Azoxymethane-induced beta-catenin-accumulated crypts in colonic mucosa of rodents as an intermediate biomarker for colon carcinogenesis

It is now well established that bile acids act as colon tumor promoters. However, a previous study provided conflicting data showing that dietary exposure of cholic acid (CHA), a primary bile acid, inhibits the carcinogen-induced formation of aberrant crypt foci (ACF), possible preneoplastic lesions, in colonic mucosa of rodents. Recently we found beta-catenin-accumulated crypts (BCAC) in colonic mucosa of rats initiated with azoxymethane (AOM) and provided evidence that BCAC might be preneoplastic lesions independent from ACF. In the present study, we investigated the modifying effects of dietary CHA on the formation of BCAC as well as ACF in male F344 rats after exposure to AOM to determine if the differences in the effect of CHA on these lesions could account for this discrepancy. The results indicate that administration of CHA (0.5%) in the diet during the post-initiation phase significantly reduced the total number, multiplicity and size of ACF (P < 0.00001) in AOM-exposed colonic mucosa as reported previously. The number of ACF even with >4 aberrant crypts/focus was also decreased significantly (P < 0.0002), suggesting that the large ACF are little resistant to continuous feeding of 0.5% CHA diet. Interestingly, the dietary CHA significantly enhanced both the multiplicity (P < 0.002) and size (P < 0.00001), but not the incidence, of AOM-induced BCAC when compared with the control diet group. Importantly, the number of large BCAC with >6 crypts/lesion was increased significantly by the dietary CHA (P < 0.003). Our results support the concept that BCAC are precursors of colon tumors and indicate the usefulness of BCAC as intermediate biomarkers for colon carcinogenesis, although the methodology for their detection requires further improvement.     

Effect of dietary galacto-oligosaccharides on azoxymethane-induced aberrant crypt foci and colorectal cancer in Fischer 344 rats

The aim of the present study was to investigate the effects of galacto-oligosaccharides (GOS, Elix'or) on the development of aberrant crypt foci (ACF) and colorectal tumours in rats treated with azoxymethane (AOM). Two groups of 102 male Fischer 344 rats were injected twice with AOM to induce colorectal tumours, and fed diets containing either a low [5% (w/w); LGOS] or a high [20% (w/w); HGOS] concentration of GOS. Four weeks after the last AOM injection, 18 animals from each group were killed and their colon was removed for scoring ACF. Half of the animals in the LGOS group were switched to an HGOS diet (L/HGOS) and half of those in the HGOS group to an LGOS diet (H/LGOS). Six weeks after the change in diet, nine animals per group were killed for scoring ACF. Ten months after the start of the study the remaining animals were killed for scoring colorectal tumours. The aberrant crypt multiplicity scored after 13 weeks and the colorectal tumour incidence in rats fed an HGOS diet were significantly lower than those in rats fed an LGOS diet. However, the induction of ACF by AOM, the proliferation rate and apoptotic index of the adenomas, and the size and multiplicity of colorectal tumours were not influenced by the amount of GOS in the diet. The aberrant crypt multiplicity, scored after 13 weeks, was predictive for the tumour outcome at the end of the study. It was concluded that an HGOS diet has a protective effect against the development of colorectal tumours in rats and that this protective effect is exerted during the promotion phase rather than the initiation phase of carcinogenesis.     

Resveratrol depresses the growth of colorectal aberrant crypt foci by affecting bax and p21(CIP) expression

We investigated whether resveratrol (RV) affects azoxymethane (AOM)-induced colon carcinogenesis, by administering RV (200 microg/kg/day in drinking water) to male F344 rats for 100 days, beginning 10 days before carcinogen treatment (two weekly doses of 15 mg/kg AOM). Aberrant crypt foci (ACF) were isolated and proliferation, apoptosis and expression of the cell cycle genes bax and p21 were determined. RV significantly reduced the number of ACF/colon [25.7 +/- 3.6 (mean +/- SEM) versus 39.4 +/- 3.3 in controls; P < 0.01] and their multiplicity (2.7 +/- 0.3 versus 4.9 +/- 0.6 in controls; P < 0.01), and also abolished large ACF. In RV-treated rats, bax expression was enhanced in ACF but not in the surrounding mucosa. In both controls and RV-treated rats, proliferation was higher in ACF than in normal mucosa. p21 was expressed in ACF of controls and of RV-treated rats and in normal mucosa of controls, but was lost in normal mucosa of RV-treated animals. In conclusion, the results suggest a protective role of RV in colon carcinogenesis with a mechanism involving changes in bax and p21 expression.     

