Japanese Journal of Clinical Oncology 32:167-171 (2002)
© 2002 Foundation for Promotion of Cancer Research
Cyclooxygenase-2 Overexpression in Colorectal Cancer is Associated with Non-polypoid Growth
1 Digestive Surgery and 2 Human Pathology, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan
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ABSTRACT |
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Background: Cyclooxygenase-2 is considered to play an important role in colorectal tumorigenesis. The purpose of this study was to clarify the relationship between cyclooxygenase-2 expression and morphology in colorectal cancer.
Methods: We investigated cyclooxygenase-2 mRNA expression and type of growth (polypoid or non-polypoid according to Shimoda’s classification) in 69 colorectal cancers by reverse transcription-polymerase chain reaction and histologic examination. Cyclooxygenase-2 mRNA expression was assessed as a ratio relative to cyclooxygenase-1 mRNA expression (cyclooxygenase-2 index).
Results: Cyclooxygenase-2 indices in normal colorectal mucosa, polypoid cancer and non-polypoid cancer were 0.7 ± 0.1, 1.0 ± 0.1 and 1.9 ± 0.1, respectively. The cyclooxygenase-2 index in non-polypoid cancer was significantly higher than in polypoid cancer (P = 0.0002) or normal mucosa (P < 0.0001). No difference in cyclooxygenase-2 index was found between polypoid cancer and normal mucosa (P = 0.67).
Conclusions: These results suggest that cyclooxygenase-2 overexpression in colorectal cancer is associated with non-polypoid growth.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Previous studies have indicated that colorectal cancers develop through accumulation of various genetic abnormalities including mutation of adenomatous polyposis coli (APC), K-ras, p53 and deleted in colorectal carcinoma (DCC) (1,2). Especially in the early stage, morphological study of these cancers reveals two distinct entities according to growth pattern: a non-polypoid (flat) type and a polypoid type (3,4). Shimoda et al. (4) showed that early colorectal cancer with non-polypoid growth was not accompanied by adenomatous areas and showed submucosal invasion even when still small. Previous analyses of the relationship between tumor morphology and genetic abnormalities (5–14) have revealed a lower prevalence of K-ras mutations in flat than in polypoid colorectal tumors (5–11). So far, K-ras mutation has been the most important molecular factor found to show a close relationship to this morphological dichotomy in colorectal cancers. Additional molecular factors, however, may be related to tumor morphology.
Epidemiological studies have shown that the regular use of aspirin-like drugs or non-steroidal anti-inflammatory drugs (NSAIDs) decreases the risk of death from colorectal cancer (15,16). A major action of these drugs is inhibition of cyclooxygenase (COX), which includes constitutive COX-1 and inducible COX-2 (17). Previous studies have found COX-2 to be overexpressed in colorectal cancer and in a subset of adenomas (18–20). Inhibition of COX-2 decreased the size and number of polyps in a mouse model of familial adenomatous polyposis (FAP) (21). These studies indicate that COX-2 expression is important in the development of colorectal tumors and may be related to the various tumor-associated genetic abnormalities.
Whether COX-2 expression is related to the morphology of colorectal cancer is still unclear. To clarify the relationship between COX-2 expression and colorectal cancer morphology, we investigated COX-2 mRNA expression and type of growth (polypoid vs non-polypoid) in 69 of these lesions.
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MATERIALS AND METHODS |
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Tissues
We studied 69 surgically resected colorectal cancers and 58 samples from normal colorectal mucosa at the resection margin. The patients underwent surgery at our hospital between January 1997 and July 1999. Two or three pieces of tumor tissue weighing about 100 mg each and a similar quantity of normal colorectal mucosa were excised from resected specimens; these samples were immediately frozen in liquid nitrogen and stored at –80°C until analysis. Remaining tissues were fixed in formalin, embedded in paraffin, routinely sectioned and examined pathologically after hematoxylin and eosin staining. In the remaining tissue, the area of tumor sampling was also histologically examined to confirm that the area actually consisted of cancer tissue.
