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. 2000 Dec;47(6):812–819. doi: 10.1136/gut.47.6.812

Sulindac and a cyclooxygenase-2 inhibitor, etodolac, increase APC mRNA in the colon of rats treated with azoxymethane

Y Kishimoto 1, N Takata 1, T Jinnai 1, T Morisawa 1, G Shiota 1, H Kawasaki 1, J Hasegawa 1
PMCID: PMC1728141  PMID: 11076880

Abstract

BACKGROUND—Non-steroidal anti-inflammatory drugs (NSAIDs) have been reported to protect against the development of colon cancer. However, the mechanism(s) by which NSAIDs exert their effects is not clear.
AIMS—The aim of this study was to examine the effects of NSAIDs on mRNA expression of tumour suppressor adenomatous polyposis coli (APC) gene in rat colon mucosa.
METHODS—Starting at six weeks of age, three groups of rats (groups 1, 2, and 3) were treated with azoxymethane (AOM), a colon specific carcinogen, and another three groups (groups 4, 5, and 6) were not given AOM. Groups 2 and 3 were given 10 mg/kg of sulindac or etodolac, respectively, three times weekly during the experiment. Groups 4 and 5 were also given sulindac or etodolac, respectively, in the same manner as in groups 2 and 3. Group 6 (untreated control) was not given any agent (AOM or NSAIDs). At 10 weeks of age, preneoplastic lesions (aberrant crypt foci (ACF)) induced by AOM in the colon were counted, and the level of expression of APC mRNA in the colonic mucosa was estimated by the reverse transcription-competitive polymerase chain reaction method and northern blot analysis.
RESULTS—Mean occurrence of ACF in rats in groups 2 and 3 was reduced to approximately 50% of that in group 1. The level of APC mRNA expression in group 1 (AOM alone) was lower than that in group 6 (untreated control) (p<0.05); however, levels of APC mRNA expression in groups 2, 3, 4, and 5, to which NSAIDs had been administered, were significantly increased compared with levels in groups 1 and 6 (p<0.01).
CONCLUSIONS—Both sulindac and etodolac reduced the occurrence of ACF and induced an increase in APC mRNA in rat colon mucosa.


Keywords: adenomatous polyposis coli; cyclooxygenase; non-steroidal anti-inflammatory drugs; aberrant crypt foci; colon; azoxymethane

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Figure 1  .

Figure 1  

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Experimental protocol. Azoxymethane (AOM) was given as 15 mg/kg by subcutaneous injection, saline as 0.3 ml subcutaneously, sulindac as 10 mg/kg in 5% gum arabic aqueous solution three times a week by oral savage, and etodolac as 10 mg/kg in 5% gum arabic aqueous solution three times a week by oral gavage.

Figure 2  .

Figure 2  

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Analysis of cyclooxygenase (COX)-2, COX-1, adenomatous polyposis coli (APC), and β-actin mRNA expression. mRNA extracted from rat colonic tissue was reverse transcribed and amplified in the presence of different amounts of mimic DNA (lanes, from left to right: 10−4, 10−5, 10−6, 10−7 ng for COX-2; 10−3, 10−4, 10−5, 10−6 ng for COX-1 and APC; 10−2, 10−3, 10−4, 10−5 ng for β-actin). (A) COX-2 expression in colonic tissue of a representative rat in group 3. The level of COX-2 expression was 10−7 ng in this case. (B) COX-1 expression in the same rat as in (A). The level of target cDNA of COX-1 was 10−5 ng in this case. (C) APC expression in colonic tissue of a representative rat in group 2. The level of target cDNA of APC was 10−5 ng in this case. (D) β-actin expression in the same rat as in (C). The level of target cDNA of β-actin was 10−3 ng in this case. The level of β-actin expression was used as an internal control.

Figure 3  .

Figure 3  

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Northern blot analysis of cyclooxygenase (COX)-1 and adenomatous polyposis coli (APC) mRNAs from each group (groups 1, 2, 3, 4, 5, and 6). β-Actin expression was used as an internal control.

Figure 4  .

Figure 4  

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Levels of COX-2 mRNA expression measured by RT-competitive PCR. Data are shown as box plots: the box covers all values (including minimum and maximum values). The broken line indicates the limit of detection for COX-2 mRNA expression by RT-PCR. Levels of COX-2 expression in each case were normalised to that of β-actin mRNA (10−3 ng of mimic DNA). There was a tendency towards increased levels of COX-2 expression in the groups administered azoxymethane (AOM). However, levels were not significantly different between groups.    

Figure 5  .

Figure 5  

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Levels of COX-1 expression measured by RT-competitive PCR. Data are shown as box plots: the box covers all values (including minimum and maximum values), and the central continuous line in each box represents the mean. Levels of COX-1 expression in each case were normalised to that of β-actin mRNA (10−3 ng of mimic DNA). There was no difference in COX-1 mRNA expression levels between groups. AOM, azoxymethane.

Figure 6  .

Figure 6  

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Levels of adenomatous polyposis coli (APC) expression measured by RT-competitive PCR. Data are shown as box plots: the box covers all values (including minimum and maximum values), and the central continuous line in each box represents the mean. Levels of APC expression in each case were normalised to that of β-actin mRNA (10−3 ng of mimic DNA). APC mRNA expression level in group 1 (azoxymethane (AOM) alone) was significantly lower than that in group 6 (untreated control) (p<0.05). Both sulindac and etodolac caused an increase in APC mRNA in the rat colonic mucosa (groups 2, 3, 4, and 5) (p<0.01). *p<0.05; **p<0.01.

Figure 7  .

Figure 7  

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COX-1 and APC mRNA expression measured by northern blot analysis. mRNA levels were measured by northern blot analysis as described in materials and methods. Values are expressed as the ratio (%) to the average of those of group 6 (no treatment). Levels of APC mRNA expression in group 1 were lower than those in group 6 (p<0.05). Levels of APC mRNA expression in groups 2, 3, 4, and 5 were significantly increased compared with those in group 6 (p<0.01). *p<0.05;**p<0.01.    

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