Calcium and 1,25-dihydroxyvitamin D3 modulate genes of immune and inflammatory pathways in the human colon: a human crossover trial.

BACKGROUND: A high dietary calcium intake with adequate vitamin D status has been linked to lower colorectal cancer risk, but the mechanisms of these effects are poorly understood. OBJECTIVE: The objective of this study was to elucidate the effects of a Western-style diet (WD) and supplemental calcium and/or 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the colorectal mucosa. DESIGN: We conducted 2 crossover trials to define molecular pathways in the human colorectum altered by 1) a 4-wk WD supplemented with and without 2 g calcium carbonate/d and 2) a 4-wk WD supplemented with 1,25(OH)2D3 (0.5 µg/d) with or without 2 g calcium carbonate/d. The primary study endpoint was genome-wide gene expression in biopsy specimens of the rectosigmoid colonic mucosa. Serum and urinary calcium concentrations were also measured. RESULTS: Changes in urinary calcium accurately reflected calcium consumption. The WD induced modest upregulation of genes involved in inflammatory pathways, including interferon signaling, and calcium supplementation reversed these toward baseline. In contrast, supplementation of the WD with 1,25(OH)2D3 induced striking upregulation of genes involved in inflammation, immune response, extracellular matrix, and cell adhesion. Calcium supplementation largely abrogated these changes. CONCLUSIONS: Supplementing 1,25(OH)2D3 to a WD markedly upregulated genes in immune response and inflammation pathways, which were largely reversed by calcium supplementation. This study provides clinical trial evidence of global gene expression changes occurring in the human colorectum in response to calcium and 1,25(OH)2D3 intervention. One action of 1,25(OH)2D3 is to upregulate adaptive immunity. Calcium appears to modulate this effect, pointing to its biological interaction in the mucosa. This trial was registered at clinicaltrials.gov as NCT00298545 Trial protocol is available at http://clinicalstudies.rucares.org (protocol numbers PHO475 and PHO554).

Effect of caloric intake on Western-style diet-induced intestinal tumors in a mouse model for hereditary colon cancer.

Increased caloric intake has been associated with increased risk for cancer of the large intestine. We studied caloric intake effect on tumor formation in Apc1638( N/+ ) mice, a preclinical model for human familial adenomatous polyposis. Mice were fed a controlled AIN-76A diet or a new Western-style diet (NWD). Intestinal tumor development was evaluated after 6 mo of feeding 1) AIN-76A diet (fed ad libitum) vs. AIN-76A (caloric intake reduced 30%); 2) NWD (fed ad libitum) vs. NWD (caloric intake reduced 30%); and 3) AIN-76A (fed ad libitum) vs. NWD (paired-fed with NWD providing equal caloric intakes to AIN-76A). Intestinal tumor incidences were 78-100% with intergroup variation P > 0.05; however, tumor multiplicity responded differently to dietary treatment: 1) Tumor multiplicity was unchanged after AIN-76A (caloric intake reduced 30% vs. mice fed AIN-76A ad libitum); 2) tumor multiplicity was unchanged after NWD (caloric intake reduced 30% vs. NWD ad libitum); and 3) tumor multiplicity increased 130% after NWD was paired-fed with the same caloric intake as mice fed AIN-76A ad libitum (P < 0.05). Body weights showed no association with tumor development. Findings indicated modified nutrients in NWD were mainly responsible for increased tumors in mice fed NWD vs. AIN-76A in this preclinical mouse model for human FAP.

Altered folate availability modifies the molecular environment of the human colorectum: implications for colorectal carcinogenesis.

