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1.
Front Pharmacol ; 10: 769, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31354485

RESUMO

Curcumin (CCM) has many potential uses in anticancer chemotherapy, but its low water solubility poses a major problem, preventing its translation into clinical use. TPGS is a water-soluble derivative of vitamin E that acts as a surfactant with the ability to form micellar nanoparticles in water. More importantly, TPGS acts as a potent antioxidant that can neutralize intracellular reactive oxygen species (ROS). In this study, we solubilized CCM with TPGS using thin-film rehydration to prepare aqueous formulations containing CCM at clinically relevant concentrations. We found that the minimal TPGS:CCM ratio for producing nanoparticles was 5:1 (w/w): at or above this ratio, stable nanoparticles formed with an average particle diameter of 12 nm. CCM was released from TPGS/CCM micelles in simulated colonic and gastric fluids. These TPGS/CCM nanoparticles were shown to decrease intracellular ROS levels and apoptosis and inhibited migration of HT-29 human colon cancer cells more potently than free CCM. Pharmacokinetic analysis showed TPGS/CCM to be more bioavailable than free CCM after oral administration to rats. Our results suggest that TPGS/CCM may increase therapeutic efficacy of CCM against colon cancer and merits further investigation in a clinical setting.

2.
Redox Biol ; 24: 101206, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31039479

RESUMO

We tested whether novel CYP11A1-derived vitamin D3- and lumisterol-hydroxyderivatives, including 1,25(OH)2D3, 20(OH)D3, 1,20(OH)2D3, 20,23(OH)2D3, 1,20,23(OH)3D3, lumisterol, 20(OH)L3, 22(OH)L3, 20,22(OH)2L3, and 24(OH)L3, can protect against UVB-induced damage in human epidermal keratinocytes. Cells were treated with above compounds for 24 h, then subjected to UVB irradiation at UVB doses of 25, 50, 75, or 200 mJ/cm2, and then examined for oxidant formation, proliferation, DNA damage, and the expression of genes at the mRNA and protein levels. Oxidant formation and proliferation were determined by the DCFA-DA and MTS assays, respectively. DNA damage was assessed using the comet assay. Expression of antioxidative genes was evaluated by real-time RT-PCR analysis. Nuclear expression of CPD, phospho-p53, and Nrf2 as well as its target proteins including HO-1, CAT, and MnSOD, were assayed by immunofluorescence and western blotting. Treatment of cells with the above compounds at concentrations of 1 or 100 nM showed a dose-dependent reduction in oxidant formation. At 100 nM they inhibited the proliferation of cultured keratinocytes. When keratinocytes were irradiated with 50-200 mJ/cm2 of UVB they also protected against DNA damage, and/or induced DNA repair by enhancing the repair of 6-4PP and attenuating CPD levels and the tail moment of comets. Treatment with test compounds increased expression of Nrf2-target genes involved in the antioxidant response including GR, HO-1, CAT, SOD1, and SOD2, with increased protein expression for HO-1, CAT, and MnSOD. The treatment also stimulated the phosphorylation of p53 at Ser-15, increased its concentration in the nucleus and enhanced Nrf2 translocation into the nucleus. In conclusion, pretreatment of keratinocytes with 1,25(OH)2D3 or CYP11A1-derived vitamin D3- or lumisterol hydroxy-derivatives, protected them against UVB-induced damage via activation of the Nrf2-dependent antioxidant response and p53-phosphorylation, as well as by the induction of the DNA repair system. Thus, the new vitamin D3 and lumisterol hydroxy-derivatives represent promising anti-photodamaging agents.

3.
Arch Biochem Biophys ; 666: 16-21, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30926433

RESUMO

25-Hydroxyvitamin D3 3-epimerase catalyzes the 3ß â†’ 3α epimerization of 25-hydroxyvitamin D3 (25(OH)D3) producing 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3). 3-Epi-25(OH)D3 is one of the most abundant forms of vitamin D present in the serum. It can be converted to 3-epi-1α,25-dihydroxyvitamin D3 by CYP27B1 which generally displays lower biological activity than 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). The 25(OH)D3 3-epimerase has been poorly characterized to date and the gene encoding it has not been identified. The 3-epimerase has been reported to be present in the microsomal fraction of cells, including liver cells, and to use NADPH as cofactor. It can also act on 1,25(OH)2D3 and 24,25(OH)2D3 forming the 3α-epimers. In this study we have characterized the activity of the 25(OH)D3 3-epimerase in rat and human liver microsomes, using 25(OH)D3 as substrate and HPLC to analyze product formation. For both rat and human liver microsomes the preferred cofactor was NADH, with the rat enzyme displaying a 6-fold greater catalytic efficiency (Vmax/Km) for NADH over that for NADPH. No activity was observed with oxidized cofactor, either NAD+ or NADP+. This was unexpected since the initial step in the epimerization, predicted to be the oxidation of the 3ß-OH to a ketone, would require oxidized cofactor. The rat 3-epimerase in microsomes gave a Km for 25(OH)D3 of 14 µM. The reverse reaction, conversion of 3-epi-25(OH)D3 to 25(OH)D3, was catalyzed by both rat and human liver microsomes but at lower rates than the forward reaction. In conclusion, both rat and human 25-hydroxyvitamin D3 3-epimerase catalyze the reversible interconversion of 25(OH)D3 and 3-epi-25(OH)D3, and use NADH as the preferred cofactor. The lack of requirement for exogenous NAD+ suggests that the enzyme has a tightly bound NAD+ in its active site that is released only upon its reduction.


