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KEY WORDS: colonic neoplasm, cyclooxygenase, immunohistochemistry, inflammatory bowel disease.

Expression of cyclooxygenase-2 protein in colon disease

LU HONG, GE ZHIZHENG, LIU WENZHONG, CHEN XIAOYU, PENG YANSHEN & XIAO SHUDONG

Department of Gastroenterology, Renji Hospital, Shanghai Second Medical University, Shanghai Institute ofDigestive Disease, Shanghai, China

INTRODUCTION

Cyclooxygenase (COX) is a rate-limiting enzymerequired for the conversion of arachidonic acid to

prostaglandins (PG). Two cyclooxygenase isoformshave been identified, COX-1 and COX-2. The COX-1enzyme is produced constitutively in normal tissues,while COX-2 is inducible at inflammation sites.1 Recentstudies indicate that it is the inducible COX-2 that playsan important role in gastrointestinal carcinogenesis.Several epidemiological studies have demonstrated a40–60% decrease in relative risk of colorectal canceramong patients with rheumatoid arthritis who havetaken long-term non-steroidal anti-inflammatory drugs(NSAIDs).2 The best-known action of NSAIDs is to

Correspondence to: Hong Lu, Department of Gastroenterology, RenjiHospital, Shanghai Second Medical University, Shanghai Institute ofDigestive Disease, Shanghai 200001, China. Email:annielu@online.sh.cn

Originally published as: Lu H, Chen XY, Liu WZ, Peng JS, Xiao SD.Expression of cyclooxygenase-2 protein in human gastric cancer.Chin J Gastroenterol 2000; 5: 150–152.

OBJECTIVE: Several epidemiological studies haveindicated that the long-term administration of non-steroidal anti-inflammatory drugs (NSAIDs) mayreduce the incidence of colorectal cancer. The bestknown action of NSAIDs is to block cyclooxygenase,the key enzyme required for the conversion ofarachidonic acid to prostaglandins. Two cyclo-oxygenase isoforms have been identified and these arereferred to as COX-1 and COX-2. Recent studiesindicate that inducible COX-2 plays an important rolein gastrointestinal inflammation and carcinogenesis.The present study was undertaken to investigate theexpression and clinical implications of COX-2 andCOX-1 in normal and diseased colons.

METHODS: COX-2 and COX-1 protein expression inspecimens from normal controls and diseased colontissues were examined semiquantitatively by usingimmunohistochemical methods.

RESULTS: By using immunohistochemical detectionmethods, low COX-2 protein expression in colonicepithelial cells was observed in 20.0% (2/10) ofnormal controls. Eighty percent (16/20) of specimensfrom inflammatory bowel disease (IBD) had a high

COX-2 expression, 46.40% (13/28) of adenomas and64.3% of (9/14) well-differentiated colonic carci-nomas had some COX-2 protein expression.Expression of COX-2 protein was increased in IBDand colonic carcinomas compared with normalcontrols. There were no significant differencesbetween colonic adenomas and colonic carcinomas.No correlation was found between COX-2 proteinexpression and patient gender, patient age, tumor size,tumor location or the degree of differentiation/metastasis of the tumor. Strong immunoreactive COX-2 was expressed in clusters in interstitial cells (mainlymononuclear cells) in 53.6% (15/28) of adenomasand 64.3% (9/14) of colonic carcinomas. StrongCOX-2 protein expression was also displayed in thenormal glands that were adjacent to the adenomasand carcinomas. Expression of COX-1 protein wasobserved in the epithelial cells and interstitial cells ortumor cells of normal colon, IBD, colonic adenomasand colonic carcinomas.

CONCLUSIONS: Our results indicated that COX-2protein overexpression may contribute to the develop-ment of IBD and colonic carcinogenesis.

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block cyclooxygenase. The present study was under-taken to investigate the expression and distribution ofthe COX-2 protein in inflammatory bowel disease(IBD), sporadic colonic adenoma and colonic adeno-carcinoma, and to investigate the role of COX-2 incolonic carcinogenesis.

