- Email: [email protected]
Animal Models for Colon Cancer Chemoprevention
Animal Models for Colon Cancer Chemoprevention Bandaru S. Reddy American Health Foundation, Valhalla, New York
lorectal cancer, the disease appears to result from an increase in the number of genetic mutations, mostly acquired, that accumulate in the genome of the evolving cancer cell. The histopathologic changes associated with the development of colorectal cancer are driven by the progressive accumulation of definable genetic changes, including the activation of one or more oncogenes plus the inactivation of several tumor suppressor genes and by endogenous and exogenous promoting agents. These genetic changes proceed from cellular hyperproliferation to small, benign adenomas to more dysplastic, larger adenomas and then can become cancer and ultimately metastasize. Identification and removal of these premalignant lesions are considered important in order to design a rational approach to reduce the incidence and mortality of colon cancer.
I. Introduction II. Preclinical Models for Colon Cancer III. Chemoprevention Studies Utilizing Preclinical Models IV. Conclusion
GLOSSARY cancer chemoprevention Intervention with chemical agents that may block the tumor initiation and promotion events that are the sequential stages of cancer development or delay the carcinogenic process. preclinical efficacy Studies involving the evaluation of agents in a realistic laboratory animal model for cancer indicating the inhibition of carcinogenesis
C olorectal cancer is a tumor of colon and rectum,
I. INTRODUCTION
which occurs with high frequency in both men and women in Western countries. Most cases of colorectal cancers arise in a benign adenoma; some evidence suggests that some cancers arise directly from the mucosal cells. With regard to genetic mechanisms of co-
Encyclopedia of Cancer, Second Edition Volume 1
In studies of various human diseases, it is critical that reliable animal models both chemically induced and transgenic are developed that demonstrate similarity
49
Copyright 2002, Elsevier Science (USA). All rights reserved.
50
ANIMAL MODELS FOR COLON CANCER CHEMOPREVENTION
to the human disease. Animal models are extremely valuable in our understanding of the human disease, but one has to be familiar with the limitations of the model system that is being used to study the human disease. Animal models should bear relevance to human colorectal cancer with similarities not only in terms of histopathology and molecular and genetic lesions during early and promotion/progression stages of carcinogenesis, but also adequacy of the model for prevention studies. The animal models should also reflect the efficacy of both effective and ineffective nutritional and chemopreventive agents that have been evaluated in humans. It should also be recognized that extrapolation of data obtained in animal model systems entails inherent sources of uncertainty that must be taken into account in predicting human responsiveness. This brief article discusses the carcinogeninduced colon cancer model to study the relationship between chemopreventive agents and colon carcinogenesis.
II. PRECLINICAL MODELS FOR COLON CANCER Animal models have been developed to study the multiple environmental factors involved in the pathogenesis of cancer of the colon. These animal models are (a) induction of colon tumors in rats through aromatic amines such as 3,2-dimethyl-4-aminobi-phenyl (DMBA); (b) derivatives and analogs of cycacin such as methyazoxymethanol (MAM), 1,2-dimethylhydrazine (DMH), and azoxymethane (AOM) in rats and mice of selected strains; (c) direct-acting carcinogens of the type of alkylureas, such as methylnitrosourea (MNU) or N-methyl-N`nitro-Nnitrosoguanidine (MNNG); and (d) heterocyclic amines such as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). The spectrum of epithelial lesions induced in the colon by these carcinogens is similar to various types of neoplastic lesions observed in the colorectum of humans. Several studies have utilized these relevant animal models to investigate the modulation of colon carcinogenesis by nutritional and chemopreventive agents.
A. Alkylnitrosoureido Compounds MNNG and MNU are direct alkylating agents, which do not require metabolic activation, and thus they are topical and potent carcinogens. Intrarectal instillation of NMU or MNNG induced colorectal tumors in rodent models. Because of the fact that biochemical activation is not required for these carcinogens, it is an ideal way of inducing colon tumors in animals and of studying modifying effects during the postinitiation stage of colon carcinogenesis without involving metabolism of the genotoxic, initiating carcinogen. Intrarectal administration of MNNG at a dose rate of 1–3 mg/rat/week for 20 weeks induced colon tumors in 100% of male F344 rats of which 43% tumors were adenocarcinomas and 57% were adenomas. The neoplasms were all located in the distal colon and rectum, as MNNG and MNU are locally acting carcinogens. No metastatic lesions were usually observed. Although MNNG and MNU given intrarectally provided the most reliable model for the topical and selective production of tumors in the distal colon and rectum, the major weakness of this model is that the technique of intrarectal injection requires highly skilled technicians and quantification of carcinogens instilled intrarectally is difficult.
B. Heterocyclic Amines 2-Amino-3,8-dimethylimidazo[4,5-f]quiniline (IQ), a heterocyclic aromatic amine produced from food pyrolysis, was first isolated from a variety of broiled or cooked fish and meat. Among a number of heterocyclic amines that have been demonstrated to be highly mutagenic and tumorigenic in rodent models, IQ and 2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine (PhIP) have attracted a lot of attention because they demonstrate a multitarget organospecificity with specific cancer induction in Zymbal gland, skin, colon, oral cavity, and mammary gland of rodents. Colon tumors were induced in male F344 rats by administering PhIP daily in the diet at 100 and 400 ppm for 52 and 104 weeks. Although the colon tumor incidences were about 43 and 55% in animals given PhIP at 100 and 400 ppm, respectively, there was severe toxicity due to PhIP. We have also utilized
ANIMAL MODELS FOR COLON CANCER CHEMOPREVENTION
heterocyclic amines such as IQ and PhIP to induce colon tumors. Results of these studies from our laboratory indicate that when these agents were administered daily in the diet for 52 weeks, the tumor incidence was very low, ranging between 5 and 28%.
C. 1,2-Dimethylhydrazine 1,2-Dimethylhydrazine is an effective carcinogen for the induction of tumors of the colon and rectum in rats and mice by systemic subcutaneous or intraperitoneal injections. The usefulness of this organospecific carcinogen, which induces selectively tumors in the colon, was confirmed by several laboratories. Despite the differences in doses, schedules, and animal strains used by different investigators, there is some consistency in their results in using DMH as a colon carcinogen. Subcutaneous injection of DMH at a dose rate of 20 mg/kg body weight, once weekly for 20 weeks induces colon adenomas and adenocarcinomas in about 60% of male F344 rats. However, the major weakness of this model is that multiple injections of DMH are required to induce colon tumors in the laboratory rodents.
