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Am J Physiol Gastrointest Liver Physiol 292: G1150-G1156, 2007. First published December 28, 2006; doi:10.1152/ajpgi.00269.2006 Free Article
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MUCOSAL BIOLOGY

Long-term feeding of various fat diets modulates azoxymethane-induced colon carcinogenesis through Wnt/beta-catenin signaling in rats

Takehiro Fujise, Ryuichi Iwakiri, Takashi Kakimoto, Ryosuke Shiraishi, Yasuhisa Sakata, Bin Wu, Seiji Tsunada, Akifumi Ootani, and Kazuma Fujimoto

Department of Internal Medicine and Gastrointestinal Endoscopy, Saga Medical School, Saga, Japan

Submitted 19 June 2006 ; accepted in final form 25 December 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The Wnt signaling pathway plays an essential role in carcinogenesis, and the amount of fat intake and composition of dietary fatty acids are crucial factors for colon carcinogenesis. We investigated whether various dietary fats affected the Wnt signaling pathway of colon tumorigenesis in azoxymethane (AOM)-treated rats. Male Sprague-Dawley rats were given intraperitoneal injections of AOM and supplemented with 10% corn, olive, beef, and fish oil for 44 wk. Aberrant crypt foci (ACF) and tumors were examined at 12 and 44 wk. Normal appearing colon mucosal proliferation and apoptosis were evaluated by 5-bromo-2'-deoxyuridine (BrdU) incorporation and percentages of fragmented DNA, respectively. Expressions of beta-catenin, cyclin D1, Wnt2, Wnt3, and Wnt5a of normal appearing colon mucosa were analyzed by Western blot analysis. Long-term dietary corn oil and beef tallow increased ACF, tumor incidence, and tumor numbers in AOM-treated rats. In contrast, both olive and fish oil inhibited them. Dietary corn oil and beef tallow increased BrdU incorporation and the expression of cytosolic beta-catenin and cyclin D1 and decreased apoptosis in the colon mucosa. Expressions of Wnt2 and Wnt3 in rats fed with beef tallow and Wnt5a in rats fed with corn oil increased with or without AOM-treatment. BrdU-incorporated cells were often observed at the tops of crypts in rats fed with beef tallow, whereas this was not observed in rats fed with the other diet. Long-term high intake of corn oil and beef tallow enhanced cell proliferation through Wnt signaling and modulated the distribution of proliferating cells, which might contribute to promoting effects in colon tumorigenesis.

fatty acid

THE ETIOLOGY of colorectal cancer is very complex, and both genetic and environmental factors are thought to be involved in this process. Among environmental factors, dietary habits play an important role. High consumption of meat and fat, together with low consumption of fruits, vegetables, vitamins, and fibers, has been suggested to increase risks of colorectal cancer (5, 13, 31, 37). Many epidemiological studies have demonstrated a positive relationship between dietary fat intake and colorectal cancer (37). Experimental studies have shown that a high-fat diet rich in n-6 polyunsaturated fatty acid (PUFA) and saturated fatty acids (SFA) promoted colon carcinogenesis, particularly in postinitiation or promotional phases and/or both (10, 26, 28, 39). On the other hand, diets rich in n-3 PUFA and n-9 monounsaturated fatty acid (MUFA) have been reported to reduce colon tumorigenesis in both the initiation and postinitiation phases (2, 26, 29), supporting epidemiological reports showing that an n-3 PUFA-rich diet suppressed the risk of colon cancer in humans (3, 7, 8). These experimental and epidemiological studies suggested that not only the amount of fat intake but also the composition of ingested dietary fatty acids are crucial factors for colon carcinogenesis.

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 (12, 30). The key molecule in this pathway is a multiprotein scaffold consisting of beta-catenin, glycogen synthase kinase (GSK)-3beta, and adenomatous polyposis coli (APC). The APC gene was first identified by genetic analysis in patients with a hereditary cancer syndrome termed familial adenomatous polyposis (13). Hereditary forms of colorectal cancer and ~85% of all sporadic colorectal cancers show loss of APC function (16). It is commonly accepted that the crucial tumor suppressor role of APC lies in its ability to destabilize cytoplasmic free beta-catenin (35). Therefore, disrupted regulation of the Wnt signaling pathway plays a central role in the etiology of colon carcinogenesis (17, 19, 35).

Aberrant crypt foci (ACFs) were first identified in the colon mucosa of rodents exposed to carcinogens (4) and have also been confirmed to be present in the human colon (21). ACFs are regarded as preneoplastic or precancerous lesions in the colorectum of humans and rodents (4, 21). The number of crypts/foci was shown to increase with time following carcinogen treatment, and ACFs demonstrate increased cell proliferation in rodents (23, 24).

