RESUMO
Melanogenesis-stimulated B16-F10 cells enter in a quiescent state, present inhibited mitochondrial respiration and increased reactive oxygen species levels. These alterations suggest that these cells may be under redox signaling, allowing tumor survival. The aim of this study was to evaluate redox-modified proteins in B16-F10 cells after melanogenesis stimulation and rose bengal-photodynamic therapy (RB-PDT). A redox proteomics label-free approach based on the biotin switch assay technique with biotin-HPDP and N-ethylmaleimide was used to assess the thiol-oxidized protein profile. Aconitase was oxidized at Cys-448 and Cys-451, citrate synthase was oxidized at Cys-202 and aspartate aminotransferase (Got2) was oxidized at Cys-272 and Cys-274, exclusively after melanogenesis stimulation. After RB-PDT, only guanine nucleotide-binding protein subunit beta-2-like 1 (Gnb2l1) was oxidized (Cys-168). In contrast, melanogenesis stimulation followed by RB-PDT led to the oxidation of different cysteines in Gnb2l1 (Cys-153 and Cys-249). Besides that, glyceraldehyde-3-phosphate dehydrogenase (Gapdh) presented oxidation at Cys-245, peptidyl-prolyl cis-trans isomerase A (Ppia) was oxidized at Cys-161 and 5,6-dihydroxyindole-2-carboxylic acid oxidase (Tyrp1) was oxidized at Cys-65, Cys-30, and Cys-336 after melanogenesis stimulation followed by RB-PDT. The redox alterations observed in murine melanoma cells and identification of possible target proteins are of great importance to further understand tumor resistance mechanisms.
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Secreted quiescin/sulfhydryl oxidase (QSOX) is overexpressed in many tumor cell lines, including melanoma, and is usually associated with a pro-invasive phenotype. Our previous work described that B16-F10 cells enter in a quiescent state as a protective mechanism against damage generated by reactive oxygen species (ROS) during melanogenesis stimulation. Our present results show that QSOX activity was two-fold higher in cells with stimulated melanogenesis when compared to control cells. Considering that glutathione (GSH) is one of the main factor responsible for controlling redox homeostasis in cells, this work also aimed to investigate the relationship between QSOX activity, GSH levels and melanogenesis stimulation in B16-F10 murine melanoma cell line. The redox homeostasis was impaired by treating cells with GSH in excess or depleting its intracellular levels through BSO treatment. Interestingly, GSH-depleted cells without stimulation of melanogenesis kept high levels of viability, suggesting a possible adaptive mechanism of survival even under low GSH levels. They also showed lower extracellular activity of QSOX, and higher QSOX intracellular immunostaining, suggesting that this enzyme was less excreted from cells and corroborating with a diminished extracellular QSOX activity. On the other hand, cells under melanogenesis stimulation showed a lower GSH/GSSG ratio (8:1) in comparison with control (non-stimulated) cells (20:1), indicating a pro-oxidative state after stimulation. This was accompanied by decreased cell viability after GSH-depletion, no alterations in QSOX extracellular activity, but higher QSOX nucleic immunostaining. We suggest that melanogenesis stimulation and redox impairment caused by GSH-depletion enhanced the oxidative stress in these cells, contributing to additional alterations of its metabolic adaptive response.
Assuntos
Melanoma , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Animais , Camundongos , Glutationa/metabolismo , Melanoma/metabolismo , Oxirredução , Oxirredutases/metabolismo , Espécies Reativas de Oxigênio , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismoRESUMO
Melanogenesis- stimulated B16-F10 cells enter in a quiescent state, present inhib-ited mitochondrial respiration and increased reactive oxygen species levels. Thesealterations suggest that these cells may be under redox signaling, allowing tumorsurvival. The aim of this study was to evaluate redox-modified proteins in B16-F10 cells after melanogenesis stimulation and rose bengal-photodynamic therapy(RB-PDT). A redox proteomics label-free approach based on the biotin switchassay technique with biotin-HPDP and N-ethylmaleimide was used to assess thethiol-oxidized protein profile. Aconitase was oxidized at Cys-448 and Cys-451,citrate synthase was oxidized at Cys-202 and aspartate aminotransferase (Got2)was oxidized at Cys-272 and Cys-274, exclusively after melanogenesis stimula-tion. After RB- PDT, only guanine nucleotide-binding protein subunit beta- 2- like1 (Gnb2l1) was oxidized (Cys-168). In contrast, melanogenesis stimulation fol-lowed by RB- PDT led to the oxidation of different cysteines in Gnb2l1 (Cys-153and Cys- 249). Besides that, glyceraldehyde-3-phosphate dehydrogenase (Gapdh)presented oxidation at Cys-245, peptidyl-prolyl cis-trans isomerase A (Ppia) wasoxidized at Cys-161 and 5,6-dihydroxyindole-2-carboxylic acid oxidase (Tyrp1)was oxidized at Cys-65, Cys-30, and Cys-336 after melanogenesis stimulation fol-lowed by RB-PDT. The redox alterations observed in murine melanoma cells andidentification of possible target proteins are of great importance to further under-stand tumor resistance mechanisms.