Low folate status increases colorectal cancer risk. Paradoxically, overly abundant folate supplementation, which is not uncommon in the United States, may increase risk. The mechanisms of these effects are unknown. We conducted two translational studies to define molecular pathways in the human colon altered either by folate supplementation or by dietary folate depletion (followed by repletion). In the first study, 10 healthy, at-risk volunteers (with documented stable/normal folate intake) received supplemental folic acid (1 mg/d) for 8 weeks. In the second study, 10 similar subjects were admitted to a hospital as inpatients for 12 weeks to study folate depletion induced by a low folate diet. A repletion regimen of folic acid (1 mg/d) was provided for the last 4 of these weeks. Both studies included an 8-week run-in period to ensure stabilized folate levels prior to intervention. We obtained 12 rectosigmoid biopsies (from 4 quadrants of normal-appearing mucosa 10-15 cm from the anal verge) at baseline and at measured intervals in both studies for assessing the primary endpoints: genome-wide gene expression, genomic DNA methylation, promoter methylation (depletion/repletion study only), and p53 DNA strand breaks. Serum and rectosigmoid folate concentrations accurately tracked all changes in folate delivery (P < 0.05). In the first study, gene array analysis revealed that supplementation upregulated multiple inflammation- and immune-related pathways in addition to altering several 1-carbon-related enzymes (P < 0.001). In the second study, folate depletion downregulated genes involved in immune response, inflammation, the cell cycle, and mitochondrial/energy pathways; repletion reversed most of these changes. However, changes in gene expression after repletion in the second study (involving immune response and inflammation) did not reach the levels seen after supplementation in the first study. Neither genomic nor promoter-specific DNA methylation changed during the course of the depletion/repletion protocol, and genomic methylation did not change with supplementation in the first study. p53 DNA strand breaks increased with depletion after 12 weeks. In sum, depletion downregulates, whereas repletion or supplementation upregulates pathways related to inflammation and immune response. These findings provide novel support to the concept that excessive folate supplementation might promote colorectal carcinogenesis by enhancing proinflammatory and immune response pathways. These results indicate that modest changes in folate delivery create substantial changes in the molecular milieu of the human colon.

Cox-2 expression, PGE(2) and cytokines production are inhibited by endogenously synthesized n-3 PUFAs in inflamed colon of fat-1 mice.

There is great interest in the role of polyunsaturated fatty acids (PUFAs) in promoting (n-6 class) or inhibiting (n-3 class) inflammation. Mammalian cells are devoid of desaturase that converts n-6 to n-3 PUFAs. Consequently, essential n-3 fatty acids must be supplied with the diet. We have studied the effect of endogenously produced n-3 PUFAs on colitis development in fat-1 transgenic mice carrying the Caenorhabditis elegans fat-1 gene encoding n-3 desaturase. Colonic cell lipid profile was measured by capillary gas chromatography in fat-1 and wild-type (WT) littermates fed standard diet supplemented with 10% (w/w) safflower oil rich (76%) in n-6 polyunsaturated linoleic acid (LA). Experimental colitis was induced by administrating 3% dextran sodium sulphate (DSS). Colitis was scored by histopatological analysis. Cyclooxygenase-2 (Cox-2) expression was evaluated by real time polymerase chain reaction. Prostaglandin E(2) (PGE(2)) levels and cytokine production were determined by enzyme and microsphere-based immunoassays, respectively. The n-6/n-3 PUFA ratios in colonic cells of fat-1 mice were markedly lower (9.83±2.62) compared to WT (54.5±9.24, P<.001). Results also showed an attenuation of colonic acute and chronic inflammation in fat-1 mice with significant decreases in PGE(2) production (P<.01) and Cox-2 expression (P<.01). High levels of colitis-induced proinflammatory cytokines, interleukin (IL)-18, IL-1α, IL-1ß, IL-6, monocytes chemotactic proteins 1, 2 and 3 (MCP 1,2,3), matrix metalloproteinase 9 and tumor necrosis factor α (TNF-α) were down-regulated in DSS acutely and chronically treated fat-1 mice. The expression of fat-1 gene in the colon was associated with endogenous n-3 PUFAs production, decreased Cox-2 expression, increased PGE(2) and cytokine production.

An Msh2 conditional knockout mouse for studying intestinal cancer and testing anticancer agents.

