Constituents of Cryptotaenia japonica Inhibit Melanogenesis via CREB- and MAPK-Associated Signaling Pathways in Murine B16 Melanoma Cells

Melanin plays an important role in protecting the skin against ultraviolet light and is responsible for skin color. However, overproduction of melanin is related to several skin disorders, such as age spots, freckles, café au lait spots, Becker’s nevus and other hyperpigmentation syndromes. The aim of this study was to identify the effects of kaempferol-7-O-β-d-glucuronide (K7G) and tilianin, isolated from Cryptotaenia japonica, on melanogenesis and their mechanisms of action in murine B16 melanoma cells. The α-melanocyte-stimulating hormone (α-MSH)-induced melanin production was significantly inhibited by K7G and tilianin in a dose-dependent manner. The effects of these compounds on the signaling pathway of melanogenesis were examined. K7G and tilianin downregulated the expression of microphthalmia-associated transcription factor (MITF) and melanocyte-specific enzymes, i.e., tyrosinase and TRP1. These compounds also inhibited the phosphorylation of cyclic adenosine monophosphate (cAMP)-response element binding protein (CREB) in a dose-dependent manner. In addition, these compounds increased the phosphorylation of extracellular signal-regulated kinase (ERK) but decreased the phosphorylation of c-Jun N-terminal kinase (JNK) in B16 cells. Based on the above results, the anti-melanogenic effects of these compounds are caused by suppression of the MAPK signaling pathway through the down-regulation of α-MSH-induced CREB accumulation. This finding suggests that K7G and tilianin may be good candidates for further research to develop therapeutic agents for hyperpigmentation diseases.


Introduction
Melanocytes are located at the epidermal-dermal junction and produce pigment granules, i.e., eumelanin and pheomelanin, which are responsible for the coloration of skin and hair [1]. Although melanin plays an important role in the protection of skin against ultraviolet (UV) light, abnormal changes in melanin, such as overproduction and accumulation of pigments, may be caused by skin irritation, hormonal changes, aging, a metabolic disorder, or other underlying problems. Melasma, age spots, freckles, café au lait spots, Becker's nevus, macules due to Peutz-Jeghers syndrome and lentigos are examples of hyperpigmentation syndromes [2][3][4]. These abnormal changes greatly influence the quality of life.
Melanin is synthesized by melanocyte-specific enzymes, such as tyrosinase and tyrosinase-related protein 1 [1]. TRP-1 forms a complex with the enzyme to induce melanin formation in murine
To investigate the anti-melanogenic effects of the compounds isolated from C. japonica, alteration of the melanin contents in B16F10 cells by the compounds was measured first. B16 melanoma is a murine tumor cell line used for research as a model for human skin [27]. There are many literatures mentioning the comparative study between B16F10 murine melanoma cells and Human melanoma cells [28]. They showed similar gene expression and previous reports had showed similarities on protein expression of both cell lines. As shown in Table 1, K7G and tilianin of C. japonica showed good inhibition of melanin production in a dose-dependent manner. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-thiazolium bromide (MTT) assay to avoid the possibility of decreased melanin due to cytotoxicity. The cytotoxicity of K7G and tilianin with higher concentration was determined and the result showed that there was no cytotoxicity caused by active compounds (Figure 2). The half-maximal inhibitory concentrations (IC 50 ) of the two compounds were 12.3 ± 3.2 µM and 15.5 ± 4.5 µM, indicating that these compounds are much more active than arbutin (149.3 ± 2.02 µM), a reference compound. compounds ( Figure 2). The half-maximal inhibitory concentrations (IC50) of the two compounds were 12.3 ± 3.2 μM and 15.5 ± 4.5 μM, indicating that these compounds are much more active than arbutin (149. 3    Cell viability (B) was reported as percentages. The data were expressed as the mean ± S.D. from three independent experiments performed in triplicate. # p < 0.0001 in comparison with the control group and * p < 0.005, ** p < 0.001 in comparison with the α-MSH group. "+" is denoted as α-MSH treated group, "-" is denoted as α-MSH untreated group.    Cell viability (B) was reported as percentages. The data were expressed as the mean ± S.D. from three independent experiments performed in triplicate. # p < 0.0001 in comparison with the control group and * p < 0.005, ** p < 0.001 in comparison with the α-MSH group. "+" is denoted as α-MSH treated group, "-" is denoted as α-MSH untreated group. Cell viability (B) was reported as percentages. The data were expressed as the mean ± S.D. from three independent experiments performed in triplicate. # p < 0.0001 in comparison with the control group and * p < 0.005, ** p < 0.001 in comparison with the α-MSH group. "+" is denoted as α-MSH treated group, "-" is denoted as α-MSH untreated group. "+" is denoted as α-MSH treated group; "-" is denoted as α-MSH untreated group.

