Shigeyuki Sugie

A novel inflammation‐related mouse colon carcinogenesis model induced by azoxymethane and dextran sodium sulfate

To develop an efficient animal model for colitis‐related carcinogenesis, male Crj: CD‐1 (ICR) mice were given a single intraperitoneal administration (10 mg/kg body weight) of a genotoxic colonic carcinogen, azoxymethane (AOM), and a 1‐week oral exposure (2% in drinking water) to a non‐genotoxic carcinogen, dextran sodium sulfate (DSS), under various protocols. At week 20, colonic neoplasms (adenocarcinomas, 100% incidence with 5.60±2.42 multiplicity; and adenomas, 38% incidence with 0.20±0.40 multiplicity) with dysplastic lesions developed in mice treated with AOM followed by DSS. Protocols in which AOM was given during or after DSS administration induced a few tubular adenomas or no tumors in the colon. Immunohistochemical investigation of such dysplasias and neoplasms revealed that all lesions were positive for β‐catenin, cyclooxygenase‐2 and inducible nitric oxide synthase, but did not show p53 immunoreactivity. The results indicate that 1‐week administration of 2% DSS after initiation with a low dose of AOM exerts a powerful tumor‐promoting activity in colon carcinogenesis in male ICR mice, and may provide a novel mouse model for investigating colitis‐related colon carcinogenesis and for identifying xenobiotics with modifying effects.

Silymarin, a naturally occurring polyphenolic antioxidant flavonoid, inhibits azoxymethane‐induced colon carcinogenesis in male F344 rats

The modifying effect of dietary administration of the polyphenolic antioxidant flavonoid silymarin, isolated from milk thistle [Silybum marianum (L.) Gaertneri], on AOM‐induced colon carcinogenesis was investigated in male F344 rats. In the short‐term study, the effects of silymarin on the development of AOM‐induced colonic ACF, being putative precursor lesions for colonic adenocarcinoma, were assayed to predict the modifying effects of dietary silymarin on colon tumorigenesis. Also, the activity of detoxifying enzymes (GST and QR) in liver and colonic mucosa was determined in rats gavaged with silymarin. Subsequently, the possible inhibitory effects of dietary feeding of silymarin on AOM‐induced colon carcinogenesis were evaluated using a long‐term animal experiment. In the short‐term study, dietary administration of silymarin (100, 500 and 1,000 ppm in diet), either during or after carcinogen exposure, for 4 weeks caused significant reduction in the frequency of colonic ACF in a dose‐dependent manner. Silymarin given by gavage elevated the activity of detoxifying enzymes in both organs. In the long‐term experiment, dietary feeding of silymarin (100 and 500 ppm) during the initiation or postinitiation phase of AOM‐induced colon carcinogenesis reduced the incidence and multiplicity of colonic adenocarcinoma. The inhibition by feeding with 500 ppm silymarin was significant (p < 0.05 by initiation feeding and p < 0.01 by postinitiation feeding). Also, silymarin administration in the diet lowered the PCNA labeling index and increased the number of apoptotic cells in adenocarcinoma. β‐Glucuronidase activity, PGE2 level and polyamine content were decreased in colonic mucosa. These results clearly indicate a chemopreventive ability of dietary silymarin against chemically induced colon tumorigenesis and will provide a scientific basis for progression to clinical trials of the chemoprevention of human colon cancer.

Inhibitory effects of plumbagin and juglone on azoxymethane-induced intestinal carcinogenesis in rats

The effects of two naphthoquinones, juglone and plumbagin, and an isocoumarin, hydrangenol, on intestinal carcinogenesis in rats were examined by dietary exposure during the initiation phase. Starting at 5 weeks of age, male F344 rats were fed the diets containing either of the test chemicals at a concentration of 200 ppm or the control diet without the compounds. At 6 weeks of age, all animals were treated with s.c. injections of azoxymethane (AOM) (15 mg/kg body weight, once weekly for 3 weeks) or saline alone. Animals fed experimental diets were changed to the control diet 1 week after the last carcinogen treatment. Animals given plumbagin together with the carcinogen had a lower incidence (41%) and smaller multiplicity (0.48 +/- 0.62) of tumors in the entire intestine compared with those exposed to carcinogen alone (68% and 1.04 +/- 0.62) (P < 0.05 and < 0.01, respectively). The incidence and multiplicity of tumors in the small intestine (7% and 0.07 +/- 0.25) and the multiplicity of tumors in the entire intestine (0.60 +/- 0.76) of animals treated with juglone and the carcinogen were significantly less than those of animals treated with carcinogen alone (P < 0.05 in each). Hydrangenol tended to decrease the incidence and the multiplicity of tumors in the entire intestine induced by AOM, but the effect was not statistically significant. The present data suggest that the naphthoquinones, juglone and plumbagin, could be promising chemopreventive agents for human intestinal neoplasia.

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.

