UP256 Inhibits Hyperpigmentation by Tyrosinase Expression/Dendrite Formation via Rho-Dependent Signaling and by Primary Cilium Formation in Melanocytes.
ABSTRACT: Skin hyperpigmentation is generally characterized by increased synthesis and deposition of melanin in the skin. UP256, containing bakuchiol, is a well-known medication for acne vulgaris. Acne sometimes leaves dark spots on the skin, and we hypothesized that UP256 may be effective against hyperpigmentation-associated diseases. UP256 was treated for anti-melanogenesis and melanocyte dendrite formation in cultured normal human epidermal melanocytes as well as in the reconstituted skin and zebrafish models. Western blot analysis and glutathione S-transferase (GST)-pull down assays were used to evaluate the expression and interaction of enzymes related in melanin synthesis and transportation. The cellular tyrosinase activity and melanin content assay revealed that UP256 decreased melanin synthesis by regulating the expression of proteins related on melanogenesis including tyrosinase, TRP-1 and -2, and SOX9. UP256 also decreased dendrite formation in melanocytes via regulating the Rac/Cdc42/?-PAK signaling proteins, without cytotoxic effects. UP256 also inhibited ciliogenesis-dependent melanogenesis in normal human epidermal melanocytes. Furthermore, UP256 suppressed melanin contents in the zebrafish and the 3D human skin tissue model. All things taken together, UP256 inhibits melanin synthesis, dendrite formation, and primary cilium formation leading to the inhibition of melanogenesis.
Project description:Ethyl linoleate is an unsaturated fatty acid used in many cosmetics for its various attributes, such as antibacterial and anti-inflammatory properties and clinically proven to be an effective anti-acne agent. In this study, we investigated the effect of ethyl linoleate on the melanogenesis and the mechanism underlying its action on melanogenesis in B16F10 murine melanoma cells. Our results revealed that ethyl linoleate significantly inhibited melanin content and intracellular tyrosinase activity in ?-MSH-induced B16F10 cells, but it did not directly inhibit activity of mushroom tyrosinase. Ethyl linoleate inhibited the expression of microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase related protein 1 (TRP1) in governing melanin pigment synthesis. We observed that ethyl linoleate inhibited phosphorylation of Akt and glycogen synthase kinase 3? (GSK3?) and reduced the level of ?-catenin, suggesting that ethyl linoleate inhibits melanogenesis through Akt/GSK3?/?-catenin signal pathway. Therefore, we propose that ethyl linoleate may be useful as a safe whitening agent in cosmetic and a potential therapeutic agent for reducing skin hyperpigmentation in clinics.
Project description:Xanthohumol (XH) is the most abundant prenylated flavonoid found in the hop plant (<i>Humulus lupulus L</i>.) and has previously been shown to have depigmenting effects in B16F10 mouse melanoma cells; however, studies of its depigmenting efficacy in human melanocytes are still lacking. In this work, we explored the effects of XH on melanogenesis in MNT-1 human melanoma cells and normal human melanocytes from darkly-pigmented skin (HEM-DP). XH was screened for cytotoxicity over 48 h, and subsequently tested on melanogenesis in MNT-1 cells. XH was further tested in HEM-DP cells for melanin synthesis and melanosome export; dendricity was quantitated to assess effects on melanosome export. Melanosome degradation was studied in human keratinocytes (HaCaT). Our results showed that XH inhibited melanin synthesis in MNT-1 cells at 30 ?M but increased intracellular tyrosinase activity without affecting ROS levels. In HEM-DP cells, XH robustly suppressed cellular tyrosinase activity at nontoxic concentrations (2.5-5 ?M) without any effect on melanin synthesis. However, XH inhibited melanosome export by reducing dendrite number and total dendrite length. Further testing in HaCaT cells demonstrated that XH induced melanosome degradation at low micromolar concentrations without any cytotoxicity. In summary, our results demonstrate that XH at low micromolar concentrations might hold promise as a potent inhibitor of human pigmentation by primarily targeting melanin export and melanin degradation. Further studies to elucidate the signaling mechanisms of action of melanosome export inhibition by XH and <i>in vivo</i> efficacy are warranted.
Project description:Tyrosinase is involved in melanin biosynthesis and the abnormal accumulation of melanin pigments leading to hyperpigmentation disorders that can be treated with depigmenting agents. A natural product T1, bis(4-hydroxybenzyl)sulfide, isolated from the Chinese herbal plant, Gastrodia elata, is a strong competitive inhibitor against mushroom tyrosinase (IC50 = 0.53??M, Ki = 58 ± 6?nM), outperforms than kojic acid. The cell viability and melanin quantification assay demonstrate that 50??M of T1 apparently attenuates 20% melanin content of human normal melanocytes without significant cell toxicity. Moreover, the zebrafish in vivo assay reveals that T1 effectively reduces melanogenesis with no adverse side effects. The acute oral toxicity study evidently confirms that T1 molecule is free of discernable cytotoxicity in mice. Furthermore, the molecular modeling demonstrates that the sulfur atom of T1 coordinating with the copper ions in the active site of tyrosinase is essential for mushroom tyrosinase inhibition and the ability of diminishing the human melanin synthesis. These results evident that T1 isolated from Gastrodia elata is a promising candidate in developing pharmacological and cosmetic agents of great potency in skin-whitening.
