Colonic Aberrant Crypt Research Articles

Abstract 2479: Leptin signaling regulates colorectal tumor growth through Stat3 signaling: Tumor growth induced by dietary intake

Abstract Background: Leptin, a product of the ob gene involved in energy balance and the regulation of food intake, is secreted predominantly in adipose tissue and serum leptin levels are elevated in obese individuals. Epidemiological studies have revealed that obesity, especially visceral fat, raises the risk of colorectal cancer (CRC). However, data concerning the effect of leptin on CRC development are contradictory and difficult to interpret. The aim of this study was to investigate the impact of leptin deficiency on CRC development and to determine whether leptin acts as a tumor promoter. Methods: We investigated chemical-induced colon tumors and aberrant crypt foci (ACF) formation using wild-type (WT) mice, leptin-deficient (ob/ob) mice and leptin receptor deficient (db/db) mice. Western blot analysis, PCR and immunohistochemistry were performed to examine the role of leptin signaling in the colonic epithelium. Results: Ob/ob and db/db mice exhibited dramatically decreased tumor sizes, despite exhibiting their severe obesity and hyperinsulinemia. Tumor cell proliferation was significantly lower in the ob/ob and db/db mice than in the WT mice. These results strongly indicated that leptin is indispensable for the regulation of colonic tumor growth irrespective of obesity. Conversely, normal mucosal cell proliferation was significantly higher in ob/ob and db/db mice than in WT mice. The number of ACF in ob/ob and db/db mice was also significantly higher than that in WT mice, suggesting that leptin is not a promoter of colon tumor initiation. Here, we noted a difference in the expression of leptin-receptor (Ob-R) between tumors and normal mucosa. Interestingly, the Ob-R expression level was markedly increased in the tumors, compared with normal mucosa. Furthermore, immunohistochemical analysis revealed the tumor cell nuclear localization of phosphorylated-Stat3, which is a downstream signaling pathway of Ob-R, in WT mice, and this signal was almost completely absent in the tumors of ob/ob mice. Conclusions: We clearly demonstrated a relationship between leptin and the growth of colon tumors in leptin- or leptin receptor-deficient mice. The dramatic suppression of colonic tumor growth induced by the inhibition of leptin signaling indicates that leptin is an indispensable growth factor for colon tumors. Based on the present results, colon tumors might have a tendency to develop in overeating obese individuals whose serum leptin levels are elevated. Our data provide novel insights into leptin signaling in CRC and suggest novel therapeutic and preventive approaches for colon polyps and cancers based on the inhibition of leptin-dependent Stat3 signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2479.

Abstract 3784: Caloric restriction inhibits 1, 2-dimethylhydrazine-induced aberrant crypt foci formation and induces differential expression of sirtuins in colonic mucosa of F344 rats

Abstract Caloric restriction (CR), a 20-40% lowering of caloric intake, is associated with longevity, and decreased incidences of age-related diseases including cancer. The sirtuins are a highly conserved family of nicotinamide adenine dinucleotide (NAD+)-dependent enzymes and have been implicated as a key mediator for the beneficial effects of CR on aging and longevity. However, the underlying mechanisms by which CR decreases cancer risk have not yet been elucidated. The present study was conducted to determine whether CR would modify the growth of preneoplastic colonic aberrant crypt foci (ACF), and whether sirtuin expression levels would be correlated with ACF formation. Five-week-old male F344 rats were randomly divided into four experimental groups. Groups 1 and 2 were given subcutaneous injection of 1, 2-dimethylhydrazine (DMH, 40 mg/Kg body weight), once a week for 2 weeks to induce ACF. Rats in groups 2 and 4 were fed a CR diet (60% of the ad libitum intake), and those in groups 1 and 3 were fed CE-2 diet (CLEA, Tokyo, Japan) ad libitum. All animals were sacrificed at 4 weeks after the first DMH injection. Rat colons were removed and fixed in 10% formalin. ACF were counted after 2% methylene blue staining. The expressions of sirt1 and sirt4 in the colonic mucosa were evaluated by RT-PCR. The number of ACF in colonic mucosa was significantly decreased in DMH-treated rats with CR (group 2) as compared to in DMH-treated rats without CR (group 1). No ACF was found in DMH-untreated animals with or without CR (groups 3 and 4). The expression levels of sirt1 and sirt4 in colonic mucosa were increased by CR (group 1 vs. group 2 and group 3 vs. group 4). Interestingly, DMH treatment also increased the expression of sirt1 and sirt4 (group 1 vs. group 3), and DMH-treated rats with CR (group 2) showed the highest expression levels of these genes. These results suggest that sirt1 and sirt4 induced by CR might inhibit colonic mucosa from formation and development of ACF. Our observations warranted further investigation of association between expression of sirtuins and prevention of colon carcinogenesis. We are currently analyzing the expression of other sirtuins and will compare the expression of all sirtuin family and ACF formation in colonic mucosa of the rats with or without CR. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3784.