Chemoprevention of aberrant crypt foci in the colon of rats by dietary onion

Onion intake might reduce the risk of colorectal cancer, according to epidemiology. However, Femia showed in 2003 that diets with a 20% onion intake increase carcinogenesis in rats. We speculated this dose was too high. Prevention of initiation was thus tested in 60 rats given a 5% dried onion diet or AIN76 diet, and initiated 12 days later with azoxymethane (AOM, 1x20 mg/kg i.p.), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ, 2x200 mg/kg p.o.), or N-nitroso-N-methylurea (2x50 mg/kg p.o.). Prevention of promotion was tested in 38 rats given AOM, then randomised to: AIN76 diet; 5% onion diet; phytochemicals diet (supplemented with propyl-disulfide, quercetine-glycosides and oligofructose); 1% pluronic F68 diet (a potent chemopreventive PEG-like block-polymer, used as a positive control). Aberrant crypt foci (ACF) were scored 30 days (initiation) or 100 days (promotion) after carcinogen injection. The onion diet given during initiation reduced the number of AOM-induced ACF (60 versus 86, p=0.03), and the size of IQ-induced ACF (1.33 versus 1.97, p=0.02). Given post-initiation, the onion diet reduced the number of ACF (34 versus 59, p=0.008) and of large ACF (6 versus 15, p=0.02). Phytochemicals diet and pluronic diet reduced ACF growth similarly. Data show that a 5% onion diet reduced carcinogenesis during initiation and promotion stages, and suggest this chemoprevention is due to known phytochemicals.     

Preventive effect of fermented brown rice and rice bran against colon carcinogenesis in male F344 rats

Epidemiological and preclinical studies demonstrate that nutrition plays an important role in the etiology of cancer. It has been reported that rice components, especially rice germ plays a key role in prevention of cancer. The experiments described here examined the potential anticancer properties of brown rice fermented by Aspergillus Oryzae (FBRA) in male F344 rats using inhibition of the formation of azoxymethene (AOM) induced aberrant crypt foci (ACF) and tumors in the colon as the measure of preventive efficacy. The agent was administered at 2.5 and 5% levels in the diet during the initiation phase (during and until 1 week after carcinogen treatment) and/or post-initiation phase (beginning 1 week after carcinogen treatment) of carcinogenesis. In the ACF and tumor studies, rats were sacrificed 5 or 40 weeks after the initiation of AOM treatment (15 mg/kg body weight, once weekly for 3 weeks), respectively. Colonic ACF and tumors were evaluated histopathologically. Administration of 2.5 and 5% FBRA in the diet continuously during initiation and post-initiation period significantly inhibited the ACF formation in rats treated with AOM, compared with rats treated with AOM alone (99+/-24.1 and 79+/-18.4 vs. 139.5+/-27.7, respectively). In addition, administration of 5% FBRA in the diet during the post-initiation phase significantly suppressed the incidence (44 vs.18%) and multiplicity (0.93+/-0.96 vs. 0.18+/-0.40) of colon adenocarcinomas as compared to those given the control diet. In addition, 5% FBRA in the diet during post-initiation phase caused significant inhibition of cell proliferation in the colonic mucosa as compared to the group fed the control diet (81% reduction, p<0.05). These observations demonstrated for the first time that FBRA inhibits colon tumor development in rats, and suggest that it is a promising dietary supplement for prevention of human colon cancer.     

