Chemoprevention of Azoxymethane-induced Rat Colon Carcinogenesis by a Xanthine Oxidase Inhibitor, 1'-Acetoxychavicol Acetate

In our studies to find natural compounds with chemopreventive efficacy in foods, using azoxymethane (AOM)-induced colonic aberrant crypt foci and colonic mucosal cell proliferation as biomarkers, a x an thine oxidase inhibitor, 1′-acetoxychavicol acetate (ACA), present in the edible plant Languas galanga from Thailand was found to be effective. This study was conducted to test the ability of ACA to inhibit AOM-induced colon tumorigenesis when it was fed to rats during the initiation or post-initiation phase. Male F344 rats were given three weekly s.c. injections of AOM (15mg/kg body weight) to induce colonic neoplasms. They were fed diet containing 100 or 500 ppm ACA for 4 weeks, starting one week before the first dosing of AOM (the initiation feeding). The other groups were fed the ACA diet for 34 weeks, starting one week after the last AOM injection (the post-initiation feeding). At the termination of the study (week 38), AOM had induced 71% incidence of colonic adenocarcinoma (12/17 rats). The initiation feeding with ACA caused significant reduction in the incidence of colon carcinoma (54% inhibition by 100 ppm ACA feeding and 77% inhibition by 500 ppm ACA feeding, P=0.03 and P=0.001, respectively). The post-initiation feeding with ACA also suppressed the incidence of colonic carcinoma (45% inhibition by 100 ppm ACA feeding and 93% inhibition by 500 ppm ACA feeding, P=0.06 and P=0.00003, respectively). Such inhibition was dose-dependent and was associated with suppression of proliferation biomarkers, such as ornithine decarboxylase activity in the colonic mucosa, and blood and colonic mucosal polyamine contents. ACA also elevated the activities of phase II enzymes, glutathione S-transferase (GST) and quinone reductase (QR), in the liver and colon. These results indicate that ACA could inhibit the development of AOM-induced colon tumorigenesis through its suppression of cell proliferation in the colonic mucosa and its induction of GST and QR. The results confirm our previous finding that ACA feeding effectively suppressed the development of colonic aberrant crypt foci. These findings suggest possible chemopreventive ability of ACA against colon tumorigenesis.     

A xanthine oxidase inhibitor 1'-acetoxychavicol acetate inhibits azoxymethane-induced colonic aberrant crypt foci in rats

The modifying effect of dietary administration of a xanthine oxidase inhibitor 1'-acetoxychavicol acetate (ACA) present in an edible plant Languas galanga in Thailand on the development of azoxymethane (AOM)- induced colonic aberrant crypt foci (ACF) was investigated in rats. Male F344 rats were given s.c. injections of AOM (15 mg/kg body wt) once a week for 3 weeks to induce colonic ACF. They were fed the diets containing 100 or 200 ppm ACA for 5 weeks, starting 1 week before the first dosing of AOM. At the termination of the study (week 5), AOM induced 118 +/- 28 ACF/colon. Dietary administration of ACA caused significant reduction in the frequency of ACF (41% inhibition by 100 ppm ACA feeding and 37% inhibition by 200 ppm ACA feeding, P<0.01). Such inhibition might be associated with suppression of the proliferation biomarkers' expression such as ornithine decarboxylase activity in the colonic mucosa, number of silver-stained nucleolar organizer regions' protein in the colonic mucosal cell nuclei and blood polyamine content. These results indicate that ACA could inhibit the development of AOM-induced ACF through its suppression of cell proliferation in the colonic mucosa and ACA might be a possible chemopreventive agent against colon tumourigenesis.      

