The calcium-sensing receptor modulates the prostaglandin E2 pathway in intestinal inflammation.

Introduction: The prostaglandin E2 (PGE2) pathway is one of the main mediators of intestinal inflammation. As activation of the calcium-sensing receptor (CaSR) induces expression of inflammatory markers in the colon, we assessed the impact of the CaSR on the PGE2 pathway regulation in colon cancer cells and the colon in vitro and in vivo. Methods and Results: We treated CaSR-transfected HT29 and Caco-2 colon cancer cell lines with different orthosteric ligands or modulators of the CaSR and measured gene expression and PGE2 levels. In CaSR-transfected HT29CaSR-GFP and Caco-2CaSR-GFP cells, the orthosteric CaSR ligand spermine and the positive allosteric CaSR modulator NPS R-568 both induced an inflammatory state as measured by IL-8 gene expression and significantly increased the expression of the PGE2 pathway key enzymes cyclooxygenase (COX)-2 and/or prostaglandin E2 synthase 1 (PGES-1). Inhibition of the CaSR with the calcilytic NPS 2143 abolished the spermine- and NPS R-568-induced pro-inflammatory response. Interestingly, we observed cell-line specific responses as e.g. PGES-1 expression was affected only in HT29CaSR-GFP but not in Caco-2CaSR-GFP cells. Other genes involved in the PGE2 pathway (COX-1, or the PGE2 receptors) were not responsive to the treatment. None of the studied genes were affected by any CaSR agonist in GFP-only transfected HT29GFP and Caco-2GFP cells, indicating that the observed gene-inducing effects of spermine and R-568 were indeed mediated by the CaSR. In vivo, we had previously determined that treatment with the clinically approved calcimimetic cinacalcet worsened symptoms in a dextran sulfate sodium (DSS)-induced colitis mouse model. In the colons of these mice, cinacalcet significantly induced gene expression of PGES-2 and the EP3 receptor, but not COX-2; while NPS 2143 increased the expression of the PGE2-degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Importantly, neither treatment had any effect on the colons of non-DSS treated mice. Discussion: Overall, we show that activation of the CaSR induces the PGE2 pathway, albeit with differing effects in vitro and in vivo. This may be due to the different microenvironment in vivo compared to in vitro, specifically the presence of a CaSR-responsive immune system. Since calcilytics inhibit ligand-mediated CaSR signaling, they may be considered for novel therapies against inflammatory bowel disease.

Impaired Mineral Ion Metabolism in a Mouse Model of Targeted Calcium-Sensing Receptor (CaSR) Deletion from Vascular Smooth Muscle Cells.

BACKGROUND: Impaired mineral ion metabolism is a hallmark of CKD-metabolic bone disorder. It can lead to pathologic vascular calcification and is associated with an increased risk of cardiovascular mortality. Loss of calcium-sensing receptor (CaSR) expression in vascular smooth muscle cells exacerbates vascular calcification in vitro. Conversely, vascular calcification can be reduced by calcimimetics, which function as allosteric activators of CaSR. METHODS: To determine the role of the CaSR in vascular calcification, we characterized mice with targeted Casr gene knockout in vascular smooth muscle cells ( SM22α CaSR Δflox/Δflox ). RESULTS: Vascular smooth muscle cells cultured from the knockout (KO) mice calcified more readily than those from control (wild-type) mice in vitro. However, mice did not show ectopic calcifications in vivo but they did display a profound mineral ion imbalance. Specifically, KO mice exhibited hypercalcemia, hypercalciuria, hyperphosphaturia, and osteopenia, with elevated circulating fibroblast growth factor 23 (FGF23), calcitriol (1,25-D3), and parathyroid hormone levels. Renal tubular α-Klotho protein expression was increased in KO mice but vascular α-Klotho protein expression was not. Altered CaSR expression in the kidney or the parathyroid glands could not account for the observed phenotype of the KO mice. CONCLUSIONS: These results suggest that, in addition to CaSR's established role in the parathyroid-kidney-bone axis, expression of CaSR in vascular smooth muscle cells directly contributes to total body mineral ion homeostasis.

