AOM-induced Aberrant Crypt Foci Formation Research Articles

Changes in the Fecal Metabolome Accompany an Increase in Aberrant Crypt Foci in the Colon of C57BL/6 Mice Fed with a High-Fat Diet.

High-fat diet (HFD)-induced obesity is a risk factor for colon cancer. Our previous data show that compared to an AIN-93 diet (AIN), a HFD promotes azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) formation and microbial dysbiosis in C57BL/6 mice. To explore the underlying metabolic basis, we hypothesize that AOM treatment triggers a different fecal metabolomic profile in C57BL/6 mice fed the HFD or the AIN. We found that 65 of 196 identified metabolites were significantly different among the four groups of mice (AIN, AIN + AOM, HFD, and HFD + AOM). A sparse partial least squares discriminant analysis (sPLSDA) showed that concentrations of nine fecal lipid metabolites were increased in the HFD + AOM compared to the HFD, which played a key role in overall metabolome group separation. These nine fecal lipid metabolite concentrations were positively associated with the number of colonic ACF, the cell proliferation of Ki67 proteins, and the abundance of dysbiotic bacteria. These data suggest that the process of AOM-induced ACF formation may increase selective fecal lipid concentrations in mice fed with a HFD but not an AIN. Collectively, the accumulation of these critical fecal lipid species may alter the overall metabolome during tumorigenesis in the colon.

Sphingosine Kinase 1 expression in peritoneal macrophages is required for colon carcinogenesis.

Accumulating evidence suggests that the sphingosine kinase 1 (SphK1)/sphingosine 1-phosphate (S1P) pathway plays a pivotal role in colon carcinogenesis. Our previous studies indicate that the SphK1/S1P pathway mediates colon carcinogenesis at least by regulating cyclooxygenase 2 (COX-2) expression and prostaglandin E2 (PGE2) production. However, the mechanisms by which this pathway regulates colon carcinogenesis are still unclear. First, we show that SphK1 deficient mice significantly attenuated azoxymethane (AOM)-induced colon carcinogenesis as measured by colon tumor incidence, multiplicity, and volume. We found that AOM activates peritoneal macrophages to induce SphK1, COX-2, and tumor necrosis factor (TNF)-α expression in WT mice. Interestingly, SphK1 knockout (KO) mice revealed significant reduction of COX-2 and TNF-α expression from AOM-activated peritoneal macrophages, suggesting that SphK1 regulates COX-2 and TNF-α expression in peritoneal macrophages. We found that inoculation of WT peritoneal macrophages restored the carcinogenic effect of AOM in Sphk1 KO mice as measured by aberrant crypt foci (ACF) formation, preneoplastic lesions of colon cancer. In addition, downregulation of SphK1 only in peritoneal macrophage by short hairpin RNA (shRNA) reduced the number of ACF per colon induced by AOM. Intraperitoneal injection of sphingolipids demonstrates that S1P enhanced AOM-induced ACF formation, while ceramide inhibited. Finally, we show that SphK inhibitor SKI-II significantly reduced the number of ACF per colon. These results suggest that SphK1 expression plays a pivotal role in the early stages of colon carcinogenesis through regulating COX-2 and TNF-α expression from activated peritoneal macrophages.

Plum polyphenols inhibit colorectal aberrant crypt foci formation in rats: potential role of the miR-143/protein kinase B/mammalian target of rapamycin axis

