminished to four, an extensive cell death was observed. We then investigated whether sensitization of cells to acid alone (pH 7, 6.5, 5) for 1 h pulse, followed by incubation in normal media (pH 7.4) with 0.3 mM DC affected to bile acid-mediated induction of EP receptor expression; however, no statistically significant differences were found. (Table 4).


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