Normal Human Melanocytes Research Articles

Methylcobalamin Protects Melanocytes from H2O2-Induced Oxidative Stress by Activating the Nrf2/HO-1 Pathway.

PurposeOxidative stress is considered a major determinant in the pathogenesis of vitiligo. Methylcobalamin (MeCbl) is an activated form of vitamin B12 that regulates inflammatory factors, counters oxidative stress, and reduces apoptosis in many disease models. However, the specific mechanism of MeCbl repigmentation against vitiligo is unknown. In this study, we explored the effect of MeCbl on melanocytes following hydrogen peroxide (H2O2)-induced oxidative stress.MethodsWe established an oxidative stress model using the immortalized human normal melanocyte cell line PIG1. We used a Cell Counting Kit-8 (CCK-8) to detect drug cytotoxicity, and we measured the melanin content of cells using the NaOH method. Intracellular oxidative damage was assessed by flow cytometry and antioxidant enzyme detection kits. In addition, we assessed the presence of apoptosis by flow cytometry and Western blots. We explored the underlying mechanisms of MeCbl during oxidative stress in melanocytes by analyzing the results of experiments based on real-time quantitative polymerase chain reaction (RT-qPCR), Western blotting, and laser scanning confocal immunofluorescence microscopy. Finally, we repeated the experiments after applying an inhibitor to block the Nrf2 pathway.ResultsWe found that MeCbl treatment enhanced cell viability, increased melanin content, reduced intracellular reactive oxygen species (ROS) accumulation, increased the activities of antioxidant enzyme superoxide dismutase (SOD) and catalase (CAT), reduced melanocyte apoptosis, and up-regulated the expression of the Nrf2/HO-1 pathway. Moreover, the protective effects of MeCbl were significantly weakened after inhibiting the Nrf2/HO-1 pathway.ConclusionOur results indicate that MeCbl attenuated the H2O2-induced oxidative stress in melanocytes by activating the Nrf2/HO-1 pathway, this suggests that MeCbl may be an effective treatment against vitiligo.

The Biochemical and Molecular Analysis of Changes in Melanogenesis Induced by UVA-Activated Fluoroquinolones-In Vitro Study on Human Normal Melanocytes.

Fluoroquinolones cause phototoxic reactions, manifested as different types of skin lesions, including hyperpigmentation. The disturbances of melanogenesis indicate that fluoroquinolones may affect cellular processes in melanocytes. It has been reported that these antibiotics may bind with melanin and accumulate in pigmented cells. The study aimed to examine the changes in melanogenesis in human normal melanocytes exposed to UVA radiation and treated with lomefloxacin and moxifloxacin, the most and the least fluoroquinolone, respectively. The obtained results demonstrated that both tested fluoroquinolones inhibited melanogenesis through a decrease in tyrosinase activity and down-regulation of tyrosinase and microphthalmia-associated transcription factor production. Only lomefloxacin potentiated UVA-induced melanogenesis. Under UVA irradiation lomefloxacin significantly enhanced melanin content and tyrosinase activity in melanocytes, although the drug did not cause an increased expression of tyrosinase or microphthalmia-associated transcription factor. The current studies revealed that phototoxic activity of fluoroquinolones is associated with alterations in the melanogenesis process. The difference in phototoxic potential of fluoroquinolones derivatives may be connected with various effects on UVA-induced events at a cellular level.

Expression analysis of candidate genes in vitiligo patients & effect of oxidative stress on melanocytes

