Mami Takahashi

Gene mutations and altered gene expression in azoxymethane-induced colon carcinogenesis in rodents.

Studies of colon carcinogenesis in animal models are very useful to elucidate mechanisms and provide pointers to potential prevention approaches in the human situation. In the rat colon carcinogenesis model induced by azoxymethane (AOM), we have documented frequent mutations of specific genes. K‐ras mutations at codon 12 were found to be frequent in hyperplastic aberrant crypt foci (ACF) and large adenocarcinomas. In addition, mutations of the β‐catenin gene in its GSK‐3β phosphorylation consensus motif could also be identified in many adenomas and adenocarcinomas, and altered cellular localization of p‐catenin protein was observed in all of the dysplastic ACF, adenomas and adenocarcinomas examined, indicating that activation of Wnt signaling by accumulation of β‐catenin is a major mechanism in the AOM‐induced colon carcinogenesis model. Frequent gene mutations of β‐catenin and altered cellular localization of the protein are also features of AOM‐induced colon tumors in mice. Expression of enzymes associated with inflammation, such as inducible nitric oxide synthase (INOS) and the inducible type of cyclooxyge‐nase (COX), COX‐2, is increased in AOM‐induced rat colon carcinogenesis, and overproduction of nitric oxide (NO) and prostaglandins is considered to be involved in colon tumor development. We have demonstrated that increased expression of INOS is an early and important event occurring in step with β‐catenin alteration in rat colon carcinogenesis. Activation of K‐ras was also found to be involved in up‐regulation of INOS in the presence of inflammatory stimuli. In addition, expression levels of prostaglandin E2 (PGE2) receptors may be altered in colon cancers. For example, the EP, and EP2 subtypes have been shown to be up‐regulated and EP3 down‐regulated in AOM‐induced colon cancers in rats and mice. EP, and EP4 appear to be involved in ACF formation, while alteration in EP2 and EP3 is considered to contribute to later steps in colon carcinogenesis. Increased expression of some other gene products, such as the targets of Wnt/β‐catenin signaling, have also been reported. The further accumulation of data with this chemically‐induced animal colon carcinogenesis model should provide useful information for understanding colorectal neoplasia in man.

Dextran sodium sulfate strongly promotes colorectal carcinogenesis in ApcMin/+ mice : Inflammatory stimuli by dextran sodium sulfate results in development of multiple colonic neoplasms

The mouse model for familial adenomatous polyposis, ApcMin/+ mouse, contains a truncating mutation in the Apc gene and spontaneously develops numerous adenomas in the small intestine but few in the large bowel. Our study investigated whether dextran sodium sulfate (DSS) treatment promotes the development of colonic neoplasms in ApcMin/+ mice. ApcMin/+ and Apc+/+ mice of both sexes were exposed to 2% dextran sodium sulfate in drinking water for 7 days, followed by no further treatment for 4 weeks. Immunohistochemistry for cyclooxygenase‐2, inducible nitric oxide synthase, β‐catenin, p53, and nitrotyrosine, and mutations of β‐catenin and K‐ras and loss of wild‐type allele of the Apc gene in the colonic lesions were examined. Sequential observation of female ApcMin/+ mice that received DSS was also performed up to week 5. At week 5, numerous colonic neoplasms developed in male and female ApcMin/+ mice but did not develop in Apc+/+ mice. Adenocarcinomas developed in ApcMin/+ mice that received DSS showed loss of heterozygosity of Apc and no mutations in the β‐catenin and K‐ras genes. The treatment also significantly increased the number of small intestinal polyps. Sequential observation revealed increase in the incidences of colonic neoplasms and dysplastic crypts in female ApcMin/+ mice given DSS. DSS treatment increased inflammation scores, associated with high intensity staining of β‐catenin, cyclooxygenase‐2, inducible nitric oxide synthase and nitrotyrosine. Interestingly, strong nuclear staining of p53 was specifically observed in colonic lesions of ApcMin/+ mice treated with DSS. Our results suggest a strong promotion effect of DSS in the intestinal carcinogenesis of ApcMin/+ mice. The findings also suggest that strong oxidative/nitrosative stress caused by DSS‐induced inflammation may contribute to the colonic neoplasms development.

Dose-dependent suppression of hyperlipidemia and intestinal polyp formation in Min mice by pioglitazone, a PPARγ ligand

In our previous study, a peroxisome proliferator‐activated receptor γ (PPARγ) agonist, pioglitazone, suppressed both hyperlipidemia and intestinal polyp formation in Apc1309 mice at doses of 100 and 200 ppm in the diet. In contrast, it has been reported that doses of 1500 or 2000 ppm of another PPARγ agonist, troglitazone, enhanced colon polyp development in Min mice. In the present study, we therefore investigated the effects of a wide range of pioglitazone doses on both hyperlipidemia and intestinal polyp formation in Min mice. Serum triglycerides and very low density lipoprotein (VLDL) cholesterol in the basal diet group were elevated to levels 13–15 times higher than those in the wild‐type counterparts at 20 weeks of age. They were reduced dose‐dependently by treatment with 100, 200, 400 and 1600 ppm pioglitazone from 6–20 weeks of age with suppression to almost the wild‐type level at the highest dose. Moreover, up‐regulation of the liver mRNA levels for lipoprotein lipase (LPL) was evident in the pioglitazone‐treated animals. Dose‐dependent reduction of intestinal polyps was observed in Min mice given 100–1600 ppm for 14 weeks, total numbers being decreased to 63–9% of the control value. A suppressive effect of pioglitazone on colon polyp formation was also found. The PPARγ agonist, pioglitazone, may thus be a promising candidate chemopreventive agent for colon cancer.

