Fermented Viola mandshurica Inhibits Melanogenesis in B16 Melanoma Cells

Yeon-Joo KWAK,1;* Kyoung-Sook KIM,1;* Kyung-Mi KIM,1 Hai Yang YU,1 Eunsook CHUNG,1

Seok-Jo KIM,1 Jae-Young CHA,2 Young-Choon LEE,1 and Jai-Heon LEE1;y

1College of Natural Resources and Life Science, BK21 Center for Silver-Bio Industrialization, Dong-A University,Busan 604-714, South Korea2Technical Research Institute, Daesun Distilling Co., Ltd., Busan 619-934, South Korea

Received September 2, 2010; Accepted January 26, 2011; Online Publication, May 20, 2011

[doi:10.1271/bbb.100641]

We assessed the effects of chloroform extract offermented Viola mandshurica (CEFV) on melanogenesisB16 melanoma cells. CEFV treatment significantlydecreased melanin content and tyrosinase activity indose-dependent manners. To elucidate the mechanismof the inhibitory effects of CEFV on melanogenesis, weperformed RT-PCR and Western blotting for melano-genesis-related genes such as tyrosinase, tyrosinase-related protein-1 (TRP-1), TRP-2, and microphthalmia-associated transcription factor (MITF). CEFV stronglyinhibited mRNA as well as the protein expression oftyrosinase and MITF, but had no significant effect onTRP-1 or TRP-2 expressions. It markedly decreased thephosphorylation of cAMP responsive element bindingprotein (CREB), and induced the duration of extrac-ellular signal-regulated kinase (ERK) activation, leadingto reduction of MITF expression and subsequently thatof tyrosinase. Therefore, we suggest that CEFV inducesdownregulation of melanogenesis through decreasedCREB phosphorylation and ERK activation.

Key words: Viola mandshurica; melanogenesis; tyrosi-nase; B16 melanoma cells; microphthalmia-associated transcription factor (MITF)

Melanin, synthesized in the melanosomes of melano-cytes, plays a crucial role in protecting the skin from theharmful effects of UV radiation and in the absorption oftoxic drugs and chemicals.1) Melanin synthesis, calledmelanogenesis, is a complicated process that is regulatedby at least three melanogenic enzymes, tyrosinase,tyrosinase-related protein 1 (TRP-1), and tyrosinase-related protein 2 (TRP-2).2–4) Tyrosinase is the rate-limiting enzyme in the process of melanin synthesis. Itcatalyzes three different reactions: the hydroxylationof tyrosine to 3,4-dihydroxyphenylalanine (DOPA); theoxidation of DOPA to DOPAquinone; and the oxidationof 5,6-dihydroxyindole (DHI) to indole-quinone.2,3)

In the absence of thiol substances, DOPAquinoneis converted to DOPAchrome and then to DHI orindol-5,6-quinone 2-carboxylic acid (DHICA). TRP-1(DHICA oxidase) catalyzes the oxidation of DHICA,and TRP-2 (DOPAchrome tautomerase) catalyzes the

conversion of DOPAchrome to DHICA.4,5) Hence, theupregulation of melanogenic enzymes is perhaps re-sponsible for increased melanin production. Micro-phthalmia-associated transcription factor (MITF) is thebasic helix-loop-helix leucine zipper (bHLH-LZ) fam-ily, and is believed to regulate melanocyte pigmentation,development, differentiation, and survival.6,7) In addi-tion, it has been reported that MITF strongly stimulatestyrosinase promoter activities, indicating that it is animportant transcriptional regulator of melanogenesis.8)