D. Azoxymethane Azoxymethane, a metabolite of DMH, has been used extensively by many investigators to induce colon tumors and to study the effects of nutritional factors and chemopreventive agents in colon carcinogenesis. AOM is a potent inducer of carcinomas of the large intestine in various strains of male and female rats. We have used a two-dose (15 mg/kg body weight, once weekly for 2 weeks sc) regimen in all our chemoprevention and nutritional studies since mid-1985. Our results and those of others indicate that the justmentioned dose regimen in male F344 rats induced colon tumors in about 80% of animals with a mean of three tumors/rat after 40–50 weeks following the second AOM treatment. Endoscopic examination of animals revealed that the first endoscopically visible colonic tumor can be detected 15 weeks after the AOM treatment and that the mean latency period of such tumors is about 20 weeks. Of all the model systems, use of Fischer (F344) rats
51
and AOM seem to be appropriate because rat colons have light and electron microscopic morphology as well as histochemical properties that are quite similar to that of humans and biological behaviors of AOMinduced rat colon carcinomas have close similarity to those of human colon carcinomas. AOM-induced carcinomas metastasize to regional lymph nodes and liver, and these carcinomas are transplantable. Epithelial neoplasms of colon induced by AOM in F344 rats include both adenomas and adenocarcinomas. Based on our past experience with this model, about 70% of colon tumors are adenocarcinomas and the rest are adenomas. Histologically, adenomas of the colon are benign, with mild or moderate epithelial atypia. Malignant neoplasms of the colon (epithelial origin) are adenocarcinomas, the majority of which are well-differentiated, frank malignant tumors showing invasion across the line of the muscularis mucosa. Some of these are poorly differentiated, which are highly infiltrative, and often reach the intestinal wall and the serosa and may even invade the neighboring organs. In our past experience with this model, the extension of lesions into the adjacent peritoneal tissues can occur, and metastic lesions, if present, can be seen in the mesenteric lymph nodes, lung, or liver. Similar to the regional distribution of tumors in human colon, AOM treatment induces colonic tumors predominantly in the distal colon. AOM treatment also induces ras oncogene mutations at codon 12 of K- and H-ras and increases the expression of the ras family of protooncogenes that have been causally associated with colon tumor development. Enhanced ras oncogene expression has been observed in a variety of human colon tumors. AOM-induced colon tumors also demonstrate enhanced cyclooxygenase-2 and inducible nitric oxide synthase expression similar to human colonic tumors. Mutations in the tumor suppression gene, APC, are known to be early events in the colon cancer process in humans. APC gene mutations have been identified in patients with familial adenomatous polyposis, who have germline mutation in one of the APC alleles, and in sporadic colorectal cancer. Evidence in humans thus implicates the APC suppressor gene as causal in large bowel carcinogenesis. Studies indicating the presence of APC mutation in AOM-induced colon tumors in mice
52
ANIMAL MODELS FOR COLON CANCER CHEMOPREVENTION
strengthens the concept that these models are appropriate for human colon cancer studies.
III. CHEMOPREVENTION STUDIES UTILIZING PRECLINICAL MODELS The developmental strategies for chemoprevention have been markedly facilitated by the use of relevant animal models mimicking the neoplastic process that occurs in humans. The F344 rat model for AOMinduced colon cancer has been used extensively to obtain critical information on the chemopreventive efficacy of several agents.
A. Phytochemicals Several studies have demonstrated that generous consumption of vegetables reduces the risk of colon cancer. Although the nature of the constituents of these vegetables and other food items that are responsible for reduced risk has not been fully elucidated, it is clear that the plant foods contain chemopreventive agents, including several micronutrients, such as vitamins, and minerals and also contain nonnutrients, such as organosulfur compounds, polyphenols, and isoflavones, to cite a few. The diversity of these compounds is a positive feature, indicating that a variety of approaches to cancer prevention by these agents may be made so that the optimal selection will emerge. Mechanisms of chemopreventive activity of these
agents range from inhibition of carcinogen activation to detoxification of the carcinogen, blockage of binding of critical carcinogen metabolites to DNA, scavenging reactive electrophiles, and inhibiting arachidonic acid (AA) metabolism. Phytochemicals and their substituted and synthetic analogs tested for their efficacy in the AOM colon cancer model assay included anethole trithione, oltipraz [5-(2-pyrazinyl)-4methyl-1,2-dithiole-3-thione], diallyl sulfide, and curcumin, to cite a few. Administration of organosulfur compounds such as diallyl sulfide, oltipraz, or anethole trithione during the initiation and/or postinitiation stage significantly suppressed the incidence and multiplicity of AOMinduced colonic adenocarcinomas in male F344 rats (Table I). The inhibition of colon carcinogenesis by these agents was associated with an increase in the activities of detoxifying enzymes, such as glutathione S-transferase, quinone reductase, and UDPglutathione transferase in the colonic mucosa and tumors. Curcumin [diferuloylmethane; 1,7-bis-(4-hydroxy3-methoxyphenyl)-1,6-heptadiene-3,5-dione)], which has been identified as the major pigment in turmeric, the powdered rhizome of Curcuma longa Lim, possesses both anti-inflammatory and antioxidant properties. Importantly, dietary administration of curcumin inhibited AOM-induced colon tumor incidence and multiplicity in a dose-dependent manner (Table I). Curcumin, given as a dietary supplement during the promotion/progression period, dramati-
TABLE I Chemopreventive Efficacy of Naturally Occurring and Synthetic Agents against Azoxymethane-Induced Colon Carcinogenesis in F344 Rats Chemopreventive agent
Dosage g/g diet (ppm)
% Inhibition
Oltipraz Curcumin Aspirin Ibuprofen Sulindac Piroxicam Celecoxib Piroxicam DL-difluoromethylornithine 1,4-Phenylenebis(methylene)selenocyanate
200 2000 200 400 320 400 1500 150 1000 20
35 42 32 45 55 64 96 80 42
ANIMAL MODELS FOR COLON CANCER CHEMOPREVENTION
cally inhibited colon tumorigenesis, suggesting that curcumin may retard growth and/or the development of existing neoplastic lesions in the colon and that this agent may be an effective chemopreventive agent for individuals at high risk for colon cancer development, such as patients with polyps.
