Nutrition and Cancer, 60(S1), 7080 Copyright 2008, Taylor & Francis Group, LLC ISSN: 0163-5581 print

t / 1532-7914 online DOI: 10.1080/01635580802381253

Citrus Compounds Inhibit Inammationand Obesity-Related Colon Carcinogenesis in Mice

Takuji Tanaka, Yumiko Yasui, Rikako Ishigamori-Suzuki, and Takeru Oyama
Kanazawa Medical University, Daigaku, Uchinada, Ishikawa, Japan

Dietary polyphenols are important potential chemopreventive natural agents. Other agents, such as citrus compounds, are also candidates for cancer chemopreventives. They act on multiple key elements in signal transduction pathways related to cellular proliferation, differentiation, apoptosis, inammation, and obesity. This short review article provides our ndings of preclinical studies on potential chemopreventive activities of dietary citrus compounds, auraptene, collinin, and citrus unshiu segment membrane (CUSM), using clitis- and obesity-related colon tumorigenesis models. Dietary feeding with auraptene and collinin at dose levels of 0.01% and 0.05% signicantly lowered the incidence (5060% reduction) and multiplicity (6780% reduction) of colonic adenocarcinomas induced by azoxymetahene [AOM, single intraperitoneal injection of 10 mg/kg body weight (bw)] and dextran sodium sulfate (1% in drinking water). Anti-inammatory potency of aurapene and collinin may contribute to the effects. Administration with CUSM at 3 doses in diet signicantly inhibited development of aberrant crypts foci induced by 5 weekly subcutaneous injections of AOM (15 mg/kg bw) in male db/db mice: 53% inhibition by 0.02% CUSM, 54% inhibition by 0.1% CUSM, and 59% inhibition by 0.5% CUSM. CUSM treatment also decreased serum level of triglycerides. Our ndings suggest that certain citrus materials are capable of inhibiting clitis- and obesity-related colon carcinogenesis.

colon cancer (9,10). There are several studies that have investigated the possible mechanism for this (11,12) Obesity is a complex, heterogeneous, and multifactorial syndrome resulting from both genetic susceptibility and environmental factors (13). Besides obesity, it is well known that several factors, including a high-fat and low-ber diet (14), low physical activity (15), IBD (16) or hereditary disorders such as familial adenomatous polyposis and nonpolyposis syndrome (17), increase the risk for development of CRC. Fighting IBD- and obesity-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 and/or obese people has recently been receiving more attention. There are several natural compounds that are able to inhibit CRC development in rodents, and thus they are candidates for cancer chemopreventive agents (1821). For ghting this malignancy in inamed colon and colon of obese people, we tried to nd natural compounds that are capable of effectively inhibiting colon carcinogenesis in a series of preclinical studies (2224). In this article, we introduce our ndings for pushing candidate materials into clinical trials. METHODS: PRECLINICAL EXPERIMENTS

INTRODUCTION Colorectal cancer (CRC) has increased in Asia owing to the changes in lifestyle such as the dietary habit of increased meat consumption (1,2). Inammation and obesity are risk for chronic diseases including cancer development (35). In fact, CRC is one of the most serious complications of inammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohns disease (6). Long-term UC patients have high risk of developing colorectal cancer compared with the general population (7). Obesity raises the risk for a variety of disease (8). Numerous epidemiological results suggest that obesity is a risk factor for

Submitted 10 July 2008; accepted in nal form 22 July 2008. Address correspondence to Takuji Tanaka, Department of Oncologic Pathology, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293, Japan. E-mail: takutt@kanazawa-med. ac.jp

