Citrus Flavonoid Nobiletin Suppresses Azoxymethane-Induced Rat

Chapter 8

Downloaded by UNIV OF MASSACHUSETTS AMHERST on February 19, 2017 | http://pubs.acs.org Publication Date: June 9, 2006 | doi: 10.1021/bk-2006-0936.ch008

Citrus Flavonoid Nobiletin Suppresses Azoxymethane-Induced Rat Colon Tumorigenesis 1

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Rikako Suzuki , Hiroyuki Kohno , Shigeyuki Sugie , Akira Murakami , Masamichi Yano , Hajime Ohigashi , and Takuji Tanaka 2

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Department of Oncologic Pathology, Kanazawa Medical University, Ishikawa, Japan Division of Applied Life Science, Graduate School of Agriculture, Kyoto University, Kyoto, Japan National Institute of Fruit Tree Science, Shizuoka, Japan 2

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We investigated the modulatory effect of citrus nobiletin on colon carcinogenesis initiated with azoxymethane (AOM) in rats. Starting one week after the initiation, rats received the diet mixed with 100 or 500 ppm nobiletin for 34 weeks. The inhibition rates of colonic adenocarcinoma in rats that received 100 or 500 ppm nobiletin after A O M exposure were 18% and 48%, respectively. Also, nobiletin feeding suppressed the prostaglandin E levels and cell proliferation activity, and enhanced apoptosis in colonic adenocarcinomas. These results suggested that citrus nobiletin might be a possible chemopreventive agent against colon cancer development. 2

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© 2006 American Chemical Society

Patil et al.; Potential Health Benefits of Citrus ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

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Introduction In a recent literature review of citrus flavonoids, a broad spectrum of biological activities including anti-carcinogenic and anti-tumor activities were discussed (7). Epidemiological studies have revealed that flavonoid intake is correlated with a reduced risk of certain types of cancer (2, 3). Nobiletin (5,6,7,8,3',4'-hexamethoxyflavone, Figure 1) is a polymethoxy flavonoid and occurs exclusively in citrus fruits. We could ingest nobiletin from not only the citrus fruits, but also orange juice, since Valencia orange juice contains 46 ppm nobiletin (4). It is known that nobiletin possesses certain biological activities such as induction of apoptosis and reduction of proliferative activity in human colon cancer cells (5). This compound is reported to suppress the production of prostaglandin (PG) E and promatrix metalloproteinase in human synovial cells (6). Previously, we found the inhibitory effect of nobiletin on phorbol esterinduced skin tumorigenesis in mice (7). Nobiletin also inhibited peritoneal dissemination of human gastric carcinoma cells in SCID mice (8). These observations encouraged us to examine the effect of nobiletin on chemically induced carcinogenesis in rodents. Colorectal cancer, a common malignancy worldwide, is an important contributor to cancer morbidity and mortality, and to overall international cancer burden. This malignancy has increased in Asia owing to the changes in life style including dietary habit of increased meat consumption (9, 10). In Japan, colon cancer is one of the most important causes of death: the mortality rates were 16.32/100,000 for men and 9.74/100,000 for women in 2000 (77). In spite of several basic or clinical challenges to control colonic cancer death, no significant prolongation of survival has been obtained (72). Although the content of nobiletin in citrus fruits is not abundant, this compound might have great potential as a cancer-preventing agent. Indeed, a variety of non-nutritive bioactive plant substances, such as flavonoids, fiber, coumarins, and limonoids are thought to have beneficial effects on human health including suppressing colonic malignancy (13, 14). We recently found that nobiletin inhibits azoxymethane (AOM)-induced colonic putative precursor lesions, aberrant crypt foci (ACF), of colonic adenocarcinoma in rats (75), suggesting that the compound might be effective in inhibiting colon carcinogenesis. In the present study, we conducted an in vivo long-term experiment to investe the modifying effect of nobiletin on AOM-induced rat colon carcinogenesis. Since cell proliferation plays an essential role in carcinogenesis (16), we measured cell proliferation activity using biomarkers such as proliferating cell nuclear antigen (PCNA)-labeling index and polyamine level in colonic tumors and non-lesional colonic mucosa. To elucidate the mechanism of the modulating effect of nobiletin on colon carcinogenesis, apoptotic index was also assayed because certain chemopreventive agents exert their inhibitory action via induction of apoptosis (14). In addition, we measured PGE levels in colonic 2

