Dodoviscin A Inhibits Melanogenesis in Mouse B16-F10 Melanoma Cells

• Introduction
• Materials and Methods
• Materials
• Cell culture
• Cell viability assay
• Determination of melanin content in B16-F10 cells
• Tyrosinase activity analysis in the cell-free system
• Docking study
• Tyrosinase activity analysis in B16-F10 cells
• Tyrosinase activity analysis by zymography
• Western blot analysis
• Statistical analysis
• Supporting information
• Results and Discussion
• Acknowledgements
• References

Abstract

Nowadays, abnormal hyperpigmentation in human skin such as melasma, freckles, and chloasma has become a serious esthetic problem. Cutaneous depigmenting agents could be used to treat these hyperpigmentation-associated dieseases. Dodoviscin A is a natural product isolated from the aerial parts of Dodonaea viscosa. In the present study, we evaluated the effect of dodoviscin A on melanin production in B16-F10 melanoma cells for the first time. We found that dodoviscin A inhibited melanin biosynthesis induced by 3-isobutyl-1-methylxanthine and PD98059 significantly, and there was no obvious effect on the viability of dodoviscin A-treated B16-F10 cells. Meanwhile, dodoviscin A could suppress the activity of mushroom tyrosinase in the cell-free assay system and also decrease 3-isobutyl-1-methylxanthine-induced tyrosinase activity and expression of mature tyrosinase protein in B16-F10 cells. Western blotting analysis showed that dodoviscin A inhibited 3-isobutyl-1-methylxanthine and forskolin-induced phosphorylation of the cAMP response element binding protein in B16-F10 cells. These results indicate that dodoviscin A may be a new promising pigmentation-altering agent for cosmetic and therapeutic applications.

Introduction

Melanin is a multifunctional biopolymer synthesized in the melanosomes of melanocytes, which has many crucial physiological functions, including contributing to the appearance of skin and protecting skin from ultraviolet radiation-induced damage [1]. However, abnormal hyperpigmentation in human skin, such as melasma, freckles, and chloasma, is also a serious esthetic problem. Several popular cutaneous depigmenting agents, for example, kojic acid, arbutin, and hydroquinone, show toxicity against melanocytes and lead to some adverse side effects [2]. Therefore, it is necessary to find safer and more effective depigmenting agents [3], [4].

Tyrosinase (EC 1.14.18.1) is a key enzyme on melanin biosynthesis pathway in melanocytes. Inhibition of tyrosinase function is an effective approach in the treatment of hyperpigmentary disorders. Tyrosinase has a binuclear type 3 copper center within its active site, which catalyzes the ortho-hydroxylation of L-tyrosine and L-4-dihydroxyphenylalanine (L-DOPA) and the subsequent two-electron oxidation to dopaquinone [5]. According to the crystal structure reported recently, mushroom tyrosinase is a H₂L₂ tetramer and the H subunit contains the binuclear copper-binding site in the deoxy state, in which three histidine residues coordinate each copper ion (His61, His85, His94 to Cu-A and His259, His263, His296 to Cu-B) [6]. The quality control of tyrosinase is associated with the process of pigmentation. Transcription, maturation via glycosylation, trafficking to melanosomes, degradation, as well as modulation of catalytic activity directly could all modulate the quality of tyrosinase [7].

Melanin synthesis is regulated by a number of physiological and pathological effectors, such as ultraviolet radiation, melanocortin, NO, sex steroids, and a wide variety of growth factors [8], [9]. The related signaling pathways including cAMP/protein kinase A (PKA), NO/PKG, mitogen-activated protein kinase (MAPK), and Wnt pathways play important roles in melanogenesis [10], [11], [12], [13]. cAMP-mediated protein kinase A signaling pathway is the predominant cascade in melanin production. MITF, a transcription factor for tyrosinase and tyrosinase-related protein, is crucial to the differentiation and melanin formation in melanocytes. In this process, cAMP promotes an increase in the expression of MITF through the activation of the PKA and cAMP response element binding protein (CREB), and finally augments the expression of tyrosinase [14]. Furthermore, cAMP could also have an effect on the transcription and activation of melanogenic enzymes in the initiation of melanogenesis. For example, tyrosinase hydroxylase isoform I (THI, EC 1.14.16.2) and phenylalanine hydroxylase (PAH, EC 1.14.16.1), which catalyze L-tyrosine to L-DOPA and L-phenylalanine to L-tyrosine, respectively [15], [16], [17]. For these reasons, some cAMP-elevating agents [forskolin, 3-isobutyl-1-methylxanthine (IBMX), and alpha-melanocyte stimulating hormone (a-MSH)] can stimulate melanin production.

