Different susceptibilities of melanoma cells to retinoic acid-induced changes in melanotic expression

The effect of retinoic acid on murine B16 melanoma cell growth, tyrosinase activity and melanin synthesis was investigated. Retinoic acid inhibited the growth of B16F1, B16F10 and B16BL6 melanoma cells, but enhanced melanin synthesis only in the B16F1 cells. The B16F10 and B16BL6 cells exhibited retinoic acid-induced suppression of tyrosinase activity and melanin synthesis, which was most apparent in the B16F10 cell variant. For comparison, Cloudman S91 melanoma cells proved to be particularly sensitive to retinoic acid-induced growth inhibition and stimulation of the expression of their melanotic phenotype. These results suggest considerable heterogeneity in the B16 melanoma with respect to their response to retinoic acid.     

Protective effects of a Ficus deltoidea (Mas cotek) extract against UVB-induced photoageing in skin cells

Continuous exposure to ultraviolet (UV) irradiation leads to a variety of skin damage, such as sunburn, pigmentation, premature ageing, and photocarcinogenesis. Various phytochemical extracts have been identified to efficiently protect sun exposed skin from UV induced photodamage. A Ficus deltoidea (Mas cotek) water extract has been widely used for women’s health in Malaysia. In a previous study from this lab, the F. deltoidea extract exhibited strong anti-melanogenic effects towards cultured B16F1 melanoma cells. Additional studies were intended to evaluate the effects of the F. deltoidea extract on antiphotoageing activity using cultured human dermal fibroblasts and immortalised human keratinocytes (HaCaT). Both TNF-α and cyclooxygenase (COX-2) play primary roles in the inflammation process upon UV irradiation and are known to be stimulated by UVB irradiation. Treatment with the F. deltoidea extract dramatically inhibited the UVinduced TNF-α, IL-1α, IL-6, and COX-2 expression. The decreased collagen synthesis of fibroblasts as a result of UVB exposure was restored to a normal level after treatment with the F. deltoidea extract. In addition, the enhanced MMP-1 expression upon UVB irradiation was downregulated by the F. deltoidea extract in a dose-dependent manner. The overall findings indicate that the F. deltoidea extract may exert a protective effect against UVB-induced damage in the skin that is useful for anti-photoageing cosmetic products.     

Depigmenting effect of Xanthohumol from hop extract in MNT-1 human melanoma cells and normal human melanocytes

Xanthohumol (XH) is the most abundant prenylated flavonoid found in the hop plant (Humulus lupulus L.) 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 μM 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 μM) 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 in vivo efficacy are warranted.     

Interaction of sesamol (3,4-methylenedioxyphenol) with tyrosinase and its effect on melanin synthesis

Sesamin, sesamolin (lignans) and sesamol - from sesame seed (Sesamum indicum L.) - are known for their health promoting properties. We examined the inhibition effect of sesamol, a phenolic degradation product of sesamolin, on the key enzyme in melanin synthesis, viz. tyrosinase, in vitro. Sesamol inhibits both diphenolase and monophenolase activities with midpoint concentrations of 1.9 μM and 3.2 μM, respectively. It is a competitive inhibitor of diphenolase activity with a K_i of 0.57 μM and a non-competitive inhibitor of monophenolase activity with a K_i of 1.4 μM. Sesamol inhibits melanin synthesis in mouse melanoma B16F10 cells in a concentration dependant manner with 63% decrease in cells exposed to 100 μg/mL sesamol. Apoptosis is induced by sesamol, limiting proliferation. This study of the chemistry and biology of lignans, in relation to the mode of action of bioactive components, may open the door for drug applications targeting enzymes.     

