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1.
J Pineal Res ; 68(2): e12626, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31770455

RESUMO

Tryptophan hydroxylase (TPH) activity was detected in cultured epidermal melanocytes and dermal fibroblasts with respective Km of 5.08 and 2.83 mM and Vmax of 80.5 and 108.0 µmol/min. Low but detectable TPH activity was also seen in cultured epidermal keratinocytes. Serotonin and/or its metabolite and precursor to melatonin, N-acetylserotonin (NAS), were identified by LC/MS in human epidermis and serum. Endogenous epidermal levels were 113.18 ± 13.34 and 43.41 ± 12.45 ng/mg protein for serotonin (n = 8/8) and NAS (n = 10/13), respectively. Their production was independent of race, gender, and age. NAS was also detected in human serum (n = 13/13) at a concentration 2.44 ± 0.45 ng/mL, while corresponding serotonin levels were 295.33 ± 17.17 ng/mL (n = 13/13). While there were no differences in serum serotonin levels, serum NAS levels were slightly higher in females. Immunocytochemistry studies showed localization of serotonin to epidermal and follicular keratinocytes, eccrine glands, mast cells, and dermal fibrocytes. Endogenous production of serotonin in cultured melanocytes, keratinocytes, and dermal fibroblasts was modulated by UVB. In conclusion, serotonin and NAS are produced endogenously in the epidermal, dermal, and adnexal compartments of human skin and in cultured skin cells. NAS is also detectable in human serum. Both serotonin and NAS inhibited melanogenesis in human melanotic melanoma at concentrations of 10-4 -10-3  M. They also inhibited growth of melanocytes. Melanoma cells were resistant to NAS inhibition, while serotonin inhibited cell growth only at 10-3  M. In summary, we characterized a serotonin-NAS system in human skin that is a part of local neuroendocrine system regulating skin homeostasis.

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.
Int J Mol Sci ; 19(10)2018 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-30297679

RESUMO

A novel pathway of vitamin D activation by CYP11A has previously been elucidated. To define the mechanism of action of its major dihydroxy-products, we tested the divergence and overlap between the gene expression profiles of human epidermal keratinocytes treated with either CYP11A1-derived 20,23(OH)2D3 or classical 1,25(OH)2D3. Both secosteroids have significant chemical similarity with the only differences being the positions of the hydroxyl groups. mRNA was isolated and examined by microarray analysis using Illumina's HumanWG-6 chip/arrays and subsequent bioinformatics analyses. Marked differences in the up- and downregulated genes were observed between 1,25(OH)2D3- and 20,23(OH)2D3-treated cells. Hierarchical clustering identified both distinct, opposite and common (overlapping) gene expression patterns. CYP24A1 was a common gene strongly activated by both compounds, a finding confirmed by qPCR. Ingenuity pathway analysis identified VDR/RXR signaling as the top canonical pathway induced by 1,25(OH)2D3. In contrast, the top canonical pathway induced by 20,23(OH)2D3 was AhR, with VDR/RXR being the second nuclear receptor signaling pathway identified. QPCR analyses validated the former finding by revealing that 20,23(OH)2D3 stimulated CYP1A1 and CYP1B1 gene expression, effects located downstream of AhR. Similar stimulation was observed with 20(OH)D3, the precursor to 20,23(OH)2D3, as well as with its downstream metabolite, 17,20,23(OH)3D3. Using a Human AhR Reporter Assay System we showed marked activation of AhR activity by 20,23(OH)2D3, with weaker stimulation by 20(OH)D3. Finally, molecular modeling using an AhR LBD model predicted vitamin D3 hydroxyderivatives to be good ligands for this receptor. Thus, our microarray, qPCR, functional studies and molecular modeling indicate that AhR is the major receptor target for 20,23(OH)2D3, opening an exciting area of investigation on the interaction of different vitamin D3-hydroxyderivatives with AhR and the subsequent downstream activation of signal transduction pathways in a cell-type-dependent manner.