Cyclic AMP-elevating agents such as �-melanocytestimulating hormone (�-MSH), isobutymethylxanthine(IBMX) and forskolin also stimulate melanin synthesis.Moreover, it is well known that �-MSH, as a signaltransducer, potently induces MITF expression andincreases melanin synthesis.9–11) The extracellular sig-nal-regulated kinase (ERK) signaling pathway plays acrucial role in cell proliferation and differentiation.12) Ithas been reported that inhibition of the ERK pathwayinduces B16 melanoma cell differentiation and increasestyrosinase activity, suggesting that the pathway regu-lates melanogenesis.13) Furthermore, several reportshave suggested that the ERK pathway is an importantregulator of melanogenesis, because ERK activationinduces MITF phosphorylation and degradation, whichresults in reduced tyrosinase level and decreasedmelanogenesis.14–16) Enhanced intracellular cAMP con-tents can activate protein kinase A (PKA), whichsubsequently phosphorylates cAMP-responsive elementbinding protein (CREB). CREB binds the cAMPresponse element (CRE) motif of the MITF promoterand activates MITF gene transcription.17) Thus thecAMP pathway, through phosphorylation of CREB,mediates the regulation of tyrosinase expression andmelanogenesis via MITF.18)

Melanogenesis inhibitors have been the focus of muchresearch, because clinically abnormal hyperpigmenta-tion conditions, such as melasma, freckles, and senilelentigines, are caused by an excessive accumulation ofmelanin and are improved by treatment with depigment-ing agents.19,20) Although well-known depigmentingagents such as kojic acid and arbutin are used ascosmetic agents for skin whitening, the dosage is limited

* These authors contributed equally to this work.

y To whom correspondence should be addressed. Tel/Fax: +82-51-200-7592; E-mail: jhnlee@dau.ac.kr

Abbreviations: �-MSH, �-melanocyte stimulating hormone; CEFV, chloroform extract from fermented Viola mandshurica; CREB, cAMPresponsive element binding protein; DMSO, dimethyl sulfoxide; DOPA, 3,4-dihydroxyphenylalanine; ERK, extracellular signal-regulated kinase;MMP, matrix metalloproteinase; MITF, microphthalmia-associated transcription factor; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide; TRP-1, tyrosinase-related protein-1; TRP-2, tyrosinase-related protein-2

Biosci. Biotechnol. Biochem., 75 (5), 841–847, 2011

due to cytotoxicity and carcinogenic potential.21,22) Fromthis point of view, safer and more effective whiteningagents are needed. Hence potent tyrosinase inhibitors,including kaemperol,23) quercetine,24) and luteolin,25)

have been isolated from natural plants.Viola mandshurica belongs to the family Violaceae,

and is founded in the southern parts of Korea. Intraditional medicine, it is used to treat various skindisorders, such as skin eruption and eczema, and isutilized as an expectorant and diuretic. Previous phy-tochemical studies of Viola species have shown that itcontains large amounts of cyclotides26,27) and severalflavone glycosides.28,29) An inhibitory activity of thisplant on matrix metalloproteinase (MMP)-1 expressioninduced by ultraviolet (UV) irradiation in culturedhuman skin fibroblasts has been demonstrated,30,31) butthere are no reports of inhibitory effects on melano-genesis regulated by melanogenic enzymes. Therefore,we investigated the potential anti-melanogenic effectsof this plant. We found that chloroform extract fromfermented V. mandshurica (CEFV) had significant in-hibitory effects on melanogenesis in �-MSH-stimulatedB16 melanoma cells. Our results suggest that thereduction in melanin production by CEFV may berelated to downregulation of MITF via the durationof ERK activation and the reduction of CREB phos-phorylation.

Materials and Methods

Cell culture. B16F10 cells, murine melanoma cell line, were

purchased from the American Type Culture Collection (ATCC;

Manassas, VA) and cultured in Dulbecco’s Modified Eagle Medium

(DMEM, Gibco-BRL, Rockville, MD) supplemented with 10%

fetal bovine serum (FBS), penicillin (100U/mL), and streptomycin

(100 mg/mL) at 37 �C under a humidified 95% air, 5% CO2

atmosphere.

Materials. Arbutin, L-DOPA, 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyl-2H-tetrazolium bromide (MTT), mushroom tyrosinase, and

�-melanocyte stimulating hormone (�-MSH) were purchased from

Sigma (St. Louis, MO). Protease and phosphatase inhibitor cocktails

were from Pierce (Rockford, IL). The antibodies used in Western blot

including, anti-tyrosinase (C-19), TRP-1 (A-20), TRP-2 (D-18),

phospho-ERK1/2, and total (non-phosphorylated) ERK1/2 were from

Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-CREB (Ser133)

and CREB (48H2) were from Cell Signaling Technology (Beverly,

MA). Anti-microphthalmia (MITF) was from Abcom (Cambridge,

UK), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) anti-

body from Millipore (Temecula, CA).

