Effect of inflammation on cyclooxygenase (COX)-2 expression in benign and malignant oesophageal cells
Salem I. Abdalla 1, 2,
Ian R. Sanderson 2 and
Rebecca C. Fitzgerald 1, *
1 Cancer Cell Unit, Hutchison/MRC Research Centre, Hills Road, Cambridge CB2 2XZ, UK and 2 Institute of Cell and Molecular Science, Adult and Paediatric Gastroenterology Department, Barts and the London School of Medicine and Dentistry, London, UK
* To whom correspondence should be addressed. Tel: 0044 1223 763287; Fax: 0044 1223 763296; Email: rcf{at}hutchison-mrc.cam.ac.uk
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Abstract
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Chronic inflammation has been linked to carcinogenesis in various tissue sites. Barrett's oesophageal epithelium (BE) is a premalignant condition in which cyclooxygenase-2 (COX-2) levels are increased. However, it is not clear whether the primary stimulus for the high COX-2 levels is related to inflammation or malignancy. The effect of exogenous cytokines (IL-1ß, IL-10 and IL-13) on COX-2 expression was assessed by western blotting in three BE cancer cell lines (SEG-1, BIC-1 and OE33) and a squamous cancer cell line (OE21). Primary tissue was assessed from 17 patients with long BE segments, 13 oesophagitis, 30 oesophageal adenocarcinoma (EAC), and 40 normal oesophageal (NE) and duodenal (DU) controls. COX-2 protein expression was determined by western blotting and its tissue localization was examined using immunohistochemistry. COX-2 protein and the neutrophil marker myeloperoxidase (MPO) were quantified along BE segments. The leukocyte marker CD45 was used to identify any correlation between COX-2 expression and leukocyte cell distribution in EAC. IL-1ß induced COX-2 expression in SEG-1 cells (P < 0.05), whereas IL-10 and IL-13 had no effect. COX-2 protein levels were found to be increased in distal BE > proximal BE > oesophagitis > NE (P < 0.001). COX-2 expression in EAC was heterogeneous and the overall levels were not significantly increased. The increased COX-2 expression in distal BE was not associated with inflammation (MPO expression). In addition, there was no correlation between COX-2 and CD45 in AC. COX-2 protein expression in the oesophagus appears to be independent of the degree of inflammation.
Abbreviations: BE, Barrett's oesophageal epithelium; COX-2, cyclooxygenase-2 enzyme; DU, duodenum; EAC, oesophageal adenocarcinoma; EGF, epidermal growth factor; GOJ, gastro-oesophageal junction; IL, interleukin; MPO, myeloperoxidase; NE, normal squamous oesophageal epithelium; NF
B, nuclear factor
-B; NSAIDs, non-steroidal anti-inflammatory drugs; PPIs, proton pump inhibitors; TGF-ß, transforming growth factor-beta; TNF-
, tumour necrosis factor-alpha.
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Introduction
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Cyclooxygenase (COX)-2 is the inducible form of the prostaglandin synthetase enzymes, COX-1 and COX-2, which catalyse the committed step in the prostaglandin production pathway (1). COX-2 expression is increased in inflammatory and malignant conditions as a result of induction by several different stimuli including proinflammatory cytokines (IL-1ß, TNF-
and EGF) and mutagenic substances (2,3). Its expression and activity is also inhibited by several compounds including anti-inflammatory cytokines (IL-4, IL-10 and TGF-ß), dexamethasone and NSAIDs.
Barrett's oesophagus (BE) is a premalignant condition with increased levels of COX-2 (4,5). The increase in COX-2 expression, compared with normal squamous oesophageal epithelium (NE), is observed throughout the metaplasiadysplasiacarcinoma sequence although the expression levels are somewhat variable between adenocarcinoma samples (5). Since BE is associated with both inflammatory and malignant complications it is not clear which of these factors is primarily responsible for inducing COX-2 expression. This is relevant because it has been suggested that COX inhibitors, such as non-steroidal anti-inflammatory drugs (NSAIDs), might play a role for chemoprevention of oesophageal adenocarcinoma (EAC) in patients with BE (6,7).
