Inhibitory Effects of Saururi chinensis Extracts on Melanin Biosynthesis in B16F10 Melanoma Cells

Abstract

Saururus chinensis has been used in folk medicine in Korea for the treatment of edema, jaundice, gonorrhea, and several inflammatory diseases. Saururi chinensis extracts (SCE) have demonstrated anti-inflammatory and anti-oxidant activities, as well as anti-asthmatic, antihypertensive, anti-angiogenic, and therapeutic activities for atopic dermatitis. However, the inhibitory activity of SCE on the melanogenesis signaling pathway is not completely understood. This study examined the effects of SCE on the melanogenesis signaling pathway activated by α-melanocyte-stimulating hormone (α-MSH). We found that SCE inhibited melanin production in a dose-dependent manner without causing cytotoxicity in B16F10 cells. Interestingly, SCE decreased α-MSH-induced tyrosinase activity in B16F10 cells but did not inhibit tyrosinase activity under cell-free conditions. The results of this study indicate that SCE may reduce pigmentation by way of an indirect, nonenzymatic mechanism. We also found that SCE decreased α-MSH-induced microphthalmia-associated transcription factor (MITF) and tyrosinase expression and induced the activation of extracellular signal-regulated kinase (ERK). These results suggest that the depigmenting effect of SCE may result from downregulation of MITF and tyrosinase expression due to increased ERK activity. Thus, our results provide evidence that SCE might be useful as a potential skin-whitening agent.

In mammals, pigmentation results from the synthesis and distribution of melanin in the skin, hair bulbs and eyes. Melanin is a pigmented polymer, which provides photoprotection to the skin against UV radiation. Various hyperpigmented disorders such as melasma can be caused by excessive synthesis of melanin. Melanogenesis comprises many enzymatic oxidation steps in which tyrosine is converted to eumelanin and pheomelanin.¹⁾ Tyrosinase, tyrosinase-related protein-1 (TRP-1), and dihydroxyphenylalaminechrome tautomerase (TRP-2) are key enzymes involved in the regulation of melanogenesis.²⁾ The expressions of tyrosinase and TRP1 and TRP2 genes are activated by microphthalmia-associated transcription factor (MITF) through cooperation with protein kinase C (PKC).³⁾ Tyrosinase catalyses the rate-limiting reaction of the melanogenic process, and melanin production is regulated mainly by the expression and activation of tyrosinase.^3–5)

Recently, the inhibition of extracellular signal-regulated kinase (ERK) signaling was reported to induce hyperpigmentation by increasing tyrosinase activity, suggesting that the activation of ERK signaling downregulates melanogenesis by inhibiting tyrosinase activity.^6–8) These reports show that the improved methods of melanogenesis inhibition do not suppress the activity of tyrosinase as much as they control the tyrosinase upstream signaling pathway related to its activation and expression.

Several known natural melanin synthesis inhibitors, including arbutin and kojic acid, have been the focus of previous studies and are currently being utilized as cosmetic additives. A great deal of attention has continuously focused on the application of natural products in the cosmetics industry.^9,10) However, it is necessary to find safer and more effective skin-whitening agents, because of the carcinogenic potential of kojic acid as well as its weak whitening effect.

This study examined the effects of Saururi chinensis extracts (SCE) on the melanogenesis signaling pathway activated by α-melanocyte-stimulating hormone (α-MSH). Saururus chinensis is a perennial herb that is distributed throughout China and Korea, and the aerial portion of Saururus chinensis has been used in folk medicine in Korea for the treatment of edema, jaundice, gonorrhea, and several inflammatory diseases. SCE have been demonstrated to have anti-inflammatory and anti-oxidant activities,^11,12) as well as anti-asthmatic,¹³⁾ antihypertensive,¹⁴⁾ anti-angiogenic,¹⁵⁾ and therapeutic activities for atopic dermatitis.¹⁶⁾ Among the bioactive compounds isolated from Saururus chinensis, lignans such as sauchinone, manassantin A and B, saucerenol D have various biological activities, including anti-inflammatory activity,^14,17–23) cardiovascular effects,²⁴⁾ hypercholesterolemic activities,²⁵⁾ and bone destruction and osteoclast formation inhibiting activities.²⁶⁾

We are interested in reevaluation of traditional Korean herbs such as Saururus chinensis on melanogenesis in the literature. In a previous screening study performed with mushroom tyrosinase, SCE showed slightly inhibitory activity. Results from this study provided important information about SCE on melanogenesis.²⁷⁾ However, despite numerous reports, the inhibitory activity of SCE on the melanogenesis signaling pathway is not completely understood.