Preparation of CEFV from the plant. The whole plant of Viola

mandshurica (Violaceae) was collected in May 2008 in Kyoungnam

Province, South Korea. Fermented V. mandshurica extract was

prepared from whole plants of V. mandshurica, mixed gently with

crude sugar (half the weight of the plant), packed in a ceramic pot, and

fermented for 6 months in a cool, dark location. To identify the main

compound responsible for anti-melanogenic activity, fermented

V. mandshurica extract (10L) was diluted with 3 volumes of distilled

water and successively partitioned with n-hexane (12 g), CHCl3 (1.5 g),

EtOAc (8.7 g), and BuOH (34 g). We examined the effect of each

fraction on tyrosinase activity, and the chloroform extract from

fermented V. mandshurica (CEFV) showing the highest inhibitory

effect on tyrosinase activity was used.

MTT assay. The viability of cultured cells was determined by

reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-

mide (MTT) to formazan as described previously.25) In brief, B16

melanoma cells were plated in 96-well plates at a density of 1� 103

cells/well. After 24 h, the cells were washed with fresh medium

and treated with various concentrations of CEFV (0–200mg/mL).

After 72 h of incubation, cells were washed with PBS. Then 100 mLof MTT reagent (5mg/mL) was added to each well. After incubation

for 4 h, DMSO (100mL) was added to dissolve the formazan

precipitates and the amount of formazan salt was determined by

measuring the OD at 570 nm using an ELISA plate reader (Bio-Rad,

Hercules, CA). Cell viability was quantified as a percentage compared

to the control.

Tyrosinase activity assay. Tyrosinase activity was determined as

DOPA oxidase activity by a method described by Takahashi et al.,32)

with slight modifications. Briefly, B16 melanoma cells were seeded in

60mm dishes (1� 105) and cultured with various concentrations of

CEFV and arbutin, which is a typical tyrosinase inhibitor, as a positive

control, in the presence of �-MSH. After incubation for 3 d, the cells

were washed twice with cold phosphate buffered saline (PBS) and

lysed with 0.1 M sodium phosphate buffer (pH 6.8) containing 1%

Triton X-100 and protease inhibitor cocktail (Pierce, Rockford, IL).

The cells were then disrupted by freeze-thawing, and the lysates were

clarified by centrifugation at 10;000� g for 10min. After quantifica-

tion of protein levels and adjusting concentration with lysis buffer,

50mL of each lysate was placed in a well of a 96-well plate, 125 mL of

0.1M sodium phosphate (pH 6.8) and 25 mL of freshly prepared

substrate solution (10mM L-DOPA in 0.1M sodium phosphate, pH 6.8)

was added. After incubation for 30min at 37 �C, tyrosinase activity

was analyzed spectrophotometrically by following the oxidation of

DOPA to DOPAchrome at 475 nm using an ELISA plate reader.

Observation of tyrosinase activity by DOPA staining in B16

melanoma cells. B16 melanoma cells were seeded in 8 chamber slide

(4� 104/well) and preincubated for 24 h. Cells were pretreated with

CEFV (50, 100 mg/mL) for 1 h, and then stimulated with �-MSH

(200 nM). After incubation for 3 d, the cells were washed twice with

PBS and fixed with 4% formaldehyde in PBS for 30min. After

washing with PBS, cells were stained with 5mM L-DOPA solution.

After the washing procedure was performed once more, cells were

dehydrated, mounted, and then observed under microscope (Olympus

Optical Co., Tokyo, Japan) and photographed using digital video

camera system (Motic Image Plus ver. 2).

Measurement of melanin contents. Melanin contents were measured

as described by Tsuboi et al.,33) with slight modifications. Briefly, cells

were treated with the test substances at the indicated concentrations in

the presence and the absence of �-MSH for 3 d. After treatment, they

were detached by a short incubation with trypsin/EDTA. After

precipitation, the cell pellets were photographed and then solubilized in

1N NaOH at 100 �C for 30min, and centrifuged for 20min at

16;000� g. The optical densities (OD) of the supernatants were

measured at 405 nm with an ELISA reader.

