AOM-induced Colonic Aberrant Crypt Foci Research Articles

Azoxymethane Alters the Plasma Metabolome to a Greater Extent in Mice Fed a High-Fat Diet Compared to an AIN-93 Diet.

Consumption of a high-fat diet (HFD) links obesity to colon cancer in humans. Our data show that a HFD (45% energy fat versus 16% energy fat in an AIN-93 diet (AIN)) promotes azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) formation in a mouse cancer model. However, the underlying metabolic basis remains to be determined. In the present study, we hypothesize that AOM treatment results in different plasma metabolomic responses in diet-induced obese mice. An untargeted metabolomic analysis was performed on the plasma samples by gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). We found that 53 of 144 identified metabolites were different between the 4 groups of mice (AIN, AIN + AOM, HFD, HFD + AOM), and sparse partial least-squares discriminant analysis showed a separation between the HFD and HFD + AOM groups but not the AIN and AIN + AOM groups. Moreover, the concentrations of dihydrocholesterol and cholesterol were inversely associated with AOM-induced colonic ACF formation. Functional pathway analyses indicated that diets and AOM-induced colonic ACF modulated five metabolic pathways. Collectively, in addition to differential plasma metabolomic responses, AOM treatment decreases dihydrocholesterol and cholesterol levels and alters the composition of plasma metabolome to a greater extent in mice fed a HFD compared to the AIN.

Azoxymethane Alters the Plasma Metabolome to a Greater Extent in Mice Fed a High Fat Diet Compared to AIN-93 Diet

Abstract Objectives Consumption of a high fat diet (HFD) links obesity to colon cancer in humans. Consistent with this observation, our data show that a HFD (45% energy fat versus 16% energy fat in AIN-93 diet, AIN) promotes azoxymethane (AOM) induced colonic aberrant crypt foci (ACF) formation in a mouse cancer model. However, the underlying metabolic basis remains to be determined. We hypothesize that AOM treatment results in different plasma metabolomic responses in AOM mouse colon cancer model fed with a HFD compared to the AIN. Methods Four-week-old male C57BL/6 mice were fed the AIN or HFD in the context of with or without AOM treatment for 16 weeks. At the end of this study, plasma samples were extracted and derivatized by silylation and methyloximation then analyzed by time-of-flight mass spectrometry using an untargeted metabolomic technique. Results Untargeted metabolomic profiling showed that 53 of 144 identified metabolites were different between the 4 groups of mice (AIN only, AIN + AOM; HFD only, HFD + AOM). Sparse partial least squares discriminant analysis (two-dimensional plots) showed a separation between the HFD and HFD + AOM groups but not AIN and AIN + AOM groups. Major metabolites responsible for this separation were beta-sitosterol, dihydrocholesterol, alpha-tocopherol, methylphosphate, citric acid, cholesterol, isocitric acid, myristic acid, arachidonic acid, and nicotinamide. Importantly, the levels of dihydrocholesterol and cholesterol were inversely associated with AOM-induced colonic ACF formation. Functional pathway and enrichment analyses indicated that diets and AOM-induced colonic ACF modulated 5 metabolic pathways: (a) citrate cycle (TCA cycle), (b) arginine biosynthesis, (c) aminoacyl-tRNA biosynthesis, (d) alanine, aspartate and glutamate metabolism, (e) glyoxylate and dicarboxylate metabolism. Conclusions Collectively, we demonstrate that AOM decreases dihydrocholesterol and cholesterol levels, and alters the composition of plasma metabolome to a greater extent in mice fed a HFD compared to the AIN. Funding Sources This work was supported by U.S. Department of Agriculture, Agricultural Research Service, research project 3062-51000-056-00D.