Assuntos
Calcifediol/metabolismo , Microssomos Hepáticos/enzimologia , Racemases e Epimerases/metabolismo , Animais , Catálise , Feminino , Humanos , Cinética , Masculino , NAD/metabolismo , NADP/metabolismo , Ratos , Ratos Wistar
4.
Sci Rep ; 7(1): 10193, 2017 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-28860545

RESUMO

1α,20S-Dihydroxyvitamin D3 [1,20S(OH)2D3], a natural and bioactive vitamin D3 metabolite, was chemically synthesized for the first time. X-ray crystallography analysis of intermediate 15 confirmed its 1α-OH configuration. 1,20S(OH)2D3 interacts with the vitamin D receptor (VDR), with similar potency to its native ligand, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] as illustrated by its ability to stimulate translocation of the VDR to the nucleus, stimulate VDRE-reporter activity, regulate VDR downstream genes (VDR, CYP24A1, TRPV6 and CYP27B1), and inhibit the production of inflammatory markers (IFNγ and IL1ß). However, their co-crystal structures revealed differential molecular interactions of the 20S-OH moiety and the 25-OH moiety to the VDR, which may explain some differences in their biological activities. Furthermore, this study provides a synthetic route for the synthesis of 1,20S(OH)2D3 using the intermediate 1α,3ß-diacetoxypregn-5-en-20-one (3), and provides a molecular and biological basis for the development of 1,20S(OH)2D3 and its analogs as potential therapeutic agents.


Assuntos
Calcifediol/análogos & derivados , Calcifediol/farmacologia , Receptores de Calcitriol/química , Receptores de Calcitriol/metabolismo , Animais , Células CACO-2 , Calcifediol/química , Linhagem Celular , Núcleo Celular/metabolismo , Cristalografia por Raios X , Humanos , Células Jurkat , Modelos Moleculares , Transporte Proteico/efeitos dos fármacos
5.
J Steroid Biochem Mol Biol ; 173: 42-56, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-27693422

RESUMO

The classical pathway of vitamin D activation follows the sequence D3→25(OH)D3→1,25(OH)2D3 with the final product acting on the receptor for vitamin D (VDR). An alternative pathway can be started by the action of CYP11A1 on the side chain of D3, primarily producing 20(OH)D3, 22(OH)D3, 20,23(OH)2D3, 20,22(OH)2D3 and 17,20,23(OH)3D3. Some of these metabolites are hydroxylated by CYP27B1 at C1α, by CYP24A1 at C24 and C25, and by CYP27A1 at C25 and C26. The products of these pathways are biologically active. In the epidermis and/or serum or adrenals we detected 20(OH)D3, 22(OH)D3, 20,22(OH)2D3, 20,23(OH)2D3, 17,20,23(OH)3D3, 1,20(OH)2D3, 1,20,23(OH)3D3, 1,20,22(OH)3D3, 20,24(OH)2D3, 1,20,24(OH)3D3, 20,25(OH)2D3, 1,20,25(OH)3D3, 20,26(OH)2D3 and 1,20,26(OH)3D3. 20(OH)D3 and 20,23(OH)2D3 are non-calcemic, while the addition of an OH at C1α confers some calcemic activity. Molecular modeling and functional assays show that the major products of the pathway can act as "biased" agonists for the VDR with high docking scores to the ligand binding domain (LBD), but lower than that of 1,25(OH)2D3. Importantly, cell based functional receptor studies and molecular modeling have identified the novel secosteroids as inverse agonists of both RORα and RORγ receptors. Specifically, they have high docking scores using crystal structures of RORα and RORγ LBDs. Furthermore, 20(OH)D3 and 20,23(OH)2D3 have been tested in a cell model that expresses a Tet-on RORα or RORγ vector and a RORE-LUC reporter (ROR-responsive element), and in a mammalian 2-hybrid model that test interactions between an LBD-interacting LXXLL-peptide and the LBD of RORα/γ. These assays demonstrated that the novel secosteroids have ROR-antagonist activities that were further confirmed by the inhibition of IL17 promoter activity in cells overexpressing RORα/γ. In conclusion, endogenously produced novel D3 hydroxy-derivatives can act both as "biased" agonists of the VDR and/or inverse agonists of RORα/γ. We suggest that the identification of large number of endogenously produced alternative hydroxy-metabolites of D3 that are biologically active, and of possible alternative receptors, may offer an explanation for the pleiotropic and diverse activities of vitamin D, previously assigned solely to 1,25(OH)2D3 and VDR.