MATERIALS AND METHODS

Patients and specimens

All tissue specimens were obtained during surgicalresections or endoscopy from patients at the Depart-ment of Surgery or Endoscopy Center at Renji Hospital(affiliated with Shanghai Second Medical University)during 1999. The specimens were from: (i) 14 patients(five men and nine women; age range: 47–81 years)with primary colonic cancers resected surgically; (ii) 28patients (16 men and 12 women; age range: 27–73years) with colorectal adenomatous polyps excised by endoscopic polypectomy or biopsy; and (iii) 20patients (eight men and 12 women; age range: 17–61years) with inflammatory bowel disease (IBD) bybiopsy. Colonic mucosal specimens from 10 subjects(three men and seven women; age range, 30–62 years)who were diagnosed with irritable bowel syndromewere taken as normal controls.

Patients with familial adenomatous polyposis (FAP), afamily history of colorectal cancer, or who had takenNSAIDs or glucocorticoids regularly were excludedfrom the study.

The tissue specimens were fixed in 10% neutralformalin, embedded in paraffin and serially sectionedto a thickness of 4–5 µm. A diagnosis was establishedby histological examination with hematoxylin andeosin (H&E) staining.

Immunohistochemistry

COX-1/COX-2 polyclonal antiserums and an avidin–biotin–peroxide complex (ABC) kit were purchasedfrom the Santa Cruz Corporation (USA). Tissuesections were dewaxed in xylene, then rehydratedthrough a graded alcohol series to distilled water.Antigen retrieval was performed by microwave treat-ment in sodium citrate buffer (10 mmol/L, pH = 6.0).The slides were cooled to room temperature, thenimmersed in 1% H2O2 in methanol for 15 min andthen immersed in normal monkey serum for 15 minto block non-specific binding sites. After decanting the normal serum, sections were incubated with goat polyclonal immunoglobulin G (IgG) specific for

human COX-1/COX-2 in a dilution of 1:100 overnightat 4°C. The sections were rinsed with phosphate-buffered saline (PBS) for 5 min three times and thesections were then treated with biotinylated secondaryantibodies at 37°C for 60 min. After again rinsing thesections three times with PBS for 5 min each, they werestained with standard ABC and visualized by using 3,3′-diaminobenzidine (DAB). The sections were counter-stained with hematoxylin.

Evaluation of immunostaining

A semiquantitative method, scoring both thepercentage of positive cells and the intensity ofimmunostaining, was used to evaluate the degree of positivity. Positive cells had cytoplasmic and peri-nuclear brownish staining. The average percentage ofpositive cells was calculated by counting at least 10fields and assigning one of the following grades: 0 (0–5%); 1 (6–30%); 2 (31–70%); or 3 (71–100%).The intensity of staining was defined as 0 (negative);1 (weak); 2 (moderate); or 3 (strong). The degree ofexpression was defined as the sum of the percentage ofpositive cells score and the immunostaining intensityscore. A score of 1–3 denotes low expression and ascore of 4–6 denotes high expression.

Statistical analysis

The Wilcoxon signed rank test was used for statisticalanalysis. Statistical significance was set at P < 0.05.

RESULTS

COX-2 protein expression in normal and diseasedcolon

COX-2 protein expression in normal colon mucosa

Low levels of COX-2 protein expression in the colonicmucosal epithelium were found in 20.0% (2/10) ofnormal subjects (Table 1). The interstitial cells hadsporadic COX-2 protein expression.

COX-2 protein expression in IBD mucosa

High levels of COX-2 protein expression in the inflam-matory mucosal epithelium were found in 80.0%(16/20) of IBD patients, significantly higher than thatfound in the normal colonic mucosa (P < 0.05). COX-2 expression could also be seen in the interstitialcells.

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COX-2 protein expression in colonic adenoma

Twenty-eight colonic adenoma specimens (10 fromendoscopic biospies, 18 from endoscopic polypec-tomies) were studied. The mean diameter of theadenomas was 12.6 ± 1.2 mm. Histological examina-tion with H&E staining revealed that there were 25tubular, one tubulo-villous and two villous adenomas.Of the 28 adenomas, three exhibited mild dysplasia,two moderate dysplasia and two severe dysplasia.

