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beta-catenin Signaling and the Effects of Olive Oil
Chapter 107
Azoxymethane-induced Colon Carcinogenesis through Wnt/beta-catenin Signaling and the Effects of Olive Oil Takehiro Fujise, Ryuichi Iwakiri, Ryosuke Shiraishi, Bin Wu and Kazuma Fujimoto Department of Internal Medicine and Gastrointestinal Endoscopy, Saga Medical School, Japan
107.1 Introduction Colon cancer is one of the leading causes of cancer death in both men and women in Western countries, and in recent years, it has increasingly become a major cause of cancer mortality in Oriental countries, including Japan. These changes are associated with the westernization of Japanese dietary habits, which involves high consumption of meat and fat, together with low consumption of fruit, vegetables, vitamins and fibers, compared with classical Japanese diets. In fact, many epidemiological studies have shown a positive relationship between dietary fat consumption and colorectal cancer. Cancer results from the accumulation of multiple independent genetic alterations. In colon cancer, these genetic changes affect colon epithelial cell proliferation, differentiation and apoptosis. In familial adenomatous polyposis (FAP), which is one of the hereditary forms of colon cancer, a gene has been identified and its protein is a key molecule in the Wnt signaling pathway. Many experiments have shown that the Wnt signaling pathway plays a crucial role in the etiology of colon cancer, including hereditary and sporadic forms. In this chapter, we focus on dysregulation of colonic mucosal homeostasis in carcinogen-induced colon cancer models and the effects of dietary fatty acid.
107.2 Wnt signaling pathway and colon cancer The intestinal tract is characterized by rapid epithelial cell turnover, which continues throughout its life. This process involves the crypts and their associated villi, and is maintained and strictly regulated by stem cells, which give rise Olives and Olive Oil in Health and Disease Prevention. ISBN: 978-0-12-374420-3
to all the intestinal epithelial cell lineages: enterocytes and absorptive and secretory cells. The position along the crypt– villous axis defines the various stages in the life of an intestinal epithelial cell. The Wnt signaling pathway regulates a wide variety of processes in embryonic development and adult homeostasis, including cell proliferation, morphology, motility, and cell fate at the cellular level (Reya and Clevers, 2005), and plays a critical role in the development of the gastrointestinal tract. The key molecules in this pathway are a multiprotein scaffold that consists of -catenin, glycogen synthase kinase-3 (GSK3), axin, and adenomatous polyposis coli (APC). The Wnt signaling pathway begins with the Wnt ligand, one of a family of at least 16 members in mammals (Cadigan and Nusse, 1997). Wnts are secreted as growth factors that act through the cell surface Frizzled (Fz) receptor to initiate the signaling cascade (Bhanot et al., 1996; He et al., 1997). Disheveled is activated upon ligand binding to Fz, which causes inhibition of GSK3. Inhibition of GSK3 activity prevents phosphorylation of -catenin, thus blocking APC and axin-mediated degradation of -catenin. -Catenin is stabilized and translocated to the nucleus to bind members of the lymphoid enhancer factor/T cell factor (LEF/TCF) family of transcription factors and induce target gene expression (Barker et al., 2000; Giles et al., 2003). Targets of Wnt-signaling-regulated transcription include the protooncogene myc (He et al., 1998), cyclooxgenase 2 (Howe et al., 1999), matrilysin (Crawford et al., 1999), cyclin D1 (Tetsu and McCormick, 1999), and a member of the LEF/TCF family (Roose et al., 1999). APC was originally identified as an intestinal tumor suppressor gene by genetic analysis in patients with FAP (Groden et al., 1991). Hereditary forms of colorectal cancer and 85% of all sporadic colorectal cancers have loss of APC function (Kinzler and Vogelstein, 1996). It is commonly accepted
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that the crucial tumor suppressor role of APC lies in its ability to destabilize cytoplasmic free -catenin (von Kries et al., 2000). Therefore, disrupted regulation of the Wnt signaling pathway plays a central role in the etiology of colon carcinogenesis (Liu et al., 2000; von Kries et al., 2000).
107.3 Carcinogen-induced colon cancer model and alteration in signaling pathway associated with colon mucosal homeostasis It is important that reliable animal models are available that demonstrate similarity to human disease in studies of various types of cancer. Elucidation of the mechanisms of colon carcinogenesis and evaluation of the effect of chemopreventive agents have been carried out using several animal models. Azoxymethane (AOM)-induced colon cancer is one of the major models. AOM is a very potent carcinogen that induces colorectal cancer with a high incidence in rats and mice. Aberrant crypt foci (ACF) induced by treatment with AOM in rodents can be used as biomarkers in shortterm experiments. ACF were first identified in the colon mucosa of rodents exposed to carcinogens (Bird, 1987), and have also been confirmed to be present in the human colon (Noda et al., 1998). ACF are regarded as preneoplastic or precancerous lesions in the colorectum of humans and rodents (Bird, 1987; Noda et al., 1998). The number of crypt/foci has been shown to increase with time following carcinogen treatment, and ACF demonstrate increased cell proliferation in rodents (Pretow et al., 1992, 1994). Thus, the formation and growth of these putative preneoplastic lesions are thought to be valuable indices of the effects of carcinogens and agents, promoting or preventing carcinogenesis in the colon cancer. Colon carcinogenesis is known to be a multistep process that involves multiple genetic alterations to K-ras. APC, DCC and P53. In AOM-induced colon cancer model of rats and mice, K-ras mutation has frequently been seen (70%) in hyperplastic ACF. -Catenin mutation has also been found frequently (66%) in dysplastic ACF and alteration of the cellular localization of -catenin has been observed in all dysplastic ACF (Boone et al., 1992). These studies have shown that there is a genetic alteration associated with Wnt/-catenin signaling and these changes may play an important role in AOM-induced colon carcinogenesis. One previous study has evaluated expression of Wnts, Bone morphogenic protein (BMP), and Fz in human tissues and colon cancer cell lines, by using in situ hybridization (Holcombe et al., 2002). Wnt 2 has been detected in colon cancer but was undetectable in normal colonic mucosa. Differential expression of Wnt 5a with increased expression at the bottom of crypts compared to luminal villi is seen in normal colonic mucosa, whereas, in colon cancer tissue, Wnt 5a expression is increased. The expression of other Wnts (1, 4, 5b, 6, 7b and 10b) does not show any marked
Section | II Cancer
difference between normal colonic mucosa and colon cancer tissue. In poorly differentiated adenocarcinoma, a high degree of Fz receptor expression was seen compared with no expression in normal colonic mucosa and well-differentiated adenocarcinoma tissue. These results indicate that Wnt ligands and Fz receptor, distinct from alterations of key molecules such as APC and -catenin in Wnt/-catenin signaling pathways, are integrally associated with colon carcinogenesis. They also suggest that Wnt 2 and 5a play an important role in the progression from normal to neoplastic colonic mucosa, and that Fz receptor is involved in processes associated with cancer invasion. Another study has shown that Wnt 2 expression and cytoplasmic/nuclear -catenin accumulation are seen in most gastric cancers, irrespective of morphological phenotype, and that Wnt 2 up-regulation and -catenin nuclear translocation signaling may play an important role in cancer formation, invasion and dissemination (Cheng et al., 2005). These studies indicate that disruption of Wnt/-catenin signaling is one of the mechanisms of carcinogenesis in AOM-induced colon cancer models.
107.4 Dietary fatty acid composition and colon cancer Apart from these genetic factors, environmental factors are thought to be involved in colon carcinogenesis, among which, dietary habits play a pivotal role. High consumption of meat and fat, together with low consumption of fruit, vegetables, vitamins and fiber, has been suggested to increase risk of colorectal cancer (Willett et al., 1990; Bostick et al., 2003; Giles et al., 2003; Riboli and Norat, 2003). Many epidemiological studies have revealed a positive relationship between dietary fat intake and colorectal cancer (Dai et al., 2002). Experimental studies have demonstrated that a highfat diet rich in n-6 polyunsaturated fatty acid (PUFA) and saturated fatty acids (SFAs) promotes colon carcinogenesis, particularly in post-initiation or promotional phases, and/or both (Rao et al., 2001; Dai et al., 2002; Wu et al., 2004); whereas diets rich in n-3 PUFAs and n-9 monounsaturated fatty acids (MUFAs) have been reported to inhibit colon carcinogenesis in both initiation and post-initiation phases (Bartoli et al., 2000a, b; Rao et al., 2001; Reddy, 2004). This supports epidemiological reports that show that an n-3 PUFA-rich diet suppresses the risk of colon cancer in humans (Caygill and Hill, 1995; Byers, 1996). These previous studies indicate that amount of fat intake and composition of ingested dietary fatty acids are important factors for colon carcinogenesis. Recently, we have investigated the effects of dietary intake of diverse fatty acids on colon carcinogenesis in an AOMinduced rat colon cancer model, and effects of various fatty acids on the Wnt signaling pathway (Fujise et al., 2007). Male Sprague-Dawley rats were given intraperitoneal injections of AOM once weekly for 2 weeks at a dose of 15 mg kg1 body
Chapter | 107 Azoxymethane-induced Colon Carcinogenesis
weight, whereas control rats were given an equal volume of physiological saline. One day after the first AOM or saline treatment, rats designed for corn oil, olive oil, beef tallow, and fish oil diets, began to be fed with diets high in n-6 PUFAs, n-9 MUFAs, SFAs and n-3 PUFAs; whereas, one group continued to be fed with standard chow for 44 weeks. Twelve weeks after the last injection of AOM or saline, some rats were sacrificed, and colon ACF formation was analyzed. The remaining rats were sacrificed at 44 weeks, and tumors were examined. Colon mucosa was collected for further analysis. Normal-appearing colon mucosal proliferation and apoptosis were evaluated by Bromodeoxyuridine (BrdU) incorporation and percentage fragmented DNA, respectively. Expression of -catenin, cyclin D1, Wnt2, Wnt3 and Wnt5a in normal-appearing colon mucosa was analyzed by Western blotting. Twelve weeks from the start of the experiment, colon ACF developed in all rats treated with AOM. Among AOM-treated rats, those fed with 10% corn oil and 10% beef tallow had significantly greater numbers of ACF per colon compared to rats fed with standard chow (standard chow group: 129 2.3/colon; corn oil group: 188 7.4/colon; beef tallow group: 206 4.0/colon; p 0.05), whereas numbers of ACF per colon significantly decreased in AOMtreated rats fed with 10% olive oil and 10% fish oil compared to standard chow-fed rats (standard chow group: 129 2.3/ colon; olive oil group: 94 3.1/colon; fish oil group: 96 4.9/colon; p 0.05). In contrast, few ACF were found in rats with saline treatment only. The number of multicrypt ACF, which were defined as those containing four or more aberrant crypts per focus, was significantly higher in rats fed with 10% corn oil and 10% beef tallow than in those fed with standard chow (p 0.05). On the other hand, rats fed with 10% olive oil and 10% fish oil showed a significant decrease in multicrypt ACF compared to standard chow-fed rats (p 0.05). At 44 weeks, no rat without AOM treatment developed colon adenoma or carcinoma. On the other hand, all rats fed with standard chow, 10% corn oil and 10% beef tallow diet developed colon cancer 44 weeks after the last injection of AOM (Figure 107.1). Two of six rats fed with 10% olive oil did not develop colon cancer, and four of six rats fed with 10% fish oil did not develop colon cancer. The number of colon tumors per rat was significantly higher in rats fed with 10% corn oil and 10% beef tallow than in those fed with standard chow (standard chow group: 2.3 0.5/ colon; corn oil group: 4.3 0.5/colon; beef tallow group: 5.2 0.5/colon; p 0.05), whereas tumor multiplicity in the colon of rats fed with 10% fish oil was significantly lower than in those fed with standard chow (standard chow group: 2.3 0.5/colon; fish oil group: 0.5 0.2/colon; p 0.05). The number of tumors in rats fed with 10% olive oil was smaller than that in standard chow-fed rats, but there was no significant difference between these two groups (olive oil group: 1.5 0.5/colon) (Figure 107.2). The number of BrdU-incorporated epithelial cells per crypt significantly increased in AOM-treated rats compared to saline-treated
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Figure 107.1 Colon tumors were not observed in rats without AOM treatment throughout the study period. At 44 weeks, almost all tumors showed well-differentiated adenocarcinomas with polypoid growth.