Chemopreventive effects of Ku-jin tea against AOM-induced precancerous colorectal lesions in rats and metabolomic analysis

Ku-jin tea (KJT) is a health beverage prepared from the leaves of the plant Acer tataricum subsp. ginnala that has been consumed in some regions of China for thousands of years. KJT contains high levels of anti-inflammatory and antioxidative compounds such as ginnalins, but little is known about the chemopreventive effect of KJT on colon cancer. In this study, we investigated the preventive effects of KJT on colon carcinogenesis using the azoxymethane (AOM)-induced precancerous colorectal lesion model in rats. The results showed that the number of aberrant crypts, aberrant crypt foci (ACF) and crypts/focus in rats of the KJT + AOM group were significantly decreased compared with rats of the AOM group (p < 0.01). Further exploration of the prevention mechanism of KJT by UPLC-QTOF/MS-based urinary metabolomics showed that 5 metabolic pathways were modulated, including purine metabolism and amino acid metabolism, in the group with KJT. In addition, the levels of the immunomodulatory cytokines IL-1α and IL-10 were significantly decreased, and the levels of IL-2 in the serum of AOM rats increased after KJT treatment. Our present data suggest that KJT can inhibit AOM-induced colonic ACF formation and might be a useful chemopreventive agent against colorectal carcinogenesis.

Abstract 907: Chemopreventive effects of a non-camellia tea against azoxymethane -induced precancerous colorectal lesions in male rats

Abstract Tea (the leaves from Camellia plants) is one of the most popular beverages in the world; substantial evidence supports its success in the prevention of carcinogenesis and other chronic diseases in animal models. Plants other than Camellia have been widely used as tea as well in various cultures. We have systematically collected and identified over 30 species of frequently used Non-Camellia teas, which have been consumed at least 300 years in China or other nations. Recently, non-Camellia teas have attracted much attentions due to high potentials of preventing and treating metabolic and inflammatory diseases, as well as cancers. Epidemiologic studies report that tea consumption significantly reduces risk of colorectal cancer (CRC). We here reported in vivo preventive effects of the Ku-jin-cha (KJC) tea for CRC. KJC tea is a kind of “Non-Camellia tea” prepared from leaves of the plant Acer ginnala and popularly used in some Chinese regions for thousands of years. KJC contains high levels of anti-inflammatory and anti-oxidative compounds such as ginnalins. However, the potential chemopreventive effect of KJC remains unknown. Here, we investigated modulatory effects of KJC on colon carcinogenesis using the rat azoxymethane (AOM) induced precancerous colorectal lesion model. Wistar rats (male, 4∼5 weeks old) were randomly divided into 4 groups (Control, KJC, AOM and AOM+KJC, 10 rats/group) and were fed either normal water or experimental water containing KJC for 12 weeks. After one week of water or KJC drinking, all rats except the Control Group were injected with 15 mg/kg AOM (s.c., once weekly for 3 weeks). At 16∼17 weeks of age, urine and sera were collected for metabolomic analysis using UPLC-QTOF/MS (ultra-HPLC coupled with time-of-flight mass spectrometry). Rats were sacrificed at the end of KJC administration, and colorectums collected for evaluation of aberrant crypt (AC), aberrant crypt foci (ACF) and Crypts/focus. Spleens, thymuses and bloods were also taken for immunological analysis. Our results show that the numbers of AC, ACF and Crypts/focus in rats of the KJC+AOM Group were significantly decreased, versus rats of the AOM Group (p &amp;lt; 0.01). No ACF was found in rats without AOM injection. In addition, significant metabolic alterations were observed in the groups with KJC. Although KJC administration did not cause marked weight changes of spleens and thymuses, the effect of KJC on levels of inflammatory cytokines and mediators were also examined. Our results indicated that KJC significantly inhibited AOM-induced colonic ACF formation, suggesting that KJC might have a chemopreventive effect against colon carcinogenesis at least in the initiation stage of CRC. Furthermore, KJC may exert the chemopreventive effects on ACF formation by changing metabolic pathways and modulating immunity and inflammatory responses. This warrants further studies on the cellular and molecular mechanisms of KJC tea. Citation Format: Chunnian He, Wu Bi, L. Harris Zhang, Li Cao, Pei-Gen Xiao. Chemopreventive effects of a non-camellia tea against azoxymethane -induced precancerous colorectal lesions in male rats. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 907. doi:10.1158/1538-7445.AM2015-907

Antioxidant and Anti-inflammatory Effects of Yam (Dioscorea batatas Decne.) on Azoxymethane-induced Colonic Aberrant Crypt Foci in F344 Rats.