Assuntos
Hidroxicolecalciferóis/metabolismo , Hidroxicolecalciferóis/farmacologia , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Receptores de Calcitriol/metabolismo , Vitaminas/metabolismo , Vitaminas/farmacologia , Animais , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Humanos , Modelos Moleculares , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Receptores de Calcitriol/agonistas
6.
Sci Rep ; 5: 14875, 2015 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-26445902

RESUMO

To investigate whether novel pathways of vitamin D3 (D3) and 7-dehydrocholesterol (7DHC) metabolism initiated by CYP11A1 and previously characterized in vitro, occur in vivo, we analyzed samples of human serum and epidermis, and pig adrenals for the presence of intermediates and products of these pathways. We extracted human epidermis from 13 individuals and sera from 13 individuals and analyzed them by LC/qTOF-MS alongside the corresponding standards. Pig adrenal glands were also analyzed for these steroids and secosteroids. Epidermal, serum and adrenal samples showed the presence of D3 hydroxy-derivatives corresponding to 20(OH)D3, 22(OH)D3, 25(OH)D3, 1,25(OH)2D3, 20,22(OH)2D3, 20,23(OH)2D3, 20,24(OH)2D3, 20,25(OH)2D3, 20,26(OH)2D3, 1,20,23(OH)3D3 and 17,20,23(OH)3D3, plus 1,20(OH)2D3 which was detectable only in the epidermis. Serum concentrations of 20(OH)D3 and 22(OH)D3 were only 30- and 15-fold lower than 25(OH)D3, respectively, and at levels above those required for biological activity as measured in vitro. We also detected 1,20,24(OH)3D3, 1,20,25(OH)3D3 and 1,20,26(OH)3D3 in the adrenals. Products of CYP11A1 action on 7DHC, namely 22(OH)7DHC, 20,22(OH)27DHC and 7-dehydropregnenolone were also detected in serum, epidermis and the adrenal. Thus, we have detected novel CYP11A1-derived secosteroids in the skin, serum and adrenal gland and based on their concentrations and biological activity suggest that they act as hormones in vivo.


Assuntos
Glândulas Suprarrenais/química , Colecalciferol/isolamento & purificação , Enzima de Clivagem da Cadeia Lateral do Colesterol/isolamento & purificação , Desidrocolesteróis/isolamento & purificação , Epiderme/química , Secoesteroides/isolamento & purificação , Glândulas Suprarrenais/metabolismo , Animais , Colecalciferol/sangue , Colecalciferol/metabolismo , Enzima de Clivagem da Cadeia Lateral do Colesterol/sangue , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Desidrocolesteróis/sangue , Desidrocolesteróis/metabolismo , Epiderme/metabolismo , Humanos , Secoesteroides/sangue , Secoesteroides/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Suínos
7.
FEBS J ; 281(14): 3280-96, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24893882

RESUMO

CYP24A1 is the multicatalytic cytochrome P450 responsible for the catabolism of vitamin D via the C23- and C24-oxidation pathways. We successfully expressed the labile human enzyme in Escherichia coli and partially purified it in an active state that permitted detailed characterization of its metabolism of 1,25-dihydroxyvitamin D3 [1,25(OH)2 D3] and the intermediates of the C24-oxidation pathway in a phospholipid-vesicle reconstituted system. The C24-oxidation pathway intermediates, 1,24,25-trihydroxyvitamin D3, 24-oxo-1,25-dihydroxyvitamin D3, 24-oxo-1,23,25-trihydroxyvitamin D3 and tetranor-1,23-dihydroxyvitamin D3, were enzymatically produced from 1,25(OH)2 D3 using rat CYP24A1. Both 1,25(OH)2 D3 and 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3 were found to partition strongly into the phospholipid bilayer when in aqueous medium. Changes to the phospholipid concentration did not affect the kinetic parameters for the metabolism of 1,25(OH)2 D3 by CYP24A1, indicating that it is the concentration of substrates in the membrane phase (mol substrate·mol phospholipid(-1) ) that determines their rate of metabolism. CYP24A1 exhibited Km values for the different C24-intermediates ranging from 0.34 to 15 mmol·mol phospholipid(-1) , with 24-oxo-1,23,25-trihydroxyvitamin D3 [24-oxo-1,23,25(OH)3 D3] displaying the lowest and 1,24,25-trihydroxyvitamin D3 [1,24,25(OH)3 D3] displaying the highest. The kcat values varied by up to 3.8-fold, with 1,24,25(OH)3 D3 displaying the highest kcat (34 min(-1) ) and 24-oxo-1,23,25(OH)3 D3 the lowest. The data show that the cleavage of the side chain of 24-oxo-1,23,25(OH)3 D3 occurs with the highest catalytic efficiency (kcat /Km ) and produces 1-hydroxy-23-oxo-24,25,26,27-tetranorvitamin D3 and not 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3, as the primary product. These kinetic analyses also show that intermediates of the C24-oxidation pathway effectively compete with precursor substrates for binding to the active site of the enzyme, which manifests as an accumulation of intermediates, indicating that they dissociate after each catalytic step.