Clinicopathologic Features
All 69 colorectal cancers studied were classified histologically as polypoid or non-polypoid as described previously (4). Microscopic appearances in representative cases of polypoid and non-polypoid are shown in Fig. 1. In brief, colorectal cancers with intramucosal proliferation of adenoma or carcinoma were classified as polypoid (Fig. 1A), whereas colorectal cancers without an intramucosal exophytic growth pattern were classified as non-polypoid (Fig. 1B). Patient age, gender, tumor location, histological type, tumor size, depth of invasion, lymph node status and Dukes stage also were recorded. At the time of histological examination, no information was available concerning COX-2 expression.
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Total RNA Extraction
Total RNA was extracted from frozen tissue according to the method of Chomczynski and Sacchi using an RNeasy kit (Qiagen, Chatsworth, CA) (22). Extracted RNA was purified and then quantified by reading the absorbance at 260 nm. Furthermore, RNA solution was electrophoresed in agarose gels, stained with ethidium bromide and photographed under ultraviolet light to qualify the total RNA extracted.
RT-PCR
Total RNA solution was subjected to reverse transcription-polymerase chain reaction (RT-PCR). A 10 µg amount of RNA was transcribed by SuperScript II RT (Life Technologies, Rockville, MD) using oligo dT primer and RNase inhibitor in the reaction buffer. The total reaction volume was 100 µl in each reaction tube. After reverse transcription, cDNA was subjected to PCR in 20 µl of reaction mixture to amplify both COX-1 and COX-2 cDNA concomitantly. Primers were designed so that the antisense primer was shared for amplification of both COX-1 and COX-2 mRNAs. Primers used were: 5'-GCT CAG GAG GAA GTT CAT ACC-3' (COX-1 sense, bases 549–569); 5'-TGC CCA GCT CCT GGC CCG CCG CTT-3' (COX-2 sense, bases 851–872); and 5'-GYA GYT CTG GGT CAA ATT TCA G-3' (common antisense, bases 1080–1101 for COX-1 and 1133–1154 for COX-2; Y indicates an equimolar mixture of T and C). The preceding base numbers are given as indicated in the GenBank database under accession numbers M59979 (COX-1) and M90100 (COX-2). To determine the optimum PCR amplification conditions in the linear range, three amounts of cDNA (10–200 ng) were tested for each sample. Each reaction tube contained 2 µl of 10x Ex Taq buffer (Takara, Shiga, Japan), 2 µl of 2.5 mM dNTP mixture, 0.1 µl of Ex Taq polymerase (Takara), 1 µl (4 µM) of each sense primer and 2 µl (4 µM) of common antisense primer. The total reaction volume was 20 µl. PCR consisted of denaturation at 94°C for 3 min, 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 1 min and a final extension at 72°C for 5 min. The PCR products were electrophoresed on 1.5% agarose gels in 1x Tris–acetate EDTA buffer at 100 V for 30 min. The gels were stained with ethidium bromide and illuminated with ultraviolet light for photography. The gels were then analyzed with a Fluor-S Max image analyzer (Bio-Rad Laboratories, Hercules, CA). Band densities corresponding to COX-1 and COX-2 were quantified using Quantity One (Bio-Rad Laboratories) and Multi-Analyst (Bio-Rad Laboratories) software and the ratio of COX-2 to COX-1 band densities (COX-2 index) was calculated as described previously (23).
Statistical Analysis
Statistical significance was determined by one-factor ANOVA with post-hoc test, Mann–Whitney U-test, chi-squared test or Fisher’s exact test. All analyses were performed with StatView software (version 5.0) (Abacus Concepts, Berkeley, CA). P values <0.01 were considered to indicate statistical significance. All results are shown as means ± SE.
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RESULTS |
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Of the 69 colorectal cancers, 11 showed a polypoid pattern (one was early and 10 were advanced) and 58 were non-polypoid (three were early and 55 were advanced). No difference was evident between polypoid and non-polypoid cancers with respect to patient age, gender, tumor location, tumor size, depth of invasion, histological type, lymph node status or Dukes stage (Table 1).