Suppression of Azoxymethane-induced Rat Colon Aberrant Crypt Foci by Dietary Protocatechuic Acid

The modifying effect of dietary exposure to protocatechuic acid (PCA) on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) was investigated in male F344 rats. The effects of PCA feeding on the silver-stained nucleolar organizer regions protein (AgNORs) count in the colonic epithelial cells and on the ornithine decarboxylase (ODC) activity in the colonic mucosa were also estimated. Animals were given weekly s.c. injections of AOM (15 mg/kg body weight) for 3 weeks to induce ACF. These rats were fed diet containing 1000 or 2000 ppm PCA for 5 weeks, starting one week before the first dosing of AOM. All rats were killed 2 weeks after the last AOM injection, to measure the number of ACF, ODC activity, and AgNORs count per nucleus in the colon. In rats given AOM and PCA, the frequency of ACF/colon was significantly decreased compared with that in rats given AOM alone ( P < 0.005 at 1000 and P < 0.05 at 2000 ppm). ODC activity in the colon of rats given AOM and PCA at both doses was also significantly lower than that of rats treated with AOM alone ( P < 0.05). Similarly, the mean AgNORs count in rats fed PCA was significantly smaller than that of rats treated with AOM alone ( P < 0.0001). Treatment with PCA alone did not affect these three biomarkers. These results provide further evidence that PCA could be a chemopreventive agent against rat colon carcinogenesis.     

Chemoprevention of azoxymethane-induced rat colon carcinogenesis by the naturally occurring flavonoids, diosmin and hesperidin

The modulating effects of dietary feeding of two flavonoids, diosmin and hesperidin, both alone and in combination, during the initiation and post-initiation phases on colon carcinogenesis initiated with azoxymethane (AOM), were investigated in male F344 rats. Animals were initiated with AOM by weekly s.c. injections of 15 mg/kg body wt for 3 weeks to induced colon neoplasms. Rats were fed the diets containing diosmin (1000 ppm), hesperidin (1000 ppm) or diosmin (900 ppm) + hesperidin (100 ppm) for 5 weeks (initiation treatment) or 28 weeks (post-initiation treatment). The others contained the groups of rats treated with diosmin, hesperidin alone or in combination, and untreated. At the end of the study (32 weeks), the incidence and multiplicity of neoplasms (adenoma and adenocarcinoma) in the large intestine of rats initiated with AOM together with, or followed by, a diet containing diosmin or hesperidin were significantly smaller than those of rats given AOM alone (P <0.001). The combination regimen during the initiation and post-initiation stages also inhibited the development of colonic neoplasms, but the tumor data did not indicate any beneficial effect of diosmin and hesperidin administered together as compared with when these agents were given individually. In addition, feeding of diosmin and hesperidin, both alone and in combination, significantly inhibited the development of aberrant crypt foci. As for cell proliferation biomarkers, dietary exposure of diosmin and hesperidin significantly decreased the 5'-bromodeoxyuridine- labeling index and argyrophilic nuclear organizer region's number in crypt cells, colonic mucosal ornithine decarboxylase activity, and polyamine levels in the blood. These results indicate that diosmin and hesperidin, both alone and in combination, act as a chemopreventive agent against colon carcinogenesis, and such effects may be partly due to suppression of cell proliferation in the colonic crypts, although precise mechanisms should be clarified.      

S-adenosyl L-methionine inhibits azoxymethane-induced colonic aberrant crypt foci in F344 rats and suppresses human colon cancer Caco-2 cell growth in 3D culture

