Dietary administration with prenyloxycoumarins, auraptene and collinin, inhibits colitis-related colon carcinogenesis in mice Hiroyuki Kohno 1 * , Rikako Suzuki 1 , Massimo Curini 2 , Francesco Epifano 2 , Federica Maltese 2 , Sylvia Prieto Gonzales 3 and Takuji Tanaka 1 1 Department of Oncologic Pathology, Kanazawa Medical University, Uchinada, Ishikawa, Japan 2 Dipartimento di Chimica e Tecnologia del Farmaco, Sezione di Chimica Organica, Universita` degli Studi, Perugia, Italy 3 Departamento de Sı`ntesis Quimica, Centro de Quı`mica Farmac eutica, Ciudad de la Habana, Cuba We previously reported the chemopreventive ability of a prenyloxy- coumarin 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 aur- aptene and its related prenyloxycoumarin collinin can inhibit coli- tis-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, cyclooxy- genase (COX)-2, inducible nitric oxide (iNOS) and nitrotyrosine in colonic epithelial malignancy. Feeding with auraptene or colli- nin, 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 in- creased 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-re- lated colon cancer development in rodents. ' 2006 Wiley-Liss, Inc. Key words: auraptene; azoxymethane; collinin; colitis-related carcino- genesis; dextran sodium sulfate Colorectal cancer (CRC) is one of the most serious complica- tions 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 carcino- genesis process are largely unclear, although it is generally as- sumed 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 inves- tigations suggest that several agents, such as folic acid, 4 conju- gated linoleic acid, 5 ursodeoxycholic acid, 6 5-aminosalicylic acid 7 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 rats 10 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 chemopre- ventive compounds against colon cancer. 14–17 Prenyloxycoumar- ins, including auraptene (Fig. 1a) and collinin (Fig. 1b), are candi- dates 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 signifi- cantly 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 E 2 (PGE 2 ), and yet suppressed the release of tumor necrosis factor (TNF)-a and IjB degradation. 19,20 Furthermore, auraptene and collinin also cause complete inhibition of platelet aggregation, in- duced by arachidonic acid and platelet activated factor in vitro. 21 We have previously found that a citrus auraptene suppresses chemically induced carcinogenesis in rodents. 22–24 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 chemopreven- tive agents against this type of malignancy, we developed a novel colitis-related mouse CRC model, using a colon carcinogen azoxy- methane (AOM) and DSS, in which large bowel adenocarcinomas develop within a short-term period, and their histology and biologi- cal 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 adenocarci- nomas. 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 Grant sponsor: Ministry of Health, Labour and Welfare, Japan; Grant number: Grant-in-Aid (13-15); Grant sponsor: Ministry of Education, Cul- ture, Sports, Science and Technology, Japan; Grant sponsor: Kanazawa Medical University; Grant number: C2005-3, H2005-6. *Correspondence to: Department of Oncologic Pathology, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293, Japan. Fax: 181-76-286-6926. E-mail: h-kohno@kanazawa-med.ac.jp Received 12 September 2005; Accepted after revision 21 October 2005 DOI 10.1002/ijc.21719 Published online 4 January 2006 in Wiley InterScience (www.interscience. wiley.com). Abbreviations: AOM, azoxymethane; CRC, colorectal cancer; COX, cyclooxygenase; DSS, dextran sodium sulfate; FAP, familial adenomatous polyposis; H & E, hematoxylin and eosin; IBD, inflammatory bowel dis- ease; iNOS, inducible nitric oxide synthase; NSAIDs, nonsteroidal anti- inflammatory drugs; PCNA, proliferating cell nuclear antigen; PGE 2 , pros- taglandin E 2 ; ssDNA, single stranded DNA; TNF, tumor necrosis factor; UC, ulcerative colitis. Int. J. Cancer: 118, 2936–2942 (2006) ' 2006 Wiley-Liss, Inc. Publication of the International Union Against Cancer