Control AIN-76A Diet Research Articles

Abstract B26: The anti-inflammatory effects of black raspberries and the anthocyanin metabolite protocatechuic acid in N-nitrosomethylbenzylamine-induced esophageal cancer in rats

Abstract Background: Black raspberries (BRB) and their component anthocyanins (AC) inhibit esophageal cancer in rats induced by N-nitrosomethylbenzylamine (NMBA). Previous studies indicate that the bioavailability of the anthocyanins is low. The majority of BRB anthocyanins are metabolized by gut bacteria to protocatechuic acid (PCA). PCA has greater bioavailability than the AC and is chemopreventive in several organ sites. The current study was undertaken to determine if PCA is also effective against NMBA-induced esophageal cancer and if it influences inflammatory cytokine expression. Cytokines were measured using a simultaneous quantification bead assay. The pro-inflammatory cytokines measured were IL-1 β, IL-5, IL-7, erythropoietin (EPO), TNF- α, & interferon-gamma (IFN- γ), and the anti-inflammatory cytokines were IL-10 and T cell-derived IL-2 and the chemokines CCL5 & CCL20. Methods and Materials: Male Fischer 344 rats aged 3-5 weeks were randomized into groups by body weight & age. Rats were given s.c. injections of either the vehicle (20% DMSO/H2O) or NMBA (0.35 mg/kg b.w.) three times a week for five weeks. All rats were fed AIN-76A control diet during injections. Starting at week 6, groups of rats were fed experimental diets. These include Group 1 (DMSO + AIN-76A diet), Group 2 (NMBA + AIN-76A), Group 3 (NMBA + 3.8 umoles AC/ g AIN-76A), Group 4 (NMBA + 6% BRB in AIN-76A), & Group 5 (NMBA + 500 ppm PCA in AIN-76A). To match previous studies, the Group 3 diet contained 3.8 umoles of AC/g diet. A 6% BRB diet for Group 4 matched the anthocyanin content of Group 3, & the Group 5 diet contained 500 ppm PCA to match the estimated amount of Group 3 AC converted to PCA in the gut. 9 animals from each group were sacrificed at weeks 15 & 25, and 30 will be sacrificed at week 35. The esophagus was taken for tumor counts & used for H&E histological scoring. Rat blood was collected in heparin coated tubes, centrifuged, & stored at −80°C. Cytokines and chemokines were quantified using a bead array. Results: The study is ongoing. At week 15 H&E slide scoring revealed hyperplasia was significantly reduced in all test diet groups (BRB, AC, PCA) when compared to the NMBA control group. No tumors were present at week 15 in any of the NMBA-treated rats. At week 25 there were significant decreases in tumor count, hyperplasia, & dysplasia in Groups 3-5 when compared to Group 2. Preliminary results showed that pro-inflammatory IL-1 β expression was significantly higher in Group 2 when compared to Groups 1, 4, & 5 at week 25. Anti-inflammatory IL-10 expression was decreased in all NMBA treated groups at weeks 15 & 25. At week 15, T-cell derived IL-2 expression was significantly lower in Groups 1, 4, &5 when compared to Group 2, whereas at week 25, IL-2 expression was significantly lower only in Groups 1 & 5 when compared to Group 2. Conclusions: PCA is chemopreventive against NMBA-induced esophageal tumorigenesis in rats. Similar to BRB and their AC, PCA significantly reduced tumor incidence and the conversion of dysplastic lesions to papillomas. Cytokine quantification assays suggest that PCA, like BRB and AC, significantly influence the expression levels of certain proinflammatory and anti-inflammatory cytokines in a protective manner. Further observations will be presented at the meeting. Citation Format: Dan Peiffer, Jibran H. Siddiqui, Chieh-Ti Kuo, Yi-Wen Huang, Noah P. Zimmerman, Li-Shu Wang, Gary D. Stoner, Steven G. Carmella, Ben Ransom, Stephen S. Hecht. The anti-inflammatory effects of black raspberries and the anthocyanin metabolite protocatechuic acid in N-nitrosomethylbenzylamine-induced esophageal cancer in rats. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B26.

Bioactive Phytochemical Proanthocyanidins Inhibit Growth of Head and Neck Squamous Cell Carcinoma Cells by Targeting Multiple Signaling Molecules

Despite advances in surgical and medical therapies, approximate 50% survival rate of head and neck squamous cell carcinoma (HNSCC) has had marginal improvement in the last 30 years. Therefore, alternative strategies are required for the management of HNSCC. Here, we report the chemotherapeutic effect of proanthocyanidins on HNSCC cells using in vitro and in vivo models. Treatment of human HNSCC cell lines from different sub-sites, such as oral cavity (SCC1), larynx (SCC5), tongue (OSC19) and pharynx (FaDu), with grape seed proanthocyanidins (GSPs) reduced their cell viability and induced cell death in a dose- and time-dependent manner. GSPs induced inhibition of cell viability was associated with: (i) G1-phase arrest, (ii) inhibition of expressions of cyclins (cyclin D1 and Cyclin D2) and cyclin-dependent kinases (Cdk), (iii) increased expression of the Cdk inhibitory proteins (Cip1/p21, Kip1/p27), enhanced binding of Cdk inhibitors to Cdks, and downregulation of E2F transcription factor. GSPs significantly (P<0.05−0.001) increased apoptosis of SCC1 and OSC19 cells with induction of Bax, reduced expression of Bcl-2, and activation of caspase-3. GSPs also reduced the expression of epidermal growth factor receptor (EGFR), and treatment of SCC1 cells with erlotinib, an EGFR-targeting small molecule tyrosine kinase inhibitor, significantly (P<0.05−0.001) reduced cell viability and increased cell death. Dietary administration of GSPs (0.5%, w/w) in supplementation with AIN76A control diet inhibited the growth of SCC1 tumor xenografts in athymic nude mice, which was associated with: (i) inhibition of cell proliferation, (ii) induction of apoptosis of tumor xenograft cells, (iii) decreased expression of cyclins and Cdks, (iv) decreased expression of EGFR, and (v) increased expression of Cip1/p21 and Kip1/p27 proteins and their increased binding to Cdks in tumor xenograft samples. Together, these results suggest that GSPs may be a promising candidate for head and neck squamous cell carcinoma therapy.