Yam (Dioscorea batatas Decne.) has long been used as a health food and oriental folk medicine because of its nutritional fortification, tonic, anti-diarrheal, anti-inflammatory, antitussive, and expectorant effects. Reactive oxygen species (ROS), which are known to be implicated in a range of diseases, may be important progenitors of carcinogenesis. The aim of this study was to investigate the modulatory effect of yam on antioxidant status and inflammatory conditions during azoxymethane (AOM)-induced colon carcinogenesis in male F344 rats. We measured the formation of aberrant crypt foci (ACF), hemolysate antioxidant enzyme activities, colonic mucosal antioxidant enzyme gene expression, and colonic mucosal inflammatory mediator gene expression. The feeding of yam prior to carcinogenesis significantly inhibited AOM-induced colonic ACF formation. In yam-administered rats, erythrocyte levels of glutathione, glutathione peroxidase (GPx), and catalase were increased and colonic mucosal gene expression of Cu/Zn-superoxide dismutase (SOD), Mn-SOD, and GPx were up-regulated compared to the AOM group. Colonic mucosal gene expression of inflammatory mediators (i.e., nuclear factor kappaB, inducible nitric oxide synthase, cyclooxygenase-2, tumor necrosis factor alpha, and interleukin-1beta) was suppressed by the yam-supplemented diet. These results suggest that yam could be very useful for the prevention of colon cancer, as they enhance the antioxidant defense system and modulate inflammatory mediators.

Chemopreventive effects of an HDAC2-selective inhibitor on rat colon carcinogenesis and APCmin/+ mouse intestinal tumorigenesis.

Epigenetic modulators, particularly histone deacetylases (HDACs), are valid targets for cancer prevention and therapy. Recent studies report that HDAC2 overexpression is associated with colon tumor progression and is a potential target for colon cancer prevention. This study tested chemopreventive and dose-response effects of Ohio State University HDAC42 (OSU-HDAC42), a selective HDAC2 inhibitor, using a rat colon carcinogenesis model to assess aberrant crypt foci inhibition and a familial adenomatous polyposis model to assess intestinal tumor inhibition. Colonic aberrant crypt foci were induced by azoxymethane (AOM) (15 mg/kg body weight, once-weekly subcutaneous injections at 8 and 9 weeks age). One week after AOM treatment, groups of rats were fed an AIN-76A diet containing 0, 75, 150, and 300 ppm OSU-HDAC42 for 8 weeks, and colonic aberrant crypt foci were evaluated. To assess the inhibitory effect of OSU-HDAC42 on small-intestinal polyps and colon tumor growth, 6-week-old male C57Bl/6J-APC(min/+)mice were fed an AIN-76A diet containing 150 ppm OSU-HADC42 or 300 ppm pan-HDAC inhibitor suberoylanilide hydroxyamic acid (SAHA) for 80 days. Our results demonstrate that dietary OSU-HDAC42 produced dose-dependent inhibition of AOM-induced colonic aberrant crypt foci formation (13-50%; P < 0.01 to < 0.0001) and reduced multiple crypts with ≥ 4 crypts per focus (25-57%; P < 0.01 to < 0.0001) in F344 rats. Our findings show that 150 ppm OSU-HDAC42 significantly inhibited small-intestinal polyps (>46%; P < 0.001), with polyp size measuring >1 mm (P < 0.001), and colon tumors (>26%) in APC(min/+)mice, whereas 300 ppm SAHA showed nonsignificant inhibition. Mice fed 150 ppm OSU-HDAC42 had significantly decreased HDAC2, proliferating cell nuclear antigen, B cell lymphoma 2, cyclin-dependent kinase 2, and cell division cycle homolog 25C expression levels and increased p53 expression levels. These observations demonstrate the chemopreventive efficacy of OSU-HDAC42 against chemically induced and polyposis models of intestinal tumorigenesis.