The nutritional prevention of aberrant crypt foci by polyphenols may be a crucial step to dietary cancer prevention. The objective of this study was to determine the underlying mechanisms that contribute to the anti-inflammatory and antitumorigenic properties of plum (Prunus salicina L.) polyphenols, including chlorogenic acid and neochlorogenic acid, in azoxymethane (AOM)-treated rats. The hypothesis was that plum polyphenolics suppress AOM-induced aberrant crypt foci formation through alterations in the protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway and relative micro-RNA expressions. Sprague-Dawley rats (n=10/group) received plum beverage (1346mg gallic acid equivalents/L) or a control beverage ad libitum for 10 weeks with subcutaneous injections of AOM (15mg/kg) at weeks 2 and 3. Results show that the consumption of the plum beverage decreased the number of dysplastic aberrant crypt foci by 48% (P<.05) and lowered proliferation of mucosal cells by 24% (P<.05). The plum beverage decreased the activity of glutathione peroxidase, superoxide dismutase, and catalase in mucosal scrapings, as well as the superoxide dismutase activity in serum. The results were accompanied by a down-regulation of proinflammatory enzymes nuclear factor κB, nitric oxide synthase, cyclooxygenase-2, and vascular cell adhesion molecule 1 messenger RNA. Plum inhibited the expression of AKT and mTOR messenger RNA, phosphorylated AKT, mTOR, and hypoxia-inducible factor-1α protein levels, and the ratio of the phosphorylated/total protein expression of mTOR. Also, the plum beverage increased the expression of miR-143, which is involved in the regulation of AKT. These results suggest that plum polyphenols may exhibit a chemopreventive potential against colon carcinogenesis by impacting the AKT/mTOR pathway and miR-143.

P0026 The chemopreventive potential of Curcuma purpurascens rhizome in reducing azoxymethane-induced aberrant crypt foci in rats

Background Curcuma purpurascens rhizome, a member of the Zingiberaceae family, is a popular spice in Indonesia and is traditionally used for assorted remedies. Dichloromethane extract of C. purpurascens rhizome (DECPR) has previously been shown to have apoptosis-inducing effect on colon cancer cells. Methods We examined the potential of DECPR to prevent colon cancer development in rats treated with azoxymethane (AOM, 15 ml/kg) by the incidence of aberrant crypt foci (ACF). Starting from the day immediately after AOM treatment, the rats were orally administered once a day for 2 months with 10% Tween-20 (5 ml/kg, cancer control), DECPR (250 mg/kg, low dose) and DECPR (500 mg/kg, high dose). Meanwhile, the control group was intra-peritoneally injected with fluorouracil (35 mg/kg) for 5 days consecutively. After euthanising the rats, the number of ACF was enumerated in colon tissues. Bax, Bcl-2, and PCNA protein levels were examined using immunohistochemical and western blot analyses. Antioxidant enzymatic activity was measured in colon tissue homogenates and associated with malondialdehyde level. Findings DECPR exposure at both doses significantly decreased AOM-induced ACF formation, which was accompanied with the reduced expression of PCNA. Up-regulation of Bax and down-regulation of Bcl-2 suggested the involvement of apoptosis in the chemopreventive effect of DECPR. In addition, the oxidative stress resulting from AOM treatment was significantly attenuated after administration of DECPR, which was shown by the elevated antioxidant enzymatic activity and reduced malondialdehyde level. Interpretation DECPR significantly inhibits ACF formation in AOM-treated rats and may offer protection against colon cancer development.

The chemopreventive potential of Curcuma purpurascens rhizome in reducing azoxymethane-induced aberrant crypt foci in rats

Curcuma purpurascens BI. rhizome, a member of the Zingiberaceae family, is a popular spice in Indonesia that is traditionally used in assorted remedies. Dichloromethane extract of C. purpurascens BI. rhizome (DECPR) has previously been shown to have an apoptosis-inducing effect on colon cancer cells. In the present study, we examined the potential of DECPR to prevent colon cancer development in rats treated with azoxymethane (AOM) (15 mg/kg) by determining the percentage inhibition in incidence of aberrant crypt foci (ACF). Starting from the day immediately after AOM treatment, three groups of rats were orally administered once a day for 2 months either 10% Tween 20 (5 mL/kg, cancer control), DECPR (250 mg/kg, low dose), or DECPR (500 mg/kg, high dose). Meanwhile, the control group was intraperitoneally injected with 5-fluorouracil (35 mg/kg) for 5 consecutive days. After euthanizing the rats, the number of ACF was enumerated in colon tissues. Bax, Bcl-2, and proliferating cell nuclear antigen (PCNA) protein expressions were examined using immunohistochemical and Western blot analyses. Antioxidant enzymatic activity was measured in colon tissue homogenates and associated with malondialdehyde level. The percentage inhibition of ACF was 56.04% and 68.68% in the low- and high-dose DECPR-treated groups, respectively. The ACF inhibition in the treatment control group was 74.17%. Results revealed that DECPR exposure at both doses significantly decreased AOM-induced ACF formation, which was accompanied by reduced expression of PCNA. Upregulation of Bax and downregulation of Bcl-2 suggested the involvement of apoptosis in the chemopreventive effect of DECPR. In addition, the oxidative stress resulting from AOM treatment was significantly attenuated after administration of DECPR, which was shown by the elevated antioxidant enzymatic activity and reduced malondialdehyde level. Taken together, the present data clearly indicate that DECPR significantly inhibits ACF formation in AOM-treated rats and may offer protection against colon cancer development.