COX-2 protein expression was found in 46.4% (13/28)of adenomas, and it also could be seen in the inter-stitial cells. No correlation was found between COX-2protein expression and patient gender, patient age,adenoma size, location or histology type (P > 0.05).COX-2 protein expression was increased in theadenoma epithelium compared with the normalcolonic mucosa, but the difference was not statisticallysignificant. High levels of COX-2 protein expressionwere also noted in the relatively normal glandularepithelium surrounding the adenoma. Stronglyimmunoreactive COX-2-expressing interstitial cells(mainly mononuclear cells) were distributed in clustersin 53.6% (15/28) of adenomas.

COX-2 protein expression in colonic carcinoma

Hematoxylin and eosin staining showed that all 14colonic carcinomas were well differentiated. COX-2protein expression was found in 64.3% (9/14) ofcolonic carcinomas. Diffuse cytoplasmic staining wasobserved in the cancer cells. No correlation was foundbetween COX-2 protein expression and patient gender,patient age, tumor location or lymph node metastasis(P > 0.05). High levels of COX-2 expression were alsonoted in the relatively normal glandular epitheliumsurrounding the carcinoma. COX-2 protein expressionwas significantly increased in cancerous tissuecompared with the normal mucosal epithelium(P < 0.05), but there was no statistically significantdifference compared with adenomas (P > 0.05).Strongly immunoreactive COX-2-expressing interstitialcells (mainly mononuclear cells) were distributed inclusters in 64.3% (9/14) of colonic carcinomas.

COX-1 protein expression in normal and diseasedcolon

COX-1 protein expression was noted occasionally inthe epithelial cells of the normal colon, IBD, colonicadenoma and colonic carcinoma. Interstitial cellsdisplayed sporadic low or moderate positive staining.

DISCUSSION

In the present study, we have shown that low ormoderate COX-1 protein expression was noted in theepithelial and interstitial cells of the normal colon,IBD, colonic adenomas and colonic carcinomas. COX-2 protein expression was significantly increasedin the epithelial cells of IBD compared with its lowexpression in normal colon. Elevated COX-2 proteinexpression was also observed in the epithelial cells ofadenomas, colonic cancerous cells and the interstitialcells. No significant difference was found betweencolonic adenomas and colonic carcinomas. IncreasedCOX-2 protein expression was also seen in the rela-tively normal glandular epithelium surrounding theadenomas and carcinomas.

Studies by Cohn et al. have demonstrated that COX-1expression is confined to the area of epithelium prolif-eration and the zone of mucus neck cells in the mousestomach.3 An absence of COX-1 expression wasobserved in well-differentiated epithelium cells such asthose in the upper half of colonic crypts and ileal villi,and COX-1 expression was not induced by inflamma-tory mediators. Eberhart and DuBois reported that theNSAIDs currently used could increase the activity andsymptoms of ulcerative colitis.4 This may be related tothe inhibition of gastrointestinal protective PGproduced through COX-1 catalysis. The COX-2 proteinwas not expressed in normal ileum and colon but wasstrongly expressed in the surface epithelium and cryptepithelium of patients with IBD, suggesting that COX-2 expression is induced by inflammatory media-tors.5 This could explain the findings of a previousreport that the level of PG in IBD mucosa and in rectaldialysates was markedly elevated in patients with IBD.6

Prostaglandin E2 (PGE2) is a mediator of increased

Table 1. Expression of COX-2 protein in normal and diseased colon

n Percentage positive No. low expression No. high expression

Normal controls 10 20.0 (2/10) 2 0IBD 20 80.0 (16/20)* 0 16Adenoma 28 46.4 (13/28)† 5 8Colon cancer 14 64.3 (9/14)*‡ 4 5

*Compared with normal controls, P < 0.05; †compared with normal controls, P = 0.08; ‡compared with adenomas, P > 0.05.

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epithelial cell proliferation, mucosal hyperemia andvasodilation in IBD. Masferrer and Izutani reportedthat the epithelial cells expressing COX-2 also expressinducible nitric oxide synthase (iNOS), and that thesetwo compounds have a synergistic action.7,8 COX-2,iNOS, and interleukin-8 are part of a group ofinducible proteins whose promoters have nuclearfactor κ B response elements (NF-κB). Interleukin-1βand tumor necrosing factor-α (TNF-α) levels areelevated in inflammatory processes such as IBD, andNF-κB is activated.