BACKGROUND & AIMS: Mutations in the DNA mismatch repair (MMR) gene MSH2 cause Lynch syndromes I and II and sporadic colorectal cancers. Msh2(null) mice predominantly develop lymphoma and do not accurately recapitulate the colorectal cancer phenotype. METHODS: We generated and examined mice with a conditional Msh2 disruption (Msh2(LoxP)), permitting tissue-specific gene inactivation. ECMsh2(LoxP/LoxP) mice carried an EIIa-Cre transgene, and VCMsh2(LoxP/LoxP) mice carried a Villin-Cre transgene. We combined the VCMsh2(LoxP) allele with either Msh2(Delta7null) (VCMsh2(LoxP/null)) or Msh2(G674D) mutations (VCMsh2(LoxP/G674D)) to create allelic phase mutants. These mice were given cisplatin or 5-fluorouracil/leucovorin and oxaliplatin (FOLFOX), and their tumors were measured by magnetic resonance imaging. RESULTS: Embryonic fibroblasts from ECMsh2(LoxP/LoxP) mice do not express MSH2 and are MMR deficient. Reverse transcription, polymerase chain reaction, and immunohistochemistry from VCMsh2(LoxP/LoxP) mice demonstrated specific loss of Msh2 messenger RNA and protein from epithelial cells of the intestinal tract. Microsatellite instability was observed in all VCMsh2 strains and limited to the intestinal mucosa. Resulting adenomas and adenocarcinomas had somatic truncation mutations to the adenomatous polyposis coli (Apc) gene. VCMsh2(LoxP/LoxP) mice did not develop lymphoma. Comparison of allelic phase tumors revealed significant differences in multiplicity and size. When treated with cisplatin or FOLFOX, tumor size was reduced in VCMsh2(LoxP/G674D) but not VCMsh2(LoxP/null) tumors. The apoptotic response to FOLFOX was partially sustained in the intestinal mucosa of VCMsh2(LoxP/G674D) animals. CONCLUSIONS: Msh2(LoxP/LoxP) mice in combination with appropriate Cre recombinase transgenes have excellent potential for preclinical modeling of Lynch syndrome, MMR-deficient tumors of other tissue types, and use in drug development.

Sulindac effects on inflammation and tumorigenesis in the intestine of mice with Apc and Mlh1 mutations.

We have previously reported that sulindac, a non-steroidal anti-inflammatory drug, inhibited tumor formation in the small intestine but increased tumors in the colon of Apc(Min/+) mice, a model of human familial adenomatous polyposis. To further explore intestinal regional responses, we studied effects of sulindac on additional gene-targeted mouse models of human intestinal tumorigenesis; these were (i) Apc(1638N/+) mouse (chain termination mutation in exon 15 of the Apc gene); (ii) Mlh1(+/-) mouse (DNA mismatch repair deficiency, a mouse model of human hereditary non-polyposis colorectal cancer) and (iii) double-heterozygous Mlh1(+/-)Apc(1638N/+) mutant mouse. Mice were fed AIN-76A control diet with or without 0.02% sulindac for 6 months. Intestinal regional tumor incidence, multiplicity, volume and degree of inflammation were used as end points. The results showed the following: (i) sulindac inhibited tumor development in the small intestine of Apc(1638N/+) mice; (ii) in contrast, sulindac increased tumors in the small intestine of Mlh1 mutant mice, a neoplastic effect which persisted in heterozygous compound Mlh1(+/-)Apc(1638N/+) mutant mice; (iii) sulindac increased tumors in the cecum of all mice regardless of genetic background; (iv) sulindac decreased inflammation in the small intestine of Apc(1638N/+) mice, but it increased inflammation in the small intestine of Mlh1(+/-) mice and Mlh1(+/-)Apc(1638N/+) mice and (v) sulindac enhanced inflammation in the cecum of all mutant mice. Findings indicate that the effects of sulindac in the intestine of these mutant mouse models are probably related to genetic background and appear to be associated with its inflammatory-inducing response.

