Antimelanogenesis Effect of Methyl Gallate through the Regulation of PI3K/Akt and MEK/ERK in B16F10 Melanoma Cells

Methyl gallate is a polyphenolic compound found in many plants, and its antioxidant, antitumor, antibacterial, and anti-inflammatory effects have been extensively studied. More recently, antidepressant-like effects of methyl gallate have been demonstrated in some studies. In the present study, we examined the effects of methyl gallate on melanogenesis, including the tyrosinase inhibitory effect, the melanin content, and the molecular signaling pathways involved in this inhibition. The results showed that methyl gallate inhibited tyrosinase activity and significantly downregulated the expressions of melanin synthesis-associated proteins, including microphthalmia-associated transcription factor (MITF), tyrosinase, dopachrome tautomerase (Dct), and tyrosinase-related protein-1 (TRP1). In conclusion, our findings indicated that activation of MEK/ERK and PI3K/Akt promoted by methyl gallate caused downregulation of MITF and triggered its downstream signaling pathway, thereby inhibiting the production of melanin. In summary, methyl gallate showed significant inhibitory activity against melanin formation, implying that it may be a potential ingredient for application in skin-whitening cosmetics.


Introduction
Melanocytes are responsible for the production of melanin, which contributes to the colored pigmentation in the skin and hair. In the epidermis of the skin, ultraviolet irradiation promotes melanocytes to generate melanin, and the dispersal of melanin via melanosomes to keratinocytes protects the human skin from extensive sunburn. Nonetheless, aberrant regulation of melanogenesis leads to pigmentation disorders such as melasma, hyperpigmentation, age spots, and blemishes [1][2][3]. Several plant-derived agents, such as kojic acid or arbutin (tyrosinase inhibitors), have been used for skin whitening, as they may regulate melanogenesis and are applicable in the treatment of hyperpigmentation [4].
Melanogenesis is a tyrosinase-initiated pigmentation process that converts L-3,4-dihydroxyphenylalanine to dopaquinone, followed by oxidation to form melanin [5]. Te pathway of melanogenesis is largely controlled by the level and activity of tyrosinase [6]. Hence, inhibition of tyrosinase has been utilized for cosmetic purposes and skin bleaching. A number of signaling cascades, such as cAMPrelated pathways, have been reported to have crucial roles in controlling melanogenesis [7,8]. Adenylyl cyclase activation induced by UV upregulates the formation of cAMP, which sequentially binds to melanocortin receptor 1 (MC1R) in melanocytes, thus triggering cAMP production and protein kinase A (PKA) activation [9]. Te process consequently leads to phosphorylation of the cAMP responsive-element binding protein (CREB), which in turn increases the transcription level of the microphthalmia-associated transcription factor (MITF) gene [10,11]. MITF is an important transcription factor for key proteins involved in melanin synthesis in melanocytes that regulates the expressions of tyrosinase, tyrosinase-related protein-1 (TRP-1), dopachrome tautomerase (Dct), and PKC-β [12,13].
MITF is a key factor in determining PKC-β-and cAMPdependent signaling in melanogenesis regulation [14,15]. Moreover, MITF is also a major element controlling DNA replication and the genome stability of melanoma cells via the downregulation of DNA alteration and inhibition of defects during cell proliferation [16,17], while DOPAchrome tautomerase reduces oxidative stress-induced cell injury and may regulate the sensitivity of melanoma cells towards certain chemotherapy drugs [18,19].
In melanocyte cells, the mitogen-activated protein kinase (MAPK) family, including p38MAPK and extracellular signal-regulated kinase (ERK), is mostly involved in regulating MITF expression [20,21]. In addition, phosphatidylinositol-3-kinase PI3K/Akt signaling has been recently identifed as regulating melanogenesis. Te upregulation of PI3K/Akt activates melanin synthesis, which is mediated by MITF expression and subsequent tyrosinase, TPR-1 and TRP-2 expressions [22,23]. Tus, based on the fndings of previous studies, these signaling pathways have the potential to be developed as a new strategic target for controlling melanin synthesis.
Methyl gallate is a gallic acid derivative and has been shown in previous studies to possess several biological properties, such as antioxidant [24], anti-infammatory [25], antibacterial [26], and antitumor efects [27,28]. Few studies have focused on the efects and molecular mechanism of methyl gallate against melanogenesis. Terefore, using an in vitro cell-culture model, this study aims to identify the antimelanogenesis mechanism of methyl gallate in B16F10 melanocyte cells.