Preclinical Chemopreventive Study 1: Citrus Auraptene and Related Compound Collinin Inhibit Azoxymethane (AOM)/Dextran Sodium Sulfate (DSS)-Induced Colitis-Related Colon Carcinogenesis in Mice Previous experimental and epidemiological investigations suggest that several agents, such as folic acid (25), conjugated linoleic acid (26), ursodeoxycholic acid (27), 5-aminoslicylic acid (28), and aspirin, may reduce the occurrence of CRC in patients with IBD (29,30). Consistent with these data, several nonsteroidal anti-inammatory drugs (NSAIDs), including cyclooxygenase (COX)-2 inhibitors, suppressed the development of chemically induced CRC in rats (31) and intestinal polyps (tubular adenomas) in Min mice with a nonsense mutation of the adenomatous polyposis coli (APC) gene (32). In addition, clinical trials demonstrated that intake of sulindac causes regression of adenomas in patients with familial adenomatous polyposis (33). Epidemiological studies indicate an inverse correlation 70

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FIG. 1. Chemical structures and biological activities of (a) auraptene and (b) collinin. Effects of dietary auraptene on chemically induced carcinogenesis in rodents are also given. DMBA (TPA), 7,12, dimethylbenze(a )anthracene (12-O-tetradecanoylphorbol-13-acetate); 4-NQO, 4-nitroquinoline 1-oxide; AOM, azoxymetahene; NMBA, N-nitrosomethylbenzylamine; DEN, diethylnitrosamine.

between the intake of fruits/vegetables and human CRC (34). Thus, primary prevention including chemoprevention using the active compounds in fruits/vegetables is also important for reducing the risk of this malignancy. Citrus fruit contains several chemopreventive compounds against CRC (20,3537). Prenyloxycoumarins including auraptene (Fig. 1a) and collinin (Fig. 1b) are candidates of such chemopeventers. 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-inammatory activity (38). Auraptene signicantly 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)- and I B degradation (39,40). Furthermore, auraptene and collinin also cause complete inhibition of platelet aggregation induced by arachidonic acid and platelet activated factor in vitro (41). We have previously found that a citrus auraptene suppresses chemically induced carcinogenesis in a variety of tissues of rodents (20,42), as shown in Fig. 1. We also demonstrated that dietary administration of a COX-2 inhibitor and peroxisome proliferator-activated receptor ligands suppressed colitis-related colonic carcinogenesis using our mouse colon carcinogenesis model (43). In this experiment (Study 1), to determine the effects of auraptene and collinin of inammation-related colon tumorigenesis, we utilized a novel colitis-related mouse CRC model using a colon carcinogen AOM and DSS in which large bowel adeno-

carcinomas develop within a short-term period and their histology and biological alteration resemble those found in humans (44). This animal model indicates that in the large bowel, inammation induced by DSS strongly promotes the development of epithelial malignant neoplasia (4550). Oxidative/nitrosative stress caused by DSS exposure may contribute the development of high incidence of colonic adenocarcinomas with certain genetic alterations (45, 6), as shown in Table 1 and Figs. 2 and 3. A total of 75 male ICR mice aged 5 wk (Charles River Japan Inc., Tokyo, Japan) were divided into 10 experimental and control groups. The study was approved by the Institutional Animal Care Guidelines, and the experiment complied with the International Guiding Principles for Biomedical Research Involving Animals established by the Council for International Organization of Medical Sciences (CIOMS). Mice in Groups 1 through 5 were given a single intraperitoneal injection of AOM (10 mg/kg body weight, Sigma Chemical Co., St. Louis, MO). Starting 1 wk after the injection, animals were administered to 1% (wt/vol) DSS (molecular weight 36,00050,000; ICN Biochemicals, Inc., Aurora, OH) in drinking water for 7 days and then followed without any further treatment for 15 wk. Mice of Group 1 were maintained on the basal diet Charles River Formula (CRF)-1 (Oriental Yeast Co., Ltd., Tokyo, Japan) throughout the study. CRF-1 consisted of 5.7% fat, 22.4% protein, 6.6% minerals, 3.1% ber, and 62.3% carbohydrate and others (3.59 kcal/g). The major fatty acids present in CRF-1 were linoleic acid, oleic acid, and palmitic acid. Mice in Groups 2 through 5 were given 0.01% auraptene in diet (Group 2),