Chlorogenic Acid Differentially Alters Hepatic and Small Intestinal Thiol Redox Status Without Protecting Against Azoxymethane-Induced Colon Carcinogenesis in Mice

Colorectal cancer (CRC) is a leading cause of cancer-related deaths in the United States. Epidemiological data have suggested that coffee consumption is inversely related to CRC risk, which may be attributed to chlorogenic acid (CGA), an ester of caffeic acid (CA) and quinic acid. This study was conducted to determine whether chronic dietary CGA supplementation would attenuate tumorigenesis and oxidative stress in a mouse model of azoxymethane (AOM)-induced colon cancer. Mice (4-wk old; n = 15/group) were fed CGA (0%, 0.01%, or 0.1%) for 20 wk and received 6 weekly intraperitoneal AOM injections (10 mg/kg). CGA and CA dose-dependently accumulated in the small intestinal mucosa. AOM induced (P < 0.05) colonic aberrant crypt foci (14.2 ± 1.9/field) and tumors (14.6 ± 1.1/colon), which were correlated (r = .677; P < 0.05), and CGA at either dose did not reduce tumorigenesis. Hepatic GSH/GSSG and Cys/CySS ratios were unaffected by AOM, but CGA at 0.1% increased these ratios by decreasing GSSG and CySS. CGA did not affect the ratios of small intestinal GSH/GSSG or Cys/CySS, which were decreased in response to AOM treatment. Collectively, these data indicated that CGA did not protect against AOM-induced tumorigenesis but affected hepatic thiol redox status in this colon cancer model.

Germinated brown rice (GBR) reduces the incidence of aberrant crypt foci with the involvement of beta-catenin and COX-2 in azoxymethane-induced colon cancer in rats.

Chemoprevention has become an important area in cancer research due to the failure of current therapeutic modalities. Epidemiological and preclinical studies have demonstrated that nutrition plays a vital role in the etiology of cancer. This study was conducted to determine the chemopreventive effects of germinated brown rice (GBR) in rats induced with colon cancer. GBR is brown rice that has been claimed to be richer in nutrients compared to the common white rice. The male Sprague Dawley rats (6 weeks of age) were randomly divided into 5 groups: (G1) positive control (with colon cancer, unfed with GBR), (G2) fed with 2.5 g/kg of GBR (GBR (g)/weight of rat (kg)), (G3) fed with 5 g/kg of GBR, (G4) fed with 10 g/kg of GBR and (G5) negative control (without colon cancer, unfed with GBR). GBR was administered orally once daily via gavage after injection of 15 mg/kg of body weight of azoxymethane (AOM) once a week for two weeks, intraperitonially. After 8 weeks of treatment, animals were sacrificed and colons were removed. Colonic aberrant crypt foci (ACF) were evaluated histopathologically. Total number of ACF and AC, and multicrypt of ACF, and the expression of β-catenin and COX-2 reduced significantly (p < 0.05) in all the groups treated with GBR (G2, G3 and G4) compared to the control group (G1). Spearman rank correlation test showed significant positive linear relationship between total β-catenin and COX-2 score (Spearman's rho = 0.616, p = 0.0001). It is demonstrated that GBR inhibits the development of total number of ACF and AC, and multicrypt of ACF, reduces the expression of β-catenin and COX-2, and thus can be a promising dietary supplement in prevention of colon cancer.

Chemoprevention of 1,2-dimethylhydrazine-induced colonic preneoplastic lesions in Fischer rats by 6-methylsulfinylhexyl isothiocyanate, a wasabi derivative