Identification of mucin-depleted foci in the unsectioned colon of azoxymethane-treated rats: correlation with carcinogenesis

We tested the association between aberrant crypt foci (ACF) and tumor induction by feeding azoxymethane-induced rats (15 mg/kg x 2, s.c.) with synbiotics (Raftilose Synergy 1, a derivative of inulin, 10% of the diet, along with lactobacilli and bifidobacteria). After 16 weeks of feeding, synbiotics significantly increased ACF multiplicity. On the contrary, after 32 weeks, synbiotics significantly decreased intestinal tumors. When the same unsectioned colon used for ACF determination was stained with high-iron diamine Alcian blue, foci of crypts with scarce or absent mucins were identified. We defined these lesions as mucin-depleted foci (MDF), and they were visible in all azoxymethane-treated rats and correlated with tumor induction (MDF/colon: 8.2 +/- 0.9 and 3.8 +/- 0.9 in controls and synbiotic-fed rats, respectively, P < 0.01; crypts/MDF: 12.2 +/- 2 and 6.4 +/- 1 in controls and synbiotic-fed rats, respectively, P < 0.05, means +/- SE, n = 7). There were fewer MDF/colon than ACF, and they were histologically more dysplastic than mucinous lesions identified as ACF in high-iron diamine Alcian blue-stained colon. In conclusion, MDF may be premalignant lesions that predict colon carcinogenesis.     

The inhibitory effects of mangiferin, a naturally occurring glucosylxanthone, in bowel carcinogenesis of male F344 rats

Mangiferin, 1,3,6,7-tetrahydroxyxanthone-C2-beta-D-glucoside, is one of xanthone derivatives and C-glucosylxanthones, is widely distributed in higher plants and is one of constituents of folk medicines. Recent studies showed that mangiferin has a potential as an anti-oxidant and an anti-viral agent. In this study, we examined the effects of mangiferin in rat colon carcinogenesis induced by chemical carcinogen, azoxymethane (AOM). We performed two experiments: a short-term assay to investigate the effects of mangiferin on the development of preneoplastic lesions by AOM, aberrant crypt foci (ACF), and the following long-term assay for the influence of mangiferin on tumorigenesis induced by AOM. In the short-term assay, 0.1% mangiferin in a diet significantly inhibited the ACF development in rats treated with AOM compared to rats treated with AOM alone (64.6+/-22.0 vs. 108.3+/-43.0). In the long-term assay, the group treated with 0.1% mangiferin in initiation phase of the experimental protocol had significantly lower incidence and multiplicity of intestinal neoplasms induced by AOM (47.3 and 41.8% reductions of the group treated with AOM alone for incidence and multiplicity, respectively). In addition, the cell proliferation in colonic mucosa was reduced in rats treated with mangiferin (65-85% reductions of the group treated with AOM alone). These results suggest that mangiferin has potential as a naturally-occurring chemopreventive agent.     

Chemopreventive effect of 2-(allylthio)pyrazine (2-AP) on rat colon carcinogenesis induced by azoxymethane (AOM)

An investigation was conducted to assess the chemopreventive effects of 2-(allylthio)pyrazine (2-AP), synthesized for potential use as a chemopreventive agent, after administration during the pre-initiation and post-initiation stages in a rat colon carcinogenesis model with azoxymethane (AOM). One hundred, 5-week-old, male F344 rats were randomly divided into two experiments (n = 50 each). Experiment 1 rats were randomly divided into three groups: Group 1 rats were pre-treated with 2-AP (25 or 50 mg/kg body weight, 3 consecutive days through the route of intragastric intubations) before AOM (20 mg/kg body weight, single subcutaneous (s.c.) injection) initiation. Group 2 rats were treated with AOM alone. Group 3 rats were given 2-AP alone without AOM initiation. The animals were killed at the end of each experiment (week 5) and the aberrant crypt foci (ACF) of the colonic mucosa were assessed after staining with methylene blue. Experiment 2 rats were randomly divided into three groups: Group 1 rats were given 2-AP (10, 25 or 50 mg/kg body weight, five-times intragastric intubations per week for 5 weeks from week 3) after AOM (15 mg/kg body weight, three s.c. injections) initiation for 2 weeks. Group 2 rats were treated with AOM alone. Group 3 rats were given 2-AP alone without AOM initiation. The animals were killed at the end of the experiment (week 8) and the ACF of the colonic mucosa were quantified. Total numbers of ACF/colon in Group 1 rats (pre-treated with 2-AP) tended to decrease (2-AP, 50 mg/kg body weight) or increase (2-AP, 100 mg/kg body weight) depending on the dose level. Total numbers of ACF/colon in Group 1 rats (treated with AOM followed by 2-AP, all subgroups; 160.8 +/- 38.0; 161.8 +/- 38.1; 137.1 +/- 48.4) were decreased significantly compared with the values in Group 2 rats (AOM alone; 214.8 +/- 48.1) (P < 0.05 or 0.01). The highest dose group (2-AP, 50 mg/kg body weight) had the lowest levels of total numbers of ACF/colon among the three subgroups. Total numbers of aberrant crypts (AC)/colon of the highest dose group (340.1+/- 117.9) decreased significantly compared with the value for Group 2 rats (AOM alone; 545.1 +/- 38.3). These results thus suggest that 2-AP may have potential as a chemopreventive agent against rat colon carcinogenesis after administration of AOM during the post-initiation stage.     

