• Biomolecules & Therapeutics
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• 제22권3호
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• pp.207-212
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• 2014

Skin hyperpigmentation is one of the most common skin disorders caused by abnormal melanogenesis. The mechanism and key factors at play are not fully understood. Previous reports have indicated that cystamine (CTM) inhibits melanin synthesis, though its molecular mechanism in melanogenesis remains unclear. In the present study, we investigated the effect of CTM on melanin production using ELISA reader and the expression of proteins involved in melanogenesis by Western blotting, and examined the involvement of transglutaminase-2 (Tgase-2) in SK-MEL-2 human melanoma cells by gene silencing. In the results, CTM dose-dependently suppressed melanin production and dendrite extension in a-MSH-induced melanogenesis of SK-MEL-2 human melanoma cells. CTM also suppressed a-MSH-induced chemotactic migration as well as the expressions of melanogenesis factors TRP-1, TRP-2 and MITF in a-MSH-treated SK-MEL-2 cells. Meanwhile, gene silencing of Tgase-2 suppressed dendrite extension and the expressions of TRP-1 and TRP-2 in a-MSH-treated SK-MEL-2 cells. Overall, these findings suggested that CTM suppresses a-MSH-induced melanogenesis via Tgase-2 inhibition and that therefore, Tgase-2 might be a new target in hyperpigmentation disorder therapy.

• 한방안이비인후피부과학회지
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• 제16권3호
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• pp.129-144
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• 2003

Recently many efforts were focused to understand the mechanical insights of melanogenesis to develop the agents for hyper-pigmentation and hypo-pigmentation. In the melanin biosynthetic pathway, tyrosinase is the rate limiting enzyme, and ${\alpha}$-melanocyte stimulating hormone(MSH) or cAMP-elevating agents stimulate melanogenesis and enhance the melanin synthesis and the tyrosinase activity. The author has analyzed the effects of Rhizoma Bletillae on the basal melanogenic activities of B16／F10 mouse melanoma cells, and on the ${\alpha}$-MSH or forskolin-induced melanogenesis. Rhizoma Bletillae alone markedly suppressed melanin content and tyrosinase activity in a dose-dependent manner. Pretreatment of the cells with Rhizoma Bletillae also suppressed the increase of ${\alpha}$-MSH (100 nM) or forskolin (20 ${\mu}M$)-induced melanin content and tyrosinase activity. The decrease in the tyrosinase activity was paralled by a decrease in the abundance of tyrosinase protein and tyrosinase promoter activity. Pretreatment of the cells with Rhizoma Bletillae also inhibited the increase of forskolin(20${\mu}M$) induced the amount of tyrosinase protein and tyrosinase promoter activity. The results of DOPA staining revealed that pretreatment of the cells with Rhizoma Bletillae showed less intensity than B16 melanoma cells stimulated with ${\alpha}$-MSH or forskolin. These results suggest that Rhizoma Bletillae inhibits melanogenesis and abrogates ${\alpha}$-MSH and cAMP-induced melanogenesis in B16 melanoma cells.

• 약학회지
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• 제47권1호
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• pp.31-36
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• 2003

To investigate the effect of protein kinase on melanin production via cAMP-dependent pathway, we measured the melanin amount and tyrosinase activity in B16 melanoma cells stimulated by alpha-melanocyte stimulating hormone (MSH), forskolin and 8-Br-cAMP. MSH, forskolin and 8-Br-cAMP significantly increased both melanin production and tyrosinase activity in B16 cells. Melanin production and tyrosinase activity by MSH are significantly inhibited by cyclic AMP-dependent protein kinase inhibitor (KT5720) and protein kinase C down-regulation treated with PMA. Bisindolmaleimide (1$\mu$M), protein kinase C inhibitor, significantly inhibited melanin production and tyrosinase activity stimulated by MSH, forskolin and 8-Br-cAMP with the following order of potency: MSH＞forskolin＞8-Br-cAMP. Tyrosine kinase inhibitor, genistein and DHC, significantly inhibited both, but the inhibitory effect was more potent in 8-Br-cAMP-stimulated B16 cells than MSH-stimulated cells. NFkB inhibitor (parthenolide) significantly inhibited melanin production and tyrosinase activity. Neither melanin production nor tyrosinase activity induced by MSH, forskolin and 8-Br-cAMP were affected by KN-62 (calmodulin-dependent protein kinase II inhibitor), PD098059 (mitogen-activated protein kinase inhibitor, MAPKK) and worthmannin (phosphatidylinositol 3-kinase inhibitor). These results suggest that both protein kinase C and tyrosine kinase are involved in melanin production by cyclic AMP-dependent pathway and NFkB pathway may play an important role in cyclic AMP-dependent melanin production in B16 melanoma cells.