Inflammation in the oesophagus, oesophagitis, commonly occurs as a benign complication of gastro-oesophageal reflux (GOR). There are conflicting data on COX-2 expression in oesophagitis compared with non-inflamed SE and BE. COX-2 and prostaglandins were found to be increased in human and animal models of reflux oesophagitis (8,9). In addition, COX-2 inhibition reduced mucosal damage in a reflux oesophagitis rabbit model (10). However, there are reports describing the worsening of reflux oesophagitis following treatment with COX-2 inhibitors (11). In patients with BE the columnar-lined segment can also be inflamed as evidenced by activation of the transcription factor, such as nuclear factor
-B (NF-
B) resulting in the expression of cytokines, such as interleukin (IL)-8 (12). Furthermore, there is a propensity for the inflammatory cell infiltrate and proinflammatory cytokines to occur maximally at the proximal end of the segment (at the neosquamo-columnar junction) (13,14). Acid and bile have also been shown to induce proinflammatory cytokines (IL-8 and IL-1ß) in BE and associated cell lines (14,15). Furthermore, these cytokines are preferentially induced in endoscopic biopsies from the proximal end of BE compared with the distal end of the same segment (14). It is the proinflammatory cytokines, such as these, which have previously been shown to induce COX-2 expression (2,16).
The variable degree of inflammation along the BE segment provides the opportunity to examine to what extent inflammation induces COX-2 expression in BE. If inflammation is the predominant factor inducing COX-2 expression, one would expect to find an increase in COX-2 levels proximally in association with inflammatory mediators. Using both oesophageal cell lines and primary oesophageal tissues we demonstrate that inflammation is not the primary stimulus for COX-2 expression in BE.
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MATERIALS AND METHODS
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Cell culture experiments
Three BE cell lines (SEG-1, BIC-1 and OE33) and one human squamous cell line (OE21) were used. SEG-1 and BIC-1 are gifts from D.Beer, University of Michigan, MI, USA (17), and OE33 is purchased from European Collection of Cell Cultures (ECACC, Wiltshire, UK). OE21 is a human oesophageal squamous cell line that forms a single layer on confluency (ECACC, Wiltshire, UK). All cells were adapted to DMEM medium before experiments. Cells were seeded into 24-well plates at a concentration of 25 x 103 cells/well and maintained in DMEM medium supplemented with 10% foetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin and 1 mM glutamine. Cells were seeded either as single cultures or as 1:1 co-cultures to mimic the squamo-columnar junction. After attachment, cells were stimulated for 24 h with media containing IL-1ß, IL-10 (0.5, 1 and 5 nM) and IL-13 (0.5, 1, 5, 10 and 50 nM). After 24 h, cells were washed and protein extracted as described above.
Patient and tissue collection
This study was approved by the research ethics committees of Addenbrookes Hospitals, and Barts & The London Hospitals NHS Trusts. Seventeen patients with long segments of BE extending
5 cm from gastro-oesophageal junction (GOJ) and containing intestinal metaplasia were included in the study. Patients with dysplasia were excluded in order to avoid confounding owing to the heterogeneity of the dysplasia distribution. In addition, 13 oesophagitis patients and 30 patients with EAC were recruited. Control tissues of NE and duodenum (DU) were obtained from 25 and 15 patients, respectively, in whom there was no endoscopic or biopsy evidence of BE or inflammation. The dose and type of any acid-suppressant medication was recorded for all patients. The presence of any macroscopic inflammation both within the BE segment and in the normal squamous oesophageal mucosa above the BE was recorded according to the revised SavaryMiller classification grades 0IV (18).
In each case biopsies were taken for routine histopathological diagnosis and additional, paired biopsies were taken for research. For BE, quadrantic biopsies were taken every 2 cm from the GOJ according to International Surveillance Guidelines (19). Biopsies referred to as distal were taken 1 cm above the GOJ and biopsies taken 1 cm below the neosquamo-columnar junction (Z-line) were referred to as proximal. Biopsies from NE oesophagitis were taken 2 cm above the GOJ. Histological diagnosis of inflammation was made using the criteria of IsmailBeigi (20) as well as assessment of the presence of an inflammatory cell infiltrate (14). For research, biopsies were either fixed in formaldehyde and embedded in paraffin for histology and immunohistochemistry, or snap-frozen in liquid nitrogen and stored in 80°C until used for protein extraction. The number of patients used for each experiment varied and is stated in each figure legend.