Suppression of azoxymethane-induced rat colon carcinoma development by a fish oil component, docosahexaenoic acid (DHA)

The effects of intragastric gavage administration of docosahexaenoic acid (DNA) , a major component of fish oil, on azoxymethane (AOM)- induced colon carcinogenesis in rats was investigated. Male F344 rats were treated with 15 mg/kg body wt of AOM once a week, for two weeks. The animals were given either 1 ml of DHA or water intragastrically 5 times a week, starting the day before the first carcinogen treatment. The numbers of AOM-induced aberrant crypt foci in the rats given DHA were 76% and 62% of the control values, at 4 and 12 weeks, respectively. After 36 weeks of DHA treatment, colon tumors were counted and examined histologically. The blood plasma levels of prostaglandin E2 (PGE2) and polyunsaturated fatty acids were also quantified. The incidences of colon cancer did not differ, being 96% and 92% in the AOM and AOM+DHA groups, respectively. Colon cancer multiplicity was, however, significantly decreased by the DHA treatment; 3.65 +/- 2.18 in the AOM group and 2.41 +/- 1.58 in the AOM+DHA group (P <0.01). Notably, the numbers of moderately differentiated adenocarcinomas in the middle and distal colon in the DHA-treated group were lower than in the AOM group. The levels of PGE2 and arachidonic acid in the blood plasma of DHA-treated rats were also significantly lower than in the AOM group. These results suggest that DHA exerts its inhibitory effect on colon carcinogenesis by modulating lipid metabolism and inhibiting the arachidonic cascade.      

Enhanced growth of colorectal aberrant crypt foci in fasted/refed rats involves changes in TGFbeta1 and p21CIP expressions

We previously demonstrated that fasting/refeeding enhances the initiation phase of liver and colorectal carcinogenesis in rats. The present study was undertaken to establish whether cycles of fasting/refeeding carried out during the promotion phase of carcinogenesis may also affect the formation of aberrant crypt foci (ACF), preneoplastic lesions induced in the colon by azoxymethane (AOM). We were also interested in studying whether this effect might be mediated by changes in the proliferation, apoptosis or expression of TGFbeta1 and p21CIP genes in the colon. 44 male Fisher 344 rats were given a single dose of AOM (20 mg/kg s.c.) and one week later, they were exposed to 5 cycles of 4 days fasting followed by 7-10 days of refeeding (refed rats); controls were regularly fed; the rats were killed 2, 8 or 30 days after the last cycle of fasting. Fasting/refeeding caused a dramatic increase in crypt multiplicity when compared with regularly fed rats (AC/ACF was 4.30 +/- 1.3 in refed and 2.38 +/- 0.4 in regularly fed rats, P < 0.005 means +/- SD), while no significant changes were observed in the number of ACF/colon. In the two experimental groups, cell proliferation was higher in ACF than in the surrounding mucosa, but proliferative indexes were higher and the apoptotic index lower in ACF of refed rats compared with regularly fed rats. TGFbeta1 expression was higher in the ACF of refed rats than in those of fully fed controls while p21CIP was less expressed in refed rats than in controls. These results suggest that fasting/refeeding is a risk factor for colon cancer and must be taken into account for cancer prevention in humans.     

