Rikako Suzuki

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.

Pomegranate seed oil rich in conjugated linolenic acid suppresses chemically induced colon carcinogenesis in rats

Pomegranate (Punica granatum L.) seed oil (PGO) contains more than 70% cis(c)9,frans(f)11,c13–18:3 as conjugated linolenic acids (CLN). Our previous short‐term experiment demonstrated that seed oil from bitter melon (Momordica charantia) (BMO), which is rich in c9,t11,t13‐CLN, inhibited the occurrence of colonic aberrant crypt foci (ACF) induced by azoxymethane (AOM). In this study, we investigated the effect of dietary PGO on the development of AOM‐induced colonic malignancies and compared it with that of conjugated linoleic acid (CLA). To induce colonic tumors, 6‐week old male F344 rats were given subcutaneous injections of AOM (20 mg/kg body weight) once a week for 2 weeks. One week before the AOM treatment they were started on diet containing 0.01%, 0.1%, or 1%±PGO or 1% CLA for 32 weeks. Upon termination of the bioassay (32 weeks) colon tumors were evaluated histopathologically. AOM exposure produced colonic adenocarcinoma with an incidence of 81% and multiplicity of 1.88±1.54 at week 32. Administration of PGO in the diet significantly inhibited the incidence (AOM+0.01% PGO, 44%, P<0.05; AOM+0.1% PGO, 38%, P<0.01; AOM+1% PGO, 56%) and the multiplicity (AOM+0.01% PGO, 0.56±0.73, P<0<01; AOM+0.1% PGO, 0.50±0.73, P<0.005; AOM+1% PGO, 0.88±0.96, P<0.05) of colonic adenocarcinomas, although a clear dose‐response relationship was not observed at these dose levels. CLA feeding also slightly, but not significantly, reduced the incidence and multiplicity of colonic adenocarcinomas. The inhibition of colonic tumors by PGO was associated with an increased content of CLA (c9,t11–18:2) in the lipid fraction of colonic mucosa and liver. Also, administration of PGO in the diet elevated expression of peroxisome proliferator activated receptor (PPAR) γ protein in the nontumor mucosa. These results suggest that PGO rich in c9,t11,c13‐CLN can suppress AOM‐induced colon carcinogenesis, and the inhibition is associated in part with the increased content of CLA in the colon and liver and/or increased expression of PPARγ protein in the colon mucosa.

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.

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)

Sequential observations on the occurrence of preneoplastic and neoplastic lesions in mouse colon treated with azoxymethane and dextran sodium sulfate

Previously, we proposed a novel mouse model for colitis‐related colon carcinogenesis using azoxymethane (AOM) and dextran sodium sulfate (DSS) (Cancer Sci 2003; 94: 965–73). In the current study, sequential analysis of pathological alterations during carcinogenesis in our model was conducted to establish the influence of inflammation caused by DSS on colon carcinogenesis in this model. Male ICR mice were given a single intraperitoneal injection of AOM (10 mg/kg body weight) and given 2% (w/v) DSS in the drinking water for 7 days, starting 1 week after the AOM injection. They were sequentially sacrificed at weeks 2, 3, 4, 5, 6, 9, 12, and 14 for histopathological and immunohistochemical examinations. Colonic adenomas were found in 2 (40% incidence and 0.40±0.49 multiplicity) of 5 mice at week 3 and colon carcinomas developed in 2 (40% incidence and 2.00±3.52 multiplicity) of 5 mice at week 4. Their incidence gradually increased with time and reached 100% (6.20±2.48 multiplicity) at week 6. At week 14, the multiplicity of adenocarcinoma was 9.75±2.49 (100% incidence). In addition, colonic dysplasia was noted at all time‐points. The scores of colonic inflammation and nitrotyrosine immunohistochemistry were extremely high at early time‐points and were well correlated. Our results suggest that combined treatment of mice with AOM and DSS generates neoplasms in the colonic mucosa via dysplastic lesions induced by nitrosative stress.