The preventive effects of dietary exposure to a wasabi derivative 6-methylsulfinylhexyl isothiocyanate (6-MSITC) during the initiation and post-initiation phases on the development of 1,2-dimethylhydrazine (DMH)-induced colonic aberrant crypt foci (ACF), and β-catenin-accumulated crypts (BCAC) were investigated in male F344 rats. To induce ACF and BCAC, rats were given four weekly subcutaneous injections of DMH (40 mg/kg body weight). The rats also received diets containing 200 or 400 ppm 6-MSITC during the initiation or post-initiation phases. The experiment was terminated 12 weeks after the start. DMH exposure produced a substantial number of ACF (323.8±69.7/colon) and BCAC (3.80±1.05/cm(2)) at the end of the study. Dietary administration of 6-MSITC at a dose of 400 ppm during the initiation phase caused a significant reduction in the total number of ACF (52% reduction, P<0.0001), larger ACF (4 or more crypt ACF) (58% reduction, P<0.001) and BCAC (76% reduction, P<0.00001). The dietary exposure to 6-MSITC significantly reduced the size (crypt multiplicity) of BCAC during both initiation and post-initiation treatment when compared to group 1 treated with DMH alone. Immunohistochemically, 6-MSITC administration lowered the proliferating cell nuclear antigen labeling index in ACF and BCAC. In addition, protein levels of hepatic cytochrome P-450 isozymes at 24 h after 6-MSITC exposure were significantly suppressed (P<0.01). The results indicated that 6-MSITC exerted chemopreventive effects in the present short-term colon carcinogenesis bioassay, through alterations in cell proliferation activity and drug metabolizing enzyme levels.

Effect of hesperetin, a citrus flavonoid, on bacterial enzymes and carcinogen-induced aberrant crypt foci in colon cancer rats: a dose-dependent study

Abstract Hesperetin, an important bioactive compound in Chinese traditional medicine, has antioxidant and anticarcinogenic properties. Hesperetin is found in abundance in orange and grape juices (200–590 mg L−1) consumed in the daily diet. We have investigated the effect of different doses of hesperetin on faecal and colonic mucosal bacterial enzymes and aberrant crypt foci (ACF) in 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in male Wistar rats. The rats were divided into six groups and were fed a modified pellet diet for 16 weeks. Group 1 served as control and group 2 received the modified pellet diet along with hesperetin (30 mg kg−1). The rats in groups 3–6 rats were given a weekly subcutaneous injection of DMH (20 mg kg−1) for the first four weeks. Hesperetin was supplemented orally at different doses (10, 20 or 30 mg kg−1) for a total of 16 weeks. At the end of the experimental period all rats were killed. In DMH-treated rats, the activity of faecal and colonic mucosal bacterial enzymes, such as β-glucuronidase, β-galactosidase, β-glucosidase, nitroreductase, sulfatase and mucinase, were significantly elevated, but in rats supplemented hesperetin along with DMH the activity was significantly lowered (P &amp;lt; 0.05). The total number of aberrant crypts was significantly increased in unsupplemented DMH-treated rats, while hesperetin supplementation to DMH-treated rats significantly reduced the total number of crypts. The results demonstrated that hesperetin supplementation at a dose of 20 mg kg−1 played a potent role in suppressing the formation of aberrant crypt foci and reducing the activity of bacterial enzymes in colon cancer.

Abstract A141: Chemopreventive properties of omeprazole against azoxymethane-induced colonic aberrant crypt foci formation in fats

Abstract Omeprazole is a proton-pump inhibitor which is widely used to treat ulcers and gastroesophageal reflux diseases (GERD). Previous studies show that omeprazole exhibits possible anti-tumorigenic properties in preclinical models of colon cancer. However, no systematic studies were carried to establish the potential anti-carcinogenic potential of omeprazole using established animal models of colon cancer. In the present study, omeprazole was evaluated for its colon cancer chemopreventive properties using azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) as a efficacy endpoint in F344 rats. In addition, we analyzed the growth inhibitory effects and induction of apoptosis in human colon cancer HCA-7 cells. We also studied its possible anti-inflammatory properties in Raw 264.7 macrophage cell line. Prior to efficacy studies we evaluated the maximum tolerated dose (MTD) of omeprazole. For MTD assay, seven week-old rats were fed either control AIN-76 diet, diets containing 5 different dose levels of omeprazole (100, 200, 400, 600 and 800 ppm) for six weeks. Based on body weight gain and toxicity symptoms, the MTD of Omeprazole is found to be ∼600 ppm. To evaluate the inhibitory properties of omeprazole on colonic ACF, seven-week-old male F344 rats were fed experimental diets containing 0, 200, and 400 ppm of omeprazole. One week later, rats received s.c. injections of azoxymethane (AOM) (15 mg/kg body wt., once weekly for two weeks) or equal volume of normal saline (vehicle). Rats were continued on experimental diets for seven weeks before being sacrificed. Colons were evaluated histopathologically for ACF and immunohistochemistry for PCNA marker to assess the crypt cell proliferation. Administration of omeprazole at 400 ppm significantly suppressed total colonic ACF formation (∼30%, p&amp;lt;0.001) when compared to the control diet group. Rats fed with 200 ppm and 400 ppm of Omeprazole showed significantly lower numbers (∼30–50%, p&amp;lt;0.05–0.001) of multi-crypt foci (≥4 or more). Overall, omeprazole produced significant dose-response effect in preventing colonic ACF formation. In in vitro assays, Omeprazole inhibited phorbol-12-myristate 13-acetate -induced iNOS expression in macrophages in a dose dependent manner. Furthermore, omeprazole induced apoptosis by suppressing survivin in HCA-7 colon cancer cells. The results from this study provides evidence for the first time that omeprazole, a proton-pump inhibitor may be used as a chemopreventive agent against colon carcinogenesis. (Supported by NOI-N01-CN-53300). Citation Information: Cancer Prev Res 2010;3(1 Suppl):A141.

