Melanogenesis In Normal Human Melanocytes Research Articles

Study of the Effects of Novel Analogs of Calebin-A on Melanogenesis

In our previous study, we documented the anti-melanogenic efficacy of calebin-A (CBA), which is a curcuminoid analog. The effects of its newly synthesized analogs, i.e., bisdemethoxy calebin (BD), demethoxycalebin-1 (DA1), demethoxycalebin-2 (DA2), and tetrahydrocalebin-A (THCBA), on melanogenesis have not been examined yet. Herein, we evaluated these four CBA analogs to determine their impacts on the enzymatic activity of mushroom tyrosinase. Additionally, we examined their effects on melanogenesis and the tyrosinase activity in B16F10 mouse and MNT-1 human melanoma cells. The antioxidant activity of the analogs was also assessed. Our results revealed that BD was ineffective, while DA1 and DA2 showed similar antioxidant activities, with THCBA exhibiting the greatest antioxidant activity. Next, the diphenolase activity assay results revealed that DA1 showed the most excellent inhibitory efficacy, DA2 and BD showed similar inhibition profiles, and THCBA was ineffective. In addition, the results of the monophenolase activity showed a similar pattern, except that THCBA suppressed the activity. The four analogs were evaluated for any cytotoxicity over a 48 h duration in B16F10 and HaCaT keratinocytes, where DA1 exerted cytotoxicity at the concentration of 50 µM. Based on this, the analogs were evaluated over a 10–50 µM concentration range, while DA1 was evaluated over 10–35 µM. BD showed the greatest efficacy at multiple concentrations in significantly diminishing melanogenesis in hormone-stimulated B16F10 cells, while DA1 and DA2 suppressed melanin at 35 and 50 µM, respectively, and THCBA stimulated melanogenesis at 50 µM. In addition, BD and DA1 suppressed tyrosinase activity in B16F10 cells, with no effect in the case of DA2 and THCBA analogs. However, in MNT-1 cells, only DA1 showed efficacy in suppressing melanin production while the other three analogs were ineffective. Interestingly, BD and DA1 suppressed MNT-1 cell tyrosinase activity. Collectively, our results indicate that of the four analogs, DA1 merits further investigation as a potential compound for hyperpigmentation skin disorders. Additional research is necessary to delineate the molecular mechanisms underlying the melanogenesis-inhibitory effect of CBA analogs and examine their efficacy in diminishing melanogenesis in normal human melanocytes.

Streptomycin generates oxidative stress in melanin-producing cells: In vitro study with EPR spectroscopy evidence

Streptomycin (STR) is an aminoglycoside antibiotic with a broad-spectrum of activity and ototoxic potential. The mechanism of STR-induced inner ear damage has not been fully elucidated. It was previously found that STR binds to melanin, which may result in the accumulation of the drug in melanin-containing tissues. Melanin pigment is present in various parts of the inner ear, including the cochlea and vestibular organ.The present study aimed to assess if streptomycin generates oxidative stress and affects melanogenesis in normal human melanocytes. Moreover the variation of free radical concentration in STR-treated melanocytes was examined by electron paramagnetic resonance spectroscopy (EPR). We found that STR decreases cell metabolic activity and reduces melanin content. The observed changes in the activity of antioxidant enzymes activity in HEMn-DPs treated with streptomycin may suggest that the drug affects redox homeostasis in melanocytes. In this work EPR study expanded knowledge about free radicals in interactions of STR and melanin in melanocytes. The results may help elucidate the mechanisms of STR toxicity on pigment cells, including melanin-producing cells in the inner ear. This is important because understanding the mechanism of STR-induced ototoxicity would be helpful in developing new therapeutic strategies to protect patients' hearing.

Effects of a Standardized Hydrogenated Extract of Curcumin (Curowhite™) on Melanogenesis: A Pilot Study