AOM/DSS Induced Colitis-Associated Colorectal Cancer in 14-Month-Old Female Balb/C and C57/Bl6 Mice-A Pilot Study.

Colitis is a major risk factor for the development of colorectal cancer, leading to colitis-associated colorectal cancer (CAC). The most commonly used animal model to study CAC is the azoxymethane-dextran sulphate-sodium (AOM/DSS) model. The ideal experimental conditions of this model depend on several factors, including the used mouse strain. No data on feasibility and conditions for older mice, e.g., for aging studies, have yet been reported. Thus, we conducted a descriptive, observational pilot study where CAC was induced in 14-month-old female Balb/C and C57/Bl6 mice using 12.5 mg/kg AOM i.p. and three different concentrations of DSS (1, 2, and 3%) in drinking water (ad. lib.). The mice were monitored regularly during the three-month experimental phase. After euthanasia, the colons of the mice were evaluated macroscopically and microscopically. Both the mouse strains showed a DSS-concentration-dependent induction of CAC. Carcinomas were only observed at 3% DSS. The DSS dose was found to be significantly correlated with the histology score and % Ki67 positive cells only in C57/Bl6 mice but not in Balb/C mice, which showed a variable response to the CAC induction. No differences in colon length, weight, or mucin content were observed. Optimal conditions for CAC induction in these aged animals are thus considered to be 3% DSS, as carcinomas did not develop when 2% DSS was used. On the other hand, Balb/C mice reacted severely to 3% DSS, indicating that 2.5% DSS may be the "sweet spot" for future experiments comparing CAC in aged Balb/C and C57/Bl6 mice. This model will allow investigation of the effect of aging on CAC development and therapy.

Stereo-Specific Modulation of the Extracellular Calcium-Sensing Receptor in Colon Cancer Cells.

Pharmacological allosteric agonists (calcimimetics) of the extracellular calcium-sensing receptor (CaSR) have substantial gastro-intestinal side effects and induce the expression of inflammatory markers in colon cancer cells. Here, we compared the effects of both CaSR-specific (R enantiomers) and -unspecific (S enantiomers) enantiomers of a calcimimetic (NPS 568) and a calcilytic (allosteric CaSR antagonists; NPS 2143) to prove that these effects are indeed mediated via the CaSR, rather than via off-target effects, e.g., on ß-adrenoceptors or calcium channels, of these drugs. The unspecific S enantiomer of NPS 2143 and NPS S-2143 was prepared using synthetic chemistry and characterized using crystallography. NPS S-2143 was then tested in HEK-293 cells stably transfected with the human CaSR (HEK-CaSR), where it did not inhibit CaSR-mediated intracellular Ca2+ signals, as expected. HT29 colon cancer cells transfected with the CaSR were treated with both enantiomers of NPS 568 and NPS 2143 alone or in combination, and the expression of CaSR and the pro-inflammatory cytokine interleukin 8 (IL-8) was measured by RT-qPCR and ELISA. Only the CaSR-selective enantiomers of the calcimimetic NPS 568 and NPS 2143 were able to modulate CaSR and IL-8 expression. We proved that pro-inflammatory effects in colon cancer cells are indeed mediated through CaSR activation. The non-CaSR selective enantiomer NPS S-2143 will be a valuable tool for investigations in CaSR-mediated processes.

Vitamin D Analogs Regulate the Vitamin D System and Cell Viability in Ovarian Cancer Cells.