Assuntos
Calcitriol/metabolismo , Esteroide Hidroxilases/metabolismo , Adrenodoxina/metabolismo , Animais , Calcitriol/análogos & derivados , Humanos , Hidroxicolecalciferóis/metabolismo , Cinética , Membranas Mitocondriais/metabolismo , Ratos , Esteroide Hidroxilases/isolamento & purificação , Vitamina D3 24-Hidroxilase
8.
Anticancer Res ; 34(5): 2153-63, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24778017

RESUMO

AIM: To discover novel [20(OH)D3] analogs as antiproliferative therapeutics. MATERIALS AND METHODS: We studied in vitro liver microsome stability, in vivo toxicity using mice, vitamin D receptor (VDR) translocation, in vitro antiproliferative effect, CYP enzyme metabolism. RESULTS: 20S- and 20R(OH)D3 had reasonable half-lives of 50 min and 30 min (average) respectively in liver microsomes. They were non-hypercalcemic at a high dose of 60 µg/kg. Three new 20(OH)D3 analogs were designed, synthesized and tested. They showed higher or comparable potency for inhibition of proliferation of normal keratinocytes and in the induction of VDR translocation from cytoplasm to nucleus, compared to 1,25(OH)2D3. These new analogs demonstrated different degrees of metabolism through a range of vitamin D-metabolizing CYP enzymes. CONCLUSION: Their lack of calcemic toxicity at high doses and their high biological activity suggest that this novel 20(OH)D3 scaffold may represent a promising platform for further development of therapeutically-useful agents.


Assuntos
Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Microssomos Hepáticos/efeitos dos fármacos , Vitamina D/análogos & derivados , Animais , Meia-Vida , Camundongos , Receptores de Calcitriol/efeitos dos fármacos , Receptores de Calcitriol/metabolismo , Vitamina D/farmacologia
9.
Mol Cell Endocrinol ; 383(1-2): 181-92, 2014 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-24382416

RESUMO

We investigated the metabolism of vitamin D2 to hydroxyvitamin D2 metabolites ((OH)D2) by human placentas ex-utero, adrenal glands ex-vivo and cultured human epidermal keratinocytes and colonic Caco-2 cells, and identified 20(OH)D2, 17,20(OH)2D2, 1,20(OH)2D2, 25(OH)D2 and 1,25(OH)2D2 as products. Inhibition of product formation by 22R-hydroxycholesterol indicated involvement of CYP11A1 in 20- and 17-hydroxylation of vitamin D2, while use of ketoconazole indicated involvement of CYP27B1 in 1α-hydroxylation of products. Studies with purified human CYP11A1 confirmed the ability of this enzyme to convert vitamin D2 to 20(OH)D2 and 17,20(OH)2D2. In placentas and Caco-2 cells, production of 20(OH)D2 was higher than 25(OH)D2 while in human keratinocytes the production of 20(OH)D2 and 25(OH)D2 were comparable. HaCaT keratinocytes showed high accumulation of 1,20(OH)2D2 relative to 20(OH)D2 indicating substantial CYP27B1 activity. This is the first in vivo evidence for a novel pathway of vitamin D2 metabolism initiated by CYP11A1 and modified by CYP27B1, with the product profile showing tissue- and cell-type specificity.