Grape Proanthocyanidin Inhibit Pancreatic Cancer Cell Growth In Vitro and In Vivo through Induction of Apoptosis and by Targeting the PI3K/Akt Pathway

Pancreatic cancer is an aggressive malignancy that is frequently diagnosed at an advanced stage with poor prognosis. Here, we report the chemotherapeutic effects of bioactive proanthocyanidins from grape seeds (GSPs) as assessed using In Vitro and In Vivo models. Treatment of human pancreatic cancer cells (Miapaca-2, PANC-1 and AsPC-1) with GSPs In Vitro reduced cell viability and increased G2/M phase arrest of the cell cycle leading to induction of apoptosis in a dose- and time-dependent manner. The GSPs-induced apoptosis of pancreatic cancer cells was associated with a decrease in the levels of Bcl-2 and Bcl-xl and an increase in the levels of Bax and activated caspase-3. Treatment of Miapaca-2 and PANC-1 cells with GSPs also decreased the levels of phosphatidylinositol-3-kinase (PI3K) and phosphorylation of Akt at ser473. siRNA knockdown of PI3K from pancreatic cancer cells also reduced the phosphorylation of Akt. Further, dietary administration of GSPs (0.5%, w/w) as a supplemented AIN76A control diet significantly inhibited the growth of Miapaca-2 pancreatic tumor xenografts grown subcutaneously in athymic nude mice, which was associated with: (i) inhibition of cell proliferation, (ii) induction of apoptosis of tumor cells, (iii) increased expression of Bax, reduced expression of anti-apoptotic proteins and activation of caspase-3-positive cells, and (iv) decreased expression of PI3K and p-Akt in tumor xenograft tissues. Together, these results suggest that GSPs may have a potential chemotherapeutic effect on pancreatic cancer cell growth.

Dose-dependent effects of calorie restriction on gene expression, metabolism, and tumor progression are partially mediated by insulin-like growth factor-1.

The prevalence of obesity, an established risk and progression factor for breast and many other cancer types, remains very high in the United States and throughout the world. Calorie restriction (CR), a reduced-calorie dietary regimen typically involving a 20–40% reduction in calorie consumption, prevents or reverses obesity, and inhibits mammary and other types of cancer in multiple tumor model systems. Unfortunately, the mechanisms underlying the tumor inhibitory effects of CR are poorly understood, and a better understanding of these mechanisms may lead to new intervention targets and strategies for preventing or controlling cancer. We have previously shown that the anticancer effects of CR are associated with decreased systemic levels of insulin-like growth factor-1 (IGF-1), the primary source of which is liver. We have also reported that CR strongly suppresses tumor development and growth in multiple mammary cancer models. To identify CR-responsive genes and pathways, and to further characterize the role of IGF-1 as a mediator of the anticancer effects of CR, we assessed hepatic and mammary gland gene expression, hormone levels and growth of orthotopically transplanted mammary tumors in control and CR mice with and without exogenous IGF-1. C57BL/6 mice were fed either control AIN-76A diet ad libitum (AL), subjected to 20%, 30%, or 40% CR plus placebo timed-release pellets, or subjected to 30% or 40% CR plus timed-release pellets delivering murine IGF-1 (mIGF-1, 20 μg/day). Compared with AL-fed controls, body weights were decreased 14.3% in the 20% CR group, 18.5% in the 30% CR group, and 38% in the 40% CR group; IGF-1 infusion had no effect on body weight. Hepatic transcriptome analyses indicated that compared with 20% CR, 30% CR significantly modulated more than twice the number of genes and 40% CR more than seven times the number of genes. Many of the genes specific to the 40% CR regimen were hepatic stress-related and/or DNA damage-related genes. Exogenous IGF-1 rescued the hepatic expression of several metabolic genes and pathways affected by CR. Exogenous IGF-1 also rescued the expression of several metabolism- and cancer-related genes affected by CR in the mammary gland. Furthermore, exogenous IGF-1 partially reversed the mammary tumor inhibitory effects of 30% CR. We conclude that several genes and pathways, particularly those associated with macronutrient and steroid hormone metabolism, are associated with the anticancer effects of CR, and that reduced IGF-1 levels can account, at least in part, for many of the effects of CR on gene expression and mammary tumor burden.

Biomarkers of Phenethyl Isothiocyanate-Mediated Mammary Cancer Chemoprevention in a Clinically Relevant Mouse Model

Phenethyl isothiocyanate (PEITC) is a natural plant compound with chemopreventative potential against some cancers and the ability to induce apoptosis in breast cancer cells. Female mouse mammary tumor virus-neu mice were fed a control AIN-76A diet (n = 35) or the same diet supplemented with 3 µmol PEITC/g diet (n = 33) for 29 weeks, at which time they were killed. Breast tissue sections were stained with hematoxylin and eosin for histopathological assessments, and incidence and size of macroscopic mammary tumors were assessed. Cell proliferation (Ki-67 staining), apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick-labeling), and neoangiogenesis (CD31 staining) were determined in tumor sections. Plasma levels of transthyretin were measured in treated and control mice. Expression of proteins in mammary tumor sections was determined by immunohistochemistry. Proteomic profiling was performed by two-dimensional gel electrophoresis followed by mass spectrometry. All statistical tests were two-sided. Administration of PEITC for 29 weeks was associated with 53.13% decreased incidence of macroscopic mammary tumors (mean tumor incidence, PEITC-supplemented diet vs control diet, 18.75% vs 40.00%, difference = -21.25%, 95% confidence interval [CI] = -43.19% to 0.69%, P = .07) and with a 56.25% reduction in microscopic mammary carcinoma lesions greater than 2 mm(2) (mean incidence, PEITC-supplemented diet vs control diet, 18.75% vs 42.86%, difference = -24.11%, 95% CI = -46.35% to -1.86%, P = .04). PEITC-mediated mammary cancer growth inhibition was not because of suppression of human epidermal growth factor receptor-2 expression but was associated with reduced cellular proliferation and neoangiogenesis, increased apoptosis, and altered expression of several proteins, including decreased ATP synthase in the tumor and increased plasma levels of transthyretin. PEITC inhibits the growth of mammary cancers in a mouse model with similarities to human breast cancer progression. ATP synthase and transthyretin appear to be novel biomarkers associated with PEITC exposure.

