Japanese Journal of Clinical Oncology 33:631-635 (2003)
© 2003 Foundation for Promotion of Cancer Research
Cyclooxygenase-2 Expression and its Relationship with Proliferation of Colorectal Adenomas
1 Digestive Surgery and 2 Surgical Pathology, Tokyo Medical and Dental University Graduate School, Tokyo and 3 Department of Surgery, Tokyo Hospital, National Printing Bureau, Tokyo, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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Background: Cyclooxygenase (COX)-2 may be linked to carcinogenesis. In the previous study, we examined COX-2 expression immunohistochemically in 95 adenomas and reported a significant correlation between its expression and the grade of dysplasia. To clarify the correlation between COX-2 expression and cell proliferation, we investigated Ki-67 labeling index using immunohistochemistry and its correlation with COX-2 expression.
Methods: Immunohistological staining for Ki-67 antigen was performed on 95 colorectal adenomas previously reported.
Results: The Ki-67 labeling index was significantly higher in the high-COX-2 group than in the low-COX-2 and negative groups in adenomas with moderate (44.5 ± 6.4% vs 33.0 ± 2.6%, 39.0 ± 6.2%; P = 0.01, P < 0.001, respectively) or severe dysplasia (47.2 ± 7.6% vs 40.3 ± 7.2%, 35.0 ± 5.4%; P = 0.02, P = 0.005, respectively). There was no correlation between Ki-67 labeling index and COX-2 expression in mild dysplasia.
Conclusions: These results suggest that COX-2 may play a causal role in cell proliferation in carcinogenesis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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Considerable advancement has been made in the field of cancer prevention during the last decade. One of the most remarkable discoveries is that cyclooxygenase (COX)-2 may play a role in colorectal carcinogenesis and the blocking of COX-2 function may prevent the development of colorectal cancer. There is much evidence to suggest a link between consumption of nonsteroidal anti-inflammatory drugs (NSAIDs) and colorectal cancer prevention from epidemiologic (1,2), clinical (3,4) and animal studies (5,6). Although the exact mechanism of the anti-neoplastic activity of NSAIDs remains unclear, one possible route is via inhibition of COX. COX is a key regulatory enzyme in prostaglandin biosynthesis (7). Two COX isozymes have been identified: the constitutive COX-1 and the inducible COX-2. COX-1 is constitutively expressed in normal human tissues including the colon, while COX-2 is not usually detectable in normal tissues but is induced by cytokines, growth factors, oncogenes, serum and tumor promoters (8,9).
Colorectal adenomas are considered to be precursor lesions for most colorectal cancers, representing an important target for cancer chemoprevention. Recent studies have shown that COX-2 is expressed at high levels in 80 to 90% of colorectal adenocarcinomas (10–14), and selective inhibition of COX-2 reduces colorectal tumorigenesis in different models of carcinogenesis (5,15,16). Thus, COX-2 has been thought to play a crucial role in colorectal carcinogenesis.
We recently examined COX-2 expression in sporadic adenoma tissue using immunohistochemistry and its correlation with clinicopathological features and have found that COX-2 expression in adenoma cells is significantly associated with the grade of dysplasia in the adenoma. As shown in Table 1, COX-2 expression increased significantly with transition from mild dysplasia to moderate and severe dysplasia. However, it is still unclear whether COX-2 expression is related to cell proliferative activity in colorectal adenomas. To clarify the relationship between COX-2 expression and cell proliferation, we evaluated the Ki-67 labeling index (LI) and COX-2 expression in colorectal adenomas by immunohistochemistry.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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Tissue Samples
In a retrospective review of pathologic reports, we selected colorectal adenomas
4 mm that had been removed by polypectomy or endoscopic mucosal resection in our department between October 1999 and February 2001. Polyps associated with inflammatory bowel disease, familial polyposis coli or those having a history of NSAIDs (including low-dose aspirin) were excluded. A total of 95 polyp specimens from 78 patients were studied. All samples included in the study contained normal epithelium adjacent to the adenomatous tissue. These tumors have previously been studied for changes in COX-2 expression (17). On the basis of Morson’s criteria (18), the dysplasia of adenoma was classified into three grades: mild, moderate and severe. Severe dysplasia is synonymous with focal carcinoma in adenoma or intramucosal carcinoma.
