Tyrosinase Expression Research Articles

Anti-Melanogenesis Activity of 6-O-Isobutyrylbritannilactone from Inula britannica on B16F10 Melanocytes and In Vivo Zebrafish Models.

A potential natural melanogenesis inhibitor was discovered in the form of a sesquiterpene isolated from the flowers of Inula britannica, specifically 6-O-isobutyrylbritannilactone (IBL). We evaluated the antimelanogenesis effects of IBL on B16F10 melanocytes and zebrafish embryos. As a result, we found that 3-isobutyl-1-methylxanthine (IBMX)-induced melanin production was reduced in a dose-dependent manner in B16F10 cells by IBL. We also analyzed B16F10 cells that were and were not treated with IBMX, investigating the melanin concentration, tyrosinase activity, mRNA levels. We also studied the protein expressions of microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related proteins (TRP1, and TRP2). Furthermore, we found that melanin synthesis and tyrosinase expression were also inhibited by IBL through the modulation of the following signaling pathways: ERK, phosphoinositide 3-kinase (PI3K)/AKT, and CREB. In addition, we studied antimelanogenic activity using zebrafish embryos and found that the embryos had significantly reduced pigmentation in the IBL-treated specimens compared to the untreated controls.

Anti-Photoaging and Anti-Melanogenesis Effects of Fucoidan Isolated from Hizikia fusiforme and Its Underlying Mechanisms.

Previous studies suggested that fucoidan with a molecular weight of 102.67 kDa, isolated from Hizikia fusiforme, possesses strong antioxidant activity. To explore the cosmeceutical potential of fucoidan, its anti-photoaging and anti-melanogenesis effects were evaluated in the present study. The anti-photoaging effect was investigated in ultraviolet (UV) B-irradiated human keratinocytes (HaCaT cells), where fucoidan effectively reduced the intracellular reactive oxygen species level and improved the viability of the UVB-irradiated cells without any cytotoxic effects. Moreover, fucoidan significantly decreased UVB-induced apoptosis in HaCaT cells by regulating the protein expression of Bax, Bcl-xL, PARP, and Caspase-3 in HaCaT cells in a concentration-dependent manner. The anti-melanogenesis effect of fucoidan was evaluated in B16F10 melanoma cells that had been stimulated with alpha-melanocyte-stimulating hormone (α-MSH), and fucoidan treatment remarkably inhibited melanin synthesis in α-MSH-stimulated B16F10 cells. Further studies indicated that fucoidan significantly suppressed the expression of tyrosinase and tyrosinase-related protein-1 and -2 (TRP-1 and-2) in B16F10 cells by down-regulating microphthalmia-associated transcription factor (MITF) through regulation of the ERK–MAPK (extracellular signal regulated kinase-mitogen activated protein kinase) pathway. Taken together, these results suggest that fucoidan isolated from H. fusiforme possesses strong anti-photoaging and anti-melanogenesis activities and can be used as an ingredient in the pharmaceutical and cosmeceutical industries.

Anti-melanogenic effect of the aqueous ethanol extract of Ginkgo biloba leaf in B16F10 cells

Ginkgo biloba leaf extract (GLE) shows diverse biological effects, and it is used as an ingredient in some skincare products. GLE and a few purified compounds are known to inhibit melanogenesis. However, there are very few reports on the molecular mechanisms underlying the regulation of melanogenesis by GLE. We investigated the anti-melanogenic effect of a GLE, an aqueous ethanol extract of Ginkgo biloba leaves, and the molecular mechanisms involved. B16F10 murine melanoma cells were treated with various concentrations of GLE (0–100 μg/mL), together with 0.1 nM α-melanocyte stimulating hormone (α-MSH), which was used to induce melanogenesis. Cell proliferation, total melanin content, cellular reactive oxygen species (ROS) content, and cellular tyrosinase activity were measured. The cellular levels of microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related protein 1 (TYRP1), and the phosphorylation levels of Akt, glycogen synthase kinase 3β (GSK3β), and β-catenin were analyzed. GLE inhibited α-MSH-induced melanogenesis and ROS generation, without causing cytotoxicity. GLE decreased cellular tyrosinase activity and the levels of MITF, tyrosinase, and TYRP1. GLE decreased the phosphorylation of Akt (Ser473) and GSK3β (Ser9) and increased the phosphorylation of β-catenin (Ser33/37/Thr41). Our results suggest that GLE can inhibit α-MSH-induced melanogenesis in B16F10 cells by downregulating the expression of melanogenic proteins, i.e., MITF, tyrosinase, and TYRP1. The downregulation of melanogenic protein expression appears to result from stimulating β-catenin degradation, which is induced by phosphorylation by GSK3β that is activated via inhibition of Akt. GLE’s antioxidant activity can also play a role in its anti-melanogenic effect.