BackgroundVitiligo is an acquired hypomelanotic autoimmune disorder characterized by depigmented macules on the skin. Oxidative stress acts as an initial trigger and autoimmunity leads to vitiligo progression. Cytokines are key mediators of immune system and imbalance between pro- and anti-inflammatory cytokines play a vital role in the pathogenesis of autoimmune diseases. Also, relationship between oxidative stress and oxidative stress induced cytokine secretion is well established. Therefore, the present study aimed to monitor the expression levels of selected cytokine genes (IL1A, IL4, IL1R1, IL1RN and IFNG) from vitiliginous skin along with the effect of oxidative stress on in vitro cultured normal human melanocyte (NHM). MethodsThe gene expression of IL1A, IL1R1, IL1RN, IL4 and IFNG was performed by real-time PCR. Effect of oxidative stress on NHM was measured by MTT assay upon H2O2 treatment. ResultsOutcome of the study revealed significant increase in IFNG transcript levels from the non lesional and lesional skin of vitiligo patients as compared to controls (p = 0.0045 and p = 0.0198, respectively). However, no significant differrence was observed in the transcript levels of IL1A, IL4, IL1R and IL1RN from non lesional and lesional skin of vitiligo patients as compared to controls (p > 0.05). A significant decrease in melanocyte viability was observed upon H2O2 treatment in a dose dependent manner (p < 0.0001). ConclusionThe increased IFNG expression in vitiliginous skin advocates IFN-γ associated melanocyte destruction in vitiligo and decreased melanocyte viability upon H2O2 treatment suggests the vulnerability of melanocytes to oxidative stress which persists in vitiligo microenvironment.

301 Comprehensive analyses on melanocytes differentiated from induced pruripotent stem cells originated from xeroderma pigmentosum complementation group A

Xeroderma Pigmentosum (XP) is a rare, autosomal recessive, hereditary DNA repair disorder characterized by extreme hypersensitivity to ultraviolet radiation (UVR). XP complementation group A (XP-A) is the most frequent type in Japan, and patients with XP-A present most severe cutaneous and neurological symptoms due to nucleotide excision repair deficiency. Here, we succeeded in generating melanocytes from induced pruripotent stem cells (iPSCs) derived from XP-A patients via melanocyte precursor cells (MPCs), and performed comprehensive analyses of gene expression to investigate the underlying molecular mechanisms of the disease. XP-A-iPSC- derived melanocytes (XP-A-iMCs) appeared morphologically similar to normal human epidermal melanocytes and expressed similarly melanocyte markers. We analyzed difference of the microarray data between XP-A-iMCs and healthy-control-iPSC-derived melanocytes (HC-iMCs) at 12 hours after high dose (150 J/m2) UV-B irradiation. Consequently we found that the major GO term categories for genes specifically upregulated in XP-A-iMCs were cell proliferation and death, and that for genes specifically downregulated in XP-A-iMCs was cell cycle regulation. These results suggest that XP-A-iMCs have increased apoptosis and cell proliferation compared to HC-iMCs. This phenotype of XP-A-iMCs may be responsible for the pigmented and depigmented maculae characteristic of XP-A patients.

Silver Nanoparticles Genotoxicity on Normal Human Melanocyte Cell Line (HBF-4 cells).

Nanotechnology has been rapidly used in diverse consumer products in both medical and industrial fields; which has raised various concerns. Silver nanoparticles (AgNPs) are one of the most widely used nanomaterials due to their excellent broad-spectrum antibacterial properties. However the studies on their cyto- and geno-toxic effects are scarce. The present study aim to evaluate the DNA damaging effects of AgNPs and estimate the release of intracellular reactive oxygen species (ROS) on HBF-4 normal human melanocyte cells. The study was conducted on HBF-4 cells, they will be treated with AgNPs at different levels (0.01 ,0.1,1, 10 and 100 μg/mL), LC50 was determined using MTT viability assay. Cells were then divided into two groups: the first was treated with the LC50 dose and the second was treated with 0.1 LC50 dose. DNA fragmentation assay using diphenylamine (DPA) technique and Intracellular ROS assay were measured and compared to control group. Significant increases in DNA damage and release of intracellular ROS were observed on treated HBF-4 cells with LC50 and its 1/10 LC50 value in comparison to the control group. Conclusion: silver nanoparticles can induce oxidative stress and DNA damage in normal human melanocyte cells with a potential carcinogenic effect to be considered. Further investigations form their genetic alterations mechanisms are required.

