Development of 5-[(3-aminopropyl)phosphinooxy]-2-(hydroxymethyl)-4H-pyran-4-one as a novel whitening agent

A stable derivative of kojic acid, 5-[(3-aminopropyl)phosphinooxy]-2-(hydroxymethyl)-4H-pyran-4-one (Kojyl-APPA), was synthesized in good yield. The effects of Kojyl-APPA on tyrosinase activity and melanin synthesis were investigated. Kojyl-APPA showed tyrosinase inhibition effect (30%) in situ, but not in vitro. Kojyl-APPA inhibited tyrosinase activity significantly at 24 h after treatment in normal human melanocytes. It means that Kojyl-APPA is not a direct inhibitor of tyrosinase itself, but it is converted to a potential inhibitor kojic acid enzymatically in cells. In addition, Kojyl-APPA decreased melanin content to 75% of control in melanoma cells and decreased neomelanin synthesis to 43% of control in normal human melanocytes. Its permeation through skin increased by about 8 times as compared with kojic acid.     

Effect of Extracorporeal Shockwave Treatment on the Melanogenic Activity of Cultured Melanocytes

In addition to the traditional lithotripsy treatment, extracorporeal shockwaves (ESWs) have been shown to be effective in the treatment of certain musculoskeletal disorders and in enhancing skin flap neovascularization. However, relatively little is known about its effect on melanocytes. To investigate its effect on the melanogenic activity of cultured melanocytes, mouse B16F10 melanocytes were treated with defocused ESWs of different energies (15, 21, and 27 kV) and at different doses (300 and 600 impulses). Cell viability was measured 1 and 24 h after treatment. Melanin content was measured and compared against a standard curve generated with fungal melanin. Cellular tyrosinase activity was calculated with the 3,4-dihydroxyphenylalanine (DOPA) oxidase assay. The results demonstrated that ESW treatment reduced cell viability. Our results also indicated that the overall decrease in cell viability lasted for 6 days. After ESW treatment with 300 or 600 impulses at 21 kV, no significant change in melanin content or tyrosinase activity of the B16F10 melanocytes was noted as compared to those of the control. The present study suggests that ESW treatment does not alter the melanogenic activity of the cultured melanocytes.     

1-(2,4-dihydroxyphenyl)-3-(2,4-dimethoxy-3-methylphenyl)propane, a novel tyrosinase inhibitor with strong depigmenting effects

A series of diarylpropane compounds was isolated by screening a plant extract library for inhibitors of mushroom tyrosinase. The most potent compound, 1-(2,4-dihydroxyphenyl)-3-(2,4-dimethoxy-3-methylphenyl)propane (UP302: CAS# 869743-37-3), was found in the medicinal plant Dianella ensifolia. Synthetic and plant-derived versions of UP302 inhibited mushroom tyrosinase with similar potencies. UP302 inhibited mushroom tyrosinase with K(i)=0.3 microM, in a competitive and reversible fashion. UP302 was 22 times more potent than Kojic acid in inhibiting murine tyrosinase, with IC(50) values of 12 and 273 microM respectively. Experiments on mouse melanoma cells B16-F1 and on human primary melanocytes demonstrated that UP302 inhibits melanin formation with IC(50) values of 15 and 8 microM respectively. Long-term treatment of cultured melanocytes with up to 62 microM of UP302 revealed no detectable cytotoxicity. In a reconstructed skin model (MelanoDerm) topical application of 0.1% UP302 resulted in significant skin lightening and decrease of melanin production without effects on cell viability, melanocyte morphology or overall tissue histology. In conclusion, UP302 is a novel tyrosinase inhibitor that suppresses melanin production in both cultured melanocytes and reconstructed skin with high potency and without adverse side effects.     

黑色素的合成及小分子对其功能的调控

黑色素作为一种天然色素, 可以大致分为真黑素和褐黑素. 它们广泛存在于微生物、 高等动物和植物体内, 具有自由基清除、 辐射防护和热调节等功能. 对人类而言, 黑色素在一定程度上影响着皮肤、 头发以及眼睛的颜色, 在保护皮肤免受紫外线照射产生有害损伤方面具有重要作用. 黑素细胞功能异常会带来一系列的皮肤问题, 如黑色素瘤和白癜风等疾病. 因此, 调控黑色素的产生是治疗色素相关疾病的重要途径. 黑色素合成过程中涉及到酪氨酸酶、 酪氨酸酶相关蛋白酶等多种酶的催化和化学反应. 通过小分子调控这些酶的催化过程, 改变其活性及表达是调控黑色素合成的有效途径. 在生物体内, 黑色素都是通过生物合成的. 由于黑色素的独特功能, 化学家也开发了一些人工化学合成黑色素的方法. 在小分子调控黑色素功能方面, 已发现多种可抑制黑色素形成的小分子, 这些小分子为黑色素相关疾病的治疗提供了新途径. 本文综合评述了黑色素的合成(包括生物合成与人工合成)、 抑制机理以及小分子化合物对黑色素的调控, 为开发安全、 高效的黑色素相关药物提供了理论基础.     