The stimulation of melanogenesis by novel natural products is desirable for cosmetic applications such as skin tanning, anti-greying, and clinical use for treating vitiligo and leukoderma disorders. Microphthalmia transcription factor (MITF) is a central transcription factor that controls the expression of tyrosinase, which is a key enzyme responsible for catalyzing the rate-limiting processes of melanin production. Tetrahydrocurcuminoids (THCr), which mostly consist of tetrahydrocurcumin (THC), are a colorless bioactive mixture derived from curcuminoids that are extracted from the Curcuma longa plant. THCr has been reported to exhibit superior properties, including antioxidant and anti-inflammatory effects. Our previous study reported the greater melanogenesis-stimulating effects of purified THC, compared to hexahydrocurcumin (HHC) or octahydrocurcumin (OHC). Curowhite™ (CW) is a proprietary extract that consists of 25% hydrogenated curcuminoids (mixture of THCr, hexahydrocurcuminoids, and octahydrocurcuminoids) encapsulated in a β-cyclodextrin (βCyD) excipient. The encapsulation of THCr in a suitable excipient, such as the widely popular cyclodextrins, helps to enhance the stability, solubility, and bioavailability of the THCr. CW is marketed as a nutraceutical with GRAS status and is safe when administered orally, as shown in vivo studies. However, the impact of CW on melanogenesis remains unexplored. Herein, the impact of CW on melanogenesis were investigated using B16F10 and MNT-1 cells. Our findings show that CW is markedly cytotoxic to B16F10 cells without affecting the cellular melanin content. However, in MNT-1 cells, CW significantly stimulated intracellular melanin content over the concentration range (20–60 µg/mL) with increased dendrite formation while being nontoxic to MNT-1 cells or HaCaT cells after a 5-day treatment. Examination of the effects of the excipient βCyD on cytotoxicity and melanogenesis confirmed that the excipient had no contribution to the biological impacts that were found to be exclusively attributable to the encapsulated mixture (THCr). The mechanisms of CW’s promelanogenic effects in MNT-1 cells were found to be related, at least in part, to an increase in tyrosinase and MITF protein levels, as CW did not alter tyrosinase activity in MNT-1 cells. Moreover, CW exhibited antioxidant activity as obtained through DPPH radical scavenging assay. Together, the findings of this pilot study indicate that CW might hold an exciting avenue as a pro-pigmenting nutraceutical for treating hypopigmentation disorders, the detailed mechanisms of which warrant further exploration. Moreover, future investigations are necessary to examine CW’s effects on melanogenesis in normal human melanocytes and in vivo studies.

Ursodeoxycholic Acid May Inhibit Environmental Aging-Associated Hyperpigmentation.

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-1α, IL-8, TNF-α, 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.

Methylquercetins stimulate melanin biosynthesis in a three-dimensional skin model.

In a previous study, we found that both synthetic 3-O-methylquercetin (3MQ) and 3,4',7-O-trimethylquercetin (34'7TMQ) increased extracellular melanin content. 34'7TMQ increased the activity of melanogenic enzymes by stimulating the p38 pathway and the expression of microphthalmia-associated transcription factor (MITF). In contrast, 3MQ increased the activity of melanogenic enzymes without the involvement of MITF, which suggests that 3MQ inhibits the degradation of melanogenic enzymes. In the present study, we investigated the effects of 3MQ and 34'7TMQ on melanogenesis in normal human melanocytes and using a commercial three-dimensional (3D) skin model system. Both 3MQ and 34'7TMQ elongated the dendrites of normal human melanocytes from a Caucasian donor, but did not stimulate melanogenesis in the melanocytes. In the 3D skin model, which included melanocytes from an Asian donor, 3MQ and 34'7TMQ increased and elongated the melanocytes and showed a tendency to stimulate melanogenesis. These results suggest that 3MQ and 34'7TMQ could be put to practical use in skin care products and agents aimed at preventing hair graying.

Effect of thioridazine on antioxidant status of HEMn-DP melanocytes

Thioridazine as an antipsychotic agent was extensively used to treat various psychotic disorders, e.g. schizophrenia. However, the therapy with this drug can induce serious side effects such as extrapyramidal symptoms or ocular and skin disorders, which mechanisms are still not fully established. To gain inside the molecular mechanisms underlying thioridazine toxicity, we examined the effect of this drug on cell viability, antioxidant defence system as well as melanogenesis in normal human melanocytes. It was demonstrated that thioridazine induces concentration-dependent loss in cell viability. The value of EC50 was calculated to be 2.24 μM. To study the effect of thioridazine on antioxidant defence system in melanocytes, the level of hydrogen peroxide and the activities of antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase were determined. The drug in concentrations of 0.1, 0.25, 1.0 and 2.5 μM caused changes in cellular antioxidant defence system indicating the induction of oxidative stress. It was also shown that the analysed neuroleptic in concentrations of 1.0 and 2.5 μM significantly inhibited melanogenesis. The observed changes in cell viability, antioxidant defence system and melanization in normal human melanocytes after thioridazine treatment may explain an important role of reactive oxygen species as well as melanin in mechanisms involved in this drug side effects directed on pigmented tissues.