Salicylic acid in ginseng root alleviates skin hyperpigmentation disorders by inhibiting melanogenesis and melanosome transport

Abnormal melanogenesis and melanosome transport can cause skin pigmentation disorders that are often treated using ginseng-based formulation. We previously found that phenolic acid compounds in ginseng root could inhibit melanin production and as a skin-whitening agents. However, mechanisms of action underlying effects of ginseng phenolic acid monomers on melanogenesis remain unclear. This study was conducted to investigate effects of salicylic acid, a main ginseng root phenolic acid component, on melanogenesis and melanosome functions in melanocytes of zebrafish and other species. Salicylic acid exhibited no cytotoxicity and reduced melanin levels and tyrosinase activity in B16F10 murine melanoma cells and normal human epidermal melanocytes regardless of prior cell stimulation with α-melanocyte stimulating hormone. Additionally, salicylic acid treatment reduced expression of melanogenic enzymes tyrosinase, tyrosinase-related protein 1 and tyrosinase-related protein 2, while reducing expression of their master transcriptional regulator, microphthalmia-associated transcription factor. Moreover, reduced phosphorylation of cAMP response-element binding protein was observed due to reduced cAMP levels resulting from salicylic acid inhibition of upstream signal regulators (adenylyl cyclase and protein kinase A). Furthermore, salicylic acid treatment suppressed expression of transport complex-associated proteins melanophilin and myosin Va in two UVB-treated melanocytic cell lines, suppressed phagocytosis of fluorescent microspheres by UVB-stimulated human keratinocytes (HaCaT), inhibited protease-activated receptor 2 activation by reducing both Ca2+ release and activation of phosphoinositide 3 kinase/AKT and mitogen-activated protein kinases and induced anti-melanogenic effects in zebrafish. Collectively, these results indicate that salicylic acid within ginseng root can inhibit melanocyte melanogenesis and melanin transport, while also suppressing keratinocyte phagocytic function.

TCF21 regulates miR-10a-5p/LIN28B signaling to block the proliferation and invasion of melanoma cells.

Background and aimSome research has suggested that miRNA-10a (miR-10a-5p) had an inhibitory function in proliferation and invasion of cancers. Whereas the role of miR-10a-5p in melanoma has not been fully explored. This study aims to confirm LIN28B as the targeted gene of miR-10a-5p which was explored in melanoma cells. In addition, upstream regulatory molecule of miR-10a-5p was also investigated in melanoma cells.MethodsReal-time Quantitative polymerase chain reaction (RT-qPCR) was adopted to analyze miR-10a-5p expression level in melanoma and the normal human epidermal melanocyte cells. Several biological assays were performed to evaluate miR-10a-5p influences on cell proliferation, migration and invasion ability in A375 and B16-F10 cells. Gene prediction of miRNA targeting and a dual luciferase assay were applied to assess miR-10a-5p-targeted LIN28B. Western blot assessed the impacts of miR-10a-5p on the protein expression of LIN28B. Western blot analyzed the TCF21 effects on the expression of LIN28B and RT-qPCR assessed the influence of TCF21 on the expression level of miRNA-10a. In addition, Chromatin Immunoprecipitation (ChIP) Assay and JASPAR databases were employed to explore the regulatory relationship between TCF21 and miR-10a-5p.ResultsWe discovered that miR-10a-5p expression was lower in melanoma cells and high expression of miR-10a-5p suppressed the proliferation, migration and invasion abilities of melanoma cells. We also discovered that miR-10a-5p targeted the LIN28B mRNA 3′UTR area and diminished LIN28B protein expression. We found that LIN28B expression was strongly decreased by TCF21 upregulation in the two melanoma cells. The qRT-PCR assay showed that miR-10a-5p expression level was obviously boosted by increased TCF21 expression. The results also demonstrated that TCF21 directly regulated miR-10a-5p at transcript levels.ConclusionTCF21 induced miRNA-10a targeting LIN28B could affect the progression and growth of melanoma.

Blue Lagoon Algae Improve Uneven Skin Pigmentation: Results from in vitro Studies and from a Monocentric, Randomized, Double-Blind, Vehicle-Controlled, Split-Face Study