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Melanogenesis is a biosynthetic pathway for the formation of the pigment melanin in human skin. A key enzyme in the process of pigmentation through melanin is tyrosinase, which catalyzes the first and only limiting step in melanogenesis. Since the discovery of its methanogenic properties, tyrosinase has been the focus of research related to the anti-melanogenesis. In addition to developing more effective and commercially safe inhibitors, more studies are required to better understand the mechanisms involved in the skin depigmentation process. However, in vivo assays are necessary to develop and validate new drugs or molecules for this purpose, and to accomplish this, zebrafish has been identified as a model organism for in vivo application. In addition, such model would allow tracking and studying the depigmenting activity of many bioactive compounds, important to genetics, medicinal chemistry and even the cosmetic industry. Studies have shown the similarity between human and zebrafish genomes, encouraging their use as a model to understand the mechanism of action of a tested compound. Interestingly, zebrafish skin shares many similarities with human skin, suggesting that this model organism is suitable for studying melanogenesis inhibitors. Accordingly, several bioactive compounds reported herein for this model are compared in terms of their molecular structure and possible mode of action in zebrafish embryos. In particular, this article described the main metabolites of Trichoderma fungi, in addition to substances from natural and synthetic sources.
Assuntos
Melaninas , Peixe-Zebra , Animais , Humanos , Melaninas/metabolismo , Peixe-Zebra/metabolismo , Monofenol Mono-Oxigenase , Pele , Estrutura MolecularRESUMO
The anti-tyrosinase activity of the leaf extract of Schinus terebinthifolius, also known as Brazilian peppertree, was evaluated using multiple inâ silico approaches, such as molecular homology, molecular docking, MM-GBSA, molecular dynamics, MM-PBSA, QSAR, and skin permeability predictions. With these computational tools, the compounds that downregulate tyrosinase enzyme activity could be evaluated, and more potent molecules could be identified. The results indicated that various compounds, especially luteolin, are accountable for the anti-tyrosinase activity of S.â terebinthifolius. For cosmetic application, further studies with luteolin are especially recommended, for having presented a good performance both in theoretical inhibition (30.92â kJ mol-1 ) and skin permeability (LogKp=-6.62â cm-1 ).
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Anacardiaceae , Humanos , Luteolina , Simulação de Acoplamento Molecular , Monofenol Mono-Oxigenase , Extratos Vegetais/farmacologiaRESUMO
OBJECTIVE: Melanin gives some natural protection against the harmful effects of ultraviolet radiation; however, excessive production of melanin causes skin hyperpigmentation. Depigmenting cosmetics can be used to control this process; however, depigmenting agents commonly used have some disadvantages, such as low bioavailability, photosensitization, cellular toxicity, and insolubility. Natural sources of melanogenic inhibitors have become important alternatives to synthetic ones. The objective of this review was to summarize the results of studies on natural extracts that have been reported in the literature to inhibit the process of melanogenesis, giving a view on their suitability for potential use in new cosmetic formulations for skin-lightening. DATA SOURCES: A systematic literature search was carried out using the descriptors: "melanogenesis", "tyrosinase", "tyrosinase inhibition", and "natural agents". STUDY SELECTION: Publications were selected based on our designated inclusion and exclusion criteria, and a total of 15 studies met these criteria. DATA EXTRACTION: The following were used in the review of each paper which met the criteria: the name of the plant (all of the natural extracts turned out to be from plants), the method used to obtain the plant extract, the method for evaluating anti-tyrosinase activity, the main results, and the conclusions. DATA SYNTHESIS: All evaluated natural agents demonstrated anti-tyrosinase effect. The species Leathesia difformis, Morus alba, Orostachys japonicus, Heracleum moellendorffii, Coix lacryma-jobi (adlay), Inula brittanica, and Gailardia aristata stood out from the others due to their application as potential inhibitors of more than three proteins related to melanogenesis, including the cyclic adenosine monophosphate response element-binding protein, microphthalmia-associated transcription factor, tyrosinase, tyrosinase-related protein-1, tyrosinase-related protein-2, and dopachrome tautomerase. CONCLUSION: The plants present an anti-tyrosinase effect that must be better explored in the new cosmetic formulations. The anti-melanogenic effects of the plant are mainly related to the presence of phenolic and antioxidant compounds.