B. Nonsteroidal Anti-inflammatory Drugs In recent years, attention has been drawn to the potential chemopreventive properties of nonsteroidal anti-inflammatory drugs (NSAIDs). Several casecontrol and cohort studies have provided unequivocal evidence for the inverse relationship between colon cancer and the use of NSAIDs, specifically aspirin. There has been ample and consistent experimental evidence from laboratory animal model studies to indicate that NSAIDs, including indomethacin, piroxicam, sulindac, aspirin, ibuprofen, and ketoprofen, inhibit chemically induced colon cancer (Table I). More importantly, piroxicam and sulindac administered during the promotion/progression stage significantly inhibit colon tumorigenesis. Results generated in this preclinical model assay provided baseline information for eventual clinical evaluation of the efficacy of NSAIDs in the late intervention/prevention protocols of colonic tumors in high-risk individuals, such as patients with sporadic colonic polyps of familial adenomatous polyposis (FAP). One of the mechanisms by which NSAIDs inhibit colon cancer is through the modulation of cyclooxygenase 1 and 2 (COX-1 and COX-2), which leads to a reduction of eicosanoid production, which in turn affects cell proliferation and tumor growth. However, these drugs can cause unwanted side effects, including gastrointestinal ulceration, bleeding, and renal toxicity, through the inhibition of constitutive COX-1 activity. Overexpression of COX-2 has been observed in colon tumors, and many commonly used NSAIDs have very little selectivity for COX-1 or COX-2; therefore, more specific yet minimally toxic inhibitors of COX-2 were developed and tested for chemopreventive efficacy. Celecoxib, a selective COX-2 inhibitor that induces very few toxic side effects, has been found to be significantly more effective than the commonly used NSAIDs in the chemoprevention of colon carcinogenesis in laboratory animal models; it
53
may thus be an effective chemopreventive agent against colon cancer (Table I). A recent clinical trial has shown a reduction in adenomas in patients with FAP-administered celecoxib.
C. Combinations of Low Doses of Various Chemopreventive Agents There is increasing interest in the use of combinations of low doses of chemopreventive agents that differ in mode of action, rather than administering single agents as a means of obtaining increased efficacy and minimized toxicity. This approach is extremely important when a promising chemopreventive agent demonstrates apparent efficacy but may produce toxic effects at higher doses. An example of combination of agents producing positive results in laboratory animal models has been a study in which the NSAID piroxicam and DFMO, a specific irreversible enzymeactivated or suicide inhibitor of ornithine decarboxylase (ODC), were evaluated for their chemopreventive efficacy. An important finding of the study was that the lowest dose levels of piroxicam (100 ppm) and DFMO (1000 ppm), when administered together, were more effective in inhibiting the incidence and multiplicity of colon adenocarcinomas than administration of these compounds as single agents, even at higher levels. These data strongly support the view that the use of combinations of chemopreventive agents having diverse actions should have beneficial applications in human cancer chemoprevention trials. This should be one of the approaches used in future research and human intervention trials.
D. Organoselenium Epidemiological studies have also pointed to an inverse association between dietary selenium intake and colon cancer risk in humans. A randomized clinical trial demonstrated that supplementation of seleniumenriched brewer’s yeast reduced the incidence and mortality from cancer of the colon. This finding was corroborated by studies with selenium supplementation of the diet in chemically induced colon carcinogenesis in laboratory animals. Humans ingest primarily organic forms of selenium, such as selenomethionine and selenocysteine, by eating grains,
54
ANIMAL MODELS FOR COLON CANCER CHEMOPREVENTION
vegetables, and animal products. Chronic feeding of inorganic and certain organic forms of selenium at levels 5 ppm produced toxic effects. Therefore, substantial efforts were made to find and/or develop forms of organic selenium compounds that have the maximal chemopreventive efficacy and lowest possible toxicity. Studies in our laboratory have indicated that certain synthetic organoselenium compounds, such as 1,4-phenyulenebis(methylene)selenocyanate (p-XSC), hold great promise as chemopreventive agents because they have been found to be superior to historically used selenium compounds, such as sodium selenite and selenomethionine. More importantly, the chemopreventive efficacy of this agent is more pronounced when given along with a low-fat diet, thus making a strong case for the use of low-fat dietary regimens along with a chemopreventive agent as a desirable approach for primary prevention in the general population and for secondary prevention of colon cancer in high-risk individuals.
IV. CONCLUSION An impressive body of observation supports the concept that chemoprevention has the potential to be a major component of colon cancer prevention and control. Accumulating evidence indicates that several NSAIDs, including aspirin, piroxicam, and sulindac, can reduce the incidence of colon cancer in laboratory animals and in humans. Celecoxib, a selective COX-2 inhibitor that induces very few toxic side effects as compared to traditional NSAIDs, has been found to be more effective than the commonly used NSAIDs against colon carcinogenesis in laboratory animal models. Studies have also indicated that the synthetic organoselenium compound p-XSC holds great promise as a chemopreventive agent because its chemopreventive index is higher than inorganic and naturally occurring organic forms of selenium. Growing knowledge of the mechanisms by which chemopreventive agents act offers opportunities to use combinations of specific chemopreventive agents, the aggregate action of which would be clinically beneficial while toxicity would be minimal. How to best use such knowledge toward prevention and control of colorectal cancer is a primary challenge for the future.