Deoxycholic acid promotes the growth of colonic aberrant crypt foci

AKR/J mice are resistant to the tumorigenic properties of the colon carcinogen, azoxymethane (AOM). Following AOM exposure, limited numbers of preneoplastic lesions, referred to as aberrant crypt foci (ACF), are formed in the colon, and their progression to tumors rarely occurs. To determine whether genetic resistance can be overcome by exposure to a dietary tumor promoter, AOM-exposed AKR/J mice were fed a diet containing 0.25% deoxycholic acid (DCA). DCA exposure was begun 1 wk prior to or 1 wk after tumor initiation with AOM. Mice placed on the DCA diet prior to AOM treatment developed a significantly higher multiplicity of ACF compared to AOM-exposed mice fed a control diet (15.50 +/- 0.96 vs. 6.17 +/- 0.48, respectively; P < 0.05). When DCA exposure was begun after AOM treatment (post-initiation), ACF formation was further enhanced (34.00 +/- 1.22). Interestingly, increased numbers of ACF were associated with the presence of nuclear beta-catenin, assessed by immunohistochemistry. While approximately 33% of ACF from mice exposed to DCA prior to AOM treatment contained positive nuclear beta-catenin staining, approximately 77% of ACF from mice fed DCA after AOM were positive. Accumulation of nuclear beta-catenin was not associated with a loss of E-cadherin from the plasma membrane, although loss of APC staining was a consistent feature of most AOM-induced ACF, regardless of DCA exposure. These results demonstrate that exposure to DCA, an important digestive component, is sufficient to sensitize the resistant AKR/J colon to formation of high-grade dysplasia, and that nuclear translocation of beta-catenin may play an important role in this process.     

Dietary polyamines promote the growth of azoxymethane-induced aberrant crypt foci in rat colon

We have examined whether dietary polyamines influence the formation and initial growth of azoxymethane (AOM)-induced aberrant crypt foci (ACF) in rat colon. Effects of a combination of dietary polyamines at three dose levels (putrescine: 50, 280, 740 nmol/g; spermidine: 10, 261, 763 nmol/g; spermine: 1, 31, 91 nmol/g) in the polyamine-poor AIN-76A diet were studied in animals in two different experimental situations: animals treated with AOM alone and animals treated with AOM + difluoromethylornithine (DFMO), a specific inhibitor of endogenous polyamine synthesis. In both experimental situations, dietary polyamines enhanced the growth of ACF, expressed as the number of large ACF (foci with three or more aberrant crypts, ACF > or = 3), whereas the formation of ACF, expressed as the number of ACF, was apparently not altered. In animals treated with AOM alone, maximal growth enhancing effect on ACF was nearly obtained with the median level of dietary polyamine. In rats fed a low polyamine diet, basic AIN-76A, DFMO reduced the growth of AOM-induced ACF by 83%. This inhibitory effect of DFMO was counteracted by dietary polyamines in a dose- dependent manner, and it was abolished at the highest level of polyamines. In conclusion, it was demonstrated that dietary polyamines are able to enhance the growth of AOM-induced ACF. Further, dietary polyamines reversed the DFMO-caused inhibition of ACF growth, probably by compensating for the DFMO-reduced endogenous polyamine synthesis.      

Chemopreventive effect of squalene on colon cancer

Epidemiologic and laboratory studies suggest a cancer protective effect and/or lack of a tumor promoting effect by dietary olive oil as compared with other types of non-marine oils. Squalene, a constituent of olive oil, and a key intermediate in cholesterol synthesis may be regarded as partially responsible for the beneficial effects of olive oil, which include decreased mortality rates among populations with high olive oil consumption. Thus, in this study we have assessed the chemopreventive efficacy of squalene on azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF). In addition, we measured the effect of squalene on serum cholesterol levels in the rats. Male F34 rats (5 weeks old) were fed the control diet (modified AIN-76A) or experimental diets containing 1% squalene or 320 p.p.m. sulindac. Two weeks later, all animals except those in vehicle (normal saline)-treated groups were s.c. injected with AOM (15 mg/kg body wt, once weekly for 2 weeks). At 16 weeks of age, all rats were killed, colons were evaluated for ACF and serum was assayed for the cholesterol levels. As expected, dietary administration of sulindac suppressed ACF development and reduced crypt multiplicity, i.e. number of aberrant crypts/focus. Administration of dietary squalene inhibited total ACF induction and crypt multiplicity by approximately >46% (P < 0.001). Further, squalene at a level of 1% did not show any significant effect on serum cholesterol levels. Our finding that squalene significantly suppresses colonic ACF formation and crypt multiplicity strengthens the hypothesis that squalene possesses chemopreventive activity against colon carcinogenesis.      