Western blotting
Protein was extracted from snap-frozen biopsies and cells using lysis buffer containing protease (Rouche, Germany) inhibitors. Protein content was measured using the BCA protein Assay kit (Sigma, UK) (21), and 25 µg of total protein was separated using 10% SDSPAGE and blotted onto Hybond-P membranes (Amersham, UK). The membranes were probed using monoclonal antibodies for COX-1 and COX-2 at a dilution of 1:1000 (Cayman Chemicals, MI). Visualization was achieved using biotinylated anti-mouse IgG, Horseradish streptavidin (Vector Laboratories, Peterborough, UK) and chemiluminescence (ECL, Amersham). Band intensity was determined using Kodak Electrophoresis Documentation and Analysis system 120 (EDAS) software (Eastman Kodak, NY). The arbitrary densitometry units were converted into ng/mg of total protein values using serial dilutions of the recombinant COX-1 and COX-2 proteins, as described previously (5).
Myeloperoxidase (MPO) assay
Myeloperoxidase was measured in homogenates from proximal and distal BE biopsies according to a modification of a previously described method (22). In Brief, 100 µl of measured protein was mixed with 200 µl of solution 1 [0.25 g of hexadecyl trimethyl ammonium bromide (Sigma, UK) in 50 ml of 50 mM potassium phosphate buffer] and the mixture was incubated on ice for 15 min. Endoscopic tissue biopsy from inflamed stomach (gastritis) was homogenized in solution 1 and used as a positive control. Horseradish peroxidase was diluted with solution 1 to obtain serial standard concentrations. An aliquot of 200 µl of solution 2 (1 ml of O-dianisidine stock and 8.3 µl of hydrogen peroxide made up to 50 ml by addition of 50 mM potassium phosphate buffer) and 50 µl of reconstituted sample or standard were mixed and incubated at room temperature for 30 min. After incubation 200 µl of the mixture was placed on a flat-topped microtitre plate and absorbance was measured at 450 nm with reference value of 620 nm. A log to log standard curve was generated and the result was expressed as MPO units per mg of protein. All tissue samples from one experiment were measured in duplicate in a single assay.
Immunohistochemistry
Duplicate slides from 30 EACs were immunostained for COX-2 and the leukocyte marker CD45. Tissue sections were processed for immunohistochemistry as described previously (23). The primary antibodies were COX-2 at a dilution of 1:150 (Cayman Chemical) and CD45 at a dilution of 1:200 (Santa Cruz Biotechnology). Visualization was performed using the 3,3'-diaminobenzidine method (DAB) (Vector Laboratories, CA). The specificity of positive antibody staining was verified by an absorption test using blocking peptide. For negative controls, phosphate buffered saline was substituted for the primary antibody. Areas of positive COX-2 and CD45 immunostaining were compared in duplicate cancer slides.
Statistical analysis
The data are presented as a mean ± SEM. Analysis of variance (ANOVA) was used to detect variability, and the
2-test and Student's t-test were used to identify specific differences between groups. P < 0.05 was required for significance.