Dietary Cholecalciferol and Calcium Levels in a Western-Style Defined Rodent Diet Alter Energy Metabolism and Inflammatory Responses in Mice ,

Male and female C57Bl6 mice were fed a control AIN76A diet, a new Western-style diet (NWD1) reflecting dietary patterns linked to elevated colon cancer incidence (higher fat, lower cholecalciferol, calcium, methyl donors, fiber), or NWD1 with elevated cholecalciferol and calcium (NWD2) from weaning. After 24 wk, serum 25-hydroxyvitamin D [25(OH)D] decreased by >80% in the NWD1 group compared with controls, but with no alteration in serum calcium or bone mineral density. The decreased serum 25(OH)D was prevented in the NWD2 group. After 32 wk, the NWD1 group compared with controls reduced overall energy expenditure by 15% without altering food consumption or physical activity and induced glucose intolerance, phenotypes associated with metabolic syndrome. These responses were unexpectedly exacerbated in the NWD2 group, further shifting mice toward greater fatty acid storage rather than oxidation compared with both control and NWD1 groups, but there was no change in physical activity, causing significant weight gain due to increased fat mass. The NWD1 group also exhibited inflammatory responses compared with controls, including macrophage-associated crown-like structures in epididymal adipose tissue and increased serum concentrations of the proinflammatory cytokine IL-1β, and of its targets, MCP-1 and Rantes, which were prevented or greatly mitigated in the NWD2 group. However, there was also elevated lipid storage in the liver and steatosis not seen in the control and NWD1 groups. Thus, elevating cholecalciferol and calcium in a Western-style diet can reduce inflammation associated with risk for colon tumor development, but interaction of nutrients in this diet can compromise liver function when fed long term.

Abstract LB-177: A supercritical turmeric extract prevents small intestinal and colonic tumors in the APCMin/+ mice by suppressing proliferation and cancer stem cell markers

Abstract CRC is one of the most commonly diagnosed cancers in the United States. Curcumin, a principle compound in turmeric, has been extensively investigated and proven to be antiinflammatory and anticarcinogenic against numerous cancers including CRC. Turmeric is consumed as a dietary ingredient by large populations and turmeric extracts as a dietary supplement by healthy as well as high-risk individuals with various chronic diseases. Turmeric, however, contains over 300 different compounds including essential oils; curcuminoids, turmerones, etc. Most turmeric extracts commonly used as dietary supplements contains &amp;lt;6% curcuminoids; however, to date no studies have been undertaken to determine whether these turmeric extracts are effective or not in comparison to curcumin in preventing colonic tumors. In this study, we tested a supercritical extract of turmeric (TurmericForce™) in comparison to an equal amount of curcumin against the APC-min/+ SI and colon polyposis model. Six-week-old male APC-Min/+ mice (n = 15 per group) were fed with control AIN-76A diet or diets containing 2,000 ppm turmeric extract or 2,000 ppm or 120 ppm curcumin for 14 weeks; intestinal tumors were then evaluated for tumor incidence and multiplicity. Mice fed with either 2,000 ppm turmeric extract or curcumin exhibited a significantly suppressed SI polyp multiplicity by 26.3 ±8.3 (Mean±SD; 45.5%, p&amp;lt;0.0001) and 32.5 ±7.8 (32.5%, P&amp;lt;0.0001), respectively. Importantly, the 2,000 ppm turmeric extract showed significantly (p&amp;lt;0.026) better inhibitory effect compared to 2,000 ppm curcumin in SI polyp suppression. Colon tumor incidences were 77%, 54%, and 50% for mice fed control, turmeric extract and curcumin, respectively. Furthermore, colonic tumor multiplicities were 1.46 ± 1.1 (control); 0.57± 0.6 (turmeric extract, 61%inhibition, p&amp;lt;0.01) and 0.61±0.65 (2,000 ppm curcumin, 56% inhibition, p&amp;lt;0.015). In contrast, 120 ppm curcumin (equal to curcumin content in turmeric extract) produced limited SI tumor inhibitory effects (14% inhibition) in APC-Min/+ mice. Both turmeric extract and curcumin showed a significant reduction is PCNA and β-catenin expression levels and an increase in p21 and TUNEL positive cells. Also, Real time PCR and IHC analysis showed a significant reduction in cancer stem (CD133 and ALDH1) marker positive cells by the turmeric extract and curcumin. These results suggest that the supercritical turmeric extract had equal or better chemopreventive properties than curcumin alone in suppressing intestinal tumorigenesis. {Supported by Kerley-Cade Endowment} Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-177. doi:1538-7445.AM2012-LB-177

Abstract 1614: Grape seed proanthocyanidins inhibit pancreatic cancer cell growth in vitro and in vivo through induction of apoptosis and inhibition of PI3K/Akt pathway