Role of UV in cutaneous melanoma

Malignant melanoma arises from epidermal melanocytes, the cells responsible for the production of the skin pigment melanin. The photoprotective role of melanin, which is transferred to neighboring keratinocytes, in UV-induced skin carcinogenesis, specifically in nonmelanoma skin cancers, has been well documented. Although melanocyte-resident melanin is expected to offer similar protection to melanocytes from UV-induced damage, UV radiation has long been suspected to have an etiologic role in cutaneous melanoma. However, nearly three decades of efforts using a variety of in vitro and in vivo models of human skin and mouse genetic models have produced conflicting data. Epidemiologic studies have also failed to establish a definitive association between UV exposure and risk of melanoma. In this review, we evaluate the dual role of the melanin pigment as a photoprotector as well as a photosensitizer and examine the evidence for association between melanin levels (constitutive and induced) and melanoma risk. We also discuss possible reasons for the lack of signature UV mutations in melanoma oncogenes known to date and potential alternative mechanisms to explain the role of UV in melanomagenesis.     

Increased melanogenesis is a risk factor for oxidative DNA damage--study on cultured melanocytes and atypical nevus cells

Melanin synthesis is an oxygen-dependent process that acts as a potential source of reactive oxygen species (ROS) inside pigment-forming cells. The synthesis of the lighter variant of melanin, pheomelanin, consumes cysteine and this may limit the capacity of the cellular antioxidative defense. We show that tyrosine-induced melanogenesis in cultured normal human melanocytes (NHM) is accompanied by increased production of ROS and decreased concentration of intracellular glutathione. Clinical atypical (dysplastic) nevi (DN) regularly contain more melanin than do normal melanocytes (MC). We also show that in these cultured DN cells three out of four exhibit elevated synthesis of pheomelanin and this is accompanied by their early senescence. By using various redox-sensitive molecular probes, we demonstrate that cultured DN cells produce significantly more ROS than do normal MC from the same donor. Our experiments employing single-cell gel electrophoresis (comet assay) usually reveal higher fragmentation of DNA in DN cells than in normal MC. Even if in some cases the normal alkaline comet assay shows no differences in DNA fragmentation between DN cells and normal MC, the use of the comet assay with formamidopyrimidine DNA glycosylase can disclose that the DNA of the cultured DN cells harbor more oxidative damage than the DNA of normal MC from the same person.     

First total synthesis of (+/-)-Linderol A,a tricyclic hexahydrodibenzofuran constituent of Lindera umbellata bark,with potent inhibitory activity on melanin biosynthesis of cultured B-16 melanoma cells

The first total synthesis of (+/-)-Linderol A, a hexahydrodibenzofuran constituent of Lindera umbellata bark, with potent inhibitory activity on the melanin biosynthesis of cultured B-16 melanoma cells, was achieved through 19 steps of reaction in 6.6% overall yield, in which the critical step was a tandem reaction of a 3-ethoxycarbonylcoumarin derivative with dimethylsulfoxonium methylide to yield the 2-ethoxycarbonylcyclopenta[b]benzofuran-3-ol derivative.     

Melanosomal damage in normal human melanocytes induced by UVB and metal uptake--a basis for the pro-oxidant state of melanoma

Melanins are ubiquitous catecholic pigments, formed in organelles called melanosomes within melanocytes, the function of which is to protect skin against harmful effects of UV radiation. Melanosomes within melanoma cells are characteristically abnormal, with fragmented melanin and disrupted membranes. We hypothesize that the disruption of melanosomal melanin might be an early event in the etiology and progression of melanoma, leading to increased oxidative stress and mutation. In this report, we examine the effect of a combination of UV treatment and metal ion exposure on melanosomes within melanocytes, as well as their ability to act as pro-oxidants in ex situ experiments, and assay the effects of this treatment on viability and cell cycle progression. UVB exposure causes morphologic changes of the cells and bleaching of melanosomes in normal melanocytes, both significantly enhanced in Cu(II) and Cd(II)-treated cells, as observed by microscopy. The promoted bleaching by Cu(II) is due to its ability to redox cycle under oxidative conditions, generating reactive oxygen species; verified by the observed enhancement of hydroxyl radical generation when isolated melanosomes were treated with both Cu(II) ions and UVB, as assayed by DNA clipping. Single-dose UVB/Cu treatment does not greatly affect cell viability or cell cycle progression in heavily pigmented cells, but did so in an amelanotic early stage melanoma cell line.     

