Melanin pigment, the primary determinant of skin and hair color in mammals, also protects the skin from injuries related to UV irradiation [1
]. However, excessive cutaneousmelanin deposition, which may occur through increases in the number of melanocytes or melanin synthesis, can lead to hyperpigmentation disorders such as melasma, actinic and senile lentigines, and post-inflammatory hyperpigmentation [2
Melanin is synthesized through the complex process of melanogenesis, which occurs within vesicles called melanosomes in melanocytes and is mediated by melanocyte-specific enzymes, such as tyrosinase and tyrosinase-related proteins (TRPs) [4
]. Tyrosinase is the rate-limiting enzyme that catalyzes the hydroxylation of tyrosine into dihydroxyphenylalanine (DOPA), followed by the subsequent oxidation of DOPA into dopaquinone. TRP1 and TRP2 are also present in the melanosome and play a critical role in melanin biosynthesis [5
Melanogenesis is mainly stimulated by α-melanocyte stimulating hormone (α-MSH), which is produced from proopiomelanocortin in response to UV irradiation [6
]. α-MSH activates the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway through the melanocortin receptor 1 (MC1R), the activation of which upregulates the transcription of microphthalmia-associated transcription factor (MITF), the master regulator in the transcription of genes encoding melanogenic enzymes [4
]. Alterations in MITF expression are highly associated with abnormal skin and hair pigmentation [7
]. Indeed, it has been reported that the downregulation of MITF expression by tea catechins was responsible for a reduction of melanin synthesis in mouse and human melanoma [8
]. Thus, MITF is an attractive molecular target for the potential treatment or prevention of hyperpigmentation disorders.
Asian pear (mostly Pyrus
spp.) belongs to the Rosaceae family and is one of the most widely consumed fruits in Eastern Asia. It is not only used as a fruit but also as a traditional medicine against cough, diuresis, and melasma [9
]. Many investigations have revealed that the presence of polyphenol-rich foods in the diet may be related to a lower risk of hyperpigmentation [3
] and various phenolic compounds from pear fruits have been identified in our previous studies [10
]. Therefore, in this study, we investigated the anti-melanogenic effects of pear fruit extract and evaluated the depigmentation mechanisms of the extract and its constituent protocatechuic acid (PCA) in mouse melanoma cells.
3. Materials and Methods
3.1. Materials and Cell Culture
Pear (Pyrus pyrifolia Nakai cv. Chuhwangbae) was cultivated in Naju and its immature fruit was collected in May 2015 after 35 days of florescence. The samples were certified by Wol-Soo Kim (Laboratory of Pomology, College of Agriculture and Life Science, Chonnam National University, South Korea). Arbutin (A4256), PCA (37580), α-MSH, melanin, forskolin, 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). B16F10 and SK-MEL-28 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum and 1% antibiotics (100 U/mL penicillin/streptomycin) in an atmosphere of 5% CO2 at 37 °C. All cell culture reagents were obtained from Gibco BRL (Grand Island, NY, USA).
3.2. Sample Preparation
Immature pear fruit (1 kg, fresh weight) was homogenized with 60% ethanol (4 L) by using a homogenizer (T50 digital ULTRA-TURRAX, IKA, Staufen, Germany). The homogenate was kept at 25 °C for 24 h and filtered under vacuum through no. 2 filter paper (Whatman, Maidstone, UK). The filtrates were concentrated, lyophilized, and stored at −20 °C until used.
3.3. Determination of Arbutin and PCA Contents in Immature Pear Fruit
Immature pear fruit extract (1 g fresh weight equivalent) was dissolved in 50% methanol (1 mL) and filtered through a 0.45 μm Millipore membrane filter. The dissolved solution was subjected to Octadecyl-silica (ODS)-HPLC analysis. Arbutin and PCA were separated on an ODS column (UG120, 4.6 mm internal diameter (ID) × 250 mm, 5 μm, Shiseido, Tokyo, Japan). The mobile phase was composed of H2O/acetic acid (98:2, v/v, eluent A) and MeOH/H2O (60:40, v/v, eluent B). The gradient program used was as follows: started at 100% A and increased to 100% B linearly over 45 min. The column temperature was maintained at 40 °C and the flow rate was 1 mL/min. Arbutin and PCA were monitored at 280 and 254 nm, respectively, using a photodiode array detector (SPD-M20D; Shimadzu, Kyoto, Japan). Arbutin and PCA contents in each sample were quantified by the chromatographic peak area of external standards. The calibration curves were plotted in the concentration range of 0.01–10 μg.
3.4. Cell Viability
The cells were seeded in 96-well plates, treated with various concentrations of PE or PCA for 72 h, and then incubated with MTT for 2 h. After the incubation, the cells were thoroughly washed with phosphate-buffered saline (PBS) and the insoluble formazan product was dissolved in dimethyl sulfoxide (DMSO). The absorbance of each well was measured at 560 nm by using a microplate reader (Biotek, Winooski, VT, USA). The absorbance of 0.1% DMSO-treated control cells was considered to represent 100% viability.