Suppression of colitis-related mouse colon carcinogenesis by a COX-2 inhibitor and PPAR ligands

BackgroundIt is generally assumed that inflammatory bowel disease (IBD)-related carcinogenesis occurs as a result of chronic inflammation. We previously developed a novel colitis-related mouse colon carcinogenesis model initiated with azoxymethane (AOM) and followed by dextran sodium sulfate (DSS). In the present study we investigated whether a cyclooxygenase (COX)-2 inhibitor nimesulide and ligands for peroxisome proliferator-activated receptors (PPARs), troglitazone (a PPARγ ligand) and bezafibrate (a PPARα ligand) inhibit colitis-related colon carcinogenesis using our model to evaluate the efficacy of these drugs in prevention of IBD-related colon carcinogenesis.MethodsFemale CD-1 (ICR) mice were given a single intraperitoneal administration of AOM (10 mg/kg body weight) and followed by one-week oral exposure of 2% (w/v) DSS in drinking water, and then maintained on the basal diets mixed with or without nimesulide (0.04%, w/w), troglitazone (0.05%, w/w), and bezafibrate (0.05%, w/w) for 14 weeks. The inhibitory effects of dietary administration of these compounds were determined by histopathological and immunohistochemical analyses.ResultsFeeding with nimesulide and troglitazone significantly inhibited both the incidence and multiplicity of colonic adenocarcinoma induced by AOM/DSS in mice. Bezafibrate feeding significantly reduced the incidence of colonic adenocarcinoma, but did not significantly lower the multiplicity. Feeding with nimesulide and troglitazone decreased the proliferating cell nuclear antigen (PCNA)-labeling index and expression of β-catenin, COX-2, inducible nitric oxide synthase (iNOS) and nitrotyrosine. The treatments increased the apoptosis index in the colonic adenocarcinoma. Feeding with bezafibrate also affected these parameters except for β-catenin expression in the colonic malignancy.ConclusionDietary administration of nimesulide, troglitazone and bezafibrate effectively suppressed the development of colonic epithelial malignancy induced by AOM/DSS in female ICR mice. The results suggest that COX-2 inhibitor and PPAR ligands could serve as an effective agent against colitis-related colon cancer development.

Chemoprevention by Nimesulide, a Selective Cyclooxygenase‐2 Inhibitor, of 2‐Amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP)‐induced Mammary Gland Carcinogenesis in Rats

Breast cancer is common in women all over the world, and exploration of chemopreventive approaches to this cancer is very important. Nimesulide, a selective inhibitor of cyclooxygenase‐2 (COX‐2), is a good candidate as a chemopreventive agent with low toxicity. We examined its effects on mammary tumor development in female Sprague‐Dawley rats induced with the environmental carcinogen 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP). Rats at 7 weeks of age received intragastric intubations of PhIP (85 mg/kg body weight) 4 times weekly for 2 weeks and were maintained on control diet (high fat diet) or experimental diet (high fat diet supplemented with 400 ppm nimesulide) throughout the experiment. COX‐2 protein was over‐expressed in epithelial cancer cells and stromal cells of the PhIP‐induced mammary carcinomas, but was weak or not apparent in normal mammary gland cells. The development of mammary carcinomas was clearly suppressed by administration of nimesulide. The carcinoma incidence was 51% as compared to 71% for the control diet group. The average multiplicity of carcinomas in the experimental diet group was 1.2±0.2 (P < 0.05), significantly smaller than the control diet group value (2.6±0.5). The size of carcinomas was also clearly decreased; 1.1±0.4 cm3/rat in experimental diet group (P < 0.05), 4.1±1.3 cm3/rat in the control diet group. The results therefore provide evidence that the selective COX‐2 inhibitor, nimesulide, possesses chemopreventive activity against PhIP‐induced mammary carcinogenesis in rats.

Modifying Effects of Fungal and Herb Metabolites on Azoxymethane-induced Intestinal Carcinogenesis in Rats

Modifying effects of a fungal product, flavoglaucin, and four plant‐derived chemicals, shikonin, gingerol, oleanolic acid and paeoniflorin, on intestinal carcinogenesis were examined in a rat model using azoxymethane (AOM). A total of 280 male F344 rats, 6 weeks old, were divided into 12 groups. Group 1 (30 rats) was given two subcutaneous injections of 15 mg/kg of AOM at the start of the experiment. Groups 2 (30 rats), 3 (20 rats), 4 (20 rats), 5 (30 rats) and 6 (30 rats) received a test chemical (flavoglaucin, shikonin, gingerol, oleanolic acid or paeoniflorin, respectively) in the diet at a concentration of 0.02% for 3 weeks, during which time AOM was applied, and then kept on basal diet until the end of experiment (one year). Groups 7–11 (each 20 rats) were given a test chemical corresponding to Groups 2–6, respectively. Group 12 (20 rats) served as a control. The incidence and average number of intestinal tumors in Group 2 (47%, 0.57 ± 0.68) were significantly less than in Group 1 (74%, 1.07 ± 0.87) (P < 0.05, respectively). Multiplicity of intestinal neoplasms of Group 3 (0.55 ± 0.60) or 4 (0.47 ± 0.51) was also significantly smaller than that of Group 1 (P < 0.05 and P < 0.01, respectively). These results suggest that flavoglaucin, shikonin and gingerol might be promising chemopreventive agents for intestinal neoplasia.