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TABLE 1 Sequential observation of AOM/DSS-induced mouse colon tumorigenesisa

Experimental Weeks Week 2 Week 3 Week 4 Week 5 Week 6 Week 9 Week 12 Week 14

Incidence (multiplicity) 0 (0) 0 (0) 40% 60% 100% 100% 100% 100% of colonic (2.00 3.52) (1.60 1.85) (4.23 2.46) (4.92 3.46) (6.92 1.85) (10.00 1.92) adenocarcinomas Immunohistochemical 7.32 1.35 5.22 0.40 5.40 1.32 3.27 0.55 2.09 0.55 2.87 0.62 2.82 0.64 2.75 0.25 nitrotyrosine-positive scores
a

Abbreviations are as follows: AOM, azoxymethane; DSS, dextran sodium sulfate.

FIG. 2. Experimental protocol of our mouse model for inammation-related mouse colon carcinogenesis. Incidence and multiplicity of colonic adenocarcinomas in mice that were initiated with azoxymethane (AOM), 1,2-dimethylhydrazine (DMH), or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhlP) and promoted by dextran sodium sulfate (DSS) and mutations in exon 3 of beta-catenin in induced epithelial malignancies are shown.

(a) Incidence (%) of colonic adenocarcinoma

120 100 80 60 40 20 0 0.1 0.25 0.5 1 2 Dose (%) of DSS in drinking water

(b) Multiplicity (no./colon) of colonic adenocarcinoma 6

5 4 3 2 1 0 0.1 0.25 0.5 1 2 Dose (%) of DSS in drinking water

FIG. 3. Effects of dose of dextran sodium sulfate (DSS) on azoxymethane (AOM)/DSS-induced mouse colonic carcinogenesis. Treatment with 1% and 2% DSS in drinking water after the initiation with AOM resulted in occurrence of colonic adenocarcinomas. The incidence (a) and multiplicity (b) of colonic adenocarcinomas in male ICR mice given 2% DSS are greater than that of mice treated with 1% DSS.

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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 wk, starting 1 wk 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. Auraptene (99.6% purity) (51) and collinin (99.8% purity) (38) were synthesized as described previously. All animals were sacriced at the end of the study (Week 20) to determine the incidence and multiplicity of colonic tumors and dysplastic crypts. In addition to histopathology, immunohistochemistry for proliferating cell nuclear antigen (PCNA), apoptotic nuclei, COX-2, iNOS, single stranded DNA (ssDNA) (52), and nitrotyrosine was done to determine the effects of dietary auraptene and collinin on their expression in the colonic epithelial malignancies. At Week 12, messenger RNA (mRNA) expression of COX2, iNOS, interleukin (IL)-1 , and TNF- was also determined in the nontumorous colonic mucosa of mice in Groups 1, 3, and 5. At sacrice (Week 20), 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, adenoma, and adenocarcinoma. The tumors were histologically diagnosed as tubular adenoma or adenocarcinoma. Animals belonging to Groups 610 did not have large bowel neoplasms in any organs examined, including the colon. 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 signicantly smaller than that of Group 1 (P < 0.05). The multiplicity of

colon adenocarcinomas in Groups 2 through 5 were also signicantly lower than that of Group 1 (P < 0.05). Colitis was present with or without colonic dysplasia in the middle or distal colon of mice treated with DSS. Colonic inammation scores in Groups 3 and 5 were signicantly decreased when compared with that in Group 1 (P < 0.05). The PCNA-labeling indexes of colonic adenocarcinomas developed in Groups 25 were signicantly smaller than Group 1 (P < 0.05), and the apoptotic index measured by ssDNA immunohistochemistry in Groups 2, 4, and 5 was signicantly greater than Group 1 (P < 0.05). COX-2 expression scores of colonic adenocarcinomas in Groups 2, 3, and 5 and that of iNOS in Groups 25 were signicantly decreased when compared with that in Group 1 (P < 0.05). The nitrotyrosine-positive scores of Groups 3 and 5 were signicantly higher than that of Group 1 (P < 0.05). The scores of Groups 2 and 4 were also lower than that of Group 1, but the differences were insignicant. mRNA expression of the nontumorous colonic mucosa of Groups 3 and 5 were signicantly lower than that of Group 1 (P < 0.05), but iNOS expression was comparable among Groups 1, 3, and 5 (Fig. 4).

