International Journal of

Molecular Sciences Article

Gomisin N Inhibits Melanogenesis through Regulating the PI3K/Akt and MAPK/ERK Signaling Pathways in Melanocytes Jae Kyoung Chae 1,† , Lalita Subedi 1,† , Minsun Jeong 1 , Yong Un Park 1 , Chul Young Kim 2 , Hakwon Kim 3 and Sun Yeou Kim 1,4,5, * 1

2 3 4 5

* †

College of Pharmacy, Gachon University, #191, Hambakmoero, Yeonsu-gu, Incheon 21936, Korea; [email protected] (J.K.C.); [email protected] (L.S.); [email protected] (M.J.); [email protected] (Y.U.P.) College of Pharmacy, Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan 15588, Korea; [email protected] Department of Applied Chemistry and Institute of Natural Sciences, Kyung Hee University, Global Campus, #1732 Deogyeong-daero, Giheung-gu, Yongin, Gyenggi-do 17104, Korea; [email protected] Gachon Medical Research Institute, Gil Medical Center, Inchon 21565, Korea Gachon Institute of Pharmaceutical Science, Gachon University; #191 Hambakmoe-ro, Yeonsu-gu, Incheon 21565, Korea Correspondence: [email protected]; Tel.: +82-32-899-6411 These authors contributed equally to this work.

Academic Editor: Manickam Sugumaran Received: 20 December 2016; Accepted: 13 February 2017; Published: 22 February 2017

Abstract: Gomisin N, one of the lignan compounds found in Schisandra chinensis has been shown to possess anti-oxidative, anti-tumorigenic, and anti-inflammatory activities in various studies. Here we report, for the first time, the anti-melenogenic efficacy of Gomisin N in mammalian cells as well as in zebrafish embryos. Gomisin N significantly reduced the melanin content without cellular toxicity. Although it was not capable of modulating the catalytic activity of mushroom tyrosinase in vitro, Gomisin N downregulated the expression levels of key proteins that function in melanogenesis. Gomisin N downregulated melanocortin 1 receptor (MC1R), adenylyl cyclase 2, microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). In addition, Gomisin N-treated Melan-A cells exhibited increased p-Akt and p-ERK levels, which implies that the activation of the PI3K/Akt and MAPK/ERK pathways may function to inhibit melanogenesis. We also validated that Gomisin N reduced melanin production by repressing the expression of MITF, tyrosinase, TRP-1, and TRP-2 in mouse and human cells as well as in developing zebrafish embryos. Collectively, we conclude that Gomisin N inhibits melanin synthesis by repressing the expression of MITF and melanogenic enzymes, probably through modulating the PI3K/Akt and MAPK/ERK pathways. Keywords: Schisandra chinensis; Gomisin N; lignan; melanogenesis; skin whitening

1. Introduction Melanin is a pigment found in most animal organs including skin, hair, eyes, inner ear, bones, heart, and brain [1,2]. Melanogenesis is a complex process where multiple signaling pathways are involved. The melanocortin 1 receptor (MC1R) is a key regulator in melanogenesis, signaling through its ligands such as melanocyte-stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH) [3]. Skin melanin is biosynthesized by melanocytes in the epidermis and then transferred to keratinocytes, where they play important roles in skin protection by absorbing UV radiation from Int. J. Mol. Sci. 2017, 18, 471; doi:10.3390/ijms18020471

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Int. J. Mol. Sci. 2017, 18, 471