3.5. Determination of Melanin Content
The cells were treated with α-MSH (1 μM) in the presence or absence of PE, PCA, or arbutin (positive control) for 72 h, trypsinized and centrifuged at 1000× g for 5 min at 4 °C. The cell pellets were photographed and then solubilized in 1 N NaOH containing 10% DMSO at 80 °C for 1 h. The melanin content was calculated by the comparison of the absorbance at 405 nm with those of a standard curve of synthetic melanin.
3.6. Measurement of Tyrosinase Activity
To measure the tyrosinase activity in a cell free system, 1250 U/mL of mushroom tyrosinase solution (20 μL) was added to a mixture of 50 mM sodium phosphate buffer, pH 6.8 (100 μL), the desired sample (5 μL), and 5 mM l-tyrosine (125 μL). After incubation for 5 min at 25 °C, the production of dopachrome was determined by monitoring the change in absorbance at 475 nm every 20 s for 30 min.
To measure cellular tyrosinase activity, the cells were first treated with PCA for 24 h and then α-MSH (1 μM) was added. After incubation for 24 h, the cells were washed with PBS and centrifuged at 1000× g for 5 min at 4 °C. The cell pellets were resuspended in radioimmunoprecipitation assay (RIPA) buffer and centrifuged at 20,000× g for 20 min at 4 °C. The supernatant was used as a crude enzyme solution. The crude enzyme solution (20 μL; equivalent to 100 μg protein) was added to a mixture of 50 mM sodium phosphate buffer, pH 6.8 (100 μL) and 5 mM l-tyrosine (30 μL). After incubation for 3 h at 37 °C, the absorbance of the solution was measured at 475 nm.
3.7. RNA Analysis
Total RNA was isolated using TRIzol™ (Invitrogen, Carlsbad, CA, USA), cDNA was synthesized using ReverTra Ace®
qPCR RT kit (Toyobo, Osaka, Japan), and quantitative PCR was performed using a Mx3000P qPCR System (Agilent Technologies, Santa Clara, CA, USA). The primer sequences used in this study are shown in Table S1
. mRNA levels were normalized to the expression of mouse ribosomal protein, Large, P0 (Rplp0), and the data were calculated by the comparative threshold cycle method.
The cells were washed with PBS and centrifuged at 1000× g for 5 min at 4 °C. The obtained pellets were resuspended in RIPA buffer with protease and proteasome inhibitors, incubated on ice for 20 min, sonicated, and centrifuged at 20,000× g for 20 min at 4 °C. The supernatants were separated using 8% or 10% SDS-PAGE and then transferred to nitrocellulose membranes. The membranes were incubated overnight at 4 °C with primary antibodies against MITF, GAPDH (Thermo Fisher Scientific, Rockford, IL, USA), tyrosinase, TRP1, TRP2, β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), cAMP-responsive element binding protein (CREB), or pCREB (Cell Signaling Technology, Danvers, MA, USA), which was followed by incubation with the appropriate secondary antibodies (Thermo Fisher Scientific, Rockford, IL, USA). The blots were developed by using the EZ-Western Lumi Femto™ western blot detection kit (Daeil Lab Service, Seoul, Korea).
3.9. Luciferase Reporter Assay
The mouse MITF-M promoter construct (−1143 to +48, wtCRE) was amplified via PCR using mouse genomic DNA as template, and cloned into the pGL3-basic firefly luciferase reporter vector (Promega, Madison, WI, USA). The mtCRE was constructed by deleting the CRE motif (−153/−146, 5′-TGACGTCA-3′) by using the site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA). All constructs were verified via DNA sequencing. The cells were seeded in 24-well plates and transfected for 24 h with pGL3-Mitf-M (wtCRE or mtCRE) luciferase reporter, using Lipofectamine® 2000 reagent (Invitrogen, Carlsbad, CA, USA), before treatment with α-MSH and/or PCA for a further 24 h. The cells were harvested and assayed using the Nano-Glo® Dual-Luciferase® Reporter Assay System (Promega, Madison, WI, USA). The pNL1.1 luciferase vector (Promega, Madison, WI, USA) was used as a normalization control.
3.10. cAMP Determination
The cells were pretreated with PCA for 24 h, exposed to α-MSH for 30 min, washed in PBS, and lysed in 0.1 M HCl. After centrifugation, the cAMP in the supernatant was measured by a cAMP assay kit (BioVision, Milpitas, CA, USA).
3.11. Statistical Analysis
All experiments were performed a minimum of three times. The data were presented as the mean ± SEM. The differences between the means of the individual groups were assessed using Student’s t-test or one-way analysis of variance (ANOVA); differences were considered significant at p < 0.05. The statistical software package Prism 6.0 (GraphPad Software, La Jolla, CA, USA) was used for these analyses.