Organomagnesium suppresses inflammation-associated colon carcinogenesis in male Crj: CD-1 mice.

Magnesium (Mg) deficiency increases genomic instability and Mg intake has been reported to be inversely associated with a risk of colorectal cancer (CRC). This study was designed to determine whether organo-Mg in drinking water suppresses inflammation-associated colon carcinogenesis in mice. Male Crj: CD-1 mice were initiated with a single i.p. injection of azoxymethane (AOM, 10mg/kg body weight) and followed by a 1 week exposure to dextran sulfate sodium (DSS, 1.5%, w/v) in drinking water to induce colonic neoplasms. They were then given the drinking water containing 7, 35 or 175 p.p.m. organo-Mg for 13 weeks. The chemopreventive efficacy of organo-Mg was determined 16 weeks after the AOM exposure. Administration with organo-Mg at all doses caused a significant inhibition of CRC development (P < 0.01 and P < 0.001). Especially, the highest dose of organo-Mg significantly suppressed the occurrence of all the colonic pathological lesions (mucosal ulcer, dysplasia, adenoma and adenocarcinoma). Organo-Mg also significantly reduced the number of mitoses/anaphase bridging, as well as proliferation of CRC. Additionally, at week 4, organo-Mg lowered the messenger RNA expression of certain proinflammatory cytokines, such as interleukin-1ß, interleukin-6, interferon-γ and inducible nitric oxide synthase in the lesion-free colorectal mucosa at week 4 but increased the Nrf-2 messenger RNA expression. Our findings that organo-Mg inhibits inflammation-related mouse colon carcinogenesis by modulating the proliferative activities and chromosomal instability of CRC and suppressing colonic inflammation may suggest potential use of organo-Mg for clinical chemoprevention trials of CRC in the inflamed colon.

Generation of a homology model for the human cytochrome P450, CYP24A1, and the testing of putative substrate binding residues by site-directed mutagenesis and enzyme activity studies.

A systematic analysis of conserved H-bonding patterns and tertiary structural motifs from 13 crystal structures was used to create a homology model for the human multicatalytic cytochrome P450, CYP24A1, involved in catabolism of 1alpha,25-dihydroxyvitamin D3. The substrate was docked in the active site and used to identify potential substrate contact residues in the B' helix, B'/C loop, F-helix and the beta-5 hairpin. Seven CYP24A1 mutants were created and studied by mammalian cell transfection and CYP24A1 activity assay. Mutants showed reduced metabolic rates and altered metabolite patterns compared to wild-type. We conclude that: Ile-131 positions substrate via A-ring and cis-triene contacts; Trp-134 and Gly-499 are determinants of substrate access; Leu-148 contacts the substrate side-chain; Met-246 is important in mediating regioselectivity. Our findings validate the new model of CYP24A1, which can now be used to predict structural modifications for rational vitamin D drug design.

Hepatic activation and inactivation of clinically-relevant vitamin D analogs and prodrugs.

Like most pharmaceutical agents, vitamin D analogs are subject to hepatic metabolism by a variety of cytochrome P450 (CYP)-based systems. Metabolism can involve activation as well as inactivation of the vitamin D analog and one of the more successful families includes the 1alpha-hydroxyvitamin D prodrugs (1alpha-OH-D2, 1alpha-OH-D3, 1alpha-OH-D4, 1alpha-OH-D5), that all require a step of activation. Some of these prodrugs are in use or clinical trial because they have a therapeutic advantage over calcitriol. However, the nature of the activation of these molecules is poorly understood, particularly with regard to the CYP isoform involved. Various transfected CYPs and hepatic cell lines combined with tandem LC-MS analysis were used to investigate the metabolism of a spectrum of vitamin D analogs, including 1alpha-OH-Ds and the topical analog, calcipotriol. In the case of the 1alpha-OH-Ds, evidence was found of multiple sites of side-chain hydroxylation consistent with the generation of more than one active form. The potential involvement of CYP27A and other putative 25-hydroxylases in 1alpha-OH-D activation was also shown, as well as the potential for CYP24 activation and inactivation. In the case of calcipotriol, the respective roles of non-vitamin D-related CYPs and CYP24 in the catabolism of this anti-psoriatic drug were dissected out using cell lines with or without CYP24 expression, allowing us to demonstrate the potential contribution of CYP24 to "vitamin D resistance". The implications of hepatic metabolism in the context of other facets thought to play a role in the mechanism of action of anticancer and antiproliferative vitamin D analogs are discussed.

Promise of vitamin D analogues in the treatment of hyperproliferative conditions.