rats at 44 weeks. In rats fed with 10% corn oil and 10% beef tallow, BrdU-incorporated cell number significantly increa sed compared to that in standard chow-fed rats regardless of AOM treatment (standard chow group: 2.7 0.15/crypt; corn oil group: 3.2 0.21/crypt; beef tallow group: 3.9 0.21/ crypt; p 0.05; standard chow AOM group: 4.5 0.22/ crypt; corn oil AOM group: 6.4 0.15/crypt, beef tallow AOM group: 6.9 0.23/crypt; p 0.05). The num ber of BrdU-incorporated cells in rats fed with 10% olive oil and 10% fish oil significantly decreased (standard chow group: 2.7 0.15/crypt; olive oil group: 1.3 0.09/crypt; fish oil group: 1.8 0.21/crypt; p 0.05; standard chow AOM group: 4.5 0.22/crypt; olive oil AOM group: 1.9 0.15/crypt; fish oil group: 3.7 0.10/crypt; p 0.05) (Figure 107.3A). BrdU-incorporated epithelial cells were normally observed at the base of crypts. Among rats fed with 10% corn oil and 10% beef tallow, BrdU-positive cells spread to the upper portion of crypts even without AOM treatment. BrdU-incorporated cells were often observed at the top of crypts in rats fed with 10% beef tallow. Changes in distribution of BrdU-positive cells were more evident in rats treated with AOM. Many epidemiological and experimental studies have demonstrated that the amount and type of dietary fat play an important role in colon carcinogenesis (Reddy, 2004; Roynette et al., 2004), while controversy still exists regarding the influence of dietary fat on colon tumorigenesis. Our study showed that any type of high-fat diet did not result in the development of ACF or colon tumors per se. With AOM treatment, both 10% corn oil and 10% beef tallow diets significantly enhanced numbers of ACF and crypt multiplicity of foci 12 weeks after AOM treatment. Furthermore, all rats fed with standard chow, 10% corn oil or 10% beef tallow developed colon tumors at 44 weeks, and the number of tumors significantly increased in rats fed with 10% corn oil and 10% beef tallow compared to rats fed
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Section | II Cancer
6
* * * BrdU incorporated cell number
7 6
AOM(−) AOM(+)
* * *
* * *
**
**
5
*
4
**
*
3 2
*
**
*
1
A
Standard chow
Corn oil
Olive oil
Beef tallow
Fish oil
* * *
* * *
* * *
* * *
* * *
10 9 8
*
AOM(−) AOM(+)
*
7 6 5
**
**
4 3
**
2
**
1 0 B
4 3 2 1
*
0 Corn oil
* * *
0
*
Standard chow
* * *
8
*
5 Tumor number/colon
at the base of crypts in the colon mucosa. In colon carcinogenesis, two main hypotheses have been proposed for morphogenesis of colon tumor: bottom-up and top-down morphogenesis. Development of human adenomatous polyps is believed to proceed through the top-down mechanism, in which genetically altered cells in the superficial portions of the mucosa spread laterally, and downward to form new
% Fragmented DNA
with standard chow. These results indicated that dietary intake of corn oil rich in n-6 PUFAs and beef tallow rich in SFAs promoted colon carcinogenesis in AOM-treated rats. In contrast to corn oil and beef tallow, olive oil rich in n-9 MUFAs and fish oil rich in n-3 PUFAs ameliorated AOMinduced ACF formation and colon carcinogenesis, compared to rats fed with standard chow. Our results were supported by previous studies that have shown that a high-fat diet rich in n-6 PUFA and SFA promotes colon tumorigenesis, particularly in post-initiation and/or promotional phases in rodents (Dai et al., 2002; Wu et al., 2004), whereas a high-fat diet rich in n-9 MUFA and n-3 PUFA inhibits colon carcinogenesis in both post-initiation and/or promotional phases (Bartoli et al., 2000; Rao et al., 2001; Reddy, 2004). Many previous studies have evaluated cancerous tissues to analyze the effects of dietary fat (Bartoli et al., 2000; Rao et al., 2001; Dai et al., 2002; Reddy, 2004; Wu et al., 2004). Our study focused on background characteristics of the colonic mucosa after long-term feeding of different types of dietary fat, and assessed colonic epithelial proliferation in normal-appearing colon mucosa in colon tumorigenesis. Few studies have investigated the effects of dietary fatty acid composition on normal-appearing colon mucosa. BrdU incorporation in normal-appearing colon mucosa surrounding colon tumors significantly increased in rats fed with 10% corn oil and 10% beef tallow, but decreased in those fed with 10% olive oil and 10% fish oil diets. These results showed that long-term feeding of 10% corn oil and 10% beef tallow accelerated the proliferation potential of the colonic mucosal epithelium, which might have promoted colon carcinogenesis after AOM treatment. Our study also showed that the range of BrdU-positive cells spread to the upper portion of crypts in rats fed with corn oil and beef tallow. Commonly, stem cells are located
Olive oil
Beef tallow
Fish oil
Figure 107.2 Tumor number in rats treated with AOM. No rat without AOM treatment showed colon tumors, including adenoma and carcinoma. Numbers of colon tumors per rat treated with AOM were significantly higher in rats fed with 10% corn oil and 10% beef tallow compared to rats fed with standard chow, whereas tumor multiplicity of the colon in rats fed with 10% fish oil diet was significantly lower than that of rats fed with standard chow. *p 0.05.
Standard chow
Corn oil
Olive oil
Beef tallow
Fish oil
Figure 107.3 Effect of dietary fatty acid components on BrdU incorporation and DNA fragmentation in the rat colon mucosa. (A) The number of BrdU-incorporated epithelial cells per crypt significantly increased in AOM-treated rats compared to rats treated with saline 44 weeks after initial treatment. Among with or without AOM-treated rats, BrdU-incorporated cell number significantly increased in rats fed with 10% corn oil and 10% beef tallow diets compared to standard chow-fed rats, and significantly decreased in rats fed with 10% olive oil and 10% fish oil. *p 0.05 compared to saline-treated rats fed with standard chow. **p 0.05 compared to AOM-treated rats fed with standard chow. ***p 0.05 compared to rats treated with saline. (B) Following AOM treatment, percentage of fragmented DNA was significantly inhibited in the colon mucosa among all dietary groups. In rats fed with 10% olive oil and 10% fish oil, % fragmented DNA was significantly higher compared to that of rats fed with standard chow regardless of AOM treatment. *p 0.05 compared to saline-treated rats fed with standard chow. **p 0.05 compared to AOMtreated rats fed with standard chow. ***p 0.05 compared to rats treated with saline.