OBJECTIF: La mélanine offre une certaine protection naturelle contre les effets nocifs des rayons ultraviolets ; cependant, une production excessive de mélanine provoque une hyperpigmentation cutanée. Les cosmétiques dépigmentants peuvent servir à contrôler ce processus ; cependant, les agents dépigmentants couramment utilisés présentent certains inconvénients, comme une biodisponibilité faible, une photosensibilité, une toxicité cellulaire et une insolubilité. Les sources naturelles d'inhibiteurs de la mélanogénèse sont devenues des alternatives importantes aux inhibiteurs synthétiques. L'objectif de cette revue était de résumer les résultats des études sur les extraits naturels signalés dans la littérature comme inhibant le processus de mélanogenèse, en donnant un aperçu de leur adéquation à une utilisation potentielle dans de nouvelles formulations cosmétiques pour l'éclaircissement de la peau. SOURCES DES DONNÉES: Une recherche systématique dans la littérature a été réalisée à l'aide des descripteurs : « mélanogenèse ¼, « tyrosinase ¼, 'inhibition de la tyrosinase ¼ et « agents naturels ¼. Sélection des études : Les publications ont été sélectionnées d'après nos critères d'inclusion et d'exclusion désignés et un total de 15 études remplissaient ces critères. EXTRACTION DES DONNÉES: Les éléments suivant ont été utilisés dans l'examen de chaque article répondant aux critères : le nom de la plante (tous les extraits naturels se sont avérés provenir des plantes), la méthode utilisée pour obtenir l'extrait végétal, la méthode d'évaluation de l'activité anti-tyrosinase, les principaux résultats et les conclusions. SYNTHÈSE DES DONNÉES: Tous les agents naturels évalués ont démontré un effet anti-tyrosinase. Les espèces Leathesia difformis, Morus alba, Orostachys japonicus, ,Heracleum moellendorffii, Coix lacryma-jobi (adlay), Inula brittanica, et Gailardia aristata se sont distinguées des autres en raison de leur application comme inhibiteurs potentiels de plus de trois protéines liées à la mélanogenèse, dont la protéine de liaison d'élément de réponse d'adénosine monophosphate cyclique, du facteur de transcription associé à la microphtalmie, la tyrosinase, la protéine liée à la tyrosinase-1, la protéine liée à la tyrosinase-2 et la dopachrome tautomérase. CONCLUSION: Les plantes présentent un effet anti-tyrosinase qui doit être exploré plus en profondeur dans les nouvelles formulations cosmétiques. Les effets inhibiteurs de la mélanogénèse des plantes sont principalement dus à la présence de composés phénoliques et antioxydants.
Assuntos
Hiperpigmentação , Melanoma Experimental , Animais , Melaninas , Monofenol Mono-Oxigenase , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Raios UltravioletaRESUMO
Coloration traits are central to animal communication; they often govern mate choice, promote reproductive isolation and catalyse speciation. Specific genetic changes can cause variation in coloration, yet far less is known about how overall coloration patterns-which involve combinations of multiple colour patches across the body-can arise and are genomically controlled. We performed genome-wide association analyses to link genomic changes to variation in melanin (eumelanin and pheomelanin) concentration in feathers from different body parts in the capuchino seedeaters, an avian radiation with diverse colour patterns despite remarkably low genetic differentiation across species. Cross-species colour variation in each plumage patch is associated with unique combinations of variants at a few genomic regions, which include mostly non-coding (presumably regulatory) areas close to known pigmentation genes. Genotype-phenotype associations can vary depending on patch colour and are stronger for eumelanin pigmentation, suggesting eumelanin production is tightly regulated. Although some genes are involved in colour variation in multiple patches, in some cases, the SNPs associated with colour changes in different patches segregate spatially. These results suggest that coloration patterning in capuchinos is generated by the modular combination of variants that regulate multiple melanogenesis genes, a mechanism that may have promoted this rapid radiation.