Molecular responses to stress induced in normal human caucasian melanocytes in culture by exposure to simulated solar UV

Melanocytes play a central role in the response of skin to sunlight exposure. They are directly involved in UV-induced pigmentation as a defense mechanism. However, their alteration can lead to melanoma, a process where the role of sun overexposure is highly probable. The transformation process whereby UV damage may result in melanoma initiation is poorly understood, especially in terms of UV-induced genotoxicity in pigmented cells, where melanin can act either as a sunscreen or as a photosensitizer. The aim of this study was to analyze the behavior of melanocytes from fair skin under irradiation mimicking environmental sunlight in terms of spectral power distribution. To do this, normal human Caucasian melanocytes in culture were exposed to simulated solar UV (SSUV, 300-400 nm). Even at relatively high doses (until 20 min exposure, corresponding to 12 kJ/m2 UV-B and 110 kJ/m2 UV-A), cell death was limited, as shown by cell viability and low occurrence of apoptosis (caspase-3 activation). Moreover, p53 accumulation was three times lower in melanocytes than in unpigmented cells such as fibroblasts after SSUV exposure. However, an important fraction of melanocyte population was arrested in G2-M phase, and this correlated well with a high induction level of the gene GADD45, 4 h after exposure. Among the genes involved in DNA repair, gene XPC was the most inducible because its expression increased more than two-fold 15 h after a 20 min exposure, whereas expression of P48 was only slightly increased. In addition, an early induction of Heme Oxygenase 1 (HO1) gene, a typical response to oxidative stress, was also observed for the first time in melanocytes. Interestingly, this induction remained significant when melanocytes were exposed to UV-A radiation only (320-400 nm), and stimulation of melanogenesis before irradiation further increased HO1 induction. These results were obtained with normal human cells after exposure to SSUV radiation, which mimicked natural sunlight. They provide new data related to gene expression and suggest that melanin in light skin could contribute to sunlight-induced genotoxicity and maybe to melanocyte transformation.     

Inhibitory Effect of Elaeagnus umbellata Fractions on Melanogenesis in α-MSH-Stimulated B16-F10 Melanoma Cells

When skin is exposed to UV radiation, melanocytes produce melanin. Excessive melanin production leads to skin pigmentation, which causes various cosmetic and health problems. Therefore, the development of safe, natural therapeutics that inhibit the production of melanin is necessary. Elaeagnus umbellata (EU) has long been widely used as a folk medicinal plant because of pharmacological properties that include anti-ulcer, antioxidant, and anti-inflammatory properties. In this study, we investigated the antioxidant activity and melanogenesis inhibitory effects of EU fractions in B16-F10 melanoma cells. EU fractions showed a dose-dependent increase in antioxidant activity in radical scavenging activity. In addition, we evaluated the effect of EU fractions on tyrosinase activity and melanogenesis in α-melanocyte-stimulating hormone-induced B16-F10 melanoma cells. EU was noncytotoxic at 12.5–50 μg/mL. EU fractions effectively inhibited tyrosinase activity and melanogenesis, suppressed the phosphorylation of CREB and ERK involved in the melanogenesis pathway, and down-regulated expression of melanogenesis-related proteins. Interestingly, the anti-melanogenesis effect was most effective at a concentration of 50 μg/mL EU, and the effects of the fractions were superior to those of the extract. Therefore, our study suggests that EU has potential as a safe treatment for excessive pigmentation or as a natural ingredient in cosmetics.     

Effects and Mechanism of Nicotinamide Against UVA‐ and/or UVB‐mediated DNA Damages in Normal Melanocytes

Melanoma incidences are increasing rapidly, and ultraviolet (UV) radiation from the sun is believed to be its major contributing factor. UV exposure causes DNA damage in skin which may initiate cutaneous skin cancers including melanoma. Melanoma arises from melanocytes, the melanin‐producing skin cells, following genetic dysregulations resulting into hyperproliferative phenotype and neoplastic transformation. Both UVA and UVB exposures to the skin are believed to trigger melanocytic hyperplasia and melanomagenesis. Melanocytes by themselves are deficient in repair of oxidative DNA damage and UV‐induced photoproducts. Nicotinamide, an active form of vitamin B3 and a critical component of the human body's defense system has been shown to prevent certain cancers including nonmelanoma skin cancers. However, the mechanism of nicotinamide's protective effects is not well understood. Here, we investigated potential protective effects and mechanism of nicotinamide against UVA‐ and/or UVB‐ induced damage in normal human epidermal melanocytes. Our data demonstrated an appreciable protective effect of nicotinamide against UVA‐ and/or UVB‐ induced DNA damage in melanocytes by decreasing both cyclobutane pyrimidine dimers and 8‐hydroxy‐2′‐deoxyguanosine levels. We found that the photoprotective response of nicotinamide was associated with the activation of nucleotide excision repair genes and NRF2 signaling. Further studies are needed to validate our findings in in vivo models.     