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sunlight and scavenging reactive free radicals [4,5]. Melanin synthesis and transfer in the skin and hair follicles is regulated by the availability of its precursors [6]. L-tyrosine and L-dihydroxyphenylalanine (L-DOPA), major substrates of melanogenic enzymes, also function as hormone-like regulators in melanogenesis [7]. On the other hand, the overproduction of melanin results in undesirable skin concerns such as freckles and melasma [8,9]. Melanogenesis can affect the behavior of normal and malignant melanocytes by modulating the elastic properties of the cells [10]. Although the receptors for serotonin and melatonin expressed in cutaneous cells play a key role in maintaining cellular homeostasis, the excessive production of melanin via uncontrolled hormonal changes may cause pathological conditions in the skin [11]. Thus, there have been extensive efforts to elucidate molecular mechanisms that control melanogenesis as the primary step for treating hyperpigmentary skin disorders. In addition, various types of skin whitening agents that inhibit melanin synthesis have been identified from plant and non-plant extracts and commercially used in cosmeceuticals [12,13]. The most widely used whitening agents include hydroquinone, mequinol, arbutin, Kojic acid, ascorbic acid, and retinoic acid [12,14]. However, there are various limitations of their use in treating acute or chronic symptoms of hyperpigmentation in humans. For example, hydroquinone, although it has long been used for depigmentation since the 1960s, can cause skin irritation and contact dermatitis [15,16]. It also leads to DNA damage by increasing the production of reactive oxygen species and the development of exogenous ochronosis in mammalian cells [17,18]. Other well-known tyrosinase inhibitors such as Kojic acid and ascorbic acid not only have poor skin penetration, stability, and whitening efficacy but also can cause cytotoxicity, dermatitis, and erythema from long-term use [19,20]. In this regard, there is an increasing need to develop safer and more effective whitening agents for treating human skin hyperpigmentation. Natural herbs used in traditional medicine may provide alternative sources for identifying novel whitening agents that control key steps in melanogenesis with less or no side effects [21]. Schisandra chinensis, also known as northern fine-flavor berry, is naturally found in northeastern China, far-east Russia, Japan, and Korea [22]. This plant has long been used for the treatment of night blindness, skin burn, aseptic inflammation, and liver diseases in traditional oriental medicine [23,24]. S. chinensis fruit extract and its lignan compounds have been shown to possess various pharmacological effects in murine cell lines. For example, schisandrin A and C have anti-oxidative effects, while schisandrin B has anti-fibrotic, anti-inflammatory, anti-oxidant, and anti-apoptotic activities [25]. Another lignan compound Gomisin N (Figure 1A) has been reported to suppress oxidative stress-induced apoptosis by inhibiting the release of cytochrome C from mitochondria to the cytoplasm, the cleavage of caspase 3 and PARP, and Ca2+ -induced mitochondrial permeability transition in H9c2 rat cardiomyocytes [7,8]. Interestingly, several studies using mouse models and human skin cell lines have revealed the therapeutic potential of S. chinensis for treating skin disorders. Lee et al. reported that the methanol extract of S. chinensis fruit alleviated contact dermatitis symptoms by reducing the production of pro-inflammatory cytokines such as TNF-α and IFN-γ when topically applied [8,24]. Kang et al. showed that Schisandra fructus water extract inhibited IκB activation, thereby suppressing the production of TNF-α, IL-6, and GM-CSF in the human mast cell line HMC-1 [26]. These findings led us to postulate that S. chinensis lignans might affect skin cell functions more broadly. In this study, we sought to investigate the putative roles of Gomisin N, one of the major lignan compounds in S. chinensis, in regulating melanogenesis, thereby evaluating its potential use as a cosmeceutical agent. Here, we report that Gomisin N inhibits melanin biosynthesis without cellular toxicity in human and mouse cells, as well as in zebrafish embryos.

was comparable with the results obtained by PTU treatment [27] (Figure 1C). To evaluate whether the reduction of melanin upon Gomisin N treatment is due to decreased activity of tyrosinase, we performed L-DOPA staining assay in normal human epidermal melanocyte (NHEM) cells. As shown in Figure 1E, NHEM cells treated with Gomisin N exhibited decreased levels of L-DOPA compared with untreated cells. However, the inhibitory effect of Gomisin N on melanin production was not Int. J. Mol. Sci. 2017,in18, 471 melanoma MNT-1 cells (Figure 1D). 3 of 13 significant human

Figure 1. Gomisin N structure and effectson onmelanin melanin production cell viability. (A) (A) Chemical Figure 1. Gomisin N structure and itsits effects productionand and cell viability. Chemical structure of Gomisin N. Melanin contents and cell viability after 72 h of Gomisin N (1, 5, 10 µM) structure of Gomisin N. Melanin contents and cell viability after 72 h of Gomisin N (1, 5, 10 µM) treatment in Melan-A cells (B), B16F10 cells (C), and in MNT1 cells (D). PTU (1, 10, 100 µM) was used treatment in Melan-A cells (B), B16F10 cells (C), and in MNT1 cells (D). PTU (1, 10, 100 µM) was used as a positive control. (E) Melanin contents in NHEM were measured by L-DOPA staining. Scale bar is as a positive control. (E) Melanin contents in NHEM were measured by L-DOPA staining. Scale bar is 100 µm. The results are presented as a percentage of the vehicle-treated control and as the mean ± SD 100 µm. The results are presented as a percentage of the vehicle-treated control and as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus the vehicle-treated of three independent * puntreated < 0.05, **control p < 0.01, and ***bar p

ERK Signaling Pathways in Melanocytes.

Gomisin N, one of the lignan compounds found in Schisandra chinensis has been shown to possess anti-oxidative, anti-tumorigenic, and anti-inflammatory...
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