Regulation of melanin synthesis by the TGF-β family in B16 melanoma cells

Effects of representative members of the transforming growth factor-β (TGF-β) family, TGF-β1, activin A and BMP-2, on melanin content and expression of pigment-producing enzymes were examined in B16 melanoma cells. Treatment with TGF-β1 or activin A but not with BMP-2 significantly decreased melanin content and expression of Tyrosinase and Tyrp-1, suggesting an inhibitory effect of TGF-β1 and activin A on melanin synthesis. TGF-β1 completely inhibited melanin synthesis induced by α-melanin stimulating hormone (α-MSH), whereas activin A only slightly did. As compared with parental B16 cells, the inhibitory effects of TGF-β1 and activin A on melanin content were relative smaller in B16 F10 cells, a subline of B16 cells that contain more pigment. The present study indicates that in addition to TGF-β, activin negatively regulates melanogenesis in the absence of α-MSH, but that the activity in the presence of α-MSH was slightly different between TGF-β and activin.     

A novel synthetic compound, (Z)-5-(3-hydroxy-4-methoxybenzylidene)-2-iminothiazolidin-4-one (MHY773) inhibits mushroom tyrosinase

As part of continued efforts for the development of new tyrosinase inhibitors, (Z)-5-(substituted benzylidene)-2-iminothiazolidin-4-one derivatives (1a – 1l) were rationally synthesized and evaluated for their inhibitory potential in vitro. These compounds were designed and synthesized based on the structural attributes of a β-phenyl-α,β-unsaturated carbonyl scaffold template. Among these compounds, (Z)-5-(3-hydroxy-4-methoxybenzylidene)-2-iminothiazolidin-4-one (1e, MHY773) exhibited the greatest tyrosinase inhibition (IC₅₀ = 2.87 μM and 8.06 μM for monophenolase and diphenolase), and outperformed the positive control, kojic acid (IC₅₀ = 15.59 and 31.61 μM). The kinetic and docking studies demonstrated that MHY773 interacted with active site of tyrosinase. Moreover, a melanin quantification assay demonstrated that MHY773 attenuates α-melanocyte-stimulating hormone (α-MSH) and 3-isobutyl-1-methylxanthine (IBMX)-induced melanin contents in B16F10 melanoma cells. Taken together, these data suggest that MHY773 suppressed the melanin production via the inhibition of tyrosinase activity. MHY773 is a promising for the development of effective pharmacological and cosmetic agents for skin-whitening.     

Evaluation of the inhibition of mushroom tyrosinase and cellular tyrosinase activities of oxyresveratrol: comparison with mulberroside A

The inhibitory effects of oxyresveratrol, the aglycone of mulberroside A, on mushroom and cellular tyrosinase activities and melanin synthesis were evaluated. Mulberroside A and oxyresveratrol showed inhibitory activity against mushroom tyrosinase, with oxyresveratrol demonstrating a greater inhibitory effect than that of mulberroside A. Oxyresveratrol and mulberroside A strongly inhibited melanin production in Streptomyces bikiniensis and exhibited dose-dependent inhibition of tyrosinase activity and inhibition of melanin synthesis in B16F10 melanoma cells. However, the compounds exhibited nearly similar inhibitory effects on the activity of cellular tyrosinase and melanin synthesis in murine melanocytes. The inhibition of melanin synthesis by mulberroside A and oxyresveratrol was involved in suppressing the expression level of melanogenic enzymes, tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). These results indicate that the inhibition rate of mushroom tyrosinase might not provide an accurate estimate of the inhibition rate of melanin synthesis in melanocytes.     

Chloroform extract from Moricandia arvensis inhibits growth of B16-F0 melanoma cells and promotes differentiation in vitro

Objectives: Poor therapeutic results have been reported for treatment of malignant melanoma; therefore in this study we have investigated inhibitory capacity of ethyl acetate, chloroform (Chl) and methanol extracts from Moricandia arvensis on mouse melanoma (B16‐F0) and human keratinocyte (HaCaT) cell proliferation. Influence of Chl extract on percentage distribution in cell cycle phases and melanogenesis was also studied. Material and methods: Cell viability was determined at various periods using the MTT assay, and flow cytometry was used to analyse effects of Chl extract on progression through the cell cycle and apoptosis. In addition, amounts of melanin and tyrosinase were measured spectrophotometrically at 475 nm. Results: Chl extract exhibited significant anti‐proliferative activity after incubation with the two types of tumour skin cells. Morphological changes in B16‐F0 cells, accompanied by increase of tyrosinase activity, and of melanin synthesis were observed, which are markers of differentiation of malignant melanoma cells. Furthermore, cell cycle analysis revealed that B16‐F0 cells treated with Chl extract were arrested predominantly in G₁ phase. Conclusion: Chl extract had the ability to reverse malignant melanoma cells from proliferative to differentiated state, thus providing a new perspective in developing novel strategies for prevention and treatment of malignant melanoma, possibly through consumption of the extract in an appropriate cancer prevention diet. Moreover, there is scope for the extract being introduced into cosmetic products as a natural tanning agent.     