BACKGROUND: Ovarian cancer (OC) is one of the most lethal cancers in women. The active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25D3, calcitriol) has anticancer activity in several cancers, including ovarian cancer, but the required pharmacological doses may cause hypercalcemia. We hypothesized that newly developed, low calcemic, vitamin D analogs (an1,25Ds) may be used as anticancer agents instead of calcitriol in ovarian cancer cells. METHODS: We used two patient-derived high-grade serous ovarian cancer (HGSOC) cell lines with low (13781) and high (14433) mRNA expression levels of the gene encoding 1,25-dihydroxyvitamin D3 24-hydroxylase CYP24A1, one of the main target genes of calcitriol. We tested the effect of calcitriol and four structurally related series of an1,25Ds (PRI-1906, PRI-1907, PRI-5201, PRI-5202) on cell number, viability, the expression of CYP24A1, and the vitamin D receptor (VDR). RESULTS: CYP24A1 mRNA expression increased in a concentration-dependent manner after treatment with all compounds. In both cell lines, after 4 h, PRI-5202 was the most potent analog (in 13781 cells: EC50 = 2.98 ± 1.10 nmol/L, in 14433 cells: EC50 = 0.92 ± 0.20 nmol/L), while PRI-1907 was the least active one (in 13781 cells: EC50 = n/d, in 14433 cells: EC50 = n/d). This difference among the analogs disappeared after 5 days of treatment. The 13781 cells were more sensitive to the an1,25Ds compared with 14433 cells. The an1,25Ds increased nuclear VDR levels and reduced cell viability, but only in the 13781 cell line. CONCLUSIONS: The an1,25Ds had different potencies in the HGSOC cell lines and their efficacy in increasing CYP24A1 expression was cell line- and chemical structure-dependent. Therefore, choosing sensitive cancer cell lines and further optimization of the analogs' structure might lead to new treatment options against ovarian cancer.

Effects of pharmacological calcimimetics on colorectal cancer cells over-expressing the human calcium-sensing receptor.

The calcium-sensing receptor (CaSR) is a ubiquitously expressed multifunctional G protein-coupled receptor. Several studies reported that the CaSR plays an anti-inflammatory and anti-tumorigenic role in the intestine, and that it is down-regulated during colorectal carcinogenesis. We hypothesized that positive allosteric CaSR modulators (type II calcimimetics) selectively targeting the intestinal cells could be used for the treatment of intestinal pathologies. Therefore, the aim of this study was to determine the effect of pharmacological stimulation of CaSR on gene expression in vitro and on tumor growth in vivo. We stably transduced two colon cancer cell lines (HT29 and Caco2) with lentiviral vectors containing either the CaSR fused to GFP or GFP only. Using RNA sequencing, RT-qPCR experiments and ELISA, we determined that CaSR over-expression itself had generally little effect on gene expression in these cells. However, treatment with 1 µM of the calcimimetic NPS R-568 increased the expression of pro-inflammatory factors such as IL-23α and IL-8 and reduced the transcription of various differentiation markers in the cells over-expressing the CaSR. In vivo, neither the presence of the CaSR nor p.o. treatment of the animals with the calcimimetic cinacalcet affected tumor growth, tumor cell proliferation or tumor vascularization of murine HT29 xenografts. In summary, CaSR stimulation in CaSR over-expressing cells enhanced the expression of inflammatory markers in vitro, but was not able to repress colorectal cancer tumorigenicity in vivo. These findings suggest potential pro-inflammatory effects of the CaSR and type II calcimimetics in the intestine.

Switching to a Healthy Diet Prevents the Detrimental Effects of Western Diet in a Colitis-Associated Colorectal Cancer Model.

Inflammatory bowel disease increases the odds of developing colitis-associated cancer. We hypothesized that Western-style diet (WD) aggravates azoxymethane (AOM)/dextran sulfate sodium salt (DSS)-induced colitis-associated tumorigenesis and that switching to the standard AIN93G diet will ameliorate disease symptoms even after cancer initiation. Female BALB/c mice received either WD (WD group) or standard AIN93G diet (AIN group) for the whole experimental period. After five weeks, the mice received 12.5 mg/kg AOM intraperitoneally, followed by three DSS cycles. In one group of mice, the WD was switched to AIN93G the day before starting the first DSS cycle (WD/AIN group). Feeding the WD during the whole experimental period aggravated colitis symptoms, shortened the colon (p < 0.05), changed microbiota composition and increased tumor promotion. On molecular level, the WD reduced proliferation (p < 0.05) and increased expression of the vitamin D catabolizing enzyme Cyp24a1 (p < 0.001). The switch to the AIN93G diet ameliorated this effect, reflected by longer colons, fewer (p < 0.05) and smaller (p < 0.01) aberrant colonic crypt foci, comparable with the AIN group. Our results show that switching to a healthy diet, even after cancer initiation is able to revert the deleterious effect of the WD and could be an effective preventive strategy to reduce colitis symptoms and prevent tumorigenesis.