The present study aimed to investigate effects of dietary intake of diverse fatty acids on colon carcinogenesis in the azoxymethane (AOM)-induced rat colon cancer model and the effects of various fatty acids on the Wnt signaling pathway.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and experimental procedures. Male Sprague-Dawley rats were used in this study. Rats were divided into five dietary groups: standard chow, 10% corn oil, 10% olive oil, 10% beef tallow, and 10% fish oil. Each dietary group was then divided into AOM-treated and vehicle-treated subgroups. Rats were housed in plastic cages with filter tops in a holding room that was maintained under controlled conditions (illumination from 8:00 to 20:00). All rats had access to water and food ad libitum.

The experimental design is summarized in Fig. 1. Beginning at 6 wk of age, all rats were fed with standard chow. At 7 wk of age, rats intended for carcinogen treatment were intraperitoneally injected with AOM (Sigma, St. Louis, MO) dissolved in 1 ml physiological saline solution (PSS) once a week for 2 wk at a dose of 15 mg/kg body wt, whereas control rats were given an equal volume of PSS alone. One day after AOM or saline treatment, groups of rats designed for the corn oil, olive oil, beef tallow, and fish oil diets began to be fed with diets high in n-6 PUFA, high n-9 MUFA, high SFA, or high n-3 PUFA, whereas one group continued to be fed with standard chow. The fatty acid composition of standard chow consisted of 14.6% palmitic acid, 24.6% oleic acid, 46.6% linoleic acid, 3.8% {alpha}-linolenic acid, and 2.6% stearic acid. The energy of standard chow was 3.6 kcal/g and that of the 10% oil diet was 4.04 kcal/g. The corn oil diet included 10.5% palmitic acid, 29.0% oleic acid, 56.8% linoleic acid, and 1.9% stearic acid. The olive oil diet included 7.0% palmitic acid, 78.0% oleic acid, 12.0% linoleic acid, and 1.0% stearic acid. The beef tallow diet included 32.5% palmitic acid, 41.2% oleic acid, 2.7% linoleic acid, and 14.5% stearic acid. The fish oil diet included 12.0% docosahexaenoic acid and 18.6% eicosapentaenoic acid. Twelve weeks after the last injection of AOM or saline, some rats were killed, and colon ACF formation was analyzed. The remaining rats were killed at 44 wk, and the colon mucosa was collected for further analysis.


Figure 1
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  		Fig. 1. Experimental design. Groups of male Sprague-Dawley rats were fed with standard chow. At the start of the experiments, when animals were 7–8 wk old, azoxymethane (AOM) was administered intraperitoneally at a dose of 15 mg/kg rat body wt. One day after AOM-treatment, rats were divided into the following respective experimental diets: standard chow, 10% corn oil, 10% olive oil, 10% beef tallow, and 10% fish oil. PUFA, polyunsaturated fatty acid; MUFA, monounsaturated fatty acid; SFA, saturated fatty acid; w, weeks.

 
Collection of colon tissue samples. Under halothane anesthesia, rats were killed. After a laparotomy, the entire colon was carefully removed, flushed thoroughly with PSS, and opened longitudinally on its antimesenteric border to expose the luminal side. The mucosa and tumors were carefully harvested, respectively.

ACF analysis. For ACF analysis, 12 wk after the last injection of AOM or saline, rats were killed, and the colon was carefully removed. The colon was flushed with ice-cold PSS, opened longitudinally, placed with the flat mucosal side up between two pieces of wet filter papers, and fixed in 10% buffered formalin for ACF analysis. After a minimum of 24 h in buffered formalin, the colon was stained with 0.2% methylene blue dissolved in the same formalin solution for 5 min and rinsed with PSS. After being stained, the colon was placed with the mucosal side up on a slide, observed using a stereoscopic microscope, and assayed for numbers and multiplicity of crypts. ACFs were distinguished from the surrounding normal crypts by their features and defined as single or multiple crypts according to 1) whether they had altered luminal openings, 2) whether they exhibited thickened epithelia, and 3) whether they were larger than adjacent normal crypts, according to Bird (4). Crypt multiplicity corresponded to the number of crypts per foci and was categorized as containing <3 or ≥4 aberrant crypts/foci. Six rats were evaluated in each group.

5-Bromo-2'-deoxyuridine incorporation assay. We evaluated cell proliferation in the surrounding normal mucosa by 5-bromo-2'-deoxyruridine (BrdU) incorporation using the Cell Proliferation Kit (Amersham Pharmacia Biotech). Briefly, rats were intraperitoneally injected with the labeling reagent, which was supplied as a concentrated aqueous solution of BrdU and 5-fluoro-2'-deoxyuridine (10:1 ratio) at a dose of 10 ml/kg body wt at 44 wk after the last injection of AOM or saline. Two hours after the injection of the labeling reagent, rats were killed, and the colon was carefully removed. The colon was immediately fixed in 10% neutral buffered formalin. Samples were then embedded in paraffin and sectioned. Specimen were dewaxed and immersed in PBS containing 0.3% hydrogen peroxide for 10 min at room temperature. After specimens had been washed three times with PBS, sufficient reconstituted nuclease/anti-BrdU solution was applied directly onto the specimen. Colons were incubated for 2 h at room temperature. After specimens had been washed three times with PBS, sufficient peroxidase-anti-mouse IgG2a solution was added onto specimens. Specimens were incubated for 1 h at room temperature. They were then soaked in PBS containing 0.02% diaminobenzidine and substrate/intensifier at a dose of 5 drops/50 ml diaminobenzidine solution for 1 min for color development. Finally, specimens were counterstained by an immersion in methyl green. A minimum of 50 crypts were randomly selected for the BrdU incorporation assay, and numbers of cells were assessed. Six rats were tested in each group.