Chapter | 107 Azoxymethane-induced Colon Carcinogenesis
crypts that connect to pre-existing normal crypts and eventually replace them (Shih et al., 2001; Wright and Poulson, 2002). Our study of BrdU-positive cells in upper portions of crypts indicated alterations in the distribution of proliferating cells in rats fed with 10% corn oil and 10% beef tallow. This suggests the involvement of top-down morphogenesis in colon carcinogenesis, which results from a high n-6 PUFA and SFA diet. In view of homeostasis of the crypt–villous axis in the colon mucosa, BMP and Hedgehog signaling without Wnt/-catenin signaling play a crucial role, including cell proliferation, differentiation and apoptosis, which is generally regulated strictly by these signal transduction pathways (Radtke and Clevers, 2005; Zhang and Li, 2005). One previous study has demonstrated altered expression of BMP in cancerous tissue compared to normal colonic mucosa (Holcombe et al., 2002). Fatty acid composition may influence the crucial signal transduction pathways, which maintain homeostasis of the crypt–villous axis in the colon mucosa, including BMP signaling and Hedgehog signaling. Apoptosis in normal-appearing colon mucosa at 44 weeks after the last injection of AOM or saline was evaluated using a DNA fragmentation assay (Figure 107.3B). Following AOM treatment, the percentage of fragmented DNA was significantly inhibited in the colon mucosa of all dietary groups. In rats fed with 10% olive oil and 10% fish oil, percentage fragmented DNA was significantly higher than that in rats fed with standard chow, with or without AOM treatment (standard chow group: 3.4 0.30%; olive oil group: 6.0 0.57%; fish oil group: 8.7 0.53%; p 0.05; standard chow AOM group: 2.6 0.33%; olive oil AOM group: 3.78 0.29%; fish oil group: 4.08 0.12%; p 0.05). In our previous study, we have revealed that a higher risk for colon cancer with dietary corn oil rich in n-6 PUFA is attributed to reduced apoptosis in the colon mucosa, and this was associated with inhibition of the tumor suppressor gene p53-mediated mitochondria-dependent apoptotic pathway (Wu et al., 2004). This study demonstrated a significant decrease in apoptosis in rats fed with 10% corn oil and 10% beef tallow, and reduction of colon mucosal apoptosis by dietary corn oil and beef tallow might be the result of accelerated cell proliferation during colon tumorigenesis. Many studies have suggested that the chemopreventive effect of n-3 PUFA was partly due to increased mucosal apoptosis (Davidson et al., 2000). This study showed that there was an apparent suppression of colon carcinogenesis, together with increased mucosal apoptosis in rats treated with AOM, by dietary intake of olive oil rich in n-9 MUFA and fish oil rich in n-3 PUFA. Increased apoptosis might be one of the crucial factors involved in tumor inhibition. Other factors, including ornithine decarboxylase activity (Rao and Reddy, 1993), diacylglycerol (Jiang et al., 1996), COX-2 and iNOS (Narayanan et al., 2003), may contribute to reduced carcinogenesis, together with n-3 PUFAs and n-9 MUFAs.
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Expression of cyclin D1 increased in AOM-treated rats. This increase was more significant in rats fed with 10% corn oil and 10% beef tallow, with or without AOM treatment, with a similar pattern as the BrdU incorporation assay. Results of the -catenin assay were similar to those with cyclin D1. Namely, accumulation of cytoplasmic catenin was significantly elevated in rats fed with 10% corn oil and 10% beef tallow, with or without AOM treatment. Thus, we investigated Wnt proteins that existed as upstream signals in the Wnt/-catenin signaling pathway. Expression of Wnt 2 and Wnt 3 was significantly higher in rats fed with 10% beef tallow, with or without AOM treatment, than that in other dietary groups. In rats fed with 10% corn oil, expression of Wnt 5a significantly increased compared to that in other rats fed with other diets. Resent studies have indicated that the Wnt signaling pathway is required during stem cell homeostasis for normal progression of intestinal epithelial cells through the crypt–villous axis (Radtke and Clevers, 2005; Reya and Clevers, 2005), and dysregulation of the Wnt signaling pathway is observed in many cancer tissues (Radtke and Clevers, 2005; Reya and Clevers, 2005). This study evaluated expression of cyclin D1, a product gene in Wnt/-catenin signaling, which is activated through accumulation of -catenin in the cytosol. Expression of cyclin D1 increased in rats fed with 10% corn oil and 10% beef tallow, and significant accumulation of -catenin in the cytosol was observed, which suggested that Wnt/-catenin signaling was activated by dietary consumption of corn oil and beef tallow. Wnt expression, an upstream signal in the Wnt/-catenin signaling pathway, increased in rats fed with corn oil; and Wnt 2 and 3 increased in rats fed with beef tallow in rat colon mucosa, regardless of AOM treatment. These results indicated that increased colon mucosal proliferation potential was, at least in part, attributed to activation of Wnt/-catenin signaling. Several studies in humans have indicated that expression of Wnt genes is accelerated in colon carcinoma tissues compared to surrounding normalappearing mucosa, using in situ hybridization (Mohammed et al., 2001; Holcombe et al., 2002). In another study of human gastric cancer, co-existence of Wnt 2 up-regulation and -catenin translocation was positively associated with lymph node metastasis (Cheng et al., 2005). In this study, it is noteworthy that up-regulation of Wnt signaling in the normal-appearing surrounding colon mucosa was observed in rats with long-term consumption of corn oil and beef tallow. Our study indicated that long-term intake of dietary SFA and n-6 PUFA accelerated colon carcinogenesis by increasing cell proliferation through up-regulating the Wnt/-catenin signaling pathway in AOM-treated rats. In contrast, n-9 MUFA and n-3 PUFA had a suppressive effect on colon carcinogenesis through decreasing cell proliferation and increasing cell apoptosis in colon mucosa. These results indicate that dietary fatty acid composition might be an important factor for modulation of mucosal proliferation potential and apoptosis.
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Many studies have investigated the inhibitory effect of olive oil on colon carcinogenesis. Dietary intake of olive oil inhibited the formation of ACF in an AOM-induced rat colon cancer model, and decreased colon mucosal arachidonate concentration compared with dietary intake of corn oil. Furthermore, AOM treatment induced a significant increase in prostaglandin E2 (PGE2) formation, but no change was found in rats fed with olive oil (Bartoli et al., 2000). The results indicated that these inhibitory effects may be due to modulation of arachidonic acid metabolism and mucosal PGE2 synthesis (Bartoli et al., 2000). Other studies using IL-10 knockout mice have shown that an olive oil diet inhibits COX-2 immunostaining in colon mucosa and decreases the risk of neoplasia-associated chronic colitis. However, a fish oil diet, which generally shows a chemopreventive effect on colon carcinogenesis, encourages chronic colitis and colitis-associated premalignant changes (Hegazi et al., 2006). An olive oil diet has an additive inhibitory effect with sulindac. These effects were mediated by regulating COX-2 expression, which is involved in prostaglandin synthesis and caspase-3, which plays a critical role in apoptosis (Schwautz et al., 2004). Recently, olive oil polyphenols have shown a strong inhibitory effect on cancer cell proliferation, which has been linked to the induction of a G2/M phase cell cycle block in cancer cell lines. These cell cycle blocks are mediated by inhibition of p38 and cyclic AMP response element binding protein (CREB) phosphorylation, which leads to a downstream reduction in COX-2 expression (Corona et al., 2007). Other studies have indicated that olive oil extract induces cell apoptosis and cell cycle arrest (Fabiani et al., 2002; Fini et al., 2008). In conclusion, many studies have indicated that olive oil has chemopreventive effects in colon carcinogenesis, through inhibition of cell proliferation and induction of apoptosis and/or cell cycle arrest (Figure 107.4). Our studies have investigated the role of the Wnt/-catenin
Section | II Cancer
signaling pathway in the colon-cancer-promoting effect of dietary fatty acid in an AOM-induced rat colon cancer model. However, the detailed mechanism associated with the inhibition of cell proliferation has not been elucidated. Further investigations are warranted to fully evaluate the mechanism of inhibitory and promoting effects of dietary fatty acid composition in colon carcinogenesis.
Summary points Many epidemiological studies have revealed a positive relationship between dietary fat intake and colorectal cancer. l Many experiments have shown that the Wnt signaling pathway plays a crucial role in the etiology of colon cancer, including hereditary and sporadic forms. l Disruption of Wnt/-catenin signaling is one of the mechanisms of carcinogenesis in AOM-induced colon cancer models. l Amount of fat intake and composition of ingested dietary fatty acids are important factors for colon carcinogenesis. l Diets rich in n-3 PUFAs and n-9 monounsaturated fatty acids (MUFAs) have been reported to inhibit colon carcinogenesis in both initiation and post-initiation phases. l Olive oil rich in n-9 MUFAs and fish oil rich in n-3 PUFAs ameliorated AOM-induced ACF formation and colon carcinogenesis, compared to rats fed with standard chow. l There was an apparent suppression of colon carcinogenesis, together with increased mucosal apoptosis in rats treated with AOM, by dietary intake of olive oil rich in n-9 MUFA and fish oil rich in n-3 PUFA. l Many studies have indicated that olive oil has chemopreventive effects in colon carcinogenesis, through inhibition of cell proliferation and induction of apoptosis and/or cell cycle arrest. l
References
Figure 107.4 Schematic design of chemopreventive effect of olive oil in colon carcinogenesis.