Suppression of Intestinal Polyp Development by Nimesulide, a Selective Cyclooxygenase-2 Inhibitor, in Min Mice

Nonsteroidal anti‐inflammatory drugs (NSAIDs) suppress colon carcinogenesis in man and experimental animals. However, conventional NSAIDs inhibit both cyclooxygenase (COX) isoforms, COX‐1 and COX‐2, and cause gastrointestinal side‐effects. Nimesulide, a selective inhibitor of COX‐2, is much less ulcerogenic. We, therefore, examined its influence on the development of intestinal polyps in Min mice. Female Min mice at 4 weeks old were given 400 ppm nimesulide in their diet for 11 weeks. This treatment resulted in a significant reduction of the numbers of both small and large intestinal polyps, the total being 52% of that in untreated control Min mice. The size of the polyps in the nimesulide‐treated group was also significantly decreased. The results suggest that nimesulide is a good candidate as a chemopreventive agent for human colon cancer with low toxicity.

Suppression of azoxymethane-induced aberrant crypt foci in rat colon by nimesulide, a selective inhibitor of cyclooxygenase 2.

Non-steroidal anti-inflammatory drugs, such as piroxicam and sulindac, are known to inhibit development of aberrant crypt foci (ACF) and cancer in the colon. However, these agents cause gastrointestinal side-effects. Nimesulide is a selective inhibitor of cyclooxygenase 2 and has been shown to have a more potent anti-inflammatory action than piroxicam, but be less ulcerogenic and, therefore, a potentially more useful chemopreventive agent. To assess this possibility the inhibitory effects of nimesulide on the formation of ACF induced by azoxymethane in rat colon were investigated, and compared with those of piroxicam and sulindac. Male F344 rats were treated s.c. with 15 mg/kg body weight azoxymethane once a week for 2 weeks and given 50, 100 or 200 ppm nimesulide, 200 ppm piroxicam, or 200 ppm sulindac in their diet from the day before the first carcinogen treatment until the end of the experiment at week 4. At this time, nimesulide at doses of 50, 100 and 200 ppm had reduced the numbers of azoxymethane-induced ACF to 75%, 71% and 65% respectively compared to the control. The number of azoxymethane-induced ACF per colon in the group given 200 ppm nimesulide was almost the same as in those given 200 ppm piroxicam, and lower than that in the group given 200 ppm sulindac. These results suggest that nimesulide, a selective cyclooxygenase 2 inhibitor, warrants attention as a candidate for chemopreventive agent with low toxicity, active against colon carcinogenesis.

Suppression of Nitric Oxide Production in Lipopolysaccharide-stimulated Macrophage Cells by ω3 Polyunsaturated Fatty Acids

Although nitric oxide (NO) is an important biological mediator, its excessive production in inflammation is thought to be a causative factor for cellular injury and, over the long term, cancer. In the present study, the effects of several fatty acids on NO production in murine macrophage cell line RAW264 cells stimulated with lipopolysaccharide were examined. Suppression of NO production was observed with the ω3 polyunsaturated fatty acids (PUFAs), docosahexaenoic acid, eicosapentaenoic acid and α‐linolenic acid, in a dose‐dependent fashion. In contrast, no inhibition was observed with ω6 PUFA (linoleic acid),ω9 PUFA (oleic acid) or a saturated fatty acid (stearic acid). Western and northern blot analyses suggested that suppression of the induction of inducible NO synthase gene expression is responsible for the inhibition of NO production by ω3 PUFAs. The inhibitory effect of ω3 PUFA on NO production in activated macrophages could contribute to their cancer chemopreven‐tive influence.

Roles of Prostanoids in Colon Carcinogenesis and their Potential Targeting for Cancer Chemoprevention

Prostanoids are produced in response to numerous growth factors and environmental stimuli. Their synthesis is dependent on two cyclooxygenase (COX) enzymes, COX-1 and COX-2, which are rate-limiting for the production of prostaglandins (PGs) and thromboxanes from free arachidonic acid. Selective inhibitors of both COX forms have the potential to inhibit colon tumorigenesis, and there is abundant documented evidence of elevated expression of COX-2 in colon tumors and a variety of other malignancies. The resultant high level PGE2 production may play an important role in cell proliferation, modulation of apoptosis, angiogenesis, inflammation and immune surveillance. Prostanoids exert their biological actions through binding to eight specific membrane receptors; the four subtypes EP1 to EP4 for PGE2; DP for PGD2; FP for PGF2; IP for PGI2; and TP for thromboxane A2. Recently, genetic and pharmacologic experiments have suggested that all PGE2 receptors can contribute to colon tumorigenesis. Moreover, it is suggested that EP1 and EP4 play roles in polyp formation independently. It is important to determine details of the down-stream signaling pathways of prostanoid receptors for further understanding of the mechanisms of cancer development. Furthermore, it is anticipated that development of specific receptor antagonists will provide new advantageous tools for chemoprevention.