Abstract B42: Lack of chemopreventive effects of metformin in azoxymethane-induced rat colon carcinogenesis

Abstract Metformin, an insulin-lowering agent, has been associated with decreased cancer risk in epidemiologic studies in diabetic patients. Short-term preclinical and clinical observations also supported inhibition of colonic precancerous lesions. However, recent meta-analysis studies show that metformin or related drugs may reduce risk of pancreatic and hepatocellular carcinoma but a non-significant inverse correlation with colon cancer risk. The present study explores a systematic analysis of chemopreventive efficacies of metformin on a well established model of colon carcinogenesis, using colonic preneoplastic lesions and adenocarcinomas as end points. Effect of dietary metformin (100 ppm, 400 ppm, 800 ppm to 1,600 ppm) on AOM-induced aberrant crypt foci (ACF) development; and a dietary metformin (500 ppm and 1,000 ppm) on AOM-induced colon adenocarcinoma was studied in F344 rats. ACF and tumor efficacy endpoints were carried out on AOM-treated 8-week-old rats (48 per group) fed the control AIN-76A diet. Either three days (ACF Study) or 4 weeks (adenocarcinoma study) after carcinogen treatment, rats were fed the diets containing different doses of metformin. ACF and colon adenocarcinomas were determined at 8 and 48 weeks after AOM-treatment, respectively. Dietary metformin at 100 and 400 ppm had no significant effect on the colonic ACF formation; whereas at higher doses a significant increase was observed in the total ACF (37%) and multicrypt ACF by 43%. In long term bioassay, dietary metformin at 500 and 1,000 ppm had no significant effect on adenocarcinoma incidence. With regard to colon adenocarcinoma multiplicity, metformin at both dose levels failed to provide any inhibitory effect, albeit, modest increase (∼20%) in adenocarcinoma multiplicity at high dose level was seen. Similarly, dietary metformin fail to show any inhibitory effect on colon tumor volume. Rats that were fed with metformin fail to show significant effects on markers of cell proliferation and apoptosis in colonic tumors. These observations show, for the first time, that metformin tested at different dose levels does not provide chemopreventive effects at the preneoplastic lesion stage or at adenocarcinoma stages in a well-established rat colon cancer model. Further studies are warranted to understand the discrepancies between preclinical models and more so in human clinical observations on the potential beneficial effects of metformin for colon cancer risk. {This work is supported by NCI-N01 CN-53300} Citation Information: Cancer Prev Res 2011;4(10 Suppl):B42.

Dietary folate protects against azoxymethane-induced aberrant crypt foci development and oxidative stress in rat colon

Azoxymethane (AOM) induces cancer and oxidative stress in rat colon. This study tested the hypothesis that dietary folate supplementation protects against AOM-induced oxidative stress and reduces aberrant crypt foci (ACF) development in rat colon. Fifty-four weanling male albino rats, with an average body weight of 50 ± 5 g, were randomly divided into three groups--A, B and C (18 rats per group)--and fed 2, 8 or 40 mg of folic acid per kg of supplemented diets, respectively, throughout the eight weeks' experimental period. The animals were supplied with diet and water ad libitum for four weeks and they reached an average body weight of 100 g. Thereafter each group was then further randomly subdivided into three subgroups (six rats per subgroup): control, vehicle and AOM-injected groups. The control group did not receive any treatment (neither AOM injection nor saline), the rats in the vehicle group were given 1 mL intraperitoneal injection of saline once a week for two weeks and the rats in the AOM-injected group were given two intraperitoneal injections of AOM dissolved in saline once a week for two weeks totaling 30 mg/kg body weight. After the last AOM injection, animals were continuously fed ad libitum their specified diet for two weeks of last AOM injection, all rats were sacrificed, and colon tissues were collected and used for ACF enumeration and measurements of glutathione (GSH) and total antioxidant capacity (TAC). The results revealed that AOM-injected rats showed lower levels of GSH and TAC as compared with control and vehicle groups. Folic acid-supplemented diets suppressed the AOM-induced ACF and GSH depletion in a dose-dependent manner and augmented the TAC. It was concluded that folic acid supplementation protects against the AOM-induced ACF formation by suppressing the AOM-induced GSH depletion in rat colon cells.