Assuntos
Plumas , Estudo de Associação Genômica Ampla , Animais , Genoma , Melaninas , Fenótipo , Pigmentação/genéticaRESUMO
Tyrosinases belong to the functional copper-containing proteins family, and their structure contains two copper atoms, in the active site, which are coordinated by three histidine residues. The biosynthesis of melanin in melanocytes has two stages depending on the actions of the natural substrates L-DOPA and L-tyrosine. The dysregulation of tyrosinase is involved in skin cancer initiation. In the present study, using molecular modeling tools, we analyzed the inhibition activity of tyrosinase activity using kojic acid (KA) derivatives designed from aromatic aldehydes and malononitrile. All derivatives showed conformational affinity to the enzyme active site, and a favorable distance to chelate the copper ion, which is essential for enzyme function. Molecular dynamics simulations revealed that the derivatives formed promising complexes, presenting stable conformations with deviations between 0.2 and 0.35 Å. In addition, the investigated KA derivatives showed favorable binding free energies. The most stable KA derivatives showed the following binding free energies: -17.65 kcal mol-1 (D6), -18.07 kcal mol-1 (D2), -18.13 (D5) kcal mol-1, and -10.31 kcal mol-1 (D4). Our results suggest that these derivatives could be potent competitive inhibitors of the natural substrates of L-DOPA (-12.84 kcal mol-1) and L-tyrosine (-9.04 kcal mol-1) in melanogenesis.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Simulação de Acoplamento Molecular/métodos , Simulação de Dinâmica Molecular , Monofenol Mono-Oxigenase/antagonistas & inibidores , Monofenol Mono-Oxigenase/química , Pironas/química , Pironas/farmacologia , Domínio Catalítico , Humanos , Levodopa/metabolismo , Melaninas/biossíntese , Melanócitos/metabolismo , Melanoma/metabolismo , Estrutura Molecular , Monofenol Mono-Oxigenase/metabolismo , Neoplasias Cutâneas/metabolismo , Relação Estrutura-Atividade , Tirosina/metabolismoRESUMO
Oculocutaneous albinism (OCA) is a genetic disorder characterized by skin, hair, and eye hypopigmentation due to a reduction or absence of melanin. Clinical manifestations include vision problems and a high susceptibility to skin cancer. In its non-syndromic form, OCA is associated with six genes and one chromosomal region. Because OCA subtypes are not always clinically distinguishable, molecular analysis has become an important tool for classifying types of OCA, which facilitates genetic counseling and can guide the development of new therapies. We studied eight Brazilian individuals aged 1.5-18 years old with clinical diagnosis of OCA. Assessment of ophthalmologic characteristics showed results consistent with albinism, including reduced visual acuity, nystagmus, and loss of stereoscopic vision. We also observed the appearance of the strabismus and changes in static refraction over a 2-year period. Dermatologic evaluation showed that no participants had preneoplastic skin lesions, despite half of the participants reporting insufficient knowledge about skin care in albinism. Whole-exome and Sanger sequencing revealed eight different mutations: six in the TYR gene and two in the SLC45A2 gene, of which one was novel and two were described in a population study but were not previously associated with the OCA phenotype. We performed two ophthalmological evaluations, 2 years apart; and one dermatological evaluation. To the best of our knowledge, this is the first study to perform clinical follow-up and genetic analysis of a Brazilian cohort with albinism. Here, we report three new OCA causing mutations.
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Vitiligo is an acquired pigmentary disorder resulting from selective destruction of melanocytes. Emerging studies have suggested that T helper cell 17 (Th17) is potentially implicated in vitiligo development and progression. It was recently discovered that metabotropic glutamate receptor 4 (mGluR4) can modulate Th17-mediated adaptive immunity. However, the influence of mGluR4 on melanogenesis of melanocytes has yet to be elucidated. In the present study, we primarily cultured mouse bone marrow-derived dendritic cells (BMDC) and then knocked down and over-expressed mGluR4 using transfection. Transduced BMDC were co-cultured with CD4+ T cells and the expression of Th17-related cytokines were measured. The morphology and melanogenesis of B16 cells were observed after being treated with co-culture medium of CD4+ T cells and transduced BMDC. We found that mGluR4 knockdown did not affect the co-stimulatory CD80 and CD86 upregulation after lipopolysaccharide stimulation but did increase the expression of Th17-related cytokines, and further down-regulated the expression of microphthalmia-associated transcription factor (MITF) and the downstream genes, decreased melanin production, and destroyed the morphology of B16 cells. Conversely, over-expression of mGluR4 reduced the expression of CD80 and CD86, suppressed the production of Th17-related cytokines, increased the expression of MITF, and did not destroy the morphology of B16 cells. Our study confirmed that mGluR4 modulated the Th17 cell polarization and resulted in the alteration of melanogenesis and morphology of B16 cells. Collectively, these findings suggest mGluR4 might be a potent target involved in the immune pathogenesis of vitiligo.