See Also the Following Articles CHEMOPREVENTION, PRINCIPLES OF • CHEMOPREVENTION TRIALS • COLORECTAL CANCER: EPIDEMIOLOGY AND TREATMENT • HEREDITARY COLON CANCER AND DNA MISMATCH REPAIR • NUTRITIONAL SUPPLEMENTS AND DIET AS CHEMOPREVENTIVE AGENTS • TRANSGENIC MICE IN CANCER RESEARCH
Bibliography Clark, L. C., Combs, G. F., Turnbull, B. W., State, E. H., Chalker, D. K., Chow, J., Gover, R. A., Graham, G. F., Gross, E. G., Krongard, A., Lesher, J. L., Park, K., Sanders, B. S., Smith, C. L., and Taylor, J. R. (1996). Effect of selenium supplementation for cancer prevention in patients with carcinoma of the skin: A randomized controlled trial. J. Am. Med. Assoc. 276, 1957–1963. Dubois, R. N., Radhika, A., Reddy, B. S., and Entingh, A. J. (1996). Increased cycloozygenase-2 levels in carcinogeninduced rat colonic tumors. Gastroenterology 110, 1259–1262. Elwell, M. R., and McConnell, E. S. (1990). Small and large intestine. In “Pathology of Fischer Rat” (G. A. Boorman, S. L. Eustis, M. R. Wlwell, C. A. Montgomer, and Mackenzie, eds.), p. 43 Academic Press, San Diego. Holt, P. R., Mokuolu, A. O., Distler, P., Liu, T., and Reddy, B. S. (1996). Regional distribution of carcinogen-induced colon neoplasia in the rat. Nutr. Cancer 25, 129–135. Ito, N., Hasegawa, R., Sano, S., Tamano, S., Esumi, H., Takayama, S., and Sugimura, T. (1991). A new colon and mammary carcinogen in cooked food, 2-amino-1-methyl-6phenylimidazo-[4,5-b]pyridine (PhIP). Carcinogenesis 12, 1503–1506. Kawamori, T., Lubet, R., Steele, V. E., Kelloff, G. J., Kaskey, R. B., Rao, C. V., and Reddy, B. S. (1999). Chemopreventive effect of curcumin, a naturally-occurring antiinflammatory agent, during the promotion/progression stages of colon cancer. Cancer Res. 59, 597–601. Kawamori, T., Rao, C. V., Siebert, K., and Reddy, B. S. (1998). Chemopreventive activity of celecoxib, a specific cyclooxygenase-2 inhibitor, against colon carcinogenesis. Cancer Res. 58, 409–412. Kensler, T. W., Tsuda, H., and Wogan, G. N. (1999). United States–Japan workshop on new rodent models for the analysis and prevention of carcinogenesis. Cancer Epidemiol. Biomark. Prevent. 8, 1033–1037. Kune, G. A., Kune, S., and Watson, L. F. (1988). Colorectal cancer risk, chronic illness, operations and mediations: Case-control results from the Melbourne Colorectal Cancer Study. Cancer Res. 48, 4399–4404. Pozharisski, K. M. (1990). Tumors of the intestines. In “Pathology of Tumors in Laboratory Animals” (V. Turuso, and U. Mohr, Eds.), Vol. 1, pp. 159–198. IARC Scientific Publication No. 99, Lyon, France.
ANIMAL MODELS FOR COLON CANCER CHEMOPREVENTION
Rao, C. V., Rivenson, A., Simi, B., and Reddy, B. S. (1995). Chemoprevention of colon carcinogenesis by dietary curcumin, a naturally occurring plant phenolic compound. Cancer Res. 55, 2219–2225. Rao, C. V., Rivenson, A., Simi, B., Zang, E., Kelloff, G., Steele, V., and Reddy, B. S. (1995). Chemoprevention of colon carcinogenesis by sulindac, a non-steroidal antiinflammatory agent. Cancer Res. 55, 1464–1472. Rao, C. V., Tokumo, K., Kelloff, G., and Reddy, B. S. (1991). Inhibition by dietary oltipraz of experimental intestinal carcinogenesis induced by azoxymethane in male F344 rats. Carcinogen. (Lond.) 12, 1051–1056. Reddy, B. S., Hirose, Y., Lubet, R. A., Steele, V. E., Kelloff, G. J., Paulson, S., Siebert, K., and Rao, C. V. (2000). Chemoprevention of colon cancer by specific cyclooxygenase-2 inhibitor, celecoxib, administered during different stages of carcinogenesis. Cancer Res. 60, 293–297. Reddy, B. S., Maruyama, H., and Kelloff, G. J. (1987). Doserelated inhibition of colon carcinogenesis by dietary piroxicam, a nonsteroidal anti-inflammatory drug, during different stages of rat colon tumor development. Cancer Res. 47, 5340–5346. Reddy, B. S., Narisawa, T., Maronpot, R., and Weisburger, J. (1975). Animal models for the study of dietary factors and cancer of the large bowel. Cancer Res. 35, 3421–3426. Reddy, B. S., Nayini, J., Tokumo, K., Rigotty, J., Zange, E., and Kelloff, G. (1990). Chemoprevention of colon carcinogenesis by concurrent administration of piroxicam, a nonsteroidal antiinflammatory drug, with D,L--difluoromethylornithine, an ornithine decarboxylase inhibitor in diet. Cancer Res. 50, 2562–2568. Reddy, B. S., Rao, C. V., Rivenson, A., and Kelloff, G. J. (1993). Inhibitory effect of aspirin on azoxymethaneinduced colon carcinogenesis in F344 rats. Carcinogen. (Lond.) 14, 1493–1497.
55
Reddy, B. S., Rao, C. V., Rivenson, A., and Kelloff, G. (1997). Chemoprevention of colon carcinogenesis by organosulfur compounds. Cancer Res. 53, 3494–3498. Reddy, B. S., and Rivenson, A. (1993). Inhibitory effect of Bifidobacterium longum on colon, mammary, and liver carcinogenesis induced by 2-amino-3-methylimidazo[4,5-f ] quinoline, a food mutagen. Cancer Res. 53, 3914–3918. Reddy, B. S., Rivenson, A., El-Bayoumy, K., Upadhyaya, P., Pittman, B., and Rao, C. V. (1997). Chemoprevention of colon cancer by synthetic organoselenium compounds, 1,4phenylenebis(methylene)selenocyanate and p-methoxy benzyl selenocyanate, in low and/or high fat fed F344 rats. J. Natl. Cancer Inst. 89, 506–516. Reddy, B. S., Tokumo, K., Kulkarni, N., Aliga, C., and Kelloff, G. (1992). Inhibition of colon carcinogenesis by prostaglandin synthesis inhibitors and related compounds. Carcinogenesis 13, 1019–1023. Reddy, B. S., Wantanabe, K., and Weisburger, J. H. (1997). Effect of high-fat diet on colon carcinogenesis in F344 rats treated with 1,2-dimethylhydrazine, methylazoxymethanol acetate, or methylnitrosourea. Cancer Res. 37, 4156–4158. Singh, J., Hamid, R., and Reddy, B. S. (1997). Dietary fat and colon cancer: Modulation of cyclooxygenase-2 by types and amount of dietary fat during the postinitiation stage of colon carcinogenesis. Cancer Res. 57, 3465–3470. Singh, J., Kulkarni, N., Kelloff, G., and Reddy, B. S. (1994). Modulation of azoxymethane-induced mutational activation of ras protooncogens of chemopreventive agents in colon carcinogenesis. Carcinogenesis 15, 1317–1323. Thun, M. H., Namboodiri, M. M., and Heath, C. W. (1991). Aspirin use and reduced risk of fatal colon cancer. N. Engl. J. Med. 325, 1593–1596.
lorectal cancer, the disease appears to result from an increase in the number of genetic mutations, mostly acquired, that accumulate in the genome of the evolving cancer cell. The histopathologic changes associated with the development of colorectal cancer are driven by the progressive accumulation of definable genetic changes, including the activation of one or more oncogenes plus the inactivation of several tumor suppressor genes and by endogenous and exogenous promoting agents. These genetic changes proceed from cellular hyperproliferation to small, benign adenomas to more dysplastic, larger adenomas and then can become cancer and ultimately metastasize. Identification and removal of these premalignant lesions are considered important in order to design a rational approach to reduce the incidence and mortality of colon cancer.