Abstract LB-21: Taraxacum officinale (common dandelion) inhibits azoxymethane-induced aberrant crypt foci formation in C67BL/6 mice and regulates apoptosis in mice and in human colorectal carcinoma HCT-116 cells

Abstract Taraxacum officinale (TO), the common dandelion, has been used traditionally as a diuretic and as a treatment for jaundice and other liver disorders. Recent studies have substantiated its diuretic effect; in addition, it exhibits considerable antimicrobial, anti-inflammatory, antioxidant, hypolipidemic, and anti-breast and anti-prostate cancer-cell properties. In this study, the effects of TO as a chemopreventive agent of early-stage colon cancer development were evaluated. In the in vivo investigation, C57BL/6 mice between 6–10 wk old were randomized into negative control (N = 10), positive control (N = 10), and experimental (N = 10) groups. Aberrant crypt foci (ACF), putative precursors of colon cancer, were induced in positive control and experimental mice by intraperitoneal injection of azoxymethane (AOM; 5 mg/kg body mass) at the beginning of the first and second weeks, while the negative control mice received none. The experimental group was daily fed TO extract (1 mL/mouse; 84.22 mg/mL) by gavage throughout the duration of the 8-wk study while the control groups received a sterilized-water placebo. Mice in all three groups were sacrificed at the end of the 8-wk period and 25 mm of the proximal colon were resected and analyzed for ACF development after staining with methylene blue. Histological preparations of the proximal colon samples were used to determine apoptotic index, mitotic index, and caspase induction. Oral TO administration significantly reduced the number of AOM-induced ACF in the proximal colon in the experimental group in comparison to the positive control group (10.67 ± 3.75 &amp;lt; 32.10 ± 9.86; P &amp;lt; 0.00016), which indicates a 68.9 % inhibition of ACF formation by TO and a potency index of 3.01. The mice in all three groups experienced weight gain (negative: 7.0% &amp;lt; positive: 7.5% &amp;lt; experimental: 8.1%). Colon samples from the experimental group revealed a higher apoptotic index (AI) (1.34 &amp;gt; 0.60; P &amp;lt; 0.0002) and a lower mitotic index (MI) (1.13 &amp;lt; 2.80; P &amp;lt; 0.25) than the positive control group (AOM only). Caspase 3 and 9 induction was detected in human colorectal carcinoma HCT-116 cells treated with TO (.05 mg/mL) in chamber slides, using a specific carboxyfluorecein (FAM) labeled peptide fluoromethyl ketone (FMK) caspase inhibitor (FAM-peptide-FMK). Caspase 3 and 9 activation was also observed in TO-treated mouse colon tissues using the same caspase inhibitor. The data suggests that TO possesses potential chemopreventive effects on colon cancer development through the inhibition of AOM-induced ACF formation via C3/C9-regulated apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-21. doi:10.1158/1538-7445.AM2011-LB-21

Abstract A50: Plantago major inhibits azoxymethane-induced aberrant crypt foci in C57BL/6 mice and modulates apoptosis in mice and human colon cancer cells