Effect of dietary oligofructose and inulin on colonic preneoplastic aberrant crypt foci inhibition

Oligofructose and inulin, naturally-occurring fermentable chicory fructans, have been shown to stimulate the growth of bifidobacteria which are regarded as beneficial strains in the colon and inhibit colon carcinogenesis in the laboratory animal models. The present study was designed to determine the effect of oligofructose and inulin on the azoxymethane (AOM)-induced preneoplastic lesions such as aberrant crypt foci (ACF) formation in the colon of male F344 rats. At 5 weeks of age, groups of animals were fed the AIN-76A (control) and the experimental diets containing 10% oligofructose or inulin. At 7 weeks of age, all animals received s.c. injection of AOM dissolved in normal saline at a dose rate of 15 mg/kg body wt, once weekly for 2 weeks. The animals were necropsied 7 weeks after the last AOM injection, and the ACF were visualized under light microscopy in the formalin-fixed, unsectioned methylene blue-stained colons. They were distinguished by their increased size, more prominent epithelial cells and pericryptal space. The feeding of oligofructose or inulin significantly inhibited the ACF formation and the crypt multiplicity in the colon. The degree of ACF inhibition was more pronounced in animals fed inulin than in those fed oligofructose. The findings suggest that chicory fructan supplements inhibit ACF formation, an early preneoplastic marker of malignant potential in the process of colon carcinogenesis.      

Chemopreventive effect of farnesol and lanosterol on colon carcinogenesis

Cholesterol metabolites play a several critical roles in regulating cell growth and function. 3-Hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, the rate-limiting enzyme for this pathway, is down regulated by feedback mechanisms due to increased levels of cholesterol and its premetabolites. Several HMG-CoA metabolites, such as farnesyl pyrophosphate and geranyl pyrophosphate are implicated in oncogene activation and tumorigenesis. Recent studies suggest that inhibition of HMG-CoA reductase by specific inhibitors or by naturally-occurring phytochemicals, such as farnesol or squalene can modulate tumor cell growth. Thus, in this study, we have assessed the chemopreventive efficacy of farnesol and lanosterol on azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) in rats. In addition, we measured the effect of farnesol and lanosterol on serum high denisity lipoprotein (HDL) and cholesterol levels in the rats. Seven-week-old male F344 rats were fed the control diet (modified AIN-76A) or experimental diets containing I or 2% lanosterol or 1.5% farnesol. One week later, all animals except those in vehicle (normal saline)-treatment groups were s.c. injected with AOM (15 mg/kg body weight, once weekly for 2 weeks). At 16 weeks of age, all rats were killed, colons were evaluated for ACF and serum was assayed for HDL and cholesterol levels. Administration of dietary farnesol significantly inhibited ACF formation by about 34% (P < 0.001) and reduced crypt multiplicity by about 44% (P < 0.0001). Also, administration of lanosterol at dose levels of I or 2 % in the diet significantly suppressed AOM-induced colonic ACF as well as multicrypt foci formation. (P < 0.01-0.001). Further, farnesol at 1.5% and lanosterol at 1% did not show any significant effect on serum HDL nor on total cholesterol levels. However, lanosterol at 2% significantly increased serum HDL (P < 0.05) and cholesterol (P < 0.01) levels. That farnesol and lanosterol significantly suppress colonic ACF formation and crypt multiplicity strengthens the hypothesis that these agents possess chemopreventive activity against colon carcinogenesis.     