1Alpha,25-dihydroxyvitamin D3 [1alpha,25-(OH)2D3; calcitriol] is best known as a hormone involved in calcium homeostasis but is also a potent antiproliferative agent in many cell types, particularly epithelial cells. 1Alpha,25(OH)2D3 mediates its actions through a classic steroid hormone-like transcriptional mechanism by influencing the expression of hundreds of genes. Effects of 1alpha,25(OH)2D3 have been observed on expression of cell cycle regulators, growth factors and their receptors, apoptotic machinery, metastatic potential, and angiogenesis; all of which have some effect on hyperproliferative conditions. This minireview focuses on the anticancer potential of 1alpha,25(OH)2D3 and its analogues by summarizing the promising data from animal and human trials of 1alpha,25(OH)2D3 and some of the more interesting synthetic vitamin D analogues in the treatment of a variety of different animal cancer models and in human patients with advanced cancer. Optimal administration of vitamin D analogues is only just being achieved with high-dose intermittent administration overcoming bioavailability and hypercalcemia problems and combination therapy with cytotoxic agents (taxols and cisplatins), antiresorptive agents (bisphosphonates), or cytochrome P450 inhibitors being attempted. Although the potential of vitamin D as an antiproliferative drug has been realized in the treatment of psoriasis and in parathyroid cell hyperplasia associated with secondary hyperparathyroidism, the search for an anticancer treatment incorporating a vitamin D analogue remains elusive.

Evidence for the activation of 1alpha-hydroxyvitamin D2 by 25-hydroxyvitamin D-24-hydroxylase: delineation of pathways involving 1alpha,24-dihydroxyvitamin D2 and 1alpha,25-dihydroxyvitamin D2.

While current dogma argues that vitamin D prodrugs require side-chain activation by liver enzymes, recent data suggest that hydroxylation may also occur extrahepatically. We used keratinocytes and recombinant human enzyme to test if the 25-hydroxyvitamin D-24-hydroxylase (CYP24A1) is capable of target cell activation and inactivation of a model prodrug, 1alpha-hydroxyvitamin D2 (1alpha(OH)D2) in vitro. Mammalian cells stably transfected with CYP24A1 (V79-CYP24A1) converted 1alpha(OH)D2 to a series of metabolites similar to those observed in murine keratinocytes and the human cell line HPK1A-ras, confirming the central role of CYP24A1 in metabolism. Products of 1alpha(OH)D2 included the active metabolites 1alpha,24-dihydroxyvitamin D2 (1alpha,24(OH)2D2) and 1alpha,25-dihydroxyvitamin D2 (1alpha,25(OH)2D2); the formation of both indicating the existence of distinct activation pathways. A novel water-soluble metabolite, identified as 26-carboxy-1alpha,24(OH)2D2, was the presumed terminal degradation product of 1alpha(OH)D2 synthesized by CYP24A1 via successive 24-hydroxylation, 26-hydroxylation and further oxidation at C-26. This acid was absent in keratinocytes from Cyp24a1 null mice. Slower clearance rates of 1alpha(OH)D2 and 1alpha,24(OH)2D2 relative to 1alpha,25(OH)2D2 and 1alpha,25(OH)2D3 were noted, arguing for a role of 24-hydroxylated metabolites in the altered biological activity profile of 1alpha(OH)D2. Our findings suggest that CYP24A1 can activate and inactivate vitamin D prodrugs in skin and other target cells in vitro, offering the potential for treatment of hyperproliferative disorders such as psoriasis by topical administration of these prodrugs.

Altered pharmacokinetics of 1alpha,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 in the blood and tissues of the 25-hydroxyvitamin D-24-hydroxylase (Cyp24a1) null mouse.

The 25-hydroxyvitamin D-24-hydroxylase (CYP24A1) plays an important role in regulating concentrations of both the precursor 25-hydroxyvitamin D3 [25(OH)D3] and the hormone 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)(2)D3]. Previous studies suggest that Cyp24a1-null mice cannot clear exogenous 1alpha,25(OH)2D3 efficiently. Here, we examined the metabolic clearance in Cyp24a1-null mice in vivo and in vitro using a physiological dose of [1beta-3H]1alpha,25(OH)2D3 or [26,27-methyl-3H]25(OH)D3. Cyp24a1-null mice showed difficulty in eliminating [1beta-3H]1alpha,25(OH)2D3 from the bloodstream and tissues over a 96-h time course, whereas heterozygotic mice eliminated the hormone within 6-12 h, although there was clearance of labeled hormone into water-soluble products involving liver in both genotypes. RT-PCR showed that Cyp24a1-null mice have decreased expression of 25-hydroxyvitamin D-1alpha-hydroxylase that must play a role in their survival. After the administration of [26,27-methyl-3H]25(OH)D3, Cyp24a1-null mice showed higher [26,27-methyl-3H]25(OH)D3 levels and no [26,27-methyl-3H]24,25(OH)2D3 formation, whereas heterozygotic mice showed significant [26,27-methyl-3H]24,25(OH)2D3 production. Based upon in vitro experiments, keratinocytes from Cyp24a1-null mice fail to synthesize [1beta-3H]calcitroic acid from [1beta-3H]1alpha,25(OH2D3 or [26,27-methyl-3H]24,25(OH)2D3 from [26,27-methyl-3H]25(OH)D3 as do control mice, confirming the target cell catabolic role of CYP24A1 in these processes. Finally, the role of vitamin D receptor (VDR) in the vitamin D catabolic cascade was examined using VDR-null mice. Keratinocytes from VDR-null mice failed to metabolize [1beta-3H]1alpha,25(OH)2D3 confirming the importance of vitamin D-inducible, VDR-mediated, C24 oxidation pathway in target cells. These results suggest that the absence of CYP24A1 or VDR retards catabolism of 1alpha,25(OH)2D3 and 25(OH)D3, reinforcing the physiological importance of CYP24A1 in vitamin D homeostasis.