Lactobacillus helveticus-Fermented Milk Whey Suppresses Melanin Production by Inhibiting Tyrosinase through Decreasing MITF Expression.

Whey obtained from milk fermented by the Lactobacillus helveticus CM4 strain (LHMW) has been shown to improve skin barrier function and increase skin-moisturizing factors. In this study, we investigated the effects of LHMW on melanin production to explore the additional impacts of LHMW on the skin. We treated mouse B16 melanoma cells with α-melanocyte-stimulating hormone (α-MSH) alone or simultaneously with LHMW and measured the amount of melanin. The amount of melanin in B16 cells treated with α-MSH significantly increased by 2-fold compared with that in control cells, and tyrosinase activity was also elevated. Moreover, treatment with LHMW significantly suppressed the increase in melanin content and elevation of tyrosinase activity due to α-MSH. LHMW also suppressed the α-MSH-induced increased expression of tyrosinase, tyrosinase-related protein 1 (TRP1), and dopachrome tautomerase (DCT) at the protein and mRNA levels. Furthermore, the mRNA and protein microphthalmia-associated transcription factor (MITF) expression levels were significantly increased with treatment with α-MSH alone, which were also suppressed by LHMW addition. LHMW suppression of melanin production is suggested to involve inhibition of the expression of the tyrosinase gene family by lowering the MITF expression level. LHMW may have promise as a material for cosmetics with expected clinical application in humans.

Melatonin Induces Melanogenesis in Human SK-MEL-1 Melanoma Cells Involving Glycogen Synthase Kinase-3 and Reactive Oxygen Species.

Melatonin is present in all living organisms where it displays a diversity of physiological functions. Attenuation of melanogenesis by melatonin has been reported in some mammals and also in rodent melanoma cells. However, melatonin may also stimulate melanogenesis in human melanoma cells through mechanisms that have not yet been revealed. Using the human melanoma cells SK-MEL-1 as a model, an increase in both tyrosinase activity and melanin was already observed at 24 h after melatonin treatment with maximal levels of both being detected at 72 h. This effect was associated with the induction in the expression of the enzymes involved in the synthesis of melanin. In this scenario, glycogen synthase kinase-3β seems to play a significant function since melatonin decreased its phosphorylation and preincubation with specific inhibitors of this protein kinase (lithium or BIO) reduced the expression and activity of tyrosinase. Blocking of PI3K/AKT pathway stimulated melanogenesis and the effect was suppressed by the inhibitors of glycogen synthase kinase-3β. Although melatonin is a recognized antioxidant, we found that it stimulates reactive oxygen species generation in SK-MEL-1 cells. These chemical species seem to be an important signal in activating the melanogenic process since the antioxidants N-acetyl-l-cysteine and glutathione decreased both the level and activity of tyrosinase stimulated by melatonin. Our results support the view that regulation of melanogenesis involves a cross-talk between several signaling pathways.