Chemopreventive Effects of Frondanol A5, a Cucumaria frondosa Extract, against Rat Colon Carcinogenesis and Inhibition of Human Colon Cancer Cell Growth

Sea cucumber extracts have been widely used to treat individuals with inflammatory conditions in East Asia. The present study has been designed to test potential colon cancer-preventive properties of Frondanol A5, a glycolipid extract from the sea cucumber, Cucumaria frondosa, using in vivo and in vitro models of colon cancer. Chemopreventive efficacy of Frondanol A5 was evaluated on azoxymethane-induced rat colon carcinogenesis using colonic aberrant crypt foci (ACF) as efficacy marker. At 7 weeks of age, groups of rats (12 per group) were fed the AIN-76A diet, and ACFs were induced by azoxymethane (15 mg/kg body weight). Three days after azoxymethane treatment, rats were fed with the diets containing 0, 150, and 450 ppm of Frondanol A5 and continued on the diets for 8 weeks, at which time ACFs were evaluated. Expression levels of proliferating cell nuclear antigen and p21(WAF1/CIP1) were determined in ACFs. Further, Frondanol A5 (10-120 microg/mL) was studied for its growth-inhibitory and apoptotic effects in the HCT-116 cell line. Dietary administration of 150 and 450 ppm of Frondanol A5 significantly suppressed azoxymethane-induced total colonic ACF formation, approximately 34% to 55% (P < 0.01 to P < 0.0001), and multicrypt aberrant foci (48-68.5%, P < 0.0001) in a dose-dependent manner. ACFs in rats treated with Frondanol A5 showed significant upregulation of p21(WAF1/CIP1) and downregulation of proliferating cell nuclear antigen compared with control group. Frondanol A5 showed growth inhibition at S and G(2)-M phase with a decrease in Cdc25c and an increase in p21(WAF1/CIP) with significant apoptosis associated with H2AX phosphorylation and caspase-2 cleavage in HCT116 cells. Overall, Frondanol A5 exhibits potential chemopreventive properties for colon carcinogenesis, which suggests further development of this sea cucumber extract.

Effects of co-treatment of dextran sulfate sodium and MeIQx on genotoxicity and possible carcinogenicity in the colon of p53-deficient mice

To investigate the effects of dextran sulfate sodium (DSS) and/or 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) on in vivo genotoxicity in the colon, male C57BL/6 p53 (+/+), p53 (+/-) or p53 (-/-) gpt delta mice were twice given 1-week treatment with DSS, 2 weeks apart, and then sacrificed after 2 and 14 weeks. Although colon length was significantly shortened after DSS treatment in all genotypes at each time point, no significant difference in gpt mutant frequency (MF) and tumorigenicity was found between DSS and control groups regardless of genotype. Then, male B6C3F(1) p53 (+/+) or p53 (+/-) gpt delta mice were given DSS as described above and/or fed 300 ppm MeIQx for 7 weeks. Colon length was significantly shortened with DSS in either genotype at weeks 7 and 26, but no effects of co-treatment with MeIQx or p53 deficiency were evident. MeIQx showed a tendency to increase gpt MF in the colon of mice with either genotype, but co-treatment with DSS did not affect these increments. Appreciable incidences of colonic aberrant crypt foci (ACFs) were reported in DSS as well as co-treatment groups of each genotype. Colonic adenomas were observed in co-treatment groups of both genotypes as well as the DSS-only group of p53 (+/+). No effects of the combination of DSS and MeIQx on colon pre- and neoplastic lesions were reported. Our results indicate that MeIQx may take more than 7 weeks to induce genotoxicity in the colon and that the co-treatment of mice did not enhance colon tumorigenicity even in p53-deficient mice.