Western-style diets induce oxidative stress and dysregulate immune responses in the colon in a mouse model of sporadic colon cancer.

A Western-style diet (WD), defined by high-fat, low-calcium, and vitamin D content, is associated with increased risk of human colorectal cancer. Understanding molecular mechanisms altered by the WD is crucial to develop preventive and therapeutic strategies. Effects of a WD on the colonic transcriptome of C57Bl/6J mice, a model for sporadic colon cancer, were studied at endpoints before tumors occur. To assess whether a WD induces inflammatory changes, expression profiles of a broad spectrum of inflammatory proteins were performed and numbers of lamina propria macrophages were determined with semiquantitative morphometry. Transcriptome changes were translated into molecular interaction network maps and pathways. Pathways related to oxidative stress response; lipid, glutathione, and xenobiotic metabolism; and the immune response were perturbed by the WD. Several nuclear factor-erythroid 2-related factor 2- and aryl hydrocarbon receptor-dependent genes, including those coding for enzymes involved in phase 1 and 2 drug metabolism and oxidative stress responses, were induced. Oxidative stress was demonstrated by measurements of endogenous colonic redox-sensitive compound concentrations. Perturbations in immune response-related pathways, expression of inflammatory proteins, and increased numbers of lamina propria macrophages showed that the WD significantly alters the local colonic immune response. Collectively, these data suggest that consumption of a WD interferes with networks of related biological response pathways involving colonic lipid metabolism, oxidative stress, and the immune response. These new findings impact our understanding of links between consumption of WD and colon carcinogenesis, providing additional information for developing preventive means for decreasing colorectal cancer risk.

Chemoprevention of colorectal neoplasia by estrogen: potential role of vitamin D activity.

Postmenopausal hormone replacement therapy lowers colon cancer incidence. In humans, the mechanism is unknown, but animal models suggest that it may involve activation of the vitamin D receptor (VDR) pathway. The aims of our study were to determine whether estrogen intervention affects global gene expression in rectal mucosal biopsies and whether vitamin D-related genes are affected. Estradiol was given to raise serum estradiol to premenopausal levels in 10 postmenopausal women under close nutritional control. Primary end points were expression of VDR, CYP24A1, CYP27B1, and E-cadherin in rectal mucosa by reverse transcription-PCR and examining response to estradiol by genome-wide arrays. Responses in gene expression in rectal biopsies to estrogen were determined in each subject individually and compared with a human estrogen response gene array database and a custom array in vitro-generated database. Cluster analysis showed that subjects maintained their overall gene expression profile and that interindividual differences were greater than intraindividual differences after intervention. Eight of 10 subjects showed significant enrichment in estrogen-responsive genes. Gene array group analysis showed activation of the VDR pathway and down-regulation of inflammatory and immune signaling pathways. Reverse transcription-PCR analysis showed significant up-regulation of VDR and E-cadherin, a downstream target of vitamin D action. These data suggest that the chemopreventive action of hormone replacement therapy on colon neoplasia results, at least in part, from changes in vitamin D activity. Evaluation of gene arrays is useful in chemopreventive intervention studies in small groups of subjects.

Western-style diet-induced colonic tumors and their modulation by calcium and vitamin D in C57Bl/6 mice: a preclinical model for human sporadic colon cancer.