Immunohistochemical Staining of Ki-67
Immunohistochemical staining of Ki-67 was performed on 3-µm thick sections of formalin-fixed, paraffin-embedded sequential tissue sections to those previously stained for COX-2, in accordance with the standard procedures.
The immunohistochemical process of Ki-67 was similar to that of COX-2 (17). Slides were incubated with Ki-67 monoclonal antibody (Novocastra, Newcastle, UK) at a 1:100 dilution for 60 min at room temperature after a 10-min microwave pretreatment. The secondary antibody was biotinylated goat anti-mouse IgG (Dako, Copenhagen, Denmark). After color development with 3,3'-diaminobenzidine tetrahydrochloride, the sections were counterstained with hematoxylin. Sections of colonic adenocarcinoma known to be positive for Ki-67 served as a positive control, and nonspecific mouse serum served as the negative control.
Evaluation of Immunostaining
The grades of epithelial dysplasia appear as a continuous spectrum from a mild change, which is closest to the normal epithelium, via moderate, to severe changes that are similar to those seen in invasive carcinoma. It is common to see different grades of dysplasia in one adenoma. Since the changes in the control of cell growth are both early and cumulative in carcinogenesis, determination of the proliferative activity of individual dysplastic areas of an adenoma throws light on some features of colorectal carcinoma biology. When one adenoma had different areas of dysplastic grades, separate evaluations were made for areas corresponding to adenoma with mild, moderate, or severe dysplasia according to the method previously described (19).
There are two counting methods of Ki-67 labeling in studies of colorectal neoplasms: counting cells in randomly selected fields (RS method) (20) and counting cells in the highest labeling areas (HL method) (21). There is a highly positive correlation between the RS and HL methods. We used the RS method as follows: three microscopic fields in each dysplastic area were selected randomly, and in each field 500 or more adenoma cells were counted; the ratio of Ki-67 positive cells to all counted cells was calculated, excluding cells with questionable nuclear staining. In each dysplastic area, the sum of the three fields was determined as the LI.
Statistical Analysis
Statistical significance was determined by the chi-squared, one-factor ANOVA and Fisher’s PLSD (post hoc) tests. Analysis was done by the Stat View 5.0 software package (Abacus Concepts, Berkeley, CA). P-values <0.05 indicated statistical significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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Ki-67 Labeling Index
Ki-67 immunostaining produced discernible diffuse or granular brown nuclear staining, more accentuated in the nucleoli, with a relatively uniform intensity. The Ki-67 LI increased significantly in correlation with the grade of dysplasia (P < 0.001, by one-factor ANOVA, Fig. 1). The Ki-67 LI was significantly greater in moderate than in mild dysplasia and also in severe as compared with mild dysplasia (P < 0.001, P < 0.001, respectively, by Fisher’s PLSD tests). However, there was no significant difference in the Ki-67 LI between moderate and severe dysplasia.
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Correlation between COX-2 Expression and Ki-67 Labeling Index
The Ki-67 LI for adenomas was significantly higher as indicated by COX-2 expression (P < 0.001, by one-factor ANOVA, Figures 2 and 4A, B). In severe dysplasia, the Ki-67 LI of the high COX-2 group was higher than that of the low and negative COX-2 groups, although there was no difference between the low-COX-2 and negative groups (P < 0.001, by one-factor ANOVA, Fig. 3A). In moderate dysplasia, the Ki-67 LI was also significantly higher with greater expression of COX-2 (P < 0.001, by one-factor ANOVA, Fig. 3B). In mild dysplasia, there was no correlation between Ki-67 LI and COX-2 expression (P = 0.066, by one-factor ANOVA, Fig. 3C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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We have demonstrated that COX-2 expression is significantly higher with an advanced dysplastic change and is associated with cell proliferation activity.