Isoprenylated flavonoids are distributed widely in plants and exhibit a variety of pharmacological and medicinal properties, including antioxidant, anti-inflammatory [18], [19], antiviral [20], cytotoxic [21], and tyrosinase inhibitory activities [22]. We recently reported that some new isoprenylated flavonoids obtained from Dodonaea viscosa, belonging to the genus Sapindaceae, could promote adipocyte differentiation [23]. In the current study, the inhibitory effect of dodoviscin A, one of these isoprenylated flavonoids, on melanogenesis in B16-F10 cells was investigated.

Materials and Methods

Materials

Mushroom tyrosinase, L-DOPA, IBMX, forskolin, and kojic acid (≥ 99 % purity) were obtained from Sigma-Aldrich. Antibodies against tyrosinase and MITF were obtained from Abcam, and all other antibodies were purchased from Cell Signaling Technology. PD98059 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Beyotime Institute of Biotechnology. Dodoviscin A was isolated from the aerial parts of Dodonaea viscosa and was identified as described previously [23]. The purity of dodoviscin A was determined to be 95.0 % by HPLC-UV (CH₃OH−H₂O 78 : 22, flow rate 1 mL/min, wavelength 349 nm) performed on an Agilent 1200 (Agilent Technologies) and a YMC C18 column (150 × 4.6 mm, 5 mm; YMC Co. Ltd.).

Cell culture

B16-F10 mouse melanoma cells were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences and were cultured in Dulbeccoʼs modified Eagleʼs medium (DMEM; Gibco) with 10 % fetal bovine serum (FBS; Gibco) at 37 °C in a humidified atmosphere containing 5 % CO₂.

Cell viability assay

The cytotoxicity of dodoviscin A was measured using the MTT assay based on the reduction of MTT to formazan. Briefly, B16-F10 cells were seeded in a 96-well plate and exposed to various concentrations of dodoviscin A for 48 h. Then MTT reagent (0.5 mg/mL final concentration) was added to each well, following incubation at 37 °C for 4 h. Finally, 100 µL of dimethylsulfoxide (DMSO) was added to dissolve formazan crystals. The absorbance was measured at 570 nm relative to 630 nm using FlexStation 3 microplate reader (Molecular Devices Co.).

Determination of melanin content in B16-F10 cells

Determination of melanin content was performed using the published protocol with minor modifications [24]. Briefly, IBMX (a cAMP-elevating agent) and PD98059 (a MEK1 inhibitor) were used to induce the melanin production during testing. B16-F10 cells (15 000/well) were seeded in 24-well plates and then treated with various concentrations of dodoviscin A for 72 h in the presence of 100 µM IBMX or 15 µM PD98059. After washing twice with phosphate buffered saline (PBS), the cells were incubated in 100 µL of 1 M NaOH at 70 °C for 15 min. The relative quantity of melanin was analyzed by the absorbance at a wavelength of 490 nm (OD₄₉₀).

Tyrosinase activity analysis in the cell-free system

The inhibitory effect of dodoviscin A on mushroom tyrosinase activity was investigated. 100 µL of PBS with or without test sample mixed with 50 µL of the mushroom tyrosinase (1.25 µg/mL final concentration) were added into 96-well plates, and then 50 µL of L-DOPA (1 mM final concentration) was mixed. The linear increase of OD₄₉₀ was measured at room temperature.

Docking study

The crystal structure of Agaricus bisporus tyrosinase was retrieved from the protein data bank (PDB, PDB ID: 2Y9X) for the docking study. Molecular docking was performed using Glide5.5 with its XP mode in a standard procedure [25], [26]. The water molecules were removed, while the copper ions were retained with a + 2 charge. Tropolone at the binding pocket was selected to define the grid box center. The compounds were sketched by Maestro and processed by LigPrep with Epik to treat protonation states. For Epik, the option “add metal binding states” was selected. The docked conformation of the compounds in the ligand binding sites was selected for further study.

Tyrosinase activity analysis in B16-F10 cells

B16-F10 cells (3000/well) were seeded in 96-well plates and treated with different concentrations of dodoviscin A in the presence of 100 µM IBMX for 48 h; the cells were washed twice with PBS and lysed with 100 µL PBS containing 1 % Triton X-100, 1 mM PMSF, and 1 mM L-DOPA. After 1 h incubation at 37 °C, the oxidation of L-DOPA was measured at 490 nm.

Tyrosinase activity analysis by zymography

B16-F10 cells were treated with dodoviscin A for 72 h and then lysed with 100 µL PBS containing 1 % Triton X-100, 1 mM PMSF, and protease inhibitor cocktail. Protein concentration was determined using the BCA protein assay kit (Beyotime Institute of Biotechnology). After the cleared cell lysates were mixed with native gel sample loading buffer and were resolved on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) by electrophoresis, the gels were equilibrated in PBS (pH 6.8) at room temperature and then soaked with 3 mM L-DOPA until colorimetric detection of the active form of tyrosinase.