Barker, N., Morin, P.J., Clevers, H., 2000. The Yin-Yang of TCF/beta-catenin signaling. Adv. Cancer Res. 77, 1–24. Bartoli, R., Fernandez-Banares, F., Navarro, E., Castella, E., Mane, J., Alvarez, M., Pastor, C., Cabre, E., Gassull, M.A., 2000. Effect of olive oil on early and late events of colon carcinogenesis in rats: modulation of arachidonic acid metabolism and local prostaglandin E2 synthesis. Gut 46, 191–199. Bartoli, R., Femandes-Banares, F., Navarro, E., Castella, E., Mane, J., Alvarez, M., Pastor, C., Cabre, E., Gassull, M.A., 2000. Effect of olive oil on early and late events of colon carcinogenesis in rats: modulation of arachidonic acid metabolism and local prostaglandin E2 synthesis. Gut 46, 191–199. Bhanot, P., Brink, M., Samos, C.H., Hsieh, J.C., Wang, Y., Macke, J.P., Andrew, D., Nathans, J., Nusse, R., 1996. A new member of the frizzled
Chapter | 107 Azoxymethane-induced Colon Carcinogenesis
family from drosophila functions as a Wingless receptor. Nature 282, 225–230. Bird, R.P., 1987. Observation and quantification of aberrant crypts in the murine colon treated with a colon carcinogen: preliminary findings. Cancer Lett. 37, 147–151. Boone, C.W., Steele, V.E., Kelloff, G.J., 1992. Screening for chenopreventive (anticarcinogenic) compoumds in rodents. Mutat Res. 267, 251–255. Bostick, R.M., Potter, J.D., McKenzie, D.R., Sellers, T.A., Kushi, L.H., Steinmetz, K.A., Folson, A.R., 1993. Reduced risk of colon cancer with high intake of vitamin E: The Iowa Women’s Health Study. Cancer Res. 53, 4230–4237. Byers, T., 1996. Nutrition and cancer among American Indians and Alaska Natives. Cancer 78, 1612–1616. Cadigan, K.M., Nusse, R., 1997. Wnt signaling: a common theme in animal development. Genes. Dev. 11, 3286–3305. Caygill, C.P., Hill, M.J., 1995. Fish n-3 fatty acids and human colorectal and breast cancer mortality. Eur. J. Cancer. Prev. 4, 329–332. Cheng, X.X., Wang, Z.C., Chen, X.Y., Sun, Y., Kong, Q.Y., Liu, J., Li, H., 2005. Correlation of Wnt-2 expression and beta-catenin intracellular accumulation in Chinese gastric cancers: relevance with tumor dissemination. Cancer Lett. 223, 339–347. Corona, G., Deiana, M., Incani, A., Vauzour, D., Dessi, M.A., Spencer, J.P., 2007. Inhibition of p38/CREB phosphorylation and COX-2 expression by olive oil polyphenols underlies their anti-proliferative effects. Biochem. Biophys. Res. Commun. 362, 606–611. Crawford, H.C., Fingleton, B.M., Rudolph-Owen, L.A., Goss, K.J., Rubinfeld, B., Polakis, P., Matrisian, L.M., 1999. The metalloproteinase matrilysin is a target of beta-catenin transactivation in intestinal tumors. Oncogene 18, 2883–2891. Dai, W., Liu, T., Wang, Q., Rao, C.V., Reddy, B.S., 2002. Down-regulation of PLK3 gene expression by types and amount of dietary fat in rat colon tumors. Int. J. Oncol. 20, 121–126. Davidson, L., Brown, R., Chang, W., Morris, J., Wang, N., Carroll, R., Turner, N., Lupton, J., Chapkin, R., 2000. Morphodensitometric analysis of protein kinase C beta (II) expression in rat colon: modulation by diet and relation to in situ cell proliferation and apoptosis. Carcinogenesis 21, 1513–1519. Fabiani, R., De Bartolomeo, A., Rosignoli, P., Servili, M., Montedoro, G.F., Morozzi, G., 2002. Cancer chemoprevention by hydroxytyrosol isolated from virgin olive oil through G1 cell cycle and apoptosis. Eur. J. Cancer Prev. 11, 351–358. Fini, L., Hotchkiss, E., Fogliano, V., Graziani, G., Romano, M., De Vol, E.B., Qin, H., Selgrad, M., Boland, C.R., Ricciardiello, L., 2008. Chemopreventive properties of pinoresinol-rich olive oil involve a selective activation of ATM-p53 cascade in colon cancer cell lines. Carcinogenesis 29, 139–146. Fujise, T., Iwakiri, R., Kakimoto, T., Shiraishi, R., Sakata, Y., Wu, B., Tsunada, S., Ootani, A., Fujimoto, K., 2007. Long-term feeding of various fat diets modulates azoxymethane-induced colon carcinogenesis through Wnt/beta-catenin signalimg in rats. Am. J. Physiol. Gastrointest. Liver Physiol. 292, G1150–G1156. Giles, R.H., van Es, J.H., Clevers, H., 2003. Caught up in a Wnt storm; Wnt signaling in cancer. Biochim. Biophys. Acta 1653, 1–24. Groden, J., Thliveris, A., Samowitz, W., Carlson, M., Gelgert, L., Albertsen, H., Joslyn, G., Stevens, J., Spilio, L., Robertson, M., 1991. Identification and characterization of the familial adenomatous polyposis coli gene. Cell 66, 589–600. He, T.C., Sparks, A.B., Rago, C., Hermeking, H., Zawel, L., da Costa, L.T., Morin, P.J., Vogelstein, B., Kinzler, K.W., 1998. Identification of c-MYC as a target of the APC pathway. Science 281, 1438–1439.
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He, X., Saint-Jeannet, J.-P., Wang, Y., Nathans, J., Dawid, I., Varmus, H., 1997. A member of the Frizzled protein family mediating axis induction by Wnt-5a. Science 275, 1652–1654. Hegazi, R.A., Saad, R.S., Mady, H., Matarese, L.E., O’Keefe, S., Kandil, H.M., 2006. Dietary fatty acids modulate chronic colitis, colitis-associated colon neoplasia and COX-2 exoression in IL-10 knock out mice. Nutrition 22, 275–282. Holcombe, R.F., Marsh, J.L., Waterman, M.L., Lin, F., Milovanovic, T., Truong, T., 2002. Expression of Wnt ligands and frizzled receptors in colonic mucosa and in colon carcinoma. J. Clin. Pathol.: Mol. Pathol. 55, 220–226. Howe, L.R., Subbaramaiah, K., Chung, W.J., Dannenberg, A.J., Brown, A.M., 1999. Transcriptional activation of cyclooxygenase-2 in Wnt1-transformed mouse mammary epithelial cells. Cancer Res. 59, 1572–1577. Jiang, Y., Lupton, J., Chapkin, R., 1996. Dietary fat and fiber differentially alter intracellular second messengers during tumor development in rat colon. Carcinogenesis 17, 1227–1233. Kinzler, K.W., Vogelstein, B., 1996. Lessons from hereditary colorectal cancer. Cell 87, 159–170. Liu, W., Dong, X., Mai, M., Seelan, R.S., Taniguchi, K., Krishnandath, K.K., Halling, K.C., Cunningham, J.M., Boardman, L.A., Qian, C., Christensen, E., Schmidt, S.S., Roche, P.C., Smith, D. I., Thibodeau, S.N., 2000. Mutations in Axin2 cause colorectal cancer with detective mismatch repair by activating beta-catenin–Tcf signaling. Nat. Genet. 26, 146–147. Mohammed, I.M., Roczo, N., Phillips, W.A., Baindur-Hudson, S., 2001. Expression of Wnt genes in human colon cancers. Cancer Lett. 166, 185–191. Narayanan, B., Narayanan, N., Simi, B., Reddy, B.S., 2003. Modulation of inducible nitric oxide synthase and related proinflammatory genes by the omega-3 fatty acid docosahexaenoic acid in human colon cancer cell. Cancer Res. 63, 972–979. Noda, T., Iwakiri, R., Fujimoto, K., Matsuo, S., Aw, T.Y., 1998. Programmed cell death induced by ischemia-reperfusion in rat intestinal mucosa. Am. J. Physiol. 274, G270–G276. Pretow, T.P., Cheyer, C., O’Riordan, M.A., 1994. Aberrant crypt foci and colon tumors in F344 rats have similar increases in proliferative activity. Int. J. Cancer 56, 599–602. Pretow, T.P., O’Riordan, M.A., Somich, G.A., Amini, S.B., Pretlow, T.G., 1992. Aberrant crypts correlate with tumor incidence in F344 rats treated with azoxymethane and phytate. Carcinogenesis 13, 1509–1512. Radtke, F., Clevers, H., 2005. Self-renewal and cancer of the gut: Two sides of a coin. Science 307, 1904–1909. Rao, C., Reddy, B.S., 1993. Modulating effect of amount and types of dietary fat on ornithine decarboxylase, tyrosine protein kinase and prostaglandins production during colon carcinogenesis in F344 rats. Carcinogenesis 14, 1327–1333. Rao, C.V., Hirose, Y., Indranie, C., Reddy, B.S., 2001. Moduration of experimental colon tumorigenesis by types and amounts of dietary fatty acids. Cancer Res. 61, 1927–1933. Reddy, B.S., 2004. Omega-3 fatty acids in colorectal cancer prevention. Int. J. Cancer 112, 1–7. Reya, T., Clevers, H., 2005. Wnt signaling in stem cells and cancer. Nature 434, 843–850. Reya, T., Clevers, H., 2005. Wnt signaling in stem cells and cancer. Nature 434, 843–850. Riboli, E., Norat, T., 2003. Epidemiologic evidence of the protective effect of fruit and vegetables on cancer risk. Am. J. Clin. Nutr. 78, 559S–569S.
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Roose, J., Huls, G., van Beest, M., Moerer, P., van der Horn, K., Goldschmeding, R., Logtenberg, T., Clevers, H., 1999. Synergy between tumor suppressor APC and the beta-catenin-Tcf4 target Tcf1. Science 285, 1923–1926. Roynette, C.E., Calder, P.C., Dupertuis, Y.M., Pichard, C., 2004. n-3 polyunsaturated fatty acids and colon cancer prevention. Clin. Nutr. 23, 139–151. Schwautz, B., Birk, Y., Raz, A., Madar, Z., 2004. Nutritional-pharmacological combinations. Eur. Nutr. 43, 221–229. Shih, I.M., Wang, T.L., Traverso, G., Romans, K., Hamilton, S.R., BenSasson, S., Kinzler, K.W., Vogelstein, B., 2001. Top-down morphogenesis of colorectal tumors. PNAS 27, 2640–2645. Tetsu, O., McCormick, F., 1999. Beta-catenin regulates expression of Cyclin D1 in colon carcinoma cells. Nature 398, 422–426. von kries, J.P., Winbeck, G., Asbrand, C., Schwarz-Romond, T., Sochnikowa, N., Dell’Oro, A., Behrens, J., Birchmeier, W., 2000.
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Hot spots in beta-catenin for interactions with LEF-1, conduction and APC. Nat. Struct. Biol. 7, 800–807. Willett, W.C., Stampfer, M.J., Colditz, G.A., Rosner, B.A., Speizer, F.E., 1990. Relation of meat, fat, and fiber intake to the risk of colon cancer in a prospective study among women. N. Eng. J. Med. 323, 1664–1672. Wright, N.A., Poulson, R., 2002. Top down or bottom up? Competing management structures in the morphogenesis of colorectal neoplasm. Gut 51, 306–308. Wu, B., Iwakiri, R., Ootani, A., Tsunada, S., Fujise, T., Sakata, Y., Sakata, H., Toda, S., Fujimoto, K., 2004. Dietary corn oil promotes colon cancer by inhibiting mitochondria-dependent apoptosis in azoxymethane-treated rats. Exp. Biol. Med. 229, 1017–1025. Zhang, J., Li, L., 2005. BMP signaling and stem cell regulation. Dev. Biol. 284, 1–11.