S-adenosyl L-methionine (SAM) is a universal methyl group donor to various intermediary metabolites, hormones, proteins, neurotransmitters, phospholipids and nucleic acids. Deficiency of folate, which plays a role in the synthesis of SAM leads to increased risk for colon cancer. This study tested the effectiveness of SAM supplementation in protecting against azoxymethane (AOM)-induced colon carcinogenesis in male F344 rats. We also tested the effect of SAM on cyclooxygenase-2 (COX-2) in a macrophage cell line. Further, we developed a 3-D culture model using Caco-2 cells to test the effect of SAM on tumor spheroid size and number. Groups of rats were given the experimental diet containing either 0-, 400- or 800-ppm SAM, 1 week before the first AOM injection and continued until 8 weeks. In the control group, AOM produced a substantial number of aberrant crypt foci (ACF) (96 +/- 8). Dietary administration of SAM significantly reduced the number of total ACF (400 ppm SAM, 68 +/- 7.3, p < 0.01 and 800 ppm SAM, 57 +/- 7.1, p < 0.001). SAM significantly decreased AOM-induced colonic multicrypt foci in a dose-dependent manner. Suppression of Lipopolysaccharide (LPS) induced COX-2 protein expression was observed in a RAW264.7 cell line. We established growth of Caco-2 cells as spheroids, in a 3D matrix of collagen and matrigel. Treatment with SAM decreased both size and number of spheroids in a dose-dependent manner (p < 0.0001). These observations demonstrate for the first time that SAM can reduce the occurrence of ACF in AOM treated male F344 rats and suppress formation of human tumor spheroids and expression of COX-2.     

Chemoprevention of azoxymethane-induced rat colon carcinogenesis by dietary capsaicin and rotenone

The modifying effects of dietary administration of capsaicin, which is the principal pungent capsicum fruit, and rotenone, which is a naturally occurring pesticide derived from Derris and Lonchorcarpus species, on azoxymethane (AOM)-induced colon tumorigenesis were investigated in male F344 rats. Gavage with capsaicin and rotenone significantly elevated phase II enzymes, glutathione S-transferase (GST) and quinone reductase (QR), in the liver and colon. In an aberrant crypt foci (ACF) bioassay, feeding of capsaicin and rotenone at a dose of 500 ppm for 4 weeks significantly inhibited ACF formation induced by AOM (20 mg/kg body weight, once a week for 2 weeks). In a subsequent long-term study designed to confirm the protective effects of both compounds on ACF development, one group was treated with AOM alone and four other groups received the carcinogen treatment plus diets containing 500 ppm test compounds for 4 weeks (initiation phase) and for 34 weeks (post-initiation phase). Two groups were treated with capsaicin or rotenone alone (500 ppm in diet) and one group was maintained on the basal diet. At the termination of the study, dietary exposure of capsaicin during the initiation phase was found to significantly reduce the incidence of colonic adenocarcinoma (60% vs. 24%, 60% reduction, P=0.0407). Rotenone feeding during the post-initiation phase also reduced the frequency of colonic adenocarcinoma (60% vs. 19%, 68% reduction, P=0.0226). Our results suggest that two natural compounds, capsaicin and rotenone, might be useful for the prevention of human colon cancers.     

Suppression of azoxymethane-induced rat colon carcinogenesis by dietary administration of naturally occurring xanthophylls astaxanthin and canthaxanthin during the postinitiation phase

The modulating effects of dietary feeding of two xanthophylls,astaxanthin (AX) and canthaxanthin (CX) during the postinitiationphase on colon carcinogenesis initiated with azoxymethane (AOM)were investigated in male F344 rats. Animals were initiatedwith AOM by weekly s.c. injections of 15 mg/kg body wt for 3weeks and then they were fed the diets containing AX or CX atconcentrations of 100 and 500 p.p.m. for 34 weeks. The otherscontained the groups of rats treated with AX or CX alone anduntreated. At the end of the study (week 37), the incidenceand multiplicity of neoplasms (adenoma and adenocarcin-oma)in the large intestine of rats initiated with AOM and followedby AX or CX containing diet at a high dose (500 p.p.m.) weresignificantly smaller than those of rats given AOM alone (P< 0.001). In addition, AX or CX feeding significantly inhibitedthe development of aberrant crypt foci induced by AOM. Dietaryexposure to AX or CX also decreased cell proliferation activityas revealed by measuring 5'-bromodeoxyuridine-labeling indexin crypt cells, colonic mucosal ornithine decarboxylase activityand blood polyamine levels. These results indicate that AX andCX are possible chemopreventers for carcinogenesis of colonin addition to urinary bladder and oral cavity and such effectsmay be partly due to suppression of cell proliferation.     