Beclin 1 controls pigmentation by changing the nuclear localization of melanogenic factor MITF

The primary contributor for the determination of skin color is melanin, a pigment that is produced in specialized cells called melanocytes. At cellular level, melanin synthesis occurs through several enzymes like tyrosinase (TYR) and tyrosinase related proteins and the expression of these proteins are regulated transcriptionally by microphthalmia associated transcription factor (MITF). Melanin pigmentation is a complex process finely regulated by different transcription factors, structural proteins and enzymes. In recent times, several autophagic genes have been implicated in the regulation of pigmentation. Though previous report observed a visible loss of coat-color in heterozygous Beclin 1 mice, the role of this protein in pigmentation is yet to study in details. In this present work we intend to study the role of Beclin 1, a central autophagic factor, in pigmentation. Using human melanoma cells and primary melanocytes, we showed that Beclin 1 downregulation significantly decreased the melanin content, tyrosinase activity and the expression of TYR and tyrosinase related protein 1 (TYRP1). These effects were recapitulated in a Beclin 1 knockdown in vivo model of zebrafish. Most importantly, re-expression of Beclin 1 rescued the pigmentation-associated defects both in cellular and in organismal level indicating the specificity. Surprisingly, Beclin 1 knockdown cells did not show significant changes in MITF expression but the nuclear localization of MITF was altered. Together, these data suggest that indeed Beclin 1 is associated with melanogenesis and this effect is more likely exerted through the subcellular distribution rather than the change in expression of MITF.

Human Melanocyte-Derived Spheroids: A Precise Test System for Drug Screening and a Multicellular Unit for Tissue Engineering.

Pigmentation is the result of melanin synthesis, which takes place in melanocytes, and its further distribution. A dysregulation in melanocytes' functionality can result in the loss of pigmentation, the appearance of pigment spots and melanoma development. Tissue engineering and the screening of new skin-lightening drugs require the development of simple and reproducible in vitro models with maintained functional activity. The aim of the study was to obtain and characterize spheroids from normal human melanocytes as a three-dimensional multicellular structure and as a test system for skin-lightening drug screening. Melanocytes are known to lose their ability to synthesize melanin in monolayer culture. When transferred under non-adhesive conditions in agarose multi-well plates, melanocytes aggregated and formed spheroids. As a result, the amount of melanin elevated almost two times within seven days. MelanoDerm™ (MatTek) skin equivalents were used as a comparison system. Cells in spheroids expressed transcription factors that regulate melanogenesis: MITF and Sox10, the marker of developed melanosomes—gp100, as well as tyrosinase (TYR)—the melanogenesis enzyme and melanocortin receptor 1 (MC1R)—the main receptor regulating melanin synthesis. Expression was maintained during 3D culturing. Thus, it can be stated that spheroids maintain melanocytes' functional activity compared to that in the multi-layered MelanoDerm™ skin equivalents. Culturing both spheroids and MelanoDerm™ for seven days in the presence of the skin-lightening agent fucoxanthin resulted in a more significant lowering of melanin levels in spheroids. Significant down-regulation of gp100, MITF, and Sox10 transcription factors, as well as 10-fold down-regulation of TYR expression, was observed in spheroids by day 7 in the presence of fucoxanthin, thus inhibiting the maturation of melanosomes and the synthesis of melanin. MelanoDerm™ samples were characterized by significant down-regulation of only MITF, Sox10 indicating that spheroids formed a more sensitive system allowed for quantitative assays. Collectively, these data illustrate that normal melanocytes can assemble themselves into spheroids—the viable structures that are able to accumulate melanin and maintain the initial functional activity of melanocytes. These spheroids can be used as a more affordable and easy-to-use test system than commercial skin equivalents for drug screening.

Hydrogen Sulfide Promotes Cell Proliferation and Melanin Synthesis in Primary Human Epidermal Melanocytes