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106 adenocarcinomas and their surrounding colonic mucosa since close association between the high level of PGE and colonic carcinogenesis has been suggested (17). 2


Materials & Methods Animals, Chemicals, and Diets. Four-week-old male F344 rats were obtained from SCL, Inc. (Shizuoka, Japan). They were maintained in the Kanazawa Medical University Animal Facility according to the Institutional Animal Care Guidelines. All animals were housed in plastic cages (3 or 4 rats/cage) with free access to drinking water and a basal diet, CE-2 (CLEA Inc., Tokyo, Japan), under controlled conditions of relative humidity (50±10%), lighting (12-h light/dark cycle), and temperature (23±2°C). AOM was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Nobiletin (>99% purity) was isolated from Citrus unshiu (7). Experimental diets were prepared by mixing nobiletin into the basal diet at a dose of 100 or 500 ppm on a weekly basis.

Experimental Procedure. Male F344 rats were divided into five groups, as shown in Figure 2. At 5 weeks of age, the rats in groups 1 through 3, designated for carcinogen treatment, were subcutaneously injected with AOM (20 mg/kg body weight) once a week for two weeks. Group 1 was fed the basal diet throughout the experiment. Groups 2 and 3 were fed the diets containing nobiletin at dose levels of 100 and 500 ppm, respectively, for 34 weeks, starting one week after the last injection of AOM. Group 4 was given 500 ppm nobiletin-containing diet alone, and group 5 served as an untreated control. The experiment was terminated at 36 weeks after the start. All rats were provided with the experimental diets and tap water ad libitum, and weighed weekly. The food intake was also recorded weekly. At the termination of the study (week 36), all rats were sacrificed by an overdose of ether. At autopsy, all organs, especially the intestine, were carefully examined grossly, and then all abnormal lesions were examined histologically. Colons were fixed in 10% buffered formalin and processed for histopathological examination by routine methods. Intestinal neoplasms were diagnosed according to the criteria described by Pozharisski (18). The weighed liver and kidney were also submitted to histological examination for checking the toxicity of nobiletin.



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36 wks

A O M (20mg/kg bw S.C.) 2 or 3

100 or 500 ppm nobiletin in diet

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Scraped colonic mucosa without tumors was stored at -70 °C until measured. Proteins were extracted from the mucosa, and then tissue polyamine levels in the extraction were determined by the method described by Koide et al U9).

Measurement of PGE Level 2

For PGE determination, the scraped colonic mucosa of 5 rats each randomly selected and colonic adenocarcinomas from the experimental groups were homogenized in a 400 pL of PBS on ice. After centrifugation at 10,000 * g for 5 minutes, the supernatants obtained were diluted at the ratio of 1:9, and measured for PGE concentration using a commercial kit (Cayman, Ann Arbor, MI, USA) according to the protocol of the manufacturer. Protein concentrations for tissue samples were determined using the Bradford method (20). 2

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Measurement of PCNA and Apoptotic Indices in Colonic Neoplasms PCNA-labeling and apoptotic indices were determined in all colonic tumors by immunohistochemistry. Tumor tissues fixed in 10% buffered formalin for 2 days were embedded in paraffin. Serial cross-sections of 3 \im were cut, and mounted onto gelatin-coated glass slides. A mouse monoclonal primary antibody against PCNA (1:50 dilution; PC 10, DAKO Japan, Kyoto, Japan) and a rabbit polyclonal primary antibody against single stranded DNA (ssDNA, 1:300 dilution; DAKO Japan, Kyoto, Japan) were applied to the sections according to the manufacturer's protocol (DAKO LSAB 2 kit/HRP, DAKO Japan, Kyoto, Japan). A l l incubation steps were carried out for 15 min at 37°C. Negative controls were prepared by omitting the primary antibodies. All nuclei which densely immunoreacted with PCNA or ssDNA antibody were regarded as PCNA or ssDNA positive. The PCNA and apoptotic indices were determined by counting the number of positive cells among at least 200 cells in the tumor, and were indicated as percentages.

Statistical evaluation Where applicable, data were analyzed using one-way ANOVA with Bonferroni correction or Fisher's exact probability test with P

Citrus Flavonoid Nobiletin Suppresses Azoxymethane-Induced Rat

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