Effects of K7G and Tilianin on the Expression of Melanogenesis-Related Proteins
The effects of K7G and tilianin on the expression of tyrosinase, TRP-1 and MITF were examined by western blot analysis. The cells were treated with K7G and tilianin, separately, and then stimulated by α-MSH for 48 h. The time interval for the measurement followed that in previous studies [5]. Tyrosinase and MITF proteins were significantly suppressed by K7G and tilianin ( Figure 3). MITF is a key transcription factor for regulating the expression of melanogenesis-related genes [29]. These two compounds also suppressed the protein expression of tyrosinase and TRP-1. These results indicate that the anti-melanogenic effects of K7G and tilianin result from the down-regulation of MITF expression.

Effects of K7G and Tilianin on CREB Phosphorylation
The cAMP-mediated signaling pathways have major roles in the regulation of skin pigmentation [30]. Specifically, cAMP activates CREB, which leads to upregulation of MITF expression. The optimized reaction time for CREB was determined by activating cells with only α-MSH ( Figure 4A). The cells were treated with K7G and tilianin for 1 h, respectively, and then stimulated by α-MSH for 1 h. As observed in Figure 4, CREB phosphorylation was significantly suppressed by K7G and tilianin. The regulation of α-MSH-induced CREB phosphorylation is known to be potentially important in regulating pigmentation. According to this experiment, the degree of CREB phosphorylation was reduced by K7G and tilianin in a dose-dependent manner ( Figure 4B). Finally, these results indicate that the anti-melanogenic activities of the two compounds derive from the down-regulation of MITF, tyrosinase and TRP-1 through the down-regulation of CREB phosphorylation.

Effect on the MAP Kinase Signaling Pathway
To investigate if the MAPK signaling pathway is involved in the inhibitory effect of K7G and tilianin on melanogenesis, B16F10 cells were treated with two compounds at various concentrations for 1 h and then stimulated by α-MSH for 1 h. The optimized reaction time for CREB was determined by activating cells with only α-MSH ( Figure 5A). K7G and tilianin enhanced the phosphorylation of ERK. On the other hand, the expression of p38 was not affected, and the phosphorylation of JNK appeared to be inhibited in tilianin-treated cells ( Figure 5B). Several reports have indicated that the MAP kinase family members, including ERK, JNK and p38, are known to be activated by various extracellular stimuli [31]. UV radiation is one such typical extracellular stimulus that induces MAPK activation. Moreover, recent reports have implicated MAPKs in mammalian melanogenesis [32]. Previous studies reported that the downregulation of ERK signaling pathway activates cAMP-induced melanogenesis in B16 melanoma cells, which means that the increase in ERK phosphorylation may decrease melanin synthesis [33]. As expected, K7G and tilianin increased the phosphorylation of ERK, and the resulting melanin synthesis decreased, although the phosphorylation of JNK was significantly decreased by tilianin only at a high dose.
Comparing the structures of K7G and tilianin, the skeleton (flavonol vs. flavone) and sugar moiety (glucuronic acid vs. glucose) are different, and they have a methoxyl group at the C-4 position. These structural differences might be responsible for the variation in the phosphorylation of JNK and other activity potencies. Although the anti-melanogenic activity of K7G and tilianin and their mechanisms of action were discovered in this study, further research on the pharmacological efficacy of these compounds should be performed to develop an anti-melanogenic drug.