Nabag (Zizyphus spina-christi) Extract Prevents Aberrant Crypt Foci Development in Colons of Azoxymethane-Treated Rats by Abrogating Oxidative Stress and inducing Apoptosis

Zizyphus spina-christi (ZSC) fruit is a rich source of bioactive compounds but any medicinal properties in chemoprevention of colon cancer have hitherto not been studied. The aim of the present study was to examine in vivo protective effects of ZSC water extract on colon carcinogenesis in azoxymethane (AOM)-treated rats. Our results showed that ZSC significantly reduced AOM-induced colonic aberrant crypt foci development and AOM-induced oxidative stress as indicated by restoration of endogenous glutathione depletion and abrogating the impairment of total antioxidant capacity. Caspase-3 cleavage, which has been considered as an apoptotic index, was almost undetectable in AOM-treated rats and ZSC exhibited pro-apoptotic effects evidenced by increased levels of cleaved caspase-3. In the studied model, our findings provide the first in vivo evidence that ZSC extract could inhibit the early stage of colon carcinogenesis by preventing oxidative stress and inducing apoptosis.

Abstract 4392: Targeting HADC-2: Chemopreventive effect of OSU-HDAC42 on rat colon carcinogenesis and APCmin/+ Mice intestinal tumorigenesis.

Abstract Epigenetic modulators particularly histone deacetylases (HDACs) are valid targets cancer for prevention and Therapy. Recent studies from our laboratory and others suggest that HDAC-2 over-expression is associated with colon tumor progression and potential target for colon cancer prevention. The present study explored the chemopreventive effects of novel HDAC-2 inhibitor, OSU-HDAC42, in a mouse model of familial adenomatous polyposis (FAP). We used a rat colon carcinogenesis model assay, inhibition of aberrant crypt foci (ACF) development, as measure of the dose-response and chemopreventive effects. Colonic ACF were induced by azoxymethane (AOM) (15 mg/kg BW once weekly for 2 weeks by s.c. injection at the age of 8 and 9 weeks). One week after AOM-treatment groups of rats were fed AIN-76A diet containing 0, 75, 150 and 300 ppm OSU-HDAC42 for eight-weeks, and then colonic ACF were evaluated. Six week-old male C57Bl/6J-APCmin/+ and wild type mice were fed AIN-76A diet containing 150 ppm OSU-HADC42 or 300 ppm SAHA for 80 days. At ∼120 days of age, all mice were killed, and the intestines were evaluated for polyps. Dietary administration of OSU-HDAC42 produced a dose-dependent inhibition of AOM-induced colonic ACF formation (by 13-50%, p&amp;lt;0.05 -0.001) and reduced the number of foci with ≥4 crypts/focus (by 25-57%, P&amp;lt;0.05-0.0001) in F344 rats. 150 ppm OSU-HDAC42 significantly inhibited development of small intestinal (&amp;gt;46%, p&amp;lt;0.001) and colon tumors (&amp;gt;26%, P≥0.05) in APCmin/+ mice. Whereas 300 ppm SAHA showed limited non-significant inhibition of intestinal polyp formation in APCmin/+ mice. Further, OSU-HDAC42 showed inhibition in small intestinal polyps size measuring 1-2-mm (P&amp;lt;0.001). Importantly, mice fed with 150ppm OSU-HDAC42 showed significant reduction in HDAC-2, PCNA, CyclinA, CDK2, CDC25C expression pattern and increased p53 expression levels. These observations demonstrate that novel HDAC inhibitor, OSU-HDAC42 potential chemopreventive against the chemically-induced and polyposis models of colon tumorigenesis. {This work is supported by NCI-N01-CN53300} Citation Format: Durgadevi Ravillah, Li Qian, Misty Brewer, Yuting Zhang, Laura Biddick, Venkateshwar Madka, Jagan M.R. Patlolla, Altaf Mohammed, Vernon Steele, Chintalapally V. Rao. Targeting HADC-2: Chemopreventive effect of OSU-HDAC42 on rat colon carcinogenesis and APCmin/+ Mice intestinal tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4392. doi:10.1158/1538-7445.AM2013-4392