We reported previously that a new Western-style diet (NWD) for 18 months, consisting of elevated lipids and decreased calcium, vitamin D and methyl-donor nutrients, induced colonic tumors in normal C57Bl/6 mice [Newmark, H.L. et al. (2001) A Western-style diet induces benign and malignant neoplasms in the colon of normal C57Bl/6 mice. Carcinogenesis, 22, 1871-1875], suggesting a new mouse model for human sporadic colon cancer. Here, we have extended this study during a longer feeding period of 2 years wherein tumor formation, tumor inhibition by addition of dietary calcium and vitamin D and their effects on gene expression were determined. We also similarly tested individual supplements of methyl donor (transfer) nutrients (folic acid, choline, methionine and dietary fiber), but these had no significant effect on colonic tumor incidence or multiplicity, whereas supplementation with combined calcium and vitamin D produced significant decrease in both colon tumor incidence and multiplicity, during 2 years of feeding. No visible colonic tumors were found at 6 months, very few at 12 months, more at 18 months and significantly at 24 months. In a related study of gene changes of the mouse colonic mucosa at 6 months of feeding taken from this study, long before any tumors were visibly detectable, indicated altered profiles of gene expression linked to later risk of dietary initiation of colon tumor formation. This type of early genetic altered profile, an indication of increased risk of later colonic tumor development, may become a useful tool for prediction of colon tumor risk while the colon grossly still appears histologically and physiologically normal.

Dietary induction of colonic tumors in a mouse model of sporadic colon cancer.

A defined rodent "new Western diet" (NWD), which recapitulates intake levels of nutrients that are major dietary risk factors for human colon cancer, induced colonic tumors when fed to wild-type C57Bl/6 mice for 1.5 to 2 years from age 6 weeks (two-thirds of their life span). Colonic tumors were prevented by elevating dietary calcium and vitamin D(3) to levels comparable with upper levels consumed by humans, but tumorigenesis was not altered by similarly increasing folate, choline, methionine, or fiber, each of which was also at the lower levels in the NWD that are associated with risk for colon cancer. The NWD significantly altered profiles of gene expression in the flat colonic mucosa that exhibited heterogeneity among the mice, but unsupervised clustering of the data and novel statistical analyses showed reprogramming of colonic epithelial cells in the flat mucosa by the NWD was similar to that initiated by inheritance of a mutant Apc allele. The NWD also caused general down-regulation of genes encoding enzymes involved in lipid metabolism and the tricarboxylic acid cycle in colonic epithelial cells before tumor formation, which was prevented by the supplementation of the NWD with calcium and vitamin D(3) that prevented colon tumor development, demonstrating profound interaction among nutrients. This mouse model of dietary induction of colon cancer recapitulates levels and length of exposure to nutrients linked to relative risk for human sporadic colon cancer, which represents the etiology of >90% of colon cancer in the United States and other Western countries.

Loss of Rb1 in the gastrointestinal tract of Apc1638N mice promotes tumors of the cecum and proximal colon.

To examine the role of Rb1 in gastrointestinal (GI) tumors, we generated mice with an Apc(1638N) allele, Rb(tm2brn) floxed alleles, and a villin-cre transgene (RBVCA). These animals had exon 19 deleted from Rb1 throughout the GI tract. We have shown previously that Rb1 deficiency is insufficient for GI tumor initiation, with inactivation of an Apc allele capable of overcoming the insufficiency. In this study we demonstrate that RBVCA mice have reduced median survival because of an increase in tumor incidence and multiplicity in the cecum and the proximal colon. Large intestinal tumors are predominantly adenomas, whereas the tumors of the small intestine are a mixture of adenomas and adenocarcinomas. We find truncation mutations to the second Apc allele in tumors of both the large and small intestine. Expression profiles of duodenal and cecal tumors relative to each other show unique gene subsets up and down regulated. Substantial expression patterns compare to human colorectal cancer, including recapitulation of embryonic genes. Our results indicate that Rb1 has significant influence over tumor location in the GI tract, and that both cecal and duodenal tumors initiate through inactivation of Apc. Expression profile analysis indicates the two tumor types differentially regulate distinct sets of genes that are over-expressed in a majority of human colorectal carcinomas.

Interaction of Muc2 and Apc on Wnt signaling and in intestinal tumorigenesis: potential role of chronic inflammation.