Background/Aim: Hydrogen sulfide (H₂S) has been found to act as a physiological intercellular messenger to regulate cell survival. In this study, we evaluated whether H₂S could promote cell proliferation and melanin synthesis in human epidermal melanocytes (HEMs). Methods: Primary HEMs were cocultured with sodium hydrosulfide (NaHS, the most widely used H₂S donor) or endogenously overexpressed with cystathionine-γ-lyase (CSE) gene, which is the most predominant H₂S-producing enzyme. Then, cell viability, intracellular melanin content, tyrosinase (TYR) activity, and expression of microphthalmia-associated transcription factor (MITF), TYR, together with TYR-related protein 1 (TRP-1) in both transcript and protein levels, were detected. Results: We first confirmed that NaHS (10–100 μm) increased cell viability, intracellular melanin content, and TYR activity in a dose-dependent manner. Then, we found that endogenous H₂S production also promoted cell proliferation, intracellular melanin content, and TYR activity. In addition, we observed the mRNA and protein expression of MITF, TYR, and TRP-1 was significantly up-regulated after NaHS treatment and CSE gene transfection. Conclusions: This study demonstrates that H₂S promotes cell proliferation and melanin synthesis in HEMs, which indicates pharmacologic regulation of H₂S may be potential treatment for skin disorders caused by loss of melanocytes or dysfunction of melanogenesis.

Melanogenesis-inhibitory activities of limonoids and tricyclic diterpenoids from Azadirachta indica.

The chemical constituents of the roots and bark of Azadirachta indica were investigated, leading to the isolation of six tricyclic diterpenoids and four limonoids including a new compound, azadirachtin J (4). The structures were elucidated on the basis of NMR spectroscopic techniques, mass spectrometry as well as comparison with the literature. Furthermore, melanogenesis-inhibitory activities of the isolated compounds were evaluated. As a result, compounds 1-3 and 10 exhibited superior inhibitory activities against melanogenesis with no, or almost no, toxicity to the cells (86.5-105.1% cell viability). Western blot analysis showed that compounds 1 and 3 exhibited melanogenesis inhibitory activities in α-MSH-stimulated B16 melanoma cells due to, at least in part, inhibition of the expression of MITF, followed by a decrease in the expression of tyrosinase, TRP-1, and TRP-2. Compounds 1 and 3 exhibited tyrosinase inhibitory activities (IC50 values of 44.86μM and 69.85μM respectively). Docking results confirm that the active inhibitors strongly interact with tyrosinase residues.

Ocean acidification inhibits initial shell formation of oyster larvae by suppressing the biosynthesis of serotonin and dopamine

Ocean acidification has severely affected the initial shell formation of marine bivalves during their larval stages. In the present study, it was found that dopamine (DA) content in early D-shape larvae was significantly higher than that in trochophore and D-shape larvae, while the serotonin (5-HT) content in early D-shape larvae and D-shape larvae was obviously higher than that in trochophore. Incubation of trochophore with 5-HT or DA could accelerate the formation of calcified shell, and the treatments with selective antagonists of receptors for 5-HT and DA (Cg5-HTR-1 and CgD1DR-1) obviously inhibited the formation of calcified shells. When oyster larvae were subjected to an experimental acidified treatment (pH 7.4), the biosynthesis of 5-HT and DA was inhibited, while the mRNA expression levels of the components in TGF-β pathway were significantly up-regulated in D-shape larvae. Moreover, the phosphorylation of TIR and the translocation of smad4 were hindered upon acidification treatments, and the expression patterns of chitinase and tyrosinase were completely reverted. These results collectively suggested that monoamine neurotransmitters 5-HT and DA could modulate the initial shell formation in oyster larvae through TGF-β smad pathway by regulating the expression of tyrosinase and chitinase to guarantee the chitin synthesis for shell formation. CO2-induced seawater acidification could suppress the biosynthesis of 5-HT and DA, as well as the activation of TGF-β smad pathway, which would subvert the expression patterns of chitinase and tyrosinase and cause the failure of initial shell formation in oyster early D-shape larvae.

지모 추출물의 피부 미백 및 항산화 효과 연구

The objective of this study is to investigate the skin whitening and antioxidant effects of the Anemarrhenae Rhizoma extract (ARE). Following the previously studied method, we examined the inhibitory effects of melanin synthesis and tyrosinase activity by using B16F10 cells. First, we measured the Diphenylpicrylhydrazyl (DPPH) assay, nitrite scavenging activity, and superoxide dismutase-like activity to verifying antioxidant efficacy according to skin whitening. In addition, we confirmed the skin whitening efficacy of ARE by measuring gene expression associated with a skin whitening by the Reverse transcription polymerase chain reaction (RT-PCR) method in B16F10 cells. In this study, we confirmed that ARE has skin whitening and antioxidant effects at high concentrations. In particular, ARE at a concentration of 500 ㎍/ml inhibited the expression of Tyrosinase, TRP-2 (tyrosinase-related protein), and MITF (microphthalmia transcription factor) genes better than Arbutin. In conclusion, our results confirmed that ARE has the potential for development as a skin whitening efficacy substance.