DNA fragmentation assay. Forty-four weeks after the last injection of AOM or saline, colonic mucosa of all groups of rats were examined. The mucosal scraping was processed immediately after collection to minimize nonspecific DNA fragmentation. The mount of fragmented DNA was determined as previously described (1, 21) with some modifications. Briefly, mucosal scrapings of different intestinal segments were homogenized in 10 volumes of lysis buffer consisting of 5 mM Tris·HCl, 20 mM EDTA, and 0.5% (wt/vol) Triton X-100 (pH 8.0). A 1-ml aliquot of each sample was centrifuged for 20 min at 27,000 g to separate intact chromatin (pellet) from fragmented DNA (supernatant) (40). The supernatant was decanted and saved, and the pellet was resuspended in 1 ml Tris buffer (pH 8.0) with 10 mM Tris·HCl and 1 mM EDTA. Pellet and supernatant fractions were assayed for DNA content using a diphenylamine reaction as previously described (6). Results are expressed as percentages of fragmented DNA. Six rats were tested in each group.

Western blot analysis. Forty-four weeks after the last injection of AOM or saline, colonic mucosa of all groups of rats were examined. The mucosal scraping was immediately washed twice with ice-cold PBS (pH 7.4) and centrifuged at 1,000 g for 5 min at 4°C. The pellet was then resuspended with 2 volumes of buffer A and lysed at 4°C for 30 min. Buffer A consisted of 250 mM sucrose (Sigma), 20 mM HEPES (Sigma)/KOH (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leuptin, and 1.8 mg/ml iodoacetamide. The homogenate was centrifuged at 1,000 g for 10 min at 4°C to remove nuclei, and the supernatant was again centrifuged at 10,000 g for 15 min at 4°C to remove mitochondria. The supernatants of the 10,000-g spin were further centrifuged at 1,000,000 g for 1 h at 4°C. The resulting supernatant was the soluble cytosolic fraction, and the pellet was the membranous fraction. The supernatant and pellet fractions of the resuspended solution in buffer A were divided into multiple samples and frozen at –80°C for immunoblot analysis.

The amounts of cytoplasmic beta-catenin, cyclin D1, Wnt2, Wnt3, and Wnt5a were determined. Equal quantities (40 µg) of protein were electrophoresed in a SDS-polyacrylamide gel. They were then electroblotted onto a polyvinylidene difluoride membrane (Trans-Blot, Bio-Rad, Hercules, CA). After being blocked with PBS containing 0.1% Tween 20 (Sigma) and 5% skim milk at 4°C overnight, the membrane was incubated with mouse monoclonal anti-beta-catenin antibody (1:500, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal anti-cyclin D1 antibody (1:500, Santa Cruz Biotechnology), goat polyclonal anti-Wnt2 antibody (1:500, Santa Cruz Biotechnology), goat polyclonal anti-Wnt3 antibody (1:500, Santa Cruz Biotechnology), and goat polyclonal anti-Wnt5a antibody (1:500, Santa Cruz Biotechnology), respectively, for 1 h. Antigen-antibody complexes were detected with horseradish peroxidase-conjugated anti-rabbit IgG (1:500, Santa Cruz Biotechnology), horseradish peroxidase-conjugated anti-mouse IgG (1:500, Santa Cruz Biotechnology), and horseradish peroxidase-conjugated anti-goat IgG (1:500, Santa Cruz Biotechnology). Detection of chemiluminescence was performed using ECL Western blotting detection reagents (Amersham Pharmacia Biotech). Densitometoric assessment of the bands on the autoradiogram was done using Image Gauge VDS (Fujifilm, Tokyo, Japan). Band intensities were quantified by measurements of absolute integrated optical intensities, estimating bands in each lane profile. Results are expressed as ratios to beta-actin densitometry units. Six rats were tested in each group.