DOPA staining of tyrosinase on polyacrylamide gel. DOPA staining

assay was performed as described by Laskin et al.,34) with slight

modifications. B16 melanoma cells were treated with CEFV (10, 50,

100mg/mL) in the presence and the absence of �-MSH for 3 d. To

identify cellular tyrosinase activity, samples lysed in 0.1 M sodium

phosphate buffer (pH 6.8) containing 1% Triton X-100, 1mM PMSF,

10mg/mL aprotinin, and 10 mg/mL leupeptin were centrifuged for

20min at 12;000� g. The protein concentration of the cell lysates was

determined using an assay kit (Bio-Rad, Hercules, CA) with bovine

serum albumin (BSA) as standard. Equal amounts (20mg each) of

the cell lysates were mixed with Laemmli sample buffer without �-mercaptoethanol, and then resolved on 10% nondenaturing polyacryl-

amide gel without sodium-dodecyl-sulfate (SDS) by electrophoresis.

The gels containing the tyrosinase bands were placed in a flat-

bottomed container with 200mL of 0.1M sodium phosphate buffer

(pH 6.8) and equilibrated at room temperature with gentle shaking.

After 30min, the rinse buffer was drained from the gels and replaced

with fresh buffer. After one repetition of the rinse procedure, the gels

were transferred into 200mL of a staining solution that contained the

rinse buffer supplemented with 5mM L-DOPA, and were incubated in

the dark for 30min at 37 �C. Protein bands that contained tyrosinase

activity were visualized as dark melanin-containing bands. The

842 Y.-J. KWAK et al.

intensities of the bands obtained from DOPA staining assay was

estimated with a Scion Image Instrument (Scion Crop., MD).

Western blotting. The cells were lysed in cold RIPA buffer (Pierce,

Rockford, IL) containing protease and phosphatase inhibitor cocktail.

The whole-cell lysates, which contained 20 mg of protein per lane, wereseparated by SDS–PAGE using a 10% resolving and a 3% acrylamide

stacking gel, and then transferred to a nitrocellulose membrane

(Millipore, Billerica, MA) in a Western blot apparatus (Bio-Rad). The

membrane was blocked with 5% skimmed milk in phosphate-buffered

saline containing 0.05% Tween-20. The tyrosinase, TRP-1, and TRP-2

bands were detected with rabbit polyclonal anti-tyrosinase antibody

(dilution 1:1,000), rabbit polyclonal anti-TRP-1 antibody (dilution

1:1,000), and rabbit polyclonal anti-TRP-2 antibody (dilution 1:500)

respectively. Bound antibodies were detected using an enhanced

chemiluminescence kit (Amersham Biosciences, Little Chalfont, UK).

Equal loading was assessed using anti-GAPDH antibody to normalize

the amounts of total protein.

Semi-quantitative and real-time reverse transcription-polymerase

chain reaction (RT-PCR). To determine the effects of CEFV on

melanogenesis-related gene expression, semi-quantitative and quanti-

tative real-time reverse transcription-polymerase chain reactions

(RT-PCR) were carried out. B16 melanoma cells were treated with

or without CEFV and stimulated with �-MSH. For analysis of the

tyrosinase, TRP-1, and TRP-2 mRNA levels, after incubation for 48 h,

total cellular RNA was prepared using Trizol solution (Invitrogen,

Paisley, UK) following the manufacturer’s instructions. For analysis of

MITF mRNA levels in the signaling pathway, after incubation for

30min, total cellular RNA was prepared. Reverse transcription and

cDNA amplification were carried out with 1mg of isolated total RNA

using a RT-PCR kit (Clontech, Mountain View, CA). The oligonu-

cleotide primers used for semi-quantitative PCR were as follows: for

tyrosinase (1,210 bp) 50-ACATTTTTGATTTGAGTGTC-30 (forward)

and 50-TGTGGTAGTCGTCTTTGTCC-30 (reverse); TRP-1 (805 bp)