Dietary administration with prenyloxycoumarins, auraptene and collinin, inhibits colitis-related colon carcinogenesis in mice

We previously reported the chemopreventive ability of a prenyloxycoumarin auraptene in chemically induced carcinogenesis in digestive tract, liver and urinary bladder of rodents. The current study was designed to determine whether dietary feeding of auraptene and its related prenyloxycoumarin collinin can inhibit colitis‐related mouse colon carcinogenesis. The experimental diets, containing the compounds at 2 dose levels (0.01 and 0.05%), were fed for 17 weeks to male CD‐1 (ICR) mice that were initiated with a single intraperitoneal injection of azoxymethane (AOM, 10 mg/kg body weight) and promoted by 1% (w/v) DSS in drinking water for 7 days. Their tumor inhibitory effects were assessed at week 20 by counting the incidence and multiplicity of colonic neoplasms and the immunohistochemical expression of proliferating cell nuclear antigen (PCNA)‐labeling index, apoptotic index, cyclooxygenase (COX)‐2, inducible nitric oxide (iNOS) and nitrotyrosine in colonic epithelial malignancy. Feeding with auraptene or collinin, at both doses, significantly inhibited the occurrence of colonic adenocarcinoma. In addition, feeding with auraptene or collinin significantly lowered the positive rates of PCNA, COX‐2, iNOS and nitrotyrosine in adenocarcinomas, while the treatment increased the apoptotic index in colonic malignancies. Our findings may suggest that certain prenyloxycoumarins, such as auraptene and collinin, could serve as an effective agent against colitis‐related colon cancer development in rodents.

Dietary seed oil rich in conjugated linolenic acid from bitter melon inhibits azoxymethane-induced rat colon carcinogenesis through elevation of colonic PPARγ expression and alteration of lipid composition

Our previous short‐term experiment demonstrated that seed oil from bitter melon (Momordica charantia) (BMO), which is rich in cis(c)9, trans(t)11, t13‐conjugated linolenic acid (CLN), inhibited the development of azoxymethane (AOM)‐induced colonic aberrant crypt foci (ACF). In our study, the possible inhibitory effect of dietary administration of BMO on the development of colonic neoplasms was investigated using an animal colon carcinogenesis model initiated with a colon carcinogen AOM. Male F344 rats were given subcutaneous injections of AOM (20 mg/kg body weight) once a week for 2 weeks to induce colon neoplasms. They also received diets containing 0.01%, 0.1% or 1% BMO for 32 weeks, starting 1 week before the first dosing of AOM. At the termination of the study (32 weeks), AOM induced 83% incidence (15/18 rats) of colonic adenocarcinoma. Dietary supplementation with 0.01% and 0.1% BMO caused significant reduction in the incidence (47% inhibition by 0.01% BMO, p<0.02; 40% inhibition by 0.1% BMO, p<0.05; and 17% inhibition by 1% BMO) and the multiplicity (64% inhibition by 0.01% BMO, p<0.005; 58% inhibition by 0.1% BMO, p<0.02; and 48% inhibition by 1% BMO, p<0.05) of colonic adenocarcinoma, though a clear dose response was not observed. Such inhibition was associated with the increased content of CLA (c9,t11‐18:2) in the lipid composition in colonic mucosa and liver. Also, BMO administration in diet enhanced expression of peroxisome proliferator‐activated receptor (PPAR) γ protein in the nonlesional colonic mucosa. These findings suggest that BMO rich in CLN can suppress AOM‐induced colon carcinogenesis and the inhibition might be caused, in part, by modification of lipid composition in the colon and liver and/or increased expression of PPARγ protein level in the colon mucosa.