Previous studies have demonstrated the importance of COX-2 in colon carcinogenesis, not only in the early stage of neoplastic evolution but also in cancer progression (5,11,15,16). Fujita et al. (22) reported that COX-2 mRNA was significantly higher in colorectal carcinomas of larger size having deeper invasions. Hasegawa et al. (23) reported that COX-2 mRNA was markedly increased in the sporadic colorectal adenomas, especially in those with larger diameters. Elder et al. (24) and Azumaya et al. (25) also reported that COX-2 expression showed a size-dependent increase in colorectal adenomas. These findings suggest an important role of COX-2 in the growth of colorectal tumors.
Our recent study (17), using immunohistochemistry in sporadic adenomas demonstrated that COX-2 expression in adenoma cells is significantly related to the grade of dysplasia. However, Einspahr et al. (26) quantitatively estimated COX-2 gene expression in 108 paraffin-embedded colorectal adenomas using real-time PCR and concluded that COX-2 expression was not related to the degree of dysplasia. The reason for this discrepancy is not clear, but could be attributed to the different methods used in these studies.
Cell proliferative activity is important for evaluating tumor growth. Monoclonal antibodies that recognize Ki-67 have shown to label all proliferating cells in G1, S, G2 and M phases of the cell cycle and are widely used to estimate the proliferative fraction of neoplasms (27,28).
We clearly demonstrated a correlation between COX-2 expression and the proliferative activity of neoplastic epithelial cells in sporadic colorectal adenomas. There are a few reports that demonstrate the relationship between COX-2 expression in colorectal adenomas and Ki-67 LI. In adenomas with severe or moderate dysplasia, the Ki-67 LI was significantly higher in the high-COX-2 group than in the low-COX-2 and negative groups. However, there was no difference in Ki-67 LI among the high-COX-2, low COX-2 and negative groups in adenomas with mild dysplasia. These results suggest that COX-2 may be related to cell proliferation of the adenoma in its progression to carcinoma. Moreover, these observations support the hypothesis that COX-2 may be involved in the growth of colorectal tumors. On the other hand, Sakuma et al. (29) have reported that colorectal carcinoma tissue overexpresses COX-2, but there is no relationship between COX-2 expression and Ki-67 labeling, suggesting that COX-2 expression may not be related to cell proliferation. This discrepancy can be possibly attributed to the different materials studied (adenoma versus carcinoma). We confirmed the relationship between COX-2 expression and cell proliferation in the progression of adenoma. However, we did not examine COX-2 and Ki-67 expression between adenoma and carcinoma in this study. It is interesting to estimate COX-2 and Ki-67 expression in the sequence from severe dysplasia via early cancer to advanced cancer. Risio and Rossini (30) reported that the Ki-67 LI progressively increased along the sequence from normal mucosa via low-grade and high-grade dysplasia adenoma to advanced cancer, whereas the Ki-67 LI of early cancer were not significantly different from that found in the low-grade dysplasia areas. The rationale for this is not clear, but it can be based on the changes occurring in the submucosal microenvironment caused by invasion.
In conclusion, these results indicate that increased COX-2 expression may be related to growth and malignant transformation of adenomas and may play an important role in the progression of colorectal adenomatous change. Hence, sporadic adenomas with upregulated COX-2 expression are potential targets for the anti-proliferative effects of NSAIDs.
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Acknowledgment |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Sciences, Sports and Culture of Japan.
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FOOTNOTES |
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+ For reprints and all correspondence: Takanobu Sato, Department of Surgery, Kyoundo Hospital, Sasaki Institute, 1–8 Kanda Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan. E-mail: sato{at}po.kyoundo.jp
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentREFERENCES |
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Received October 17, 2003; accepted October 26, 2003
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