Preclinical Chemopreventive Study 2: Citrus Unshiu Segment Membranes (CUSM) Suppresses Development of Preneoplastic Lesions in db/db Mice Treated With AOM The modern Western lifestyle, such as a high caloric intake, high-fat diets, and physical inactivity, results in a positive energy balance, diabetes, and obesity. These lifestyle patterns might also be risk factors for the development of CRC (8), which is 1 of the major causes of morbidity and mortality inthe Western world (1). This malignancy has also increased in Asia owing to the changes in lifestyle such as the dietary habit of increased

FIG. 4. Messenger RNA (mRNA) expression of cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), interleukin-1beta (IL-1 ), and tumor necrosis factor-alpha (TNF- ) in the colonic mucosa (Study 1). Data are mRNA/glyderaldehyde-3-phosphate dehydrogenase mRNA rations (103 ).

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meat consumption (1,2). Several prospective and case-control studies have addressed the relationship between obesity/diabetes and CRC (8,53,54). Certain food components are known to exert a cancer chemopreventive activity against CRC development (55). However, few studies have so far been performed on the preventive effect of food components on obesity- and diabetes-related colon carcinogenesis (56,57). We recently have made CUSM that are rich in soluble and insoluble ber and separate the juice vesiculates from Satsuma mandarin (Citrus unshiu Marc.). Mandarin orange fruit constitutes 9 to 13 segments (juice sacs) that contain juice vesicles, and a membrane that wraps the segment is called the segment membrane. Although CUSM is waste product that remains after squeezing citrus unshiu for fruit juice, it contains biologically active compounds such as avonoids, including hesperidin and auraptene. Citrus bers and avonoids have been reported to inhibit colon carcinogenesis in rodents (20,58,59). Obese individuals are thus often recommended to consume such diet low-energy foods rich in ber with a possibly specic hypolipidemic effect such as pectin-enriched dishes, fruit purees and juices, and wheat bran biscuits (60). Supplementationwith avonoids (hesperidin or naringin) improves the hyperglycemia in db/db mice (61). Although the biological activity of CUSM has yet to be elucidated, we suspected that CUSM might have a preventive effect on obesity- and diabetes-related colon carcinogenesis. C57BL/KsJ-db/db (db/db) mice are used as a genetically altered animal model with genotypes of obesity and diabetes mellitus (62). A disruption of the leptin receptor [obese mutation (Ob)-R] gene in these mice leads to an overexpression of leptin in the adipose tissue and a concomitantly high serum concentration of leptin (63,64). The synthesis of leptin in adipocytes is inuenced by insulin (65), TNF- (66), glucocorticoids (67), reproductive hormones (68), and prostaglandins (69) that may be involved in the neoplastic processes (70). In addition, leptin can act as a growth factor in colonic epithelial cells (71) whereas modulating the proliferation of colonic cryptal cells (72). In contrast, more leptin in the blood clearly decreased colon carcinogenesis in 3 different animal models (73,74). Thus, leptin might be 1 of the biological factors involved in the development of CRC associated with obesity/diabetes. The db/db mouse, therefore, is a very useful model for elucidating the relationship between colon carcinogenesis and obesity/diabetes. In this study (Study 2), we determined the possible modulatory effects of dietary CUSM on the occurrence of AOMinduced aberrant crypt foci (ACF) and -catenin accumulated crypts (BCAC), which are putative precursor lesions for colonic adenocarcinoma (49,75), in db/db, db/+, and +/+ male mice. A total of 36 homozygous db/db, 40 heterozygous db/+, and 40 littermate controls (+/+) male mice aged 4 wk (Japan SLC, Inc., Shizuoka, Japan) were divided into 4 groups, respectively. The study was approved by the Institutional Animal Care Guidelines, and the experiment complied with the International Guiding Principles for Biomedical Research Involving Animals es-