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RT-PCR results in representative cases are shown in Fig. 2. COX-2 indices in all specimens are shown in Fig. 3. Significant differences in the COX-2 index were found between normal colorectal mucosa, polypoid cancer and non-polypoid cancer (0.7 ± 0.1, 1.0 ± 0.1, 1.9 ± 0.1, respectively; P < 0.0001, one-factor ANOVA). The COX-2 index in non-polypoid cancer was significantly higher than in polypoid cancer (P = 0.0002, Sheffe’s test) or normal mucosa (P < 0.0001, Sheffe’s test). However, no difference in COX-2 index was seen between polypoid cancer and normal mucosa (P = 0.67, Sheffe’s test). The range of COX-2 indices in normal mucosa was 0.1–1.8. Defining COX-2 overexpression as a COX-2 index >1.8, 29 cancers showed overexpression; all were non-polypoid.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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In the present study, COX-2 overexpression was observed only in non-polypoid colorectal cancers, while expression in polypoid cancers did not differ from that in normal colorectal mucosa. These results suggest that COX-2 is related to tumorigenesis in non-polypoid but not polypoid lesions.
Previous studies have investigated relationships between the morphology of colorectal cancer and abnormalities of various genes including K-ras (5–9), APC (9,10), p53 (9,11–13) and DCC (9,14). In addition, microsatellite instability (24), chromosomal aberration (25) and expression of sialyl Tn antigen (26), Ki-67 (11,12,27), c-myc (12) and bcl-2 (11) have been investigated with reference to colorectal tumor morphology. Among these, only K-ras mutation had a relatively close association with tumor morphology that was confirmed by many authors; K-ras mutation was less frequent in flat (non-polypoid) colorectal tumors than in the polypoid type (5–11). Accordingly, non-polypoid colorectal cancer is considered to develop via a different pathway to polypoid cancer.
All colorectal cancers with COX-2 overexpression showed non-polypoid growth in the present study. Although the way in which COX-2 affects the morphogenesis of colorectal cancer is not clear, Tsujii et al. (28) reported that transfection of the COX-2 gene increased invasiveness in Caco-2, a human colon cancer cell line, in a simple invasion chamber assay. Increased invasiveness of cancer cells resulting from COX-2 overexpression may contribute to formation of a non-polypoid configuration.
Both advanced and early colorectal cancers are morphologically classified into polypoid or non-polypoid growth types (4). In the present study, the ratio of polypoid growth type in advanced colorectal cancers was 15%, similar to that of 21.8% reported by Shimoda et al. (4) Lack of COX-2 overexpression in polypoid colorectal cancers suggests that polypoid cancers have different biological characteristics to non-polypoid cancers. Differences in the molecular pathway of carcinogenesis may lead to these contrasting growth patterns.
Recent studies have shown that K-ras mutation was associated with COX-2 overexpression in experimental colon cancer in rat (29), colon cancer cell line (30) and human colorectal adenomas (31). A lower prevalence of K-ras mutation has been reported in non-polypoid colorectal tumors (5–11). These findings, taken together, indicate that COX-2 overexpression is likely to be found in polypoid colorectal cancer with K-ras mutation. Although K-ras mutation was not examined in the present study, higher COX-2 expression was detected in non-polypoid colorectal cancer. The reasons for this discrepancy are not clear, but the different materials and methods used in these studies may have led to the different results.
In the present study, we used RT-PCR for the semiquantitative analysis of COX-2 mRNA expression. Immunohistochemical staining is another useful method to study COX-2 expression in colorectal cancer with different morphology. Because large advanced colorectal cancers may have genetic heterogeneity, analysis of early cancer may be more helpful in clarifying the role of COX-2 in tumorigenesis of polypoid and non-polypoid colorectal cancer. However, the number of early colorectal cancers was only four and the difference in COX-2 index between polypoid and non-polypoid early cancer did not reach statistical significance (data not shown). Further studies with immunohistochemistry including a larger series of tissue samples are required to confirm the present findings of non-involvement of COX-2 in polypoid colorectal cancer.
We found COX-2 expression to be significantly higher in non-polypoid than in polypoid colorectal cancer and COX-2 overexpression was limited to non-polypoid cancers. These results suggest that cyclooxygenase-2 overexpression is associated with non-polypoid growth.
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Acknowledgments |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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This work should be attributed to Digestive Surgery, Graduate School, Tokyo Medical and Dental University. We thank Professor Makoto M. Taketo, Kyoto University, for useful advice.
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FOOTNOTES |
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+ For reprints and all correspondence: Hidefumi Tsunozaki, Surgery, Tokyo Metropolitan Ohtsuka Hospital, 2–8–1 Minami-Ohtsuka, Toshima-ku, Tokyo 170-8476, Japan
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Received December 3, 2001; accepted February 26, 2002
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