Assuntos
Animais , Masculino , Vitiligo/imunologia , Células Dendríticas/citologia , Células da Medula Óssea/citologia , Diferenciação Celular/fisiologia , Receptores de Glutamato Metabotrópico/fisiologia , Células Th17/imunologia , Vitiligo/genética , RNA Interferente Pequeno/imunologia , Células Th17/citologia , Citometria de Fluxo , Melaninas/biossíntese , Melanócitos/citologia , Camundongos Endogâmicos C57BLRESUMO
The cytotoxicity of a dinuclear imine-copper (II) complex 2, and its analogous mononuclear complex 1, toward different melanoma cells, particularly human SKMEL-05 and SKMEL-147, was investigated. Complex 2, a tyrosinase mimic, showed much higher activity in comparison to complex 1, and its reactivity was verified to be remarkably activated by UVB-light, while the mononuclear compound showed a small or negligible effect. Further, a significant dependence on the melanin content in the tumor cells, both from intrinsic pigmentation or stimulated by irradiation, was observed in the case of complex 2. Similar tests with keratinocytes and melanocytes indicated a much lower sensitivity to both copper (II) complexes, even after exposition to UV light. Clonogenic assays attested that the fractions of melanoma cells survival were much lower under treatment with complex 2 compared to complex 1, both with or without previous irradiation of the cells. The process also involves generation of reactive oxygen species (ROS), as verified by EPR spectroscopy, and by using fluorescence indicators. Autophagic assays indicated a remarkable formation of cytoplasmic vacuoles in melanomas treated with complex 2, while this effect was not observed in similar treatment with complex 1. Monitoring of specific protein LC3 corroborated the simultaneous occurrence of autophagy. A balance interplay between different modes of cell death, apoptosis and autophagy, occurs when melanomas were treated with the dinuclear complex 2, in contrast to the mononuclear complex 1. These results pointed out to different mechanisms of action of such complexes, depending on its nuclearity.
Assuntos
Complexos de Coordenação/química , Cobre/química , Iminas/química , Monofenol Mono-Oxigenase/metabolismo , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Complexos de Coordenação/metabolismo , Complexos de Coordenação/farmacologia , Espectroscopia de Ressonância de Spin Eletrônica , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos da radiação , Humanos , Melaninas/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tubulina (Proteína)/metabolismo , Raios UltravioletaRESUMO
The llama (Lama glama) is a fiber-producing species that presents a wide range of coat colors, among which white is one of the most important for the textile industry. However, there is little information about the molecular mechanisms that control the white phenotype in this species. In domestic mammals, a white coat is usually produced by mutations in the KIT proto-oncogene receptor tyrosine kinase (KIT) and microphthalmia-associated transcription factor (MITF) genes. In this work we have sequenced and described the coding regions of KIT and MITF-M, the melanocyte-specific isoform, and the two transcriptional variants MITF-M(-) and MITF-M(+). Moreover, we studied the expression of these genes in the skin of white and colored llamas. Although no variants were revealed to be associated with white coat color, significant differences between phenotypes were observed in the expression levels of KIT and MITF-M. Interestingly, white llamas expressed less MITF-M(+) than did colored ones, which is consistent with a consequent reduction in the synthesis of melanin. Even though our results indicate that downregulation of KIT and MITF-M expression is involved in white phenotype production in llamas, the causative gene of white coat color remains unknown.
Assuntos
Camelídeos Americanos/genética , Regulação da Expressão Gênica , Variação Genética , Fator de Transcrição Associado à Microftalmia/genética , Fases de Leitura Aberta/genética , Proteínas Proto-Oncogênicas c-kit/genética , Animais , Camelídeos Americanos/fisiologia , Cabelo/química , Cor de Cabelo/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Análise de Sequência de DNA/veterináriaRESUMO
Abstract Melanogenesis is a biological process which led to the synthesis of melanin pigment. Abnormal melanin production results in melasma, solar lentigo, post inflammatory melanoderma, etc. In this study, we examined the potential inhibitory effects of 17 brown macroalgae from Persian Gulf on melanogenesis. The effects of various concentrations (100, 250 and 500 µg/mL) of methanolic extracts of macroalgae belonging to four genera (including: Padina, Colpomonia, Cystoseira and Sargassum) were studied on oxidation of L-Dopa by mushroom tyrosinase. Subsequently, the activity of macroalgae with high inhibitory effect on monophenolase activity of mushroom tyrosinase and zebrafish was investigated using L-tyrosine as a substrate. Anti-melanogenesis effects of algae extracts were studied on zebrafish as an alternative in vivo model. Kojic acid was used as a positive control. All the tested macroalgae showed inhibitory effect on activities of diphenolase and monophenolase (of mushroom tyrosinase). P. boergesinii exhibited the most in vivo anti-tyrosinase activity compared with other samples. P. boergesenii inhibited zebrafish tyrosinase more potent than kojic acid (83% vs 50% inhibition for kojic acid). Moreover, it reduced melanin synthesis in zebrafish 42% (kojic acid: 50%).