Assuntos
Glândulas Suprarrenais/metabolismo , Epiderme/metabolismo , Ergocalciferóis/metabolismo , Queratinócitos/metabolismo , Placenta/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Glândulas Suprarrenais/citologia , Glândulas Suprarrenais/efeitos dos fármacos , Animais , Células CACO-2 , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Células Epidérmicas , Epiderme/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica , Humanos , Hidroxicolesteróis/farmacologia , Hidroxilação , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Cetoconazol/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Especificidade de Órgãos , Placenta/citologia , Placenta/efeitos dos fármacos , Gravidez , Ratos , Ratos Wistar , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Técnicas de Cultura de Tecidos
10.
Drug Metab Dispos ; 41(5): 1112-24, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23454830

RESUMO

CYP11A1 can hydroxylate vitamin D3 at carbons 17, 20, 22, and 23, producing a range of secosteroids which are biologically active with respect to their ability to inhibit proliferation and stimulate differentiation of various cell types, including cancer cells. As 1α-hydroxylation of the primary metabolite of CYP11A1 action, 20S-hydroxyvitamin D3 [20(OH)D3], greatly influences its properties, we examined the ability of both human and mouse CYP27B1 to 1α-hydroxylate six secosteroids generated by CYP11A1. Based on their kcat/Km values, all CYP11A1-derived metabolites are poor substrates for CYP27B1 from both species compared with 25-hydroxyvitamin D3. No hydroxylation of metabolites with a 17α-hydroxyl group was observed. 17α,20-Dihydroxyvitamin D3 acted as an inhibitor on human CYP27B1 but not the mouse enzyme. We also tested CYP27B1 activity on 20,24-, 20,25-, and 20,26-dihydroxyvitamin D3, which are products of CYP24A1 or CYP27A1 activity on 20(OH)D3. All three compounds were metabolized with higher catalytic efficiency (kcat/Km) by both mouse and human CYP27B1 than 25-hydroxyvitamin D3. CYP27B1 action on these new dihydroxy derivatives was confirmed to be 1α-hydroxylation by mass spectrometry and nuclear magnetic resonance analyses. Both 1,20,25- and 1,20,26- trihydroxyvitamin D3 were tested for their ability to inhibit melanoma (SKMEL-188) colony formation, and were significantly more active than 20(OH)D3. This study shows that CYP11A1-derived secosteroids are 1α-hydroxylated by both human and mouse CYP27B1 with low catalytic efficiency, and that the presence of a 17α-hydroxyl group completely blocks 1α-hydroxylation. In contrast, the secondary metabolites produced by subsequent hydroxylation of 20(OH)D3 at C24, C25, or C26 are very good substrates for CYP27B1.


Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Colecalciferol/metabolismo , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/antagonistas & inibidores , Animais , Colecalciferol/análogos & derivados , Inibidores Enzimáticos/farmacologia , Humanos , Hidroxilação , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Camundongos
11.
Biochem Pharmacol ; 84(12): 1696-704, 2012 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-23041230

RESUMO

20-Hydroxyvitamin D(3) (20(OH)D(3)), the major product of CYP11A1 action on vitamin D(3), is biologically active and is produced in vivo. As well as potentially having important physiological actions, it is of interest as a therapeutic agent due to its lack of calcemic activity. In the current study we have examined the ability of CYP24A1, the enzyme that inactivates 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), to metabolize 20(OH)D(3). Rat CYP24A1 was expressed in Escherichia coli, purified by Ni-affinity chromatography and assayed with substrates incorporated into phospholipid vesicles which served as a model of the inner mitochondrial membrane. In this system CYP24A1 metabolized 1,25(OH)(2)D(3) with a catalytic efficiency 1.4-fold higher than that seen for 25-hydroxyvitamin D(3) (25(OH)D(3)). CYP24A1 hydroxylated 20(OH)D(3) to several dihydroxy-derivatives with the major two identified by NMR as 20,24-dihydroxyvitamin D(3) (20,24(OH)(2)D(3)) and 20,25-dihydroxyvitamin D(3) (20,25(OH)(2)D(3)). The catalytic efficiency of CYP24A1 for 20(OH)D(3) metabolism was more than 10-fold lower than for either 25(OH)D(3) or 1,25(OH)(2)D(3) and no secondary metabolites were produced. The two major products, 20,24(OH)(2)D(3) and 20,25(OH)(2)D(3), caused significantly greater inhibition of colony formation by SKMEL-188 melanoma cells than either 1,25(OH)(2)D(3) or the parent 20(OH)D(3), showing that CYP24A1 plays an activating, rather than an inactivating role on 20(OH)D(3).