Inhibitory Effect of Yogurt on Aberrant Crypt Foci Formation in the Rat Colon and Colorectal Tumorigenesis in RasH2 Mice

The inhibitory effects of yogurt consisting of milk fermented by Lactobacillus delbrueckii subsp. bulgaricus strain 2038 and Streptococcus salivarius subsp. thermophilus strain 1131 on formation of colonic aberrant crypt foci (ACF) in rats and also on development of colorectal tumors in transgenic mice harboring human prototype c-Ha-ras genes (rasH2 mice) were examined. F344 rats and rasH2 mice were fed commercial diet containing freeze-dried yogurt or starter medium (non-fermented milk). Rats were inoculated orally with heterocyclic amine 2-amino-methyl-6-phenylimidazo[4,5-b]pyridine hydrochloride (PhIP) for two weeks. The rats were necropsied 14 days after the PhIP treatment, and ACF in the colon and rectum were counted. RasH2 mice were injected with 1,2-dimethylhydrazine dihydrochloride (DMH) for 20 weeks. Three weeks after the last injection of DMH, rasH2 mice were necropsied to determine the number and the size of colorectal tumors. Yogurt supplementation in diet significantly reduced the number of ACF and aberrant crypts (ACs) in rats fed control diet (P<0.01), but not in rats fed non-fermented milk diet. On the other hand, rasH2 mice receiving the yogurt-supplemented diet had significantly reduced numbers of tumors induced by DMH compared with those fed the non-fermented milk-supplemented diet (P<0.05). These results demonstrate that the yogurt used in this study appears to have tumor-suppressing properties, and rasH2 mice are a useful model for the evaluation of antitumor activities of foods.

Abstract A94: Preventive effects of naproxen and NO-naproxen in carcinogen induced models of urinary bladder, colon, and mammary cancers

Abstract Nonsteroidal anti-inflammatory drugs (NSAIDs) have been highly effective in preventing colon, urinary bladder, and skin cancer preclinically; and also in clinical trials of colon adenoma formation. However, certain NSAIDs cause gastrointestinal (GI) ulceration and may increase cardiovascular (CV) events. Naproxen appears to cause the lowest CV events of the common NSAIDs other than aspirin. NO-naproxen was tested based on the finding that adding a nitric oxide (NO) group to NSAIDs may help alleviate GI toxicity as well as the expectation that the production of NO itself might be therapeutically effective. When examined in the hydroxybutyl(butyl)nitrosamine (OH-BBN) rat urinary bladder cancer model, naproxen at doses of either 400 or 200 ppm in the diet greatly decreased the development of large invasive bladder cancers when treatment was initiated shortly after the last OH-BBN dosing. In a second study, naproxen at 133 ppm or NO-naproxen at 183 or 550 ppm caused striking decreases in the incidence of large tumors. Surprisingly, if treatments were started three months after the last OH-BBN (when multiple microscopic lesions already exist) both NO-naproxen (550 ppm) and naproxen (400 ppm) were still highly effective (91–96% decreases); implying that most of their effects are late. In the azoxymethane (AOM)-induced rat colon aberrant crypt foci (ACF) model, naproxen administered at 200 and 400 ppm reduced mean ACFs in the colon by approximatley 45–60%. NO-naproxen administered at roughly equimolar doses (300 and 600 ppm), reduced total ACF by 20–40%, respectively. In contrast, in the methylnitrosourea (MNU)-induced ER+ mammary cancer model in rats (where most NSAIDs are relatively ineffective) neither NO-naproxen nor naproxen showed significant inhibition at doses of 550 and 400 ppm. These data show that both naproxen and NO-naproxen are effective agents against urinary bladder and colon (but not mammary) carcinogenesis. Additional data showing the effects of sulindac and NO-sulindac in the urinary bladder model will also be presented. Data also imply that both naproxen and NO-naproxen are effective, but that the NO-derivative does not appear to be more effective. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A94.

Variational Image Segmentation for Endoscopic Human Colonic Aberrant Crypt Foci

The aim of this paper is to introduce a variational image segmentation method for assessing the aberrant crypt foci (ACF) in the human colon captured in vivo by endoscopy. ACF are thought to be precursors for colorectal cancer, and therefore their early detection may play an important clinical role. We enhance the active contours without edges model of Chan and Vese to account for the ACF's particular structure. We employ level sets to represent the segmentation boundaries and discretize in space by finite elements and in (artificial) time by finite differences. The approach is able to identify the ACF, their boundaries, and some of the internal crypts' orifices.

Chemopreventive efficacy of naproxen and nitric oxide-naproxen in rodent models of colon, urinary bladder, and mammary cancers.