Assuntos
Calcitriol/farmacologia , Di-Hidroxicolecalciferóis/farmacologia , Queratinócitos/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Sítios de Ligação , Células Cultivadas , Humanos , Queratinócitos/efeitos dos fármacos , Simulação de Acoplamento Molecular , Ligação Proteica , Receptores de Hidrocarboneto Arílico/química
4.
J Pineal Res ; 65(2): e12501, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29702749

RESUMO

Melatonin and its derivatives (N1 -acetyl-N2 -formyl-5-methoxykynurenine [AFMK] and N-acetyl serotonin [NAS]) have broad-spectrum protective effects against photocarcinogenesis, including both direct and indirect antioxidative actions, regulation of apoptosis and DNA damage repair; these data were primarily derived from in vitro models. This study evaluates possible beneficial effects of melatonin and its active derivatives against ultraviolet B (UVB)-induced harm to human and porcine skin ex vivo and to cultured HaCaT cells. The topical application of melatonin, AFMK, or NAS protected epidermal cells against UVB-induced 8-OHdG formation and apoptosis with a further increase in p53ser15 expression, especially after application of melatonin or AFMK but not after NAS use. The photoprotective action was observed in pre- and post-UVB treatment in both human and porcine models. Melatonin along with its derivatives upregulated also the expression of antioxidative enzymes after UVB radiation of HaCaT cells. The exogenous application of melatonin or its derivatives represents a potent and promising tool for preventing UVB-induced oxidative stress and DNA damage. This protection results in improved genomic, cellular, and tissue integrity against UVB-induced carcinogenesis, especially when applied prior to UV exposure. In addition, our ex vivo experiments provide fundamental justification for further testing the clinical utility of melatonin and metabolites as protectors again UVB in human subjects. Our ex vivo data constitute the bridge between vitro to vivo translation and thus justifies the pursue for further clinical utility of melatonin in maintaining skin homeostasis.


Assuntos
Dano ao DNA , Desoxiguanosina/análogos & derivados , Melatonina/farmacologia , Estresse Oxidativo , Pele/metabolismo , Raios Ultravioleta/efeitos adversos , Animais , Linhagem Celular , Desoxiguanosina/metabolismo , Humanos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Pele/patologia , Suínos
5.
Sci Rep ; 8(1): 1478, 2018 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-29367669

RESUMO

20S-hydroxyvitamin D3 [20S(OH)D3] is anti-inflammatory and not hypercalcemic, suggesting its potential as a lead compound. In this study, side chain modified 20S(OH)D3 analogs (4, 13, 23 and 33) together with their 1α-OH derivatives were synthesized and their metabolism and biological activities tested. 4, 13 and 23 are good substrates for CYP27B1, enabling enzymatic synthesis of their 1α-OH derivatives 5, 14 and 24. However, 33 could not be hydroxylated by CYP27B1 and acts as an inhibitor. All analogs were poorer substrates for CYP24A1 than calcitriol, indicating improved catabolic stability. While the parent analogs showed minimal VDR stimulating activity, their 1α-OH derivatives were potent VDR agonists. 4, 5, 14 and 24 significantly upregulated the expression of CYP24A1 at the mRNA level, consistent with their VDR activation abilities and indicating that 1α-hydroxylation is required to produce analogs with strong activity. These analogs have anti-inflammatory activities that are influenced by side chain composition and by 1α-hydroxylation. To understand their molecular interactions with the VDR, 20S(OH)D3, 4 and 33 were co-crystalized with the VDR ligand binding domain, which revealed subtle differences to the calcitriol-bound receptor. This study demonstrates the potential of the 20S(OH)D3 scaffold for the development of novel anti-inflammatory agents.


Assuntos
Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Calcifediol/análogos & derivados , Proliferação de Células/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Receptores de Calcitriol/agonistas , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Calcifediol/química , Calcifediol/farmacologia , Células Cultivadas , Humanos , Hidroxilação , Queratinócitos/citologia , Queratinócitos/metabolismo , Vitamina D3 24-Hidroxilase/metabolismo
6.
J Steroid Biochem Mol Biol ; 177: 159-170, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28676457