Global gene expression analysis of the mouse colonic mucosa treated with azoxymethane and dextran sodium sulfate

BackgroundChronic inflammation is well known to be a risk factor for colon cancer. Previously we established a novel mouse model of inflammation-related colon carcinogenesis, which is useful to examine the involvement of inflammation in colon carcinogenesis. To shed light on the alterations in global gene expression in the background of inflammation-related colon cancer and gain further insights into the molecular mechanisms underlying inflammation-related colon carcinogenesis, we conducted a comprehensive DNA microarray analysis using our model.MethodsMale ICR mice were given a single ip injection of azoxymethane (AOM, 10 mg/kg body weight), followed by the addition of 2% (w/v) dextran sodium sulfate (DSS) to their drinking water for 7 days, starting 1 week after the AOM injection. We performed DNA microarray analysis (Affymetrix GeneChip) on non-tumorous mucosa obtained from mice that received AOM/DSS, AOM alone, and DSS alone, and untreated mice at wks 5 and 10.ResultsMarkedly up-regulated genes in the colonic mucosa given AOM/DSS at wk 5 or 10 included Wnt inhibitory factor 1 (Wif1, 48.5-fold increase at wk 5 and 5.7-fold increase at wk 10) and plasminogen activator, tissue (Plat, 48.5-fold increase at wk 5), myelocytomatosis oncogene (Myc, 3.0-fold increase at wk 5), and phospholipase A2, group IIA (platelets, synovial fluid) (Plscr2, 8.0-fold increase at wk 10). The notable down-regulated genes in the colonic mucosa of mice treated with AOM/DSS were the peroxisome proliferator activated receptor binding protein (Pparbp, 0.06-fold decrease at wk 10) and the transforming growth factor, beta 3 (Tgfb3, 0.14-fold decrease at wk 10). The inflammation-related gene, peroxisome proliferator activated receptor γ (Pparγ 0.38-fold decrease at wk 5), was also down-regulated in the colonic mucosa of mice that received AOM/DSS.ConclusionThis is the first report describing global gene expression analysis of an AOM/DSS-induced mouse colon carcinogenesis model, and our findings provide new insights into the mechanisms of inflammation-related colon carcinogenesis and the establishment of novel therapies and preventative strategies against carcinogenesis.

Ursodeoxycholic Acid versus Sulfasalazine in Colitis-Related Colon Carcinogenesis in Mice

Purpose: Inflammation influences carcinogenesis. In the current study, we investigated whether ursodeoxycholic acid (UDCA) can inhibit colitis-related mouse colon carcinogenesis and compared it with the effects of sulfasalazine. Experimental Design: Male CD-1 mice were given a single i.p. injection of azoxymethane followed by 1-week oral exposure of 1% dextran sodium sulfate in drinking water. They are then maintained on a basal diet mixed with UDCA (0.016%, 0.08%, or 0.4%) or sulfasalazine (0.05%) for 17 weeks. At week 20, the tumor-inhibitory effects of both chemicals were assessed by counting the incidence and multiplicity of colonic neoplasms. The immunohistochemical expression of the proliferating cell nuclear antigen labeling index in colonic epithelial malignancies was also assessed. Finally, at week 5, the mRNA expressions for cyclooxygenase-2, inducible nitric oxide synthase, peroxisome proliferator-activated receptor-γ, and tumor necrosis factor-α were measured in nontumorous mucosa. Results: Feeding the mice with UDCA at all doses significantly inhibited the multiplicity of colonic adenocarcinoma. The treatment also significantly lowered the proliferating cell nuclear antigen labeling index in the colonic malignancies. UDCA feeding reduced the expression of inducible nitric oxide synthase and tumor necrosis factor-α mRNA in the colonic mucosa, while not significantly affecting the expression of cyclooxygenase-2 mRNA and peroxisome proliferator-activated receptor-γ mRNA. Sulfasalazine caused a nonsignificant reduction in the incidence and multiplicity of colonic neoplasia and did not affect these mRNA expression. Conclusions: Our findings suggest that UDCA rather than sulfasalazine could serve as an effective suppressing agent in colitis-related colon cancer development in mice.