Abstract The American Cancer Society reported that 35,420 Americans died in 2009 because of pancreatic cancer. It is an aggressive malignancy that is frequently diagnosed at an advanced stage with poor prognosis. Phosphoinositide 3-kinase (PI3K)/Akt is a potent survival pathway that may mediate resistance to chemotherapeutic drugs and conventional therapies in pancreatic cancer patients. Thus, PI3K/Akt is considered as a promising therapeutic target for this cancer. Dietary phytochemicals offer promising options for the development of effective strategies for the prevention of cancer, and thus can be utilized as complementary and alternative medicine. Therefore, to develop newer and more effective chemotherapeutic agent for pancreatic cancer, we have examined the effect of grape seed proanthocyanidins (GSPs) on the growth of pancreatic cancer cell lines and the molecular mechanisms underlying this effect using PANC-1, AsPC-1 and Miapaca-2 cell lines as a model. The effect of GSPs on pancreatic cancer cell lines was studied on cell proliferation, cell cycle regulation, apoptosis and PI3K/Akt pathway using MTT assay, western blotting and FACS analysis. In vitro treatment of pancreatic cancer cells (Miapaca-2, PANC-1, and AsPC-1) with GSPs resulted in: (i) growth inhibition, which was associated with G2/M-phase arrest, and includes the inhibition of the expressions of cyclin B1, Cdc25B and Cdc25c proteins. (ii) Induction of apoptosis of pancreatic cancer cells by GSPs was associated with the enhanced expression of Bax, and caspase-3 activation while reduced levels of anti-apoptotic proteins (Bcl2, Bcl-xl). (iii) Blocking of PI3K/Akt pathway. Exposure of Miapaca-2 and PANC-1 cancer cells to PI3K inhibitor (wortmannin) also resulted in a dose-dependent induction of apoptosis in these cells. The in vitro data were further verified using in vivo tumor xenograft model. Administration of GSPs (0.2% and 0.5%, w/w) as a supplement of an AIN76A control diet resulted in a dose-dependent inhibition of the growth of Miapaca-2 tumor xenografts in athymic nude mice (35-65%; P&amp;lt;0.05-0.01) compared to non-GSPs-treated controls. The growth inhibitory effect of dietary GSPs on the pancreatic xenograft tumors was associated with the induction of apoptotic cell death of tumor cells, and induction of Bax expression and activation of caspase-3 while decreased the expression of anti-apoptotic proteins. Further, the levels of phospho-Akt and the proteins of PI3K family (PI3K 110 and PI3K 85) were markedly decreased in the xenograft tumors treated with GSPs compared with controls. Together, this preclinical study provides evidence that the chemotherapeutic effect of GSPs on the growth of pancreatic cancer cells in vitro and in vivo is mediated, at least in part, through the induction of apoptosis, G2/M phase arrest and blocking of PI3K/Akt cell survival pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1614. doi:1538-7445.AM2012-1614

Abstract 4104: Moderate calorie restriction activates autophagy during tumor growth suppression

Abstract Multiple mouse models of pancreatic, colon, prostate, skin and breast cancer have shown that calorie restriction (CR) prevents obesity and suppresses tumor development and growth. In normal muscle, studies have shown that mild CR attenuates the age-related impairment of autophagy, cellular damage, and cell death. Autophagy is a survival response in solid tumors that co-localizes with hypoxic regions, allowing tolerance to metabolic stress. Autophagy also protects cells from genome damage and limits both inflammation and cell death as possible means to tumor suppression. Thus, we hypothesized that autophagy plays a major role in CR-mediated tumor inhibition. To test this hypothesis, we utilized immortalized baby mouse kidney epithelium (iBMK) tumor cell lines. iBMK cells were first immortalized by E1A and dominant negative p53 (p54DD) transfection, and rendered apoptosis-deficient by Bcl-2 overexpression. They were then transfected with oncogenic Ras and EGFP-LC3 plasmids. Cell lines were either autophagy deficient through knockout of the essential autophagy gene Atg5 (Atg5 -/-) or were autophagy competent (Atg5 +/+). Female nude mice were fed either control AIN-76A diet ad-libitum or a CR regimen (isonutrient; 30% reduction in calories relative to control group) for 8 weeks prior to subcutaneous flank injection of cell lines (n=15 per group). CR mice were significantly lighter, had decreased body fat percentage, and had lower fasting glucose levels (p&amp;lt;0.001 for all). Additionally, CR mice had lower serum levels of IGF-1 (p&amp;lt;0.001), insulin (p&amp;lt;0.001), leptin (p&amp;lt;0.001), and resistin (p=0.01), and higher serum adiponectin (p&amp;lt;0.001). We found that autophagy competent tumors, transplanted into female nude mice (5x10^4 cells per mouse), grew at significantly different growth rates depending on the diet treatment (Control &amp;gt; CR; p&amp;lt;0.001), while autophagy deficient tumors were unaffected by CR (no difference between control and CR tumors). In order to determine the effect of CR on LC3 localization and activation of autophagy, we repeated the injections in a separate cohort of control and CR mice (n=2 per timepoint). Tumors were collected at 1, 3, 8, and 15 days post-injection. In CR mice injected with autophagy competent cells, we saw LC3 translocate to the autophagosomes, demonstrated by punctate perinuclear localization, as early as 1d post-injection. In contrast, in control mice injected with autophagy competent cells, and in all mice injected with autophagy deficient cells, we saw a diffuse cytoplasmic LC3 localization. In conclusion, we found that CR is capable of activating autophagy as a mechanism of tumor growth suppression and increases LC3 autophagosome localization in tumors. Taken together, these findings suggest that autophagy may play a key role in the anticancer effects of CR and may be an important intervention target for the prevention and treatment of obesity-related cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4104. doi:1538-7445.AM2012-4104

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.