RESUMO

Melanoma represents a significant clinical problem affecting a large segment of the population with a relatively high incidence and mortality rate. Ultraviolet radiation (UVR) is an important etiological factor in malignant transformation of melanocytes and melanoma development. UVB, while being a full carcinogen in melanomagenesis, is also necessary for the cutaneous production of vitamin D3 (D3). Calcitriol (1,25(OH)2D3) and novel CYP11A1-derived hydroxyderivatives of D3 show anti-melanoma activities and protective properties against damage induced by UVB. The former activities include inhibitory effects on proliferation, plating efficiency and anchorage-independent growth of cultured human and rodent melanomas in vitro, as well as the in vivo inhibition of tumor growth by 20(OH)D3 after injection of human melanoma cells into immunodeficient mice. The literature indicates that low levels of 25(OH)D3 are associated with more advanced melanomas and reduced patient survivals, while single nucleotide polymorphisms of the vitamin D receptor or the D3 binding protein gene affect development or progression of melanoma, or disease outcome. An inverse correlation of VDR and CYP27B1 expression with melanoma progression has been found, with low or undetectable levels of these proteins being associated with poor disease outcomes. Unexpectedly, increased expression of CYP24A1 was associated with better melanoma prognosis. In addition, decreased expression of retinoic acid orphan receptors α and γ, which can also bind vitamin D3 hydroxyderivatives, showed positive association with melanoma progression and shorter disease-free and overall survival. Thus, inadequate levels of biologically active forms of D3 and disturbances in expression of the target receptors, or D3 activating or inactivating enzymes, can affect melanomagenesis and disease progression. We therefore propose that inclusion of vitamin D into melanoma management should be beneficial for patients, at least as an adjuvant approach. The presence of multiple hydroxyderivatives of D3 in skin that show anti-melanoma activity in experimental models and which may act on alternative receptors, will be a future consideration when planning which forms of vitamin D to use for melanoma therapy.


Assuntos
Melanoma/metabolismo , Neoplasias Cutâneas/metabolismo , Vitamina D/metabolismo , Animais , Humanos , Melanoma/patologia , Neoplasias Cutâneas/patologia
7.
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
8.
Sci Rep ; 7(1): 11434, 2017 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-28900196

RESUMO

Using LC/qTOF-MS we detected lumisterol, 20-hydroxylumisterol, 22-hydroxylumisterol, 24-hydroxylumisterol, 20,22-dihydroxylumisterol, pregnalumisterol, 17-hydroxypregnalumisterol and 17,20-dihydroxypregnalumisterol in human serum and epidermis, and the porcine adrenal gland. The hydroxylumisterols inhibited proliferation of human skin cells in a cell type-dependent fashion with predominant effects on epidermal keratinocytes. They also inhibited melanoma proliferation in both monolayer and soft agar. 20-Hydroxylumisterol stimulated the expression of several genes, including those associated with keratinocyte differentiation and antioxidative responses, while inhibiting the expression of others including RORA and RORC. Molecular modeling and studies on VDRE-transcriptional activity excludes action through the genomic site of the VDR. However, their favorable interactions with the A-pocket in conjunction with VDR translocation studies suggest they may act on this non-genomic VDR site. Inhibition of RORα and RORγ transactivation activities in a Tet-on CHO cell reporter system, RORα co-activator assays and inhibition of (RORE)-LUC reporter activity in skin cells, in conjunction with molecular modeling, identified RORα and RORγ as excellent receptor candidates for the hydroxylumisterols. Thus, we have discovered a new biologically relevant, lumisterogenic pathway, the metabolites of which display biological activity. This opens a new area of endocrine research on the effects of the hydroxylumisterols on different pathways in different cells and the mechanisms involved.


Assuntos
Ergosterol/metabolismo , Redes e Vias Metabólicas , Animais , Biomarcadores , Linhagem Celular Tumoral , Cromatografia Líquida , Relação Dose-Resposta a Droga , Epiderme/efeitos dos fármacos , Epiderme/metabolismo , Ergosterol/química , Ergosterol/farmacologia , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Receptores de Calcitriol/química , Receptores de Calcitriol/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Suínos
9.
Cell Mol Life Sci ; 74(21): 3913-3925, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28803347