• 대한수의학회지
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• 제35권2호
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• pp.375-381
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• 1995

Two intraocular tumors were identified in low and medium dosed groups of a carcinogenicity study using 200 males and 200 females Sprague-Dawley rats in Screening & Toxicology Research Center, Korea Research Institute of Chemical Technology. The tumors were grossly observed as white or yellow, unilateral nodules. They were approximately $1-2{\times}3-5mm$ in size. The tumors located in the region of iris and/or ciliary body invaded peripheral cornea. The microscopic features were usually composed of spindle cells arranged in parallel, forming gently curving bundles or whorls. The spindle cells had poorly defined cell boundaries, scant to moderate cytoplasm, fusiform nuclei and indistinct nucleoli. Mitotic figures were rare and areas of necrosis were present. The spindle cells had positive immunoreactivity for S-100 protein and vimentin but negative for desmin, collagen and HMB-45 antibody. In special histochemical studies, the spindle cells react with Gomori's stain for argyrophih fibers, Prussian blue stain for iron but negative with Masson-Fontana's stain for melanin granules. Ultrastructurally, cytoplasmic premelanosomes were not observed in the tumor cells due to the poor preservation of tumor masses. Based on the results, the tumors were diagnosed as amelanotic melanoma.

• 한방안이비인후피부과학회지
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• 제16권2호
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• pp.57-77
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• 2003

Objectives: The aim of this study was to investigate the depigmentation effects of Barley, Unpolished rice, Job's-tear. Metbods: We investigated that the extracts of Barley, Unpolished rice, Job's-tear inhibit activity of tyrosinase, the enzyme which convert ts 3-(3,4-dihydroxyphenyl) alanine to dopachrom in the biosynthetic process of melanin, the UV absorbance of the extracts in the UV-A region and UV-B region was measured by UV scanning, the effect of extracts on cell viability and melanin production in cultured B16 mouse melanoma cells were measured, and cytoprotective effects of extracts on PC12 cells injured by hydrogen peroxide measured by MTT assay. Results: The extracts of Barley, Unpolished rice, Job's-tear inhibited activity of tyrosinase in low density. The Barley, Job's-tear extracts not only showed inhibitory effects on melanin production in cultured B16 mouse melanoma cells, but also exhibited cytoprotective effects on PC12 cells injured by hydrogen peroxide in low density. Unpolished rice extract showed inhibitory effect on melanin production in cultured B16 mouse melanoma cells, but did not showed cytoprotective effect Barley, Unpolished rice, Job's-tear extracts did not showed an absorbance effect in the UV-A region and UV-8 region. Conclusions: These results suggest that Barley, Unpolished rice, Job's-tear inhibit melanin biosynthesis which is involved in hyperpigmentation and could be used as a whitening agent.

• 약학회지
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• 제55권6호
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• pp.485-491
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• 2011

Previously, we have reported that apigenin, a natural flavonoid found in a variety of vegetables and fruits, stimulated melanogenesis through the activation of $K^+-Cl^-$-cotransport (KCC) in B16 melanoma cells. In this study we investigated the possible involvement of reactive oxygen species (ROS) in the mechanism of apigenin-induced melanogenesis in B16 cells. Apigenin elevated intracellular ROS level in a dose-dependent manner. Treatment with various inhibitors of NADPH oxidase, diphenylene iodonium (DPI), apocynin (Apo) and neopterine (NP) significantly inhibited both the generation of ROS and melanogenesis induced by apigenin. In addition these inhibitors profoundly inhibited apigenin-induced $Cl^-$-dependent $K^+$ efflux, a hallmark of KCC activity. However, the apigenin-induced ROS generation was not significantly affected by treatment with a specific KCC inhibitor R-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid (DIOA). These results indicate that the ROS production may be a upstream regulator of the apigenin-induced KCC stimulation, and in turn, melanogenesis in the B16 cells. Taken together, these results suggest that the NADPH oxidase-mediated ROS production may play an important role in the apigenin-induced melanogenesis in B16 cells. These results further suggest that NADPH oxidase may be a good target for the management of hyperpigmentation disorders.

• 약학회지
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• 제47권6호
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• pp.432-437
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• 2003

It has been known that quercetin, a bioflavonoid widely distributed in fruits and vegetables as dietary-derived flavonoid and exert significant multiple biological effects such as antioxidant and anti-inflammatory, anti-tumor effects. In addition, it has been shown to have a chemoprotective role in cancer, though complex effects on signal transduction involved in cell proliferation and angiogenesis. The present study investigated whether quercetin can enhance antioxidant enzyme activity (glutathione peroxidase: GPx, superoxide dismutase: SOD, catalase: CAT) and regulate the reactive oxygen species (ROS) generation in the presence of vitamin E, L-ascorbic acid, reduced glutathione (GSH) on B16F10 murine melanoma cells. After 48h treatment of cells with quercetin in the presence of vitamin E, L-ascorbic acid, GSH, we measured the cytotoxicities by MTT assay. The cells exhibited a dose-dependent inhibition in their proliferation in the presence of vitamin E, L-ascorbic acid, GSH respectively. We also investigated the effects of antioxidant enzyme activity and ROS generation. The antioxidant enzyme activity of quercetin in the presence of vitamin E was stronger than GSH, L-ascorbic acid, the same treatments decreased ROS generation in B16F10 murine melanoma cells. Taken together, these result demonstrate that the antioxidant effect of quercetin can enhanced in the presence of vitamin E and it might plays an important role in anti-oxidative effects.