A Coculture Based Tyrosine-Tyrosinase Electrochemical Gene Circuit for Connecting Cellular Communication with Electronic Networks.

There is a growing interest in mediating information transfer between biology and electronics. By the addition of redox mediators to various samples and cells, one can both electronically obtain a redox "portrait" of a biological system and, conversely, program gene expression. Here, we have created a cell-based synthetic biology-electrochemical axis in which engineered cells process molecular cues, producing an output that can be directly recorded via electronics-but without the need for added redox mediators. The process is robust; two key components must act together to provide a valid signal. The system builds on the tyrosinase-mediated conversion of tyrosine to L-DOPA and L-DOPAquinone, which are both redox active. "Catalytic" transducer cells provide for signal-mediated surface expression of tyrosinase. Additionally, "reagent" transducer cells synthesize and export tyrosine, a substrate for tyrosinase. In cocultures, this system enables real-time electrochemical transduction of cell activating molecular cues. To demonstrate, we eavesdrop on quorum sensing signaling molecules that are secreted by Pseudomonas aeruginosa, N-(3-oxododecanoyl)-l-homoserine lactone and pyocyanin.

7,8-Dimethoxycoumarin stimulates melanogenesis via MAPKs mediated MITF upregulation.

Background: Melanin in the skin is the defense against the harmful UV radiation, which is considered as one of the major risk factors for skin cancer. The compound 7,8-dimethoxycoumarin (DMC, C11H10O₄), a natural coumarin molecule present in several medicinal plants, possesses antioxidant and anti-inflammatory activities. However, the mechanism underlying its effects on melanogenesis in melanocytes is unclear. Therefore, we investigated the effect of DMC on melanogenesis activation in B16F10 melanoma cells. Methods: We examined the cytotoxic range of DMC on B16F10 melanoma cells and increased effects of melanogenesis, and intracellular tyrosinase activity. In addition, regulation mechanisms were assessed by Western blot analysis. Results: The results showed that DMC significantly increased melanin content and tyrosinase activity in the cells without being cytotoxic. Furthermore, DMC stimulated the expression of tyrosinase, TRP-1, TRP-2, and MITF thereby activating melanin production and Akt phosphorylation was increased in the Akt signaling pathway. on the contrary, interfering with the phosphorylation of ERK in the MAPKs pathway. Conclusions: These results suggest that DMC may serve as a candidate for potential melanin-producing activator and anti-gray hair applications.

Inhibitory Effects of Pigmentation in B16F10 Melanoma Cells from Schizandra chinensis Fractions

The Melanin synthesis is controlled by a series of enzymatic reactions regulated at the level of tyrosinase in melanocytes and melanoma cells. In an ongoing search for new tyrosinase inhibitors from natural sources, the dried leaves of Schisandra chinensis were extracted with 70% acetone and separated into fractions 1-3 (Fr.1-Fr.3). All fractions were evaluated for their inhibitory activity potency against the tyrosinase, and Fr.2 showed potent tyrosinase activities with 34.7% inhibition concentration 1,000 μg/mL compared to the positive control, albutin (23.5%). Finally, melanin contents was measured to determine the whitening effect. Fr.2 was inhibited at 19.3% at 50μg/mL, respectively. We investigated the changes in protein level of tyrosinase, Melanocyte Inducing Transcription Factor (MITF) by using western blotting. Fr.2 was able to significantly inhibit tyrosinase expression in protein level. These results suggest that Fr.2 inhibited melanin production which may be dependent on tyrosinase activity and expression in B16F10 melanoma cells, and an effective whitening agent for the skin.