Cell Shape Normalization,Dendrite Orientation,and Melanin Production of Normal and Genetically Altered (Haploinsufficient NF1)‐Melanocytes by Microstructured Substrate Interactions

Little is known about how functional regulation failure in genetically altered cells is influenced by topographical confinement of cells, a situation often present in tissues in vivo. We used cultured melanocytes derived from human skin samples as a model system for such investigations. Normal melanocytes have a very well defined shape consisting of a cell body and two dendrites arranged 180° relative to each other. In contrast, neurofibromin 1‐melanocytes (NF1‐melanocytes) have up to a 50 % reduction of neurofibromin 1, which results in an altered morphology that can be easily measured. NF1‐melanocytes deviate from the defined structure of normal melanocytes by forming more than two dendrites per cell. We show that morphology consequences of genetically altered melanocytes can be canceled if cells interact with substrates microstructured by stripes that apply mechanophysical signals in the form of physical topography. The strength of the mechanophysical signal was varied systematically by increasing the height of the microstructures. Melanocytes respond to surface topographical features that are larger than 50 nm and have lateral confinements smaller 4 μm. The response of normal and NF1‐melanocytes to different topographies was analyzed quantitatively by determining density distributions for the number of dendrites per cell, the angles between dendrites, and the orientation imprinted in the substrate. The synthesis of melanin, a pigment produced by melanocytes, differs in the case of genetically altered NF1‐ and normal melanocytes. In both cases, the interaction with microstripes enhanced melanin production significantly. This enhanced melanin production is speculated to be caused by the mechanical stabilization of the dendrites by substrate guidance.     

Modulation of Melanogenesis and Antioxidant Status of Melanocytes in Response to Phototoxic Action of Doxycycline

Doxycycline is a commonly used tetracycline antibiotic showing the broad spectrum of antibacterial action. However, the use of this antibiotic is often connected with the risk of phototoxic reactions that lead to various skin disorders. One of the factors influencing the photosensitivity reactions is the melanin content in melanocytes. In this study, the impact of doxycycline and UVA irradiation on cell viability, melanogenesis and antioxidant defense system in cultured normal human epidermal melanocytes (HEMn‐DP) was examined. The exposure of cells to doxycycline and UVA radiation resulted in concentration‐dependent loss in melanocytes viability and induced melanin biosynthesis. Significant changes were stated in cellular antioxidant enzymes activity: SOD, CAT and GPx, which indicates alterations of antioxidant defense system. The results obtained in vitro may explain the mechanisms of phototoxic reactions that occur in normal human epidermal melanocytes in vivo after exposure of skin to doxycycline and UVA radiation.     

Artocarpus plants as a potential source of skin whitening agents

Artocarpus plants have been a focus of constant attention due to the potential for skin whitening agents. In the in vitro experiment, compounds from the Artocarpus plants, such as artocarpanone, norartocarpetin, artocarpesin, artogomezianol, andalasin, artocarbene, and chlorophorin showed tyrosinase inhibitory activity. Structure-activity investigations revealed that the 4-substituted resorcinol moiety in these compounds was responsible for their potent inhibitory activities on tyrosinase. In the in vitro assay, using B16 melanoma cells, the prenylated polyphenols isolated from Artocarpus plants, such as artocarpin, cudraflavone C, 6-prenylapigenin, kuwanon C, norartocarpin, albanin A, cudraflavone B, and brosimone I showed potent inhibitory activity on melanin formation. Structure-activity investigations revealed that the introduction of an isoprenoid moiety to a non-isoprenoid-substituted polyphenol enhanced the inhibitory activity of melanin production in B16 melanoma cells. In the in vivo investigation, the extract of the wood of Artocarpus incisus and a representative isolated compound from it, artocarpin had a lightening effect on the skin of guinea pigs' backs. Other in vivo experiments using human volunteers have shown that water extract of Artocarpus lakoocha reduced the melanin formation in the skin of volunteers. These results indicate that the extracts of Artocarpus plants are potential sources for skin whitening agents.     