Somatic mutations of the adenomatous polyposis coli (APC) gene are initiating events in the majority of sporadic colon cancers. A common characteristic of such tumors is reduction in the number of goblet cells that produce the mucin MUC2, the principal component of intestinal mucus. Consistent with these observations, we showed that Muc2 deficiency results in the spontaneous development of tumors along the entire gastrointestinal tract, independently of deregulated Wnt signaling. To dissect the complex interaction between Muc2 and Apc in intestinal tumorigenesis and to elucidate the mechanisms of tumor formation in Muc2(-/-) mice, we crossed the Muc2(-/-) mouse with two mouse models, Apc(1638N/+) and Apc(Min/+), each of which carries an inactivated Apc allele. The introduction of mutant Muc2 into Apc(1638N/+) and Apc(Min/+) mice greatly increased transformation induced by the Apc mutation and significantly shifted tumor development toward the colon as a function of Muc2 gene dosage. Furthermore, we showed that in compound double mutant mice, deregulation of Wnt signaling was the dominant mechanism of tumor formation. The increased tumor burden in the distal colon of Muc2/Apc double mutant mice was similar to the phenotype observed in Apc(Min/+) mice that are challenged to mount an inflammatory response, and consistent with this, gene expression profiles of epithelial cells from flat mucosa of Muc2-deficient mice suggested that Muc2 deficiency was associated with low levels of subclinical chronic inflammation. We hypothesize that Muc2(-/-) tumors develop through an inflammation-related pathway that is distinct from and can complement mechanisms of tumorigenesis in Apc(+/-) mice.

Dietary calcium and cholecalciferol modulate cyclin D1 expression, apoptosis, and tumorigenesis in intestine of adenomatous polyposis coli1638N/+ mice.

Both epidemiological and experimental findings have indicated that components of Western diets influence colonic tumorigenesis. Among dietary constituents, calcium and cholecalciferol have emerged as promising chemopreventive agents. We have demonstrated that a Western-style diet (WD) with low levels of calcium and cholecalciferol and high levels of (n-6) PUFA, increased the incidence of neoplasia in mouse intestine compared with a standard AIN-76A diet; models included wild-type mice and mice with targeted mutations. In the present study, adenomatous polyposis coli (Apc)(1638N/+) mice carrying a heterozygous Apc mutation were fed either an AIN-76A diet, a WD, or a WD supplemented with calcium and cholecalciferol (WD/Ca/VitD3). Diets were fed for 24 wk and effects on cellular and molecular events were assessed by performing immunohistochemistry in colonic epithelium along the crypt-to-surface continuum. Feeding WD to Apc(1638N/+) mice not only enhanced cyclin D1 expression in colonic epithelium compared with AIN-76A treatment as previously reported but also significantly increased the expression of the antiapoptotic protein B-cell lymphoma 2 (Bcl-2) concomitantly with a decrease in the proapoptotic Bcl2-associated X protein and the number of apoptotic epithelial cells. WD treatment enhanced mutant Apc-driven small intestinal carcinogenesis and also resulted in the formation of a small number of colonic adenomas (0.16 +/- 0.09; P < 0.05). By contrast, the WD/Ca/VitD3 diet reversed WD-induced growth, promoting changes in colonic epithelium. Importantly, Apc(1638N/+) mice fed the WD/Ca/VitD3 diet did not develop colonic tumors, further indicating that dietary calcium and cholecalciferol have a key role in the chemoprevention of colorectal neoplasia in this mouse model of human colon cancer.

Growth inhibition of colon cancer cells by polyisoprenylated benzophenones is associated with induction of the endoplasmic reticulum response.