Introduction: Bathing in the Blue Lagoon (BL) in Iceland benefits patients with psoriasis. Accordingly, the BL water contains algae with biological activities that improve skin barrier function and affect T-cell responses relevant for psoriasis. Bathing in the BL is also becoming increasingly popular among healthy individuals and anecdotal evidence suggests positive effects on uneven skin pigmentation. Objective: The aim of the study was to address the impact of BL algae on skin pigmentation. Methods: In this work, in vitro gene expression studies in melanocytes and a noninvasive in vivo study were conducted. Results: We here report that normal human epidermal melanocytes, which had been treated with nontoxic concentrations of BL algae, show a significantly reduced expression of α melanocyte-stimulating hormone-induced expression of genes important for melanin synthesis, such as tyrosinase, tyrosinase-related protein 1, dopachrome tautomerase, melan A protein, and pre-melanosome protein. This in vitro observation prompted us to conduct a randomized, double-blind, intra-individual, comparative split-face in vivo study, in which 60 volunteers with pre-existing facial pigment spots were treated twice daily with a BL algae containing serum or a vehicle control. We found that constitutive skin pigmentation as determined by colorimetry (individual typology angle and luminescence) did not differ significantly between vehicle- and serum-treated skin sites. In marked contrast, digital photography under cross-polarized lighting and RBX technology (VISIA CR) revealed that the number of pigment spots in the serum-treated face decreased significantly compared to the vehicle-treated side. Conclusion: Thus, BL algae can affect human melanocyte function in vitro and reduce uneven facial skin pigmentation in vivo.

Identification of L-Cysteinamide as a Potent Inhibitor of Tyrosinase-Mediated Dopachrome Formation and Eumelanin Synthesis.

The purpose of this study is to identify amino acid derivatives with potent anti-eumelanogenic activity. First, we compared the effects of twenty different amidated amino acids on tyrosinase (TYR)-mediated dopachrome formation in vitro and melanin content in dark-pigmented human melanoma MNT-1 cells. The results showed that only L-cysteinamide inhibited TYR-mediated dopachrome formation in vitro and reduced the melanin content of cells. Next, the antimelanogenic effect of L-cysteinamide was compared to those of other thiol compounds (L-cysteine, N-acetyl L-cysteine, glutathione, L-cysteine ethyl ester, N-acetyl L-cysteinamide, and cysteamine) and positive controls with known antimelanogenic effects (kojic acid and β-arbutin). The results showed the unique properties of L-cysteinamide, which effectively reduces melanin content without causing cytotoxicity. L-Cysteinamide did not affect the mRNA and protein levels of TYR, tyrosinase-related protein 1, and dopachrome tautomerase in MNT-1 cells. L-Cysteinamide exhibited similar properties in normal human epidermal melanocytes (HEMs). Experiments using mushroom TYR suggest that L-cysteinamide at certain concentrations can inhibit eumelanin synthesis through a dual mechanism by inhibiting TYR-catalyzed dopaquinone synthesis and by diverting the synthesized dopaquinone to the formation of DOPA-cysteinamide conjugates rather than dopachrome. Finally, L-cysteinamide was shown to increase pheomelanin content while decreasing eumelanin and total melanin contents in MNT-1 cells. This study suggests that L-cysteinamide has an optimal structure that can effectively and safely inhibit eumelanin synthesis in MNT-1 cells and HEMs, and will be useful in controlling skin hyperpigmentation.

MiR-383-5p inhibits human malignant melanoma cells function via targeting CENPF

Human malignant melanoma (MM), is a type of skin cancer with high morbidity and mortality. In this study, we investigated the role of miR-383-5p in human MM cells in vitro. miR-383-5p expression was downregulated in MM cell lines compared with the human normal melanocyte cell line, and miR-383-5p overexpression inhibited the proliferation, migration, and invasion of M14 and A375 cells. Furthermore, miR-383-5p was able to effectively bind to the 3'UTR of CENPF mRNA. miR-383-5p expression was negatively correlated with CENPF expression and miR-383-5p overexpression inhibited CENPF protein expression in M14 and A375 cells. The overexpression of CENPF could effectively rescue the inhibitory effect on proliferation and invasion caused by miR-383-5p. Additionally, using publicly available databases, we showed that CENPF expression was upregulated in human MM tissues and could predict the prognosis of MM. In conclusion, miR-383-5p acts as a tumor suppressor in human MM by targeting CENPF, suggesting CENPF as a potential therapeutic target for human MM.

Antimelanogenesis Effects of Theasinensin A.

