Colorectal cancer (CRC) is one of the most serious complications of inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease.1 Long-term UC patients have high risk of developing CRC, when compared with the general population.2 The precise mechanisms of the IBD-related carcinogenesis process are largely unclear, although it is generally assumed that chronic inflammation influences the development of IBD-related carcinogenesis.3
Fighting IBD-related CRC as well as sporadic CRC, by cancer chemoprevention strategy, is important to reduce the risk, and thus primary prevention of CRC in IBD has recently been receiving more attention. Previous experimental and epidemiological investigations suggest that several agents, such as folic acid,4 conjugated linoleic acid,5 ursodeoxycholic acid,6 5-aminosalicylic acid7 and aspirin, may reduce the occurrence of CRC in patients with IBD.8, 9 Consistent with these data, several nonsteroidal anti-inflammatory drugs (NSAIDs), including cyclooxygenase (COX)-2 inhibitors, suppressed the development of chemically induced colon carcinomas in rats10 and intestinal polyps in Min mice, with a nonsense mutation of the Apc gene.11 In addition, clinical trials demonstrated that intake of a NSAID, sulindac, causes regression of adenomas in patients with familial adenomatous polyposis.12
Epidemiological studies indicate an inverse correlation between the intake of fruits/vegetables and human colon cancer.13 Thus, primary prevention, including chemoprevention, using the active compounds in fruits and vegetables is also important for reducing the risk of this malignancy. Citrus fruit contains several chemopreventive compounds against colon cancer.14, 15, 16, 17 Prenyloxycoumarins, including auraptene (Fig. 1a) and collinin (Fig. 1b), are candidates of such chemopreventers. They are secondary metabolites, mainly found in plants belonging to the families of Rutaceae and Umbelellierae. Several of these coumarins were shown to possess valuable pharmacological properties. These compounds were reported to have anti-inflammatory activity.18 Auraptene significantly attenuated the lipopolysaccharide (LPS)-induced protein expression of inducible nitric oxide synthase (iNOS) and COX-2, with decreases in production of nitric anion and prostaglandin E2 (PGE2), and yet suppressed the release of tumor necrosis factor (TNF)-α and IκB degradation.19, 20 Furthermore, auraptene and collinin also cause complete inhibition of platelet aggregation, induced by arachidonic acid and platelet activated factor in vitro.21 We have previously found that a citrus auraptene suppresses chemically induced carcinogenesis in rodents.22, 23, 24
For understanding the pathogenesis of IBD and IBD-related CRC, several animal models have been established. Most used is a mouse model with dextran sodium sulfate (DSS).25 Modifying effects of several xenobiotics on CRC-related colon carcinogenesis have been reported,26, 27 using this model. However, this colitis model using DSS, with or without carcinogen, needs to a long period repeated administration of DSS to induce colitis and colitis-related CRC that mimic human UC. To investigate the pathogenesis in IBD-related CRC and search novel and effective chemopreventive agents against this type of malignancy, we developed a novel colitis-related mouse CRC model, using a colon carcinogen azoxymethane (AOM) and DSS, in which large bowel adenocarcinomas develop within a short-term period, and their histology and biological alteration resemble to those found in humans.28 Our animal model indicates that in the large bowel, inflammation induced by DSS strongly promotes the development of epithelial malignant neoplasia. Oxidative/nitrosative stress caused by DSS exposure may contribute the development of high incidence of colonic adenocarcinomas.29, 30 Recently, we demonstrated that dietary administration of COX-2 inhibitor and peroxisome proliferator-activated receptor ligands suppressed colitis-related colonic carcinogenesis, using our mouse colon carcinogenesis model.31
As a part of our search for safer chemopreventive agents against colitis-related colon cancer, we examined, in the present study, the effects of auraptene and collinin on our mouse colon carcinogenesis model.28