RESUMO

The skin being a protective barrier between external and internal (body) environments has the sensory and adaptive capacity to maintain local and global body homeostasis in response to noxious factors. An important part of the skin response to stress is its ability for melatonin synthesis and subsequent metabolism through the indolic and kynuric pathways. Indeed, melatonin and its metabolites have emerged as indispensable for physiological skin functions and for effective protection of a cutaneous homeostasis from hostile environmental factors. Moreover, they attenuate the pathological processes including carcinogenesis and other hyperproliferative/inflammatory conditions. Interestingly, mitochondria appear to be a central hub of melatonin metabolism in the skin cells. Furthermore, substantial evidence has accumulated on the protective role of the melatonin against ultraviolet radiation and the attendant mitochondrial dysfunction. Melatonin and its metabolites appear to have a modulatory impact on mitochondrion redox and bioenergetic homeostasis, as well as the anti-apoptotic effects. Of note, some metabolites exhibit even greater impact than melatonin alone. Herein, we emphasize that melatonin-mitochondria axis would control integumental functions designed to protect local and perhaps global homeostasis. Given the phylogenetic origin and primordial actions of melatonin, we propose that the melatonin-related mitochondrial functions represent an evolutionary conserved mechanism involved in cellular adaptive response to skin injury and repair.


Assuntos
Antioxidantes/farmacologia , Melatonina/farmacologia , Mitocôndrias/metabolismo , Pele/metabolismo , Animais , Humanos , Mitocôndrias/efeitos dos fármacos , Pele/efeitos dos fármacos , Fenômenos Fisiológicos da Pele
10.
Sci Rep ; 7(1): 1274, 2017 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-28455491

RESUMO

Ultraviolet light (UV) is an inducer of reactive oxygen species (ROS) as well as 6-4-photoproducts and cyclobutane pyrimidine dimers (CPD) in the skin, which further cause damage to the skin cells. Irradiation of cultured human melanocytes with UVB stimulated ROS production, which was reduced in cells treated with melatonin or its metabolites: 6-hydroxymelatonin (6-OHM), N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), N-acetylserotonin (NAS), and 5-methoxytryptamine (5-MT). Melatonin and its derivatives also stimulated the expression of NRF2 (nuclear factor erythroid 2 [NF-E2]-related factor 2) and its target enzymes and proteins that play an important role in cell protection from different damaging factors including UVB. Silencing of NRF2 using siRNA diminished the protective effects of melatonin, while the membrane melatonin receptors (MT1 or MT2) did not change the activities of either melatonin or its derivatives. Melatonin and its metabolites enhanced the DNA repair in melanocytes exposed to UVB and stimulated expression of p53 phosphorylated at Ser-15. In conclusion, melatonin and its metabolites protect melanocytes from UVB-induced DNA damage and oxidative stress through activation of NRF2-dependent pathways; these actions are independent of an effect on the classic membrane melatonin receptors. Thus, melatonin and its derivatives can serve as excellent protectors of melanocytes against UVB-induced pathology.


Assuntos
Melanócitos/fisiologia , Melanócitos/efeitos da radiação , Melatonina/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Protetores contra Radiação/metabolismo , Raios Ultravioleta , Células Cultivadas , Reparo do DNA/efeitos dos fármacos , Humanos , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/toxicidade
11.
Anticancer Res ; 36(3): 877-86, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26976974

RESUMO

Vitamin D3 (D3) can be metabolized by cytochrome P450scc (CYP11A1) into 20S-hydroxyvitamin D3 (20D3) as a major metabolite. This bioactive metabolite has shown strong antiproliferative, antifibrotic, pro-differentiation and anti-inflammatory effects while being non-toxic (non-calcemic) at high concentrations. Since D3 analogs with two symmetric side chains (Gemini analogs) result in potent activation of the vitamin D receptor (VDR), we hypothesized that the chain length and composition of these types of analogs also containing a 20-hydroxyl group would affect their biological activities. In this study, we designed and synthesized a series of Gemini 20D3 analogs. Biological tests showed that some of these analogs are partial VDR activators and can significantly stimulate the expression of mRNA for VDR and VDR-regulated genes including CYP24A1 and transient receptor potential cation channel V6 (TRPV6). These analogs inhibited the proliferation of melanoma cells with potency comparable to that of 1α,25-dihydroxyvitamin D3. Moreover, these analogs reduced the level of interferon γ and up-regulated the expression of leukocyte associated immunoglobulin-like receptor 1 in splenocytes, indicating that they have potent anti-inflammatory activities. There are no clear correlations between the Gemini chain length and their VDR activation or biological activities, consistent with the high flexibility of the ligand-binding pocket of the VDR.