Nonsteroidal anti-inflammatory drugs (NSAID) have been highly effective in preventing colon, urinary bladder, and skin cancer preclinically, and also in clinical trials of colon adenoma formation. However, certain NSAIDs cause gastrointestinal ulceration and may increase cardiovascular events. Naproxen seems to cause the lowest cardiovascular events of the common NSAIDs other than aspirin. Nitric oxide (NO)-naproxen was tested based on the finding that adding a NO group to NSAIDs may help alleviate GI toxicity. In the azoxymethane-induced rat colon aberrant crypt foci (ACF) model, naproxen administered at 200 and 400 ppm in the diet reduced mean ACFs in the colon by about 45% to 60%, respectively. NO-naproxen was likewise administered in the diet at roughly equimolar doses (300 and 600 ppm) and reduced total ACF by 20% to 40%, respectively. In the hydroxybutyl (butyl) nitrosamine rat urinary bladder cancer model, NO-naproxen was given at 183 or 550 ppm in the diet, and naproxen at 128 ppm. The NO-naproxen groups had 77% and 73% decreases, respectively, in the development of large urinary bladder tumors, whereas the 128 ppm naproxen group also showed a strong decrease (69%). If treatments were started 3 months after hydroxybutyl (butyl) nitrosamine, NO-naproxen (550 ppm) and naproxen (400 ppm) were also highly effective (86-94% decreases). In the methylnitrosourea-induced mammary cancer model in rats, NO-naproxen and naproxen showed nonsignificant inhibitions (12% and 24%) at 550 and 400 ppm, respectively. These data show that both naproxen and NO-naproxen are effective agents against urinary bladder and colon, but not mammary, carcinogenesis.

Chemopreventive effect of raw and cooked lentils (Lens culinaris L) and soybeans (Glycine max) against azoxymethane-induced aberrant crypt foci

Although lentils (Lens culinaris L) contain several bioactive compounds that have been linked to the prevention of cancer, the in vivo chemopreventive ability of lentils against chemically induced colorectal cancer has not been examined. Our present study examined the hypothesis that lentils could suppress the early carcinogenesis in vivo by virtue of their bioactive micro- and macroconstituents and that culinary thermal treatment could affect their chemopreventive potential. To accomplish this goal, we used raw whole lentils (RWL), raw split lentils (RSL), cooked whole lentils (CWL), and cooked split lentils (CSL). Raw soybeans (RSB; Glycine max) were used for the purpose of comparison with a well-studied chemopreventive agent. Sixty weanling Fischer 344 male rats, 4 to 5 weeks of age, were randomly assigned to 6 groups (10 rats/group): the control group (C) received AIN-93G diet, and treatment leguminous groups of RWL, CWL, RSL, CSL, and RSB received the treatment diets containing AIN-93G+5% of the above-mentioned legumes. After acclimatization for 1 week (at 5th to 6th week of age), all animals were put on the control and treatment diets separately for 5 weeks (from 6th to 11th week of age). At the end of the 5th week of feeding (end of 11th week of age), all rats received 2 subcutaneous injections of azoxymethane carcinogen at 15 mg/kg rat body weight per dose once a week for 2 consecutive weeks. After 17 weeks of the last azoxymethane injection (from 12th to 29th week of age), all rats were euthanized. Chemopreventive ability was assessed using colonic aberrant crypt foci and activity of hepatic glutathione-S-transferases. Significant reductions (P < .05) were found in total aberrant crypt foci number (mean +/- SEM) for RSB (27.33 +/- 4.32), CWL (33.44 +/- 4.56), and RSL (37.00 +/- 6.02) in comparison with the C group (58.33 +/- 8.46). Hepatic glutathione-S-transferases activities increased significantly (P < .05) in rats fed all treatment diets (from 51.38 +/- 3.66 to 67.94 +/- 2.01 micromol mg(-1) min(-1)) when compared with control (C) diet (26.13 +/- 1.01 micromol mg(-1) min(-1)). Our findings indicate that consumption of lentils might be protective against colon carcinogenesis and that hydrothermal treatment resulted in an improvement in the chemopreventive potential for the whole lentils.

Conversion of the Common Food Constituent 5-Hydroxymethylfurfural into a Mutagenic and Carcinogenic Sulfuric Acid Ester in the Mouse in Vivo