Statistical analysis. Results are expressed as means ± SE. Data were evaluated by ANOVA in which multiple comparisons were performed using the least-significant difference method. Colon cancer incidence was expressed as percentages of animals with cancer, and results were statistically analyzed using the {chi}2-test. Differences were considered significant if the probability of the difference occurring by chance was <5 in 100 (P < 0.05).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
ACF formation at 12 wk. ACFs showed dilated irregular luminal openings, thicker epithelial linings, and protrusions toward the lumen. Twelve weeks from the start of the experiment series, few ACFs were found in rats without AOM treatment. In contrast, colon ACFs developed in all rats treated with AOM (Fig. 2A). Among AOM-treated rats, rats fed with 10% corn oil and 10% beef tallow diets had significantly greater numbers of ACFs per colon compared with rats fed with standard chow (P < 0.05). On the other hand, numbers of ACFs per colon significantly decreased in AOM-treated rats fed with 10% olive oil and 10% fish oil diets compared with standard chow-fed rats (P < 0.05).


Figure 2
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  		Fig. 2. Effects of dietary fatty acid components on aberrant crypt foci (ACF) formation in AOM-treated rats. A: mean numbers of total colon ACFs formed in male AOM-treated rats. B: mean numbers of aberrant crypt foci containing 4 or more aberrant crypts in male AOM-treated rats. Values are means ± SD; 6 rats were studied in each group. *P < 0.05 compared with AOM-treated rats fed with standard chow.

 
Crypt multiplicity of ACFs in rats treated with AOM is shown in Fig. 2B. Numbers of multicrypt ACFs, which were determined as containing up to 4 or more aberrant crypts per focus, were significantly higher in rats fed with 10% corn oil and 10% beef tallow diets than that of rats fed with standard chow (P < 0.05), whereas rats fed with 10% olive oil and 10% fish oil diets showed a significant decrease in numbers of multicrypt ACFs compared with standard chow-fed rats (P < 0.05).

Tumor incidence and tumor multiplicity at 44 wk. Results of tumor incidence and tumor multiplicity are summarized in Table 1. At 44 wk, 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 diets developed colon cancer 44 wk after the last injection of AOM. 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 numbers of colon tumors per rat were significantly higher in rats fed with 10% corn oil and 10% beef tallow than those of rats fed with standard chow (P < 0.05), whereas tumor multiplicity in colons of rats fed with 10% fish oil was significantly lower than that of rats fed with standard chow (P < 0.05). The number of tumors in rats fed with 10% olive oil was smaller than that of standard chow-fed rats, but there was no statistical significance between these two groups.


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  		Table 1. Tumor incidence and tumor multiplicity in rats treated with AOM

 
BrdU incorporation by the normal mucosal epithelium surrounding tumors. We evaluated BrdU incorporation of tumor tissues surrounding normally appearing mucosa to evaluate proliferation potential. The numbers of BrdU-incorporated epithelial cells per crypt significantly increased in AOM-treated rats compared with saline-treated rats at 44 wk (Fig. 3). In rats fed with 10% corn oil and 10% beef tallow, BrdU-incorporated cell numbers significantly increased compared with standard chow-fed rats regardless of AOM treatment (P < 0.05). BrdU-incorporated cell numbers of rats fed with 10% olive oil and 10% fish oil significantly decreased (P < 0.05). BrdU-incorporated epithelial cells were normally observed at the base of crypts (Fig. 4A). 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 (Fig. 4B). BrdU-incorporated cells were often observed at the top of crypts in saline-treated rats fed with 10% beef tallow (Fig. 4C). Changes in the distribution of BrdU-positive cells were more evident in rats treated with AOM (not shown).


Figure 3
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  		Fig. 3. Effects of various fat diets on numbers of 5-bromo-2'-deoxyuridine (BrdU)-incorporated cells in surrounding normal appearing mucosa. Numbers of BrdU-incorporated epithelial cells per crypt significantly increased in AOM-treated rats compared with rats treated with saline 44 wk after the initial treatment. Among with or without AOM-treated rats, BrdU-incorporated cell numbers significantly increased in rats fed with 10% corn oil and 10% beef tallow diets compared with standard chow-fed rats and significantly decreased in rats fed with 10% olive oil and 10% fish oil diets. Open bars, rats treated with saline; solid bars, rats treated with AOM. Values are means ± SD; 6 rats were studied in each group. *P < 0.05 compared with saline-treated rats fed with standard chow; **P < 0.05 compared with AOM-treated rats fed with standard chow; ***P < 0.05 compared with rats treated with saline.

 

Figure 4
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  		Fig. 4. Histological findings of surrounding normal appearing mucosa. A: saline-treated rats fed with standard chow. B and C: saline-treated rats fed with 10% beef tallow. Magnifications: x100. BrdU-incorporated colon epithelial cells were normally observed mainly at the base of crypts (A). Among rats without AOM treatment, the distribution of BrdU-positive cells showed spreading into the upper portion of crypts in rats fed with 10% corn oil (data was not shown) and 10% beef tallow (B). Interestingly, BrdU-positive cells were often observed at the top of crypts in rats fed with 10% beef tallow (C).