tablished by the CIOMS. Animals were free access to drinking water and a basal diet, MF (Oriental Yeast Co., Ltd.). At 5 wk of age, all mice were subcutaneously injected with AOM (15 mg/kg body weight) once a week for 5 wk. Group 1 was fed the basal diet, MF (Oriental Yeast Co., Ltd.), throughout the experiment. Groups 2 through 4 were fedthe diets containing CUSM at dose levels of 0.02, 0.1, and 0.5%, respectively, for 7 wk, starting 1 wk after the lastinjection of AOM. The experiment was terminated 12 wk after the start. Powdered CUSM was obtained from Ehime Beverage Inc. (Matsuyama, Japan). The composition of CUSM (100 g) was as follows: 2.4 g moisture; 5.5 g protein; 0.3 g fat; 51 g ber (22.0 g soluble and 29.0 g insoluble); 2.3 g ash; 26.6 g saccharide (6.1 g D-ucutose, 5.5 g glucose, and 15.0 g D-suclose); 2.2 g hesperidin; and 9.7 g others that include avonoids, carotenoids, and unknown components. At the termination of the study (Week 12), all mice were sacriced by an overdose of ether to analyze the number of AFC and BCAC. At autopsy, all organs, including the intestine, were carefully examined grossly and then were examined histopathologically. The weighed liver and kidney were also submitted for histological examinations to investigate the toxicity of CUSM. At Week 12, mRNA expression of COX-2, iNOS, IL-1 , and TNF- was also determined in the nonlesional colonic mucosa of mice in Groups 1 through 4. The presence of ACF and BCAC were determined in the distal part (1 cmfrom the anus) of the colon, according to the standard procedures that areroutinely used in our laboratory (59,76). To identify BCAC intramucosal lesions, the colon (0.580.87 cm2 /colon) was embeddedin parafn, and then a total of20 serial sections (4 mthick each) per mouse were made by an en face preparation (77,78). Tissue sections were subjected to hematoxylin and eosin (H & E) staining for histopathology and -catenin immunohistochemistry to count the number of colonic BCAC (77,78), and others were used for Ob-R, insulin-like growth factor-1 receptor (IGF-1R), and PCNA immunohistochemistry. Serial sections from the liver of all mice were also made for histopathology (H & E stain) and morphometric analysis of liver brosis (Sirius-red) and fatty change (H & E stain) using an image analysis software NIH Image v.1.63. At sacrice, blood to measure the serum concentrations of glucose, leptin, insulin, cholesterol, and triglyceride levels was collected from 5 mice each of genotypes +/+, db/+, and db/db. They were starved overnight prior to blood collection for clinical chemistry. At the end of the study (Week 12), all the mice that received AOM developed colonic ACF and BCAC. Regarding the mean number of ACF/colon in the AOM alone groups, the mean number of db/db mice (147 23, P < 0.05) was signicantly higher than that of db/+ (77 19) or +/+ mice (69 12). In comparison to the AOM alone group, the dietary administration with CUSM signicantly reduced the number of ACF in all the genotypes (P < 0.05): db/db mice, 53% reduction by 0.02% CUSM, 54% reduction by 0.1% CUSM, and 59% reduction by 0.5% CUSM; db/+ mice, 48% reduction by 0.1% CUSM and 38% reduction by 0.5% CUSM; and +/+ mice, 45% reduction

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FIG. 5. messenger RNA expression of cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), interleukin-1beta (IL-1 ), and tumor necrosis factoralpha (TNF- ) in the colonic mucosa (Study 2). Data are messenger RNA(mRNA)/glyderaldehyde-3-phosphate dehydrogenase mRNA rations (103 ).