Cell Culture and Treatment with Methyl Gallate.
B16F10 mouse melanoma cells were purchased from the Taiwan Food Industry Research and Development Institute (Hsinchu, Taiwan). Te cells were cultured and maintained in Dulbecco's modifed Eagle's medium (DMEM; Gibco Life Technologies, Carlsbad, CA, USA) contained with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C with 5% CO 2 atmosphere. Methyl gallate dissolved in DMSO and further diluted with DMEM to achieve the indicated fnal concentrations (50, 100, 200, 300, 400, 500, and 600 μM). Cells were cultured with diferent concentrations of methyl gallate and harvested after 24 h of incubation. Experiments were performed in triplicate and repeated multiple times.

Cell Viability
Assay. MTT cell viability assay was assessed to determine the viability of methyl gallate against B16F10 cells. Cells were seeded at 1 × 10 4 cells/well in 96 well plates. After treatment with various concentrations of methyl gallate, the cells were incubated at 37°C for 24 h. Te cell viability was determined using 3-(4,5-cimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) solution (1 mg/ml, 50 μl/well) and then added to each well, and cells were incubated at 37°C for 4 h. After the removal of the MTT solution, 100 μl DMSO was added to the well and incubated for 10 min. Absorbance was determined at 595 nm using a microtiter plate ELISA reader.

Melanin Content
Analysis. B16F10 cells (1 × 10 5 cells/ well) were incubated in 24 well plates. In brief, B16F10 cells were treated with methyl gallate for 24 h. Te cells were washed with PBS and then dissolved in 200 μl of 1N NaOH at 80°C for 2 h. Te samples were centrifuged for 30 min at 12,000 rpm to collect the supernatant. Te relative melanin content was determined by measuring the absorbance at 475 nm on a microtiter plate ELISA reader. Te melanin content was determined using standard curves using synthetic melanin solutions (0∼200 μg/ml). Melanin production was calculated as the μg of melanin/μg of total proteins in a cell extract.

Tyrosinase Activity Assay.
Tyrosinase activity was estimated as L-DOPA oxidase activity. B16F10 cells (2 × 10 6 cells/well) were incubated in 24 well plates. After treatment with diferent concentrations of methyl gallate (0, 10, 50, 100, and 200 μM) for 24 h, cells were washed with PBS and lysed with RIPA bufer containing protease inhibitor cocktail. Te cell lysates were then centrifuged at 12,000 rpm for 15 min to collect the supernatant. Te sodium phosphate bufer (0.1 M, pH 7.0) reacted with an equal volume of 1 mg/ ml L-DOPA. After incubation at 37°C for 2 h. Absorbance was then measured at 405 nm in an ELISA reader. Each measured result was expressed as the percentage change from the control [12].
2.6. Tyrosinase Activity Staining. Te tyrosinase activity staining was analyzed according to a previously described method [12]. B16F10 cells were incubated in 10 cm dish plates. In brief, the cells were lysed with RIPA bufer containing protease inhibitor cocktail. Te total protein 2 Evidence-Based Complementary and Alternative Medicine concentration was determined with Bradford Assay (Bio-Rad, Hercules, CA, USA), after which 25 μg of total protein were loaded onto and separated by 10% SDS-PAGE. Te sample was prepared in 0.1% SDS and β-mercaptoethanol and heat treatment was avoided. After electrophoresis, the gels were soaked in 10 mM Na 2 HPO 4 bufer (pH 6.2) for 30 min, followed by incubation in the same bufer containing 2 mM L-DOPA at 37°C.

Statistical Analysis.
Te results of the MTT assay were subjected by Student's t-test (Sigma-Stat 2.0, San Rafael, CA, USA). Results with p < 0.05 were considered statistically signifcant.  (Figure 1(a)). We also observed the cell type using a microscope and found that when up to 400 μM methyl gallate was added, there was no signifcant change in cell type (Figure 1(b)).