The Dietary Effect of Monoglucosyl-rutin on Azomethane-Induced Colon Carcinogenesis

The dietary effect of monoglucosyl-rutin (M-R), a flavonoid, on azoxymethane (AOM)-induced colon carcinogenesis ‍was investigated in two experiments with 5 week old, F344 male rats. In the first experiment (5 weeks study), effects ‍of MR on AOM (15 mg/kg body weight 3 times weekly)-induced formation of aberrant crypt foci (ACF) in five ‍groups were assessed. In this experiment, group 3 given 500 ppm M-R with AOM had a significantly smaller number ‍of ACF containing 4 or more aberrant crypts than group 1 with AOM alone, and groups 2 and 3 given 100 ppm or ‍500 ppm M-R respectively had significantly lower BrdU labeling indices in the epithelial cells of large bowel than ‍group 1. For the second experiment, rats were divided into 8 groups. Groups 1-5 were given AOM as in the first ‍experiment. Groups 2-5 were fed diets containing 100ppm or 500ppm M-R for 4 weeks in the initiation phase or 36 ‍weeks in the post-initiation phase. Group 6 was given 500ppm M-R throughout the experiment, and group 7 was ‍kept on the basal diet and served as a control. At the termination of the experiment (40 weeks after the start), groups ‍2-5 had significantly smaller numbers of positive cells with anti-proliferating cell nuclea antigen (PCNA) antibody ‍than group 1. Furthermore, group 5 treated with 500ppm M-R for 36 weeks demonstrated tendencies for decrease in ‍the incidence and multiplicity of colon tumors. These data suggest that M-R has the potential to inhibit AOMinduced ‍colon carcinogenesis.     

SHORT COMMUNICATION: Piroxicam-induced regression of azoxymethane-induced aberrant crypt foci and prevention of colon cancer in rats

Piroxicam has been shown to prevent azoxymethane (AOM)-inducedaberrant crypt foci and colon cancer in rats. In this communicationwe evaluate whether piroxicam can also cause regression of precancerouslesions identified as aberrant crypt foci, thus preventing theoccurrence of cancer. Male Fischer-344 rats were administered0.125g/kg piroxicam in theirdiet starting either 1 week priorto or 12 weeks after a single subcutaneous injection of AOM(30 mg/kg body wt). The yield of aberrant crypt foci and ofcolon adenomas and adenocarcinomas was determined at 5, 12,27 and 37 weeks after administering the AOM respectively. Whenpiroxicam was administered starting 1 week prior to AOM theyield of aberrant crypt foci at the three initial time pointswas reduced. When the administration of piroxicam was delayeduntil 12 weeks afterAOM the yield of aberrant crypt foci wasreduced from 53.8±8.1 foci/colon at 12 weeks to 11.1±2.0at 27 weeks. At 37 weeks after administering AOM the yield ofcolon tumors was 0.59±0.11 tumors/animal, while in ratsadministered piroxicam beginning either 1 week prior to or 12weeks after AOM the yield was similarly reduced to 0.14±0.07and 0.17±0.07 tumors/animal respectively. Thus piroxicamwas demonstrated not only to prevent, but also to cause regressionof aberrant crypt foci, both of which were associated with theprevention of colon tumors.     

Anti-carcinogenic properties of omeprazole against human colon cancer cells and azoxymethane-induced colonic aberrant crypt foci formation in rats