Western blot analysis

The cells were harvested and lysed with RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 % NP-40, 0.1 % SDS, and 0.5 % sodium deoxycholate) containing 1 mM PMSF and protease inhibitor cocktail. The protein samples were separated by 10 % SDS-PAGE gels and then transferred onto PVDF membranes (Bio-Rad). The membranes were blocked with 1 % BSA in 0.1 % Tween 20, 0.01 M Tris-buffered saline (TBST) at room temperature for 1 h and then incubated with primary antibodies overnight at 4 °C. The membranes were then incubated with appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. After multiple washes, the immunoblots were detected using the chemiluminescence detection system (Pierce).

Statistical analysis

The data are expressed as mean ± standard deviation (SD). Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Dunnettʼs test. P < 0.05 was considered statistically significant.

Supporting information

The antioxidant activities and part of the effect on the signaling pathways of dodoviscin A are depicted in Supporting Information.

Results and Discussion

Dodonaea viscosa has been reported to exhibit many medicinal properties and is widely used in traditional medicine [27], [28], [29], [30], [31], [32]. Dodoviscin A is a nature isoprenylated flavonoid compound first derived from Dodonaea viscosa by us ([Fig. 1]). There are few published data about its biological and pharmacological activities, except for the promotion of adipocyte differentiation in our previous report [23]. In this present study, we provide the key evidences that dodoviscin A could effectively suppress melanin formation in B16-F10 melanoma cells.

The results of the MTT assay showed that dodoviscin A has no cytotoxic effect on B16-F10 cells at concentrations of 0.625–2.5 µM ([Fig. 2 A]). [Fig. 2 B] and [C] showed that treatment of dodoviscin A for 72 h could significantly inhibit IBMX or PD98059-induced melanogenesis of B16-F10 cells in a dose-dependent manner, and the inhibitory effect of dodoviscin A on melanogenesis was more effective than the well-known melanogenic inhibitor kojic acid.

Tyrosinase catalyzes the rate-limiting step of melanin biosynthesis. There are many cosmetic materials that regulate melanogenesis by inhibiting tyrosinase catalytic activity primarily [33]. To elucidate the mechanism of the depigmenting effect of dodoviscin A, the direct effect of dodoviscin A on mushroom tyrosinase activity was investigated in a cell-free system. Kojic acid was used as a positive control at the same concentration. It was shown that dodoviscin A could suppress L-DOPA oxidation activity dose-dependently, with a 75 % decrease at the concentration of 200 µM compared to the vehicle-treated controls. However, kojic acid exhibited a stronger inhibitory effect on mushroom tyrosinase with an IC₅₀ value of 6.22 ± 1.71 µM ([Fig. 3 A]).

To investigate the binding mode of dodoviscin A on mushroom tyrosinase (PDB ID: 2Y9X), a docking study was performed ([Fig. 3 B]). It was shown that dodoviscin A resided in the ligand binding sites and could establish van der Waals interactions with His263. However, in addition to the van der Waals interactions, phi-phi interactions were also established between kojic acid and His263. Most importantly, kojic acid could also strongly interact with two copper ions with interaction distances of 2.7 Å and 3.4 Å, respectively. The results of the docking study indicated that the interaction between dodoviscin A and the key amino acids in the binuclear copper-binding site was much weaker than that of kojic acid, which could explain the above results of the different inhibitory effect of dodoviscin A and kojic acid on mushroom tyrosinase activity. However, dodoviscin A could decrease equivalent melanin content in B16-F10 cells at a concentration of 0.625 µM, which is significantly lower than kojic acid (50 µM). Thus, there might be other important mechanisms involved in the dodoviscin A-induced depigmenting effect besides its direct tyrosinase-inhibitory effects.

In addition, antioxidant activities of dodovicin A were evaluated by the DPPH free radical scavenging and metal chelating assay in light of the phenolic structure of dodovicin A. Dodovicin A has no effective antioxidant activities up to 400 µM (Fig. 1S, Supporting Information), suggesting that the depigmenting effects of dodoviscin A were independent from its antioxidant activities.

Tyrosinase is firstly synthesized as a 55 kDa polypeptide. This immature polypeptide undergoes glycosylation events in the ER and Golgi apparatus to form mature tyrosinase (65–80 kDa) for further inclusion into the melanosome [34]. In this study, Western blotting analyses were performed to examine the effects of dodoviscin A on tyrosinase protein expression after 72 h treatment. We found that IBMX-induced increasing levels of mature tyrosinase protein were significantly suppressed by dodoviscin A in a dose-dependent manner ([Fig. 4 A]). This corresponded to the result of mature tyrosinase protein analysis by the L-DOPA oxidation zymography assay ([Fig. 4 B]). [Fig. 4 C] showed that dodoviscin A could also dose-dependently suppress cellular tyrosinase activity significantly, which may explain the decreasing effect of dodoviscin A on mature tyrosinase protein expression ([Figs. 4 A] and [B]). Therefore, dodoviscin A might generate the depigmenting efficacy through its downregulatory effect of mature tyrosinase protein expression in B16-F10 cells.