Male Crj: CD-1 (ICR) (Charles River Japan, Tokyo, Japan), aged 5 weeks, were used in this study. They were maintained at Kanazawa Medical University Animal Facility, according to the Institutional Animal Care Guidelines. All animals were housed in plastic cages (5 or 6 mice/cage), with free access to drinking water and a pelleted basal diet, CRF-1 (Oriental Yeast Co., Tokyo, Japan), under controlled conditions of humidity (50 ± 10)%, light (12/12 hr light/dark cycle) and temperature (23 ± 2)°C. They were quarantined for the first 7 days, and then randomized by body weight into experimental and control groups. A colonic carcinogen AOM was purchased from Sigma Chemical Co. (St. Louis, MO). DSS with a molecular weight of 36,000–50,000 was purchased from ICN Biochemicals (Aurora, OH). DSS for induction of colitis was dissolved in water at a concentration of 1% (w/v). Auraptene (99.6% purity)32 and collinin (99.8% purity)18 were synthesized, as described previously. Experimental diet, containing auraptene or collinin, was prepared every week by mixing the respective compound in powdered basal diet CRF-1, at a concentration (w/w) of 0.01 or 0.05%. The dose levels of the 2 compounds were selected on the basis of our previous experiments.18, 22, 23, 24
A total of 75 male ICR mice were divided into 10 (experimental and control) groups (Fig. 2). Mice in groups 1 through 5 were given a single intraperitoneal injection of AOM (10 mg/kg body weight). Starting 1 week after the injection, animals were administered to 1% DSS in drinking water for 7 days, and then followed without any further treatment for 15 weeks. Mice of group 1 were maintained on basal diet, throughout the study. Mice in groups 2 through 5 were given 0.01% auraptene in diet (group 2), 0.05% auraptene in diet (group 3), 0.01% collinin in diet (group 4) or 0.05% collinin in diet (group 5), respectively, for 17 weeks, starting 1 week after the stop of DSS administration. Group 6 was given a single dose of AOM. Group 7 was given 1% DSS for 7 days. Animals in groups 8 and 9 were given the diets containing 0.05% auraptene and 0.05% collinin alone, respectively. Group 10 consisted of untreated mice. All animals were killed at the end of the study (week 20). Their large bowels were flushed with saline, excised, their length measured (from ileocecal junction to the anal verge) and cut open longitudinally along the main axis, and then washed with saline. The large bowels were macroscopically inspected, cut and fixed in 10% buffered formalin, for at least 24 hr. Histological examination was performed on paraffin-embedded sections, after hematoxylin and eosin (H & E) staining. Colonic neoplasms were diagnosed, according to the description by Ward.33 Grade of colitis was scored, and the sections were stained with H & E,29, 34 from all groups.
Immunohistochemistry for proliferating cell nuclear antigen (PCNA), apoptotic nuclei, COX-2, iNOS and nitrotyrosine was performed on 4-μm-thick paraffin-embedded sections, from the colons of mice in each group by the labeled streptavidin biotin method, using a LSAB KIT (DAKO Japan, Kyoto, Japan), with microwave accentuation. The paraffin-embedded sections were heated for 30 min at 65°C, deparaffinized in xylene and rehydrated through graded ethanols at room temperature. A 0.05 M Tris HCl buffer (pH 7.6) was used to prepare solutions, and for washes between various steps. Incubations were performed in a humidified chamber. For the determination of PCNA-incorporated nuclei, the PCNA-immunohistochemistry was performed.35 Apoptotic index was also evaluated by immunohistochemistry for single stranded DNA (ssDNA).35 Sections were treated for 40 min at room temperature, with 2% BSA, and incubated overnight at 4°C with primary antibodies, such as anti-PCNA mouse monoclonal antibody (diluted 1:50; PC10, DAKO Japan), anti-ssDNA rabbit polyclonal antibody (diluted 1:300, DAKO Japan), anti-COX-2 rabbit polyclonal antibody (diluted 1:50, IBL Co., Gunma, Japan), anti-iNOS rabbit polyclonal antibody (diluted 1:1,000, Wako Pure Chemical Industries, Osaka, Japan), and anti-nitrotyrosine rabbit polyclonal antibody (diluted 1:500, Upstate Biotechnology, Lake Placid, NY). To reduce the nonspecific staining of mouse tissue by the mouse antibodies, a Mouse On Mouse IgG blocking reagent (Vector Laboratories, Burlingame, CA) was applied for 1 hr. Horseradish peroxidase activity was visualized by treatment with H2O2 and 3,3′-diaminobenzidine for 5 min. At the last step, the sections were weakly counterstained with Mayer's hematoxylin (Merck, Tokyo, Japan). For each case, negative controls were performed on serial sections. On the control sections, incubation with the primary antibodies was omitted.