Project description:Skin color/pigmentation is regulated through melanogenesis process in specialized melanin-producing cells, melanocytes, involving multiple signaling pathways. It is highly influenced by intrinsic and extrinsic factors such as oxidative, ultraviolet radiations and other environmental stress conditions. Besides determining the color, it governs response and tolerance of skin to a variety of environmental stresses and pathological conditions including photodamage, hyperpigmentation, and skin cancer. Depigmenting reagents have been deemed useful not only for cosmetics but also for pigmentation-related pathologies. In the present study, we attempted modulation of 1-oleoyl-2-acetyl-glycerol- (OAG-) induced melanogenesis in human melanoma and primary melanocytes. In both cell types, OAG-induced melanogenesis was associated with increase in enhanced expression of melanin, tyrosinase, as well as stress chaperones (mortalin and HSP60) and Reactive Oxygen Species (ROS). Treatment with TXC (trans-4-(Aminomethyl) cyclohexanecarboxylic acid hexadecyl ester hydrochloride) and 5/40 natural compounds resulted in their reduction. The data proposed an important role of mortalin and oxidative stress in skin pigmentation and the use of TXC and natural extracts for modulation of pigmentation pathways in normal and pathological conditions.
Project description:Melanogenesis is the process of production of melanin pigments that are responsible for the colors of skin, eye, and hair and provide protection from ultraviolet radiation. However, excessive levels of melanin formation cause hyperpigmentation disorders such as freckles, melasma, and age spots. Liver X receptors (LXR) are nuclear oxysterol receptors belonging to the family of ligand-activated transcription factors and physiological regulators of lipid and cholesterol metabolism. In the skin, activation of LXRs stimulates differentiation of keratinocytes and augments lipid synthesis in sebocytes. However, the function of LXRs in melanogenesis has not been clearly elucidated. In addition, although beauvericin, a well-known mycotoxin primarily isolated from several fungi, has various biological properties, its involvement in melanogenesis has not been reported. Therefore, in this study, we examined the effects of beauvericin on melanogenesis and its molecular mechanisms. Beauvericin decreased melanin content and tyrosinase activity without any cytotoxicity. Beauvericin also reduced protein levels of MITF, tyrosinase, TRP1, and TRP2. In addition, beauvericin suppressed cAMP-PKA-CREB signaling and upregulated expression of LXR-?, resulting in the suppression of p38 MAPK. Our results indicate that beauvericin attenuates melanogenesis by regulating both cAMP/PKA/CREB and LXR-?/p38 MAPK pathways, consequently leading to a reduction of melanin levels.
Project description:Tyrosinase plays an essential role in melanogenesis. Excess production of melanin can be a reason for hyperpigmentation skin disorders in mammals and enzymatic browning in plant-derived foods. Catalyzing the rate-limiting step of melanin synthesis, tyrosinase has become the most studied target for melanogenesis inhibition. Over the past ten years, a number of synthetic thiosemicarbazone derivatives have been reported to possess strong tyrosinase inhibitory properties with IC50 values below 1 ?M, placing them among the most potent tyrosinase inhibitors. This review gives an overview of tyrosinase activity and describes tyrosinase-inhibiting thiosemicarbazones in terms of their structure-activity relationships, kinetics of enzyme inhibition and mechanism of action. Results of the studies of thiosemicarbazones as tyrosinase inhibitors from over 20 research articles have been analyzed, compared and summarized in the present paper. Using thiosemicarbazones as tyrosinase inhibitors is a promising approach in developing anti-melanogenetic agents for skin-whitening cosmetics and anti-browning agents for food.