Azoxymethane-induced Colon Carcinogenesis through Wnt/beta-catenin Signaling and the Effects of Olive Oil Takehiro Fujise, Ryuichi Iwakiri, Ryosuke Shiraishi, Bin Wu and Kazuma Fujimoto Department of Internal Medicine and Gastrointestinal Endoscopy, Saga Medical School, Japan
107.1 Introduction Colon cancer is one of the leading causes of cancer death in both men and women in Western countries, and in recent years, it has increasingly become a major cause of cancer mortality in Oriental countries, including Japan. These changes are associated with the westernization of Japanese dietary habits, which involves high consumption of meat and fat, together with low consumption of fruit, vegetables, vitamins and fibers, compared with classical Japanese diets. In fact, many epidemiological studies have shown a positive relationship between dietary fat consumption and colorectal cancer. Cancer results from the accumulation of multiple independent genetic alterations. In colon cancer, these genetic changes affect colon epithelial cell proliferation, differentiation and apoptosis. In familial adenomatous polyposis (FAP), which is one of the hereditary forms of colon cancer, a gene has been identified and its protein is a key molecule in the Wnt signaling pathway. Many experiments have shown that the Wnt signaling pathway plays a crucial role in the etiology of colon cancer, including hereditary and sporadic forms. In this chapter, we focus on dysregulation of colonic mucosal homeostasis in carcinogen-induced colon cancer models and the effects of dietary fatty acid.
107.2 Wnt signaling pathway and colon cancer The intestinal tract is characterized by rapid epithelial cell turnover, which continues throughout its life. This process involves the crypts and their associated villi, and is maintained and strictly regulated by stem cells, which give rise Olives and Olive Oil in Health and Disease Prevention. ISBN: 978-0-12-374420-3
to all the intestinal epithelial cell lineages: enterocytes and absorptive and secretory cells. The position along the crypt– villous axis defines the various stages in the life of an intestinal epithelial cell. The Wnt signaling pathway regulates a wide variety of processes in embryonic development and adult homeostasis, including cell proliferation, morphology, motility, and cell fate at the cellular level (Reya and Clevers, 2005), and plays a critical role in the development of the gastrointestinal tract. The key molecules in this pathway are a multiprotein scaffold that consists of -catenin, glycogen synthase kinase-3 (GSK3), axin, and adenomatous polyposis coli (APC). The Wnt signaling pathway begins with the Wnt ligand, one of a family of at least 16 members in mammals (Cadigan and Nusse, 1997). Wnts are secreted as growth factors that act through the cell surface Frizzled (Fz) receptor to initiate the signaling cascade (Bhanot et al., 1996; He et al., 1997). Disheveled is activated upon ligand binding to Fz, which causes inhibition of GSK3. Inhibition of GSK3 activity prevents phosphorylation of -catenin, thus blocking APC and axin-mediated degradation of -catenin. -Catenin is stabilized and translocated to the nucleus to bind members of the lymphoid enhancer factor/T cell factor (LEF/TCF) family of transcription factors and induce target gene expression (Barker et al., 2000; Giles et al., 2003). Targets of Wnt-signaling-regulated transcription include the protooncogene myc (He et al., 1998), cyclooxgenase 2 (Howe et al., 1999), matrilysin (Crawford et al., 1999), cyclin D1 (Tetsu and McCormick, 1999), and a member of the LEF/TCF family (Roose et al., 1999). APC was originally identified as an intestinal tumor suppressor gene by genetic analysis in patients with FAP (Groden et al., 1991). Hereditary forms of colorectal cancer and 85% of all sporadic colorectal cancers have loss of APC function (Kinzler and Vogelstein, 1996). It is commonly accepted
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that the crucial tumor suppressor role of APC lies in its ability to destabilize cytoplasmic free -catenin (von Kries et al., 2000). Therefore, disrupted regulation of the Wnt signaling pathway plays a central role in the etiology of colon carcinogenesis (Liu et al., 2000; von Kries et al., 2000).
107.3 Carcinogen-induced colon cancer model and alteration in signaling pathway associated with colon mucosal homeostasis It is important that reliable animal models are available that demonstrate similarity to human disease in studies of various types of cancer. Elucidation of the mechanisms of colon carcinogenesis and evaluation of the effect of chemopreventive agents have been carried out using several animal models. Azoxymethane (AOM)-induced colon cancer is one of the major models. AOM is a very potent carcinogen that induces colorectal cancer with a high incidence in rats and mice. Aberrant crypt foci (ACF) induced by treatment with AOM in rodents can be used as biomarkers in shortterm experiments. ACF were first identified in the colon mucosa of rodents exposed to carcinogens (Bird, 1987), and have also been confirmed to be present in the human colon (Noda et al., 1998). ACF are regarded as preneoplastic or precancerous lesions in the colorectum of humans and rodents (Bird, 1987; Noda et al., 1998). The number of crypt/foci has been shown to increase with time following carcinogen treatment, and ACF demonstrate increased cell proliferation in rodents (Pretow et al., 1992, 1994). Thus, the formation and growth of these putative preneoplastic lesions are thought to be valuable indices of the effects of carcinogens and agents, promoting or preventing carcinogenesis in the colon cancer. Colon carcinogenesis is known to be a multistep process that involves multiple genetic alterations to K-ras. APC, DCC and P53. In AOM-induced colon cancer model of rats and mice, K-ras mutation has frequently been seen (70%) in hyperplastic ACF. -Catenin mutation has also been found frequently (66%) in dysplastic ACF and alteration of the cellular localization of -catenin has been observed in all dysplastic ACF (Boone et al., 1992). These studies have shown that there is a genetic alteration associated with Wnt/-catenin signaling and these changes may play an important role in AOM-induced colon carcinogenesis. One previous study has evaluated expression of Wnts, Bone morphogenic protein (BMP), and Fz in human tissues and colon cancer cell lines, by using in situ hybridization (Holcombe et al., 2002). Wnt 2 has been detected in colon cancer but was undetectable in normal colonic mucosa. Differential expression of Wnt 5a with increased expression at the bottom of crypts compared to luminal villi is seen in normal colonic mucosa, whereas, in colon cancer tissue, Wnt 5a expression is increased. The expression of other Wnts (1, 4, 5b, 6, 7b and 10b) does not show any marked
Section | II Cancer
difference between normal colonic mucosa and colon cancer tissue. In poorly differentiated adenocarcinoma, a high degree of Fz receptor expression was seen compared with no expression in normal colonic mucosa and well-differentiated adenocarcinoma tissue. These results indicate that Wnt ligands and Fz receptor, distinct from alterations of key molecules such as APC and -catenin in Wnt/-catenin signaling pathways, are integrally associated with colon carcinogenesis. They also suggest that Wnt 2 and 5a play an important role in the progression from normal to neoplastic colonic mucosa, and that Fz receptor is involved in processes associated with cancer invasion. Another study has shown that Wnt 2 expression and cytoplasmic/nuclear -catenin accumulation are seen in most gastric cancers, irrespective of morphological phenotype, and that Wnt 2 up-regulation and -catenin nuclear translocation signaling may play an important role in cancer formation, invasion and dissemination (Cheng et al., 2005). These studies indicate that disruption of Wnt/-catenin signaling is one of the mechanisms of carcinogenesis in AOM-induced colon cancer models.
107.4 Dietary fatty acid composition and colon cancer Apart from these genetic factors, environmental factors are thought to be involved in colon carcinogenesis, among which, dietary habits play a pivotal role. High consumption of meat and fat, together with low consumption of fruit, vegetables, vitamins and fiber, has been suggested to increase risk of colorectal cancer (Willett et al., 1990; Bostick et al., 2003; Giles et al., 2003; Riboli and Norat, 2003). Many epidemiological studies have revealed a positive relationship between dietary fat intake and colorectal cancer (Dai et al., 2002). Experimental studies have demonstrated that a highfat diet rich in n-6 polyunsaturated fatty acid (PUFA) and saturated fatty acids (SFAs) promotes colon carcinogenesis, particularly in post-initiation or promotional phases, and/or both (Rao et al., 2001; Dai et al., 2002; Wu et al., 2004); whereas diets rich in n-3 PUFAs and n-9 monounsaturated fatty acids (MUFAs) have been reported to inhibit colon carcinogenesis in both initiation and post-initiation phases (Bartoli et al., 2000a, b; Rao et al., 2001; Reddy, 2004). This supports epidemiological reports that show that an n-3 PUFA-rich diet suppresses the risk of colon cancer in humans (Caygill and Hill, 1995; Byers, 1996). These previous studies indicate that amount of fat intake and composition of ingested dietary fatty acids are important factors for colon carcinogenesis. Recently, we have investigated the effects of dietary intake of diverse fatty acids on colon carcinogenesis in an AOMinduced rat colon cancer model, and effects of various fatty acids on the Wnt signaling pathway (Fujise et al., 2007). Male Sprague-Dawley rats were given intraperitoneal injections of AOM once weekly for 2 weeks at a dose of 15 mg kg1 body
Chapter | 107 Azoxymethane-induced Colon Carcinogenesis
weight, whereas control rats were given an equal volume of physiological saline. One day after the first AOM or saline treatment, rats designed for corn oil, olive oil, beef tallow, and fish oil diets, began to be fed with diets high in n-6 PUFAs, n-9 MUFAs, SFAs and n-3 PUFAs; whereas, one group continued to be fed with standard chow for 44 weeks. Twelve weeks after the last injection of AOM or saline, some rats were sacrificed, and colon ACF formation was analyzed. The remaining rats were sacrificed at 44 weeks, and tumors were examined. Colon mucosa was collected for further analysis. Normal-appearing colon mucosal proliferation and apoptosis were evaluated by Bromodeoxyuridine (BrdU) incorporation and percentage fragmented DNA, respectively. Expression of -catenin, cyclin D1, Wnt2, Wnt3 and Wnt5a in normal-appearing colon mucosa was analyzed by Western blotting. Twelve weeks from the start of the experiment, colon ACF developed in all rats treated with AOM. Among AOM-treated rats, those fed with 10% corn oil and 10% beef tallow had significantly greater numbers of ACF per colon compared to rats fed with standard chow (standard chow group: 129 2.3/colon; corn oil group: 188 7.4/colon; beef tallow group: 206 4.0/colon; p 0.05), whereas numbers of ACF per colon significantly decreased in AOMtreated rats fed with 10% olive oil and 10% fish oil compared to standard chow-fed rats (standard chow group: 129 2.3/ colon; olive oil group: 94 3.1/colon; fish oil group: 96 4.9/colon; p 0.05). In contrast, few ACF were found in rats with saline treatment only. The number of multicrypt ACF, which were defined as those containing four or more aberrant crypts per focus, was significantly higher in rats fed with 10% corn oil and 10% beef tallow than in those fed with standard chow (p 0.05). On the other hand, rats fed with 10% olive oil and 10% fish oil showed a significant decrease in multicrypt ACF compared to standard chow-fed rats (p 0.05). At 44 weeks, no rat without AOM treatment developed colon adenoma or carcinoma. On the other hand, all rats fed with standard chow, 10% corn oil and 10% beef tallow diet developed colon cancer 44 weeks after the last injection of AOM (Figure 107.1). Two of six rats fed with 10% olive oil did not develop colon cancer, and four of six rats fed with 10% fish oil did not develop colon cancer. The number of colon tumors per rat was significantly higher in rats fed with 10% corn oil and 10% beef tallow than in those fed with standard chow (standard chow group: 2.3 0.5/ colon; corn oil group: 4.3 0.5/colon; beef tallow group: 5.2 0.5/colon; p 0.05), whereas tumor multiplicity in the colon of rats fed with 10% fish oil was significantly lower than in those fed with standard chow (standard chow group: 2.3 0.5/colon; fish oil group: 0.5 0.2/colon; p 0.05). The number of tumors in rats fed with 10% olive oil was smaller than that in standard chow-fed rats, but there was no significant difference between these two groups (olive oil group: 1.5 0.5/colon) (Figure 107.2). The number of BrdU-incorporated epithelial cells per crypt significantly increased in AOM-treated rats compared to saline-treated
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Figure 107.1 Colon tumors were not observed in rats without AOM treatment throughout the study period. At 44 weeks, almost all tumors showed well-differentiated adenocarcinomas with polypoid growth.