I. Introduction II. Preclinical Models for Colon Cancer III. Chemoprevention Studies Utilizing Preclinical Models IV. Conclusion
GLOSSARY cancer chemoprevention Intervention with chemical agents that may block the tumor initiation and promotion events that are the sequential stages of cancer development or delay the carcinogenic process. preclinical efficacy Studies involving the evaluation of agents in a realistic laboratory animal model for cancer indicating the inhibition of carcinogenesis
C olorectal cancer is a tumor of colon and rectum,
I. INTRODUCTION
which occurs with high frequency in both men and women in Western countries. Most cases of colorectal cancers arise in a benign adenoma; some evidence suggests that some cancers arise directly from the mucosal cells. With regard to genetic mechanisms of co-
Encyclopedia of Cancer, Second Edition Volume 1
In studies of various human diseases, it is critical that reliable animal models both chemically induced and transgenic are developed that demonstrate similarity
49
Copyright 2002, Elsevier Science (USA). All rights reserved.
50
ANIMAL MODELS FOR COLON CANCER CHEMOPREVENTION
to the human disease. Animal models are extremely valuable in our understanding of the human disease, but one has to be familiar with the limitations of the model system that is being used to study the human disease. Animal models should bear relevance to human colorectal cancer with similarities not only in terms of histopathology and molecular and genetic lesions during early and promotion/progression stages of carcinogenesis, but also adequacy of the model for prevention studies. The animal models should also reflect the efficacy of both effective and ineffective nutritional and chemopreventive agents that have been evaluated in humans. It should also be recognized that extrapolation of data obtained in animal model systems entails inherent sources of uncertainty that must be taken into account in predicting human responsiveness. This brief article discusses the carcinogeninduced colon cancer model to study the relationship between chemopreventive agents and colon carcinogenesis.
II. PRECLINICAL MODELS FOR COLON CANCER Animal models have been developed to study the multiple environmental factors involved in the pathogenesis of cancer of the colon. These animal models are (a) induction of colon tumors in rats through aromatic amines such as 3,2-dimethyl-4-aminobi-phenyl (DMBA); (b) derivatives and analogs of cycacin such as methyazoxymethanol (MAM), 1,2-dimethylhydrazine (DMH), and azoxymethane (AOM) in rats and mice of selected strains; (c) direct-acting carcinogens of the type of alkylureas, such as methylnitrosourea (MNU) or N-methyl-N`nitro-Nnitrosoguanidine (MNNG); and (d) heterocyclic amines such as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). The spectrum of epithelial lesions induced in the colon by these carcinogens is similar to various types of neoplastic lesions observed in the colorectum of humans. Several studies have utilized these relevant animal models to investigate the modulation of colon carcinogenesis by nutritional and chemopreventive agents.
A. Alkylnitrosoureido Compounds MNNG and MNU are direct alkylating agents, which do not require metabolic activation, and thus they are topical and potent carcinogens. Intrarectal instillation of NMU or MNNG induced colorectal tumors in rodent models. Because of the fact that biochemical activation is not required for these carcinogens, it is an ideal way of inducing colon tumors in animals and of studying modifying effects during the postinitiation stage of colon carcinogenesis without involving metabolism of the genotoxic, initiating carcinogen. Intrarectal administration of MNNG at a dose rate of 1–3 mg/rat/week for 20 weeks induced colon tumors in 100% of male F344 rats of which 43% tumors were adenocarcinomas and 57% were adenomas. The neoplasms were all located in the distal colon and rectum, as MNNG and MNU are locally acting carcinogens. No metastatic lesions were usually observed. Although MNNG and MNU given intrarectally provided the most reliable model for the topical and selective production of tumors in the distal colon and rectum, the major weakness of this model is that the technique of intrarectal injection requires highly skilled technicians and quantification of carcinogens instilled intrarectally is difficult.
B. Heterocyclic Amines 2-Amino-3,8-dimethylimidazo[4,5-f]quiniline (IQ), a heterocyclic aromatic amine produced from food pyrolysis, was first isolated from a variety of broiled or cooked fish and meat. Among a number of heterocyclic amines that have been demonstrated to be highly mutagenic and tumorigenic in rodent models, IQ and 2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine (PhIP) have attracted a lot of attention because they demonstrate a multitarget organospecificity with specific cancer induction in Zymbal gland, skin, colon, oral cavity, and mammary gland of rodents. Colon tumors were induced in male F344 rats by administering PhIP daily in the diet at 100 and 400 ppm for 52 and 104 weeks. Although the colon tumor incidences were about 43 and 55% in animals given PhIP at 100 and 400 ppm, respectively, there was severe toxicity due to PhIP. We have also utilized
ANIMAL MODELS FOR COLON CANCER CHEMOPREVENTION
heterocyclic amines such as IQ and PhIP to induce colon tumors. Results of these studies from our laboratory indicate that when these agents were administered daily in the diet for 52 weeks, the tumor incidence was very low, ranging between 5 and 28%.
C. 1,2-Dimethylhydrazine 1,2-Dimethylhydrazine is an effective carcinogen for the induction of tumors of the colon and rectum in rats and mice by systemic subcutaneous or intraperitoneal injections. The usefulness of this organospecific carcinogen, which induces selectively tumors in the colon, was confirmed by several laboratories. Despite the differences in doses, schedules, and animal strains used by different investigators, there is some consistency in their results in using DMH as a colon carcinogen. Subcutaneous injection of DMH at a dose rate of 20 mg/kg body weight, once weekly for 20 weeks induces colon adenomas and adenocarcinomas in about 60% of male F344 rats. However, the major weakness of this model is that multiple injections of DMH are required to induce colon tumors in the laboratory rodents.