• 대한화장품학회지
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• 제31권3호
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• pp.265-271
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• 2005

Citrus essential oil (bergamot, grapefruit, lemon, mandarin, petigrain)이 B16 melanoma 세포에서 melanin 생성에 미치는 영향과 RBL 2H3 비만세포에서 ROS 생성에 미치는 영향을 관찰하였다 5 종류의 citrus essential oil은 DPPH 라디칼 소거와 세포 증식 및 독성에서 대조군에 비해 유의한 변화를 일으키질 않았다. 정제한 tyrosinase 활성에 있어서 mandarin과 petigrain essential oil은 유의하게 tyrosinase 활성을 억제하였으나 bergamot은 전혀 억제하지 않았다. 이러한 결과는 mandarin과 petigrain essential oil이 tyrosinase 효소 억제 기전은 설명하지 못하지만 tyrosinase 효소에 직접적으로 작용하여 억제하는 것으로 보인다. B16 melanoma 세포에서 MSH에 의한 melanin 생성이 5 종류의 citrus essential oil에 의해 모두 농도 의존적으로 억제되었다. Bergamot essential oil은 tyrosinase 효소 활성을 직접적으로 억제하지는 못했지만 melanoma 세포에서 MSH에 의한 melanin 생성은 억제하는 것으로 나타났다. 다른 4종류의 citrus essential oil도 모두 tyrosinase 효소 활성을 억제하였으며, MSH에 의한 melanin 생성도 억제하였다. 한편 DCF-DA를 이용한 ROS 생성에 있어서는 madarin essential oil은 ROS 생성에 이렇다 할 영향을 주지 않았지만 다른 4개의 essential oil (Bergamot, Grapefruit, Lemon, Petigrain)은 농도 의존적으로 ROS 생성을 증가시켰다. 이상의 결과를 종합하여 볼 때 citrus essential oil은 MSH에 의한 melanin 생성을 억제하는 것으로 보아 미백제로서의 개발 가능성이 있는 것으로 사료된다.

• BMB Reports
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• 제51권11호
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• pp.572-577
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• 2018

Recent studies showed that the PD-1/PD-L1 checkpoint blockade is a dramatic therapy for melanoma by enhancing antitumor immune activity. Currently, major strategies for the PD-1/PD-L1 blockade have mainly focused on the use of antibodies and compounds. Seeking an alternative approach, others employ endogenous proteins as blocking agents. The extracellular domain of PD-1 (ePD1) includes the binding site with PD-L1. Accordingly, we constructed a PD-1-based recombinantly tailored fusion protein (dFv-ePD1) that consists of bivalent variable fragments (dFv) of an MMP-2/9-targeted antibody and ePD1. The melanoma-binding intensity and antitumor activity were also investigated. We found the intense and selective binding capability of the protein dFv-ePD1 to human melanoma specimens was confirmed by a tissue microarray. In addition, dFv-ePD1 significantly suppressed the migration and invasion of mouse melanoma B16-F1 cells, and displayed cytotoxicity to cancer cells in vitro. Notably, dFv-ePD1 significantly inhibited the growth of mouse melanoma B16-F1 tumor cells in mice and in vivo fluorescence imaging showed that dFv-ePD was gradually accumulated into the B16-F1 tumor. Also the B16-F1 tumor fluorescence intensity at the tumor site was stronger than that of dFv. This study indicates that the recombinant protein dFv-ePD1 has an intensive melanoma-binding capability and exerts potent therapeutic efficacy against melanoma. The novel format of the PD-L1-blocked agent may play an active role in antitumor immunotherapy.

• Preventive Nutrition and Food Science
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• 제20권1호
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• pp.73-77
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• 2015

As an approach to search for chemopreventive agents, we tested p-coumaric acid, 3-methoxy-p-coumaric acid (ferulic acid), and 3,5-dimethoxy-p-coumaric acid (sinapic acid) in B16/F10 melanoma cells. Intracellular melanin contents were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and cytotoxicity of the compounds were examined by lactate dehydrogenase (LDH) release. p-Coumaric acid showed inhibitory effect on melanogenesis, but ferulic acid increased melanin content, and sinapic acid had almost no effect on melanogenesis. Treatment with ferulic acid resulted in a 2 to 3 fold elevation in the production of melanin. Correlatively, cell viability decreased in a dose-dependent manner when treated with ferulic acid. However, ferulic acid did not affect the LDH release from the cells. Treatment with sinapic acid resulted in a 50~60% elevation in the release of LDH when treated with a $200{\mu}g/mL$ concentration and showed neither cytostasis nor increase of melanin synthesis in a dose-dependent manner. Taken together, p-coumaric acid inhibits melanogenesis, ferulic acid induces melanogenesis, and sinapic acid exerts cytotoxic effects in B16/F10 murine melanoma cells. The results indicate that the addition of methoxy groups to p-coumaric acid shows the melanogenic or cytotoxic effects in melanoma cells compared to the original compound. Therefore, this study suggests the possibility that methoxylated p-coumaric acid, ferulic acid can be used as a chemopreventive agent.