Tyrosinase Activity and Melanin Production in B16F10
Melanocytes after Methyl Gallate Treatment. We used an in vitro tyrosinase activity assay to study the inhibitory efect of methyl gallate. Te results showed that 100 μM or higher concentrations of methyl gallate signifcantly reduced the tyrosinase activity as compared with the controls; treatment with 200 μM methyl gallate was observed to inhibit tyrosinase activity by about 50% (Figure 2(a)). In addition, we used tyrosinase activity gel stain analysis to confrm the fnding. Following the addition of diferent concentrations of methyl gallate (10, 50, 100, and 200 μM), tyrosinase activity staining analysis showed that with the increase of methyl gallate concentration, the inhibition of tyrosine activity was greater (Figure 2(b)). Subsequently, we examined melanin synthesis in B16F10 melanocytes. When the cells were treated with 50 μM or higher concentrations of methyl gallate, there were significant diferences in melanin synthesis as compared with the control. When 200 μM methyl gallate was added, melanin synthesis was inhibited by approximately 50%. In addition, we used positive control groups treated with 200 μM kojic acid and 2 mM arbutin for inhibition of melanin synthesis and observed that 100 μM methyl gallate had a better efect than kojic acid and arbutin, while 200 μM methyl gallate was even more efective (Figure 2(c)).

Efect of Methyl Gallate on Melanin Synthesis-Related
Pathway Proteins. From the above experimental results, it was found that methyl gallate inhibited the activity of intracellular tyrosinase and melanin production. To understand whether it also afects the expressions of other melanin synthesis-related proteins, we used diferent concentrations of methyl gallate (50, 100, and 200 μM) for treatment. After 24 hours of reaction, western blotting analysis was used to determine diferences in protein expressions. From the experimental results, it was observed that the expressions of melanin-related proteins, such as MC1R, were decreased with increasing methyl gallate concentrations; decreases in the expressions of MITF, phosphorylated CREB, tyrosinase, TRP-1, and Dct were also observed with increasing methyl gallate concentrations ( Figure 3). p38MAPK, MEK/ERK, and PI3K/Akt are known to be associated with melanin synthesis-related pathways. Te expression levels of these proteins after the addition of methyl gallate are shown in Figure 3. Te expressions of p-MEK, p-ERK, p-RSK1, and p-AKT increased with an increasing concentration of methyl gallate, while p-p38MAPK was downregulated, and the expressions of MEK, ERK, RSK1, and Akt did not change (Figure 3). Terefore, it was speculated that methyl gallate may also afect B16F10 melanocytes melanogenesis through the activation of MEK/ ERK and PI3K/Akt signaling pathways.

Efects of MEK/ERK and PI3K/Akt Inhibition on Methyl Gallate-Repressed
Melanogenesis-Related Protein Expressions. After adding MEK/ERK and PI3K/Akt inhibitors (PD98059 and LY294002, respectively), the activity of tyrosinase and the content of melanin in the cells were studied. It was found that the activity of tyrosinase and the melanin content decreased signifcantly by approximately 50% after methyl gallate treatment in B16F10 melanocytes (Figure 4(a)). To confrm whether methyl gallate inhibited melanin synthesis through PI3K/Akt and MEK/ERK, we employed western blotting to examine melanin synthesisrelated protein expressions in MEK/ERK and PI3K/Akt signaling. Te results showed that the inhibition of MEK/ ERK and PI3K/Akt led to higher expressions of tyrosinase, TRP-1, and Dct than methyl gallate treatment alone. Both PD98059 and LY294002 treatments had signifcant efects in terms of the degree of recovery of MITF, tyrosinase, TRP-1, and Dct protein expressions as compared with cells treated with methyl gallate only. Tese results demonstrated that   Evidence-Based Complementary and Alternative Medicine methyl gallate activates the MEK/ERK and PI3K/Akt signaling pathways, which in turn afect melanin synthesis in B16F10 cells (Figure 4(b)).