Abstract Plantago major (PM), commonly known as common plantain, is a temperate perennial herb. Traditionally, its seeds and leaves have been ingested or applied externally to treat inflammation and infection. Studies showed PM possesses inhibitory effects towards a variety of tumors and cancer cell lines, including mice Ehrlich ascites carcinoma and breast adenocarcinoma, gastric, leukaemia, ovary, melanoma, and renal human cell lines. While PM has been shown to exhibit selective cytotoxic activity against cancer cells, its quantitative potential as a chemopreventive agent on colon cancer has yet to be elucidated. In this study, C57BL/6 mice (N = 30) of 9 wk old were randomized into three groups (N = 10). Positive and experimental mice groups were injected with Azoxymethane (AOM; 5 mg/kg body weight) at the onset and second wk of the 8-wk duration of the in vivo study while the negative group received no injection. The mice in the experimental group were given daily oral feedings of 20 mg/ml of PM from the onset of the study while the negative and positive control groups were given a sterilized water placebo. All groups were sacrificed after 8 wks and 25 mm cuts of the proximal colon were analyzed for aberrant crypt foci (ACF). PM feeding significantly reduced the number of AOM-induced ACF in the proximal colon compared to the water-fed positive control AOM-induced group (7.02 ± 2.61 &amp;lt; 32.1 ± 9.86; P &amp;lt; 0.0003). The negative control, water placebo group had minimal ACF (2.0 ± 1.56) compared to the experimental and positive control groups. The potency index of the herb was calculated as 4.57. Percent inhibition of AOM-induced ACF formation of 78.1% was obtained with the PM treatment. On average, all three groups gained weight (calculated as % increase) through the course of the experiment (experimental, 6.93 &amp;lt; negative control 15.6 &amp;lt; positive control 39.4). Histological (H&amp;E) slides of the proximal colon displayed a significantly higher apoptotic index than the positive control (AOM only) (2.0 &amp;gt; 0.6) and a lower mitotic index (MI) than control (1.5 &amp;lt; 2.8). Caspase 3 induction was identified in human colon cancer cells (COLO-205) treated with PM (5mg/ml) in chamber slides, using a specific carboxyfluorecein (FAM) labeled peptide fluoromethyl ketone (FMK) caspase inhibitor (FAM-Peptide-FMK). Caspase 3 activation was also observed in immunofluorescent slides of PM treated proximal colon tissues. Through inhibition of AOM-induced ACF via apoptosis, our data suggests PM to be a prospective chemopreventive and potential therapeutic agent for colon cancer. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A50.

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 beta-catenin and NF-kappaB 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 beta-catenin and NF-kappaB signaling, and suggest its potential usefulness for the prevention of human CRC.

Alkylation-induced colon tumorigenesis in mice deficient in the Mgmt and Msh6 proteins

O6-methylguanine DNA methyltransferase (MGMT) suppresses mutations and cell death that result from alkylation damage. MGMT expression is lost by epigenetic silencing in a variety of human cancers including nearly half of sporadic colorectal cancers, suggesting that this loss maybe causal. Using mice with a targeted disruption of the Mgmt gene we tested whether Mgmt protects against azoxymethane (AOM) induced colonic aberrant crypt foci (ACF), against AOM and dextran sulfate sodium (DSS) induced colorectal adenomas, and against spontaneous intestinal adenomas in ApcMin mice. We also examined the genetic interaction of the Mgmt null gene with a DNA mismatch repair null gene, namely Msh6. Both Mgmt and Msh6 independently suppress AOM-induced ACF, and combination of the two mutant alleles had a multiplicative effect. This synergism can be explained entirely by the suppression of alkylation-induced apoptosis when Msh6 is absent. In addition, following AOM+DSS treatment Mgmt protected against adenoma formation to the same degree as it protected against AOM-induced ACF formation. Finally, Mgmt deficiency did not affect spontaneous intestinal adenoma development in ApcMin/+ mice, suggesting that Mgmt suppresses intestinal cancer associated with exogenous alkylating agents, and that endogenous alkylation does not contribute to the rapid tumor development seen in ApcMin/+ mice.

Inhibition of Azoxymethane-Induced Colonic Aberrant Crypt Foci Formation by Silibinin in Male Fisher 344 Rats