by 0.1% CUSM and 62% reduction by 0.5% CUSM. In addition, the percentages of ACF consisting of more than 4 aberrant crypts (ACs) in all the CUSM-feeding groups in the db/db mice (36% reduction by 0.02% CUSM, 30% reduction by 0.1% CUSM, and 47% reduction by 0.5% CUSM) were signicantly smaller (P < 0.05) than that of AOM alone group (77 11). Dietary administration with CUSM reduced the percentages of ACF consisting of more than 4 ACs in the db/+ and +/+ mice; the reduction rates were insignicant. BCAC also developed in the colon of all the genotypes of mice that received AOM alone, and the frequency per cm2 of colonic mucosa was high in order of db/db, +/+, and db/+ mice. The dietary administration with CUSM at the highest dose (0.5%) signicantly (P < 0.05) reduced the number of BCAC in the +/+ (65% reduction) and db/db mice (74% reduction). CUSM at a dose of 0.1% also signicantly lowered the number of BCAC in db/db mice (53% reduction, P < 0.05). The immunohistochemical expression of Ob-R and IGF-1R was observed in the cytoplasm and nuclei of cryptal cells. Their expression was relatively strong in the nuclei of atypical cells in BCAC when compared to their surrounding cryptal cells. Feeding with CUSM did not inuence the stainability of Ob-R and IGF-1R. PCNA-labeling index was determined in BCAC that developed in the db/db mice (Groups 912). The mean PCNA-labeling indexes of Group 11 (AOM/0.1% CUSM) and Group 12 (AOM/0.5% CUSM) were signicantly lower than that of Group 9 (AOM alone; P < 0.05). The values of Groups 9 and 10 (AOM/0.02% CUSM) were comparable. A histopathological examination of the liver revealed the occurrence of fatty metamorphosis and brosis in the db/db mice that received AOM alone in contrast to the +/+ and db/+ mice. When the db/db mice were fed with 0.5% CUSM, these histopathological alterations were signicantly inhibited fatty metamorphosis (P < 0.05) and liver brosis (P < 0.05). All the measurements of total cholesterol, triglycerides, glucose, insulin, and leptin in the db/db mice were higher than those

of db/+ and +/+ mice. The dietary administration with CUSM did not signicantly affect the serum levels of total cholesterol, glucose, insulin, and leptin in all the genotypes. However, the serum level of triglycerides signicantly decreased in the db/db mice (P < 0.05) when fed the diet containing 0.5% CUSM. mRNA expression of the nonlesional colonic mucosa of Groups 24 were signicantly lower than that of Group 1. iNOS expression of Groups 24 was also smaller than Group 1, but the differences were insignicant (Fig. 5).

DISCUSSION The results of the Study 1 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 epithelial malignancies. These ndings 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 step-wise progression from inamed and hyperplastic cryptal cells through at dysplasia to nally adenocarcinoma (79), but the mechanism is still unclear. However, mucosal inammation may result in colonic carcinogenesis through several proposed mechanisms such as induction of genetic mutations, increased-cryptal cell proliferation, changes in crypt cell metabolism, bile acid enterohepatic circulation, and alterations in bacteria ora (80). 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