 
Mucosal apoptosis. Apoptosis in tumor tissues surrounding normal appearing colon mucosa at 44 wk after the last injection of AOM or saline was evaluated using a DNA fragmentation assay (Fig. 5). Following AOM treatment, percentages of fragmented DNA were significantly inhibited in the colon mucosa of all dietary groups. In rats fed with 10% olive oil and 10% fish oil, percentages of fragmented DNA were significantly higher than those in rats fed with standard chow with or without AOM treatment. In AOM-treated rats, the villous height decreased in rats fed with 10% olive oil and 10% fish oil compared with rats fed with a normal diet, but no significant differences were found in rats fed with 10% corn oil and 10% beef tallow compared with rats fed with standard chow (villous height: fish oil, 1,615 ± 35 µm; olive oil, 1,714 ± 33.3 µm; normal chow, 1,961 ± 34.6 µm; corn oil, 2,051 ± 31.7 µm; and beef tallow, 2,010 ± 50.8 µm; P < 0.05 compared with rats fed with standard chow).


Figure 5
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  		Fig. 5. Effects of various fat diets on percentages of fragmented DNA in surrounding normal appearing mucosa. Following AOM treatment, percentages of fragmented DNA were significantly inhibited in the colon mucosa among all dietary groups. In rats fed with 10% olive oil and 10% fish oil, percentages of fragmented DNA were significantly higher than that of rats fed with standard chow regardless AOM treatment. Open bars, rats treated with saline; solid bars, rats treated with AOM. Values are means ± SD; 6 rats were studied in each group. *P < 0.05 compared with saline-treated rats fed with standard chow; **P < 0.05 compared with AOM-treated rats fed with standard chow; ***P < 0.05 compared with rats treated with saline.

 
Western blot analysis of proteins associated with the cell cycle and Wnt signaling. First, we evaluated the expression of cyclin D1 and accumulation of beta-catenin in the cytosol of the mucosal epithelium to evaluate cell cycle activity. As shown in Fig. 6, A and B, the 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, showing similar patterns as the BrdU incorporation assay. Results of the beta-catenin assay were similar to those of cyclin D1, namely, accumulation of cytoplasmic beta-catenin was significantly elevated in rats fed with 10% corn oil and 10% beef tallow with or without AOM treatment (P < 0.05). Second, we investigated Wnt proteins, which existed as upstream signals in the Wnt/beta-catenin signaling pathway. As shown in Fig. 7, expressions of Wnt2 and Wnt3 were significantly higher in rats fed with 10% beef tallow with or without AOM treatment than those in other dietary groups (P < 0.05 in each; Fig. 7, A and B). In rats fed with 10% corn oil, the expression of Wnt5a significantly increased compared with other rats fed with other diets (P < 0.05 in each; Fig. 7C).


Figure 6
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  		Fig. 6. Effects of various fat diets on expressions of cyclin D1 and cytosolic beta-catenin in surrounding normal appearing mucosa. A: cyclin D1. B: cytosolic beta-catenin. The expression of cyclin D1 increased in AOM-treated rats. This increase was observed more significantly in rats fed with 10% corn oil and 10% beef tallow regardless of AOM treatment. Results for beta-catenin were similar to those with cyclin D1. The accumulation of cytosolic beta-catenin was significantly elevated in rats fed with 10% corn oil and 10% beef tallow regardless of AOM treatment. Open bars, rats treated with saline; solid bars, rats treated with AOM. Values are means ± SD; 6 rats were studied in each group. *P < 0.05 compared with saline-treated rats fed with standard chow; **P < 0.05 compared with AOM-treated rats fed with standard chow; ***P < 0.05 compared with rats treated with saline.

 

Figure 7
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  		Fig. 7. Effects of various fat diets on expressions of Wnts in surrounding normal appearing mucosa. A: Wnt2. B: Wnt3. C: Wnt5a. Expressions of Wnt2 and Wnt3 were significantly higher in rats fed with 10% beef tallow regardless of AOM treatment. In rats fed with 10% corn oil, the expression of Wnt5a was significantly increased compared with other dietary groups. Open bars, rats treated with saline; solid bars, rats treated with AOM. Values are means ± SD; 6 rats were studied in each group. *P < 0.05 compared with saline-treated rats fed with standard chow; **P < 0.05 compared with AOM-treated rats fed with standard chow; ***P < 0.05 compared with rats treated with saline.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Many epidemiological and experimental studies have demonstrated that not only the amount but also the type of dietary fats differing in fatty acid composition were important in colon tumorigenetic processes (29, 32, 36), whereas controversies still exist regarding the influence of dietary fat on colon carcinogenesis. This study demonstrated that any type of high-fat diet did not result in the development of ACFs or colon tumors per se. With AOM treatment, both 10% corn oil and 10% beef tallow diets significantly enhanced the numbers of ACF and multiplicity of foci 12 wk after the start of experiments. In addition, all experimental rats fed with standard chow, 10% corn oil, and 10% beef tallow developed colon tumors 44 wk after AOM treatment, and the numbers of tumors significantly increased in rats fed with corn oil and beef tallow compared with standard chow-fed animals. These results indicated that the dietary intake of corn oil (rich in n-6 PUFA) and beef tallow (rich in SFA) promoted colon carcinogenesis in AOM-treated rats. In contrast to corn oil and beef tallow, olive oil (rich in n-9 MUFA) and fish oil (rich in n-3 PUFA) ameliorated AOM-induced ACF formation and colon carcinogenesis in this study. Our results were supported by those of previous studies showing that a high-fat diet rich in n-6 PUFA and SFA promoted colon carcinogenesis, particularly in the postinitiation and/or promotional phases in rodents (10, 28, 39), whereas a high-fat diet rich in n-9 MUFA and n-3 PUFA inhibited colon tumorigenesis in both the initiation and postinitiation phases (2, 26, 29).