Chemoprevention is a practical approach to control colorectal cancer, which is one of the major causes of cancer mortality in the United States. Based on our recent silibinin efficacy studies in human colorectal cancer cells, we investigated the effects of its dietary feeding on azoxymethane (AOM)-induced aberrant crypt foci (ACF) formation and associated biomarkers in male Fisher 344 rats. Five-week-old male Fisher 344 rats were fed control or silibinin-supplemented (0.033%, 0.1%, 0.33%, or 1%, w/w) diet. After 2 weeks, AOM was injected once a week for 2 weeks while silibinin treatments were continued. In another protocol, identical silibinin treatments were done but started 2 weeks post-AOM initiation. All rats were sacrificed at 16 weeks of age, and colon samples were evaluated for ACF, followed by proliferation, apoptosis, and inducible nitric oxide synthase and cyclooxygenase-2, by immunohistochemistry and/or immunoblotting. Silibinin significantly (P < 0.001) reduced dose-dependently the number and multiplicity of AOM-induced ACF formation. Silibinin feeding in pre- and post-AOM initiation decreased mean number of ACF by 39% to 65% and in post-AOM initiation by 29% to 55%. Silibinin dose-dependently decreased AOM-induced colonic cell proliferation, evidenced by proliferative cell nuclear antigen and cyclin D1 immunohistochemical staining, and induced apoptosis in these colon tissues, evidenced by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling staining and cleaved poly(ADP-ribose) polymerase. Furthermore, silibinin significantly decreased AOM-induced inducible nitric oxide synthase- and cyclooxygenase-2-positive cells in colon tissues. The present findings show possible beneficial activity of silibinin at least in early stage of colon tumorigenesis, suggesting that silibinin might be an effective natural agent for colorectal cancer chemoprevention.

Effects of resistant starch on colonic preneoplastic aberrant crypt foci in rats

This study was designed to determine the effects of resistant starch (RS) at different levels on the azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) at the pre-initiation (PI) and promotion (P) stages in Wistar rats. According to the consuming assigned diets, all animals received AOM at a dosage of 15 mg/kg once a week for two consecutive weeks. In experiment 1, four groups of rats ( n = 12) were given AOM after 3 weeks of consuming the AIN-76 (control group) and RS diets. In experiment 2, four groups of rats ( n = 12) were given AOM before 3 weeks of consuming the AIN-76 and RS diets. Rats were killed after 13 weeks of initial injecting AOM. Colons were fixed in formalin and ACF were quantified after staining. In experiment 1, rats fed RS had more ACF than that of the control fed rats. In experiment 2, rats fed RS had fewer ACF than that of the control fed rats. The results indicate that dietary RS suppresses AOM-induced ACF formation, only at the P stage. A significant dose-response effect was observed between suppression of ACF formation and dietary RS amount. However, RS promote the formation of ACF at the PI stage.

Susceptibility of Snark‐deficient mice to azoxymethane‐induced colorectal tumorigenesis and the formation of aberrant crypt foci

SNF-1/5'-AMP-activated kinase (AMPK)-related kinase (SNARK) is a member of the AMPK-related kinases. Snark(+/-) mice exhibited mature-onset obesity and related metabolic disorders. Obesity is regarded as a risk factor for colorectal cancer. To investigate whether Snark deficiency is involved in tumorigenesis in the large intestine, obese Snark(+/-) mice were treated with a chemical carcinogen, azoxymethane (AOM). The incidences of both adenomas and aberrant crypt foci (ACF) were significantly higher in Snark(+/-) mice than in their wild-type counterparts 28 weeks after the completion of AOM treatment (10 mg/kg/week for 8 weeks). Furthermore, ACF formation was enhanced in Snark(+/-) mice treated with AOM for 2 weeks, suggesting that Snark deficiency contributed to the early phase of tumorigenesis. The total number of ACF was correlated with bodyweight in Snark(+/-) and Snark(+/+) mice, suggesting that obesity was a risk factor for colorectal tumorigenesis in this model. However, the correlation coefficient was higher in Snark(+/-) mice. Moreover, AOM-induced ACF formation was also enhanced in preobese Snark(+/-) mice. Together, these findings suggest that AOM-induced tumorigenesis in Snark(+/-) mice was enhanced via obesity-dependent and -independent mechanisms.