β‐Catenin mutations in a mouse model of inflammation‐related colon carcinogenesis induced by 1,2‐dimethylhydrazine and dextran sodium sulfate

In a previous study, we developed a novel mouse model for colitis‐related carcinogenesis, utilizing a single dose of azoxymethane (AOM) followed by dextran sodium sulfate (DSS) in drinking water. In the present study, we investigated whether colonic neoplasms can be developed in mice initiated with a single injection of another genotoxic colonic carcinogen 1,2‐dimethylhydrazine (DMH), instead of AOM and followed by exposure of DSS in drinking water. Male crj: CD‐1 (ICR) mice were given a single intraperitoneal administration (10, 20 or 40 mg/kg body weight) of DMH and 1‐week oral exposure (2% in drinking water) of a non‐genotoxic carcinogen, DSS. All animals were killed at week 20, histological alterations and immunohistochemical expression of β‐catenin, cyclooxygenase (COX‐2) and inducible nitric oxide synthase (iNOS) were examined in induced colonic epithelial lesions (colonic dysplasias and neoplasms). Also, the β‐catenin gene mutations in paraffin‐embedded colonic adenocarcinomas were analyzed by the single strand conformation polymorphism method, restriction enzyme fragment length polymorphism and direct sequencing. The incidences of colonic neoplasms with dysplastic lesions developed were 100% with 2.29 ± 0.95 multiplicity, and 100% with 10.38 ± 4.00 multiplicity in mice given DMH at doses of 10 mg/kg or 20 mg/kg and 2%DSS, respectively. Although approximately half of the mice given DMH at a dose of 40 mg/kg bodyweight were dead after 2–3 days after the injection, mice who received DMH 40 mg/kg and 2%DSS had 100% incidence of colonic neoplasms with 9.75 ± 6.29 multiplicity. Immunohistochemical investigation revealed that adnocarcinomas, induced by DMH at all doses and 2%DSS, showed positive reactivities against β‐catenin, COX‐2 and iNOS. In DMH/DSS‐induced adenocarcinomas, 10 of 11 (90.9%) adenocacrcinomas had β‐catenin gene mutations. Half of the mutations were detected at codon 37 or 41, encoding serine and threonine that are direct targets for phosphorylation by glycogen synthase kinase‐3β. The present results suggests that, as in the previously reported model (AOM/DSS) our experimental protocol, DMH initiation followed by DSS, may provide a novel and useful mouse model for investigating inflammation‐related colon carcinogenesis and for identifying xenobiotics with modifying effects. (Cancer Sci 2005; 96: 69–76)

Induction of intestinal tumours in rats by chrysazin.

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Chemopreventive Effect of Curcumin on N‐Nitrosomethylbenzylamine‐induced Esophageal Carcinogenesis in Rats

Modifying effects of curcumin (derived from the rhizome of Curcuma longa L.) during the initiation or post‐initiation phase of N‐nitrosomethylbenzylamine (NMBA)‐induced esophageal carcinogenesis were investigated in male F344 rats. Five‐week‐old rats were divided into 5 groups, and groups 1, 2 and 3 were given intraperitoneal injections of NMBA (0.5 mg/kg body weight/injection 15 times) for 5 weeks from 7 weeks old to induce esophageal neoplasms. Groups 2 and 3 were fed the diet containing 500 ppm curcumin during the initiation and post‐initiation phases, respectively. Group 4 was given the diet containing curcumin throughout the experiment, and group 5 was kept on the basal diet alone and served as an untreated control. Incidence and multiplicity of esophageal neoplasms of group 1 (NMBA alone) were 66.7% and 0.83 ±0.70, respectively. Those of groups 2 and 3 were significantly less than those of group 1 (39.3%, 0.46±0.64, P < 0.05; 33.3%, 0.36±0.56, P < 0.05, respectively). Furthermore, the incidence and multiplicity of esophageal preneoplastic lesions (moderate or severe epithelial dysplasia) of group 2 (57.1%, 0.61±0.57; 40%, 0.29±0.46) or 3 (56.7%, 0.67±0.66; 23.3%, 0.23±0.43) were less than those of group 1 (100%, 1.67±0.70; 70.8%, 0.92±0.72) (P < 0.05). In this experiment, feeding of curcumin significantly decreased the expression of cell proliferation biomarkers (5‐bromo‐2′‐deoxyuridine labeling index) in the non‐lesional esophageal epithelium (P < 0.01). These findings indicate that curcumin inhibits NMBA‐induced esophageal carcinogenesis when given during the post initiation as well as initiation phase. This inhibition may be related to suppression of the increased cell proliferation induced by NMBA in the esophageal epithelium.