Intensity and localization of immunoreactivities, against all primary antibodies used, were examined on all sections using a microscope (Olympus BX41, Olympus Optical Co., Tokyo, Japan). The PCNA and apoptotic indices were determined, by counting the number of positive cells among at least 200 cells in the lesion, and were indicated as percentages. Each slide for COX-2, iNOS and nitrotyrosine was evaluated for intensity of immunoreactivity on a 0 to 4+ scale. The overall intensity of the staining reaction was scored, with 0 indicating no immunoreactivity and no positive cells, 1+ weak immunoreactivity and <10% of positive cells, 2+ mild immunoreactivity and 10–30% of positive cells, 3+ moderate immunoreactivity and 31–60% of positive cells, and 4+ strong immunoreactivity and 61–100% of positive cells.
Measurements were compared by Bonferroni multiple comparison post test or Fisher's exact probability test. Differences were considered statistically significant at p < 0.05.
Bloody stool was observed in a few mice, which received 1% DSS, and their body weight gains were slightly decreased during the period of treatment. However, thereafter no such clinical symptoms were noted. Body weights, liver weights and relative liver weights, in all groups at the end of the study, are shown in Table I. The mean body weights, liver weights and relative liver weights did not significantly differ among the groups. The mean length of large bowel in groups 2–5 was lower than group 1, but the differences did not reach statistical significance. Histologically, there were no pathological alterations suggesting toxicity of auraptene and collinin in the liver, kidneys, lung and heart. Food consumption (g/day/mice) did not significantly differ among the groups (data not shown).
Table I. Body, Liver, Relative Liver Weights and Length of Large Bowel
Body wt (g)
Liver wt (g)
Relative liver wt (g/100 g body wt)
Length of colon (cm)
Values in parentheses indicate the numbers of mice examined.
Macroscopically, nodular or polypoid colonic tumors were observed in the middle and distal colon of mice in groups 1 through 5. Histopathologically, AOM/DSS treated mice showed dysplasia (Fig. 3a), adenoma (Fig. 3b) and adenocarcinoma (Fig. 3c). These tumors histologically diagnosed as tubular adenoma or well-/moderately-differentiated tubular adenocarcinoma. Animals of groups 6–10 did not have large bowel neoplasms in any organs examined, including the colon. The incidences and multiplicity of colon neoplasma are shown in Table II, respectively. Group 1 (AOM/DSS) induced 100% incidence of colon adenocarcinomas, with a multiplicity of 3.00 ± 1.41. The incidences of colorectal adenocarcinomas in groups 2 (AOM/DSS/0.01% auraptene), 3 (AOM/DSS/0.05% auraptene), 4 (AOM/DSS/0.01% collinin) and 5 (AOM/DSS/0.05% collinin) were significantly smaller than that of group 1 (p < 0.02, p < 0.01, p < 0.01 and p < 0.01, respectively). The multiplicity of colon adenocarcinomas in groups 2, 3, 4 and 5 were also significantly lower than that of group 1 (p < 0.005, p < 0.001, p < 0.005 and p < 0.001, respectively). Colitis was present with or without colonic dysplasia in the middle or distal colon of mice treated with DSS. As shown in Fig. 4, colonic inflammation scores in groups 3 (p < 0.05) and 5 (p < 0.05) were significantly decreased, when compared with that in group 1.