Flax seed oil and flax seed meal reduce the formation of aberrant crypt foci (ACF) in azoxymethane-induced colon cancer in Fisher 344 male rats

Flax seed oil and flax seed meal are good sources of omega-3 fatty acids. The objective of this study was to explicate the effects of feeding flax seed oil and flax seed meal on AOM-induced aberrant crypt foci (ACF) in Fisher 344 male rats. Following an acclimatization period, rats were divided into six groups and fed AIN 93G diet Control (C), C + 7 and 14% soybean oil (SBO), C + 7 and 14% flax seed oil (FSO) and C + 10 and 20% flax seed meal (FSM). All rats received 16 mg/kg body weight of AOM at 7 and 8 weeks of age. The rats were euthanized with CO 2 at 17 weeks of age. FSM and FSO reduced the incidence of ACF which are putative precursor lesions in the development of colon cancer in the distal colon by 88% and 77%, in the proximal colon by 86% and 87% with a total reduction of 87.5% and 84%, respectively. Glutathione- S-transferase (GST) activities were significantly ( P < 0.05) higher in rats fed C + 7 and 14% FSO and C + 10 and 20% FSM, as compared to rats fed C + SBO diets. Results of this study showed that FSO and FSM reduced the incidence of AOM-induced ACF formation and may therefore be effective chemopreventive agents.

Dietary Inulin Suppresses Azoxymethane-Induced Preneoplastic Aberrant Crypt Foci in Mature Fisher 344 Rats

Preneoplastic aberrant crypt foci (ACF) are generally accepted as reliable markers for colon carcinogenesis in animal models. Rat model ACF studies, however, use younger rats, and there are no published reports on the suitability of adult rats for ACF studies. In this study, inulin, a known suppressor of azoxymethane (AOM)-induced ACF, was tested for its ability to suppress ACF formation in mature rats. After a 2-wk acclimation period, 12-mo-old Fisher 344 retired male breeders received two subcutaneous injections of AOM dissolved in saline at weekly intervals. In experiment 1, six groups received 0, 4, 8, 10, 12 and 16 mg AOM/kg body at each injection and were fed AIN-93M diet. In experiment 2, four groups of rats were fed 10 mg AOM/kg body at each injection based on the results of experiment 1, and were fed 0, 2.5, 5 and 10 g long-chain inulin diets/100 g. All the rats were killed after 11-wk feeding periods. In experiment 1, there was a significant (P < 0.05) AOM dose response on ACF formation. Rats fed >10 mg of AOM had greater (P < 0.05) mortality. In experiment 2, there was a significant increase in cecal weight and a decrease in cecal pH from 7.17 in the control group to 6.87, 6.61 and 5.76 in the groups fed inulin at 2.5, 5.0 and 10 g/100 g, respectively. Long-chain inulin dose-dependently reduced ACF incidence in the colon (P < 0.01). Compared with rats fed the control diet, the percentage reductions of ACF in rats fed 2.5, 5.0 and 10 g inulin diets/100 g were 25, 51, and 65, respectively. The results of this study indicate that mature rats can be used as models in ACF studies, and dietary long-chain inulin dose-dependently suppresses AOM-induced ACF formation in Fisher 344 mature male rats.

Dietary Inulin Suppresses Azoxymethane-Induced Aberrant Crypt Foci and Colon Tumors at the Promotion Stage in Young Fisher 344 Rats

This study was designed to determine the effect of 10% dietary long-chain inulin on the azoxymethane (AOM)-induced colonic preneoplastic aberrant crypt foci (ACF) and small intestinal and colon tumors at the initiation (I), promotion (P) and I + P stages (20 rats per treatment) in Fisher 344 male weanling rats. After an acclimatization period of 1 wk, groups of Fisher 344 male weanling rats were assigned to consume AIN 93G diet (control) or AIN 93G diet containing 10% inulin. All the rats received 16 mg/kg body AOM dissolved in saline subcutaneously at 7 wk of age followed by a second injection at 8 wk of age. An additional group of five rats received only saline and consumed the control diet. The rats received the assigned diets until asphyxiation by CO(2) at 16 wk of age for the ACF experiment and 45 wk for the end-point tumor experiment. Feed intake, weight gain, diarrheal index, cecal weight, cecal pH, ACF and tumors in the colon were determined. Rats fed inulin had diarrhea after 2 wk of feeding and recovered by approximately 4 wk. Cecal weight was greater in rats fed inulin and cecal pH was lower. The inulin group had more than 66% fewer aberrant crypts and 60% fewer ACF compared with the control group. Tumor incidences in the small intestine and colon of rats in the control, I, P and I + P groups were: 78, 31, 0 and 11% and 90, 73, 69 and 50%, respectively. The corresponding values for the distal portion of the colon were 87, 63, 45 and 33%, respectively. Colon tumors per tumor-bearing rat were 4.2, 3.09, 1.36 and 1.2 for the control, I, P and I + P groups, respectively. All groups differed, P < 0.05. The results of this study indicate that dietary long-chain inulin suppresses AOM-induced ACF formation, an early preneoplastic marker of colon tumorigenesis in rats, and colon tumors, particularly at the promotion stage.