Assuntos
Monofenol Mono-Oxigenase/análise , Microalgas/química , Peixe-Zebra , Oceano ÍndicoRESUMO
BACKGROUND: Hyperpigmentation disorders such as post-inflammatory hyperpigmentation are major concerns not only in light-skinned people but also in Asian populations with darker skin. The anti-tyrosinase and immunomodulatory effects of sericin have been known for decades. However, the therapeutic effects of sericin on hyperpigmentation disorders have not been well documented. METHODS: In this study, we used an in vitro model to study the anti-tyrosinase, tolerogenic, and anti-melanogenic effects of sericin on Staphylococcus aureus peptidoglycan (PEG)-stimulated melanocytes, dendritic cells (DCs), and artificial skin (MelanoDerm™). Enzyme-linked immunosorbent assay, conventional and immunolabeled electron microscopy, and histopathological studies were performed. RESULTS: The results revealed that urea-extracted sericin has strong anti-tyrosinase properties as shown by a reduction of tyrosinase activity in melanin pigments both 48 h and 10 days after allergic induction with PEG. Anti-inflammatory cytokines including interleukin (IL)-4, IL-10, and transforming growth factor-ß were upregulated upon sericin treatment (10, 20, and 50 µg/mL), whereas production of allergic chemokines, CCL8 and CCL18, by DCs was diminished 48 h after allergic induction with PEG. Moreover, sericin lowered the expression of micropthalmia-associated transcription factor (MITF), a marker of melanogenesis regulation, in melanocytes and keratinocytes, which contributed to the reduction of melanin size and the magnitude of melanin deposition. However, sericin had no effect on melanin transport between melanocytes and keratinocytes, as demonstrated by a high retention of cytoskeletal components. CONCLUSION: In summary, sericin suppresses melanogenesis by inhibition of tyrosinase activity, reduction of inflammation and allergy, and modulation of MITF function.
Assuntos
Hiperpigmentação/tratamento farmacológico , Queratinócitos/efeitos dos fármacos , Melanócitos/efeitos dos fármacos , Monofenol Mono-Oxigenase/antagonistas & inibidores , Sericinas/farmacologia , Células Cultivadas , Humanos , Hipersensibilidade , Inflamação , Queratinócitos/ultraestrutura , Melanócitos/ultraestrutura , Fator de Transcrição Associado à Microftalmia , Microscopia Eletrônica , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/efeitos dos fármacosRESUMO
Previous studies showed that P1G10, a proteolytic fraction from Vasconcellea cundinamarcensis latex, reduced the tumor mass in animals bearing melanoma, increased in vitro DNA fragmentation and decreased cell adhesion. Here, we present some molecular and cellular events related to the antimetastatic effect induced by the CMS-2 fraction derived from P1G10 in metastatic melanoma B16-F10 and melanocyte Melan-a. Using difference gel electrophoresis and mass spectrometry, we identified four proteins overexpressed in tumor cells, all of them related to proliferation, survival, migration and cell invasion, that had their expression normalized upon treatment with CMS-2: nucleophosmin 1, heat shock protein 65, calcyclin binding protein and eukaryotic translation initiation factor 4H. In addition, some antioxidant and glycolytic enzymes show increased expression after exposure to CMS-2, along with an induction of melanogenesis (differentiation marker). The down regulation of cofilin 1, a protein involved in cell motility, may explain the inhibition of cell migration and dendritic-like outgrowth in B16-F10 and Melan-a, observed after CMS-2 treatment. Taken together, it is argued that CMS-2 modulates the expression of proteins related to metastatic development, driving the cell to a more differentiated-like state. These effects support the CMS-2 antimetastatic activity and place this fraction in the category of anticancer agent.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Caricaceae/enzimologia , Cisteína Proteases/farmacologia , Melanoma/tratamento farmacológico , Metástase Neoplásica/tratamento farmacológico , Proteínas de Plantas/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cisteína Proteases/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Melanoma/patologia , Camundongos , Metástase Neoplásica/patologia , NucleofosminaRESUMO
BACKGROUND: Hyperpigmentation disorders such as post-inflammatory hyperpigmentation are major concerns not only in light-skinned people but also in Asian populations with darker skin. The anti-tyrosinase and immunomodulatory effects of sericin have been known for decades. However, the therapeutic effects of sericin on hyperpigmentation disorders have not been well documented. METHODS: In this study, we used an in vitro model to study the anti-tyrosinase, tolerogenic, and anti-melanogenic effects of sericin on Staphylococcus aureus peptidoglycan (PEG)-stimulated melanocytes, dendritic cells (DCs), and artificial skin (MelanoDerm™). Enzyme-linked immunosorbent assay, conventional and immunolabeled electron microscopy, and histopathological studies were performed. RESULTS: The results revealed that urea-extracted sericin has strong anti-tyrosinase properties as shown by a reduction of tyrosinase activity in melanin pigments both 48 h and 10 days after allergic induction with PEG. Anti-inflammatory cytokines including interleukin (IL)-4, IL-10, and transforming growth factor-p were upregulated upon sericin treatment (10, 20, and 50 µg/mL), whereas production of allergic chemokines, CCL8 and CCL18, by DCs was diminished 48 h after allergic induction with PEG. Moreover, sericin lowered the expression of micropthalmia-associated transcription factor (MITF), a marker of melanogenesis regulation, in melanocytes and keratinocytes, which contributed to the reduction of melanin size and the magnitude of melanin deposition. However, sericin had no effect on melanin transport between melanocytes and keratinocytes, as demonstrated by a high retention of cytoskeletal components. CONCLUSION: In summary, sericin suppresses melanogenesis by inhibition of tyrosinase activity, reduction of inflammation and allergy, and modulation of MITF function.