Theasinensin A (TSA) is a major group of catechin dimers mainly found in oolong tea and black tea. This compound is also manufactured with epigallocatechin gallate (EGCG) as a substrate and is refined after the enzyme reaction. In previous studies, TSA has been reported to be effective against inflammation. However, the effect of these substances on skin melanin formation remains unknown. In this study, we unraveled the role of TSA in melanogenesis using mouse melanoma B16F10 cells and normal human epidermal melanocytes (NHEMs) through reverse transcription polymerase chain reaction (RT-PCR), Western blotting analysis, luciferase reporter assay, and enzyme-linked immunosorbent assay analysis. TSA inhibited melanin formation and secretion in α-melanocyte stimulating hormone (α-MSH)-induced B16F10 cells and NHEMs. TSA down-regulated the mRNA expression of tyrosinase (Tyr), tyrosinase-related protein 1 (Tyrp1), and Tyrp2, which are all related to melanin formation in these cells. TSA was able to suppress the activities of certain proteins in the melanocortin 1 receptor (MC1R) signaling pathway associated with melanin synthesis in B16F10 cells: cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB), protein kinase A (PKA), tyrosinase, and microphthalmia-associated transcription factor (MITF). We also confirmed α-MSH-mediated CREB activities through a luciferase reporter assay, and that the quantities of cAMP were reduced by TSA in the enzyme linked immunosorbent assay (ELISA) results. Based on these findings, TSA should be considered an effective inhibitor of hyperpigmentation.

Synthesis and characterization of gold nanoparticles biosynthesised from Aspalathus linearis (Burm.f.) R.Dahlgren For progressive macular hypomelanosis

IntroductionThe study investigated a natural solution for the hypopigmentary disorder, progressive macular hypomelanosis (PMH). The aim was to determine whether gold nanoparticles synthesized from Aspalathus linearis (Burm.f.) R.Dahlgren could be effective for PMH. MethodsGold nanoparticles (ALAuNPs) were synthesized using the ethanolic extract of A. linearis (ALEtOH) and tested, along with the extract for its regulatory effect of melanogenesis in human melanocytes and its antibacterial activity against Cutibacterium acnes (ATCC 6919 strain). Melanosome transfer was determined in a co-culture of normal human melanocytes and keratinocytes and was quantified using flow cytometry. ResultsThe ALAuNPstab (AuNPs stabilized with gum arabic) and ALAuNPnon-stab (non-stabilized) exhibited significantly (P < 0.001) higher antibacterial activity against C. acnes with 50 % inhibition at a concentration of 66.05 ± 1.10 μg/mL and 68.12 ± 1.05 μg/mL, respectively, as compared to that of ALEtOH (271.20 ± 2.32 μg/mL). The ALEtOH exhibited 50 % reduction in cell proliferation (IC50 value) in human melanocytes at 345.5 ± 2.47 μg/mL, whilst ALAuNPstab and ALAuNPnon-stab exhibited an IC50 value of 67.51 ± 1.12 and 69.63 ± 1.07 μg/mL, respectively. The ALEtOH and ALAuNPstab tested at a concentration of 62.5 μg/mL, exhibited a significant increase (P < 0.001) in the total number of melanin present in human melanocytes compared to the untreated (control) cells. The cells treated with ALEtOH and ALAuNPstab led to a significant (P < 0.001) increase in the number of melanosomes transferred as compared to the untreated cells. ConclusionThis study confirmed that A. linearis and its biosynthesised gold nanoparticles stimulated melanin synthesis.

Novel Chemically Modified Curcumin (CMC) Analogs Exhibit Anti-Melanogenic Activity in Primary Human Melanocytes.