Effect of Grape Seed Proanthocyanidins on Colon Aberrant Crypts and Breast Tumors in a Rat Dual-Organ Tumor Model

Cancers of the colon and breast are two of the most prevalent cancers in developed countries. The present experiments were conducted to determine the influence of several dietary doses of grape seed proanthocyanidins on 7,12-dimethylbenz[a]anthracene-induced mammary tumorigenesis and azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) formation in a dual-organ tumor model. In addition, the effects of the grape seed proanthocyanidins on liver cytochrome P-450 1A and 2E1 and glutathione S-transferase activities and on colonic ornithine decarboxylase activity were examined to determine possible mechanisms of action. Feeding female rats diets containing 0.1-1.0% grape seed proanthocyanidins was associated with a significant 72-88% inhibition of AOM-induced aberrant crypt foci formation and a 20-56% inhibition of ornithine decarboxylase activity in the distal third of the colon. Feeding the grape proanthocyanidins resulted in no significant effect on the activity of liver cytochrome P-450 2E1. There was no effect of feeding these doses of proanthocyanidins on 7,12-dimethylbenz[a]anthracene-induced rat mammary tumorigenesis. This lack of action on mammary tumorigenesis in part may be due to lack of effect of dietary proanthocyanidins on the liver carcinogen-metabolizing enzymes cytochrome P-450 1A and glutathione S-transferase. These results indicate that grape polyphenolics warrant further evaluation as potential colon cancer chemopreventive agents.

Chemopreventive effects of coffee bean and rice constituents on colorectal carcinogenesis

Polyphenolic compound chlorogenic acid (CGA) known to be much contained in coffee beans was found to have a regressive effect on induced aberrant crypt foci (ACF) as well as on development of ACF in azoxymethane (AOM)-induced colorectal carcinogenesis in rats. Rice germ and gamma-aminobutyric acid-enriched defatted rice germ inhibited AOM-induced ACF formation and colorectal carcinogenesis in rats. Ferulic acid (FA) also known to be contained in coffee beans and rice prevented AOM-induced ACF formation and intestinal carcinogenesis in rats. Both of food factors, coffee and rice may be of benefit to prevention of human colorectal cancers.

Inhibitory effect of Coptidis Rhizoma and Scutellariae Radix on azoxymethane-induced aberrant crypt foci formation in rat colon.

This study was conducted to obtain effective cancer chemopreventive agents with low toxicity from medicinal herbs. The effect of aqueous extracts from 9 medicinal herbs with antiinflammatory effect were examined on the formation of azoxymethane (AOM)-induced aberrant crypt foci (ACF), putative preneoplastic lesions of the colon. Male F344 rats were treated with 15 mg/kg body weight of AOM once a week for two weeks. Herbal extract consisting of 2% of the diet was administered from 1 d prior to the first carcinogen treatment. The number of AOM-induced ACF per colon was counted at 4 week. Extracts of Coptidis Rhizoma and Scutellariae Radix significantly inhibited AOM-induced ACF formation. The number of ACF was decreased to 54% and 78% of that of the control by 2% Coptidis Rhizoma and Scutellariae Radix extract in the diet, respectively. Berberine and Baicalin, major ingredients of Coptidis Rhizoma and Scutellariae Radix, inhibited ACF formation at a dose equivalent to the amount in each herbal extract. Therefore, to investigate the mechanisms of action of berberine and baicalein which is the active substances of orally administered baicalin, their effects on cyclooxygenase 1 and 2 activities were studied. Berberine was found to inhibit cyclooxygenase 2 activity without inhibition of cyclooxygenase 1 activity, and baicalein inhibited cyclooxygenase 1 activity. Thus, Coptidis Rhizoma and Scutellariae Radix suppressed experimental colon carcinogenesis, and their chemopreventive effects were explained from the inhibition of berberine on cyclooxygenase 2 activity and baicalein on cyclooxygenase 1 activity.