rats at 44 weeks. In rats fed with 10% corn oil and 10% beef tallow, BrdU-incorporated cell number significantly increa sed compared to that in standard chow-fed rats regardless of AOM treatment (standard chow group: 2.7 0.15/crypt; corn oil group: 3.2 0.21/crypt; beef tallow group: 3.9 0.21/ crypt; p 0.05; standard chow AOM group: 4.5 0.22/ crypt; corn oil AOM group: 6.4 0.15/crypt, beef tallow AOM group: 6.9 0.23/crypt; p 0.05). The num ber of BrdU-incorporated cells in rats fed with 10% olive oil and 10% fish oil significantly decreased (standard chow group: 2.7 0.15/crypt; olive oil group: 1.3 0.09/crypt; fish oil group: 1.8 0.21/crypt; p 0.05; standard chow AOM group: 4.5 0.22/crypt; olive oil AOM group: 1.9 0.15/crypt; fish oil group: 3.7 0.10/crypt; p 0.05) (Figure 107.3A). BrdU-incorporated epithelial cells were normally observed at the base of crypts. Among rats fed with 10% corn oil and 10% beef tallow, BrdU-positive cells spread to the upper portion of crypts even without AOM treatment. BrdU-incorporated cells were often observed at the top of crypts in rats fed with 10% beef tallow. Changes in distribution of BrdU-positive cells were more evident in rats treated with AOM. Many epidemiological and experimental studies have demonstrated that the amount and type of dietary fat play an important role in colon carcinogenesis (Reddy, 2004; Roynette et al., 2004), while controversy still exists regarding the influence of dietary fat on colon tumorigenesis. Our study showed that any type of high-fat diet did not result in the development of ACF or colon tumors per se. With AOM treatment, both 10% corn oil and 10% beef tallow diets significantly enhanced numbers of ACF and crypt multiplicity of foci 12 weeks after AOM treatment. Furthermore, all rats fed with standard chow, 10% corn oil or 10% beef tallow developed colon tumors at 44 weeks, and the number of tumors significantly increased in rats fed with 10% corn oil and 10% beef tallow compared to rats fed
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Section | II Cancer
6
* * * BrdU incorporated cell number
7 6
AOM(−) AOM(+)
* * *
* * *
**
**
5
*
4
**
*
3 2
*
**
*
1
A
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Corn oil
Olive oil
Beef tallow
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* * *
* * *
* * *
* * *
* * *
10 9 8
*
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*
7 6 5
**
**
4 3
**
2
**
1 0 B
4 3 2 1
*
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5 Tumor number/colon
at the base of crypts in the colon mucosa. In colon carcinogenesis, two main hypotheses have been proposed for morphogenesis of colon tumor: bottom-up and top-down morphogenesis. Development of human adenomatous polyps is believed to proceed through the top-down mechanism, in which genetically altered cells in the superficial portions of the mucosa spread laterally, and downward to form new
% Fragmented DNA
with standard chow. These results indicated that dietary intake of corn oil rich in n-6 PUFAs and beef tallow rich in SFAs promoted colon carcinogenesis in AOM-treated rats. In contrast to corn oil and beef tallow, olive oil rich in n-9 MUFAs and fish oil rich in n-3 PUFAs ameliorated AOMinduced ACF formation and colon carcinogenesis, compared to rats fed with standard chow. Our results were supported by previous studies that have shown that a high-fat diet rich in n-6 PUFA and SFA promotes colon tumorigenesis, particularly in post-initiation and/or promotional phases in rodents (Dai et al., 2002; Wu et al., 2004), whereas a high-fat diet rich in n-9 MUFA and n-3 PUFA inhibits colon carcinogenesis in both post-initiation and/or promotional phases (Bartoli et al., 2000; Rao et al., 2001; Reddy, 2004). Many previous studies have evaluated cancerous tissues to analyze the effects of dietary fat (Bartoli et al., 2000; Rao et al., 2001; Dai et al., 2002; Reddy, 2004; Wu et al., 2004). Our study focused on background characteristics of the colonic mucosa after long-term feeding of different types of dietary fat, and assessed colonic epithelial proliferation in normal-appearing colon mucosa in colon tumorigenesis. Few studies have investigated the effects of dietary fatty acid composition on normal-appearing colon mucosa. BrdU incorporation in normal-appearing colon mucosa surrounding colon tumors significantly increased in rats fed with 10% corn oil and 10% beef tallow, but decreased in those fed with 10% olive oil and 10% fish oil diets. These results showed that long-term feeding of 10% corn oil and 10% beef tallow accelerated the proliferation potential of the colonic mucosal epithelium, which might have promoted colon carcinogenesis after AOM treatment. Our study also showed that the range of BrdU-positive cells spread to the upper portion of crypts in rats fed with corn oil and beef tallow. Commonly, stem cells are located
Olive oil
Beef tallow
Fish oil
Figure 107.2 Tumor number in rats treated with AOM. No rat without AOM treatment showed colon tumors, including adenoma and carcinoma. Numbers of colon tumors per rat treated with AOM were significantly higher in rats fed with 10% corn oil and 10% beef tallow compared to rats fed with standard chow, whereas tumor multiplicity of the colon in rats fed with 10% fish oil diet was significantly lower than that of rats fed with standard chow. *p 0.05.
Standard chow
Corn oil
Olive oil
Beef tallow
Fish oil
Figure 107.3 Effect of dietary fatty acid components on BrdU incorporation and DNA fragmentation in the rat colon mucosa. (A) The number of BrdU-incorporated epithelial cells per crypt significantly increased in AOM-treated rats compared to rats treated with saline 44 weeks after initial treatment. Among with or without AOM-treated rats, BrdU-incorporated cell number significantly increased in rats fed with 10% corn oil and 10% beef tallow diets compared to standard chow-fed rats, and significantly decreased in rats fed with 10% olive oil and 10% fish oil. *p 0.05 compared to saline-treated rats fed with standard chow. **p 0.05 compared to AOM-treated rats fed with standard chow. ***p 0.05 compared to rats treated with saline. (B) Following AOM treatment, percentage of fragmented DNA was significantly inhibited in the colon mucosa among all dietary groups. In rats fed with 10% olive oil and 10% fish oil, % fragmented DNA was significantly higher compared to that of rats fed with standard chow regardless of AOM treatment. *p 0.05 compared to saline-treated rats fed with standard chow. **p 0.05 compared to AOMtreated rats fed with standard chow. ***p 0.05 compared to rats treated with saline.