Nutritional and Pharmacological Targeting of the Calcium-Sensing Receptor Influences Chemically Induced Colitis in Mice.

The calcium-sensing receptor (CaSR) is the main regulator of extracellular Ca2+ homeostasis. It has diverse functions in different tissues, including the intestines. Intestine-specific knockout of the CaSR renders mice more susceptible to dextran sulphate sodium (DSS)-induced colitis. To test our hypothesis that the CaSR reduces intestinal inflammation, we assessed the effects of nutritional and pharmacological agonists of the CaSR in a colitis model. We treated female Balb/C mice with dietary calcium and protein (nutritional agonists of the CaSR) or pharmacological CaSR modulators (the agonists cinacalcet and GSK3004774, and the antagonist NPS-2143; 10 mg/kg), then induced colitis with DSS. The high-protein diet had a strong pro-inflammatory effect-it shortened the colons (5.3 ± 0.1 cm vs. 6.1 ± 0.2 cm normal diet, p < 0.05), lowered mucin expression and upregulated pro-inflammatory cytokines, such as interferon-γ, (4.2-fold, p < 0.05) compared with the normal diet. Cinacalcet reduced mucin expression, which coincided with an increase in tumor necrosis factor-α (4.4-fold, p < 0.05) and IL-6 (4.9-fold, p < 0.05) in the plasma, compared with vehicle. The CaSR antagonist, NPS-2143, significantly reduced the cumulative inflammation score compared with the vehicle control (35.3 ± 19.1 vs. 21.9 ± 14.3 area under the curve, p < 0.05) and reduced infiltration of inflammatory cells. While dietary modulation of the CaSR had no beneficial effects, pharmacological inhibition of the CaSR may have the potential of a novel add-on therapy in the treatment of inflammatory bowel diseases.

Expression profiling of colorectal cancer cells reveals inhibition of DNA replication licensing by extracellular calcium.

Colorectal cancer is one of the most common cancers in industrialised societies. Epidemiological studies, animal experiments, and randomized clinical trials have shown that dietary factors can influence all stages of colorectal carcinogenesis, from initiation through promotion to progression. Calcium is one of the factors with a chemoprophylactic effect in colorectal cancer. The aim of this study was to understand the molecular mechanisms of the anti-tumorigenic effects of extracellular calcium ([Ca2+]o) in colon cancer cells. Gene expression microarray analysis of colon cancer cells treated for 1, 4, and 24h with 2mM [Ca2+]o identified significant changes in expression of 1571 probe sets (ANOVA, p<10-5). The main biological processes affected by [Ca2+]o were DNA replication, cell division, and regulation of transcription. All factors involved in DNA replication-licensing were significantly downregulated by [Ca2+]o. Furthermore, we show that the calcium-sensing receptor (CaSR), a G protein-coupled receptor is a mediator involved in this process. To test whether these results were physiologically relevant, we fed mice with a standard diet containing low (0.04%), intermediate (0.1%), or high (0.9%) levels of dietary calcium. The main molecules regulating replication licensing were inhibited also in vivo, in the colon of mice fed high calcium diet. We show that among the mechanisms behind the chemopreventive effect of [Ca2+]o is inhibition of replication licensing, a process often deregulated in neoplastic transformation. Our data suggest that dietary calcium is effective in preventing replicative stress, one of the main drivers of cancer and this process is mediated by the calcium-sensing receptor.

Role of proinflammatory cytokines on expression of vitamin D metabolism and target genes in colon cancer cells.