Association of stem-like cells in gender-specific chemoprevention against intestinal neoplasia in MIN mouse

This study was undertaken to examine the gender-sensitivity and chemopreventive responsiveness of celecoxib on intestinal stem-like cells as a biomarker of colon carcino-genesis, using the MIN mouse model. Male and female MIN mice (6-7-weeks old) were randomized to either control diet or to a diet supplemented with celecoxib (1,500 ppm). The animals were euthanized ten weeks later and the intestines were flushed and opened longitudinally to assess tumor count. Small intestinal segments were formalin-fixed and tissue sections were subjected to immunohistochemical evaluation of DCAMKL1, a known marker of stem-like cells. We found that in animals receiving control (AIN 76A diet) alone, female MIN mice had a higher polyp count than males (52.32 ± 13.89 vs. 35.43 ± 16.05; p<0.0005). However, compared to control diet groups, celecoxib supplementation caused a larger reduction in the number of polyps in females than their male cohorts (6.38 ± 1.43 vs. 12.83 ± 6.74; a reduction of 88% in females to 64% in males). Significant differences (p=0.013) were observed in the number of DCAMKL1-stained cells in the crypts of the wild-type (WT) (10.01 ± 1.07 stem cells per high powered field; HPF) compared to the MIN mice (24.15 ± 8.08 stem cells per HPF), illustrating increased stem-like cells in animals that are more prone to neoplasia. DCAMKL1 labeled stem-like cells were equal in number in the male and female groups receiving the control AIN 76A diet alone (females, 25.73 stem-like cells/HPF); males, 24.15 stem-like cells/HPF). However, females showed a greater reduction in the number of DCAMKL1-labeled stem-like cells with celecoxib supplementation than the respective males (16.63 ± 4.23 vs. 21.56 ± 9.06; a reduction of 35.4% in females to 10.7% in males). We conclude that a higher number of stem-like cells in the uninvolved mucosa paralleled tumorigenesis and mirrored greater chemopreventive responsiveness of female MIN mice compared to males.

Abstract 808: Chemoprevention of colon cancer by DFMO, sulindac and NO-sulindac administered individually or in combinations in male F344 rats

Abstract Different mode of action exerted by low-dose combination of agents represents a practical approach for improving the chemopreventive efficacy and minimizing unwanted toxicities. Recent clinical studies in high-risk sporadic colorectal cancer patients proven that DFMO combined with COX-1/COX-2 inhibitor, sulindac dramatically reduced polyps formation. Present study designed to address whether i) low-dose combinations DFMO and sulindac would provide the efficacy against rat colon adenocarcinomas in rat model representing human sporadic colon cancers; ii) test the advantages of nitric-oxide releasing (NO)-sulindac would provide colon cancer chemopreventive activity alone or combined with DFMO; and iii) understand the molecular changes associated in colon tumor inhibition by these combinations. Seven-week old, male F344 rats (36 rats/group) were fed the control AIN-76A diet and AOM (15 mg/kg b.w., once weekly for 2 weeks, at the age of 8 and 9 weeks) administered by s.c. Eight weeks after the last carcinogen treatment, rats were fed the diets containing 0 ppm, 500 ppm DFMO, 150 ppm Sulindac, 200 ppm NO-Sulindac individually or in combinations for 40 weeks. Rats were sacrificed and colon tumors were evaluated by histopathologically and colonic tissues were assayed for expression levels of COX-2, iNOS, p21WAF1/CIP1, apoptotic markers, and cell proliferation (BrdU) and ODC enzymatic activity. Bioassay results suggest that administration of 500 ppm DFMO and 150 ppm sulindac significantly suppressed total adenocarcinoma multiplicity by 50% (p&amp;lt;0.003) and 35.4% (p&amp;lt;0.03), respectively; whereas NO-sulindac had no significant effect on AOM-induced colon cancers. Importantly, combination of DFMO with sulindac or NO-Sulindac significantly suppressed colon adenocarcinoma incidence (42%p&amp;lt;0.0004; 32% p&amp;lt;0.001), and multiplicity (58%, p&amp;lt;0.0001; 52%, p&amp;lt;0.0018). We observed greater inhibitory effects of DFMO (63%), sulindac (52%) alone and more pronounced inhibitory DFMO combined with sulindac (78%) on adenocarcinomas with size of &amp;gt;0.5-mm. Colonic adenocarcinomas harvested from rats fed DFMO, sulindac or combinations showed significant inhibition of COX-2 and iNOS expression and activity compared to control diet fed rat colonic tumors. Also, DFMO and sulindac or combinations showed considerable enhancement of p21WAF1/CIP1, caspase-3 cleavage, down regulation of bcl-xL, bcl-2 and survivin in rat colonic tumors. Rats that were fed with the combination DFMO and sulindac showed reduced tumor cell proliferation (BrdU labeling index by 16-28%, p&amp;lt;0.05-0.01) in colonic tumors. These observations show that combination of low-dose DFMO and sulindac possesses significant colon tumor inhibitory properties; whereas 200 ppm NO-sulindac alone or combined with DFMO had modest colon tumor inhibitory effects. [Supported by NIH, NCI grants NO1-CN-35110 and R01 CA-102942)] 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 808. doi:10.1158/1538-7445.AM2011-808

Abstract 2368: Reversal of epithelial to mesenchymal transition in non-small cell lung cancer cells by grape seed proanthocyanidins leads to decreased cancer cell migration