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RESULTS
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IL-1ß stimulated COX-2 expression in oesophageal cell lines
As an initial test for the hypothesis that inflammatory cytokines may induce COX-2 expression in the oesophagus, we used a simplified tissue culture model. SEG-1, BIC-1, OE33 and OE21 cells were seeded as individual cultures or as 1:1 co-cultures to represent the squamo-columnar junction (Figure 1A). IL-1ß induced COX-2 expression in the SEG-1 Barrett's cell line (P < 0.05) and to a lesser degree in the squamous cell carcinoma cell lines OE21 (P = ns, not significant) at concentrations of 0.5 nM (Figure 1BD and Figure 2) (densitometric scans of blots represented in Figure 1). On the other hand, there was no effect of IL-1ß on the low basal COX-2 levels in the Barrett's BIC-1 and OE33 cell lines, (Figure 1BD and Figure 2). IL-1ß also induced COX-2 expression in the 1:1 columnar: squamous co-culture (SEG-1/OE21 and BIC-1/OE21) (Figures 1D and 2), but this was not significantly different from the effect on the same cell lines individually. The anti-inflammatory cytokines IL-10 and IL-13 had no significant effect on COX-2 expression in oesophageal cell lines (data not shown). Hence, the in vitro data suggested that the proinflammatory cytokine IL-1ß was capable of inducing COX-2 in one-third of Barrett's cell lines. Next, we went on to assess the relationship between inflammation and COX-2 expression using primary human samples.

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Fig. 1. A representative western blot from cell culture experiments. Barrett's cancer cell lines (SEG-1, BIC-1 and OE33) and a squamous cell line (OE21) were used. In addition, 1:1 columnar/squamous co-cultures were used to represent the neosquamo-columnar junction (A). Exogenous IL-1ß caused a dose-dependant increase in COX-2 protein expression in SEG-1 and OE21 oesophageal cell lines and in the columnar/squamous co-culture (BD). Western blots are representative of three separate experiments. M is a protein marker, S is the recombinant COX-2 standard and C is the non-stimulated control. 1, 2 and 3 are 0.5, 1 and 5 nM of IL-1ß, respectively. See online Supplementary material for a colour version of this figure.
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Fig. 2. Cumulative data showing the effect of IL-1ß on COX-2 expression in oesophageal cell lines. 0.5 nM of IL-1ß induced COX-2 protein expression in SEG-1 and OE21 oesophageal cell lines as well as in the co-cultures. Data pooled from three separate experiments and all experiments were performed in triplicate. *P < 0.05.
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Patient characteristics
The study included a total of 100 patients from four groups (BE, oesophagitis, EAC and control groups). The demographics of the BE patients are in keeping with the previous studies and are summarized in Table I (24). All BE patients had intestinal metaplasia but none had dysplasia. The mean length of the BE segment was 7 cm (range 512 cm). In the oesophagitis group the level of inflammation varied from grade IIII (Revised SavaryMiller classification). Endoscopic evidence of BE was seen in 55% of EAC patients. All NE and DU control samples were histologically normal. The percentage of patients on a proton pump inhibitors (PPIs) was similar for control and oesophagitis patients (25 and 30%, respectively) and for BE and EAC patients (88 and 80%, respectively). However, it is unlikely that our results are confounded by PPI treatment since all patients were asked to stop PPIs 2 weeks before endoscopy.
COX-2 expression is upregulated in biopsies from BE and oesophagitis
BE expressed high levels of COX-2 compared with the NE controls (P < 0.0001). COX-2 expression was also increased in the inflamed oesophageal biopsies (oesophagitis) in comparison with the NE controls (Figure 3A and B). However, this increase was not statistically significant and was lower than COX-2 expression in the BE biopsies. Of note, COX-2 expression was also increased in DU compared with NE biopsies (5). Indeed, the columnar epithelium (BE and DU) expressed higher levels of COX-2 compared with the inflamed and non-inflamed squamous epithelium (P<0.0004) (Figure 3C). However, densitometry confirmed that COX-1 was expressed equally in all tissue biopsies studied in keeping with its role as a house-keeping gene (Figure 3A).

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Fig. 3. (A) Representative western lots showing COX-1 and COX-2 protein (molecular weight, 70 and 72 kDa, respectively) from oesophagitis (oesoph), Barrett's epithelium (BE) and normal oesophageal squamous epithelium (NE). Each lane represents a different patient. The double bands seen in COX-1 are as a result of N-glycosylation of the protein. (B) Cumulative data obtained from densitometric analysis of western blots for NE (n = 25), oesoph (n = 13), BE (n = 34, biopsies from 17 patients), ***P < 0.0001 BE versus NE. (C) Shows that columnar epithelium (including BE and DU) expressed increased levels of COX-2 compared with squamous epithelium (NE and oesophagitis), ***P < 0.0004. COX-1 is equally expressed in all tissue biopsies.