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cell death that maintains homeostasis (81). In neoplastic tissues, changes in cell proliferation and apoptosis are regarded as a common denominator in the pathogenesis of tumor formation (82). It is thought that intermittent colonic epithelial damage and restitution caused by chronic inammation 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 (83). In this 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 inammation is recognized as one of the major causes of human cancer (84). Inammation-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 human promyelocytic leukemia-60 cells; attenuates inammatory leukocyte activation in vivo; and decreases inammation, hydrogen dioxide production, and cell proliferation (85). In addition, auraptene likely reduces the production of lipid peroxidation products in rat colon carcinogenesis (42). These ndings suggest that auraptene mitigates oxidative stress by suppressing oxygen radical generation by inammatory leukocytes. Because nitrotyrosine production may involve in CRC development in this colitisrelated mouse colon carcinogenesis model (43,59), our results suggesting potential use of the antioxidants collinin and auraptene in prevention of IBD-associated cancer may be caused by their suppression of oxidative/nitrosative cellular damage in our model. Given the correlation between increased COX-2 (86,87) or iNOS (88) expression and cancer occurrence in the inamed colon of IBD patients, the chemopreventive effect of NSAIDs and iNOS inhibitors seems to be mediated, at least in part, by COX and iNOS inhibition (89). We (90) and others (91,92) have demonstrated that COX-2 inhibitors inhibited colon tumorigenesis as well as colitis induced by naturally occurring carcinogen. A specic inhibitor of iNOS is able to inhibit colon carcinogenesis in ApcMin/+ mice that received DSS (93). Suh et al. (94) synthesized novel synthetic triterpenoids that suppressed iNOS and COX-2 protein expression and demonstrated their potent differentiating, antiproliferating, and antiinammatory activities (95). Auraptene also can inhibit iNOS and COX-2 expression in RAW 264.7 cells treated with LPS and TNF- (39). Our recent study (59) indicated that changes of inammation scores paralleled with those of the nitrotyrosine immunohistochemical scores in the colonic mucosa, and these alterations in the inamed colon resulted in powerful promotion effect of DSS in the AOM/DSS-induced mouse colon carcinogenesis. In this study, suppressing effects of dietary feeding with auraptene and collinin after the treatment with AOM and DSS

might be mainly due to their inhibition of inammation and oxidative/nitrosative stress in the colon. The results of the Study 2 conrmed the high susceptibility of AOM-induced colon carcinogenesis in the obese/diabetic db/db mice in our previous ndings (11). The high susceptibility in the db/db mice may be related to the increases in the body weight and the serum levels of total cholesterol, triglycerides, glucose, insulin, and leptin, thus suggesting a positive association between obesity/diabetes and colon tumorigenesis. Our ndings also suggest that insulin resistance involves CRC development (96). The main purpose of this study was to investigate the effect of CUSM on the early phase of AOM-induced colon carcinogenesis in the db/db mice. Because these lesions are considered to be putative precursor lesions of colonic adenocarcinoma (49), the results obtained clearly indicate the inhibitory effects of the dietary administration of CUSM on the development of AOMinduced ACF and BCAC in the db/db mice as well as the +/+ and db/+ mice. In this experiment, all the serum measurements of total cholesterol, triglycerides, glucose, insulin, and leptin were greater in the db/db mice than those of db/+ and +/+ mice, thus suggesting that these measurements may contribute to the high susceptibility of db/db mice to AOM-induced colon tumorigenesis. However, among the chemical proles, only the triglyceride level lowered by feeding with CUSM correlated with a lower incidence of colonic preneoplastic lesions in the db/db mice. These ndings suggest that a high level of serum triglyceride is the most important biological effect for developing colonic tumors in db/db mice, and a modication (lowering) of this value may thus lead to the inhibition of colon tumorigenesis. In fact, a positive association between the serum triglyceride levels and the risk of CRC development was found in humans (97). This association was also suspected by the ndings in animal experiments (98) in which model animals for human familial adenomatous polyposis were used (76). An association between diabetes and cancer was suggested over 100 years ago (99). The increased incidence of CRC in the Type 2 diabetic patients has been supported by a recent prospective, population-based cohort, case-control, and metaanalysis studies (54). Thus, there is an attractive hypothesis of insulin resistance CRC in which insulin resistant may thus be associated with the development of CRC (100), and this malignancy may therefore become a modiable disease (101). Regarding the mechanism of action, insulin resistance is associated with hyperinsulinemia, increased levels of growth factors including IGF-1, and alterations in nuclear factor kappa B (NF- B) and peroxisome proliferator-activated receptors signaling, which may promote CRC through their effect on the colonic cryptal cell kinetics (57). Insulin and the IGF axis may be related to CRC development (102). Recently, an interesting nding indicated that leptin may interact with IGFs to promote survival and the expansion of colonic epithelial cells that were APC decient, but not those expressing wild type APC (103). We recently observed increased immunohistochemical expression of NF- B (Fig. 6a) in the colonic neoplasms