Prevention of Familial Adenomatous Polyp Development in APCmin Mice and Azoxymethane-Induced Colon Carcinogenesis in F344 Rats by ω-3 Fatty Acid Rich Perilla Oil

The present study explored the preventive effects of perilla oil, rich in α-linolenic acid, in rodent models of colon tumorigenesis. Six-week-old male F344 rats were fed diets containing 5% corn oil or 10 or 20% perilla oil. Colonic aberrant crypt foci (ACF) were induced by azoxymethane (AOM) and colonic ACF were evaluated. In familial adenomatous polyposis mode, APCmin mice fed with 20% corn oil or perilla oil for 80 days and intestines were evaluated for polyps. Multiple colonic mucosal and polyp samples were assayed for the expression and activity of cyclooxygenase COX-isoforms. Dietary perilla oil produced a dose-dependent inhibition of AOM-induced colonic ACF formation (by 35–53%, P < 0.01–0.005) and reduced the number of foci with ≥4 crypts/focus (by 38–50%, P < 0.01–0.001) in F344 rats. Dietary perilla oil significantly inhibited development of small intestinal (>69%, P < 0.0001) and colon tumors (>52%, P < 0.03) in APCmin mice. Administration of perilla oil produced lower levels of type-2 prostaglandins (38–53%) from COX-activities in polyps of APCmin mice. These observations demonstrate that dietary perilla oil rich in ω-3 fatty acids possesses preventive activity against intestinal neoplastic lesions, both in FAP in genetically-predisposed tissues, as well as against chemically induced preneoplastic lesions in the colon.

Cocoa‐rich diet prevents azoxymethane‐induced colonic preneoplastic lesions in rats by restraining oxidative stress and cell proliferation and inducing apoptosis

Cocoa is a rich source of bioactive compounds with potential chemopreventive ability but up to date its effectiveness in animal models of colon carcinogenesis has not been addressed. Herein, we investigated the in vivo effect of a cocoa-rich diet in the prevention of azoxymethane (AOM)-induced colon cancer and the mechanisms involved. Our results showed that cocoa feeding significantly reduced AOM-induced colonic aberrant crypt foci formation and crypt multiplicity. Oxidative imbalance in colon tissues seems to be prevented by cocoa as indicated by reduced oxidation markers levels and increased enzymatic and non-enzymatic endogenous defences. Cocoa-rich diet also exhibited antiproliferative effects by decreasing the levels of extracellular regulated kinases, protein kinase B and cyclin D1 together with pro-apoptotic effects evidenced by reduced Bcl-x(L) levels and increased Bax levels and caspase-3 activity. Our findings provide the first in vivo evidence that a cocoa-rich diet may inhibit the early stage of colon carcinogenesis probably by preventing oxidative stress and cell proliferation and by inducing apoptosis.

Abstract A35: Chemoprevention of colon carcinogenesis in F344 rats by Se,Se′-1,4-phenylenebis(1,2-ethanediyl)bis-isoselenourea (PBISe) a novel analog of PBIT, an iNOS inhibitor

