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

Disclosures

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

In This Article

Materials and Methods

Patients

A total of 85 patients were included in the study. Patients were stratified in the following groups: Control group (14), patients with oesophagitis (7), Barrett's oesophagus (25), low-grade intraepithelial neoplasia (21), high-grade intraepithelial neoplasia and oesophageal adenocarcinoma (18). The demographic characteristics of patients are summarized in Table 1. Of the seven cases with oesophagitis; five cases were grade B and two were grade C of the LA classification.[23] Barrett's oesophagus was endoscopically reported according to the Prague classification[24] and suspected when islands, tongues, or circular extension of salmon-pink epithelium was seen above the gastro-oesophageal junction. When a hiatal hernia was present, Barrett's oesophagus was considered if the epithelium was seen 1 cm above the gastric folding. The diagnosis was always confirmed by histological examination and defined as the presence of specialized columnar epithelium with mucin-containing globet cells in the oesophagus. Only patients with Barrett's mucosa ≥2 cm were included in this study. The range of circular (C) metaplasia went from 1 to 8 cm and of longitudinal metaplasia (L) from 2 to 8, having >70% a degree ≥C2M3.

For immunohistochemical studies, 68 patients were included in the study. Samples of patients with oesophagitis, Barrett's oesophagus, intraepithelial neoplasia (low- and high-grade) and oesophageal adenocarcinoma, were selected from paraffin tissue samples and the histological diagnosis was confirmed by the same pathologist prior to all immunohistochemical studies. Control biopsies were obtained from patients with no evidence of oesophageal lesions and no clinical symptoms of GERD and who were referred to endoscopy from different reasons. These was a standardized interview based on the absence/presence and frequency of typical reflux symptoms which included heartburn and regurgitation, but also chest pain, epigastric pain, eructation, etc. Control biopsies were taken of the normal squamous epithelium, ≥3 cm above the mucosal transition (cardia).

For real-time PCR studies, we analysed oesophageal biopsies obtained from 17 patients with Barrett's oesophagus or adenocarcinoma attending in the Endoscopy Unit of the Service of Digestive Diseases of the University Hospital of Zaragoza (Video Endoscope Olympus Exera). Biopsy samples were obtained from pathological mucosa, normal duodenal mucosa and from normal squamous oesophageal epithelium, which was used as a control. All biopsies were immediately placed into liquid nitrogen and stored at −80 °C until analysis.

The study protocol was approved by the Institutional Review Board Committee of the University Hospital of Zaragoza and written informed consent was obtained from all patients.

Immunohistochemistry

Formalin-fixed, paraffin-embedded tissue samples were sectioned and applied to slides. Slides were subsequently deparaffinized with xylene for 15 min, hydrated gradually in a graded series of ethanol (100%, 96%, 70%, twice for 3 min in each) and washed in deionized water. Slides were treated with a 0.3% H2O2–methanol solution for 5 min to quench endogenous peroxidase activity and then washed in PBS (twice for 5 min each). Next, slides were incubated for 30 min in 1.5% normal blocking serum in PBS.

The sections were incubated with the primary antibody for 30 min at room temperature. Optimal concentrations for all antibodies used were determined in pilot experiments. Thus, tissue sections were incubated with mouse monoclonal anti-human COX-1 (1:10 dilution) or COX-2 (1:100) antibodies and rabbit polyclonal antibodies against human EP1 (1:50), EP2 (1:100), EP3 (1:500) or EP4 receptor (1:1000). All antibodies and blocking peptides were purchased from Cayman Chemical (Nottingham, UK). Additionally, another polyclonal COX-1 antibody (sc-1752, Santa Cruz Biotechnology, CA, USA) was tested. Immunohistochemistry was performed using the ChemMate™ DAKO Envision system (Dako Cytomation). Slides were washed with three changes of PBS for 5 min each. The chromogen used was 3, 3'-diaminobenzidine tetrahydrochloride (10 min of incubation). Finally, the sections were washed in deionized H2O, counterstained with Harri's haematoxylin solution and dehydrated through ethanol (65% and 100%, twice each) and xylene (three times for 5 min each). Specificity of antibodies was validated by neutralization with the respective blocking peptide in a 4-fold excess of peptide for 1 h at room temperature and run in parallel with antibody alone. Incubation of sections with the blocking peptide resulted in negative staining (Figure 1).

Figure 1.

A representative example of positive EP2 subtype receptor protein staining (a) vs. negative staining (b) when blocking peptide EP2 receptor was added as described in Material and methods.