Polyisoprenylated benzophenones derived from Garcinia xanthochymus have cytotoxic activity in vitro and antitumor activity in rodent models, but the mechanism is unknown. The purpose of our study was to examine in parallel molecular pathways that are targeted by 3 Garcinia-derived benzophenones-xanthochymol (X), guttiferone E (GE) and guttiferone H (GH), in 3 human colon cancer cell lines, HCT116, HT29 and SW480. The IC50 concentrations were determined and the cells were then treated with X, GE or GH at their respective IC50 or IC50x2 concentrations. Effects on the cell cycle, mitochondrial membrane potential and apoptosis were assessed by flow cytometry and caspase activation. Changes in gene expression were assessed with Illumina 24 K gene arrays. We found that X, GE and GH induced loss of mitochondrial membrane potential and G1 arrest at their IC50 concentrations and induced caspase activation at IC50 x 2 concentrations. An analysis of the changes in gene expression revealed that with all 3 compounds and all 3 cell lines there was a marked increase in expression of several genes, including XBP1, ATF4 and DDIT3/CHOP, which are components of the endoplasmic reticulum stress response. The DDIT4/REDD1 gene, an inhibitor of the mTOR survival pathway, was also up-regulated. Therefore, X, GE and GH appear to inhibit the growth of human colon cancer cells, at least in part, by activating the endoplasmic reticulum stress response and inhibiting the mTOR cell survival pathway. These combined effects may contribute to the anticancer activity of these novel compounds.

Effect of a Western-style diet fortified with increased calcium and vitamin D on mammary gland of C57BL/6 mice.

We previously reported hyperproliferation and hyperplasia in C57Bl/6 mouse mammary gland after feeding a Western-style diet (WD); these findings decreased after supplementing WD with increased calcium and vitamin D(3). We now again fortified WD with increased calcium and vitamin D(3) from two sources: (1) a food source, calcium- and vitamin D(3)-enriched yogurt (WD(y) diet) or (2) adding calcium and vitamin D(3) directly to WD (WD(CaD) diet). After 6 months of feeding the number of mammary ducts was higher in mice consuming WD compared to WD(y) (216.0 vs. 202.7, P <.05) and WD(CaD) (216.0 vs. 194.9, P <.001). The percentage of small ducts increased in WD compared to AIN-76A controls (23.3% vs. 17.4%) but was lower in the WD(y) (17.1%) and WD(CaD) (14.8%) groups. WD mice had higher numbers of epithelial cells per duct than WD(y) (33.2 vs. 27.4, P <.001) and WD(CaD) (33.2 vs. 27.8, P <.001) mice, and AIN-76A-fed mice had higher numbers than WD(y) (31.1 vs. 27.4, P <.005) or WD(CaD) (31.1 vs. 27.8, P <.01) mice. Mitotic index was higher in WD than in WD(CaD) mice (0.0020 vs. 0.0009, P <.001). Thus, small mammary gland ductules and mitosis increased after feeding WD and decreased after supplementing the diets with increased calcium and vitamin D(3), administered either in a dairy food (yogurt) or directly as calcium carbonate plus vitamin D(3) in WD, suggesting further study of these nutrients for their possible relationship to breast cancer prevention.

The untapped potential of genetically engineered mouse models in chemoprevention research: opportunities and challenges.

The past decade has witnessed the unveiling of a powerful new generation of genetically engineered mouse (GEM) models of human cancer, which are proving to be highly effective for elucidating cancer mechanisms and interrogating novel experimental therapeutics. This new generation of GEM models are well suited for chemoprevention research, particularly for investigating progressive stages of carcinogenesis, identifying biomarkers for early detection and intervention, and preclinical assessment of novel agents or combinations of agents. Here we discuss opportunities and challenges for the application of GEM models in prevention research, as well as strategies to maximize their relevance for human cancer.

Vitamin D and prevention of breast cancer: pooled analysis.