Insights into Vitamin D metabolism using cyp24 over-expression and knockout systems in conjunction with liquid chromatography/mass spectrometry (LC/MS).

The development of novel gene expression systems for cytochrome P450s (CYPs) together with a revolution in analytical mass spectrometry with the emergence of liquid chromatography/mass spectrometry (LC/MS) has opened the door to answering some long-standing questions in Vitamin D metabolism. Our studies focused on: (1) elucidating the role of CYP24 in 25-OH-D3 and 1alpha,25-(OH)2D3 metabolism; (2) exploring how DBP influences this process; (3) measuring 25-OH-D3 metabolism in CYP24-knockout (CYP24-XO) cells and; (4) comparing 1alpha-OH-D2 metabolism in the CYP24-XO mouse in vivo and in vitro. Methodology employed CYP24 over-expression and knockout systems in conjunction with state-of-the-art analytical LC/MS, diode array, and radioisotopic detection methods. We found that CYP24 metabolizes 25-OH-D3 and 1alpha,25-(OH)2D3 at similar rates in vitro, but that for 25-OH-D3 but not 1alpha,25-(OH)2D3, this rate is strongly influenced by the concentration of DBP. Unlike their wild type littermates, the administration of 25-OH-D3 to CYP24-XO mice results in no measurable 24,25-(OH)2D3 production. When neonatal murine keratinocytes are prepared from wild type and CYP24-XO mice there was no measurable production of 24,25-(OH)2D3 or 1alpha,24,25-(OH)2D3 in CYP24-XO mice. Similar experiments using the same wild type and CYP24-XO animals and cells and [3H] 1alpha-OH-D2 resulted in the apparent paradox that the Vitamin D prodrug was 25-hydroxylated in vivo but 24-hydroxylated in vitro.

Importance of cytochrome P450-mediated metabolism in the mechanism of action of vitamin D analogs.

The elucidation of the metabolic pathway for vitamin D, including the delineation of the specific cytochrome P450s (CYPs) involved in activation and catabolism, has emphasized the overall importance of metabolic considerations in vitamin D analog design. This short review attempts to summarize recent findings with isolated CYPs and animal models in which CYPs are genetically manipulated to draw attention to structural features of vitamin D analogs that make them more or less resistant to metabolic enzymes. We conclude by placing metabolic considerations in the context of the other important aspects of vitamin D analogs.

Vitamin D analogs--drug design based on proteins involved in vitamin D signal transduction.

Vitamin D analogs have proven to be very valuable tools for the treatment of calcium-related diseases and certain hyperproliferative conditions such as renal osteodystrophy, psoriasis and cancer. In general, vitamin D analogs exploit the enzymic and receptor machinery of the 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) signal transduction pathway. Key proteins in this cascade include the vitamin D receptor (VDR), the vitamin D-binding protein (DBP) and three cytochrome P450s (CYP27A, CYP27B and CYP24) which effect the synthesis and breakdown of the natural hormone, 1alpha,25(OH)(2)D(3). Analogs have been designed which reduce or enhance the importance of each of these proteins in the signal transduction pathway. Vitamin D prodrugs require one or more steps of activation and overcome congenital or acquired blocks in the 1alpha-hydroxylation step. By far the biggest class of vitamin D analogs are the VDR agonists which directly mimic 1alpha,25(OH)(2)D(3) and trigger protein conformational changes in the receptor which lead to changes in the transcriptional machinery at vitamin D-responsive genes. Other emerging classes of molecules include the VDR antagonists and CYP24 inhibitors which target different events in the cascade. This review assesses the relative importance of each of the proteins of the vitamin D cascade, evaluates the success of these modifications in tailoring drugs in all classes for selected disease states and contemplates future directions for the field.