Abstract Inducible nitric oxide synthase (iNOS) is potential target for inflammation and cancer. Previously, we have shown that S,S′-1,4-Phenylenebis(1,2-ethanediyl)bisisothiourea (PBIT) inhibit colon carcinogenesis induced by azoxymethane (AOM). Although, colon cancer inhibitory efficacy of PBIT has been significant, selective iNOS inhibitors do not completely abrogate NO production due to the exogenous bioavailability and NO generation by eNOS in tumor tissues. To create an iNOS selective and multi-targeted molecule, we have developed a novel isosteric analogue of PBIT, namely PBISe in which sulfur was replaced with selenium. We examined the chemopreventive efficacy of PBISe on AOM-induced rat colon carcinogenesis model using aberrant crypt foci (ACF) as end point. At seven weeks of age, rats (12/group) were fed the control diet (AIN 76A) and colonic ACF were induced by AOM. Three days after second AOM treatment, rats were fed the diets containing 0, 10 and 20 ppm of PBI-Se and continued on the respective diets for 8 weeks before ACF were evaluated histopathologically. Furthermore, PBISe (0–12 µM) and PBIT (0 – 100 µM) were studied for the markers of growth inhibition and induction of apoptosis in the human colon cancer CaCo2 cell line. In addition, inhibitory effect of PBISe and PBIT on proinflammatory signaling molecules iNOS, IL-8 and IL-6 were studied by RT-PCR. Results suggest that dietary administration of 10 and 20ppm of PBISe significantly suppressed AOM-induced total colonic ACF formation (32 – 41%, p&amp;lt; 0.001), and multi crypt (4 or more) aberrant foci (29 – 47%, p&amp;lt; 0.001) respectively. Dietary PBISe suppressed AOM-induced colonic ACF in a dose-dependent manner and it required less than half the dose of PBIT to inhibit total ACFs in rats. Both PBIT and PBISe in CaCo2 cells induced dose-dependent apoptosis. PBIT and PBISe treatment on CaCo2 cells showed significant decrease in cell cycle protein cyclin D1 (∼70%, p&amp;lt;0.0001) and iNOS (∼99%, p&amp;lt;0.0001). Also, treatment with PBIT (30 and 60 M) significantly decreased cytokine induced IL-6 at both the doses whereas PBISe had no such effect. PBIT significantly attenuated the LPS-induced IL-6 and IL-8 production in a dose dependent manner. Whereas, PBISe significantly increased IL-8 and attenuated IL-6 production in CaCo2 cells suggesting differential modulatory effects on immune cells/inflammatory pathways. Incorporation of selenium into the structure of PBIT has provided the agent with additional novel cytotoxic and immunologic properties. Results from in vitro and in vivo bioassay clearly suggest that PBI-Se could be further developed for prevention and treatment of colon cancer. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A35.

An n-3 PUFA-rich microalgal oil diet protects to a similar extent as a fish oil-rich diet against AOM-induced colonic aberrant crypt foci in F344 rats

The chemopreventive effects of high fat microalgal oil diet on azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were studied in male Fischer 344 rats following 8 weeks of dietary treatment. These effects were compared to the effects of high fat fish oil and high fat corn oil diets to determine whether microalgal oil is a good alternative for fish oil regarding protection against colorectal cancer. Despite the difference in fatty acid composition and total amount of n-3 polyunsaturated fatty acids (PUFAs) between microalgal oil and fish oil, both these oils gave the same 50% reduction of AOM-induced ACF when compared to corn oil. To determine whether oxidative stress could play a role in the chemoprevention of colorectal cancer by n-3 PUFAs, feces and caecal content were examined using the TBA assay. The results showed that lipid peroxidation does occur in the gastrointestinal tract. As several lipid peroxidation products of n-3 PUFAs can induce phase II detoxifying enzymes by an EpRE-mediated pathway, the in vivo results suggest that this route may contribute to n-3 PUFA-mediated chemoprevention. All in all, n-3 PUFA-rich oil from microalgae is as good as fish oil regarding chemoprevention in the colon of the rat.