Abstract Epithelial to mesenchymal transition (EMT) in cancer endows tumor cells with enhanced migratory and invasive property. EMT is considered as a major event during distant tumor metastasis and is mostly regulated by chronic and sustained inflammation. Cyclooxygenase-2 (COX-2) overexpression has been shown in &amp;gt;70% of lung adenocarcinomas and has reciprocal relationship with E-cadherin expression, an epithelial marker. Since, lung cancer is a highly malignant cancer with a potent capacity to metastasize distantly, an approach that decreases its invasive potential or the ability of cell migration may facilitate the development of an effective strategy for its treatment and/or prevention. Dietary phytochemicals offer promising new options for the development of more effective strategies for the prevention of cancer cell invasion and metastasis, and thus can be utilized as complementary and alternative medicine. We hypothesize that reduction of basal level of COX-2, a biomarker of inflammation, in non-small cell lung cancer (NSCLC) cells by grape seed proanthocyanidins (GSPs) should lead to reversal of EMT and that will result in reduced capacity of cell migration. Here we report that treatment of NSCLC A549 cells with various concentrations of GSPs (0, 10, 20, 40 µg/ml) for 24 h resulted in a dose-dependent reduction of COX-2 expression. Using an in vitro cell migration assay, we found that treatment of cells with GSPs (0, 10, 20, 40 µg/ml) inhibited tumor cell migration from 25±5%-55±5% compared to non-treated control cells. Western blot analysis revealed that treatment of A549 cells with GSPs also resulted in upregulation of epithelial markers (E-cadherin, Keratin-8 and Keratin-18) concomitant with decrease in the levels of mesenchymal markers (Fibroncetin, N-cadherin and Vimentin). Further, treatment of cells with phorbol-12-myristate 13-acetate, an inducer of COX-2, resulted in increased cell migration (118±5%-140±8%) compared to control cells and simultaneously reduced the levels of E-cadherin. Treatment of cells with celecoxib, an inhibitor of COX-2, rescued E-cadherin expression and inhibited cell migration (21±5%-63±5%). Dietary administration of GSPs (0.2% w/w) with control AIN76A-diet to athymic nude mice resulted in reduced xenograft growth and reduced expression of COX-2 in A549 tumor xenografts, and that resulted in upregulation of epithelial markers (E-cadherin, Kertain-8 and Keratin-18) while downregulation of mesenchymal biomarkers (N-cadherin and Fibronectin) in xenograft tissues. Together these data suggest that reversal of epithelial to mesenchymal transition in human NSCLC cells by GSPs is associated with the down regulation of COX-2, and that leads to inhibition of cancer cell migration. 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 2368. doi:10.1158/1538-7445.AM2011-2368

Loss of JNK2 increases intestinal tumor susceptibility in Apc1638+/- mice with dietary modulation

A recent study has shown that c-Jun NH2-terminal kinases (JNKs) 2 interacts with and inhibits β-catenin signaling in vitro. To determine the role of genetic interaction between JNK2 and β-catenin in vivo and to elucidate JNK2-mediated intestinal carcinogenesis, we crossed the JNK2-/- mice with Apc1638+/- mice that carry inactivated Apc allele and develop intestinal tumor due to β-catenin activation. We found that the introduction of mutant JNK2 into Apc1638+/- mice did not increase intestinal tumorigenesis when the mice were fed a defined AIN-76A control diet. However, loss of JNK2 significantly increased animal body weight in the Apc/JNK2+/- and Apc/JNK2-/- mice. Surprisingly, JNK2 loss was synergistic with a Western-style high-risk diet (high fat and phosphate and low calcium and vitamin D) to accelerate intestinal tumorigenesis. Tumor number increased to 3.56 from 1.89 (on AIN-76A diet) in the Apc/JNK2+/- mice (P<0.01) and increased to 4.14 from 1.92 (on AIN-76A diet) in the Apc/JNK2-/- mice (P<0.01) although there was a slight increase of tumor formation in Apc/JNK2+/+ mice. Intestinal tumorigenesis in Apc/JNK2 double-mutant mice with high-risk diet modulation was associated with β-catenin signaling, peroxisome proliferator-activated receptor-γ and inflammation pathway. Collectively, we concluded that JNK2 may function in controlling fat metabolism and loss of JNK2 increases the risk of obesity, the latter synergizes with high-fat diet to increase intestinal tumor susceptibility. This data strongly suggests the importance of JNK2 in intestinal carcinogenesis and the importance of dietary manipulation for cancer prevention in the population whose JNK2 is inactivated.

Genistein and genistein-containing dietary supplements accelerate the early stages of cataractogenesis in the male ICR/f rat

Cataract-related loss of vision affects large numbers of people in today’s aging populations and presents a healthcare burden to many nations. The role of dietary supplements within the lens is largely unknown, although benefits from dietary anti-oxidants are expected. In this study, the effects of genistein as its aglycone, a genistein-containing dietary supplement (Novasoy ®200), and a genistein-containing food (soy protein isolate, PRO-FAM 932) on the development of lens opacity were examined in the hereditary cataractous ICR/f rat. These studies were carried out in a background diet of semi-purified, isoflavone-free AIN-76A with casein as its protein source. The amount of genistein for the experimental diets was standardized to its concentration (as genistein aglycone as well as simple and complex β-glucoside conjugates) in the soy protein isolate supplement. Also tested was a high-dose genistein diet containing an 11-fold higher amount of genistein aglycone. The composition of each diet was verified by reverse-phase HPLC and blood plasma isoflavone concentrations were determined by LC-tandem mass spectrometry. The development of opacity in each lens was monitored and digitally recorded using slit-lamp examination over the course of the study. Each of the genistein-containing diets caused a significantly more rapid development of fibrous opacification in the anterior cortical region and development of apparent water clefts or vacuoles in the posterior subcapsular region than the AIN-76A control diet; however, the establishment of dense lens opacification was not significantly different between each of the diets. There was also no significant difference observed between the low-dose and high-dose genistein aglycone groups. These data suggest that genistein-containing dietary supplements accelerate the early stages of cataractogenesis in the male ICR/f rat, with no dose-dependent effects.