Assuntos
Humanos , Queratinócitos/efeitos dos fármacos , Monofenol Mono-Oxigenase/antagonistas & inibidores , Hiperpigmentação/tratamento farmacológico , Sericinas/farmacologia , Melanócitos/efeitos dos fármacos , Fatores de Transcrição/efeitos dos fármacos , Microscopia Eletrônica , Transdução de Sinais/efeitos dos fármacos , Queratinócitos/ultraestrutura , Células Cultivadas , Fator de Transcrição Associado à Microftalmia , Hipersensibilidade , Inflamação , Melanócitos/ultraestruturaRESUMO
Melanoma is a rare and aggressive skin tumor; the survival of patients diagnosed late is fairly low. This high mortality rate is due to the characteristics of the cells that allow them to be resistant to radiotherapy and conventional chemotherapy, besides of being able to evade the immune system. Melanin, the pigment responsible for skin, hair and eye color, seems to be involved in this resistance. The main function of melanin is to protect the cells against ultraviolet (UV) light by absorbing this radiation and reactive oxygen species (ROS) scavenging. But this pigment may have also a role as photosensitizer, because when it is irradiated with UVA light (320-400 nm), the generation of ROS was detected. Besides, the melanogenesis stimulation on B16-F10 cells resulted in cell cycle arrest, induction of a quiescent state, change in the expression of several proteins and alterations on ADP/ATP ratio. The present study aimed to investigate the influence of melanogenesis stimulation in mitochondrial function of B16-F10 melanoma cells. Therefore, we analyzed cells respiration, mitochondrial membrane potential (Δψm) and mitochondria mass in B16-F10 melanoma cells stimulated with 0.4mML-tyrosine and 10mM NH4Cl. Our results showed that the induction of melanin synthesis was able to reduce significantly the oxygen consumption after 48h of stimulation, without changes of mitochondrial membrane potential when compared to non-stimulated cells. Despite of respiration inhibition, the mitochondria mass was higher in cells with melanogenesis stimulation. We suggest that the stimulation in the melanin synthesis might be promoting the inhibition of electrons transport chain by some intermediate compound from the synthesis of the pigment and this effect could contribute to explain the entry in the quiescent state.
Assuntos
Melaninas/biossíntese , Melanoma Experimental/metabolismo , Mitocôndrias/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Monofenol Mono-Oxigenase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tirosina/metabolismoRESUMO
El melanoma humano (Cáncer a la piel), se origina por muchos factores, uno de ellos la sobreexposición solar o las mutaciones que esta pueda generar en diversos genes los cuales transcriben una sobreproducción de melanina, otros factores son los antecedentes familiares, ya que el gen mutado podría ser o no expresado en las siguientes generaciones. La melanina proviene de la melanogénesis en la cual juega un rol muy importante la enzima tirosinasa, la sobreexpresión de esta puede ocasionar una serie de desórdenes en el organismo, como la producción excesiva e innecesaria de melanina y esto conducir a trastornos y/o patologías de hiperpigmentación, desde simples problemas estéticos como las pecas hasta graves problemas clínicos como el melanoma humano. En los últimos años el uso de plantas medicinales han sido un gran aliado de la salud humana, para contrarrestar un sin número de enfermedades, y han sido una alternativa para evitar el consumo de productos sintetizados químicamente, el cedrón (Lippia citriodora) es utilizado como medicina alternativa en Perú para trastornos digestivos, trastornos en el sistema nervioso como un relajante (en insomnio y ansiedad) y en estados gripales, por lo que el tema principal en este estudio es la evaluación del extracto metanólico de cedrón (Lippia citriodora) como inhibidor de tirosinasa en la melanogénesis, los ensayos mostraron que el extracto no es citotóxico sin embargo no presento una actividad inhibitoria significativa como se esperaban, pero son un indicio de que podría utilizarse como un ayudante en los tratamientos de melanoma humano. Por lo que se concluye que a una concentración de 40 µg/ml del extracto metanólico de cedrón (Lippia citriodora) se obtuvo un mejor resultado, logrando inhibir la acción de la enzima tirosinasa en comparación de las otras concentración estudiadas en este trabajo.