Chapter | 107 Azoxymethane-induced Colon Carcinogenesis
crypts that connect to pre-existing normal crypts and eventually replace them (Shih et al., 2001; Wright and Poulson, 2002). Our study of BrdU-positive cells in upper portions of crypts indicated alterations in the distribution of proliferating cells in rats fed with 10% corn oil and 10% beef tallow. This suggests the involvement of top-down morphogenesis in colon carcinogenesis, which results from a high n-6 PUFA and SFA diet. In view of homeostasis of the crypt–villous axis in the colon mucosa, BMP and Hedgehog signaling without Wnt/-catenin signaling play a crucial role, including cell proliferation, differentiation and apoptosis, which is generally regulated strictly by these signal transduction pathways (Radtke and Clevers, 2005; Zhang and Li, 2005). One previous study has demonstrated altered expression of BMP in cancerous tissue compared to normal colonic mucosa (Holcombe et al., 2002). Fatty acid composition may influence the crucial signal transduction pathways, which maintain homeostasis of the crypt–villous axis in the colon mucosa, including BMP signaling and Hedgehog signaling. Apoptosis in normal-appearing colon mucosa at 44 weeks after the last injection of AOM or saline was evaluated using a DNA fragmentation assay (Figure 107.3B). Following AOM treatment, the percentage of fragmented DNA was significantly inhibited in the colon mucosa of all dietary groups. In rats fed with 10% olive oil and 10% fish oil, percentage fragmented DNA was significantly higher than that in rats fed with standard chow, with or without AOM treatment (standard chow group: 3.4 0.30%; olive oil group: 6.0 0.57%; fish oil group: 8.7 0.53%; p 0.05; standard chow AOM group: 2.6 0.33%; olive oil AOM group: 3.78 0.29%; fish oil group: 4.08 0.12%; p 0.05). In our previous study, we have revealed that a higher risk for colon cancer with dietary corn oil rich in n-6 PUFA is attributed to reduced apoptosis in the colon mucosa, and this was associated with inhibition of the tumor suppressor gene p53-mediated mitochondria-dependent apoptotic pathway (Wu et al., 2004). This study demonstrated a significant decrease in apoptosis in rats fed with 10% corn oil and 10% beef tallow, and reduction of colon mucosal apoptosis by dietary corn oil and beef tallow might be the result of accelerated cell proliferation during colon tumorigenesis. Many studies have suggested that the chemopreventive effect of n-3 PUFA was partly due to increased mucosal apoptosis (Davidson et al., 2000). This study showed that there was an apparent suppression of colon carcinogenesis, together with increased mucosal apoptosis in rats treated with AOM, by dietary intake of olive oil rich in n-9 MUFA and fish oil rich in n-3 PUFA. Increased apoptosis might be one of the crucial factors involved in tumor inhibition. Other factors, including ornithine decarboxylase activity (Rao and Reddy, 1993), diacylglycerol (Jiang et al., 1996), COX-2 and iNOS (Narayanan et al., 2003), may contribute to reduced carcinogenesis, together with n-3 PUFAs and n-9 MUFAs.
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Expression of cyclin D1 increased in AOM-treated rats. This increase was more significant in rats fed with 10% corn oil and 10% beef tallow, with or without AOM treatment, with a similar pattern as the BrdU incorporation assay. Results of the -catenin assay were similar to those with cyclin D1. Namely, accumulation of cytoplasmic catenin was significantly elevated in rats fed with 10% corn oil and 10% beef tallow, with or without AOM treatment. Thus, we investigated Wnt proteins that existed as upstream signals in the Wnt/-catenin signaling pathway. Expression of Wnt 2 and Wnt 3 was significantly higher in rats fed with 10% beef tallow, with or without AOM treatment, than that in other dietary groups. In rats fed with 10% corn oil, expression of Wnt 5a significantly increased compared to that in other rats fed with other diets. Resent studies have indicated that the Wnt signaling pathway is required during stem cell homeostasis for normal progression of intestinal epithelial cells through the crypt–villous axis (Radtke and Clevers, 2005; Reya and Clevers, 2005), and dysregulation of the Wnt signaling pathway is observed in many cancer tissues (Radtke and Clevers, 2005; Reya and Clevers, 2005). This study evaluated expression of cyclin D1, a product gene in Wnt/-catenin signaling, which is activated through accumulation of -catenin in the cytosol. Expression of cyclin D1 increased in rats fed with 10% corn oil and 10% beef tallow, and significant accumulation of -catenin in the cytosol was observed, which suggested that Wnt/-catenin signaling was activated by dietary consumption of corn oil and beef tallow. Wnt expression, an upstream signal in the Wnt/-catenin signaling pathway, increased in rats fed with corn oil; and Wnt 2 and 3 increased in rats fed with beef tallow in rat colon mucosa, regardless of AOM treatment. These results indicated that increased colon mucosal proliferation potential was, at least in part, attributed to activation of Wnt/-catenin signaling. Several studies in humans have indicated that expression of Wnt genes is accelerated in colon carcinoma tissues compared to surrounding normalappearing mucosa, using in situ hybridization (Mohammed et al., 2001; Holcombe et al., 2002). In another study of human gastric cancer, co-existence of Wnt 2 up-regulation and -catenin translocation was positively associated with lymph node metastasis (Cheng et al., 2005). In this study, it is noteworthy that up-regulation of Wnt signaling in the normal-appearing surrounding colon mucosa was observed in rats with long-term consumption of corn oil and beef tallow. Our study indicated that long-term intake of dietary SFA and n-6 PUFA accelerated colon carcinogenesis by increasing cell proliferation through up-regulating the Wnt/-catenin signaling pathway in AOM-treated rats. In contrast, n-9 MUFA and n-3 PUFA had a suppressive effect on colon carcinogenesis through decreasing cell proliferation and increasing cell apoptosis in colon mucosa. These results indicate that dietary fatty acid composition might be an important factor for modulation of mucosal proliferation potential and apoptosis.
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Many studies have investigated the inhibitory effect of olive oil on colon carcinogenesis. Dietary intake of olive oil inhibited the formation of ACF in an AOM-induced rat colon cancer model, and decreased colon mucosal arachidonate concentration compared with dietary intake of corn oil. Furthermore, AOM treatment induced a significant increase in prostaglandin E2 (PGE2) formation, but no change was found in rats fed with olive oil (Bartoli et al., 2000). The results indicated that these inhibitory effects may be due to modulation of arachidonic acid metabolism and mucosal PGE2 synthesis (Bartoli et al., 2000). Other studies using IL-10 knockout mice have shown that an olive oil diet inhibits COX-2 immunostaining in colon mucosa and decreases the risk of neoplasia-associated chronic colitis. However, a fish oil diet, which generally shows a chemopreventive effect on colon carcinogenesis, encourages chronic colitis and colitis-associated premalignant changes (Hegazi et al., 2006). An olive oil diet has an additive inhibitory effect with sulindac. These effects were mediated by regulating COX-2 expression, which is involved in prostaglandin synthesis and caspase-3, which plays a critical role in apoptosis (Schwautz et al., 2004). Recently, olive oil polyphenols have shown a strong inhibitory effect on cancer cell proliferation, which has been linked to the induction of a G2/M phase cell cycle block in cancer cell lines. These cell cycle blocks are mediated by inhibition of p38 and cyclic AMP response element binding protein (CREB) phosphorylation, which leads to a downstream reduction in COX-2 expression (Corona et al., 2007). Other studies have indicated that olive oil extract induces cell apoptosis and cell cycle arrest (Fabiani et al., 2002; Fini et al., 2008). In conclusion, many studies have indicated that olive oil has chemopreventive effects in colon carcinogenesis, through inhibition of cell proliferation and induction of apoptosis and/or cell cycle arrest (Figure 107.4). Our studies have investigated the role of the Wnt/-catenin
Section | II Cancer
signaling pathway in the colon-cancer-promoting effect of dietary fatty acid in an AOM-induced rat colon cancer model. However, the detailed mechanism associated with the inhibition of cell proliferation has not been elucidated. Further investigations are warranted to fully evaluate the mechanism of inhibitory and promoting effects of dietary fatty acid composition in colon carcinogenesis.
Summary points Many epidemiological studies have revealed a positive relationship between dietary fat intake and colorectal cancer. l Many experiments have shown that the Wnt signaling pathway plays a crucial role in the etiology of colon cancer, including hereditary and sporadic forms. l Disruption of Wnt/-catenin signaling is one of the mechanisms of carcinogenesis in AOM-induced colon cancer models. l Amount of fat intake and composition of ingested dietary fatty acids are important factors for colon carcinogenesis. l Diets rich in n-3 PUFAs and n-9 monounsaturated fatty acids (MUFAs) have been reported to inhibit colon carcinogenesis in both initiation and post-initiation phases. l Olive oil rich in n-9 MUFAs and fish oil rich in n-3 PUFAs ameliorated AOM-induced ACF formation and colon carcinogenesis, compared to rats fed with standard chow. l There was an apparent suppression of colon carcinogenesis, together with increased mucosal apoptosis in rats treated with AOM, by dietary intake of olive oil rich in n-9 MUFA and fish oil rich in n-3 PUFA. l Many studies have indicated that olive oil has chemopreventive effects in colon carcinogenesis, through inhibition of cell proliferation and induction of apoptosis and/or cell cycle arrest. l
References
Figure 107.4 Schematic design of chemopreventive effect of olive oil in colon carcinogenesis.