5-Hydroxymethylfurfural (HMF), formed by acid-catalyzed dehydration and in the Maillard reaction from reducing sugars, is found at high levels in numerous foods. It was shown to initiate colon aberrant crypt foci in rats and skin papillomas and hepatocellular adenomas in mice. HMF is inactive in in vitro genotoxicity tests using standard activating systems but is activated to a mutagen by sulfotransferases. The product, 5-sulfoxymethylfurfural (SMF), is a stronger carcinogen than HMF. SMF has not been detected in the biotransfomation experiments conducted on HMF in humans and animals in vivo up to date. Here, we report pharmacokinetic properties of HMF and SMF in FVB/N mice. Sensitive assays for the quantification of HMF and SMF by LC-MS/MS multiple reaction monitoring were devised. SMF, intravenously injected (4.4 micromol/kg body mass), showed first-order elimination kinetics in blood plasma (t(1/2) = 7.9 min). HMF, injected intravenously (793 micromol/kg body mass), demonstrated biphasic kinetics in plasma (t(1/2) = 1.7 and 28 min for the initial and terminal elimination phases, respectively); the volume of distribution of the central compartment corresponded approximately to the total body water. The maximum SMF plasma level was observed at the first sampling time, 2.5 min after HMF administration. On the basis of these kinetic data, it was estimated that between 452 and 551 ppm of the initial HMF dose was converted to SMF and reached the circulation. It is likely that additional SMF reacted with cellular structures at the site of generation and thus is ignored in this balance. Our work supports the hypothesis that HMF-related carcinogenicity may be mediated by its reactive metabolite SMF.

Pleurotus ostreatus suppresses food‐borne carcinogen‐ and inflammation‐induced colon carcinogenesis and inhibits endotoxemia in mice

The association among diet, colon cancer risk and mortality is well established, and epidemiological studies suggest that mushroom intake may prevent the development of cancer. In the present study we evaluated whether edible osyter mushroom (Pleurotus ostreatus) affects the development of food‐borne carcinogen‐ (2‐amino‐1‐methyl‐6‐phenylimidazo [4‐5‐b] pyridine, PhIP) and inflammation‐ (dextran sodium sulfate, DSS) induced colon carcinogenesis in mice. Here we show that an oral administration of P. ostreatus extract (POE) for 16 weeks (3 times per week at 0, 100 and 500 mg/kg of body weight) significantly decreased the PhIP/DSS‐induced formation of colonic tumors, and reduced the numbers of aberrant colonic crypts in a dose‐dependent manner. Anti‐inflammatory properties of the oyster mushroom were evaluated in macrophages treated with P. ostreatus powder (POP). Thus, POP suppressed LPS‐induced secretion of TNF‐α, IL‐6, IL‐12p40, and nitric oxide (NO), and down‐regulated expression of iNOS and COX‐2 in RAW264.7 cells. These effects were mediated by the inhibition of AP‐1 but not NF‐κB activity in macrophages challenged with LPS. Finally, POP suppressed LPS‐induced secretion of TNF‐α and IL‐6 in the endotoxemia mice model. In summary, our data suggest that the dietary oyster mushroom can be used for the prevention against colon carcinogenesis and inflammation.

Rats consuming bran from black and brown sorghums have lower short chain fatty acid concentrations and fewer aberrant colonic crypts

Diets containing bran isolated from brown and black sorghum grain produce fewer (68 and 38%, respectively, P &lt; 0.04) high multiplicity aberrant crypt foci (HMACF) in azoxymethane‐ (AOM) injected rats, relative to a cellulose diet. Because of the potential for short chain fatty acids (SCFA) to influence colon carcinogenesis, we determined SCFA concentrations in feces (reported as µM/g wet feces) from rats (n=10/diet) consuming diets containing 6% fiber from either cellulose or bran isolated from white, brown (contains tannins), or black (contains anthocyanins) sorghum. Diets were fed for 10 wk, with two AOM injections (15 mg/kg BW) given in wk 3 and 4. Total SCFA were affected (P &lt; 0.0001) by diet; greatest for white sorghum bran (32.0 µM), intermediate for cellulose (22.6 µM), and lowest for black or brown sorghum bran (15.4 or 14.7 µM, respectively). Butyrate concentrations were affected (P &lt; 0.0001) by diet; highest for white sorghum bran (7.8 µM), lowest for brown sorghum bran (1.07 µM), while concentrations were intermediate and similar for black sorghum bran and cellulose (2.7 and 3.6 µM, respectively). Differences among diets were the same when butyrate was expressed as a percentage of SCFA. These data suggest that there are significant changes either in the microbiota within the lumen or in the transport of SCFA into colonocytes, both of which can influence colon carcinogenesis. Funded by USDA 58‐5430‐5‐339 and NIEHS P30‐ES09106.