It was reported that phosphorylation of CREB is involved in melanogenesis [15], [16]. We found that the phosphorylation level of CREB protein was suppressed in B16-F10 cells by dodoviscin A in a dose- and time-dependent manner ([Fig. 5 A] and [B]), however, no visible changes were observed in the phosphorylation of GSK3α/β, MEK, ERK, and AKT after different times of dodoviscin A treatment (Fig. 2S, Supporting Information). Meanwhile, although it acts as an important transcription factor regulating the expression of tyrosinase, there was no statistically significant difference in MITF expression levels between dodoviscin A and the vehicle-treated group ([Fig. 5 C]). However, IBMX-induced mature tyrosinase protein levels were significantly decreased by dodoviscin A in B16-F10 cells ([Fig. 4 A]). As reported, MITF was crucial but not sufficient to promote transcription of melanogenic genes [35], [36]. Post-transcriptional regulation is important in cAMP-mediated increased activity of some melanogenic enzymes. However, in the process, the expression of tyrosinase in the mRNA level is unchanged in human melanocytes, which is evidenced by some effects of melanotropic peptides or resveratrol on melanocytes [36], [37]. We found that the actions of dodoviscin A were at least partially similar to that of melanotropic peptides or resveratrol. Therefore, it can be speculated that dodoviscin A may inhibit the expression of post-transcriptional mature tyrosinase through inhibition of CREB phosphorylation. However, whether the degradation of tyrosinase is involved in the effect of dodoviscin A on the melanogenesis still requires further investigation.

It was reported that the transcription and the activation of THI were regulated by cAMP/PKA/CREB but independent of MITF [15], [16], [38]. In addition, the activating phosphorylation of PAH is required for cAMP-dependent protein kinase [17]. Thus, the depigmenting effect of dodoviscin A might be possibly related to cAMP/PKA/CREB-regulated key enzymes such as THI and PAH. Nevertheless, to confirm this conjecture, further studies are needed to evaluate the depigmenting mechanisms of dodoviscin A in B16-F10 cells, human melanocytes, or animal models.

In conclusion, the effects of dodoviscin A on melanogenesis were first evaluated in B16-F10 cells. Our results demonstrate that dodoviscin A is an effective melanin synthesis inhibitor in B16-F10 melanoma cells and may potentially be used as a therapeutic agent for treatment of skin hyperpigmentation.

Acknowledgements

This work was supported by grant 81 072 681 of the National Natural Science Foundation of China, by grant 2011AA09070102 of the National Marine “863′′ Project, and by grant 2012ZX09301001–001 of the National Science and Technology Major Project “Key New Drug Creation and Manufacturing Program”.

Conflict of Interest

All authors here declare that there are no conflicts of interest.

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Correspondence

• References

• 1 Lin JY, Fisher DE. Melanocyte biology and skin pigmentation. Nature 2007; 445: 843-850
  CrossrefPubMedSearch in Google Scholar
• 2 Maeda K, Fukuda M. Arbutin: mechanism of its depigmenting action in human melanocyte culture. J Pharmacol Exp Ther 1996; 276: 765-769
  PubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 5 Rolff M, Schottenheim J, Decker H, Tuczek F. Copper-O2 reactivity of tyrosinase models towards external monophenolic substrates: molecular mechanism and comparison with the enzyme. Chem Soc Rev 2011; 40: 4077-4098
  CrossrefPubMedSearch in Google Scholar
• 6 Ismaya WT, Rozeboom HJ, Weijn A, Mes JJ, Fusetti F, Wichers HJ, Dijkstra BW. Crystal structure of Agaricus bisporus mushroom tyrosinase: identity of the tetramer subunits and interaction with tropolone. Biochemistry 2011; 50: 5477-5486
  CrossrefPubMedSearch in Google Scholar
• 7 Ando H, Kondoh H, Ichihashi M, Hearing VJ. Approaches to identify inhibitors of melanin biosynthesis via the quality control of tyrosinase. J Invest Dermatol 2007; 127: 751-761
  CrossrefPubMedSearch in Google Scholar
• 8 Slominski A, Tobin DJ, Shibahara S, Wortsman J. Melanin pigmentation in mammalian skin and its hormonal regulation. Physiol Rev 2004; 84: 1155-1228
  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar
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