Interleukin 6 (IL-6) and tumor necrosis factor alpha (TNFα) are proinflammatory cytokines that play a critical role in inflammatory bowel disease, as well as in colorectal tumorigenesis. We hypothesize that these cytokines modulate the expression and thus activity of the vitamin D system in colonic epithelial cells. We treated the colon cancer cell line COGA-1A for 6, 12, and 24h with 1,25-dihydroxyvitamin D3 (1,25-D3), IL-6, TNFα, and with combinations of these compounds. Using quantitative RT-PCR, we analyzed mRNA expression of genes activating and catabolizing 1,25-D3 (1α-hydroxylase (CYP27B1), 24-hydroxylase (CYP24A1)), expression of several vitamin D target genes, as well as expression of cyclooxygenase 2 (COX-2) and 15-hydroxyprostaglandin dehydrogenase. As expected, treatment with 1,25-D3 resulted in an upregulation of CYP24A1, whereas expression of CYP27B1 was not affected. Treatment with TNFα and IL-6 led to decreased expression of the vitamin D activating enzyme CYP27B1. The strong inflammatory property of TNFα was mirrored by its activation of COX-2 and inhibition of prostaglandin E2 (PGE2) catabolism. Interestingly, expression of the calcium ion channel TRPV6 was markedly decreased by TNFα. We conclude from these results that the presence of proinflammatory cytokines might impair activation of 1,25-D3, limiting its anti-inflammatory action. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

Regulation of the calcium-sensing receptor expression by 1,25-dihydroxyvitamin D3, interleukin-6, and tumor necrosis factor alpha in colon cancer cells.

Anti-proliferative effects of calcium in the colon are mediated, at least in part, via the calcium-sensing receptor (CaSR), a vitamin D target gene. The expression of CaSR decreases during colorectal tumor progression and the mechanisms regulating its expression are poorly understood. The CaSR promoter harbors vitamin D elements responsive to 1,25-dihydroxyvitamin D3 (1,25D3) and NF-κB, STAT, and SP1 binding sites accounting for responsiveness to proinflammatory cytokines. Therefore, in the current study we investigated the impact of 1,25D3, tumor necrosis factor alpha (TNFα), and interleukin (IL)-6 on CaSR expression in a differentiated (Caco2/AQ) and in a moderately differentiated (Coga1A) colon cancer cell line. 1,25D3 induced CaSR expression in both cell lines. Treatment with TNFα was accompanied by a 134-fold induction of CaSR in Coga1A (p<0.01). In Caco2/AQ cells the expression of CaSR was upregulated also by IL-6 (3.5-fold). Our data demonstrated transcriptional and translational activation of the CaSR by 1,25D3, TNFα, and IL-6 in a time- and cell line-dependent manner. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

Epigenetic regulation of the 1,25-dihydroxyvitamin D3 24-hydroxylase (CYP24A1) in colon cancer cells.

Calcitriol is the hormonally active form of vitamin D and has anti-proliferative and pro-apoptotic effects. Calcitriol and its precursor calcidiol (25(OH)D3) are degraded by the 1,25-dihydroxyvitamin D3 24-hydroxylase (CYP24A1). This enzyme is overexpressed in colorectal tumors, however, the mechanisms of this overexpression remain to be elucidated. CYP24A1 mRNA level differs among colorectal cancer cell lines and range from almost undetectable to high. Since DNA methylation and histone acetylation regulate CYP24A1 gene expression in prostate cancer cell lines, we investigated whether epigenetic mechanisms could explain the differences in basal expression of CYP24A1 in colon cancer cells. Methyltransferase inhibitor 5-aza-2'-deoxycytidine (DAC) treatment resulted in an over 50-fold induction of CYP24A1 mRNA expression in Coga1A and HT-29 cells but in no response in Caco2/AQ and Coga13 cells. This finding is supported by a strong increase in CYP24A1 activity after DAC treatment in Coga1A (35%). In addition, calcitriol and DAC had synergistic effects on CYP24A1 gene transcription. Interestingly, the CYP24A1 promoter was not methylated in Coga1A and HT-29 (<5%), while in Caco2/AQ it was 62% methylated. This suggests that DNA demethylation must activate genes upstream of CYP24A1 rather than act on the gene itself. However, transcriptional regulators of CYP24A1 such as vitamin D receptor (VDR), retinoid X receptor (RXR), specificity protein 1 (SP1), or mediator complex subunit 1 (MED1) were not upregulated. We conclude that in colon cancer cells, CYP24A1 gene expression is inducible by methyltransferase and some histone deacetylase inhibitors in a cell line-dependent manner. This effect does not correlate with the methylation state of the promoter and therefore must affect genes upstream of CYP24A1. This article is part of a Special Issue 'Vitamin D Workshop'.