The intensity and extent of the staining were evaluated blinded simultaneously by an arbitrary scale that ranged from 0 to 3; 0 = no staining; 1+ = weak staining; 2+ = moderate staining and 3+ = strong staining. In normal oesophagus, we scored the staining in the superficial/medium and/or basal epithelium as the degree of intensity (0–3). In the columnar epithelium, the intensity and extent of the staining was scored along the gland. Thus, the staining was evaluated in the base of the gland, the isthmus, the pit and/or surface epithelium. The final score was the mean of all them (0 if mean <0.5; 1 if mean between 0.5 and <1.5; 2 if mean between 1.5 and <2.5 and 3 if mean between 2.5 and 3). Adenocarcinoma sections were evaluated taking into account both the presence of staining as the percentage of positive cells. Thus, the staining was scored as 0 with 0% positive cells, 1 when 1–30% cells were positive, 2 when 31–70% cells were positive cells and 3 when 70–100% were positive cells in the areas of the greater intensity.

Real Time RT-PCR Analysis of mRNA Expression

Total RNA was extracted from biopsies and OE33 cells using an RNeasy Fibrous Tissue Kit (Quiagen, Crawley, Surrey, UK) according to the manufacturer's instructions. Real-time PCR analysis was performed on cDNA generated by reverse transcription using an ABI PRISM 7000 Sequence Detection System using standardized TaqMan assay reagents (Applied Biosystems, Foster City, CA, USA): Hs00377721_m1 for COX-1 gene, Hs00153133_m1 for COX-2, Hs00168752_m1 for EP1, Hs00168754_m1 for EP2, Hs00168755_m1 for EP3 and Hs00168761_m1 for EP4. The PCR reactions were performed according to the manufacturer's protocol.

Relative quantification was calculated using the comparative threshold (Ct) cycle method. Results were expressed as ΔCt values [ΔCt (problem)−ΔCt (reference)]. The housekeeping TATA-box binding protein (human TBP control reagent kit, Applied Biosystems) was used as reference gene for normalization of the amount of sample and represented as ΔCt values. Comparison of gene expression was obtained from subtraction of normal mucosa ΔCt values from Barrett's oesophagus or adenocarcinoma ΔCt values to give ΔΔCt. Relative quantification was expressed as fold-induction or repression of the gene of interest compared to the control condition (values = 1) according to the formula 2−ΔΔCt.

Western Blotting for COX-1 Isoenzyme

Oesophageal biopsies were sonicated in RIPA buffer containing protease inhibitors, as previously described.[25] Briefly, supernatants were collected by centrifugation and protein concentrations were measured. Extracted proteins (50 μg) were separated by SDS/PAGE and transferred electrophoretically to PDVF membranes, which were blocked with 5% blocking agent in TBST. Membranes were incubated with a mouse monoclonal anti-COX-1 antibody (1:1000 dilution). After, the membrane was probed with 1:2000 dilution of peroxidase-conjugated secondary antibodies in TBST with 1% blocking agent. Immunoreactive protein was detected using enhanced chemilumiscence. Membranes were then stripped and incubated with a rabbit polyclonal anti-actin antibody, as an internal loading control.

Cell Culture

Barrett's associated adenocarcinoma cells (cell line OE33) were purchased from European Collection of Cell Cultures (EACC, Salisbury, UK). The cells (passages 7–20) were cultured as previously described.[25] Subconfluent cells were treated as follows: (a) medium (1% FCS) at physiologic pH (pH 7.4) containing 0.3 mM deoxycholate (DC) for 12 h; (b) medium (1% FCS) adjusted to different pH values (7, 6,5, 5, 4) for 1 h and followed by incubation in normal media (pH 7.4) to complete 12 h of cell culture and (c) medium (1% FCS) adjusted to different pH values (7, 6,5, 5, 4) for 1 h and followed by incubation in fresh medium (pH 7.4) containing DC (0.3 mM) to complete 12 h of cell culture. Acidified medium was obtained by addition of 0.1–1N HCl to fresh medium until achieving the pH desired and then filtered. Previous studies have demonstrated that the deoxycolate (0.3 mM) produced a marked increase in the production of prostaglandin E2, which was associated with an increase in the levels of COX-2 mRNA with maximal effects at 12–24 h.[26]

Statistical Analysis

For immunohistochemical data, we analysed median values and the percentage of positive staining. Statistical significance was analysed using Mann–Whitney tests.

For statistical evaluation of mRNA expression levels, mean values and range were calculated. Values obtained were compared between groups by nonparametric Mann–Whitney tests.

For cell culture experiments, an analysis of variance between groups was developed. Statistical significance between control group and experimental groups was analysed using Dunnett's test. All of these tests were two-sided and differences were considered statistically significant when P values were <0.05.

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