Nrf2 negatively regulates melanogenesis by modulating PI3K/Akt signaling.

Nrf2 plays a role in protection of cells against oxidative stress and xenobiotic damage by regulating cytoprotective genes. In this study, we investigated the effect of Nrf2 on melanogenesis in normal human melanocytes (NHMCs). When NHMCs were transduced with a recombinant adenovirus expressing Nrf2, melanin synthesis was significantly decreased. Consistent with this result, overexpression of Nrf2 decreased the expression of tyrosinase and tyrosinase-related protein 1. The inhibitory effect of Nrf2 was reversed by overexpression of Keap1, an intracellular regulator of Nrf2. Interestingly, Nrf2 overexpression resulted in marked activation of PI3K/Akt signaling. Conversely, inhibition of PI3K activity by treatment with wortmannin reversed the depigmentary effects of Nrf2. Taken together, these results strongly suggest that Nrf2 negatively regulates melanogenesis by modulating the PI3K/Akt signaling pathway.

Wnt inhibitory factor (WIF)‐1 promotes melanogenesis in normal human melanocytes

Wnt signaling plays a role in the differentiation as well as the development of melanocytes. Using a microarray analysis, hyperpigmentary skin of melasma expressed high levels of Wnt inhibitory factor-1 (WIF-1) compared with perilesional normal skin. In this study, the expression and functional roles of WIF-1 on melanocytes were investigated. WIF-1 was expressed both in the melanocytes of normal human skin and in cultured melanocytes. The upregulation of WIF-1 on cultured normal human melanocytes significantly induced expressions of MITF and tyrosinase, which were associated with increased melanin content and tyrosinase activity. Consistent with the stimulatory effect of WIF-1, WIF-1 siRNA reduced melanogenesis in the cells. Moreover, WIF-1 increases pigmentation in melanocytes co-cultured with WIF-1-overexpressed fibroblasts and of organ-cultured human skin. These findings suggest that melanocytes express WIF-1 constitutively in vivo and in vitro and that WIF-1 promotes melanogenesis in normal human melanocytes.

IL-4 Inhibits the Melanogenesis of Normal Human Melanocytes through the JAK2–STAT6 Signaling Pathway

Skin diseases can be characterized by their predominant CD4-positive T-helper (Th) cell profiles. Chronic dermatological conditions often give rise to abnormal skin pigmentation. To understand the role of Th cells in pigmentation, the effects of the major Th cell cytokines, IFNγ, IL-4, and IL-17A, on melanogenesis were evaluated using cultured normal human melanocytes (NHMs) instead of relying on transformed melanoma cell lines. IL-4 directly inhibited melanogenesis in NHMs and downregulated both transcription and translation of melanogenesis-associated genes, such as microphthalmia-associated transcription factor (MITF) and dopachrome tautomerase. Despite the lack of a direct inhibition of melanin pigment synthesis, IFNγ and IL-17A increased the synthesis of an antimelanogenic cytokine IL-6 in NHMs. IFNγ activated signal transducers and activators of transcription 1 (STAT1) and STAT3 phosphorylation in NHMs, and IL-4 increased the STAT3 and STAT6 phosphorylation. The differential phosphorylation profile of STAT proteins between IFNγ and IL-4 may explain the difference in their effect on melanogenesis in NHMs. The IL-4-induced downregulation of melanogenesis was inhibited by treating NHMs with a JAK2 inhibitor AG490 or STAT6 siRNA. In conclusion, the involvement of the IL-4-induced JAK2-STAT6 signaling and the IFNγ- or IL-17A-dependent antimelanogenic IL-6 production should be considered as one of the mechanisms explaining the association with hypopigmention in skin diseases.

Lotus (Nelumbo nuficera) flower essential oil increased melanogenesis in normal human melanocytes

In this study, the essential oil from lotus flower extract, including petals and stamens, was assessed with regard to its effects on melanogenesis in human melanocytes. The lotus flower essential oil was shown to stimulate melanin synthesis and tyrosinase activity in a dose-dependent manner. The lotus flower essential oil induced the expression of tyrosinase, microphthalmia-associated transcription factor M (MITF-M), and tyrosinase-related proten-2 (TRP-2) proteins, but not tyrosinase mRNA. Moreover, it increased the phosphorylation of ERK and cAMP response element binding protein (CREB). In order to verify the effective components of the lotus flower oil, its lipid composition was assessed. It was found to be comprised of palmitic acid methyl ester (22.66%), linoleic acid methyl ester (11.16%), palmitoleic acid methyl ester (7.55%) and linolenic acid methyl ester (5.16%). Among these components, palmitic acid methyl ester clearly induced melanogenesis as the result of increased tyrosinase expression, thereby indicating that it may play a role in the regulation of melanin content. Thus, our results indicate that lotus flower oil may prove useful in the development of gray hair prevention agents or tanning reagents.