Assuntos
Anti-Inflamatórios/síntese química , Anti-Inflamatórios/farmacologia , Calcifediol/análogos & derivados , Citostáticos/síntese química , Citostáticos/farmacologia , Animais , Anti-Inflamatórios/química , Calcifediol/síntese química , Calcifediol/química , Calcifediol/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citostáticos/química , Desenho de Drogas , Humanos , Células Jurkat , Queratinócitos/efeitos dos fármacos , Queratinócitos/imunologia , Camundongos , Receptores de Calcitriol/metabolismo , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologia
12.
Int J Mol Sci ; 16(4): 6645-67, 2015 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-25811927

RESUMO

Deregulated melanogenesis is involved in melanomagenesis and melanoma progression and resistance to therapy. Vitamin D analogs have anti-melanoma activity. While the hypercalcaemic effect of the active form of Vitamin D (1,25(OH)2D3) limits its therapeutic use, novel Vitamin D analogs with a modified side chain demonstrate low calcaemic activity. We therefore examined the effect of secosteroidal analogs, both classic (1,25(OH)2D3 and 25(OH)D3), and novel relatively non-calcemic ones (20(OH)D3, calcipotriol, 21(OH)pD, pD and 20(OH)pL), on proliferation, colony formation in monolayer and soft-agar, and mRNA and protein expression by melanoma cells. Murine B16-F10 and hamster Bomirski Ab cell lines were shown to be effective models to study how melanogenesis affects anti-melanoma treatment. Novel Vitamin D analogs with a short side-chain and lumisterol-like 20(OH)pL efficiently inhibited rodent melanoma growth. Moderate pigmentation sensitized rodent melanoma cells towards Vitamin D analogs, and altered expression of key genes involved in Vitamin D signaling, which was opposite to the effect on heavily pigmented cells. Interestingly, melanogenesis inhibited ligand-induced Vitamin D receptor translocation and ligand-induced expression of VDR and CYP24A1 genes. These findings indicate that melanogenesis can affect the anti-melanoma activity of Vitamin D analogs in a complex manner.


Assuntos
Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Vitamina D/análogos & derivados , Animais , Calcitriol/análogos & derivados , Calcitriol/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cricetinae , Melaninas/metabolismo , Melanoma/patologia , Camundongos , Receptores de Calcitriol/genética , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/metabolismo , Vitamina D3 24-Hidroxilase/genética
13.
J Steroid Biochem Mol Biol ; 148: 52-63, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25617667

RESUMO

CYP11A1 hydroxylates the side chain of vitamin D3 (D3) in a sequential fashion [D3→20S(OH)D3→20,23(OH)2D3→17,20,23(OH)3D3], in an alternative to the classical pathway of activation [D3→25(OH)D3→1,25(OH)2D3]. The products/intermediates of the pathway can be further modified by the action of CYP27B1. The CYP11A1-derived products are biologically active with functions determined by the lineage of the target cells. This pathway can operate in epidermal keratinocytes. To further define the role of these novel secosteroids we tested them for protective effects against UVB-induced damage in human epidermal keratinocytes, melanocytes and HaCaT keratinocytes, cultured in vitro. The secosteroids attenuated ROS, H2O2 and NO production by UVB-irradiated keratinocytes and melanocytes, with an efficacy similar to 1,25(OH)2D3, while 25(OH)D3 had lower efficacy. These attenuations were also seen to some extent for the 20(OH)D3 precursor, 20S-hydroxy-7-dehydrocholesterol. These effects were accompanied by upregulation of genes encoding enzymes responsible for defense against oxidative stress. Using immunofluorescent staining we observed that the secosteroids reduced the generation cyclobutane pyrimidine dimers in response to UVB and enhanced expression of p53 phosphorylated at Ser-15, but not at Ser-46. Additional evidence for protection against DNA damage in cells exposed to UVB and treated with secosteroids was provided by the Comet assay where DNA fragmentation was markedly reduced by 20(OH)D3 and 20,23(OH)2D3. In conclusion, novel secosteroids that can be produced by the action of CYP11A1 in epidermal keratinocytes have protective effects against UVB radiation. This article is part of a special issue entitled '17th Vitamin D Workshop'.