To our knowledge, this study is the first to show the inhibitory activity of SCE on the melanogenesis signaling pathways, including the expression of MITF and tyrosinase and the phosphorylation of ERK.

Materials and Methods

Preparation of SCE

The aerial portion of Saururus chinensis was purchased from a local herbal store, Kyungdong Market (Seoul, South Korea). The dried plant material (60 g) was cut into small pieces and extracted repeatedly with 600 mL of methanol at 60°C. The combined methanolic extract was processed through filter paper, evaporated in vacuo, and freeze-dried to give a powdered extract (3.5 g).

Materials and Reagents

Mushroom tyrosinase, α-melanocyte-stimulating hormone (α-MSH), arbutin, 3,4-dihydroxy-l-phenylalanine (l-DOPA), methylthiazolyldiphenyl-tetrazolium bromide (MTT), and PD98059 were purchased from Sigma-Aldrich Co. (St. Louis, MO, U.S.A.). Dulbecco’s modified Eagle’s medium (DMEM), trypsin–ethylenediaminetetraacetic acid (EDTA), phosphate-buffered saline (PBS), and penicillin/streptomycin were purchased from WelGENE Biopharmaceuticals (Daegu, Korea). Fetal bovine serum (FBS) was purchased from Life Technologies Co. (Gibco, Life Technologies, NY, U.S.A.). Goat polyclonal actin, goat polyclonal glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and goat polyclonal tyrosinase antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, U.S.A.). Mouse monoclonal microphthalmia (MITF) antibody-1 (Ab-1) was purchased from NeoMarkers (Fremont, CA, U.S.A.). Rabbit monoclonal phosphor-cAMP response element binding protein (CREB), rabbit monoclonal CREB, rabbit polyclonal phosphor-ERK, rabbit polyclonal ERK, rabbit polyclonal GSK3β, rabbit polyclonal phosphor-Akt and rabbit polyclonal Akt antibodies were purchased from Cell Signaling Technology Inc. (Beverly, MA, U.S.A.). Rabbit polyclonal phosphor-GSK3β was purchased from Dawinbio Inc. (Abcam, Hanam, Korea).

Cell Culture

B16F10 murine melanoma cells (CRL-6475) were purchased from the American Type Culture Collection (Rockville, MD, U.S.A.). Cells were maintained in DMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin–streptomycin in 5% CO₂ at 37°C. Cells were passaged every 3 d until a maximal passage number of 20 was achieved.

Cell Viability Assay

To determine the safety of the various extracts, the viability of cells following treatment with extracts was determined by the MTT assay. This method is based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to formazan by mitochondrial enzymes in viable cells.²⁸⁾ The quantity of formazan formed is proportional to the number of viable cells present and can be measured spectrophotometrically. Briefly, B16F10 cells were incubated at a density of 2×10⁵ cells in 6-well plates overnight. A test sample was then added to each well and incubated for 24 h and 72 h at 37°C in an atmosphere containing 5% CO₂. Next, the treated cells were labeled with MTT dye reagent (Applichem, Denmark) in PBS (1 mg/mL) for 3 h. The cells were then incubated at 37°C for 3 h, and dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. The absorbance was measured at 570 nm using a spectrophotometer. Cell viability was calculated using the following formula: cell viability (%)=(A_sample/A_control)×100, where A_sample and A_control are the absorbance values from the mixture with or without the addition of test reagent, respectively.

Measurement of Melanin Content

B16F10 cells were incubated at a density of 2×10⁵ cells in 6-well plates overnight. α-MSH (1 µm) was then added, and cells were treated with increasing concentrations of substance in phenol red-free DMEM for 3 d. Two hundred microliter aliquots of media were then placed in 96-well plates, and the optical density of each culture well was measured using an enzyme-linked immunosorbent assay (ELISA) reader (VERSAMax; Molecular Devices, Sunnyvale, CA, U.S.A.) at 405 nm. Cell numbers were then counted using a hemocytometer. Melanin production was expressed as the percentage of living cells of the controls.