Most of the previous studies have examined cancerous tissues themselves to evaluate the effects of dietary fat (2, 10, 26, 28, 29, 39). This study focused on background characteristics of the colon mucosa after long-term dietary fat and evaluated proliferation states in normally appearing colon mucosa for carcinogenetic processes, because few studies have investigated the effects of dietary fat intake and fatty acid composition on normally appearing colon mucosa. BrdU incorporation of normally appearing colon mucosa surrounding colon tumors significantly increased in rats fed with 10% corn oil and 10% beef tallow but decreased in rats fed with 10% olive oil and 10% fish oil diet. These results indicated that long-term intake of 10% corn oil and 10% beef tallow accelerated the proliferation potential of the colon mucosa, which might promote colon carcinogenesis after AOM treatment. This study also demonstrated that the range of BrdU-positive cells spread to the upper potion of crypts in rats fed with corn oil and beef tallow. Two main hypotheses have been proposed for morphogenesis in a colon tumor: bottom-up morphogenesis and top-down morphogenesis. The 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 crypts that would connect to preexisting normal crypts and eventually replaced them (33, 38). Our observation of BrdU-positive cells in upper portions of the crypts indicated alterations in the distribution of proliferating cells in rats fed with corn oil and beef tallow, suggesting the involvement of top-down morphogenesis in carcinogenesis resulting from a high-fat diet.

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 (25, 30), and dysregulation of the Wnt signaling pathway has been observed in many cancer tissues (25, 30). This study evaluated the expression of cyclin D1, a product gene in Wnt/beta-catenin signaling activated through accumulation of beta-catenin in the cytosol. The expression of cyclin D1 increased in rats fed with 10% corn oil and 10% beef tallow, and a significantly high accumulation of beta-catenin in the cytosol was observed, suggesting that Wnt/beta-catenin signaling was activated by dietary consumption of corn oil and beef tallow. Wnt expression, an upstream signal in the Wnt/beta-catenin signaling pathway, increased in rats fed with corn oil; and Wnt2 and Wnt3 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 the activation of Wnt/beta-catenin signaling. Several studies in humans have indicated that expressions of Wnt genes were accelerated in colon carcinoma tissues compared with surrounding normally appearing mucosa using in situ hybridization (14, 18). In another study of human gastric cancer, coexistence of Wnt2 upregulation and beta-catenin translocation was positively associated with lymph node metastasis (9). In this study, it is noteworthy that the upregulation of Wnt signaling in the normally appearing surrounding colon mucosa was observed in rats with long-term consumption of corn oil and beef tallow. In this study, Wnt2 and Wnt3 were upregulated in beef tallow-fed rats, and Wnt5a was upregulated in corn oil-fed rats. This kind of difference between these Wnt proteins has not been clearly demonstrated in previous publications, and this point warrants further exploration.

We previously demonstrated that a higher risk for colon cancer by dietary corn oil rich in n-6 PUFA was attributed to the reduction of apoptosis in the colon mucosa, and decreased apoptosis in the colon mucosa has been associated with the inhibition of the tumor suppressor gene p53-mediated mitochondria-dependent apoptotic pathway (39). This study showed a significant decrease in apoptosis in rats fed with corn oil and beef tallow, and a reduction of colon mucosal apoptosis by dietary corn oil and beef tallow might be the result of accelerated cell proliferation during the cancer promoting process. Many reports have suggested that the chemopreventive effect of n-3 PUFA was partly due to increased mucosal apoptosis (11). The present study indicated 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. The increase in apoptosis might be one of the factors involved in tumor-inhibiting processes. In addition to the results of this study, other factors including ornithine decarboxylase activity (27), diacylglycerol (15), cyclooxygenase 2 (26, 34), and inducible nitric oxide synthase (20) may contribute to the reduced carcinogenesis together with n-3 PUFA and n-9 MUFA.