Assuntos
Dano ao DNA/efeitos dos fármacos , Epiderme/metabolismo , Hipercalcemia/prevenção & controle , Queratinócitos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Secoesteroides/farmacologia , Raios Ultravioleta/efeitos adversos , Células Cultivadas , Dano ao DNA/efeitos da radiação , Células Epidérmicas , Epiderme/efeitos da radiação , Humanos , Hipercalcemia/etiologia , Queratinócitos/citologia , Queratinócitos/efeitos da radiação , Estresse Oxidativo/efeitos da radiação
14.
Int J Mol Sci ; 15(10): 17705-32, 2014 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-25272227

RESUMO

The human skin is not only a target for the protective actions of melatonin, but also a site of melatonin synthesis and metabolism, suggesting an important role for a local melatoninergic system in protection against ultraviolet radiation (UVR) induced damages. While melatonin exerts many effects on cell physiology and tissue homeostasis via membrane bound melatonin receptors, the strong protective effects of melatonin against the UVR-induced skin damage including DNA repair/protection seen at its high (pharmocological) concentrations indicate that these are mainly mediated through receptor-independent mechanisms or perhaps through activation of putative melatonin nuclear receptors. The destructive effects of the UVR are significantly counteracted or modulated by melatonin in the context of a complex intracutaneous melatoninergic anti-oxidative system with UVR-enhanced or UVR-independent melatonin metabolites. Therefore, endogenous intracutaneous melatonin production, together with topically-applied exogenous melatonin or metabolites would be expected to represent one of the most potent anti-oxidative defense systems against the UV-induced damage to the skin. In summary, we propose that melatonin can be exploited therapeutically as a protective agent or as a survival factor with anti-genotoxic properties or as a "guardian" of the genome and cellular integrity with clinical applications in UVR-induced pathology that includes carcinogenesis and skin aging.


Assuntos
Melatonina/metabolismo , Pele/metabolismo , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Potencial da Membrana Mitocondrial/efeitos da radiação , Estresse Oxidativo/efeitos da radiação , Receptores de Melatonina/metabolismo , Pele/efeitos da radiação , Raios Ultravioleta
15.
Int J Mol Sci ; 15(10): 19000-17, 2014 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-25334067

RESUMO

The major role of 24-hydroxylase (CYP24A1) is to maintain 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) homeostasis. Recently, it has been discovered that CYP24A1 also catalyses the hydroxylation of 20(OH)D3, producing dihydroxy-derivatives that show very effective antitumorigenic activities. Previously we showed a negative correlation of vitamin D receptor (VDR) and CYP27B1 expression with progression, aggressiveness and overall or disease-free survivals of skin melanomas. Therefore, we analyzed CYP24A1 expression in relation to clinicopathomorphological features of nevi, skin melanomas and metastases. In melanocytic tumors, the level of CYP24A1 was higher than in the normal epidermis. The statistically highest mean CYP24A1 level was found in nevi and early stage melanomas. With melanoma progression, CYP24A1 levels decreased and in advanced stages were comparable to the normal epidermis and metastases. Furthermore, the CYP24A1 expression positively correlated with VDR and CYP27B1, and negatively correlated with mitotic activity. Lower CYP24A1 levels correlated with the presence of ulceration, necrosis, nodular type and amelanotic phenotypes. Moreover, a lack of detectable CYP24A1 expression was related to shorter overall and disease-free survival. In conclusion, the local vitamin D endocrine system affects melanoma behavior and an elevated level of CYP24A1 appears to have an important impact on the formation of melanocytic nevi and melanomagenesis, or progression, at early stages of tumor development.