Barker, N., Morin, P.J., Clevers, H., 2000. The Yin-Yang of TCF/beta-catenin signaling. Adv. Cancer Res. 77, 1–24. Bartoli, R., Fernandez-Banares, F., Navarro, E., Castella, E., Mane, J., Alvarez, M., Pastor, C., Cabre, E., Gassull, M.A., 2000. Effect of olive oil on early and late events of colon carcinogenesis in rats: modulation of arachidonic acid metabolism and local prostaglandin E2 synthesis. Gut 46, 191–199. Bartoli, R., Femandes-Banares, F., Navarro, E., Castella, E., Mane, J., Alvarez, M., Pastor, C., Cabre, E., Gassull, M.A., 2000. Effect of olive oil on early and late events of colon carcinogenesis in rats: modulation of arachidonic acid metabolism and local prostaglandin E2 synthesis. Gut 46, 191–199. Bhanot, P., Brink, M., Samos, C.H., Hsieh, J.C., Wang, Y., Macke, J.P., Andrew, D., Nathans, J., Nusse, R., 1996. A new member of the frizzled
Chapter | 107 Azoxymethane-induced Colon Carcinogenesis
family from drosophila functions as a Wingless receptor. Nature 282, 225–230. Bird, R.P., 1987. Observation and quantification of aberrant crypts in the murine colon treated with a colon carcinogen: preliminary findings. Cancer Lett. 37, 147–151. Boone, C.W., Steele, V.E., Kelloff, G.J., 1992. Screening for chenopreventive (anticarcinogenic) compoumds in rodents. Mutat Res. 267, 251–255. Bostick, R.M., Potter, J.D., McKenzie, D.R., Sellers, T.A., Kushi, L.H., Steinmetz, K.A., Folson, A.R., 1993. Reduced risk of colon cancer with high intake of vitamin E: The Iowa Women’s Health Study. Cancer Res. 53, 4230–4237. Byers, T., 1996. Nutrition and cancer among American Indians and Alaska Natives. Cancer 78, 1612–1616. Cadigan, K.M., Nusse, R., 1997. Wnt signaling: a common theme in animal development. Genes. Dev. 11, 3286–3305. Caygill, C.P., Hill, M.J., 1995. Fish n-3 fatty acids and human colorectal and breast cancer mortality. Eur. J. Cancer. Prev. 4, 329–332. Cheng, X.X., Wang, Z.C., Chen, X.Y., Sun, Y., Kong, Q.Y., Liu, J., Li, H., 2005. Correlation of Wnt-2 expression and beta-catenin intracellular accumulation in Chinese gastric cancers: relevance with tumor dissemination. Cancer Lett. 223, 339–347. Corona, G., Deiana, M., Incani, A., Vauzour, D., Dessi, M.A., Spencer, J.P., 2007. Inhibition of p38/CREB phosphorylation and COX-2 expression by olive oil polyphenols underlies their anti-proliferative effects. Biochem. Biophys. Res. Commun. 362, 606–611. Crawford, H.C., Fingleton, B.M., Rudolph-Owen, L.A., Goss, K.J., Rubinfeld, B., Polakis, P., Matrisian, L.M., 1999. The metalloproteinase matrilysin is a target of beta-catenin transactivation in intestinal tumors. Oncogene 18, 2883–2891. Dai, W., Liu, T., Wang, Q., Rao, C.V., Reddy, B.S., 2002. Down-regulation of PLK3 gene expression by types and amount of dietary fat in rat colon tumors. Int. J. Oncol. 20, 121–126. Davidson, L., Brown, R., Chang, W., Morris, J., Wang, N., Carroll, R., Turner, N., Lupton, J., Chapkin, R., 2000. Morphodensitometric analysis of protein kinase C beta (II) expression in rat colon: modulation by diet and relation to in situ cell proliferation and apoptosis. Carcinogenesis 21, 1513–1519. Fabiani, R., De Bartolomeo, A., Rosignoli, P., Servili, M., Montedoro, G.F., Morozzi, G., 2002. Cancer chemoprevention by hydroxytyrosol isolated from virgin olive oil through G1 cell cycle and apoptosis. Eur. J. Cancer Prev. 11, 351–358. Fini, L., Hotchkiss, E., Fogliano, V., Graziani, G., Romano, M., De Vol, E.B., Qin, H., Selgrad, M., Boland, C.R., Ricciardiello, L., 2008. Chemopreventive properties of pinoresinol-rich olive oil involve a selective activation of ATM-p53 cascade in colon cancer cell lines. Carcinogenesis 29, 139–146. Fujise, T., Iwakiri, R., Kakimoto, T., Shiraishi, R., Sakata, Y., Wu, B., Tsunada, S., Ootani, A., Fujimoto, K., 2007. Long-term feeding of various fat diets modulates azoxymethane-induced colon carcinogenesis through Wnt/beta-catenin signalimg in rats. Am. J. Physiol. Gastrointest. Liver Physiol. 292, G1150–G1156. Giles, R.H., van Es, J.H., Clevers, H., 2003. Caught up in a Wnt storm; Wnt signaling in cancer. Biochim. Biophys. Acta 1653, 1–24. Groden, J., Thliveris, A., Samowitz, W., Carlson, M., Gelgert, L., Albertsen, H., Joslyn, G., Stevens, J., Spilio, L., Robertson, M., 1991. Identification and characterization of the familial adenomatous polyposis coli gene. Cell 66, 589–600. He, T.C., Sparks, A.B., Rago, C., Hermeking, H., Zawel, L., da Costa, L.T., Morin, P.J., Vogelstein, B., Kinzler, K.W., 1998. Identification of c-MYC as a target of the APC pathway. Science 281, 1438–1439.
1003
He, X., Saint-Jeannet, J.-P., Wang, Y., Nathans, J., Dawid, I., Varmus, H., 1997. A member of the Frizzled protein family mediating axis induction by Wnt-5a. Science 275, 1652–1654. Hegazi, R.A., Saad, R.S., Mady, H., Matarese, L.E., O’Keefe, S., Kandil, H.M., 2006. Dietary fatty acids modulate chronic colitis, colitis-associated colon neoplasia and COX-2 exoression in IL-10 knock out mice. Nutrition 22, 275–282. Holcombe, R.F., Marsh, J.L., Waterman, M.L., Lin, F., Milovanovic, T., Truong, T., 2002. Expression of Wnt ligands and frizzled receptors in colonic mucosa and in colon carcinoma. J. Clin. Pathol.: Mol. Pathol. 55, 220–226. Howe, L.R., Subbaramaiah, K., Chung, W.J., Dannenberg, A.J., Brown, A.M., 1999. Transcriptional activation of cyclooxygenase-2 in Wnt1-transformed mouse mammary epithelial cells. Cancer Res. 59, 1572–1577. Jiang, Y., Lupton, J., Chapkin, R., 1996. Dietary fat and fiber differentially alter intracellular second messengers during tumor development in rat colon. Carcinogenesis 17, 1227–1233. Kinzler, K.W., Vogelstein, B., 1996. Lessons from hereditary colorectal cancer. Cell 87, 159–170. Liu, W., Dong, X., Mai, M., Seelan, R.S., Taniguchi, K., Krishnandath, K.K., Halling, K.C., Cunningham, J.M., Boardman, L.A., Qian, C., Christensen, E., Schmidt, S.S., Roche, P.C., Smith, D. I., Thibodeau, S.N., 2000. Mutations in Axin2 cause colorectal cancer with detective mismatch repair by activating beta-catenin–Tcf signaling. Nat. Genet. 26, 146–147. Mohammed, I.M., Roczo, N., Phillips, W.A., Baindur-Hudson, S., 2001. Expression of Wnt genes in human colon cancers. Cancer Lett. 166, 185–191. Narayanan, B., Narayanan, N., Simi, B., Reddy, B.S., 2003. Modulation of inducible nitric oxide synthase and related proinflammatory genes by the omega-3 fatty acid docosahexaenoic acid in human colon cancer cell. Cancer Res. 63, 972–979. Noda, T., Iwakiri, R., Fujimoto, K., Matsuo, S., Aw, T.Y., 1998. Programmed cell death induced by ischemia-reperfusion in rat intestinal mucosa. Am. J. Physiol. 274, G270–G276. Pretow, T.P., Cheyer, C., O’Riordan, M.A., 1994. Aberrant crypt foci and colon tumors in F344 rats have similar increases in proliferative activity. Int. J. Cancer 56, 599–602. Pretow, T.P., O’Riordan, M.A., Somich, G.A., Amini, S.B., Pretlow, T.G., 1992. Aberrant crypts correlate with tumor incidence in F344 rats treated with azoxymethane and phytate. Carcinogenesis 13, 1509–1512. Radtke, F., Clevers, H., 2005. Self-renewal and cancer of the gut: Two sides of a coin. Science 307, 1904–1909. Rao, C., Reddy, B.S., 1993. Modulating effect of amount and types of dietary fat on ornithine decarboxylase, tyrosine protein kinase and prostaglandins production during colon carcinogenesis in F344 rats. Carcinogenesis 14, 1327–1333. Rao, C.V., Hirose, Y., Indranie, C., Reddy, B.S., 2001. Moduration of experimental colon tumorigenesis by types and amounts of dietary fatty acids. Cancer Res. 61, 1927–1933. Reddy, B.S., 2004. Omega-3 fatty acids in colorectal cancer prevention. Int. J. Cancer 112, 1–7. Reya, T., Clevers, H., 2005. Wnt signaling in stem cells and cancer. Nature 434, 843–850. Reya, T., Clevers, H., 2005. Wnt signaling in stem cells and cancer. Nature 434, 843–850. Riboli, E., Norat, T., 2003. Epidemiologic evidence of the protective effect of fruit and vegetables on cancer risk. Am. J. Clin. Nutr. 78, 559S–569S.
1004
Roose, J., Huls, G., van Beest, M., Moerer, P., van der Horn, K., Goldschmeding, R., Logtenberg, T., Clevers, H., 1999. Synergy between tumor suppressor APC and the beta-catenin-Tcf4 target Tcf1. Science 285, 1923–1926. Roynette, C.E., Calder, P.C., Dupertuis, Y.M., Pichard, C., 2004. n-3 polyunsaturated fatty acids and colon cancer prevention. Clin. Nutr. 23, 139–151. Schwautz, B., Birk, Y., Raz, A., Madar, Z., 2004. Nutritional-pharmacological combinations. Eur. Nutr. 43, 221–229. Shih, I.M., Wang, T.L., Traverso, G., Romans, K., Hamilton, S.R., BenSasson, S., Kinzler, K.W., Vogelstein, B., 2001. Top-down morphogenesis of colorectal tumors. PNAS 27, 2640–2645. Tetsu, O., McCormick, F., 1999. Beta-catenin regulates expression of Cyclin D1 in colon carcinoma cells. Nature 398, 422–426. von kries, J.P., Winbeck, G., Asbrand, C., Schwarz-Romond, T., Sochnikowa, N., Dell’Oro, A., Behrens, J., Birchmeier, W., 2000.
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Hot spots in beta-catenin for interactions with LEF-1, conduction and APC. Nat. Struct. Biol. 7, 800–807. Willett, W.C., Stampfer, M.J., Colditz, G.A., Rosner, B.A., Speizer, F.E., 1990. Relation of meat, fat, and fiber intake to the risk of colon cancer in a prospective study among women. N. Eng. J. Med. 323, 1664–1672. Wright, N.A., Poulson, R., 2002. Top down or bottom up? Competing management structures in the morphogenesis of colorectal neoplasm. Gut 51, 306–308. Wu, B., Iwakiri, R., Ootani, A., Tsunada, S., Fujise, T., Sakata, Y., Sakata, H., Toda, S., Fujimoto, K., 2004. Dietary corn oil promotes colon cancer by inhibiting mitochondria-dependent apoptosis in azoxymethane-treated rats. Exp. Biol. Med. 229, 1017–1025. Zhang, J., Li, L., 2005. BMP signaling and stem cell regulation. Dev. Biol. 284, 1–11.