In a rat model of trinitrobenzene sulfonic acid (TNBS)-induced colitis reported by Reuter et al., there was asixfold increase in COX-2 mRNA 72 h after the admin-istration of TNBS.9 COX-2 expression occurred mainlyin smooth muscle, infiltrating inflammatory cells andsubmucosal vessels but not in epithelial cells. Treat-ment with a selective COX-2 inhibitor resulted in aworsening of the colitis and perforation of the colon.This suggests that PG produced through COX-2promote wound healing in gastrointestinal mucosalinjury. COX-2 expression in epithelial cells in IBD is aprotective response of the wound healing process. Non-steroidal anti-inflammatory drugs may exacerbateulcerative colitis. The study by Reuter et al. also raisesthe possibility that besides the inhibition of COX-1, theability of NSAIDs to exacerbate clinical activity inulcerative colitis could also result from the inhibitionof COX-2 in epithelial cells and the resultant impair-ment of wound healing.

The adenomatous polyposis coli (APC) knockoutmouse is a model of human familial adenomatouspolyposis (FAP). The APC gene knockout mice have alarge number of COX-2-expressing polyps. COX-2-deficient mice cross-breed with APC gene knockoutmice. Mice heterozygous for the APC gene knockoutthat have a disrupted COX-2 gene have far fewerpolyps, suggesting that COX-2 may play a role in theincreased risk of colon cancer. This suggests the possi-bility that inhibition of COX-2 may reduce the risk ofmalignancy.

In a rodent model study of FAP by Oshima et al.,intestinal epithelial cells overexpressing COX-2 wereresistant to apoptosis induced by butyrate, and thiseffect was able to be reversed by NSAIDs.10 SelectiveCOX-2 inhibitors could induce apoptosis in H-ras-transformed rat intestinal epithelial cells. This suggeststhat COX-2 may inhibit resistance to apoptosis, andthat the prevention of colon carcinogenesis by NSAIDsmay be related to the induction of apoptosis throughthe inhibition of COX-2. However, there is some

conflicting evidence.11 For example, the administrationof NSAIDs (sulindac) in FAP patients did not result inany significant changes in cell kinetics or cell cycles,and no correlation was found between COX-2 proteinexpression and apoptotic index in either adenomas orcarcinomas. Also, NSAIDs have been shown to induceapoptosis independently.

Studies of animal models have shown that elevatedlevels of PGE2 and COX-2 protein expression occur inadenomatous and carcinomatous tissue and blood.Non-steroidal anti-inflammatory drugs inhibited thedevelopment of colonic adenomas and reduced bloodlevels of PG (thromboxane B2) and COX-2 expression.

Co-culture studies with human colorectal cancer celllines and in vivo studies in animals demonstrated thatmacrophages were able to stimulate cancer cell prolif-eration.12,13 The source of PG in colon cancer tissue hasbeen suggested to be the macrophages that expressCOX-2. Prostaglandins may play an important role incolon carcinogenesis. The findings of the present studydemonstrate that COX-2 is also strongly expressed inthe interstitial cells (mononuclear cells, fibroblasts,endothelial cells and smooth muscle cells) of colonicadenomatous and carcinomatous tissues. It suggeststhat a paracrine-signaling pathway between macro-phages and the neighboring epithelial cells may play arole in the early stages of colon carcinogenesis.14 Itremains unclear what induces COX-2 in macrophagesand how macrophages expressing high levels of COX-2 affect colon carcinogenesis.

Familial adenomatous polyposis and adenoma areassociated with the early onset of colon cancer. Thedata indicate that COX-2 expression is upregulated incolonic adenoma and carcinoma. This upregulation ofCOX-2 is an early event in colonic oncogenesis. Inhi-bition of COX-2 may play a prophylactic role in theadenoma–carcinoma sequence. Specific inhibitors ofCOX-2 may be more effective than non-selectiveNSAIDs for the chemoprevention of colonic carcino-genesis.

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