Cell-free Enzymatic Assay for Tyrosinase Activity

Tyrosinase activity was assayed as a function of DOPA oxidase activity. A total of 70 µL of 0.1 m phosphate buffer (pH 6.8) containing SCE was mixed with 20 µL of 10 µg/mL mushroom tyrosinase in a well of a 96-well plate, and 10 µL of 10 mm l-DOPA was added. Absorbance values were measured every 10 min for at least 1 h at 475 nm using an ELISA reader at an incubation temperature of 37°C. The quantity of dopachrome formed in the reaction mixture was determined against a blank (solution without enzyme) at 475 nm in the ELISA reader. The percentage of tyrosinase activity was calculated as follows: tyrosinase activity (%)=[(A−B)/(C−D)]×100, where A is the absorbance of reaction mixture containing test sample and mushroom tyrosinase, B is the absorbance of blank sample containing test sample but no mushroom tyrosinase, C is the absorbance of reaction mixture without test sample and with mushroom tyrosinase, and D is the absorbance of the well lacking both the test sample and mushroom tyrosinase (l-DOPA alone).

Assay of Cellular Tyrosinase Activity

Tyrosinase activity was estimated by measuring the rate of l-DOPA oxidation. B16F10 cells were incubated at a density of 2×10⁵ cells in 6-well plates and treated as indicated for 3 d in DMEM. The cells were then washed in ice-cold PBS and lysed in 100 µL of phosphate buffer (0.1 m), pH 6.8, containing 1% (w/v) Triton X-100. The cellular extract was clarified by centrifugation at 15000 rpm for 20 min. The tyrosinase substrate, l-DOPA (2 mg/mL), was prepared in the same lysis buffer. Two hundred microliters of each extract was then placed in a 96-well plate, and the enzymatic assay was initiated by adding 2 µL of an l-DOPA solution at 37°C. The control wells contained 200 µL of the lysis buffer. The absorbance at 405 nm was read every 10 min for at least 1 h at 37°C using an ELISA reader. Tyrosinase activity in the protein was calculated by the following formula: tyrosinase activity (%)=OD₄₇₅ of sample/OD₄₇₅ of control×100

Western Blot Analysis

Cells were lysed in cell lysis buffer [62.5 mm Tris–HCl (pH 6.8), 2% sodium dodecyl sulfate (SDS), 5% β-mercaptoethanol, 2 mm phenylmethylsulfonyl fluoride, protease inhibitors (Complete™; Roche, Mannheim, Germany), 1 mm Na₃VO₄, 50 mm NaF, and 10 mm EDTA]. Twenty micrograms of protein per lane was separated by SDS-polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride membranes, which were then saturated with 5% dried milk in Tris-buffered saline containing 0.5% Tween 20 (TBST). Blots were then incubated with appropriate primary antibodies at a dilution of 1 : 1000 and further incubated with horseradish peroxidase-conjugated secondary antibody. Bound antibodies were detected using an enhanced chemiluminescence plus kit (Amersham International, Little Chalfont, U.K.).

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

To determine the effects of SCE on melanogenesis-related gene expression, reverse transcription-polymerase chain reaction (RT-PCR) was carried out. B16F10 melanoma cells were treated with or without SCE and stimulated with α-MSH. For analysis of the MITF and tyrosinase mRNA levels, total cellular RNA was prepared after incubation for 24, 48, and 72 h. Total RNA was isolated from the cells using an RNeasy Mini Kit (Qiagen, Valencia, CA, U.S.A.). Next, 1 µg RNA was reverse-transcribed using a Primescript™ 1st strand cDNA synthesis kit (Takara Bio Inc., Japan). The cDNA obtained was amplified with the following primers: MITF (998 bp) forward, 5′-CCA ACT GTG AAA AAG AGG CAT T-3′ and reverse, 5′-TTC TTC TGC GCT CAT ACT GC-3′; tyrosinase (990 bp) forward, 5′-TTC TGC CTT GGC ACA GAC TT-3′ and reverse, 5′-AGG ATG TTC ACA GAT GGC TCT -3′; GAPDH (451 bp) forward, 5′-ACC ACA GTC CAT GCC ATC AC-3′ and reverse, 5′-TCC ACC ACC CTG TTG CTG TA-3′. The PCR conditions were 30 cycles for 1 min at 95°C, 1 min at 52°C, and 1 min at 72°C (for MITF), 30 cycles for 1 min at 95°C, 1 min at 58°C, and 1 min at 72°C (for tyrosinase), and 35 cycles for 1 min at 95°C, 1 min at 60°C, 1 min at 72°C (for GAPDH). The resulting PCR products were visualized by electrophoretic separation on 1% agarose gels with Safe-Pinky DNA gel staining solution (Gendepot, TX, U.S.A.). Specific primers for GAPDH were added as a control.