Melanocyte-stimulating Properties of Secretory Phospholipase A2

Abstract— Phospholipase A₂ (PLA₂) catalyzes the release of free fatty acids from membrane phospholipids, and its products derived from these fatty acids, such as prostaglandins and leukotrienes, significantly up-regulate the key mela-nogenic enzyme, tyrosinase, in melanocytes. This has led to suggestions that PLA₂ itself triggers melanin synthesis in melanogenesis following UV irradiation or inflammation.
We have examined the effect of secretory PLA₂ (sPLA₂) on melanogenesis in cultured human melanocytes. Secretory PLA₂ stimulated DNA synthesis and melanin synthesis, and these phenomena were completely inhibited by treatment with a phospholipase inhibitor, p- bromophenacyl bromide, demonstrating that the catalytic activity of sPLA₂ is required for melanogenesis. Secretory PLA₂ also stimulated tyrosinase activity, increased the amount of tyrosinase-related protein-1 and up-regulated the expression of both mRNA. These findings suggest that sPLA₂ is an important mediator of UV-induced or postinflammatory pigmentation.     

The importance of the depth distribution of melanin in skin for DNA protection and other photobiological processes

Melanin pigments are important regulators for the evolution of essential functions of human skin. The concentration of melanin, as well as its depth distribution, is strongly affected by ultraviolet radiation. In un-tanned skin, melanin pigments are found only in the basal layer of the epidermis, while in tanned skin it is distributed throughout the epidermis. So far, mainly the amount of melanin, and not its distribution, has been considered in view of skin photobiology. With an advanced radiative transfer model we investigate, for the first time, how the depth distribution of melanin influences the amount of ultraviolet radiation that reaches living cells in the epidermis, and thus can damage the DNA in the cells. The simulations are performed for average pigmented skins (type III-IV). A surprisingly large factor, up to 12, is found between the ultraviolet protection of skin with melanin distributed throughout the epidermis, and skin with melanin only in the basal layer of the epidermis. We also show that the synthesis of previtamin D3, in skin, can vary with more than 100% if the depth distribution of melanin is changed, while the degradation of folate in dermal blood is almost un-affected by variations in the melanin depth distribution.     

COMPARATIVE STUDIES ON THE CORRELATION BETWEEN PYRIMIDINE DIMER FORMATION and TYROSINASE ACTIVITY IN CLOUDMAN S91 MELANOMA CELLS AFTER ULTRAVIOLET-IRRADIATION

We compared the induction of pyrimidine dimer densities after UV-irradiation in mouse melanoma cells before and after treatment with cholera toxin. Treatment with cholera toxin stimulated tyrosinase activity up to 50-fold, leading to a marked, visually apparent increase in cellular melanin concentrations. Irradiation of treated and untreated cells was therefore designed to establish whether intracellular melanin protected cells from UV-induced DNA damage. In experiments described here, we determined cytosine-thymine (C-T) as well as thymine-thymine dimer levels (T-T) by high pressure liquid chromatography in cholera toxin-treated and untreated Cloudman S91 mouse melanoma cells after irradiation with UVC (less than 290 nm) and UVB light (290-320 nm). Surprisingly, induction of melanization had no effect on the formation of pyrimidine dimers by UVC or UVB irradiation. These results indicate that de novo melanin pigmentation induced via the c-AMP pathway is not involved in protection against UV-induced thymine-containing pyrimidine dimers. In separate experiments, irradiation of toxin-treated and untreated mouse melanoma cells with UVC or UVB light produced a 20-30% lower dimer density compared to irradiated human skin fibroblasts. This finding suggests that melanin has some protection properties against UV-induced pyrimidine dimers, although the exact defense mechanism seems highly complex.     

Placental extracts induce the expression of antioxidant enzyme genes and suppress melanogenesis in B16 melanoma cells

One of the activities of placental extracts (PEs) is skin-whitening effect, but the physiological and genetic mechanism for this effect has not yet been clarified. Here, we focus on PE as a regulator of antioxidant enzyme genes. Porcine PE was prepared, and its activity was investigated in B16 melanoma cells. PE treatment decreased the melanin content of UV-irradiated B16 cells in a dose-dependent manner. PE directly reduced the enzyme activity of tyrosinase in a cell-free assay. In addition, PE treatment increased the gene expression of cytosolic superoxide dismutase (SOD-1), extracellular SOD (SOD-3) and catalase but did not affect the expression of tyrosinase. Moreover, PE protected the B16 cells from H₂O₂-induced cell death. Taken together, our data suggest that PEs could play a role not only as a suppressor of melanin synthesis but also as a regulator of antioxidant genes and might protect the skin against oxidative stress.