D. Azoxymethane Azoxymethane, a metabolite of DMH, has been used extensively by many investigators to induce colon tumors and to study the effects of nutritional factors and chemopreventive agents in colon carcinogenesis. AOM is a potent inducer of carcinomas of the large intestine in various strains of male and female rats. We have used a two-dose (15 mg/kg body weight, once weekly for 2 weeks sc) regimen in all our chemoprevention and nutritional studies since mid-1985. Our results and those of others indicate that the justmentioned dose regimen in male F344 rats induced colon tumors in about 80% of animals with a mean of three tumors/rat after 40–50 weeks following the second AOM treatment. Endoscopic examination of animals revealed that the first endoscopically visible colonic tumor can be detected 15 weeks after the AOM treatment and that the mean latency period of such tumors is about 20 weeks. Of all the model systems, use of Fischer (F344) rats
51
and AOM seem to be appropriate because rat colons have light and electron microscopic morphology as well as histochemical properties that are quite similar to that of humans and biological behaviors of AOMinduced rat colon carcinomas have close similarity to those of human colon carcinomas. AOM-induced carcinomas metastasize to regional lymph nodes and liver, and these carcinomas are transplantable. Epithelial neoplasms of colon induced by AOM in F344 rats include both adenomas and adenocarcinomas. Based on our past experience with this model, about 70% of colon tumors are adenocarcinomas and the rest are adenomas. Histologically, adenomas of the colon are benign, with mild or moderate epithelial atypia. Malignant neoplasms of the colon (epithelial origin) are adenocarcinomas, the majority of which are well-differentiated, frank malignant tumors showing invasion across the line of the muscularis mucosa. Some of these are poorly differentiated, which are highly infiltrative, and often reach the intestinal wall and the serosa and may even invade the neighboring organs. In our past experience with this model, the extension of lesions into the adjacent peritoneal tissues can occur, and metastic lesions, if present, can be seen in the mesenteric lymph nodes, lung, or liver. Similar to the regional distribution of tumors in human colon, AOM treatment induces colonic tumors predominantly in the distal colon. AOM treatment also induces ras oncogene mutations at codon 12 of K- and H-ras and increases the expression of the ras family of protooncogenes that have been causally associated with colon tumor development. Enhanced ras oncogene expression has been observed in a variety of human colon tumors. AOM-induced colon tumors also demonstrate enhanced cyclooxygenase-2 and inducible nitric oxide synthase expression similar to human colonic tumors. Mutations in the tumor suppression gene, APC, are known to be early events in the colon cancer process in humans. APC gene mutations have been identified in patients with familial adenomatous polyposis, who have germline mutation in one of the APC alleles, and in sporadic colorectal cancer. Evidence in humans thus implicates the APC suppressor gene as causal in large bowel carcinogenesis. Studies indicating the presence of APC mutation in AOM-induced colon tumors in mice
52
ANIMAL MODELS FOR COLON CANCER CHEMOPREVENTION
strengthens the concept that these models are appropriate for human colon cancer studies.
III. CHEMOPREVENTION STUDIES UTILIZING PRECLINICAL MODELS The developmental strategies for chemoprevention have been markedly facilitated by the use of relevant animal models mimicking the neoplastic process that occurs in humans. The F344 rat model for AOMinduced colon cancer has been used extensively to obtain critical information on the chemopreventive efficacy of several agents.
A. Phytochemicals Several studies have demonstrated that generous consumption of vegetables reduces the risk of colon cancer. Although the nature of the constituents of these vegetables and other food items that are responsible for reduced risk has not been fully elucidated, it is clear that the plant foods contain chemopreventive agents, including several micronutrients, such as vitamins, and minerals and also contain nonnutrients, such as organosulfur compounds, polyphenols, and isoflavones, to cite a few. The diversity of these compounds is a positive feature, indicating that a variety of approaches to cancer prevention by these agents may be made so that the optimal selection will emerge. Mechanisms of chemopreventive activity of these
agents range from inhibition of carcinogen activation to detoxification of the carcinogen, blockage of binding of critical carcinogen metabolites to DNA, scavenging reactive electrophiles, and inhibiting arachidonic acid (AA) metabolism. Phytochemicals and their substituted and synthetic analogs tested for their efficacy in the AOM colon cancer model assay included anethole trithione, oltipraz [5-(2-pyrazinyl)-4methyl-1,2-dithiole-3-thione], diallyl sulfide, and curcumin, to cite a few. Administration of organosulfur compounds such as diallyl sulfide, oltipraz, or anethole trithione during the initiation and/or postinitiation stage significantly suppressed the incidence and multiplicity of AOMinduced colonic adenocarcinomas in male F344 rats (Table I). The inhibition of colon carcinogenesis by these agents was associated with an increase in the activities of detoxifying enzymes, such as glutathione S-transferase, quinone reductase, and UDPglutathione transferase in the colonic mucosa and tumors. Curcumin [diferuloylmethane; 1,7-bis-(4-hydroxy3-methoxyphenyl)-1,6-heptadiene-3,5-dione)], which has been identified as the major pigment in turmeric, the powdered rhizome of Curcuma longa Lim, possesses both anti-inflammatory and antioxidant properties. Importantly, dietary administration of curcumin inhibited AOM-induced colon tumor incidence and multiplicity in a dose-dependent manner (Table I). Curcumin, given as a dietary supplement during the promotion/progression period, dramati-
TABLE I Chemopreventive Efficacy of Naturally Occurring and Synthetic Agents against Azoxymethane-Induced Colon Carcinogenesis in F344 Rats Chemopreventive agent
Dosage g/g diet (ppm)
% Inhibition
Oltipraz Curcumin Aspirin Ibuprofen Sulindac Piroxicam Celecoxib Piroxicam DL-difluoromethylornithine 1,4-Phenylenebis(methylene)selenocyanate
200 2000 200 400 320 400 1500 150 1000 20
35 42 32 45 55 64 96 80 42
ANIMAL MODELS FOR COLON CANCER CHEMOPREVENTION
cally inhibited colon tumorigenesis, suggesting that curcumin may retard growth and/or the development of existing neoplastic lesions in the colon and that this agent may be an effective chemopreventive agent for individuals at high risk for colon cancer development, such as patients with polyps.
