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).

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.

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.

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.

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 role of vitamin D in cancer prevention.

Vitamin D status differs by latitude and race, with residents of the northeastern United States and individuals with more skin pigmentation being at increased risk of deficiency. A PubMed database search yielded 63 observational studies of vitamin D status in relation to cancer risk, including 30 of colon, 13 of breast, 26 of prostate, and 7 of ovarian cancer, and several that assessed the association of vitamin D receptor genotype with cancer risk. The majority of studies found a protective relationship between sufficient vitamin D status and lower risk of cancer. The evidence suggests that efforts to improve vitamin D status, for example by vitamin D supplementation, could reduce cancer incidence and mortality at low cost, with few or no adverse effects.

Colonic epithelial cell proliferation decreases with increasing levels of serum 25-hydroxy vitamin D.

Epidemiological evidence suggests a potential role for vitamin D in colon cancer prevention. Vitamin D, absorbed from the intestine or derived from solar ultraviolet light, is metabolized in the liver to 25-hydroxyvitamin D (25-OH D(3)). Previous studies examining effects of vitamin D upon carcinogenesis have focused upon the active metabolite 1,25-dihydroxyvitamin D [1,25-(OH)(2) D(3)], which interacts with nuclear vitamin D receptors in several organs. Until recently, the metabolism of 25-OH D(3) to 1,25-(OH)(2) D(3) was believed to occur only in the kidney, but more recent studies have shown that 25-OH D(3) conversion to 1,25-(OH)(2) D(3) can occur in other tissues. We examined the association between fasting levels of 25-OH D(3), 1,25-(OH)(2) D(3), and BsmI polymorphism of the vitamin D receptor (VDR) gene with indices of colonic epithelial cell proliferation and differentiation in a chemoprevention study, after giving vitamin D or calcium and taking rectal biopsies that were incubated with bromodeoxyuridine. Vitamin D receptor polymorphism was determined by genotyping of the 3' BsmI polymorphism in intron eight of the VDR gene. No significant changes in cell proliferation or in differentiation were found in subjects between study start and end. However, fasting serum levels of 25-OH D(3) showed a highly significant decrease with whole crypt labeling index and the size of the proliferative compartment (phi h). There was no correlation between serum levels of 1,25-(OH)(2) D(3) and the proliferative parameters. Calcium supplementation induced a significant effect upon the relationship between serum 25-OH D(3) and rectal epithelial cell labeling index and phi h when studied by covariance analysis without a relationship with 1,25-(OH)(2) D(3) levels. VDR genotype did not influence the effects of serum 25-OH D(3) or serum 1,25-(OH)(2) D(3) levels upon proliferation. These data suggest that there might be a local effect of 25-OH D(3) on colonic epithelial cells through conversion of 25-OH D(3) to 1,25-(OH)(2) D(3). Subsequent studies have demonstrated the presence of 1alpha-hydroxylase mRNA in normal colorectal epithelium and in colorectal cancer. Thus, vitamin D may have an important role in determining the effects of calcium on colorectal epithelial proliferation and may explain some of the discrepancies found previously in studies that examine the direct role of calcium on the colorectal epithelium.

Early development of cancer chemoprevention clinical trials: studies of dietary calcium as a chemopreventive agent for human subjects.

Early cancer chemoprevention clinical trials in human subjects had to be carried out with large numbers of subjects studied for long durations, measuring cancer as an end point. However new findings on abnormal epithelial cell growth and development during the multistage evolution of colonic tumors made it possible to carry out chemoprevention clinical trials in several stages, with fewer subjects studied for shorter durations, thus enabling investigators to analyze increasing numbers of chemopreventive agents and nutritional regimens in clinical trials. Supplemental dietary calcium was the first candidate chemopreventive agent studied in this multistage approach in human subjects, as a putative agent for colon cancer prevention. Early- and late-stage intermediate biomarker studies in humans have strongly suggested utility for supplemental dietary calcium to inhibit the development of benign and subsequent malignant colonic neoplasms. Preclinical experimental studies have further demonstrated the ability of increased dietary calcium to inhibit the evolution of colonic tumors when they were induced by targeted mutations, dietary factors, and particularly when given over a long duration of lifespan.