Anti-Melanogenic Effect of Ethanolic Extract of Sorghum bicolor on IBMX-Induced Melanogenesis in B16/F10 Melanoma Cells.

To evaluate possibility as a skin whitening agent of Sorghum bicolor (S. bicolor), its antioxidant activity and anti-melanogenic effect on 3-isobutyl-1-methylxanthine (IBMX)-induced melanogenesis in B16/F10 melanoma cells were investigated. The result of total phenolic contents (TPC) indicated that 60% ethanol extract of S. bicolor (ESB) has the highest contents than other ethanol extracts. Antioxidant activity was evaluated using the 2,2’-azino-bis-(3-ethylbenzothiazolin-6-sulfonic acid) diammonium salt (ABTS)/1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging activities and malondialdehyde (MDA) inhibitory effect. These results showed ESB has significant antioxidant activities. Inhibitory effect against tyrosinase was also assessed using L-tyrosine (IC50 value = 89.25 μg/mL) and 3,4-dihydroxy-L-phenylalanine (L-DOPA) as substrates. In addition, ESB treatment effectively inhibited melanin production in IBMX-induced B16/F10 melanoma cells. To confirm the mechanism on anti-melanogenic effect of ESB, we examined melanogenesis-related proteins. ESB downregulated melanogenesis by decreasing expression of microphthalmia-associated transcription factor (MITF), tyrosinase and tyrosinase-related protein (TRP)-1. Finally, 9-hydroxyoctadecadienoic acid (9-HODE), 1,3-O-dicaffeoylglycerol and tricin as the main compounds of ESB were analyzed using the ultra-performance liquid chromatography-ion mobility separation-quadrupole time of flight/tandem mass spectrometry (UPLC-IMS-QTOF/MS2). These findings suggest that ESB may have physiological potential to be used skin whitening material.

Pigmentation formation and expression analysis of tyrosinase in Siniperca chuatsi.

Animal pigmentation primarily depends on the presence and mixing ratio of chromatophores, functioning in animal survival and communication. For the benthic and carnivorous Siniperca chuatsi, pigmentation pattern is key to concealment and predation. In this study, the formation, distribution, and main pattern of chromatophores were observed in the embryos, larvae, skins, and visceral tissues from S. chuatsi. Melanophores were firstly visualized in the yolk sac at segmentation stage, and then they were migrated to the whole body and further clustered into the black stripes, bands, and patches. In adult S. chuatsi, the head, black band, and body side skins mainly contained melanophores, showing as deep or light black. The abdomen skin mainly contained iridophores, showing as silvery. In the eye, the pigment layers were located in the epithelial layers of iris and retina and shown as black. Then, the pigmentation-related gene, tyrosinase gene from S. chuatsi (Sc-tyr) was analyzed by bioinformatics and quantitative methods. The Sc-tyr gene encoded a protein with 540 amino acids (Sc-TYR). The Sc-TYR contained two copper ion binding sites, which were coordinated by six conserved histidines (H182, H205, H214, H366, H370, H393) and necessary for catalytic activity. The Sc-TYR was well conserved compared with TYR of various species with higher degree of sequence similarity with other fishes (77.6-98.3%). The qRT-PCR test showed that the Sc-tyr mRNA reached the peak value at segmentation stage in the embryo development, the black skins displayed a higher expression level than that in silvery skin, and the eye had the highest expression level compared with other tissues. Further research on enzyme activity showed that the expression patterns of tyrosinase activity were similar to that of the Sc-tyr mRNA. Comparing with the results of molecular and phenotype, it was found that the temporal and spatial distributions of tyrosinase corresponded well with changes in pigmentation patterns and the intensity of skin melanization. This study initially explored the pigmentation formation and tyrosinase expression, which served as a foundation for further insight into the genetics mechanism of body color formation in S. chuatsi.