Plasma membrane‐associated sialidase (NEU3) promotes formation of colonic aberrant crypt foci in azoxymethane‐treated transgenic mice

Human plasma membrane-associated sialidase (NEU3) specifically hydrolyzes gangliosides, and it is up-regulated in colon cancer and plays an essential role in the expression of malignant phenotypes. To clarify the role of NEU3 in tumorigenesis in vivo, we examined the susceptibility of NEU3 transgenic mice to induction of colonic aberrant crypt foci (ACF) by azoxymethane. Mice were injected with azoxymethane (i.p., 15 mg/kg/week) for 6 weeks, and 4 weeks later ACF had formed in the NEU3 transgenic mice significantly more than in the control wild-type mice. Enhanced phosphorylation of epidermal growth factor (EGF) receptor, Akt and ERK and up-regulation of Bcl-xL protein were observed in the transgenic colon mucosa, but no changes were found in cell proliferation, suggesting that the increased ACF formation is due to suppression of apoptosis. Immunohistological analysis with anti-cleaved caspase 3 antibody showed an actual reduction in apoptotic cells in the transgenic mucosa at 6 h after the first azoxymethane injection, when apoptosis in the colonic crypt occurs. Consistent with our previous observations of human colon cancer, thin-layer chromatography of the gangliosides from the transgenic colon mucosa revealed decreased GM3 and increased lactosylceramide as compared to those from the control mucosa, probably because of catalysis of gangliosides by NEU3. The results of this study provide the first evidence that NEU3 essentially increases azoxymethane-induced ACF formation in colon mucosa by suppression of apoptosis, possibly via activation of the EGF signaling pathway, and thus indicate that up-regulation of NEU3 is important to the promotion stage of colorectal carcinogenesis in vivo.

Soy Isoflavones Modulate Azoxymethane-Induced Rat Colon Carcinogenesis Exposed Pre- and Postnatally and Inhibit Growth of DLD-1 Human Colon Adenocarcinoma Cells by Increasing the Expression of Estrogen Receptor-β

We studied the effects of lifetime exposure to dietary soy isoflavones in an azoxymethane (AOM)-induced rat colon cancer model. Male pups born to Sprague-Dawley rats exposed (including during pregnancy and lactation) to soy isoflavones at either no (0 mg = control), low (40 mg), or high (1000 mg) doses/kg diet were weaned and continued receiving their respective parental diets until the end of the study. Weaned rats received 2 subcutaneous injections (15 mg/kg body weight) of AOM 1 wk apart. After 26 wk, rats were killed and the coordinates of colon aberrant crypt foci (ACF) and tumors were determined. Expression of estrogen receptor (ER)-β was assessed in rat colon tumors and in DLD-1 human colon adenocarcinoma cells exposed to soy isoflavones. Compared with the control, soy isoflavones did not affect incidences or multiplicities of colon ACF or tumors. Low-dose soy isoflavones decreased tumor burden and size compared with the control (P < 0.05). Expression of ERβ increased in colon tumors of soy isoflavone-treated groups compared with the control. Soy isoflavones dose-dependently arrested the growth of DLD-1 cells and at subcytotoxic levels increased the expression of ERβ. Our results suggest that pre- and postnatal exposure to dietary soy isoflavones suppresses the growth of colon tumors in male rats. The overexpression of ERβ in both rat colon tumors and DLD-1 cells caused by soy isoflavones suggests that ERβ is a critical mediator in mitigating its cancer-preventive effects.

Simultaneous exposure to dietary acrylamide and corn oil developed carcinogenesis through cell proliferation and inhibition of apoptosis by regulating p53-mediated mitochondria-dependent signaling pathway

To investigate whether simultaneous exposure to acrylamide (ACR) and long-term dietary corn oil induces colon cancer by inhibiting the tumor suppressor gene p53-mediated mitochondria-dependent apoptosis in rats. Male Sprague-Dawley rats were given intraperitoneal injections of ACR at dose of 10 mg/kgbw and diets supplemented with 10% corn oil for 8 wks; and then rats were still fed with diets supplemented with 10% oil for other 48 wks. Colonic aberrant crypt foci (ACF) and tumors, including adenomas and carcinomas, were examined at 12, 24, 36, 48 week post ACR-exposure. Colonic apoptosis and cell proliferation, expression of Wild type (wt) p53, Bcl-2, Bax and caspase-3, were detected at 48 week post ACR-exposure. ACF was found at 12 week and colon cancer invasion was found at 48 week in ACR rats on long-term dietary corn oil. Apoptosis was decreased and cell proliferation was increased in colonic mucosa in ACR-treated rats on dietary corn oil compared to vehicle rats on basal diet (P < 0.05). In ACR rats on dietary corn oil, mitochondrial wt p53 was significantly inhibited through decreased mitochondrial localization of wt p53 and increased cytosolic p53, resulting in the up-regulation of Bcl-2 and the down-regulation of Bax in the mitochondria, also inhibition of the release of cytochrome-c from the mitochondria into the cytosol and protein level of caspase-3 (P < 0.05). Results suggest that simultaneous exposure to ACR and long-term dietary corn oil induces development of colon cancer partly by inhibiting the tumor suppressor gene p53-mediated mitochondria-dependent apoptosis.