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FIG. 6. Immunohistcochemistry of nuclear factor kappa B (NF- B) in the colonic adenocarcinoma. a: Strong immunohistochemical expression of NF- B in the nuclei of colonic adenocarcinoma in a mouse treated with azoxymethane (AOM)/dextran sodium sulfate (DSS). b: Decreased immunohistochemical expression of NF- B is observed in an adenocarcinoma of a mouse that received AOM/DSS and 0.05% auraptene (AUR). c: Immunohistochemical scores of NF- B-intensity in the adenocarcinomas developed in mice treated with AOM/DSS, AOM/DSS + 0.05% AUR in diet, and AOM/DSS + 0.05% nobiletin (NOB) in diet. Dietary feeding with AUR and NOB decreases NF- B-positivity of colonic adenocarcinomas induced by AOM/DSS.

induced by the AOM/DSS treatment, suggesting that NF- B is 1 of the good molecular targets of cancer chemoprevention (104) in the inamed colon. In addition, certain citrus compounds, auraptene (22) and nobiletin (105), in diet decreased NF- Bimmunohistochemical expression in the malignancies (Figs. 6b and 6c; unpublished work). In this study, feeding with CUSM did not inuence the serum level of insulin and immunoreactivities of IGF-1R and Ob-R in the BCAC in the db/db mice. However, the treatment reduced cell proliferation activity in the BCAC by estimating PCNA-labeling index. CUSM could reduce the occurrence or progression of BCAC through lowering the cell proliferation, although the exact mechanism(s) should be elucidated. Feeding with a high-fat diet, which is implicated to play a role in the stimulation of colonic cryptal cell proliferation while also promoting colon carcinogenesis (106), thus increases the circulating leptin level (107). In addition, dietary ber has been reported to the decrease serum leptin concentration while reducing colon carcinogenesis by lowering the degree of cryptal cell proliferation (108). However, CUSM feeding did not affect serum leptin levels in the db/db mice. Thus, CUSM constituents other than ber, that is, avonoids, may have contributed to the reduction in the occurrence of putative precursor lesions, ACF and BCAC, in the colon of the db/db mice. Our recent study aimed to determine whether auraptene suppresses ACF and BCAC development in the db/db female mice initiated with AOM and indicated that dietary auraptene is able to inhibit the development of both precursor lesions (22). The suppression by

dietary auraptene is considered to be caused by reducing serum triglycerides, inducing apoptosis, and/or reducing cell proliferation. Taken together, we suspected that hesperidin and auraptene in CUSM may be responsible for the inhibition of preneoplasia development in male db/db mice because this chemical can inhibit chemically induced colon carcinogenesis in rodents (109). In conclusion, the results of Study 1 and Study 2 conrmed the high susceptibility of the inamed mouse colon induced by DSS and obese/diabetic db/db mice to AOM-induced colon carcinogenesis. Our data provide further evidence that citrus compounds, including auraptene, collinin, and CUSM, are able to inhibit inammation- and obesity-related colon carcinogenesis. Further studies focusing the detailed mechanisms of inhibitory effects of citrus materials on the development of CRC in the inamed colon and colon of obese mice should be carried out for the prevention and treatment of the colonic malignancies associated with these conditions.

ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid for Cancer Research, for the Third-Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labour and Welfare of Japan; a Grant-in-Aid (No. 18592076 to T. Tanaka, 17015016 to T. Tanaka, and 18880030 to Y. Yasui) for Scientic Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan; and a grant (H2008-12 to T. Tanaka and S2006-9 to Y. Yasui) for the Project Research

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