Project description:CMT-308 is a nonantimicrobial chemically-modified tetracycline (CMT), which we have previously shown exhibits antifungal activity and pleiotropic anti-inflammatory activities, including inhibition of the enzymatic activity of matrix metalloproteinases (MMPs). Based on its chemical structure, we hypothesized that CMT-308 could inhibit melanogenesis and might be a candidate for the treatment of skin hyperpigmentation disorders which occur due to unregulated melanin biosynthesis and/or transport. CMT-308 was first studied for any effects on activity of the enzyme tyrosinase in vitro using a purified preparation of mushroom tyrosinase; the mode of inhibition of the soluble fungal enzyme was evaluated by Lineweaver-Burk and Dixon plots as well as by non-linear least squares fitting. Next, the effects of CMT-308 were tested in mammalian cell cultures using B16F10 mouse melanoma cells and further validated in darkly-pigmented human melanocytes (HEMn-DP). Our results showed that micromolar concentrations of CMT-308 inhibited mushroom tyrosinase enzyme activity, using the first two substrates in the melanogenesis pathway (l-tyrosine and l-3,4-dihydroxyphenylalanine (l-DOPA)); CMT-308 inhibited mushroom tyrosinase primarily via a mixed mode of inhibition, with the major contribution from a competitive mode. In B16F10 cell cultures, CMT-308 (10 µM) significantly diminished total melanin levels with a selective reduction of extracellular melanin levels, under both basal and hormone-stimulated conditions without any cytotoxicity over a duration of 72 h. Studies of potential mechanisms of inhibition of melanogenesis in B16F10 cells showed that, in mammalian cells, CMT-308 did not inhibit intracellular tyrosinase activity or the activity of ?-glucosidase, an enzyme that regulates maturation of tyrosinase. However, CMT-308 suppressed MITF protein expression in B16F10 cells and showed copper chelating activity and antioxidant activity in a cell-free system. The significantly lower extracellular melanin levels obtained at 10 µM indicate that CMT-308's anti-melanogenic action may be attributed to a selective inhibition of melanosome export with the perinuclear aggregation of melanosomes, rather than a direct effect on the tyrosinase-catalyzed steps in melanin biosynthesis. These results were validated in HEMn-DP cells where CMT-308 suppressed dendricity in a fully reversible manner without affecting intracellular melanin synthesis. Furthermore, the capacity of CMT-308 to inhibit melanosome export was retained in cocultures of HEMn-DP and HaCaT. In summary, our results offer promise for therapeutic strategies to combat the effects of hyperpigmentation by use of CMT-308 at low micromolar concentrations.
Project description:Extrinsic aging of the skin caused by ultraviolet (UV) light or particulate matter is often manifested by hyperpigmentation due to increased melanogenesis in senescent skin. Ursodeoxycholic acid (UDCA), which has been commonly used as a health remedy for liver diseases, is known to possess antioxidant properties. This study was done to investigate whether UDCA inhibits cellular aging processes in the cells constituting human skin and it reduces melanin synthesis. ROS, intracellular signals, IL-1?, IL-8, TNF-?, cyclooxygenase (COX)-2, type I collagen, and matrix metalloproteinases (MMPs) levels were measured in human dermal fibroblasts treated with or without UDCA after UV exposure. Melanin levels and mechanistic pathways for melanogenesis were investigated. UDCA decreased ROS, senescence-associated secretory phenotype (SASP), and proinflammatory cytokines induced by UV treatment. UDCA reduced melanogenesis in normal human melanocytes cocultured with skin constituent cells. Our results suggest that UDCA could be a comprehensive agent for the treatment of environmental aging-associated hyperpigmentation disorders.
Project description:Although autophagy plays a role in melanogenesis by regulating melanosome degradation and biogenesis in melanocytes, a detailed understanding of the regulatory functions of autophagy factors is lacking. Here, we report a mechanistic link between microtubule-associated protein light chain 3 (LC3) activation and melanogenesis. We observed high expression of LC3 in melanosome-associated pigment-rich melanocytic nevi of sun-exposed skin, as indicated by patterns of melanosomal protein MART1 expression. Rapamycin-induced autophagy significantly increased the melanin index, tyrosinase activity and expression of several proteins linked to melanosome biogenesis, including microphthalmia transcription factor (MITF), pre-melanosome protein and tyrosinase, in Melan-a melanocytes. siRNA-mediated knockdown of LC3, but not beclin-1 or ATG5, decreased melanin content and tyrosinase activity. LC3 knockdown also markedly inhibited MITF expression and subsequent rapamycin-induced melanosome formation. More importantly, LC3 knockdown suppressed α-MSH-mediated melanogenesis by attenuating cAMP response element-binding protein (CREB) phosphorylation and MITF expression in Melan-a cells via decreased extracellular signal-regulated kinase (ERK) activity. Overexpression of constitutively active ERK reversed the effect of LC3 knockdown on CREB phosphorylation and MITF expression. These findings demonstrate that LC3 contributes to melanogenesis by increasing ERK-dependent MITF expression, thereby providing a mechanistic insight into the signaling network that links autophagy to melanogenesis.
Project description:There is a continual need to develop novel and effective melanogenesis inhibitors for the prevention of hyperpigmentation disorders. The plant Artemisia capillaris Thunberg (Oriental Wormwood) was screened for antipigmentation activity using murine cultured cells (B16-F10 malignant melanocytes). Activity-based fractionation using HPLC and NMR analyses identified the compound 4,5-O-dicaffeoylquinic acid as an active component in this plant. 4,5-O-Dicaffeoylquinic acid significantly reduced melanin synthesis and tyrosinase activity in a dose-dependent manner in the melanocytes. In addition, 4,5-O-dicaffeoylquinic acid treatment reduced the expression of tyrosinase-related protein-1. Significantly, we could validate the antipigmentation activity of this compound in vivo, using a zebrafish model. Moreover, 4,5-O-dicaffeoylquinic acid did not show toxicity in this animal model. Our discovery of 4,5-O-dicaffeoylquinic acid as an inhibitor of pigmentation that is active in vivo shows that this compound can be developed as an active component for formulations to treat pigmentation disorders.