Hyperpigmentation is a dermatological condition characterized by the overaccumulation and/or oversecretion of melanin pigment. The efficacy of curcumin as an anti-melanogenic therapeutic has been recognized, but the poor stability and solubility that have limited its use have inspired the synthesis of novel curcumin analogs. We have previously reported on comparisons of the anti-melanogenic activity of four novel chemically modified curcumin (CMC) analogs, CMC2.14, CMC2.5, CMC2.23 and CMC2.24, with that of parent curcumin (PC), using a B16F10 mouse melanoma cell model, and we have investigated mechanisms of inhibition. In the current study, we have extended our findings using normal human melanocytes from a darkly pigmented donor (HEMn-DP) and we have begun to study aspects of melanosome export to human keratinocytes. Our results showed that all the CMCs downregulated the protein levels of melanogenic paracrine mediators, endothelin-1 (ET-1) and adrenomedullin (ADM) in HaCaT cells and suppressed the phagocytosis of FluoSphere beads that are considered to be melanosome mimics. All the three CMCs were similarly potent (except CMC2.14, which was highly cytotoxic) in inhibiting melanin production; furthermore, they suppressed dendricity in HEMn-DP cells. CMC2.24 and CMC2.23 robustly suppressed cellular tyrosinase activity but did not alter tyrosinase protein levels, while CMC2.5 did not suppress tyrosinase activity but significantly downregulated tyrosinase protein levels, indicative of a distinctive mode of action for the two structurally related CMCs. Moreover, HEMn-DP cells treated with CMC2.24 or CMC2.23 partially recovered their suppressed tyrosinase activity after cessation of the treatment. All the three CMCs were nontoxic to human dermal fibroblasts while PC was highly cytotoxic. Our results provide a proof-of-principle for the novel use of the CMCs for skin depigmentation, since at low concentrations, ranging from 5 to 25 µM, the CMCs (CMC2.24, CMC2.23 and CMC2.5) were more potent anti-melanogenic agents than PC and tetrahydrocurcumin (THC), both of which were ineffective at melanogenesis at similar doses, as tested in HEMn-DP cells (with PC being highly toxic in dermal fibroblasts and keratinocytes). Further studies to evaluate the efficacy of CMCs in human skin tissue and in vivo studies are warranted.

A carbazole compound, 9-ethyl-9H-carbazole-3-carbaldehyde, plays an antitumor function through reactivation of the p53 pathway in human melanoma cells

p53, the major tumor suppressor, is frequently mutated in many cancers, and up to 84% of human melanomas harbor wild-type p53, which is considered to be an ideal target for melanoma therapy. Here, we evaluated the antitumor activity of a carbazole derivative, 9-ethyl-9H-carbazole-3-carbaldehyde (ECCA), on melanoma cells. ECCA had a selectively strong inhibitory activity against the growth of BRAF-mutated and BRAF-wild-type melanoma cells but had little effect on normal human primary melanocytes. ECCA inhibited melanoma cell growth by increasing cell apoptosis, which was associated with the upregulation of caspase activities and was significantly abrogated by the addition of a caspase inhibitor. In vivo assays confirmed that ECCA suppressed melanoma growth by enhancing cell apoptosis and reducing cell proliferation, and importantly ECCA did not have any evident toxic effects on normal tissues. RNA-Seq analysis identified several pathways related to cell apoptosis that were affected by ECCA, notably, activation of the p53 signaling pathway. Biochemical assays demonstrated that ECCA enhanced the phosphorylation of p53 at Ser15 in melanoma cells harboring wild-type p53, and importantly, the knockdown or deletion of p53 in those cells counteracted the ECCA-induced apoptosis, as well as senescence. Further investigations revealed that ECCA enhanced the phosphorylation of p38-MAPK and c-Jun N-terminal kinase (JNK), and treatment with either a p38-MAPK or a JNK inhibitor rescued the cell growth inhibition elicited by ECCA, which depended on the expression of the p53 gene. Finally, the combination of ECCA with a BRAF inhibitor significantly enhanced the growth inhibition of melanoma cells. In summary, our study demonstrates that the carbazole derivative, ECCA, induces melanoma cell apoptosis and senescence through the activation of p53 to significantly and selectively suppress the growth of melanoma cells without affecting normal human melanocytes, suggesting its potential to develop a new drug for melanoma therapy.