Assuntos
Regulação Neoplásica da Expressão Gênica , Melanoma/genética , Melanoma/patologia , Pele/patologia , Vitamina D3 24-Hidroxilase/análise , Vitamina D3 24-Hidroxilase/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Receptores de Calcitriol/análise , Pele/metabolismo , Neoplasias Cutâneas , Adulto Jovem
16.
J Pineal Res ; 57(1): 90-102, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24867336

RESUMO

We investigated the protective effects of melatonin and its metabolites: 6-hydroxymelatonin (6-OHM), N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), N-acetylserotonin (NAS), and 5-methoxytryptamine (5-MT) in human keratinocytes against a range of doses (25, 50, and 75 mJ/cm2) of ultraviolet B (UVB) radiation. There was significant reduction in the generation of reactive oxygen species (50-60%) when UVB-exposed keratinocytes were treated with melatonin or its derivatives. Similarly, melatonin and its metabolites reduced the nitrite and hydrogen peroxide levels that were induced by UVB as early as 30 min after the exposure. Moreover, melatonin and its metabolites enhanced levels of reduced glutathione in keratinocytes within 1 hr after UVB exposure in comparison with control cells. Using proliferation assay, we observed a dose-dependent increase in viability of UVB-irradiated keratinocytes that were treated with melatonin or its derivatives after 48 hr. Using the dot-blot technique and immunofluorescent staining we also observed that melatonin and its metabolites enhanced the DNA repair capacity of UVB-induced pyrimidine photoproducts (6-4)or cyclobutane pyrimidine dimers generation in human keratinocytes. Additional evidence for induction of DNA repair in cells exposed to UVB and treated with the indole compounds was shown using the Comet assay. Finally, melatonin and its metabolites further enhanced expression of p53 phosphorylated at Ser-15 but not at Ser-46 or its nonphosphorylated form. In conclusion, melatonin, its precursor NAS, and its metabolites 6-OHM, AFMK, 5-MT, which are endogenously produced in keratinocytes, protect these cells against UVB-induced oxidative stress and DNA damage.


Assuntos
Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiação , Melatonina/farmacologia , Raios Ultravioleta , Linhagem Celular , Dano ao DNA/efeitos dos fármacos , Humanos , Cinuramina/farmacologia , Melatonina/análogos & derivados , Serotonina/análogos & derivados , Serotonina/farmacologia
17.
FASEB J ; 28(7): 2775-89, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24668754

RESUMO

RORα and RORγ are expressed in human skin cells that produce the noncalcemic 20-hydroxyvitamin D3 [20(OH)D3] and 20,23-dihydroxyvitamin D3 [20,23(OH)2D3]. Chinese hamster ovary (CHO) cells stably expressing a Tet-on RORα or RORγ expression vector and a ROR-responsive element (RORE)-LUC reporter, and a mammalian 2-hybrid model examining the interaction between the ligand binding domain (LBD) of RORα or RORγ with an LBD-interacting LXXLL-peptide, were used to study ROR-antagonist activities. These assays revealed that 20(OH)D3 and 20,23(OH)2D3 function as antagonists of RORα and RORγ. Moreover, 20(OH)D3 inhibited the activation of the promoter of the Bmal1 and G6pase genes, targets of RORα, and 20(OH)D3 and 20,23(OH)2D3 inhibited Il17 promoter activity in Jurkat cells overexpressing RORα or RORγ. Molecular modeling using crystal structures of the LBDs of RORα and RORγ revealed docking scores for 20(OH)D3, 20,23(OH)2D3 and 1,25(OH)2D3 similar to those of the natural ligands, predicting good binding to the receptor. Notably, 20(OH)D3, 20,23(OH)2D3, and 1,25(OH)2D3 inhibited RORE-mediated activation of a reporter in keratinocytes and melanoma cells and inhibited IL-17 production by immune cells. Our study identifies a novel signaling pathway, in which 20(OH)D3 and 20,23(OH)2D3 act as antagonists or inverse agonists of RORα and RORγ, that opens new possibilities for local (skin) or systemic regulation.-Slominski, A. T., Kim, T.-K., Takeda, Y., Janjetovic, Z., Broz˙yna, A. A., Skobowiat, C., Wang, J., Postlethwaite, A., Li, W., Tuckey, R. C., Jetten, A. M. RORα and ROR γ are expressed in human skin and serve as receptors for endogenously produced noncalcemic 20-hydroxy- and 20,23-dihydroxyvitamin D.