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Immunohistochemistry demonstrated that COX-2 protein is expressed by both epithelial and lamina propria cells in BE and DU biopsies (Figure 4A and C). In contrast, COX-2 expression in EAC was heterogeneous although glandular malignant cells seem to express more COX-2 protein compared with lamina propria cells (Figure 4B).

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Fig. 4. Immunohistochemistry representative of 10 BE, 30 EAC, and 8 NE and DU patients. COX-2 protein was expressed in BE and DU both in the surface epithelium and the lamina propria (A and C); however, expression was more homogeneous in the surface epithelium and glands. On the other hand, COX-2 expression was heterogeneous in EAC (B). (D) Shows negative COX-2 staining in an NE section. Original magnifications 20x.
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COX-2 expression does not correlate with MPO levels
Interestingly, COX-2 protein was not expressed equally along the length of BE. In long BE segments, there was increased COX-2 expression in the distal end of the segment near the GOJ compared with the proximal end of the same segment (Figure 5A and B) (P < 0.001 proximal versus distal BE). Overall, COX-2 expression was increased in distal BE > proximal BE > oesophagitis > NE (Figure 5B). Our previous work suggested that inflammation is increased proximally (14). In order to correlate inflammation and COX-2 levels in the same patients we measured MPO activity as a marker of neutrophil infiltration. We found that there was a trend towards an increased median MPO expression in the proximal end of the BE segment as expected (Figure 5C), although this did not reach statistical significance owing to the wide variation between patients.

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Fig. 5. (A) Western blots on biopsies from the proximal (P) and distal (D) ends of long BE segments showing increased COX-2 protein expression in the distal ends of BE compared with the proximal ends of the same BE segment. (B) Cumulative data from western blots of 25 NE, 13 oesophagitis and proximal and distal ends of 17 long BE segments. COX-2 protein was increased in distal BE > proximal BE > oesophagitis > NE. **P = 0.001 proximal versus distal BE, ¶P < 0.02 BE versus oesophagitis. (C) Shows cumulative data from MPO assays (inflammation marker) on biopsies from distal and proximal ends of long BE segments (n = 17). There was a non-significant increase in MPO expression between proximal and distal ends of long BE segments.
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COX-2 expression in EAC was not influenced by presence or absence of leukocytes
Owing to the heterogeneous nature of COX-2 expression in oesophageal tumours, we determined whether leukocyte infiltration correlated with areas of increased COX-2 expression. We demonstrated that CD45 positive immunostaining was not consistent with the areas of COX-2 positivity and was generally variable (Figure 6). In addition, COX-2 expression was mainly localized to the malignant cells.

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Fig. 6. Representative immunohistochemistry demonstrating the lack of correlation between COX-2 and the leukocytes marker CD45 staining in 30 patients with EAC. (A) Shows positive COX-2 and negative CD45 staining in the same section and (B) shows areas of negative COX-2 but positive CD45 staining. Original magnifications 20x.
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DISCUSSION
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We have demonstrated that COX-2 expression is increased in BE > reflux oesophagitis > NE. Despite the fact that both oesophagitis and BE are characterized by an inflammatory infiltrate (14), the level of COX-2 expression in BE did not correspond with the degree of inflammation as determined by MPO activity. In EAC samples the pattern of COX-2 expression is heterogeneous and does not correspond to areas of macrophage infiltration. The discrepancy between COX-2 and MPO levels along the BE segment (Figure 5) excludes acute inflammation, characterized by neutrophil infiltration, as a cause of increased COX-2 expression. Overall, these data suggest that there may be other non-inflammatory factors that determine COX-2 expression levels in BE.