50-GCTGCAGGAGCCTTCTTTCTC-30 (forward) and 50-AAGACGC-

TGCACTGCTGGTCT-30 (reverse); TRP-2 (591 bp) 50-GGATGACC-

GTGAGCAATGGCC-30 (forward) and 50-CGGTTGTGACCAAT-

GGGTGCC-30 (reverse); MITF (910 bp) 50-GTATGAACACGCACT-

CTCTCGA-30 (forward) and 50-CTTCTGCGCTCATACTGCTC-30

(reverse); and �-actin (285 bp) 50-TCATGAAGTGTGACGTTGA-

CATCCGT-30 (forward) and 50-CCTAGAAGCATTTGCGGTGCAC-

GATG-30 (reverse). The reaction was cycled 28 times for tyrosinase,

25 times for TRP-1 and TRP-2, and 32 times for MITF for 30 s at

94 �C, 30 s at 56 �C, and 60 s at 72 �C. The reaction for �-actin

amplification was cycled 30 times for 30 s at 94 �C, 30 s at 58 �C, and

45 s at 72 �C. After amplification, 50% of the reaction mixture was

analyzed by electrophoresis on 1% agarose gels and stained with

ethidium bromide. Specific primers for �-actin were used as controls.

Real-time PCR was performed on a GeneAmp5700 sequence detection

system (Applied Biosystems, Foster City, CA) using SYBER Green

PCR Master Mix (Applied Biosystems, Foster City, CA). The primers

used for real-time PCR were as follows: tyrosinase (111 bp) 50-

GTCGTCACCCTGAAAATCCTAACT-30 (forward) and 50-CATCG-

CATAAAACCTGATGGC-30 (reverse); TRP-1 (164 bp) 50-CTTTCT-

CCCTTCCTTACTGG-30 (forward) and 50-TCGTACTCTTCCAAGG-

ATTCA-30 (reverse); TRP-2 (176 bp) 50-TTATATCCTTCGAAA-

CCAGGA-30 (forward) and 50-GGGAATGGATATTCCGTCTTA-30

(reverse); MITF (135 bp) 50-GTATGAACACGCACTCTCGA-30

(forward) and 50-GTAACGTATTTGCCATTTGC-30 (reverse), and

�-actin (87 bp) 50-ACTATTGGCAACGAGCGGTT-30 (forward) and

50-ATGGATGCCACAGGATTCCA-30 (reverse). After pre-denaturing

for 5min at 95 �C, the reaction was cycled 40 times for all of the genes:

30 s at 95 �C, 30 s at 55 �C, and 30 s at 72 �C. �-Actin used as internal

standard.

Statistical analysis. Values were expressed as mean� SD for three

independent experiments. Statistical significance for pairwise compar-

ison was evaluated using Student’s t-test, and multiple comparisons

were analyzed using SPSS package software (version 18.0; Statistical

Package for Social Sciences; SPSS Inc., Chicago, IL), and were

evaluated by one-way analysis of variance (ANOVA) followed by

Tukey’s multiple comparison. Differences were considered significant

at p < 0:05.

Results

Effects of CEFV on cell viabilityPrior to investigation into the inhibitory effect of

CEFV on melanogenesis, we first the cytotoxicity ofCEFV in B16 melanoma cells by MTT assay. Relativecell viability was determined by the amount of MTTconverted into formazan salt. B16 melanoma cells weretreated with CEFV at various concentrations for 3 d. Asshown Fig. 1, CEFV at concentrations of �100 mg/mLhad a modest cytotoxic effect on cells, but there wasan approximately 45% decrease in cell viability at aconcentration of 200 mg/mL. Hence we used CEFV atconcentrations of �100 mg/mL to determine its effect onmelanin production in B16 melanoma cells.