Proanthocyanidins Inhibit Photocarcinogenesis through Enhancement of DNA Repair and Xeroderma Pigmentosum Group A–Dependent Mechanism

Dietary grape seed proanthocyanidins (GSP) inhibit photocarcinogenesis in mice; however, the molecular mechanisms underlying this effect have not been fully elucidated. As ultraviolet B (UVB)-induced DNA damage in the form of cyclobutane pyrimidine dimers (CPDs) has been implicated in skin cancer risk, we studied whether dietary GSPs enhance repair of UVB-induced DNA damage and, if so, what is the potential mechanism? Supplementation of GSPs (0.5%, w/w) with AIN76A control diet significantly reduced the levels of CPD(+) cells in UVB-exposed mouse skin; however, GSPs did not significantly reduce UVB-induced CPD(+) cells in the skin of interleukin-12p40 (IL-12) knockout (KO) mice, suggesting that IL-12 is required for the repair of CPDs by GSPs. Using IL-12 KO mice and their wild-type counterparts and standard photocarcinogenesis protocol, we found that supplementation of control diet with GSPs (0.5%, w/w) significantly reduced UVB-induced skin tumor development in wild-type mice, which was associated with the elevated mRNA levels of nucleotide excision repair genes, such as XPA, XPC, DDB2, and RPA1; however, this effect of GSPs was less pronounced in IL-12 KO mice. Cytostaining analysis revealed that GSPs repaired UV-induced CPD(+) cells in xeroderma pigmentosum complementation group A (XPA)-proficient fibroblasts from a healthy individual but did not repair in XPA-deficient fibroblasts from XPA patients. Furthermore, GSPs enhance nuclear translocation of XPA and enhanced its interactions with other DNA repair protein ERCC1. Together, our findings reveal that prevention of photocarcinogenesis by GSPs is mediated through enhanced DNA repair in epidermal cells by IL-12- and XPA-dependent mechanisms.

Abstract 1875: Dietary grape seed proanthocyanidins induce rapid repair of DNA damage via nucleotide excision repair genes in preventing UV-induced immunosuppression

Abstract Ultraviolet (UV) radiation-induced immunosuppression has been implicated in the development of skin cancers, including melanoma and non-melanoma. UV-induced DNA damage, predominantly the formation of cyclobutane pyrimidine dimers (CPDs), has been recognized as an important molecular trigger for the initiation of UVB-induced immunosuppression and skin carcinogenesis. We have shown earlier that administration of dietary grape (Vitis vinifera) seed proanthocyanidins (GSPs) supplemented with AIN76A control diet inhibit photocarcinogenesis in mice; however, the molecular mechanism of chemopreventive effects of GSPs are not clearly understood. To investigate the possible mechanism of action of GSPs we used genetically modified human cells and animal model. We observed that administration of dietary GSPs (0.2 and 0.5%, w/w) with supplementation of AIN76A control diet inhibited (52-65%, P&amp;lt;0.001) UV-induced suppression of contact hypersensitivity response to a contact sensitizer, 2,4-dinitrofluorobenzene, in a contact hypersensitivity mouse (C3H/HeN) model. Dietary GSPs repaired UV-induced DNA damage (CPD-positive cells) faster in the skin of mice as demonstrated by reduced number of CPD-positive cells (59%, p&amp;lt;0.001), and reduced the migration of CPD-positive cells from the skin to draining lymph nodes compared to non-GSPs-fed control mice, which was associated with the elevated levels of nucleotide excision repair (NER) genes. GSPs did not prevent UV-induced immunosuppression in NER-deficient mice but significantly prevented in NER-proficient mice (p&amp;lt;0.001) concomitantly repaired UV-induced DNA damage in NER-proficient mice (p&amp;lt;0.01) but did not repair in NER-deficient mice as indicated by immunohistochemical analysis of CPD-positive cells. Further, southwestern dot-blot analysis revealed that GSPs repaired UV-induced CPDs in xeroderma pigmentosum complementation group A (XPA)-proficient cells obtained from healthy person but did not repair in XPA-deficient cells obtained from the patients suffering from xeroderma pigmentosum disease, indicating that NER mechanism is involved in DNA repair. Together, these data identify a novel mechanism by which dietary GSPs prevent UV-induced immunosuppression which is mediated through rapid repair of damaged DNA, and that these effects lead to the prevention of UV radiation-induced skin tumors in mice. 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 1875.

Abstract 4163: JNK2 reduction increases intestinal tumor susceptibility in Apc+/− mice

Abstract Previous studies have shown distinct functions between c-jun NH2 terminal kinases (JNK) 1 and 2. Indeed, loss of JNK1 causes spontaneous intestinal tumor formation in mice (Tong et al, Am. J. Pathol, 2007; Hu et al, Carcinogenesis, 2008), but loss of JNK2 does not have any phenotype in intestine, although in vitro studies have shown that JNK2, like JNK1, interacts with and inhibits β-catenin signaling, in which GSK3β and proteasome pathway play a critical role (Hu et al, LoS One, 2009). To determine the role of genetic interaction between JNK2 and beta-catenin in intestinal tumorigenesis and to elucidate JNK2-mediated intestinal carcinogenesis, we crossed the JNK2−/−mouse with Apc+/− mice that carry inactivated Apc allele and develops intestinal tumor due to beta-catenin activation and nuclear accumulation. We found that the introduction of mutant JNK2 into Apc+/− did not increase intestinal tumorigenesis when the mice were fed a defined AIN-76A control diet. However, JNK2 reduction significantly increased animal body weight in the Apc+/−/JNK2+/− and Apc+/−/JNK2−/− mice. Interestingly, JNK2 reduction was synergistic with a Western-style high-risk diet (high fat and phosphate, low calcium and vitamin D)(WD) to accelerate intestinal tumorigenesis. Tumor number was significantly increased from 1.89 (AIN-76A diet) to 3.56 (WD) in the Apc+/−/JNK2+/− mice (P&amp;lt;0.01) and to 4.14 (WD) in the Apc+/−/JNK2−/− mice from 1.92 (AIN-76A diet) (P&amp;lt;0.01), even though there was a slight increase of tumor formation in JNK2 wild-type mice (Apc+/−/JNK2+/+) by such a diet (1.55 v.s. 2.0, p=0.4). Therefore, we concluded that JNK2 might have function in controlling fat metabolism, and that loss of JNK2 increases the risk of obesity, the latter synergists with high-fat diet to increase intestinal tumor susceptibility. This data strongly suggests the important role of JNK2 in intestinal carcinogenesis and the essential of dietary manipulation for cancer prevention in the population if their JNK2 is inactivated. The underlying mechanisms are under investigation. 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 4163.