B. Nonsteroidal Anti-inflammatory Drugs In recent years, attention has been drawn to the potential chemopreventive properties of nonsteroidal anti-inflammatory drugs (NSAIDs). Several casecontrol and cohort studies have provided unequivocal evidence for the inverse relationship between colon cancer and the use of NSAIDs, specifically aspirin. There has been ample and consistent experimental evidence from laboratory animal model studies to indicate that NSAIDs, including indomethacin, piroxicam, sulindac, aspirin, ibuprofen, and ketoprofen, inhibit chemically induced colon cancer (Table I). More importantly, piroxicam and sulindac administered during the promotion/progression stage significantly inhibit colon tumorigenesis. Results generated in this preclinical model assay provided baseline information for eventual clinical evaluation of the efficacy of NSAIDs in the late intervention/prevention protocols of colonic tumors in high-risk individuals, such as patients with sporadic colonic polyps of familial adenomatous polyposis (FAP). One of the mechanisms by which NSAIDs inhibit colon cancer is through the modulation of cyclooxygenase 1 and 2 (COX-1 and COX-2), which leads to a reduction of eicosanoid production, which in turn affects cell proliferation and tumor growth. However, these drugs can cause unwanted side effects, including gastrointestinal ulceration, bleeding, and renal toxicity, through the inhibition of constitutive COX-1 activity. Overexpression of COX-2 has been observed in colon tumors, and many commonly used NSAIDs have very little selectivity for COX-1 or COX-2; therefore, more specific yet minimally toxic inhibitors of COX-2 were developed and tested for chemopreventive efficacy. Celecoxib, a selective COX-2 inhibitor that induces very few toxic side effects, has been found to be significantly more effective than the commonly used NSAIDs in the chemoprevention of colon carcinogenesis in laboratory animal models; it
53
may thus be an effective chemopreventive agent against colon cancer (Table I). A recent clinical trial has shown a reduction in adenomas in patients with FAP-administered celecoxib.
C. Combinations of Low Doses of Various Chemopreventive Agents There is increasing interest in the use of combinations of low doses of chemopreventive agents that differ in mode of action, rather than administering single agents as a means of obtaining increased efficacy and minimized toxicity. This approach is extremely important when a promising chemopreventive agent demonstrates apparent efficacy but may produce toxic effects at higher doses. An example of combination of agents producing positive results in laboratory animal models has been a study in which the NSAID piroxicam and DFMO, a specific irreversible enzymeactivated or suicide inhibitor of ornithine decarboxylase (ODC), were evaluated for their chemopreventive efficacy. An important finding of the study was that the lowest dose levels of piroxicam (100 ppm) and DFMO (1000 ppm), when administered together, were more effective in inhibiting the incidence and multiplicity of colon adenocarcinomas than administration of these compounds as single agents, even at higher levels. These data strongly support the view that the use of combinations of chemopreventive agents having diverse actions should have beneficial applications in human cancer chemoprevention trials. This should be one of the approaches used in future research and human intervention trials.
D. Organoselenium Epidemiological studies have also pointed to an inverse association between dietary selenium intake and colon cancer risk in humans. A randomized clinical trial demonstrated that supplementation of seleniumenriched brewer’s yeast reduced the incidence and mortality from cancer of the colon. This finding was corroborated by studies with selenium supplementation of the diet in chemically induced colon carcinogenesis in laboratory animals. Humans ingest primarily organic forms of selenium, such as selenomethionine and selenocysteine, by eating grains,
54
ANIMAL MODELS FOR COLON CANCER CHEMOPREVENTION
vegetables, and animal products. Chronic feeding of inorganic and certain organic forms of selenium at levels 5 ppm produced toxic effects. Therefore, substantial efforts were made to find and/or develop forms of organic selenium compounds that have the maximal chemopreventive efficacy and lowest possible toxicity. Studies in our laboratory have indicated that certain synthetic organoselenium compounds, such as 1,4-phenyulenebis(methylene)selenocyanate (p-XSC), hold great promise as chemopreventive agents because they have been found to be superior to historically used selenium compounds, such as sodium selenite and selenomethionine. More importantly, the chemopreventive efficacy of this agent is more pronounced when given along with a low-fat diet, thus making a strong case for the use of low-fat dietary regimens along with a chemopreventive agent as a desirable approach for primary prevention in the general population and for secondary prevention of colon cancer in high-risk individuals.
IV. CONCLUSION An impressive body of observation supports the concept that chemoprevention has the potential to be a major component of colon cancer prevention and control. Accumulating evidence indicates that several NSAIDs, including aspirin, piroxicam, and sulindac, can reduce the incidence of colon cancer in laboratory animals and in humans. Celecoxib, a selective COX-2 inhibitor that induces very few toxic side effects as compared to traditional NSAIDs, has been found to be more effective than the commonly used NSAIDs against colon carcinogenesis in laboratory animal models. Studies have also indicated that the synthetic organoselenium compound p-XSC holds great promise as a chemopreventive agent because its chemopreventive index is higher than inorganic and naturally occurring organic forms of selenium. Growing knowledge of the mechanisms by which chemopreventive agents act offers opportunities to use combinations of specific chemopreventive agents, the aggregate action of which would be clinically beneficial while toxicity would be minimal. How to best use such knowledge toward prevention and control of colorectal cancer is a primary challenge for the future.