Tyrosinase activities of cell-free extracts and living cells of cultured melanoma cells

Melanogenesis in the course of monolayer culture of a stably melanotic clonal line C₂M, derived from a mouse melanoma B 16, was investigated. Tyrosinase activity per cell of cell-free extracts was highest when the extract was prepared from cells in the mid-exponential phase of growth, when it was more than 6 times the activity of that prepared from a fully grown culture or a culture in the very early phase. On the other hand, the enzyme activity per cell of living cells in culture was highest in the early phase of culture and decreased rapidly to a level of less than one tenth of the maximum activity, in the stationary phase.The upper limit of population density of cultured melanoma cells permissive for melanin synthesis (2 to 3 × 10⁵ cells/cm²) was much higher than that of normal (nonneoplastic) melanocytes, which had been reported to produce melanin only under conditions of clonal growth.The relative efficiency of tyrosinase activity in situ, expressed by the ratio of tyrosinase activity in culture to that of cell-free extract, decreased rapidly in the exponential phase of growth. This decrease correlates to the cell density in the culture, and little if at all to the division rate, and suggests a suppressing mechanism of melanin synthesis working at the enzyme level.     

Daphne odora Exerts Depigmenting Effects via Inhibiting CREB/MITF and Activating AKT/ERK-Signaling Pathways

Daphne odora, a blooming shrub, has been traditionally used for various medicinal purposes. However, information on its anti-melanogenic activity and dermal application is limited. In this study, the Daphne odora extract (DOE), with constituents including daphnetin, was used to investigate depigmenting activity and the underlying mechanism of Daphne odora. DOE inhibited in vitro and cellular tyrosinase activity in a dose-dependent manner, and reduced the α-MSH-induced melanin biosynthesis to a control level. The protein expressions of melanin synthesis-related enzymes were also significantly reduced by DOE. Moreover, DOE decreased the phosphorylation of cAMP-response element binding proteins (CREBs) induced by α-MSH in B16F10 cells, while it activated phosphorylated extra-cellular signal-regulated kinases (ERKs) and protein kinase B (AKT) expression. These results suggest that DOE might inhibit the melanogenesis signaling pathways by activating ERK- and AKT-signaling pathways to regulate the expression of CREB and MITF and its downstream pathways. Therefore, DOE could potentially be developed as a depigmenting agent.     

Inhibitory effects of N-(acryloyl)benzamide derivatives on tyrosinase and melanogenesis

Targeting of tyrosinase has proven to be the best means of identifying safe, efficacious, and potent tyrosinase inhibitors for whitening skin. We designed and synthesized ten NAB (N-(acryloyl)benzamide) derivatives (1a–1j) using the Horner-Wadsworth-Emmons olefination of diethyl (2-benzamido-2-oxoethyl)phosphonate and appropriate benzaldehydes. A mushroom tyrosinase inhibitory assay showed compounds 1a (36.71 ± 2.14% inhibition) and 1j (25.99 ± 2.77% inhibition) inhibited tyrosinase more than the other eight NAB derivatives and kojic acid (21.56 ± 2.93% inhibition), and docking studies indicated 1a (−6.9 kcal/mole) and 1j (−7.5 kcal/mole) had stronger binding affinities for tyrosinase than kojic acid (−5.7 kcal/mole). At a concentration of 25 μM, 1a and 1j were nontoxic in B16F10 melanoma cells and exhibited stronger tyrosinase inhibition (59.70% and 76.77%, respectively) than kojic acid (50.30% inhibition) or arbutin (41.78% inhibition at 400 μM). Similarly, in B16F10 melanoma cells, compounds 1a and 1j at 25 μM decreased total melanin content by 47.97% and 61.77%, respectively (kojic acid; 38.98%). Similarities between inhibitions of tyrosinase activity and melanin contents suggested the anti-melanogenic effects of 1a and 1j were due to tyrosinase inhibition. The excellent DPPH scavenging activity of 1j suggests it might enhance in vivo effect on melanin contents. The study suggests compound 1j offers a potential starting point for the development of safe, potent tyrosinase inhibitors.     