Tyrosine levels regulate the melanogenic response to alpha-melanocyte-stimulating hormone in human melanocytes: implications for pigmentation and proliferation.

Melanocyte-stimulating hormone (alpha-MSH) increases cytosolic levels of cAMP as well as tyrosinase activity in murine melanocytes. These activities depend upon the presence of melanin precursors and may differ in human melanocytes. In this study, we demonstrate that high levels of tyrosine (3.7 mM), the chief melanin precursor, reduced the proliferative effect of alpha-MSH and altered human melanocyte morphology as compared to treatment with low (25-30 microM, half-physiological) levels of tyrosine. The anti-proliferative effect of high levels of tyrosine was not restricted to alpha-MSH; tyrosine also reduced proliferation induced by forskolin, a direct activator of the cAMP pathway. Exposure to low tyrosine levels and alpha-MSH induced a dendritic morphology; in the presence of high tyrosine and alpha-MSH, melanocytes displayed large, pigmented cell bodies and less dendricity. Exposure to alpha-MSH in the presence of low tyrosine for up to 9 days did not appreciably increase melanin levels, but culturing the human melanocytes in high levels of tyrosine with alpha-MSH increased melanin levels 10-50-fold, depending on the pigmentation background of the donor. A greater induction of melanin accumulation was observed in melanocytes derived from light-skinned donors than was observed in cells obtained from dark-skinned donors. The poor ability of alpha-MSH to stimulate melanin synthesis was not caused by a lack of induction of melanogenic proteins, as alpha-MSH increased the expression of microphthalmia (MITF), tyrosinase, dopachrome tautomerase (DCT), and Pmel-17, compared to untreated cells or cells stimulated by phorbol ester alone, regardless of tyrosine levels. DCT levels were greatly induced by low tyrosine with alpha-MSH, but were dramatically decreased by high tyrosine with alpha-MSH. Interestingly, in this same medium (high tyrosine), MITF levels also decreased after 2 weeks and were barely detectable by the third week. Despite the absence of MITF at 3 weeks of treatment in high tyrosine medium, tyrosinase levels remained high, thereby suggesting that additional factors must be responsible for tyrosinase transcription in human melanocytes. Our results indicate that tyrosine levels can regulate the proliferative activity induced by alpha-MSH, as well as the extent of melanogenesis in normal human melanocytes. The significance of this work is that tyrosine levels may be part of the mechanism that switches melanocytes out of a proliferative status and into a melanin-synthesizing, terminally differentiated phenotype.

Retinoic acid as modulator of UVB-induced melanocyte differentiation. Involvement of the melanogenic enzymes expression.

Retinoic acid (RA) is a hormone-like agent involved in the control of cell differentiation. The most characteristic feature of melanocyte differentiation, melanogenesis, is stimulated by UV radiations. Excessive chronic sun exposure results in irregular skin hypermelanosis that can be partially corrected by topical RA. The basic mechanisms underlying this effect of RA are unknown. To determine whether RA can directly modulate excessive melanin synthesis, we analyzed the in vitro effect of cis- and trans-RA on UVB-induced melanogenesis in S91 mouse melanoma cells and in normal human melanocytes (NHM). In both cells types, the two RA isoforms significantly decreased the UVB-stimulated melanogenesis in term of tyrosinase activity and melanin neosynthesis. To correlate changes in melanogenesis with the expression of melanogenic enzymes, we determined the neosynthesis rate of tyrosinase, tyrosinase-related protein-1 (TRP-1/gp 75) and tyrosinase-related protein-2 (TRP-2/DOPAchrome tautomerase). Here we show that UVB-induced melanogenesis in NHM is related to an increased synthesis of tyrosinase and TRP-1 and to a dramatic decrease of TRP-2 expression. RA inhibition of UVB-induced melanogenesis acts at the post-transcriptional level leading to a decreased tyrosinase and TRP-1 synthesis. We also show that in NHM, inhibition of TRP-2 following UVB-treatment is significantly reversed by RA. This demonstrates a negative correlation between melanogenesis and TRP-2 expression.