Suppression of azoxymethane-induced colon cancer development in rats by a cyclooxygenase-1 selective inhibitor, mofezolac

We demonstrated recently that mofezolac, a cyclooxygenase-1 (COX-1) selective inhibitor, suppresses the development of azoxymethane (AOM)-induced colonic aberrant crypt foci in F344 rats and intestinal polyps in APC1309 mice. In the present study, we therefore investigated the effects of mofezolac on colon cancer development. Male F344 rats were injected subcutaneously with 15 mg/kg body weight of AOM in the back twice at 7-day intervals from 5 weeks of age, and fed a diet containing 600 or 1200 ppm mofezolac for 32 weeks, starting 1 day before the first dosing of AOM. Treatment with 1200 ppm mofezolac significantly reduced the incidence, multiplicity and volume of colon carcinomas to 79%, 2.15 +/- 1.65 and 7.5 +/- 11.8 mm3, respectively, compared with 94%, 3.19 +/- 1.87 and 23.7 +/- 31.2 mm3 in the AOM treatment alone. Administration of 600 ppm mofezolac showed only a slight reduction. No side effects were observed in any of the groups. These results confirm that COX-1, as well as COX-2, contributes to colon carcinogenesis and that mofezolac may be a good chemopreventive agent for human colon cancer.     

Chemopreventive effect of N-(2-cyclohexyloxy-4-nitrophenyl)methane sulfonamide (NS-398), a selective cyclooxygenase-2 inhibitor, in rat colon carcinogenesis induced by azoxymethane.

Non-steroidal anti-inflammatory drugs (NSAIDs) such as sulindac and indomethacin inhibit colon carcinogenesis, and selective cyclooxygenase (COX)-2 inhibitors are considered to be potential chemopreventive agents without the side effects of usual NSAIDs. We reported that NS-398, N-(2-cyclohexyloxy-4-nitrophenyl)methane sulfonamide, suppressed the formation of preneoplastic lesions, aberrant crypt foci (ACF), induced by azoxymethane (AOM) in a short-term assay of rat colon carcinogenesis. In this study, we examined the effects of long-term NS-398 administration on rat colon carcinogenesis. After three AOM treatments at weekly intervals, a dose of 10 mg/kg of NS-398 in 5% Arabic gum solution was administered by gavage three times per week in group 2 until the termination of the experiment. Rats in group 1 were fed in a basal diet and given 5% Arabic gum solution alone after AOM treatment. At 40 weeks after the first AOM treatment, all rats were killed and the whole intestines including colon were examined. While the incidences of whole intestinal and colon neoplasms in group 1 were 84.6% and 80.8%, respectively, those in group 2 (given NS-398) were 51.9% and 44.4% respectively (P=0.0177 and P=0.0103 by Fisher's exact test, respectively). The multiplicities in group 2 (0.67+/-0.78 and 0.48+/-0.58) were also decreased significantly compared with those (1.39+/-1.10 and 1.08+/-0.74) in group 1 (P<0.01 by Welch's method and P<0.002 by Student's t test, respectively). In immunohistochemistry for proliferative cell nuclear antigen (PCNA), the PCNA-stained cell index (7.40+/-0.5) in group 2 was significantly decreased from that in group 1 (14.03+/-0.82) (P<0.001 by Welch's method). The results suggest that NS-398, a selective COX inhibitor, has a chemopreventive activity against colon carcinogenesis without side-effects such as gastric ulceration.     

Effect of dietary excess of inorganic selenium during initiation and postinitiation phases of colon carcinogenesis in F344 rats