Immunocytochemistry/Immunofluorescence Assay (ICC/IF)

B16F10 melanoma cells were seeded onto slides. After incubation at 37°C in a CO₂ incubator, ICC/IF was performed. Each slide was fixed with 4% paraformaldehyde for 10 min at room temperature and then incubated with 0.01% Triton X-100 for 5 min. Finally, the slide was incubated in NH₄Cl for 5 min. After blocking the reaction with blocking solution, the slide was incubated with tyrosinase antibody at 4°C overnight. Slides were incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibody, mounted using fluorescent mounting medium with 4′,6-diamidino-2-phenylindole (DAPI), and observed by fluorescence microscopy.

Statistical Analysis

One-way ANOVA followed by Dunnett’s T3 test was used to assess statistical significance with thresholds of *p≤0.05, **p≤0.01 and ***p≤0.001 for significant and highly significant, respectively.

Results

Effect of SCE on B16F10 Cell Viability

In order to determine if SCE could inhibit melanogenesis, B16F10 cells were treated with extract before α-MSH stimulation. We first tested the cytotoxicity of SCE in B16F10 melanoma cells by MTT assay. B16F10 cells were treated with SCE at concentrations of 1–500 ng/mL for 24 h and 72h. As shown in Fig. 1A and B, SCE was found to have no cytotoxic effect on B16F10 cells at the concentrations used.

Fig. 1. Cell Viability of B16F10 Melanoma Cells Treated with SCE at Different Concentrations

Data represent the mean±S.D. Cells were treated with SCE at 1–500 ng/mL for 24 h and 72h. Cell viability was determined using MTT assays. Results are expressed as a percent viability relative to vehicle-treated controls. Most of the melanoma B16F10 cells were viable at concentrations in the range of 10–500 ng/mL (viability > 90%). Each measurement was made in triplicate, and data represent the mean±S.D.

Effect of SCE on Melanogenesis in B16F10 Cells

The effect of SCE on the melanogenesis of B16F10 cells was examined. To determine whether SCE inhibits α-MSH-induced melanin synthesis in B16F10 cells, we measured α-MSH-induced melanin production of B16F10 cells after 3 d of SCE treatment at 10–500 ng/mL. SCE at concentrations greater than 10 ng/mL significantly inhibited melanin synthesis (Fig. 2). SCE treatment also reduced α-MSH-induced melanin release in a dose-dependent manner. In addition, the inhibitory effect for melanin synthesis by SCE was more effective compared with that of arbutin, a well-known tyrosinase inhibitor.

Fig. 2. Effect of SCE on Melanin Content in B16F10 Melanoma Cells

SCE was tested at 10, 50, 100, and 500 ng/mL in B16F10 melanoma cells. B16F10 cells were co-cultured with α-MSH (1 µm) and SCE at 10–500 ng/mL for 72 h as compared with untreated control and α-MSH stimulated cells. Levels of inhibition of melanogenesis were expressed as percentages of the control. The results are averages of three independent experiments, and the data are expressed as mean±S.D. Inhibition of melanin content was related to exposure to SCE in a dose-dependent manner. In addition, the inhibitory effect of melanin synthesis by SCE was more effective than arbutin at 55000 ng/mL concentration (200 µm). *** p<0.001 as compared with α-MSH-treated controls and compared with the untreated control.

Effects of SCE on Tyrosinase Activity in B16F10 Cells

Melanogenesis is known to be controlled by an enzymatic cascade, which is regulated by the level of tyrosinase. To investigate the mechanism of depigmenting action shown by SCE, we carried out the measurement of tyrosinase activity in both cell-based and cell-free assay systems. As shown in Fig. 3A, α-MSH-induced tyrosinase activity was inhibited in a dose-dependent manner (10–500 ng/mL). We also found that 100 ng/mL SCE significantly reduced tyrosinase activity. Specifically, as compared with untreated cells, treatment with SCE at a concentration of 100 ng/mL resulted in an approximately 40% inhibition of activity of the intracellular tyrosinase in B16F10 melanoma cells.