Table II. Incidence and Multiplicity of Colonic Neoplasia
Immunohistochemistry for PCNA, ssDNA, COX-2, iNOS and nitrotyrosine in colonic adenocarcinoma
As summarized in Table III, PCNA-labeling index of colonic adenocarcinomas developed in groups 2 (p < 0.01), 3 (p < 0.01), 4 (p < 0.01) and 5 (p < 0.05) was significantly smaller than group 1 (Figs. 5a–5c), and apoptotic index, measured by ssDNA immunohistochemistry in groups 2 (p < 0.05), 4 (p < 0.05) and 5 (p < 0.01), was significantly greater than group 1 (Figs. 5d–5f). Scores for COX-2 and iNOS expression in colonic adenocarcinomas is also given in Table III. In the positive cases of COX-2 and iNOS expression in the dysplasia and adenocarcinoma, the staining pattern was granular and localized to cytoplasm or nuclei or both. Slight immunoreactivity for COX-2 and iNOS was observed in the superficial layers of the nonlesional colonic mucosa and in parts of basal layer, in all groups. COX-2 expression scores of colonic adenocarcinomas in groups 2 (p < 0.01), 3 (p < 0.05) and 5 (p < 0.05) and that of iNOS in groups 2 (p < 0.001), 3 (p < 0.001), 4 (p < 0.01) and 5 (p < 0.01) were significantly decreased, when compared with that in group 1 (Figs. 5g–5i for COX-2 and Figs. 5j–5l for iNOS). Nitrotyrosine immunoreactivity (Figs. 5m–5o) was mainly observed in mononuclear cells infiltrated in the colonic mucosa with the lesions, and the stainability was relatively weak in the neoplastic cells. The score of nitrotyrosine is also given in Table III. The scores of groups 3 (p < 0.05) and 5 (p < 0.05) were significantly higher than that of group 1. The scores of groups 2 and 4 were also lower than that of group 1, but the differences were insignificant.
Table III. PCNA and Apoptosis Indices and Scores of COX-2, INOS and Nitrotyrosine Expression in Colonic Adenocarcinomas
Treatment (no. of mice examined)
PCNA-labeling index (%)
Apoptotic index (%)
Mean ± SD.
Numbers in parentheses are the numbers of lesions examined.
Significantly different from group 1 by Bonferroni multiple comparison post test. (p < 0.01).
Significantly different from group 1 by Bonferroni multiple comparison post test. (p < 0.05).
Significantly different from group 1 by Bonferroni multiple comparison post test. (p < 0.001).
The results of the present work clearly indicated that 2 prenyloxycoumarins, auraptene and collinin, effectively inhibited AOM/DSS-induced colitis-related colonic carcinogenesis, without any adverse effects in mice. The suppressive effect of auraptene and collinin on the development of colonic adenocarcinoma was well correlated with the inhibition of cell proliferation activity, induction of apoptosis and inhibition of imumunoreactivity of COX-2 and iNOS in the colonic malignancies. These findings may suggest that dietary auraptene and collinin suppress IBD-associated colon carcinogenesis and are possibly applicable in human clinical trials.
The pathogenesis of IBD-associated colorectal carcinogenesis is widely believed to involve a stepwise progression from inflamed and hyperplastic cryptal cells, through flat dysplasia, to finally adenocarcinoma,36 but the mechanism is still unclear. However, mucosal inflammation may result in colonic carcinogenesis through several proposed mechanisms, such as induction of genetic mutations, increased-cryptal cell proliferation, changes in crypt cell metabolism and bile acid enterohepatic circulation, and alterations in bacteria flora.37, 38 These events are considered to promote IBD-associated CRC development. In the colon, the number of epithelial cells in the crypts is strictly regulated by a balance between cell proliferation and cell death that maintains homeostasis.39 In neoplastic tissues, changes in cell proliferation and apoptosis are regarded as a common denominator in the pathogenesis of tumor formation.40 It is thought that intermittent colonic epithelial damage and restitution caused by chronic inflammation contribute to the increased cancer risk in the long-term UC patients. The elevated rate of cell turnover associated with the epithelial damage–restitution cycle may increase the occurrence of mitotic aberrations and other genetic and epigenetic changes, as well as take part in the promotion stage of cancer development.41 In the present study, the modifying effects of auraptene and collinin on the cellular proliferation and apoptosis may contribute to their lowering activity in the incidence and multiplicity of colon adenocarcinomas.