Discussion
Methyl gallate is a polyphenolic compound derived from plants and has been reported to possess a variety of bioactivities. In this study, we investigated the molecular mechanism of methyl gallate inhibition of melanin production in melanocytes. According to the results of cellsurvival experiments, 0-600 μM methyl gallate had no signifcant cytotoxicity against B16F10 melanocytes. In addition, methyl gallate treatment caused signifcant dosedependent inhibition of intracellular tyrosinase and melanin contents (Figure 1). Methyl gallate at 200 μM signifcantly inhibited the activity of tyrosinase in vitro and also inhibited the tyrosinase activity in a staining experiment. Kojic acid and arbutin (a tyrosinase inhibitor) are used in the treatment or prevention of abnormal skin pigmentation [29,30], Te experimental results showed that the cell viability following the treatment with 200 μM methyl gallate was higher than that with the treatment with 200 μM kojic acid and 2 mM arbutin, and the inhibition of melanin by 200 μM methyl gallate was better than that of 200 μM kojic acid and 2 mM arbutin. Methyl Evidence-Based Complementary and Alternative Medicine gallate is a potent compound that inhibits melanin and was proven to be more efective than kojic acid and arbutin in cell experiments.
In addition to the cAMP regulatory pathway as the major signal transduction pathway, activation of MEK/ERK and PI3K/Akt signaling pathways (also) regulates melanin synthesis [12,31]. Te phosphorylated p38MAPK activates MITF to ultimately stimulate melanin synthesis, while the activation of ERK 1/2 and JNK leads to a decrease in melanogenesis via MITF degradation [32].
Study results have indicated that the MEK/ERK pathway regulates the phosphorylation of MITF. When the MEK/ ERK pathway is activated, it will promote the phosphorylation of MITF, followed by ubiquitination, and degradation. Te downstream expressions of melanin-related proteins such as tyrosinase, TRP1, and Dct are decreased, reducing melanin synthesis [33,34]. Activation of the PI3K/Akt signaling pathway is therefore subjected to a strictlyregulated signal-dependent approach, which in turn affects the phosphorylation of MITF and regulates melanin biosynthesis [22,35]. In addition to transcriptional regulation, MITF is also phosphorylated by various posttranslational modifcations, in addition to ERK; in particular, ribosomal S6 kinase (RSK) and glycogen synthase kinase-3β (GSK3β) [11] have been used to phosphorylate MITF.
Te phosphorylated active CREB further binds MITF, which in turn stimulates the transcription of the key melanogenic enzymes [36]. CREB phosphorylation induces transcription of MITF. Te p-CREB expression decreased after treatment with methyl gallate, and p-MITF protein fragmentation increased with increasing concentrations of methyl gallate, as did the expressions of p-MEK and p-ERK. Te protein expression levels of p-RSK1 and p-AKT increased signifcantly. It was speculated that methyl gallate may activate p-MITF via the MEK/ERK and PI3K/Akt signaling pathways to down-regulate tyrosinase, TRP-1, and DCT in B16F10 melanocytes [12].
Previous studies have demonstrated that melanogenesis is mediated by the regulation of MITF activation via phosphorylation of p38 MAPK [10]. Activation of the p38 MAPK signaling pathway increases the transcription of tyrosinase, which activates melanin synthesis [37]. In this study, methyl gallate inhibited the phosphorylation level of p38MAPK, which could participate in and inhibit melanin production.
We used a MEK/ERK inhibitor (PD98059) and a PI3K/ Akt inhibitor (LY294002) to verify whether methyl gallate activates the MEK/ERK and PI3K/Akt signaling pathways, which in turn afects tyrosinase activity and melanin synthesis and decreases the expressions of proteins involved in melanin synthesis. First, we added a MEK/ERK inhibitor (PD98059) and a PI3K/Akt inhibitor (LY294002) and recorded the activity of tyrosinase and the content of melanin. Ten, we used immunostaining analysis to verify the melanin synthesis pathways of MEK/ERK and PI3K/Akt. Te results suggested that methyl gallate does activate the MEK/ERK and PI3K/Akt signaling pathways, thereby reducing the expressions of tyrosinase and melanin-related proteins and inhibiting the synthesis of melanin [12].
Te previous study showed that methyl gallate decreased melanin pigmentation in a concentration-dependent manner but did not directly inhibit tyrosinase activity. Further analysis showed that methyl gallate had no efect on extracellular signal-regulated kinase (ERK) activation but induced phosphorylation of glycogen synthase kinase 3β (GSK3β) [40]. Te results of the current study difered from the previous fndings, in that our results validated the ability of methyl gallate to inhibit melanin synthesis.

Conclusion
We demonstrate that methyl gallate suppresses the tyrosinase activity in B16F10 cells and reveal the molecular mechanism involved in melanogenesis. Our data suggest that methyl gallate may regulate melanogenesis by restraining the production of MITF, tyrosinase, TRP1, and Dct, and the process is associated with the phosphorylation of PI3K/Akt or MEK/ERK. According to our results, methyl gallate reduces melanin synthesis and could be a useful agent for skin whitening; it may also have the potential for application in cosmeceuticals in the future.

Data Availability
Data generated or analyzed during this study are provided in full within the published article.

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Authors' Contributions
Yu-Jen Wu and Ching-Chyuan Su conceived, designed, and performed the experiments; Zhi-Jiao Cheng, Guo-Fong Dai, Jue-Liang Hsu, and Jen-Jie Lin performed the experiments and analyzed the data; and Yu-Jen Wu and Wen-Tung Wu wrote the paper. All authors read and approved the fnal manuscript. Ching Chyuan Su and Yu Jen Wu contributed equally to this work.