The propensity for inflammation to occur more proximally in long BE segments may be explained by differences in the local environment. For example, the proximal epithelium is exposed intermittently to the gastric refluxate in comparison with the more constant exposure in the distal end. Indeed, intermittent (pulsatile) but not continuous acid and bile exposure has been associated with increased proinflammatory cytokines in BE (14,23). In addition, it is possible that squamo-columnar cross-talk leading to paracrine signaling, e.g. through the release of cytokines and growth factors, may play a role. In our cell culture model, squamo-columnar co-cultures did not lead to an increase in COX-2 expression. However, IL-1ß stimulated COX-2 expression in oesophageal cell lines in keeping with previous in vitro data showing an upregulation of COX-2 by proinflammatory cytokines (2,3,25).
A functional link between chronic inflammation and cancer has long been suspected (2628). Population-based studies show that susceptibility to cancer increases when tissues are chronically inflamed; and long-term use of NSAIDs reduces the risk of several cancers (2931). In the gastrointestinal tract, chronic inflammation also plays an important role in carcinogenesis. For example, ulcerative colitis and Crohn's disease are chronic inflammatory conditions associated with an increased risk of colon cancer. Refractory and severe inflammation in these conditions is successfully treated with TNF-
antibodies (e.g. Infliximab). However, there are no data on Infliximab treatment and risk of cancer in inflammatory bowel disease. Helicobacter pylori infection is another gut condition that may lead to inflammation followed either by ulcers or by atrophy and gastric cancer (32,33). Furthermore, an individual's gastric response to infection may be determined in part by polymorphisms in genes encoding cytokines, such as IL-1ß (3436). Therefore, it is likely that there are important genetic factors and molecular pathways that permit communication between cancer cells and the inflammatory cells that infiltrate tumours, although these are not well understood. Indeed, it has been proposed that inflammation is the fuel to the process of carcinogenesis triggered by genetic factors (37,38).
The role of inflammation in BE is complex (23). We have previously demonstrated that BE is characterized by increased levels of anti-inflammatory cytokines, such as IL-10 and IL-4 (23), which have been linked to human carcinogenesis (3941). Furthermore, the maximal degree of inflammation, including the expression of proinflammatory cytokines like IL-1ß, is often concentrated in the adjacent squamous mucosa separate from the tumour (14). In an animal model of reflux, Buttar et al. (42) were able to induce BE and EAC using an oesophago-jejunostomy, and COX inhibitors (NSAIDs) reduced the incidence of cancer development. Although the development of AC in this model was associated with inflammation this was only quantified at the end of the experiment and it is not known whether the inflammation had a causal role or developed as a consequence of the carcinoma. Furthermore, there are various mechanisms by which NSAIDs can prevent cancer, in addition to their anti-inflammatory activity (43,44). Although it is difficult to extrapolate from ex vivo and in vitro studies, the experiments presented here do not support a causative role for inflammation in the expression of COX-2 in BE and cancer. In keeping with this, previous studies have demonstrated that COX-2 expression was maximal during the resolution phase of inflammatory pleurisy (45). We have demonstrated that COX-2 protein was increased in BE near the GOJ and away from the site of maximal inflammation. In addition, its distribution in the adenocarcinoma tissue was concentrated in the cancerous cells and was not related to the distribution of the inflammatory infiltrate. Hence, one can postulate that the expression of COX-2 in the oesophagus may be more directly associated with signalling pathways involved in cancer progression rather than inflammation per se. For example, COX-2 and derived prostaglandins bind to the nuclear peroxisome proliferation activator receptors which act directly as transcription factors upon ligand binding. Activation of such pathways may, in turn, promote angiogenesis, inhibit immune surveillance, increase cell proliferation as well as reduce apoptosis and cell adhesion (4648). Our observations are, therefore, in keeping with the suggestion that COX inhibitors, including aspirin, may be a useful adjunct in BE chemoprevention programmes (49,50). The specific stimuli driving COX-2 expression in oesophageal disease warrant further investigation.
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SUPPLEMENTARY MATERIAL
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Supplementary material is available at http://www.carcin.oupjournals.org/
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Acknowledgments
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We thank the patients who participated in this study as well as the staff of the endoscopy and histopathology departments for their assistance. This study was funded by the Medical Research Council. Dr S.A. was funded by a scholarship from the Libyan government.
Conflict of Interest Statement: None declared.
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Received March 10, 2005;
revised April 23, 2005;
accepted April 26, 2005.