Omeprazole is a proton pump inhibitor, a widely used drug to treat ulcers and gastroesophageal refluxdisease. We have evaluated colon cancer chemopreventive properties of omeprazole using azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) in male F344 rats and analyzed cell growth inhibition and apoptosis induction in human colon cancer cells. Five-week-old male F344 rats were fed a control or experimental diet containing two doses of omeprazole (200 and 400?ppm). After one week, all animals were s.c. injected with AOM (15?mg/kg body weight, once weekly for two weeks). Rats continued on experimental diets for seven more weeks before being sacrificed. Colons were histopathologically evaluated for ACF. Human colon cancer HCT-116 and HCA-7 cells treated with omeprazole were evaluated for different markers associated with proliferation and apoptotic markers using Western blot technique. Rats fed with 200 and 400?ppm of omeprazole significantly suppressed total colonic ACF formation (~30%, P<0.001) and showed significant suppression of multi-crypt foci (~30-50%, P<0.05-0.001). Omeprazole produced significant dose-response effects on inhibition of multi-crypt foci (≥4). Omeprazole treatment in human colon cancer cell lines HCT-116 and HCA-7 cells resulted in induction of p21waf1/cip1 and decreased the expression of anti-apoptotic proteins Bcl-2, Bcl-XL and survivin in a dose-dependent manner. Anticancer properties observed in colon cancer cell lines suggest that omeprazole may induce key signaling molecules of antiproliferation and inhibition of anti-apoptotic proteins.     

Inhibition of azoxymethane-induced aberrant crypt foci in rats by natural compounds, caffeine, quercetin and morin.

The modifying effects of dietary administration of natural compounds, caffeine, quercetin and morin, which are present in our daily food, on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in rats and compared to that of a metabolic inhibitor of AOM, disulfiram. Male F344 rats were given s. c. injections of AOM (15 mg/kg body weight) once a week for 3 weeks to induce ACF. They also received the experimental diets containing one of test compounds (500 ppm) for 5 weeks, starting one week before the first dosing of AOM. At the termination of the study (week 5), AOM exposure produced 101.0+/-10.2 ACF/rat. Disulfiram almost completely inhibited ACF development (0.60+/-0.90, 99% reduction). Dietary administration of test compounds caused significant reduction in the frequency of ACF: caffeine (70.4+/-16.6, 30% reduction), quercetin (53.0+/-8.4, 48% reduction) and morin (37. 6+/-18.1, 63% reduction). Numbers of cells positive for proliferative cell nuclear antigen in ACF and surrounding crypts were lowered by feeding of test compounds. Feeding of these test compounds also suppressed polyamine content in the colonic mucosa and blood as did disulfiram. These findings might indicate possible chemopreventive effects of caffeine, quercetin and morin, through their modulation of cell proliferation activity in crypt cells, on colon tumorigenesis.     

Suppressing Effects of 6-(2,5-Dichlorophenyl)-2,4-diamino-1,3,5-triazine and Related Synthetic Compounds on Azoxymethane-induced Aberrant Crypt Foci in Rat Colon

The modifying effects of dietary administration of 6-(2,5-dichlorophenyl)-2,4-diamino-1,3,5-triazine and 5 related compounds on the occurrence of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in rats. Male F344 rats were given s.c. injections of AOM (15 mg/kg body weight) once a week for 3 weeks to induce ACF. They also received the diet containing 200 ppm test compound for 5 weeks, starting one week before the first dosing of AOM. At the termination of experiment, all of the compounds had caused a significant reduction in ACF frequency, which might be associated with suppression of the expression of proliferation biomarkers. The apoptotic index in the colonic mucosal epithelium of rats killed at 6 h after the first AOM exposure revealed no blocking activity of the compounds.     

Citrus auraptene inhibits chemically induced colonic aberrant crypt foci in male F344 rats

The modifying effect of dietary administration of auraptene isolated from the peel of citrus fruit (Citrus natsudaidai Hayata) on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) was investigated in rats. Male F344 rats were given s.c. injections of AOM (15 mg/kg body wt) once a week for 3 weeks to induce ACF. They also received diets containing 100 or 500 p.p.m. auraptene for 5 weeks, starting 1 week before the first dose of AOM. At termination of the study (week 5) dietary administration of auraptene caused a significant reduction in the frequency of ACF in a dose- dependent manner (P < 0.05). Feeding of auraptene suppressed expression of cell proliferation biomarkers (5-bromo-2'-deoxyuridine labeling- index, ornithine decarboxylase activity, polyamine content and number of silver stained nucleolar organizer region protein particles) in the colonic mucosa and the occurrence of micronuclei caused by AOM. Also, auraptene increased the activities of phase II enzymes (glutathione S- transferase and quinone reductase) in the liver and colon. These findings might suggest that inhibition of AOM-induced ACF may be associated, in part, with increased activity of phase II enzymes in the liver and colon and suppression of cell proliferation in the colonic mucosa.      

