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).

Dietary methyl donor depletion protects against intestinal tumorigenesis in Apc(Min/+) mice.

Despite recent population data, the influence of dietary folate supplementation on colon cancer risk remains controversial. This study examines the effects of folate deficiency, in combination with choline, methionine, and vitamin B12 depletion, on intestinal tumorigenesis in Apc(Min/+) mice. Methyl donor sufficient (MDS) and deficient (MDD) diets were started at five or 10 weeks of age and tumors evaluated at 16 weeks. MDD suppressed intestinal tumor formation in Apc(Min/+) mice (~80%) when started at five weeks of age. The protective effect was lost when MDD was initiated at 10 weeks of age, indicating an important time dependency on cancer suppression. Concomitant with cancer protection, MDD restricted body weight gain. Therefore, a second study was conducted in which MDS was given ad libitum or pair-fed with MDD. Although small intestinal tumors were reduced 54% in pair-fed MDS mice, MDD caused a further reduction (96%). In colon, although MDD did not affect tumor numbers, tumor size was reduced. Gene expression profiling of normal-appearing colonic mucosa after 11 weeks on MDD identified a total of 493 significantly downregulated genes relative to the MDS group. Pathway analysis placed many of these genes within general categories of inflammatory signaling and cell-cycle regulation, consistent with recently published human data obtained during folate depletion. Further studies are warranted to investigate the complex interplay of methyl donor status and cancer protection in high-risk populations.

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.

Antioxidant and cytotoxic isoprenylated coumarins from Mammea americana.

Antioxidant-guided fractionation of Mammea americana L. seeds resulted in the identification of three new isoprenylated coumarins, mammea B/BA hydroxycyclo F (1), mammea E/BC (2), and mammea E/BD (3). In addition, twelve known isoprenylated coumarins, mammea A/AA (4), mammea A/AA cyclo D (5), mammea A/AA cyclo F (6), mammea A/AC cyclo D (7), mammea A/AD cyclo D (8), mammea B/BA (9), mammea B/BA cyclo F (10), mammea B/BB (11), mammea B/BC (12), mammea B/BD (13), mammea E/BA (14), and mammea E/BB (15), as well as two known flavanols, (+)-catechin (16) and (-)-epicatechin (17) were identified. The fifteen isoprenylated coumarins were screened for their cytotoxicity in the SW-480, HT-29, and HCT-116 human colon cancer cell lines and antioxidant capacities in the DPPH (1,1-diphenyl-2-picrylhydrazyl) free-radical assay. Compounds 1 - 15 exhibited significant cytotoxic activities in the SW-480, HT-29, and HCT-116 human colon cancer cell lines (IC50 ranges 13.9 - 88.1, 11.2 - 85.3, and 10.7 - 76.7 microM, in the three cell lines, respectively) at concentrations comparable to 5-fluorouracil (IC50 = 53.0, 46.1, and 45.1 microM), a drug frequently used for human colon cancer treatment. Compounds 2 - 4, 9, and 11 - 15 displayed high antioxidant activity in the DPPH assay (IC50 range 86 - 135 microM), compounds 1, 5 - 8, and 10, however, had no antioxidant activity (IC50 > 200 microg/mL) in the DPPH assay. The results of these assays were used to study the structure-activity relationships for this class of compounds. In the SW-480 cell line, the three new coumarins, 1 - 3, also exhibited dose-dependent increases in sub-diploid cells by flow cytometry, indicating that they induce apoptosis.

Bioactive benzophenones from Garcinia xanthochymus fruits.

A MeOH extract of Garcinia xanthochymus fruits was subjected to activity-guided fractionation, yielding two new benzophenones, guttiferone H (1) and gambogenone (2). Compound 1 contains a seven-membered ring attached to the bicyclo[3.3.1]nonane system at positions 7 and 8 and displayed cytotoxicity in the SW-480 colon cancer cell line (IC(50) = 12 microM). Compound 2 has a novel benzophenone bicyclo[3.3.2]decane system and displayed cytotoxicity in the SW-480 colon cancer cell line (IC(50) = 188 microM). Both 1 and 2 induced apoptosis in SW-480 colon cancer cells and displayed antioxidant activity in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay (IC(50) = 64 and 38.7 microM, respectively). The structures of 1 and 2 were established by 1D and 2D NMR data analysis. Eleven known compounds, aristophenone A, alloathyriol, amentoflavone, 3,8' '-biapigenin, cycloxanthochymol, (+/-)-fukugetin, (+/-)-fukugiside, guttiferone E, isoxanthochymol, (+/-)-volkensiflavone, and xanthochymol, were also obtained. The 11 known compounds were also tested against SW-480 colon cancer cells and in the DPPH assay.