55 Melanocytes in Hypopigmented Burn Scar Can Be Stimulated to Produce Melanin

Abstract Introduction Currently, there are limited clinical treatments for dyschromia in burn hypertrophic scars (HTSs). Initial pilot work in a Duroc pig model showed that melanocyte quantity does not influence pigmentation. Regions of scar that are light or dark have equal numbers of melanocytes. This study aims to confirm melanocyte presence in regions of hypo- and hyper-pigmentation. If melanocytes are present, pigmentation stimulators may be a useful therapy. Methods Following IRB approval, patients with dyschromic HTSs were enrolled and demographic, injury and treatment details, and melanin indices by Skin Color Catch probes (SCC) were collected. Punch biopsies were taken of distinct regions of hyper-, hypo-, or normally pigmented scar and skin. Biopsies were processed to obtain epidermal sheets (ESs). A subset of ESs were stained using en face staining with melanocyte marker, S100b. Melanocytes were isolated from a different subset. Melanocytes were treated with NDP a-MSH, a pigmentation stimulator. mRNA was isolated from cells, and qRT-PCR was used to evaluate gene expression of melanin-synthetic genes, tyrosinase (TYR), tyrosinase-related protein-1 (TYRP1) and dopachrome tautomerase (DCT). Results 8 patients were enrolled and the cohort included patients with Fitzpatrick skin types 5 (n=6) and 3 (n=2). HTSs ranged from 4 to 395 months old and dyschromia developed mainly due to healing by re-epithelialization and was refractory to prior scar treatments. By SCC, regions of hyper-, hypo-, and normal pigmentation had significantly different melanin indices (864.9 ± 19.8 vs. 627.1 ± 30.3 vs. 786.5 ± 21.4, p< 0.01). S100b en face staining showed that regions of hyper- and hypo-pigmentation contained the same number of melanocytes (14.6 ± 3.1 vs. 14.5 ± 3.2 melanocytes/HPF (n=5, p=0.99), but these cells had different dendricity, or activity (3.9 ± 0.5 vs. 0.4 ± 0.2 dendrites/cell (n=5, p< 0.0001). When hypo-pigmented melanocytes were treated with NDP a-MSH, cells produced melanin visible by bright-field microscopy (n=3). TYR, TYRP1, and DCT gene expression were upregulated in treated cells compared to controls. Conclusions While traditionally, it may be thought that hypo-pigmented regions of burn HTS display this phenotype because of the absence of pigment-producing cells, these data show that melanocytes are present in these scar regions. The cells are present, but they are inactive in pigment production. By treating the cells with a pigment stimulator, cells can be induced to re-pigment. Applicability of Research to Practice Patients were asked, on a scale of 1–10 with 1 being “no, not at all”, and 10 being “yes, very much”, “Is the scar color different from the color of your normal skin at present?” All patients rated this question as 10/10, indicating that scar dyschromia is a pervasive problem. Patients’ quality of life may be improved with the development of treatments for hypopigmentation.

Cambios en el perfil de compuestos fenólicos, actividad antioxidante y antimelanogénica de órganos de Petasites japonicas bajo diferentes métodos de extracción

Petasites japonicus, a perennial flowering plant belonging to the Asteraceae family, has long been used in traditional herbal medicine for treating various diseases. The present study was conducted to investigate the effect of extraction methods including microwave-assisted extraction, autoclave-assisted extraction, and hot water extraction on the polyphenolic composition, antioxidant activity, and anti-melanogenic activity of P. japonicus leaves, stems, and roots. In comparison with other samples, leaf extracts exhibited strong DPPH radical scavenging activity, reducing power activity, and hydrophilic oxygen radical scavenging activity. In addition, the leaf extracts significantly suppressed α-MSH-induced melanin synthesis in B16F10 cells. Quantitative real-time PCR, western blot, and cellular reactive oxygen species (ROS) analyses indicated that the anti-melanogenic effect of leaf extracts obtained by hot water extraction may result from the inhibition of ROS generation and the downregulation of tyrosinase expression in B16F10 cells. Furthermore, the extraction methods differentially affected the content of polyphenolic compounds and bioavailability of P. japonicus extracts. Taken together, the antioxidant activity and anti-melanogenic effect of leaf hot water extracts suggest that the leaves of P. japonicus could be a beneficial source of natural antioxidants for skincare products.