Fig. 3. Inhibitory Effects of SCE on Tyrosinase Activity in Cell and Cell-Free Assays

Influence of SCE on the tyrosinase activity and expression of B16F10 cells after treatment for 72 h. B16F10 cells were treated with SCE at 10–500 ng/mL and stimulated with α-MSH (1 µm). Data shown represent the mean±S.D. SCE inhibit tyrosinase activity in cells in a dose-dependent manner (A), but did not appear to inhibit mushroom tyrosinase activity (B). Each measurement was made in triplicate, and data shown represent the mean±S.D. ** p<0.01, *** p<0.001 as compare to control.

Many skin-whitening agents directly inhibit tyrosinase. Thus, to investigate the direct effects of SCE on tyrosinase, we measured the tyrosinase activities of mushroom tyrosinase in a cell-free system, as described in Materials and Methods. However, SCE was found to have no direct inhibitory effect on mushroom tyrosinase activity at concentrations ranging from 1 to 500 ng/mL (Fig. 3B). Thus, our results indicate that SCE may reduce pigmentation by way of an indirect nonenzymatic mechanism.

Effect of SCE on Melanogenesis Pathways in B16F10 Cells

In order to determine if the inhibitory activity of SCE was related to melanogenesis pathways involving the expressions of tyrosinase and MITF, B16F10 cells were treated with the extract prior to stimulation with α-MSH. The resulting cell lysates were subjected to SDS-PAGE and Western blot analysis. As shown in Fig. 4A, MITF levels were clearly reduced after 24–72 h of SCE treatment; tyrosinase levels also decreased in a time-dependent manner. Additionally, We determined MITF expression after 6–24 h of SCE treatment. However, the MITF level was not changed by treatment with SCE at 6–12 h (Fig. 4B). These results suggest that SCE decreases melanin synthesis by downregulating MITF and tyrosinase.

Fig. 4. Effects of SCE on the Expression of the Melanogenesis-Related Proteins in B16F10 Melanoma Cells

(A) Cells were exposed to α-MSH (1 µm) in the presence of SCE (100 ng/mL) for 24, 48 and 72 h. The expression levels of the tyrosinase and MITF proteins were examined by Western blot analysis as described in Materials and Methods section. Equal protein loading was confirmed by actin expression. (CTL, control) (B) Additionally, We determined MITF expression after 6–24 h of SCE treatment. (C) The effect of tyrosinase expression in the cells was confirmed by immunocytochemistry/immunofluorescence (×400).

Furthermore, the effect of SCE on tyrosinase expression in the cells was confirmed by immunocytochemistry/immunoflourence (ICC/IF) (Fig. 4C). This experiment showed that the tyrosinase levels in cells were reduced by SCE after 72 h in the presence of α-MSH.

To examine whether the inhibition of tyrosinase and MITF protein expression by SCE was due to a decreased level of transcription, we performed RT-PCR using specific primers. However, the mRNA levels of tyrosinase and MITF showed no significant change at 48 h and 72 h compared to controls with α-MSH (Fig. 5), indicating that SCE inhibits the expression of tyrosinase and MITF, which plays a pivotal role in melanogenesis, at the translational, but not transcriptional, level.

Fig. 5. Effects of SCE on the Expression of the Melanogenesis-Related mRNAs in B16F10 Melanoma Cells

Cells were exposed to α-MSH (1 µm) in the presence of SCE (100 ng/mL) for 24, 48 and 72 h. The expression levels of the tyrosinase and MITF mRNAs were examined by reverse transcription-polymerase chain reaction as described in Materials and Methods. Equal RNA loading was confirmed using GAPDH. (CTL, control)

Effect of SCE on Inhibitory Signaling Pathways of Melanogenesis in B16F10 Cells

To elucidate the mechanism underlying the hypopigmentary effect of SCE, changes in melanogenesis-related signals induced by SCE were analyzed by Western blotting in a time-course experiment. Because the phosphorylation of ERK has been reported to trigger MITF degradation,²⁹⁾ we examined whether SCE induces ERK activation. As shown in Fig. 6, the activation of the ERK1/2 in the cells was induced by SCE at 30 min after the α-MSH treatment. This experiment showed that SCE strongly induced ERK activation in B16F10 melanoma cells.