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.     

Catalpa seed oil rich in 9t,11t,13c-conjugated linolenic acid suppresses the development of colonic aberrant crypt foci induced by azoxymethane in rats

Catalpa (Catalpa ovata) seed oil (CPO) is a unique oil that contains a high amount of 9trans,11trans,13cis-conjugated linolenic acid. In the present study, we investigated whether dietary administration with CPO affects the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) in male F344 rats to elucidate its possible cancer chemopreventive efficiency. Also, the effect of CPO on the fatty acid composition of liver tissue and colonic mucosa, the serum levels of total cholesterol and triglyceride, and the mRNA expression of cyclooxygenase (COX)-2 in the colonic mucosa were measured. In addition, the cell proliferation activity and apoptotic index in the colonic mucosa were estimated immunohistochemically. Animals were given two weekly subcutaneous injections of AOM (20 mg/kg body weight). They also received the experimental diet containing 0.01%, 0.1% or 1% CPO for 4 weeks, starting one week before the first dosing of AOM. AOM exposure produced a substantial number of ACF (99+/-28) at the end of the study (week 4). Dietary administration of CPO reduced the number of ACF (AOM + 0.01% CPO, 32+/-11, P<0.001; AOM + 0.1% CPO, 35+/-18, P<0.001; AOM + 1% CPO, 18+/-10, P<0.001). 9t,11t-conjugated linoleic acid was detected in the liver tissue and colonic mucosa of rats fed the CPO-containing diet. Additionally, dietary administration with CPO decreased the serum triglyceride level and the expression of COX-2 mRNA in the colonic mucosa. The indices of cell proliferation and apoptosis in the colonic mucosa of rats treated with AOM and 1% CPO have significant differences when compared with the AOM alone group. These findings suggest the possible chemopreventive activity of CPO in the early phase of colon carcinogenesis.     

A rapid dual organ rat carcinogenesis bioassay for evaluating the chemoprevention of breast and colon cancer

In this study we evaluated the effect of dietary administration of a high fat, low fiber diet (HRD) with or without 2% phytic acid (PA) on the development of mammary cancer and/or colon cancer in rats exposed to methylnitrosourea (MNU), azoxymethane (AOM) or MNU + AOM. The rats were fed a HRD alone or a HRD + 2% PA. At the end of week 2, the rats were given either a s.c. injection of MNU (50 mg/kg body wt) or one of normal saline (vehicle). At the end of weeks 3 and 4, the rats were given either a s.c. injection of AOM (15 mg/kg body wt per week) or one of normal saline (vehicle). Nine weeks after the injection of MNU or saline, 10 rats from each group were sacrificed and the mammary tumor incidence and the number of colonic aberrant crypt foci (ACF) were compared between different groups. The administration of different diets was continued for an additional 21 weeks and the mammary tumor and colon tumor incidence between different groups were compared. Results showed that rats injected with MNU alone did not develop ACF or colon tumors while those injected with AOM alone did not develop mammary tumors. Linear regression analysis of the number of ACF at 11 weeks versus colonic tumor incidence at 32 weeks, and the linear regression analysis of mammary tumor incidence at 11 weeks versus mammary tumor incidence at 32 weeks, both showed good linear correlation. These results demonstrate the potential value of the short term dual organ carcinogenesis bioassay for screening chemopreventive agents for their relative ability to inhibit the development of mammary cancer and/or colon cancer while on high risk diet.     

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.      