See Also the Following Articles CHEMOPREVENTION, PRINCIPLES OF • CHEMOPREVENTION TRIALS • COLORECTAL CANCER: EPIDEMIOLOGY AND TREATMENT • HEREDITARY COLON CANCER AND DNA MISMATCH REPAIR • NUTRITIONAL SUPPLEMENTS AND DIET AS CHEMOPREVENTIVE AGENTS • TRANSGENIC MICE IN CANCER RESEARCH
Bibliography Clark, L. C., Combs, G. F., Turnbull, B. W., State, E. H., Chalker, D. K., Chow, J., Gover, R. A., Graham, G. F., Gross, E. G., Krongard, A., Lesher, J. L., Park, K., Sanders, B. S., Smith, C. L., and Taylor, J. R. (1996). Effect of selenium supplementation for cancer prevention in patients with carcinoma of the skin: A randomized controlled trial. J. Am. Med. Assoc. 276, 1957–1963. Dubois, R. N., Radhika, A., Reddy, B. S., and Entingh, A. J. (1996). Increased cycloozygenase-2 levels in carcinogeninduced rat colonic tumors. Gastroenterology 110, 1259–1262. Elwell, M. R., and McConnell, E. S. (1990). Small and large intestine. In “Pathology of Fischer Rat” (G. A. Boorman, S. L. Eustis, M. R. Wlwell, C. A. Montgomer, and Mackenzie, eds.), p. 43 Academic Press, San Diego. Holt, P. R., Mokuolu, A. O., Distler, P., Liu, T., and Reddy, B. S. (1996). Regional distribution of carcinogen-induced colon neoplasia in the rat. Nutr. Cancer 25, 129–135. Ito, N., Hasegawa, R., Sano, S., Tamano, S., Esumi, H., Takayama, S., and Sugimura, T. (1991). A new colon and mammary carcinogen in cooked food, 2-amino-1-methyl-6phenylimidazo-[4,5-b]pyridine (PhIP). Carcinogenesis 12, 1503–1506. Kawamori, T., Lubet, R., Steele, V. E., Kelloff, G. J., Kaskey, R. B., Rao, C. V., and Reddy, B. S. (1999). Chemopreventive effect of curcumin, a naturally-occurring antiinflammatory agent, during the promotion/progression stages of colon cancer. Cancer Res. 59, 597–601. Kawamori, T., Rao, C. V., Siebert, K., and Reddy, B. S. (1998). Chemopreventive activity of celecoxib, a specific cyclooxygenase-2 inhibitor, against colon carcinogenesis. Cancer Res. 58, 409–412. Kensler, T. W., Tsuda, H., and Wogan, G. N. (1999). United States–Japan workshop on new rodent models for the analysis and prevention of carcinogenesis. Cancer Epidemiol. Biomark. Prevent. 8, 1033–1037. Kune, G. A., Kune, S., and Watson, L. F. (1988). Colorectal cancer risk, chronic illness, operations and mediations: Case-control results from the Melbourne Colorectal Cancer Study. Cancer Res. 48, 4399–4404. Pozharisski, K. M. (1990). Tumors of the intestines. In “Pathology of Tumors in Laboratory Animals” (V. Turuso, and U. Mohr, Eds.), Vol. 1, pp. 159–198. IARC Scientific Publication No. 99, Lyon, France.
ANIMAL MODELS FOR COLON CANCER CHEMOPREVENTION
Rao, C. V., Rivenson, A., Simi, B., and Reddy, B. S. (1995). Chemoprevention of colon carcinogenesis by dietary curcumin, a naturally occurring plant phenolic compound. Cancer Res. 55, 2219–2225. Rao, C. V., Rivenson, A., Simi, B., Zang, E., Kelloff, G., Steele, V., and Reddy, B. S. (1995). Chemoprevention of colon carcinogenesis by sulindac, a non-steroidal antiinflammatory agent. Cancer Res. 55, 1464–1472. Rao, C. V., Tokumo, K., Kelloff, G., and Reddy, B. S. (1991). Inhibition by dietary oltipraz of experimental intestinal carcinogenesis induced by azoxymethane in male F344 rats. Carcinogen. (Lond.) 12, 1051–1056. Reddy, B. S., Hirose, Y., Lubet, R. A., Steele, V. E., Kelloff, G. J., Paulson, S., Siebert, K., and Rao, C. V. (2000). Chemoprevention of colon cancer by specific cyclooxygenase-2 inhibitor, celecoxib, administered during different stages of carcinogenesis. Cancer Res. 60, 293–297. Reddy, B. S., Maruyama, H., and Kelloff, G. J. (1987). Doserelated inhibition of colon carcinogenesis by dietary piroxicam, a nonsteroidal anti-inflammatory drug, during different stages of rat colon tumor development. Cancer Res. 47, 5340–5346. Reddy, B. S., Narisawa, T., Maronpot, R., and Weisburger, J. (1975). Animal models for the study of dietary factors and cancer of the large bowel. Cancer Res. 35, 3421–3426. Reddy, B. S., Nayini, J., Tokumo, K., Rigotty, J., Zange, E., and Kelloff, G. (1990). Chemoprevention of colon carcinogenesis by concurrent administration of piroxicam, a nonsteroidal antiinflammatory drug, with D,L--difluoromethylornithine, an ornithine decarboxylase inhibitor in diet. Cancer Res. 50, 2562–2568. Reddy, B. S., Rao, C. V., Rivenson, A., and Kelloff, G. J. (1993). Inhibitory effect of aspirin on azoxymethaneinduced colon carcinogenesis in F344 rats. Carcinogen. (Lond.) 14, 1493–1497.
55
Reddy, B. S., Rao, C. V., Rivenson, A., and Kelloff, G. (1997). Chemoprevention of colon carcinogenesis by organosulfur compounds. Cancer Res. 53, 3494–3498. Reddy, B. S., and Rivenson, A. (1993). Inhibitory effect of Bifidobacterium longum on colon, mammary, and liver carcinogenesis induced by 2-amino-3-methylimidazo[4,5-f ] quinoline, a food mutagen. Cancer Res. 53, 3914–3918. Reddy, B. S., Rivenson, A., El-Bayoumy, K., Upadhyaya, P., Pittman, B., and Rao, C. V. (1997). Chemoprevention of colon cancer by synthetic organoselenium compounds, 1,4phenylenebis(methylene)selenocyanate and p-methoxy benzyl selenocyanate, in low and/or high fat fed F344 rats. J. Natl. Cancer Inst. 89, 506–516. Reddy, B. S., Tokumo, K., Kulkarni, N., Aliga, C., and Kelloff, G. (1992). Inhibition of colon carcinogenesis by prostaglandin synthesis inhibitors and related compounds. Carcinogenesis 13, 1019–1023. Reddy, B. S., Wantanabe, K., and Weisburger, J. H. (1997). Effect of high-fat diet on colon carcinogenesis in F344 rats treated with 1,2-dimethylhydrazine, methylazoxymethanol acetate, or methylnitrosourea. Cancer Res. 37, 4156–4158. Singh, J., Hamid, R., and Reddy, B. S. (1997). Dietary fat and colon cancer: Modulation of cyclooxygenase-2 by types and amount of dietary fat during the postinitiation stage of colon carcinogenesis. Cancer Res. 57, 3465–3470. Singh, J., Kulkarni, N., Kelloff, G., and Reddy, B. S. (1994). Modulation of azoxymethane-induced mutational activation of ras protooncogens of chemopreventive agents in colon carcinogenesis. Carcinogenesis 15, 1317–1323. Thun, M. H., Namboodiri, M. M., and Heath, C. W. (1991). Aspirin use and reduced risk of fatal colon cancer. N. Engl. J. Med. 325, 1593–1596.