Nitroprusside induces melanoma ferroptosis with serum supplementation and prolongs survival under serum depletion or hypoxia

BackgroundWhen proliferating tumor cells expand to areas distant from vascular sites, poor diffusion of oxygen and nutrients occur, generating a restrictive hypoxic gradient in which susceptible tumor cells die. The heterogeneous population surviving hypoxia and metabolic starvation include de-differentiated cancer stem cells (CSC), capable of self-renewing tumor-initiating cells (TICs), or those that divide asymmetrically to produce non-tumor-initiating differentiated (NTI-D) cell progeny. Under such restrictive conditions, both populations slowly proliferate, entering quiescence or senescence, when exiting from cell cycle progression. This may drive chemoresistance and tumor recurrence, since most anti-cancer treatments target rapidly proliferating cells. PurposeSince persistent or additional stress may increase NTI-D cells conversion to TICs, we investigated whether nutrient depletion or hypoxia influence expression of tyrosinase, a crucial enzyme for melanin synthesis, and B16 melanoma survival, when exposed to iron-dependent cell death oxidative stress produced by the Fenton reaction, resembling ferroptosis. Results-a) proliferating B16 melanoma with 10% serum-supplementation (10%S) normoxically express hypoxia inducible factor 1α (HIF1α) but lose tyrosinase, in contrast to those transiently exposed to (SF) serum-free medium, in which both HIF1α and tyrosinase are co-expressed; b) in contrast to the resistance to SNP toxicity in (SF) cells with higher tyrosinase expression, those in (10%S) are killed by iron from nitroprusside/ferricyanide (SNP) irrespective of exogenous H2O2, in a reaction antagonized by the anti-oxidant and MEK inhibitor UO126; c) Moreover, under transient serum depletion, SNP cooperates with hypoxia (1.5% oxygen), prolonging B16 melanoma (SF) survival; d) the hypoxia mimetic CoCl2 inhibits proliferation-associated cyclin A, irrespective of SNP, in (10%S) cells or in transiently serum-depleted (SF) cells. However, only in the latter cells, CoCl2 but not SNP, induce loss of HIF1α and apoptosis-associated PARP cleavage; e) longer term adaptation to survive serum depletion, generates (SS) cells resistant to SNP toxicity, which aerobically co-express HIF1α and tyrosinase. In SS B16 melanoma, exogenous non-toxic 100 μM H2O2 super-induces the ratio of tyrosinase to HIF1α. However, co-treatment of SS-B16 cells with SNP plus exogenous H2O2, partly increases PARP cleavage by reciprocally decreasing tyrosinase expression. SIGNIFICANCE.- These results suggest that a phenotypic plasticity in response to depletion of nutrients and/or oxygen, helps decide whether melanoma cells undergo either death by ferroptosis, or resistance to it, when challenged by the same exogenous oxidative stress (iron ± H2O2).

The expression of miR-129-5p and its target genes in the skin of goats

Coat color is one of the major quality traits of animals, and miR-129-5p acts as an important regulator for melanin biosynthesis in mammals. In this study, real-time PCR and western blotting were used to examine the expression of miR-129-5p and its targets genes in the skin of different coat color goats. The results showed that the expression of miR-129-5p in the skin samples of Inner Mongolia cashmere goats (IMCG) was higher than that of Dazu black goat (DBG). Also, the target genes (tyrosinase (TYR), frizzled 6 (FZD6) and glycogen synthase kinase 3β (GSK3β)) of miR-129-5p was highly expressed in the skin samples of DBG. The expression of miR-129-5p firstly increased and then decreased with age in F1 hybrid generation of DBG and IMCG. In addition, the expression of TYR decreased with age, while the expression of MITF increased with age but then decreased. The expression of FZD6 and GSK3β in the skin samples of F1 of different ages were irregular. Our results indicated that miR-129-5p mainly affects the formation of coat color of goats by decreasing the expression of TYR. This study suggests that miR-129-5p can act as a suppressor in the formation of coat color to lay the foundation for studying the effect of miR-129-5p on melanin synthesis.