Assuntos
Calcifediol/análogos & derivados , Esteroide Hidroxilases/metabolismo , Animais , Calcifediol/metabolismo , Hidroxilação , Espectroscopia de Ressonância Magnética , Ratos , Espectrometria de Massas por Ionização por Electrospray , Vitamina D3 24-Hidroxilase
12.
FEBS J ; 279(19): 3749-3761, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22862690

RESUMO

CYP27B1 is a mitochondrial cytochrome P450 that catalyses the hydroxylation of 25-hydroxyvitamin D3 at the C1α-position to give the hormonally active form of vitamin D3, 1α,25-dihydroxyvitamin D3. We successfully expressed human CYP27B1 in Escherichia coli and partially purified this labile enzyme and carried out a detailed characterization of its kinetic properties in a reconstituted membrane environment. The phospholipid concentration did not affect the enzyme activity in the vesicle-reconstituted system, although it was influenced by the phospholipid composition, with the addition of cardiolipin lowering the K(m) for 25-hydroxyvitamin D3. These data are consistent with the enzyme accessing substrate from the hydrophobic domain of the vesicle membrane. Cardiolipin also caused the appearance of inhibition of activity at high substrate concentrations. This substrate inhibition fitted a model for one catalytic and two inhibitory sites on the enzyme for the binding of substrate. The K(m) for human adrenodoxin was observed to decrease with decreasing substrate concentration, with the catalytic efficiency (k(cat) /K(m) ) being largely independent of adrenodoxin concentration. Human CYP27B1 was also active on 25-hydroxyvitamin D(2) and on intermediates of the CYP24A1-mediated inactivation pathway, 24R,25-dihydroxyvitamin D3, 24-oxo-25-hydroxyvitamin D3 and 24-oxo-23,25-dihydroxyvitamin D3, with all these substrates showing comparable k(cat) values of 50-71 min(-1) , similar to 25-hydroxyvitamin D3. The latter two substrates gave higher K(m) values than that for 25-hydroxy-vitamin D3. The present study shows that human CYP27B1 can be partially purified in an active form with the enzyme displaying high activity towards a range of substrates in a phospholipid vesicle-reconstituted system that mimics the inner-mitochondrial membrane.


Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Adrenodoxina/farmacologia , Escherichia coli/enzimologia , Fosfolipídeos/metabolismo , 24,25-Di-Hidroxivitamina D 3/metabolismo , Western Blotting , Calcifediol/metabolismo , Cardiolipinas/metabolismo , Ergocalciferóis/metabolismo , Humanos , Cinética , Especificidade por Substrato
13.
FASEB J ; 26(9): 3901-15, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22683847

RESUMO

We define previously unrecognized in vivo pathways of vitamin D(3) (D3) metabolism generating novel D3-hydroxyderivatives different from 25-hydroxyvitamin D(3) [25(OH)D3] and 1,25(OH)(2)D3. Their novel products include 20-hydroxyvitamin D(3) [20(OH)D3], 22(OH)D3, 20,23(OH)(2)D3, 20,22(OH)(2)D3, 1,20(OH)(2)D3, 1,20,23(OH)(3)D3, and 17,20,23(OH)(3)D3 and were produced by placenta, adrenal glands, and epidermal keratinocytes. We detected the predominant metabolite [20(OH)D3] in human serum with a relative concentration ∼20 times lower than 25(OH)D3. Use of inhibitors and studies performed with isolated mitochondria and purified enzymes demonstrated involvement of the steroidogenic enzyme cytochrome P450scc (CYP11A1) as well as CYP27B1 (1α-hydroxylase). In placenta and adrenal glands with high CYP11A1 expression, the predominant pathway was D3 → 20(OH)D3 → 20,23(OH)(2)D3 → 17,20,23(OH)(3)D3 with further 1α-hydroxylation, and minor pathways were D3 → 25(OH)D3 → 1,25(OH)(2)D3 and D3 → 22(OH)D3 → 20,22(OH)(2)D3. In epidermal keratinocytes, we observed higher proportions of 22(OH)D3 and 20,22(OH)(2)D3. We also detected endogenous production of 20(OH)D3, 22(OH) D3, 20,23(OH)(2)D3, 20,22(OH)(2)D3, and 17,20,23(OH)(3)D3 by immortalized human keratinocytes. Thus, we provide in vivo evidence for novel pathways of D3 metabolism initiated by CYP11A1, with the product profile showing organ/cell type specificity and being modified by CYP27B1 activity. These findings define the pathway intermediates as natural products/endogenous bioregulators and break the current dogma that vitamin D is solely activated through the sequence D3 → 25(OH)D3 → 1,25(OH)(2)D3.


Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Colecalciferol/metabolismo , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Animais , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Queratinócitos/enzimologia , Queratinócitos/metabolismo , Reação em Cadeia da Polimerase , Ratos , Ratos Wistar , Espectrometria de Massas em Tandem
14.
Mol Cell Endocrinol ; 361(1-2): 143-52, 2012 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-22546549

RESUMO

To define the interaction of novel secosteroids produced by the action of cytochrome P450scc with vitamin D receptor (VDR), we used a human melanoma line overexpressing VDR fused with enhanced green fluorescent protein (EGFP) and tested the ligand induced translocation of VDR from the cytoplasm to the nucleus. Hydroxyderivatives of vitamin D(3) with a full length (D(3)) side chain and hydroxy-secosteroids with a shortened side chain (pD) stimulated VDR translocation and inhibited proliferation, however, with different potencies. In general the D(3) were more potent than pD analogues. Molecular modeling of the binding of the secosteroids to the VDR genomic binding pocket (G-pocket) correlated well with the experimental data for VDR translocation. In contrast, docking scores for the non-genomic binding site of the VDR were poor. In conclusion, both the length of the side chain and the number and position of hydroxyl groups affect the activation of VDR by novel secosteroids.


Assuntos
Simulação por Computador , Melanoma/metabolismo , Modelos Moleculares , Receptores de Calcitriol/metabolismo , Secoesteroides/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Colecalciferol/farmacologia , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Células Epidérmicas , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Melanoma/patologia , Simulação de Acoplamento Molecular , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Secoesteroides/química
15.
J Med Chem ; 55(7): 3573-7, 2012 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-22404326

RESUMO

The non-naturally occurring 20R epimer of 20-hydroxyvitamin D3 is synthesized based on chemical design and hypothesis. The 20R isomer is separated by semipreparative HPLC, and its structure is characterized. A comparison of 20R isomer to its 20S counterpart in biological evaluation demonstrates that they have different behaviors in antiproliferative and metabolic studies.


Assuntos
Calcifediol/análogos & derivados , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/química , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Calcifediol/síntese química , Calcifediol/química , Calcifediol/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Enzima de Clivagem da Cadeia Lateral do Colesterol/química , Desenho de Drogas , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Estereoisomerismo , Relação Estrutura-Atividade
16.
Am J Physiol Cell Physiol ; 300(3): C526-41, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21160030

RESUMO

20-hydroxyvitamin D(2) [20(OH)D(2)] inhibits DNA synthesis in epidermal keratinocytes, melanocytes, and melanoma cells in a dose- and time-dependent manner. This inhibition is dependent on cell type, with keratinocytes and melanoma cells being more sensitive than normal melanocytes. The antiproliferative activity of 20(OH)D(2) is similar to that of 1,25(OH)(2)D(3) and of newly synthesized 1,20(OH)(2)D(2) but significantly higher than that of 25(OH)D(3). 20(OH)D(2) also displays tumorostatic effects. In keratinocytes 20(OH)D(2) inhibits expression of cyclins and stimulates involucrin expression. It also stimulates CYP24 expression, however, to a significantly lower degree than that by 1,25(OH)(2)D(3) or 25(OH)D(3). 20(OH)D(2) is a poor substrate for CYP27B1 with overall catalytic efficiency being 24- and 41-fold lower than for 25(OH)D(3) with the mouse and human enzymes, respectively. No conversion of 20(OH)D(2) to 1,20(OH)(2)D(2) was detected in intact HaCaT keratinocytes. 20(OH)D(2) also demonstrates anti-leukemic activity but with lower potency than 1,25(OH)(2)D(3). The phenotypic effects of 20(OH)D(2) are mediated through interaction with the vitamin D receptor (VDR) as documented by attenuation of cell proliferation after silencing of VDR, by enhancement of the inhibitory effect through stable overexpression of VDR and by the demonstration that 20(OH)D(2) induces time-dependent translocation of VDR from the cytoplasm to the nucleus at a comparable rate to that for 1,25(OH)(2)D(3). In vivo tests show that while 1,25(OH)(2)D(3) at doses as low as 0.8 µg/kg induces calcium deposits in the kidney and heart, 20(OH)D(2) is devoid of such activity even at doses as high as 4 µg/kg. Silencing of CY27B1 in human keratinocytes showed that 20(OH)D(2) does not require its transformation to 1,20(OH)(2)D(2) for its biological activity. Thus 20(OH)D(2) shows cell-type dependent antiproliferative and prodifferentiation activities through activation of VDR, while having no detectable toxic calcemic activity, and is a poor substrate for CYP27B1.