Assuntos
Animais , Camundongos , Monofenol Mono-Oxigenase , Lippia , Melanoma , Peru , Técnicas In Vitro , Extratos Vegetais , Medicina TradicionalRESUMO
Skin aging is a natural process of the human body that may be accelerated due to extrinsic causes. Libidibia ferrea, popularly known as jucá, is a small tree, which possesses an abundant phenolic composition with potential antioxidant and enzymatic inhibition activities. Thus, this work aimed to investigate the anti-wrinkle and anti-whitening potentials of jucá trunk bark (LFB) and pod (LFP) extracts. A comprehensive analysis of LFB and LFP phenolic composition was accomplished by means of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Effects on skin degradation were assessed by inhibitory enzymatic activity against elastase, hyaluronidase and collagenase through colorimetric assays. Cellular viability in B16F10 and primary fibroblasts were determined by Trypan Blue exclusion assay. Anti-melanogenic effects on B16F10 cells were evaluated using cellular tyrosinase, melanin content, western blot, and RT-qPCR analyses. Inhibition of matrix metalloproteinase-2 and metalloproteinase-9 (MMP-2 and MMP-9) was determined by gelatin zymography and western blot methodologies. LC-MS/MS analyses of LFB and LFP extracts allowed the characterization of 18 compounds, among them, flavonoids, phenolic acids, and secoridoids. Additionally the pod and trunk bark compositions were compared. Hyaluronidase inhibitory activity for both extracts, LFB (IC50 = 8.5 ± 0.8 µg/mL) and LFP (IC50 = 16 ± 0.5 µg/mL), was stronger than standard rutin (IC50 = 27.6 ± 0.06). Pro-MMP-2 was significantly inhibited by both extracts. LFB and LFP decreased the melanin content in B16F10 due to tyrosinase inhibitory activity. L. ferrea extracts has high potential as a cosmetic ingredient due to its anti-wrinkle and depigmentant effects.
Assuntos
Caesalpinia/química , Melaninas/metabolismo , Extratos Vegetais/farmacologia , Envelhecimento da Pele/efeitos dos fármacos , Animais , Antioxidantes/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular , Cosméticos/farmacologia , Precursores Enzimáticos/metabolismo , Fibroblastos , Flavonoides/farmacologia , Gelatinases/metabolismo , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Monofenol Mono-Oxigenase/metabolismo , Fenóis/farmacologia , Casca de Planta , Cultura Primária de Células , Espectrometria de Massas em TandemRESUMO
Melanin production within melanocytes is regulated, among others, by estradiol, whose effects on melanogenesis are still not completely elucidated. Here we show that although 10(-7) M 17ß-estradiol (E2) increased tyrosinase mRNA levels in B16-F10 malignant melanocytes, there was a transient decrease and abolishment of the temporal variation of melanin content. Both parameters were much higher in the malignant than in normal Melan-a cells. Considering that silencing clock machinery in human melanocytes increases melanogenesis, we investigated clock gene expression in those cell lines. Except for Melan-a Bmal1 and B16-F10 Per2 expression of control cells, Per1, Per2, and Bmal1 expression increased independently of cell type or E2 treatment after 24 h. However, melanoma cells showed a marked increase in Per1 and Bma11 expression in response to E2 at the same time points, what may rule out E2 as a synchronizer agent since the expression of those genes were not in antiphase. Next, we investigated the expression of Xpa, a clock-controlled gene, which in Melan-a cells, peaked at 18 h, and E2 treatment shifted this peak to 24 h, whereas B16-F10 Xpa expression peaked at 24 h in both control and E2 group, and it was higher compared to Melan-a cells in both groups. Therefore, malignant and normal melanocytes display profound differences on core elements of the local clock, and how they respond to E2, what is most probably determinant of the differences seen on melanin synthesis and Tyrosinase and Xpa expression. Understanding these processes at the molecular level could bring new strategies to treat melanoma.