Assuntos
Calcifediol/análogos & derivados , Di-Hidroxicolecalciferóis/metabolismo , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Pele/metabolismo , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Animais , Células CHO , Calcifediol/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Cricetulus , Feminino , Glucose-6-Fosfatase/antagonistas & inibidores , Glucose-6-Fosfatase/genética , Humanos , Interleucina-17/genética , Interleucina-17/metabolismo , Células Jurkat , Queratinócitos/metabolismo , Melanoma/genética , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos DBA , Regiões Promotoras Genéticas/genética
18.
Exp Dermatol ; 23(1): 15-7, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24372648

RESUMO

Evidence is accumulating that skin can act as an independent steroidogenic organ. It can respond to various stresses including UV light, trauma and oncogenesis by upregulating glucocorticoid production via elements of the local hypothalamic-pituitary-adrenal (HPA) axis. Recent data by Takei and collaborators provided in this issue of Experimental Dermatology included dryness to the list of stressors stimulating cutaneous cortisol synthesis with a possible involvement of IL-1ß as a mediator of this regulation. Thus, the last decade of research has not only documented that skin can produce cortisol, but that levels of its production change in response to environmental stress. The role of this regulated steroidogenic system in physiological or pathological outcomes requires further studies with focus on cutaneous homeostasis, formation of epidermal barrier, antimicrobial activity and display of immune (both pro- and anti-inflammatory) properties.


Assuntos
Exposição Ambiental , Epiderme/metabolismo , Regulação da Expressão Gênica , Hidrocortisona/metabolismo , Técnicas de Cultura de Órgãos/métodos , Humanos
19.
FASEB J ; 27(7): 2742-55, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23620527

RESUMO

Indolic and kynuric pathways of skin melatonin metabolism were monitored by liquid chromatography mass spectrometry in human keratinocytes, melanocytes, dermal fibroblasts, and melanoma cells. Production of 6-hydroxymelatonin [6(OH)M], N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) and 5-methoxytryptamine (5-MT) was detected in a cell type-dependent fashion. The major metabolites, 6(OH)M and AFMK, were produced in all cells. Thus, in immortalized epidermal (HaCaT) keratinocytes, 6(OH)M was the major product with Vmax = 63.7 ng/10(6) cells and Km = 10.2 µM, with lower production of AFMK and 5-MT. Melanocytes, keratinocytes, and fibroblasts transformed melatonin primarily into 6(OH)M and AFMK. In melanoma cells, 6(OH)M and AFMK were produced endogenously, a process accelerated by exogenous melatonin in the case of AFMK. In addition, N-acetylserotonin was endogenously produced by normal and malignant melanocytes. Metabolites showed selective antiproliferative effects on human primary epidermal keratinocytes in vitro. In ex vivo human skin, both melatonin and AFMK-stimulated expression of involucrin and keratins-10 and keratins-14 in the epidermis, indicating their stimulatory role in building and maintaining the epidermal barrier. In summary, the metabolism of melatonin and its endogenous production is cell type-dependent and expressed in all three main cell populations of human skin. Furthermore, melatonin and its metabolite AFMK stimulate differentiation in human epidermis, indicating their key role in building the skin barrier.


Assuntos
Melatonina/metabolismo , Redes e Vias Metabólicas , Pele/metabolismo , 5-Metoxitriptamina/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Células Epidérmicas , Epiderme/efeitos dos fármacos , Epiderme/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Queratina-10/metabolismo , Queratina-14/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Cinética , Cinuramina/análogos & derivados , Cinuramina/metabolismo , Cinuramina/farmacologia , Melanócitos/citologia , Melanócitos/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Melatonina/análogos & derivados , Melatonina/farmacologia , Serotonina/análogos & derivados , Serotonina/metabolismo , Pele/citologia , Espectrometria de Massas por Ionização por Electrospray , Suínos
20.
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
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