Assuntos
25-Hidroxivitamina D 2/análogos & derivados , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Inibidores do Crescimento/farmacologia , Queratinócitos/metabolismo , Melanócitos/metabolismo , Neoplasias/patologia , 25-Hidroxivitamina D 2/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Células HL-60 , Humanos , Queratinócitos/citologia , Queratinócitos/patologia , Melanócitos/citologia , Melanócitos/patologia , Camundongos , Neoplasias/tratamento farmacológico
17.
Drug Metab Dispos ; 38(9): 1553-9, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20554701

RESUMO

20,23-Dihydroxyvitamin D(3) [20,23(OH)(2)D(3)] is a biologically active metabolite produced by the action of cytochrome P450scc (CYP11A1) on vitamin D(3). It inhibits keratinocyte proliferation, stimulates differentiation, and inhibits nuclear factor-kappaB activity, working as a vitamin D receptor agonist. We have tested the ability of purified mouse 25-hydroxyvitamin D(3) 1alpha-hydroxylase (CYP27B1) to add a 1alpha-hydroxyl group to this vitamin D analog and determined whether this altered its biological activity. 20,23(OH)(2)D(3) incorporated into phospholipid vesicles was converted to a single product by CYP27B1, confirmed to be 1alpha,20,23-trihydroxyvitamin D(3) [1,20,23(OH)(3)D(3)] by mass spectrometry and NMR. The 20,23(OH)(2)D(3) was a relatively poor substrate for CYP27B1 compared with the normal substrate, 25-hydroxyvitamin D(3), displaying a 5-fold higher K(m) and 8-fold lower k(cat) value. Both 20,23(OH)(2)D(3) and 1,20,23(OH)(3)D(3) decreased neonatal human epidermal keratinocyte proliferation, showing significant effects at a lower concentration (0.1 nM) than that seen for 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] at 24 h of treatment. Both compounds also decreased cell biomass relative to that of control cells, measured by staining with sulforhodamine B. They caused little stimulation of the expression of the vitamin D receptor at the mRNA level compared with the 30-fold induction observed with the same concentration (100 nM) of 1,25(OH)(2)D(3) at 24 h. Addition of a 1alpha-hydroxyl group to 20,23(OH)(2)D(3) greatly enhanced its ability to stimulate the expression of the CYP24 gene but not to the extent seen with 1,25(OH)(2)D(3). This study shows that purified CYP27B1 can add a 1alpha-hydroxyl group to 20,23(OH)(2)D(3) with the product showing altered biological activity, especially for the stimulation of CYP24 gene expression.


Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Colecalciferol/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Colecalciferol/análogos & derivados , Cromatografia Líquida , Humanos , Hidroxilação , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Camundongos
18.
J Steroid Biochem Mol Biol ; 119(3-5): 171-9, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20193763

RESUMO

CYP27B1 catalyzes the 1alpha-hydroxylation of 25-hydroxyvitamin D3 to 1alpha,25-dihydroxyvitamin D3, the hormonally active form of vitamin D3. To further characterize mouse CYP27B1, it was expressed in Escherichia coli, purified and its activity measured on substrates incorporated into phospholipid vesicles, which served as a model of the inner mitochondrial membrane. 25-Hydroxyvitamin D3 and 25-hydroxyvitamin D2 in vesicles underwent 1alpha-hydroxylation with similar kinetics, the catalytic rate constants (k(cat)) were 41 and 48mol/min/mol P450, respectively, while K(m) values were 5.9 and 4.6mmol/mol phospholipid, respectively. CYP27B1 showed inhibition when substrate concentrations in the membrane were greater than 4 times K(m), more pronounced with 25-hydroxyvitamin D3 than 25-hydroxyvitamin D2. Higher catalytic efficiency was seen in vesicles prepared from dioleoyl phosphatidylcholine and cardiolipin than for dimyristoyl phosphatidylcholine vesicles. CYP27B1 also catalyzed 1alpha-hydroxylation of vesicle-associated 24R,25-dihydroxyvitamin D3 and 20-hydroxyvitamin D3, and 25-hydroxylation of 1alpha-hydroxyvitamin D3 and 1alpha-hydroxyvitamin D2, but with much lower efficiency than for 25(OH)D3. This study shows that CYP27B1 can hydroxylate 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 associated with phospholipid membranes with the highest activity yet reported for the enzyme. The expressed enzyme has low activity at higher concentrations of 25-hydroxyvitamin D in membranes, revealing that substrate inhibition may contribute to the regulation of the activity of this enzyme.


Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Calcifediol/química , Calcifediol/metabolismo , Fosfolipídeos/química , Lipossomas Unilamelares/química , 24,25-Di-Hidroxivitamina D 3/metabolismo , 25-Hidroxivitamina D 2/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/química , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/isolamento & purificação , Adrenodoxina/química , Adrenodoxina/isolamento & purificação , Adrenodoxina/metabolismo , Animais , Calcifediol/análogos & derivados , Cardiolipinas/química , Dimiristoilfosfatidilcolina/química , Cinética , Camundongos , Membranas Mitocondriais/enzimologia , Membranas Mitocondriais/metabolismo , Tamanho da Partícula , Fosfatidilcolinas/química , Fosfolipídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Espectrofotometria , Especificidade por Substrato
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