An in Vitro Study Elucidating the Effect of Oxidative Stress on Melanocytes

Oxidative stress plays a major role in melanocyte destruction in vitiligo, however the exact mechanism responsible for melanocyte death remains uncertain. We aimed to examine the effect of oxidative stress on melanocyte viability by MTT assay and expression of antioxidant genes (CAT, GPX1, G6PD and PRDX3), stress related genes (HSP60, HSP70, SERP1, SIRT1 and POLH) and melanocyte specific genes (MITF, TYR, TYRP1, TRPM1, EDN1, EZR and LAMP1) by real-time PCR upon exposing the normal human melanocytes (NHM), immortalized melanocytes derived from healthy human (PIG1) and from vitiligo patient (PIG3V) to cumene hydroperoxide (CHP). The transcript levels of selected genes were estimated by using real-time PCR. The NHM, PIG1 and PIG3V melanocytes showed significant decrease in viability under CHP (10-100μM) induced oxidative stress. PIG3V displayed significantly increased expression of PRDX3, HSP70, SERP1, POLH as well as decreased expression of CAT, MITF, TYR, TYRP1, TRPM1, EDN1 and LAMP1 under CHP (10 &amp; 20μM) treatment, as compared to NHM and/or PIG1 melanocytes. These results suggest that vitiligo melanocytes are more sensitive to CHP induced oxidative stress, as compared to normal melanocytes. The present study demonstrates that vitiligo may result from an insufficient response of melanocytes to oxidative stress induced by high H2O2 levels. Keywords: Vitiligo; melanocyte; PIG1; PIG3V; oxidative stress; cumene hydroperoxide (CHP).

Learning from Embryogenesis-A Comparative Expression Analysis in Melanoblast Differentiation and Tumorigenesis Reveals miRNAs Driving Melanoma Development.

Malignant melanoma is one of the most dangerous tumor types due to its high metastasis rates and a steadily increasing incidence. During tumorigenesis, the molecular processes of embryonic development, exemplified by epithelial–mesenchymal transition (EMT), are often reactivated. For melanoma development, the exact molecular differences between melanoblasts, melanocytes, and melanoma cells are not completely understood. In this study, we aimed to identify microRNAs (miRNAs) that promote melanoma tumorigenesis and progression, based on an in vitro model of normal human epidermal melanocyte (NHEM) de-differentiation into melanoblast-like cells (MBrCs). Using miRNA-sequencing and differential expression analysis, we demonstrated in this study that a majority of miRNAs have an almost equal expression level in NHEMs and MBrCs but are significantly differentially regulated in primary tumor- and metastasis-derived melanoma cell lines. Further, a target gene analysis of strongly regulated but functionally unknown miRNAs yielded the implication of those miRNAs in many important cellular pathways driving malignancy. We hypothesize that many of the miRNAs discovered in our study are key drivers of melanoma development as they account for the tumorigenic potential that differentiates melanoma cells from proliferating or migrating embryonic cells.

TGFβ2 Upregulates Tyrosinase Activity through Opsin-3 in Human Skin Melanocytes In Vitro

Opsin-3 (OPN3) is a potential key regulator of human melanocyte melanogenesis. How OPN3-mediated regulation of melanocyte melanogenesis is triggered is largely unclear. TGFβ can inhibit the growth of human melanocytes and reduce melanin synthesis in melanocytes. However, whether TGFβ2 can modulate pigmentation in normal human primary melanocytes through OPN3 is entirely unknown. In this study, we constructed a coculture model with human epidermal melanocytes and keratinocytes. OPN3, tyrosinase (TYR), tyrosinase-related protein (TRP)-1, and TRP-2 expression and TYR activity were detected to be higher in cocultured cells than in monocultured cells. Moreover, elevated levels of TGFβ2 were detected in the culture supernatant of melanocytes cocultured with keratinocytes. OPN3 inhibition in melanocytes decreased TYR, TRP-1, and TRP-2 expression and downregulated TYR activity. Our findings indicate that TGFβ2 upregulates TYR activity and TRP-1 and TRP-2 expression in human melanocytes through OPN3 and downstream calcium-dependent G-protein coupled signaling pathways to induce melanogenesis. Interestingly, treatment with the TGFβ2 receptor inhibitor LY2109761 (10 μM) did not inhibit TGFβ2-induced melanocyte melanogenesis though OPN3. Collectively, our data suggest that TGFβ2 upregulates TYR activity through OPN3 through a TGFβ2 receptor-independent and calcium-dependent G-protein coupled signaling pathway.