BACKGROUND: Inadequate photosynthesis or oral intake of Vitamin D are associated with high incidence and mortality rates of breast cancer in ecological and observational studies, but the dose-response relationship in individuals has not been adequately studied. METHODS: A literature search for all studies that reported risk by of breast cancer by quantiles of 25(OH)D identified two studies with 1760 individuals. Data were pooled to assess the dose-response association between serum 25(OH)D and risk of breast cancer. RESULTS: The medians of the pooled quintiles of serum 25(OH)D were 6, 18, 29, 37 and 48 ng/ml. Pooled odds ratios for breast cancer from lowest to highest quintile, were 1.00, 0.90, 0.70, 0.70 and 0.50 (p trend<0.001). According to the pooled analysis, individuals with serum 25(OH)D of approximately 52 ng/ml had 50% lower risk of breast cancer than those with serum <13 ng/ml. This serum level corresponds to intake of 4000 IU/day. This exceeds the National Academy of Sciences upper limit of 2000 IU/day. A 25(OH)D level of 52 ng/ml could be maintained by intake of 2000 IU/day and, when appropriate, about 12 min/day in the sun, equivalent to oral intake of 3000 IU of Vitamin D(3). CONCLUSIONS: Intake of 2000 IU/day of Vitamin D(3), and, when possible, very moderate exposure to sunlight, could raise serum 25(OH)D to 52 ng/ml, a level associated with reduction by 50% in incidence of breast cancer, according to observational studies.

Optimal vitamin D status for colorectal cancer prevention: a quantitative meta analysis.

BACKGROUND: Previous studies, such as the Women's Health Initiative, have shown that a low dose of vitamin D did not protect against colorectal cancer, yet a meta-analysis indicates that a higher dose may reduce its incidence. METHODS: Five studies of serum 25(OH)D in association with colorectal cancer risk were identified using PubMed. The results of all five serum studies were combined using standard methods for pooled analysis. The pooled results were divided into quintiles with median 25(OH)D values of 6, 16, 22, 27, and 37 ng/mL. Odds ratios were calculated by quintile of the pooled data using Peto's Assumption-Free Method, with the lowest quintile of 25(OH)D as the reference group. A dose-response curve was plotted based on the odds for each quintile of the pooled data. Data were abstracted and analyzed in 2006. RESULTS: Odds ratios for the combined serum 25(OH)D studies, from lowest to highest quintile, were 1.00, 0.82, 0.66, 0.59, and 0.46 (p(trend)<0.0001) for colorectal cancer. According to the DerSimonian-Laird test for homogeneity of pooled data, the studies were homogeneous (chi(2)=1.09, df=4, p=0.90. The pooled odds ratio for the highest quintile versus the lowest was 0.49 (p<0.0001, 95% confidence interval, 0.35-0.68). A 50% lower risk of colorectal cancer was associated with a serum 25(OH)D level > or =33 ng/mL, compared to < or =12 ng/mL. CONCLUSIONS: The evidence to date suggests that daily intake of 1000-2000 IU/day of vitamin D(3) could reduce the incidence of colorectal with minimal risk.

Piroxicam increases colon tumorigenesis and promotes apoptosis in Mlh1 +/- /Apc1638(N/+) mice.

BACKGROUND: The present study examines the effect of piroxicam, a non-steroidal anti-inflammatory drug, on tumor development in Mlh1+/- /Apc1638(N/+) mice, in a preclinical model of human colon cancer. MATERIALS AND METHODS: Mice were fed AIN-76A diet alone or premixed with piroxicam (60 ppm) for 9 weeks. The number, location and volume of tumors, and apoptosis in the flat mucosa were determined in small and large intestine. RESULTS: Piroxicam reduced the number of tumors per mouse by 80% in the small intestine (0.1 vs. 0.5, p < 0.05). In contrast, piroxicam increased tumor incidence (82% vs. 10%, p < 0.01), tumor multiplicity (1.2 vs. 0.1, p < 0.01) and tumor volume (2.1 vs. 0.2 mm3, p < 0.01) in the colon. Apoptosis increased in the epithelium of the small intestine. CONCLUSION: Consistent with the increased apoptosis, piroxicam reduced tumors in the small intestine. In the cecum, piroxicam increased tumorigenesis but apoptosis was not decreased, suggesting that other mechanisms besides apoptosis are involved in the differential organ-specific effect on tumorigenesis of piroxicam in this colon cancer model.