The effect of supplemental inorganic selenium given during the initiation or postinitiation phase of colon carcinogenesis induced by azoxymethane [(AOM)CAS:25843-45-2] was studied in male F344 rats. Weanling animals were raised on AIN-76A semipurified (control) diet. Starting at 4 wk of age, groups of animals intended for initiation study were fed the semipurified diets containing 0.5 and 2.5 ppm selenium in the form of sodium selenite, and those intended for postinitiation study were continued on the control diet. At 7 wk of age, all animals except the vehicle-treated controls were injected s.c. with AOM (15 mg/kg body weight, once weekly for 2 wk). One wk following AOM treatment, animals in the initiation study receiving the supplemental selenium were transferred to the control diet whereas those in the postinitiation study receiving the control diet were transferred to the diets containing 0.5 and 2.5 ppm selenium. These animals were continued on this regimen until the termination of the experiment at 34 wk post-AOM injection. Tissue and blood glutathione peroxidase activity was measured in vehicle-treated animals fed the control and selenium-supplemented diets. The results indicate that body weights were comparable among the various dietary groups. Feeding of diets containing 0.5 and 2.5 ppm selenium during the initiation phase had no effect on colon tumor incidence, but the multiplicity of adenomas was slightly inhibited in animals fed the 2.5 ppm selenium diet. The incidence and multiplicity of colon adenocarcinomas and the multiplicity of colon adenomas were inhibited in animals fed the 2.5-ppm selenium diet during the postinitiation phase of carcinogenesis. The incidence of small intestinal tumors was higher in animals fed the 2.5-ppm selenium diet during the initiation phase than in animals fed the control diet and 0.5-ppm selenium diet. Selenium-dependent glutathione peroxidase activity was increased in kidneys and small and large intestinal mucosae of animals fed the 2.5-ppm selenium diet compared to those fed the 0.5-ppm selenium and control diets.     

Dietary‐feeding of grape seed extract prevents azoxymethane‐induced colonic aberrant crypt foci formation in fischer 344 rats

Chemoprevention by dietary agents/supplements has emerged as a novel approach to control various malignancies, including colorectal cancer (CRC). This study assessed dietary grape seed extract (GSE) effectiveness in preventing azoxymethane (AOM)‐induced aberrant crypt foci (ACF) formation and associated mechanisms in Fischer 344 rats. Six‐week‐old rats were injected with AOM, and fed control diet or the one supplemented with 0.25% or 0.5% (w/w) GSE in pre‐ and post‐AOM or only post‐AOM experimental protocols. At 16 wk of age, rats were sacrificed and colons were evaluated for ACF formation followed by cell proliferation, apoptosis, and molecular analyses by immunohistochemistry. GSE‐feeding caused strong chemopreventive efficacy against AOM‐induced ACF formation in terms of up to 60% (P < 0.001) reduction in number of ACF and 66% (P < 0.001) reduction in crypt multiplicity. Mechanistic studies showed that GSE‐feeding inhibited AOM‐induced cell proliferation but enhanced apoptosis in colon including ACF, together with a strong decrease in cyclin D1, COX‐2, iNOS, and survivin levels. Additional studies showed that GSE‐feeding also decreased AOM‐caused increase in β‐catenin and NF‐κB levels in colon tissues. Compared to control animals, GSE alone treatment did not show any considerable change in these biological and molecular events in colon, and was nontoxic. Together, these findings show the chemopreventive efficacy of GSE against the early steps of colon carcinogenesis in rats via likely targeting of β‐catenin and NF‐κB signaling, and suggest its potential usefulness for the prevention of human CRC. © 2010 Wiley‐Liss, Inc.     

Suppression of Azoxymethane-induced Colonic Premalignant Lesion Formation by Coenzyme Q10 in Rats

Reactive oxygen species cause damage to proteins, lipids and DNA. Coenzyme Q10 (CoQ10) is a compound withmitochondrial bioenergetic functions. The reduced form of CoQ10 shows antioxidant activity. In the present study,effects of CoQ10 on development of azoxymethane (AOM)-induced aberrant crypt foci (ACF) and mucin-depletedfoci (MDF) in F344 male rats were investigated. To induce ACF and MDF, 6-week old rats were given two weeklysubcutaneous injections of AOM (15 mg/kg body weight) and also received a control diet or experimental dietscontaining CoQ10 (200 or 500 ppm) for 4 weeks, starting one day before the first dose of AOM. At 10 weeks of age,all animals were sacrificed and their colons were evaluated for numbers and sizes of ACF and MDF. Administrationof 200 and 500 ppm CoQ10 resulted in reduction of ACF numbers, to 77% and 68% of the carcinogen control value,respectively. The percentages of ACF consisting of more than 4 crypts in these groups were also significantly lowerthan in the controls. Treatment with 500 ppm CoQ10 furthermore decreased the number of sialomucin-producingACF and MDF per colon to 42% and 38% of the carcinogen control value without CoQ10, respectively. Theseresults suggest that CoQ10 may be an effective chemopreventive agent against colon carcinogenesis.