In conclusion, this study indicated that long-term intake of dietary SFA and n-6 PUFA accelerated colon carcinogenesis by increasing cell proliferation through the upregulation of the Wnt/beta-catenin signaling pathway in AOM-treated rats. In contrast, n-9 MUFA and n-3 PUFA had suppressive effects on colon carcinogenesis. These results indicated that dietary fatty acid composition might be an important factor for the modulation of mucosal proliferation potential.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by the president's expenditure (research project expenditure) of Saga University and Ministry of Education, Science and Culture in Japan Grants-In-Aid for Scientific Research 15590658 and 18590690 (to K. Fujimoto) and 16590605 and 18590691 (to R. Iwakiri).


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Fujimoto, Dept. of Intestinal Medicine, Saga Medical School, 5-1-1 Nabeshima, Saga 849-8501, Japan (e-mail: fujimoto{at}med.saga-u.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Aw TY, Nicotera P, Manzo L, Orrenius S. Tributyltin stinulates apoptosis in rat thymocytes. Arch Biochem Biophys 283: 46–50, 1990.[CrossRef][ISI][Medline]
  2. Bartoli R, Femandes-Banares F, Navarro E, Castella E, Mane J, Alvarez M, Pastor C, Cabre E, Gassull MA. 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, 2000.[Abstract/Free Full Text]
  3. Bingham SA. Epidemiology of colorectal cancer. In: Encyclopedia of Human Nutrition, edited by Strain J, Sadler M, Caballero B. New York: Academic, 1998, vol. 1, p. 230–235.
  4. Bird RP. Observation and quantification of aberrant crypts in the murine colon treated with a colon carcinogen: preliminary findings. Cancer Lett 37: 147–151, 1987.[CrossRef][ISI][Medline]
  5. Bostick RM, Potter JD, McKenzie DR, Sellers TA, Kushi LH, Steinmetz KA, Folson AR. Reduced risk of colon cancer with high intake of vitamin E: the Iowa Women's Health Study. Cancer Res 53: 4230–4237, 1993.[Abstract/Free Full Text]
  6. Burton K. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J 62: 315–323, 1956.[ISI][Medline]
  7. Byers T. Nutrition and cancer among American Indians and Alaska Natives. Cancer 78: 1612–1616, 1996.[Medline]
  8. Caygill CP, Hill MJ. Fish n-3 fatty acids and human colorectal and breast cancer mortality. Eur J Cancer Prev 4: 329–332, 1995.[ISI][Medline]
  9. Cheng XX, Wang ZC, Chen XY, Sun Y, Kong QY, Liu J, Li H. Correlation of Wnt-2 expression and beta-catenin intracellular accumulation in Chinese gastric cancers: relevance with tumor dissemination. Cancer Lett 223: 339–347, 2005.[CrossRef][ISI][Medline]
  10. Dai W, Liu T, Wang Q, Rao CV, Reddy BS. Down-regulation of PLK3 gene expression by types and amount of dietary fat in rat colon tumors. Int J Oncol 20: 121–126, 2002.[ISI][Medline]
  11. Davidson L, Brown R, Chang W, Morris J, Wang N, Carroll R, Turner N, Lupton J, Chapkin R. 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, 2000.[Abstract/Free Full Text]
  12. Giles RH, van Es JH, Clevers H. Caught up in a Wnt storm: Wnt signaling in cancer. Biochim Biophys Acta 1653: 1–24, 2003.[Medline]
  13. Groden J, Thliveris A, Samowitz W, Carlson M, Gelgert L, Albertsen H, Joslyn G, Stevens J, Spilio L, Robertson M, et al. Identification and characterization of the familial adenomatous polyposis coli gene. Cell 66: 589–600, 1991.[CrossRef][ISI][Medline]
  14. Holcombe RF, Marsh JL, Waterman ML, Lin F, Milovanovic T, Truong T. Expression of Wnt ligands and Frizzled receptors in colonic mucosa and in colon carcinoma. J Clin Pathol 55: 220–226, 2002.
  15. Jiang Y, Lupton J, Chapkin R. Dietary fat and fiber differentially alter intracellular second messengers during tumor development in rat colon. Carcinogenesis 17: 1227–1233, 1996.[Abstract/Free Full Text]
  16. Kinzler KW, Vogelstein B. Lessons from hereditary colorectal cancer. Cell 87: 159–170, 1996.[CrossRef][ISI][Medline]
  17. Liu W, Dong X, Mai M, Seelan RS, Taniguchi K, Krishnandath KK, Halling KC, Cunningham JM, Boardman LA, Qian C, Christensen E, Schmidt SS, Roche PC, Smith DI, Thibodean SN. Mutations in Axin2 cause colorectal cancer with detective mismatch repair by activating beta-catenin-Tcf signaling. Nat Genet 26: 146–147, 2000.[CrossRef][ISI][Medline]