Effect on the MAP Kinase Signaling Pathway
To investigate if the MAPK signaling pathway is involved in the inhibitory effect of K7G and tilianin on melanogenesis, B16F10 cells were treated with two compounds at various concentrations for 1 h and then stimulated by α-MSH for 1 h. The optimized reaction time for CREB was determined by activating cells with only α-MSH ( Figure 5A). K7G and tilianin enhanced the phosphorylation of ERK. On the other hand, the expression of p38 was not affected, and the phosphorylation of JNK appeared to be inhibited in tilianin-treated cells ( Figure 5B). Several reports have indicated that the MAP kinase family members, including ERK, JNK and p38, are known to be activated by various extracellular stimuli [31]. UV radiation is one such typical extracellular stimulus that induces MAPK activation. Moreover, recent reports have implicated MAPKs in mammalian melanogenesis [32]. Previous studies reported that the downregulation of ERK signaling pathway activates cAMP-induced melanogenesis in B16 melanoma cells, which means that the increase in ERK phosphorylation may decrease melanin synthesis [33]. As expected, K7G and tilianin increased the phosphorylation of ERK, and the resulting melanin synthesis decreased, although the phosphorylation of JNK was significantly decreased by tilianin only at a high dose.
Comparing the structures of K7G and tilianin, the skeleton (flavonol vs. flavone) and sugar moiety (glucuronic acid vs. glucose) are different, and they have a methoxyl group at the C-4′ position. These structural differences might be responsible for the variation in the phosphorylation of JNK and other activity potencies. Although the anti-melanogenic activity of K7G and tilianin and their mechanisms of action were discovered in this study, further research on the pharmacological efficacy of these compounds should be performed to develop an anti-melanogenic drug.
(A) The expression levels of the MAPK proteins were examined by western blot analysis. # p < 0.01 in comparison with the control group and * p < 0.05, ** p < 0.01 in comparison with β-actin. "+" is denoted as α-MSH treated group, "-" is denoted as α-MSH untreated group.

General Experimental Procedure
The NMR spectra were recorded on a Varian UNITY 400 FT-NMR spectrometer (Varian Inc., Palo Alto, CA, USA) using tetramethylsilane as an internal standard. HR-ESI and EI mass spectra were measured using a Waters Q-ToF Premier spectrometer (Micromass UK limited, Manchester, UK) and a JEOL JMS 700 spectrometer (JEOL, Tokyo, Japan), respectively. TLC was performed on precoated silica gel 60 F254 and silica gel 60 RP-18F254 (0.25 mm, Merck, Darmstadt, Germany), and the spots were visualized by using UV irradiation (254 nm) and spraying the plates with a 10% H2SO4 solution, followed by heating. Column chromatography was performed over Sephadex LH-20 (25-  The expression levels of the MAPK proteins were examined by western blot analysis. # p < 0.01 in comparison with the control group and * p < 0.05, ** p < 0.01 in comparison with β-actin. "+" is denoted as α-MSH treated group, "-" is denoted as α-MSH untreated group.

Plant Material
The aerial parts of C. japonica were collected in Namyang-ju, Kyungki Province, Korea, in August 2013. A voucher specimen (KRIB0071766) was deposited at the herbarium of the Korea Research Institute of Bioscience and Biotechnology.

Cell Culture
The murine B16F10 cells, derived from murine melanoma cells, were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with glutamine (1 mM), 10% heat-inactivated fetal bovine serum (FBS), penicillin (50 U/mL) and streptomycin (50 µg/mL) and incubated at 37 • C in a humidified, 5% CO 2 incubator. For the stimulation, the medium was replaced with DMEM, and the cells were then stimulated with α-MSH in the presence of the experimental compounds for the indicated periods.

Cytotoxicity Assay
The cell viability was examined using the MTT assay. The murine B16F10 cells were seeded at densities of 2 × 10 4 cells/well in 96-well plates (Thurmo Scientific, Roskilde, Denmark). The compounds were added to each well and incubated for 48 h at 37 • C in 5% CO 2 . The MTT solution (5 µL of 5 mg/mL) was added to each well, and the cells were cultured for another 4 h. The supernatant was discarded, and 100 µL of dimethyl sulfoxide (DMSO) was added to each well. The optical density of formazan was measured using a microplate reader (VeersaMax, Molecular Devices, Sunnyval, CA, USA) at 590 nm. The level of formazan generated by the untreated cells was chosen as the 100% value.