Chronic inflammation is recognized as one of the major causes of human cancer.42, 43 Inflammation-caused oxidative/nitrosative cellular damage is suspected to be responsible for the development of IBD-associated colorectal neoplasms. Therefore, certain antioxidants are effective as cancer chemopreventive agents. Auraptene suppresses 12-O-tetradecanoylphorbol-13-acetate-induced superoxide in HL-60 cells, attenuates inflammatory leukocyte activation in vivo, and decreases inflammation, H2O2 production and cell proliferation.44 In addition, auraptene quite likely reduces the production of lipid peroxidation products in rat colon carcinogenesis.24 These findings suggest that auraptene mitigates oxidative stress by suppressing oxygen radical generation by inflammatory leukocytes. Since nitrotyrosine production may involve in CRC development in this colitis-related mouse colon carcinogenesis model,29, 30 our results suggesting potential use of the antioxidants, collinin and auraptene, in the prevention of IBD-associated cancer may be caused by their suppression of oxidative/nitrosative cellular damage in our model.
There are an increasing number of reports showing that the expression of COX-2 and iNOS is closely associated with the development of cancers.45, 46 We also observed increased expression of COX-2 and iNOS in colon adenocarcinomas in this animal model.28 The increases in the reaction products of iNOS and COX-2, nitric oxide and PGE2 respectively, could contribute to colon tumorigenesis. Expression and activity of iNOS are increased in the colonic mucosa in patients with IBD47 and colonic adenomas.48 Several studies, using experimental colon carcinogenesis models, indicate that chemically induced colon tumors have higher expression or activity of iNOS or both, when compared with those found in the adjacent colonic tissue.26, 49 An iNOS-selective inhibitor could suppress the development of AOM-induced colonic preneoplastic lesions by inhibition of iNOS activity.50 Likewise, an increased COX-2 expression is reported in human and rodent CRC,51, 52 and its overexpression may confer a survival advantage on cells by inhibition apoptosis and a change in cellular adhesion to the extracellular matrix.53 Given the correlation between increased COX-2 expression and cancer occurrence in the inflamed colon, the chemopreventive effect of NSAIDs seems to be mediated, at least in part, by COX inhibition.54 Our previous study55 and those of others56, 57 shows that COX-2 inhibitors inhibited colon tumorigenesis as well as colitis, induced by naturally occurring carcinogen. Suh et al.58 synthesized novel synthetic triterpenoids that suppressed iNOS and COX-2 protein expression, and demonstrated their potent differentiating, antiproliferating and anti-inflammatory activities.59 Auraptene also can inhibit iNOS and COX-2 expression in RAW 264.7 cells treated with LPS and TNF-α.19 Our recent study29 indicated that changes of inflammation scores paralleled with those of the nitrotyrosine immunohistochemical scores in the colonic mucosa, and these alterations in the inflamed colon resulted in powerful promotion effect of DSS in the AOM/DSS-induced mouse colon carcinogenesis. In the current study, suppressing effects of dietary feeding with auraptene and collinin after treatment with AOM and DSS might be mainly due to their inhibition of inflammation and oxidative/nitrosative stress in the colon.
In conclusion, dietary administration with prenyloxycoumarins, auraptene and collinin, could effectively suppress colitis-related colon carcinogenesis, induced by AOM and DSS in male ICR mice. Our on-going study on molecular profiles in colonic samples from the current experiment will provide precise molecular mechanisms involved in their inhibitory action in AOM/DSS-induced mouse colon carcinogenesis.
We express our thanks to the staff of the Research Animal Facility. We also thank Mrs. Sotoe Yamamoto for her secretarial assistance.