  18. Mohammed IM, Roczo N, Phillips WA, Baindur-Hudson S. Expression of Wnt genes in human colon cancers. Cancer Lett 166: 185–191, 2001.[CrossRef][ISI][Medline]
  19. Morin PJ, Sparks AB, Korinek V, Barker N, Clevers H, Vogelstein B, Kinzler KW. Activation of beta-catenin-Tcf signaling in colon cancer by mutation in beta catenin or APC. Science 275: 1787–1790, 1997.[Abstract/Free Full Text]
  20. Narayanan B, Narayanan N, Simi B, Reddy BS. 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, 2003.[Abstract/Free Full Text]
  21. Noda T, Iwakiri R, Fujimoto K, Matsuo S, Aw TY. Programmed cell death induced by ischemia-reperfusion in rat intestinal mucosa. Am J Physiol Gastrointest Liver Physiol 274: G270–G276, 1998.[Abstract/Free Full Text]
  22. Pretlow TP, Barrow BJ, Ashton WS, O'Riordan MA, Pretlow TG, Jurcisek JA, Stellato TA. Aberrant crypts: putative preneoplastic foci in human colonic mucosa. Cancer Res 51: 1564–1567, 1991.[Abstract/Free Full Text]
  23. Pretow TP, Cheyer C, O'Riordan MA. Aberrant crypt foci and colon tumors in F344 rats have similar increases in proliferative activity. Int J Cancer 56: 599–602, 1994.[ISI][Medline]
  24. Pretow TP, O'Riordan MA, Somich GA, Amini SB, Pretlow TG. Aberrant crypts correlate with tumor incidence in F344 rats treated with azoxymethane and phytate. Carcinogenesis 13: 1509–1512, 1992.[Abstract/Free Full Text]
  25. Radtke F, Clevers H. Self-renewal and cancer of the gut: two sides of a coin. Science 307: 1904–1909, 2005.[Abstract/Free Full Text]
  26. Rao CV, Hirose Y, Indranie C, Reddy BS. Moduration of experimental colon tumorigenesis by types and amounts of dietary fatty acids. Cancer Res 61: 1927–1933, 2001.[Abstract/Free Full Text]
  27. Rao C, Reddy BS. Mudulating 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, 1993.[Abstract/Free Full Text]
  28. Reddy BS. Novel approaches to the prevention of colon cancer by nutritional manipulation and chemoprevention. Cancer Epidemiol Biomarkers Prev 9: 239–247, 2000.[Abstract/Free Full Text]
  29. Reddy BS. Omega-3 fatty acids in colorectal cancer prevention. Int J Cancer 112: 1–7, 2004.[CrossRef][ISI][Medline]
  30. Reya T, Clevers H. Wnt signaling in stem cells and cancer. Nature 434: 843–850, 2005.[CrossRef][Medline]
  31. Riboli E, Norat T. Epidemiologic evidence of the protective effect of fruit and vegetables on cancer risk. Am J Clin Nutr 78. 559S–569S, 2003.[Abstract/Free Full Text]
  32. Roynette CE, Calder PC, Dupertuis YM, Pichard C. n-3 Polyunsaturated fatty acids and colon cancer prevention. Clin Nutr 23: 139–151, 2004.[CrossRef][ISI][Medline]
  33. Shih IM, Wang TL, Traverso G, Romans K, Hamilton SR, Ben-Sasson S, Kinzler KW, Vogelstein B. Top-down morphogenesis of colorectal tumors. Proc Natl Acad Sci USA 27: 2640–2645, 2001.
  34. Singh J, Hamid R, Reddy BS. 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, 1997.[Free Full Text]
  35. von Kries JP, Winbeck G, Asbrand C, Schwarz-Romond T, Sochnikowa N, Dell'Oro A, Behrens J, Birchmeier W. Hot spots in beta-catenin for interactions with LEF-1, conduction and APC. Nat Struct Biol 7: 800–807, 2000.[CrossRef][ISI][Medline]
  36. Willett WC. Diet and cancer. Oncologist 5: 393–404, 2000.[Abstract/Free Full Text]
  37. Willett WC, Stampfer MJ, Colditz GA, Rosner BA, Speizer FE. Relation of meat, fat, and fiber intake to the risk of colon cancer in a prospective study among women. N Engl J Med 323: 1664–1672, 1990.[Abstract]
  38. Wright NA, Poulson R. Top down or bottom up? Competing management structures in the morphogenesis of colorectal neoplasm. Gut 51: 306–308, 2002.[Free Full Text]
  39. Wu B, Iwakiri R, Ootani A, Tsunada S, Fujise T, Sakata Y, Sakata H, Toda S, Fujimoto K. Dietary corn oil promotes colon cancer by inhibiting mitochondria-dependent apoptosis in azoxymethane-treated rats. Exp Biol Med 229: 1017–1025, 2004.[Abstract/Free Full Text]
  40. Wyllie AH. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284: 555–556, 1980.[CrossRef][Medline]



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