Relative Expression of Vitamin D Hydroxylases, CYP27B1 and CYP24A1, and of Cyclooxygenase-2 and Heterogeneity of Human Colorectal Cancer in Relation to Age, Gender, Tumor Location, and Malignancy: Results from Factor and Cluster Analysis.

Previous studies on the significance of vitamin D insufficiency and chronic inflammation in colorectal cancer development clearly indicated that maintenance of cellular homeostasis in the large intestinal epithelium requires balanced interaction of 1,25-(OH)2D3 and prostaglandin cellular signaling networks. The present study addresses the question how colorectal cancer pathogenesis depends on alterations of activities of vitamin D hydroxylases, i.e., CYP27B1-encoded 25-hydroxyvitamin D-1a-hydroxylase and CYP24A1-encoded 25-hydroxyvitamin D-24-hydroxylase, and inflammation-induced cyclooxygenase-2 (COX-2). Data from 105 cancer patients on CYP27B1, VDR, CYP24A1, and COX-2 mRNA expression in relation to tumor grade, anatomical location, gender and age were fit into a multivariate model of exploratory factor analysis. Nearly identical results were obtained by the principal factor and the maximum likelihood method, and these were confirmed by hierarchical cluster analysis: Within the eight mutually dependent variables studied four independent constellations were found that identify different features of colorectal cancer pathogenesis: (i) Escape of COX-2 activity from restraints by the CYP27B1/VDR system can initiate cancer growth anywhere in the colorectum regardless of age and gender; (ii) variations in COX-2 expression are mainly responsible for differences in cancer incidence in relation to tumor location; (iii) advancing age has a strong gender-specific influence on cancer incidence; (iv) progression from well differentiated to undifferentiated cancer is solely associated with a rise in CYP24A1 expression.

Parallel elevation of colonic 1,25-dihydroxyvitamin D3 levels and apoptosis in female mice on a calcium-deficient diet.

BACKGROUND: Epidemiology suggests that nutritional calcium and vitamin D together prevent colorectal tumor progression. 1,25(OH)2D3 is synthesized and degraded in colonocytes and, when bound to its receptor, has antiproliferative activity. MATERIALS AND METHODS: 1,25(OH)2D3 levels have been successfully measured in cell culture, but this is technically difficult in tissues. Double extraction coupled to an enzyme immunoassay was used to determine 1,25(OH)2D3 concentration in colon mucosa. RESULTS: In a mouse model fed low (0.04%) nutritional calcium, expression of the vitamin D catabolizing CYP24A1, of the synthesizing CYP27B1 hydroxylase and of the vitamin D receptor was induced in the right colon only. While CYP24A1 mRNA was raised in both genders, raised CYP27B1 and VDR was found in females only. Levels of 1,25(OH)2D3 were significantly higher in the right colon of females fed 0.04% calcium compared with the control group on 0.9% calcium, and with males fed either diet. Parallel to increased 1,25(OH)2D3, the intrinsic apoptotic pathway was enhanced in the right colon of females only. CONCLUSION: This demonstrates the significance of high nutritional calcium for colonic accumulation of 1,25(OH)2D3 and suggests that female sex hormones may protect against mitotic action of low nutritional calcium by inducing 1,25(OH)2D3 synthesis.

A 24-phenylsulfone analog of vitamin D inhibits 1alpha,25-dihydroxyvitamin D(3) degradation in vitamin D metabolism-competent cells.