Fig. 6. Effects of SCE on the Signal Transduction Pathways in B16F10 Melanoma Cells

After 24 h of serum starvation, cells were treated with SCE (100 ng/mL) for the indicated times. The expression levels of phosphor-CREB, CREB, phosphor-ERK, ERK, phosphor-Akt, AKT, phosphor-GSK3β and GSK3β proteins were examined by Western blot analysis, as described in Materials and Methods section. Equal protein loadings were confirmed ERK, CREB, Akt, GSK3β, or actin expression. (CTL, control)

In addition, we examined cyclic AMP response element-binding protein (CREB), which is known to activate the MITF promoter.³⁰⁾ However, SCE had no effects the phosphorylation status of CREB. Several studies have found that the Akt pathway is involved in the melanogenesis of G361 and B16F10 melanoma cells.^31,32) However, we found no evidence for Akt pathway activation by SCE in these cells, nor were any changes in phospho-Akt levels observed. Lastly, GSK3β has been widely implicated in the regulation of cell homeostasis by virtue of its ability to phosphorylate a broad range of substrates, including glycogen synthase, MITF, and β-catenin.³³⁾ However, the GSK3β level remained unchanged after treatment with SCE.

Inhibition of the ERK Pathway by PD98059 Restored SCE-Induced Hypopigmentation

The activation of ERK1/2 was reported to inhibit tyrosinase expression, which subsequently decreases cellular melanin synthesis. Therefore, experiments were carried out to determine whether the SCE-activated ERK1/2 signaling pathways were related to the cellular melanin synthesis in B16F10 cells treated with SCE for 1 h in the presence or absence of PD98059 (selective inhibitor of MEK).

As shown in Fig. 7A, the cellular melanin content in cells co-treated with α-MSH and PD98059 were higher than in the cells treated with α-MSH alone. In addition, this synergistic effect of α-MSH and PD98059 on cellular melanin content was offset by SCE treatment. We next examined whether PD98059 inhibits the ERK pathway in B16F10 cells and found that PD98059 does in fact block ERK activation in SCE-treated B16F10 cells (Fig. 7B). Thus, these results suggest that the reduction of melanogenesis by SCE may be mediated by the ERK signaling pathway.

Fig. 7. Effects of Inhibition of the ERK Pathway on Melanin Content and Melanogenic Protein Expression

B16F10 cells were cultured with SCE (100 ng/mL) for 1 h in the presence or absence of PD98059 (10 µm). (A) The cellular melanin content was determined and expressed as a percentage. Each percentage in the treated cells is reported relative to that of the control cells. The data are reported as the mean±S.D. of three independent experiments carried out in triplicate. (B) Whole cell lysates were subjected to Western blot analysis using antibodies against phosphor-specific ERK. Equal protein loading was confirmed using actin antibody. Each measurement was made in triplicate, and data shown represent the mean±S.D. ** p<0.01 as compared to control.

Discussion

To our knowledge, this is the first study to report the potent inhibitory effect of SCE on melanogenesis in α-MSH-induced B16F10 melanoma cells. This study demonstrates that SCE is a skin-whitening agent that acts via down-regulation of tyrosinase expression. The inhibitory effects of SCE were dose-dependent and did not incur significant cytotoxicity (Figs. 1A, B). SCE-induced melanin reduction was also accompanied by a corresponding decrease in tyrosinase activity, suggesting a possible mechanism of SCE action (Fig. 2).

Tyrosinase has been demonstrated to catalyze the rate-limiting step of melanin biosynthesis, and it is the primary target of arbutin, which is currently used as a cosmetic and medical material with efficient depigmenting effects.^34–36) SCE strongly inhibits intracellular tyrosinase activity in α-MSH-induced B16F10 melanoma cells, as demonstrated by cellular tyrosinase (Fig. 3A). However, as shown in Fig. 3B, SCE did not directly inhibit tyrosinase activity, indicating its involvement in different mechanisms. These results suggest that reduced pigmentation by SCE might be attributed to the effect of SCE upon the signaling pathways regulating tyrosinase.