Effects of high fat fish oil and high fat corn oil diets on initiation of AOM-induced colonic aberrant crypt foci in male F344 rats

Modulating effects of high fat fish oil (HFFO) and high fat corn oil (HFCO) diets on azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were studied in male F344 rats following 8 weeks of dietary treatment. The incidence of AOM-induced ACF was significantly lower in the proximal colon of rats fed the HFFO diets compared with rats fed the HFCO diets. No differential effects were found on enzyme activities that are involved in metabolic activation and detoxification of AOM. Activities of hepatic P450 IAI and P450 IIBI and hepatic and feacal levels of lipid peroxidation were increased by feeding the HFFO diet. Hepatic GST activity and plasma levels of PGE 2 were significantly lower in rats fed the HFFO diets compared with those fed the HFCO diets. These observations demonstrate that HFFO diets with high levels of n-3 PUFAs are also protective against preneoplastic lesions in the early stages of chemically induced colon carcinogenesis. It seems unlikely from our results that the inhibitory effect of a HFFO diet can be attributed to an altered metabolic activation and detoxification of AOM. Other mechanisms such as oxidative stress or reduction of PGE 2 levels may play an important role in the anticarcinogenic effects of n-3 PUFAs.

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 1 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 1 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.

Effect of lactobacilli, bifidobacteria and inulin on the formation of aberrant crypt foci in rats

Our studies were aimed at investigating the effect of lactic acid producing bacteria (LAB) or inulin, a natural source of non-digestible oligosaccharides derived from chicory, on the induction by carcinogens of aberrant crypt foci (ACF) in the colon, which are considered to be early precursor lesions of neoplasia. Strains of Bifidobacterium longum, Lactobacillus casei and Lactobacillus acidophilus were administered to rats fed a purified high starch diet, under a variety of treatment protocols including daily gavage, via the drinking water and in the diet. The rats were treated with methyl-N-nitrosourea, 1,2-dimethylhydrazine, or azoxymethane (AOM) to induce ACF. In general, no consistent significant changes in ACF numbers were detected in these experiments. In one study, the basal diet of the rats was changed to one containing a higher level of fat (corn oil). Under these conditions, a significant decrease in AOM-induced colonic ACF was seen in rats given L. acidophilus or inulin. In a concurrent group of animals fed a low fat diet, no significant decrease in ACF was observed. The results indicate that the type of diet fed can influence the detection of protective effects of LAB and oligosaccharides and that against the background of a diet with a level of fat typical of a Western diet, evidence for a protective effect of L. acidophilus and inulin towards colon cancer was obtained

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.

Evaluation of benzyl selenocyanate glutathione conjugate for potential chemopreventive properties in colon carcinogenesis.

Observational, clinical and experimental studies have suggested that dietary supplementation with selenium can inhibit the development of colon cancer. Since toxicity and chemopreventive efficacy of selenium compounds depend to a large extent, on the form of selenium the development of efficacious organoselenium compounds with low toxicity is being pursued in our laboratory. We have assessed the chemopreventive properties of a newly synthesized organoselenium compound, benzyl selenocyanate glutathione conjugate (BSeSG), and of benzyl selenocyanate (BSC), as a positive control, using azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) as a measure of efficacy. Five-week-old male F344 rats were fed the control diet (modified AIN-76A) or experimental diets containing 10 or 20 ppm BSeSG (1.7 and 3.4 ppm as Se, respectively), or 10 ppm BSC (4.1 ppm as Se). One week later, all animals except those in vehicle (normal saline)-treated groups were s.c. injected with AOM (15 mg/kg of body weight, once weekly for 2 weeks). All animals were sacrificed 7 weeks after the last AOM injection, and the ACF, levels of prostaglandin E2 (PGE2), cyclooxygenase protein expression (COX-1 and -2), and glutathione S-transferase type mu (GST-mu) were determined in the colon. As expected, dietary administration of BSC suppressed ACF development by about 37%. In rats administered 10 or 20 ppm BSeSG, the frequencies of AOM-induced colonic ACF were significantly decreased compared to those of rats given AOM and control diet by about 41% (P<0.01) and 61% (P<0.001), respectively. Administration of BSeSG inhibited PGE2 production (81-88% inhibition) via COX-2 synthesis in the colonic mucosa (18-60% inhibition). Also, BSeSG increased GST-mu protein activity in colonic mucosa (30-32% increase). These data suggest that a newly synthesized organoselenium compound, BSeSG might be a promising chemopreventive agent against colon carcinogenesis.