The antimitotic, prodifferentiating, and proapoptotic steroid hormone, 1alpha,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)], at supraphysiological levels has potential for tumor therapy. However, epithelial cells from tumor-prone organs such as colon, prostate, and breast express not only the vitamin D receptor, but also vitamin D hydroxylases. In contrast to normal cells, malignant cells have high basal levels of the hydroxylase 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24) and, in addition, have the potential to induce CYP24 in response to 1alpha,25-(OH)(2)D(3). Because 24-hydroxylation by CYP24 would rapidly degrade the steroid hormone in the course of therapy, the enzyme activity in tumor cells should be inhibited. We demonstrate that a 24-phenylsulfone analog of 1alpha,25-(OH)(2)D(3), KRC-24SO(2)Ph-1 (S-4a), rapidly and potently inhibits 24-hydroxylase activity in human tumor cells derived from colon, prostate, and mammary gland. Although enzymatic inhibition is a consequence of direct interaction, S-4a as a vitamin D analog apparently binds to the vitamin D receptor and induces CYP24 mRNA, which, however, is not translated into increased enzymatic activity. 25-Hydroxyvitamin D(3)-1alpha-hydroxylase expression is not affected at all by S-4a. When both 1alpha,25-(OH)(2)D(3) and S-4a are added to the cell culture, transcription of CYP24 is increased, possibly because of an increase in the half-life of the hormone. The colon cell line COGA-13 has very high levels of CYP24 and is, therefore, resistant to the action of vitamin D. Yet, S-4a imparts antimitotic activity to 1alpha,25-(OH)(2)D(3) and may therefore constitute a therapeutic to stimulate the antiproliferative potential of vitamin D-based antitumor activity.

The Vitamin D endocrine system of the gut--its possible role in colorectal cancer prevention.

While Vitamin D insufficiency in the US and European population is rising, epidemiological studies suggest an inverse correlation between low serum levels of 25-hydroxyvitamin D(3) (25-OH-D(3)) and colorectal cancer incidence. The antimitotic, prodifferentiating and proapoptotic active metabolite 1alpha,25-dihydroxyvitamin D(3) (1,25-(OH)(2)-D(3)) is synthesized also by colonocytes, since these possess Vitamin D synthesizing (CYP27B1) and catabolic (CYP24) hydroxylases similar to the kidney. Early during colon tumor progression, expression of CYP27B1 and of the Vitamin D receptor increases, suggesting an autocrine/paracrine growth control in colon tissue as a physiological restriction against tumor progression. However, in human adenocarcinomas expression of the catabolic CYP24 is also enhanced when compared with adjacent normal mucosa. Therefore, to maintain colonic accumulation of 1,25-(OH)(2)-D(3) its catabolism needs to be restricted. Our studies in mice show that low nutritional calcium causes hyperproliferation of colon crypts and significant elevation of CYP24 expression, which can be completely abrogated by soy feeding. We suggest that phytoestrogens in soy, known to be estrogen receptor modulators, are responsible for decreased CYP24 expression. These results and our observation that 17beta-estradiol can elevate CYP27B1 expression in rectal tissue of postmenopausal women, may underlie the observed protective effect of estrogens against colorectal cancer in females.

Clonal differences in expression of 25-hydroxyvitamin D(3)-1alpha-hydroxylase, of 25-hydroxyvitamin D(3)-24-hydroxylase, and of the vitamin D receptor in human colon carcinoma cells: effects of epidermal growth factor and 1alpha,25-dihydroxyvitamin D(3).

Human colon carcinoma cells express 25-hydroxyvitamin D(3)-1alpha-hydroxylase (CYP27B1) and thus produce the vitamin D receptor (VDR) ligand 1alpha,25-dihydroxyvitamin D(3) (1,25-D3), which can be metabolized by 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24). Expression of VDR, CYP27B1, and CYP24 determines the efficacy of the antimitotic action of 1,25-D3 and is distinctly related to the degree of differentiation of cancerous lesions. In the present study we addressed the question of whether the effects of epidermal growth factor (EGF) and of 1,25-D3 on VDR, CYP27B1, and CYP24 gene expression in human colon carcinoma cell lines also depend on the degree of cellular differentiation. We were able to show that slowly dividing, highly differentiated Caco-2/15 cells responded in a dose-dependent manner to both EGF and 1,25-D3 by up-regulation of VDR and CYP27B1 expression, whereas in highly proliferative, less differentiated cell lines, such as Caco-2/AQ and COGA-1A and -1E, negative regulation was observed. CYP24 mRNA was inducible in all clones by 1,25-D3 but not by EGF. From the observed clonal differences in the regulatory effects of EGF and 1,25-D3 on VDR and CYP27B1 gene expression we suggest that VDR-mediated growth inhibition by 1,25-D3 would be efficient only in highly differentiated carcinomas even when under mitogenic stimulation by EGF.