Prevention of colonic preneoplastic lesions by the probiotic Lactobacillus acidophilus NCFMTM in F344 rats

The experiments described here were aimed at developing novel probiotic strains that may aid in the reduction of colon cancer risk. We assessed the potential anticancer properties of Lactobacillus acidophilus NCFMTM in male F344 rats using inhibition of the formation of azoxymethane (AOM)-induced aberrant crypt foci (ACF) in the colon as the measure of preventive efficacy. At 6 weeks of age, groups of rats were fed the experimental diets containing 0, 2% or 4% lyophilized cultures of L. acidophilus NCFMTM. At 7 weeks of age, all animals in each dietary group, except the vehicle-treated rats, were s.c. injected with AOM (15 mg/kg body weight) once weekly for two weeks. The vehicle-treated groups were given s.c. injections of normal saline. All rats were necropsied 10 weeks after the last AOM injection and ACF in formalin-fixed, methylene blue-stained colonic tissues were counted under the light microscope. The contents of the cecum were analyzed for bacterial beta-glucuronidase activity. Diet supplementation with the probiotic strain NCFMTM significantly suppressed AOM-induction of colonic ACF, in terms of total number, as well as crypt multiplicity and number of ACF/cm2 colon (P<0.01 - 0.001). NCFMTM inhibited AOM-induced colonic ACF formation in a dose-dependent manner (P<0.01). A significant dose-dependent reduction of cecal beta-glucuronidase activities was observed in the rats fed 2% (P<0.04) and 4% (P<0.0001) NCFMTM. These results suggest that Lactobacillus acidophilus NCFMTM may potentially prevent colon cancer development. Further studies are warranted to determine the full potential of this probiotic strain in preclinical efficacy studies.     

Pomegranate seed oil rich in conjugated linolenic acid suppresses chemically induced colon carcinogenesis in rats

Pomegranate (Punica granatum L.) seed oil (PGO) contains more than 70% cis(c)9,trans(t)11,c13-18:3 as conjugated linolenic acids (CLN). Our previous short-term experiment demonstrated that seed oil from bitter melon (Momordica charantia) (BMO), which is rich in c9,t11,t13-CLN, inhibited the occurrence of colonic aberrant crypt foci (ACF) induced by azoxymethane (AOM). In this study, we investigated the effect of dietary PGO on the development of AOM-induced colonic malignancies and compared it with that of conjugated linoleic acid (CLA). To induce colonic tumors, 6-week old male F344 rats were given subcutaneous injections of AOM (20 mg/kg body weight) once a week for 2 weeks. One week before the AOM treatment they were started on diet containing 0.01%, 0.1%, or 1% PGO or 1% CLA for 32 weeks. Upon termination of the bioassay (32 weeks) colon tumors were evaluated histopathologically. AOM exposure produced colonic adenocarcinoma with an incidence of 81% and multiplicity of 1.88 +/- 1.54 at week 32. Administration of PGO in the diet significantly inhibited the incidence (AOM + 0.01% PGO, 44%, P < 0.05; AOM + 0.1% PGO, 38%, P < 0.01; AOM + 1% PGO, 56%) and the multiplicity (AOM + 0.01% PGO, 0.56 +/- 0.73, P < 0.01; AOM + 0.1% PGO, 0.50 +/- 0.73, P < 0.005; AOM + 1% PGO, 0.88 +/- 0.96, P < 0.05) of colonic adenocarcinomas, although a clear dose-response relationship was not observed at these dose levels. CLA feeding also slightly, but not significantly, reduced the incidence and multiplicity of colonic adenocarcinomas. The inhibition of colonic tumors by PGO was associated with an increased content of CLA (c9,t11-18:2) in the lipid fraction of colonic mucosa and liver. Also, administration of PGO in the diet elevated expression of peroxisome proliferator-activated receptor (PPAR) gamma protein in the non-tumor mucosa. These results suggest that PGO rich in c9,t11,c13-CLN can suppress AOM-induced colon carcinogenesis, and the inhibition is associated in part with the increased content of CLA in the colon and liver and/or increased expression of PPARgamma protein in the colon mucosa.