Determination of the Extracellular Melanin Content
Extracellular melanin release was measured as described previously [5]. In brief, B16F10 cells were seeded in a 96-well plate at a density of 2 × 10 4 cells/well. The next day, the cells were pretreated with the compounds (5, 10 and 20 µM) isolated from C. japonica for 1 h and then stimulated with 200 nM α-MSH for 72 h. The supernatant was collected. The relative melanin content was determined by measuring the absorbance at 405 nm using a microplate reader.

Western Blot Analysis
To determine the amount of melanogenesis-related proteins, Western blot analysis was performed. To estimate the amount of MITF, tyrosinase, and TRP-1, the cells were cultured in 60 mm dishes and pretreated with K7G and tilianin (2.5, 5, 10 and 20 µM, each); after 1 h, the cells were stimulated using α-MSH (200 nM) and then incubated for the next 48 h. To estimate the amount of ERK, JNK, and p38, the cells were cultured in 60 mm dishes and pretreated with K7G and tilianin (2.5, 5, 10 and 20 µM, each); after 1 h, the cells were stimulated using α-MSH (200 nM) and then incubated for the next 1 h. To estimate the amount of CREB, the cells were cultured in 60 mm dishes and pretreated with K7G and tilianin (2.5, 5, 10 and 20 µM, each); after 1 h, the cells were stimulated using α-MSH (200 nM) and then incubated for the next hour. The cells were treated with lysis buffer containing protease inhibitors (50 mM Tris-HCl, pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, 0.1% SDS, 1 mM EGTA, 100 µg/mL PMSF, 10 µg/mL pepstatin A, and 100 µM Na 3 VO 3 ). The homogenates were centrifuged at 14,000 rpm and 4 • C for 15 min, and the protein concentrations in the supernatants were determined using Bradford reagent (Bio-Rad, Hercules, CA, USA). The total proteins (20 µg) in the supernatants were separated by SDS-PAGE at 100 V for 90 min on a 10% gel and blotted to a PVDF (Millipore Corporation, Billerica, MA, USA) membrane. The blotted membrane was incubated with a blocking solution (5% skim milk), followed by overnight incubation at 4 • C with an appropriate primary antibody. The following primary antibodies and dilutions were used: anti-tyrosinase (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-TRP-1 (Santa Cruz Biotechnology), anti-MITF (Santa Cruz Biotechnology), anti-p-ERK, ERK (Santa Cruz Biotechnology), anti-p-JNK, JNK (Santa Cruz Biotechnology), anti-p-p38, p38 (Cell Signaling Technology, Beverly, MA, USA), anti-pCREB, CREB (Cell Signaling), anti-α-tubulin (Millipore, Billerica, CA, USA) and anti-β-actin (Cell Signaling). The membranes were washed three times with a Tris-buffered saline solution containing 0.1% Tween 20 (TBST) and then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology) for 1 h at room temperature. The membranes were washed three times with TBST and visualized with an enhanced chemiluminescence (ECL) kit (Thermo, Carlsbad, CA, USA). For quantitative analysis, the densitometric band values were determined by a bio-imaging analyzer (LAS-4000 mini, Fujifilm, Tokyo, Japan).

Conclusions
K7G and tilianin along with 11 other compounds were isolated from C. japonica. The melanin content and cell viability of these compounds were determined using murine B16 cells. K7G and tilianin had inhibitory effects on melanogenesis. To understand the mechanism of action of the compounds, we examined several key factors related to the signaling pathways of melanogenesis in melanoma cells. K7G and tilianin significantly inhibited MITF, tyrosinase and TRP-1 protein expression in murine B16 cells. Moreover, CREB phosphorylation was suppressed by these two compounds. Our results also demonstrated that K7G and tilianin enhanced the phosphorylation of ERK. Hereafter, the pharmacological effects of C. japonica along with K7G and tilianin should be performed in various animal models to develop an anti-melanogenic drug.