Investigation of the Regulation of Pigmentation in α-Melanocyte-Stimulating Hormone Responsive and Unresponsive Cultured B16 Melanoma Cells

In vitro melanocyte-stimulating hormone (MSH) stimulates melanogenesis in some, but not all, melanocytes and melanoma cells. In an attempt to explain this variation in response to αMSH, we examined cyclic adenosine monophosphate (cAMP) accumulation, tyrosinase activity, and melanin production in primary (1°) murine B16 melanoma cells and in two B16 cell lines (B16 F1 and B16 F10) that are known to respond to αMSH. In vivo all three B16 melanoma cell types produced pigmented tumours. In vitro αMSH increased tyrosinase activity and melanin content in the F1 and F10 cells but not in the B16 1° cells. αMSH, however, increased cAMP production in all three cell types, confirming that the inability of B16 1° cells to produce melanin in response to αMSH is not due to a lack of αMSH receptors or cAMP response to αMSH. Further, we present evidence for a separate pathway of melanogenesis that is independent of cAMP as calmodulin antagonists, which do not elevate cAMP, increased tyrosinase activity, and melanin production in both 1° and F1 cells.     

Four new sesquiterpenes from the rhizomes of Curcuma wenyujin

Four new sesquiterpenes, namely wenyujinols I – L (1 – 4), along with eleven known ones were isolated from the rhizomes of Curcuma wenyujin. The structures of new compounds were elucidated using spectroscopic and spectrometric data analyses. All isolated compounds were evaluated for their inhibitory effects on melanin production and tyrosinase activity in B16F10 cells. Compound 1 exhibited significant inhibition against melanin production.     

Antioxidant,anti-tyrosinase and anti-melanogenic effects of (E)-2,3-diphenylacrylic acid derivatives

During our continued search for strong skin whitening agents over the past ten years, we have investigated the efficacies of many tyrosinase inhibitors containing a common (E)-β-phenyl-α,β-unsaturated carbonyl scaffold, which we found to be essential for the effective inhibition of mushroom and mammalian tyrosinases. In this study, we explored the tyrosinase inhibitory effects of 2,3-diphenylacrylic acid (2,3-DPA) derivatives, which also possess the (E)-β-phenyl-α,β-unsaturated carbonyl motif. We synthesized fourteen (E)-2,3-DPA derivatives 1a–1n and one (Z)-2,3-DPA-derivative 1l′ using a Perkin reaction with phenylacetic acid and appropriate substituted benzaldehydes. In our mushroom tyrosinase assay, 1c showed higher tyrosinase inhibitory activity (76.43 ± 3.53%, IC₅₀ = 20.04 ± 1.91 µM) with than the other 2,3-DPA derivatives or kojic acid (21.56 ± 2.93%, IC₅₀ = 30.64 ± 1.27 μM). Our mushroom tyrosinase inhibitory results were supported by our docking study, which showed compound 1c (−7.2 kcal/mole) exhibited stronger binding affinity for mushroom tyrosinase than kojic acid (−5.7 kcal/mole). In B16F10 melanoma cells (a murine cell-line), 1c showed no cytotoxic effect up to a concentration of 25 μM and exhibited greater tyrosinase inhibitory activity (68.83%) than kojic acid (49.39%). In these cells, arbutin (a well-known tyrosinase inhibitor used as the positive control) only inhibited tyrosinase by 42.67% even at a concentration of 400 μM. Furthermore, at 25 µM, 1c reduced melanin contents in B16F10 melanoma cells by 24.3% more than kojic acid (62.77% vs. 38.52%). These results indicate 1c is a promising candidate treatment for pigmentation-related diseases and potential skin whitening agents.