For a better understanding of the depigmenting action of SCE targeting tyrosinase synthesis, Western immunoblot analysis was carried out. Our data confirmed that MITF and tyrosinase protein levels were attenuated by SCE in a time-dependent manner (Fig. 4A). In addition, SCE-induced hypopigmentation correlated with reduced tyrosinase activity (Fig. 3A), which could be responsible for the hypopigmentation of SCE-treated cells. Furthermore, the effect of SCE on tyrosinase expression in the B16F10 melanoma cells was confirmed by ICC/IF (Fig. 4C). This experiment showed that the tyrosinase levels in cells were reduced by SCE after 72 h in the presence of α-MSH.

To elucidate the mechanism underlying the hypopigmentary effect of SCE, changes in melanogenesis-related signals induced by SCE were analyzed by Western blotting in a time-course experiment (Fig. 6). It has been reported that ERK is an important regulator of melanogenesis because ERK activation induces MITF phosphorylation and its subsequent degradation and thus reduces melanin synthesis.^29,37,38) Previous studies have shown that ERK activation is related to cAMP-induced melanogenesis in B16F10 melanoma cells.³⁹⁾ However, other studies found that constitutive mutants of Ras and MEK inhibit tyrosinase transcription.³⁷⁾ Activated ERK is known to phosphorylate MITF at serine 73, and RSK-1 (activated by ERK) phosphorylates MITF at serine 409. Phosphorylated MITF has also been reported as a target for proteolysis through the ubiquitin-dependent proteasome pathway.^29,40)

We used a specific ERK pathway inhibitor, PD98059, and found that SCE-induced hypopigmentation was restored by PD98059 treatment (Fig. 7). The results of our study also showed that SCE activated ERK and reduced MITF protein levels. To our knowledge, this is the first demonstration of the activation of ERK by SCE. To investigate the relationship between ERK activation and MITF downregulation, we pretreated with PD98059 before SCE treatment, re-examined phosphor-ERK levels, and found that ERK activation suppressed melanogenesis in B16F10 melanoma cells.

It has been reported that sustained activation of ERK by sphingosine-1-phosphate (SIP) and C₂-ceramide induces MITF phosphorylation and subsequent degradation, resulting in reduced tyrosinase and melanin levels.^41,42) Consistent with these reports, our results indicate that SCE induced MITF degradation through sustained ERK activation. In our results, the mRNA levels of tyrosinase and MITF showed no significant change at 48 h or 72 h compared to the controls with α-MSH (Fig. 5). These results indicate that SCE inhibits the expression of tyrosinase and MITF, which plays a pivotal role in melanogenesis at the translational, but not transcriptional level. Furthermore, out results indicated that the reduction of the MITF protein level by SCE was not due to decreased level of MITF mRNA, suggesting that, while SCE induces MITF degradation, it does not suppress MITF gene expression, similar to SIP.⁴¹⁾ Thus far, we provided evidence that SCE inhibits melanin synthesis through the degradation of MITF by ERK activation in B16F10 melanoma cells, it is still unclear how ERK is activated by SCE. To resolve this issue, further studies such as MITF degradation, MITF promoter assay, and evaluation of ERK upstream kinase activity are required.

Other several signaling pathways, such as the CREB, Akt, and GSK3β, regulate melanogenesis.^30–33) Several studies have found that these pathways is involved in the melanogenesis of B16F10 melanoma cells. However, we found no difference in the phosphorylated CREB levels, the phosphorylated Akt levels, and the levels of GSK3β after SCE treatment, which suggests that these pathways does not participate in the SCE-induced inhibition of melanogenesis.

In conclusion, SCE suppressed cellular melanin biosynthesis and tyrosinase activity in α-MSH-induced B16F10 melanoma cells by inhibiting the expression of MITF and tyrosinase, thereby increasing ERK activity. These results further suggest that SCE inhibits melanogenesis by activating the MEK/ERK pathway-mediated suppression of MITF and its downstream target genes including tyrosinase. It is especially notable that the depigmenting effects of SCE on melanogenesis were stronger than those of arbutin, which is widely used as an ingredient in whitening cosmetics. Furthermore, the SCE did not incur significant cytotoxicity in the effective dose, unlike the carcinogenic potential of kojic acid as well as its weak whitening effect. Thus, SCE may be useful as a potential skin-whitening agent.

Acknowledgment

This research was financially supported by the Ministry of Knowledge Economy (MKE), Korea Institute for Advancement of Technology (KIAT) through the Inter-ER Cooperation Projects.

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