Abstract 954: A selective iNOS inhibitor N6-iminoethyl-lysine tetrazoleamide (NILT), suppress invasive colonic cancers and improves preventive efficacy of low-dose COX-2 inhibitor, celecoxib in F344 rats

Abstract Clinical and preclinical studies suggest that NSAIDs and cyclooxygenase (COX)-2 inhibitors such as celecoxib, reduce the risk of colorectal cancer. However, at high doses celecoxib causes gastrointestinal toxicity and an increased cardiovascular risk. Inducible nitric oxide synthase (iNOS), similar to COX-2, is overexpressed in colon tumors, and nitric oxide is shown to stimulate COX-2 activity and contributes to the tumor invasiveness. Thus, in the present study we tested a novel iNOS-selective inhibitor NILT for colon cancer inhibition and invasiveness in F344 rats and evaluated the combined low dose effects of NILT and celecoxib, to improve the chemopreventive efficacy in rats. Seven week old male F344 rats (36/group) were fed control AIN-76A diet and colon cancer was induced by administering azoxymethane (AOM), s.c., a single dose at week 8 and 9. Four weeks after the AOM treatment, groups of rats were fed experimental diets containing either 0, 100, 200 ppm of NILT or 250 and 500 ppm of celecoxib or a combination of 100 ppm NILT and 250 ppm celecoxib. Forty eight weeks after AOM treatment, rats were killed and the colonic tumors were evaluated. Multiple samples of colonic tumors from each group were assayed for activity levels of iNOS, COX-2, and expression levels of cytokines, chemokine and markers of apoptosis and cell proliferation. We found that dietary administration of 200 ppm NILT suppressed both colon adenocarcinoma incidence by 40% (p&amp;lt;0.02) and multiplicity by 46 % (p&amp;lt;0.02); whereas 100 ppm NILT showed ∼33% of colon adenocarcinoma incidence and multiplicity (p&amp;lt;0.06). Importantly, 100 ppm and 200 ppm iNOS inhibitor suppressed invasive adenocarcinomas multiplicities by 52% and 67% (P&amp;lt;0.007). Celecoxib at both the doses (250 and 500 ppm) significantly suppressed colon adenocarcinoma incidence (55 − 77%, p&amp;lt;0.002 − 0.0001) and multiplicity (66 − 82% (p&amp;lt;0.002 − 0.0002). Furthermore, low dose combination of 100 ppm NILT plus 250 ppm celecoxib suppressed colon adenocarcinoma incidence by 70% and multiplicity by 77%. Low dose combinations of NILT and celecoxib showed improved efficacy over NILT alone. In colonic tumors of rats fed NILT and/or combination of 100 ppm NILT plus 250 ppm celecoxib showed a significantly low levels of iNOS and COX-2 activities and increased p53, p21, BAX, Caspase −3 and PARP expressions, along with significant decreased levels of Bcl-2, inflammatory cytokines and chemokine expression. Significant levels of apoptotic cells were also observed in treated tumors. These results demonstrate the chemopreventive efficacy of novel iNOS inhibitor against the colon adenocarcinoma, in particular, invasive cancers and increased efficacy when combined with low-dose celecoxib. 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 954.

Abstract 4157: Genetic and dietary complementation in risk for mouse intestinal tumorigenesis

Abstract Dietary factors are significant in determining probability for intestinal tumor development even in the presence of strong genetic initiation. For example, a rodent Western-style diet (WD1), modeled to reproduce intake of nutrient risk factors in the human diet associated with sporadic colon cancer, potentiates the formation of intestinal tumors in Apc1638N/+ mice, even when the genetic initiation by Apc is augmented by targeted inactivation of the p21Waf1/cip1 locus. To investigate mechanisms of this synergistic effect, we profiled gene expression along the mouse crypt-villus axis (CVA) using cells isolated as a function of their position along this axis from WT, Apc1638N/+ and p21−/− mice fed control AIN76A diet, and WT mice fed the WD1. Modulation of sequence expression by the WD1 was enriched in the villus compartment, while changes induced by targeting either genetic locus (Apc1638N/+ or p21−/−) were largely in the crypt, where the overlap in altered gene sets was greatest for the genetic models. There was greater overlap among expression changes between Apc1638N/+ and p21−/− mice, than between these genetic mice models and mice fed the WD1, in both the villus and crypt. Paneth cell markers, whose deregulation has been reported in intestinal tumors, were up-regulated in the p21−/− mouse crypt and in WD1 mouse villus. Moreover, there was attenuated expression of secretory cell lineage markers (ie goblet and enteroendocrine cells), but enhanced expression of enterocyte markers, for both the genetic and dietary models. Genes that encode tricarboxylic acid (TCA) cycle enzymes were uniformly decreased in the crypt of both genetic models, with fewer changes in this pathway altered in the WD1 mouse. Gene ontology and biological pathway analysis also showed that Wnt pathway and c-myc target genes were overrepresented in the villus of the WD1 mouse. Supplementation of WD1 with calcium and vitamin D (WD2) reversed aberrantly up-regulated cyclin D1, cyclin D2, defensins and lysozymes in the WD1 villus back to control levels. In summary, complementary changes in cell reprogramming contributed by a western style diet and targeted inactivation of the p21 gene are important in the synergy of these factors in elevating probability of intestinal tumorigenesis. 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 4157.