Loss of Adiponectin Promotes Intestinal Carcinogenesis in Min and Wild-type Mice

BACKGROUND & AIMS Metabolic syndrome- and obesity-associated cancers, including colon cancer, are common in Western countries. Visceral fat accumulation and decreased levels of plasma adiponectin (APN) have been associated with development of human colorectal adenoma. We investigated the function of APN in intestinal carcinogenesis. METHODS APN+/+, APN+/-, or APN-/- mice (C57BL/6J) were given injections of azoxymethane (AOM), which led to development of intestinal tumors; these strains of mice were also crossed with Min mice to assess polyp formation. Adipocytokine levels and phosphorylation/activation of AMP-activated protein kinase (AMPK) were evaluated to investigate the mechanisms of APN in tumor growth. RESULTS The total number of polyps in the intestines of male APN+/-Min and APN-/-Min mice increased 2.4- and 3.2-fold, respectively, by the age of 9 weeks and 3.2- and 3.4-fold, respectively, by 12 weeks, compared with those of APN+/+Min mice. Similar results were obtained from female mice. AOM induced colon tumor formation in 40% of APN+/+, 50% of APN+/-, and 71% of APN-/- (P<.05) mice, respectively; mean values for tumor multiplicity of each genotype were 0.5, 0.6, and 1.1 (P<.05), respectively. Phosphorylation of AMPK decreased in intestinal epithelial cells of APN-/- mice compared with APN+/+ mice. Among serum adipocytokines, plasminogen activator inhibitor-1 levels increased in APN-/-Min mice and APN-/- mice that received injections of AOM. Activation of AMPK suppressed expression of plasminogen activator inhibitor-1 in Min mice. CONCLUSIONS Mice with disruptions in APN develop more intestinal tumors and have decreased activation (phosphorylation) of AMPK and increased levels of plasminogen activator inhibitor-1, compared with wild-type mice. APN and its receptor might be developed as targets for cancer chemopreventive agents.

Effect of docosahexaenoic acid on azoxymethane-induced colon carcinogenesis in rats

The effect of intragastric gavage administration of docosahexaenoic acid (DHA) on azoxymethane (AOM)-induced colon carcinogenesis in rats was studied. Male F344 rats were treated s.c. with 15 mg/kg of AOM once a week for 2 weeks and were given either 0.7 ml of DHA or water intragastrically twice a week starting the day before the first carcinogen treatment. The number of crypt multiplicity (number of crypts/focus) of aberrant crypt foci (ACF) in the colon were measured after 4, 12, and 36 weeks. The numbers and average crypt multiplicities of ACF induced by AOM were significantly lower after 12 and 36 weeks in animals given DHA. DHA also reduced the incidence of spontaneous ACF in animals without carcinogen treatment. Colorectal tumor incidence and number of tumors per rat after 36 weeks were slightly, but not significantly, lower in the DHA-treated group. These results suggest that DHA slightly suppresses colon carcinogenesis, and a possibility that warrants further study.

Plasminogen activator inhibitor-1 (Pai-1) blockers suppress intestinal polyp formation in Min mice

Obesity and hyperlipidemia are known to increase colorectal tumor risk. We noticed that Min mice, featuring a defect in the adenomatous polyposis coli (Apc) gene, develop intestinal polyps along with high serum triglyceride (TG) levels up to 10-fold those observed in wild-type mice. In these mice, messenger RNA (mRNA) expression of lipoprotein lipase, which catalyzes hydrolysis of TG, is downregulated. In the present study, we focused on adipocytokines, especially plasminogen activator inhibitor-1 (Pai-1), which is involved in hyperlipidemic status and may promote intestinal polyp formation in Min mice. Serum Pai-1 levels in the 15-week-old male Min mice were eight times higher than in wild-type mice and hepatic Pai-1 mRNA levels were 11-fold increased. In addition, Pai-1 immunostaining was strong in small intestinal epithelial cells of Min mice. Administration of a PAI-1 inhibitor, SK-216, at 25, 50 and 100 p.p.m. doses in the diet for 9 weeks reduced serum Pai-1 levels and hepatic Pai-1 mRNA levels of Min mice to the wild-type levels. Moreover, SK-216 at 50 and 100 p.p.m. significantly reduced total numbers of intestinal polyps to 64 and 56% of the untreated group value, respectively. Serum TG levels were also decreased by 43% at the dose of 100 p.p.m. Administration of 50 p.p.m. SK-116, another PAI-1 inhibitor, for 9 weeks similarly reduced serum Pai-1 levels and total numbers of intestinal polyps to 70% of the untreated group value. These results indicate that Pai-1 induction associated with hypertriglyceridemia may contribute to intestinal polyp formation with Apc deficiency, and PAI-1 could thus be a novel target for colorectal chemopreventive agents.