Chemometric-enhanced metabolic profiling of five Pinus species using HPLC-MS/MS spectrometry: Correlation to in vitro anti-aging, anti-Alzheimer and antidiabetic activities

Detailed metabolic profiling of needles of five Pinus species was investigated using complementary HPLC-MS/MS techniques together with supervised and unsupervised chemometric tools. This resulted in putative identification of 44 compounds belonging to flavonoids, phenolics, lignans, diterpenes and fatty acids. Unsupervised principal component analysis showed that differences were maintained across the metabolites characteristic of each Pinus species, are mainly related to di-O-p-coumaroyltrifolin, p-coumaroyl quinic acid derivative, arachidonic acid, hydroxypalmitic acid, isopimaric acid and its derivative. A supervised Partial Least Squares regression analysis was performed to correlate HPLC-MS/MS profiles with the variation observed in the in vitro anticholinesterase, antiaging and anti-diabetic potential. All investigated Pinus extracts exerted their antiaging activity via increasing telomerase and TERT levels in normal human melanocytes cells compared to the control (untreated cells). Profound inhibition activities of acetylcholinesterase and dipeptidyl peptidase-4 were also observed with P. pinea and P. canariensis extracts having comparable antidiabetic activities to sitagliptin as a standard antidiabetic drug. Our findings suggested that pine needles are a good source of phenolics and diterpenoids that have possible health promoting activities in management and alleviation of diabetic conditions and Alzheimer disease.

Cattleianal and Cattleianone: Two New Meroterpenoids from Psidium cattleianum Leaves and Their Selective Antiproliferative Action against Human Carcinoma Cells.

The Myrteacae family is known as a rich source of phloroglucinols, a group of secondary metabolites with notable biological activities. Leaves of Psidium cattleianum were extracted with chloroform: methanol 8:2 to target the isolation of phloroglucinol derivatives. Isolated compounds were characterized using different spectroscopic methods: nuclear magnetic resonance (NMR), ultra-violet (UV) and mass spectrometry (MS). Two new phloroglucinols were evaluated for cytotoxicity against a panel of six human cancer cell lines, namely colorectal adenocarcinoma cells (HT-29 and HCT-116); hepatocellular carcinoma cells (HepG-2); laryngeal carcinoma (Hep-2); breast adenocarcinoma cells (MCF7 and MDA-MB231), in addition to normal human melanocytes HFB-4. Additionally, cell cycle analysis and annexin-V/FITC-staining were used to gain insights into the mechanism of action of the isolated compounds. The new phloroglucinol meroterpenoids, designated cattleianal and cattleianone, showed selective antiproliferative action against HT-29 cells with IC50’s of 35.2 and 32.1 μM, respectively. Results obtained using cell cycle analysis and annexin-V/FITC-staining implicated both necrosis and apoptosis pathways in the selective cytotoxicity of cattleianal and cattleianone. Our findings suggest that both compounds are selective antiproliferative agents and support further mechanistic studies for phloroglucinol meroterpenoids as scaffolds for developing new selective chemotherapeutic agents.

Molecular and Biochemical Basis of Minocycline-Induced Hyperpigmentation-The Study on Normal Human Melanocytes Exposed to UVA and UVB Radiation.

Minocycline is a drug which induces skin hyperpigmentation. Its frequency reaches up to 50% of treated patients. The adverse effect diminishes the great therapeutic potential of minocycline, including antibacterial, neuroprotective, anti-inflammatory and anti-cancer actions. It is supposed that an elevated melanin level and drug accumulation in melanin-containing cells are related to skin hyperpigmentation. This study aimed to evaluate molecular and biochemical mechanism of minocycline-induced hyperpigmentation in human normal melanocytes, as well as the contribution of UV radiation to this side effect. The experiments involved the evaluation of cyto- and phototoxic potential of the drug using cell imaging with light and confocal microscopes as well as biochemical and molecular analysis of melanogenesis. We showed that minocycline induced melanin synthesis in epidermal melanocytes. The action was intensified by UV irradiation, especially with the UVB spectrum. Minocycline stimulated the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase (TYR) gene. Higher levels of melanin and increased activity of tyrosinase were also observed in treated cells. Moreover, minocycline triggered the supranuclear accumulation of tyrosinase, similar to UV radiation. The decreased level of premelanosome protein PMEL17 observed in all minocycline-treated cultures suggests disorder of the formation, maturation or distribution of melanosomes. The study revealed that minocycline itself was able to enhance melanin synthesis. The action was intensified by irradiation, especially with the UVB spectrum. Demonstrated results confirmed the potential role of melanin and UV radiation minocycline-induced skin hyperpigmentation.