Inhibitory effects of CEFV on cellular tyrosinaseactivity and melanin formationTo examine whether CEFV inhibits melanogenesis in

B16 melanoma cells, tyrosinase activity and melanincontent were determined. Because tyrosinase is the rate-limiting enzyme for melanin biosynthesis, we measuredmushroom tyrosinase activity and cell-free tyrosinaseactivity using L-tyrosine or L-DOPA as substrate.Lysates of �-MSH-stimulated B16 melanoma cells wereused as enzyme sources of cell-free tyrosinase. CEFVhad no an inhibitory effect on either mushroomtyrosinase or cell-free tyrosinase activities, suggestingthat it does not directly affect tyrosinase activity (datanot shown). Hence we determined cellular tyrosinaseactivity in �-MSH-stimulated B16 melanoma cells(Fig. 2A). The cells were incubated with CEFV orarbutin, a typical tyrosinase inhibitor as positive control,at concentrations of 10, 50, 100 mg/mL for 3 d. CEFVinduced a significant reduction in tyrosinase activity,corresponding to 70% of the control treated with only�-MSH at 10 mg/mL, 58% at 50 mg/mL, and 35% at100 mg/mL (Fig. 2A). The melanin contents of the cellswere also markedly decreased by treatment with CEFV,in a dose-dependent manner, corresponding to 60% at10 mg/mL, 33% at 50 mg/mL, and 24% at 100 mg/mLcompared to the control treated only with �-MSH(Fig. 2B). These results indicate that melanin reductionby CEFV was accompanied by a parallel decrease intyrosinase activity. When B16 melanoma cells wereincubated with �-MSH, a cAMP-elevating agent, thecolor of the cell pellet was black, indicating increased

Fig. 1. Effects of CEFV on Cell Viability of B16 Melanoma Cells.Cells were treated with various concentrations of CEFV (0–

200 mg/mL) for 48 h. Cell viability was measured by MTT assay.Values were expressed as means� SD for three independentexperiments, and were represented as % of control cell vi-ability.��p < 0:01 vs. control, not treated with CEFV.

Viola mandshurica Inhibits Melanogenesis 843

cellular melanogenesis. The intensity of the black colorof the cell pellet was markedly decreased by treatmentwith arbutin or CEFV (Fig. 2C). Moreover, the depig-menting effects of CEFV on melanogenesis werestronger than those of arbutin used as control at100 mg/mL. This suggests that CEFV regulates tyrosi-nase activity and subsequently inhibits melanogenesis inB16 melanoma cells. To examine the depigmentingeffect of CEFV on intracellular tyrosinase activity inB16 melanoma cells, cells were incubated with CEFVand �-MSH for 3 d, and then stained with DOPA(Fig. 3). The cells stimulated with �-MSH alone weremarkedly pigmented as compared to untreated controlcells. CEFV treatment (100 mg/mL) resulted in asignificant depigmenting effect on the �-MSH-stimu-lated B16 melanoma cells.

Effects of CEFV on the amount of intracellular activetyrosinase on zymography

To determine the mechanism underlying the depig-menting effect of CEFV, we carried out DOPA stainingzymography of tyrosinase by polyacrylamide gel elec-trophoresis (Fig. 4). Since this assay directly detects theintracellular active tyrosinase amount in each proteinband separated by electrophoresis, the active form oftyrosinase generated by de novo synthesis and subse-quently removed by proteolysis can be quantified. B16melanoma cells were treated with CEFV in the presenceor absence of �-MSH, and the lysates were resolved byelectrophoresis. Bands exhibiting active tyrosinase weredetected by DOPA staining. Tyrosinase intensity in the

control B16 melanoma cells without �-MSH treatmentwas very low, whereas it was dramatically increased bystimulation of the cells with �-MSH. Treatment withCEFV significantly inhibited �-MSH-enhanced tyrosi-nase intensity in a dose-dependent manner, correspond-ing to a 9.8% inhibition at a concentration of 10 mg/mL,66.4% at 50 mg/mL, and 93.5% at 100 mg/mL, with anIC50 value of 37.3 mg/mL. These results were consistentwith those on tyrosinase activity assay (Fig. 2A) and themelanin content assay (Fig. 2B).

Effects of CEFV on protein and mRNA expression ofmelanogenic enzymesTo elucidate whether CEFV influences the protein

expression of melanogenic enzymes such as tyrosinase,TRP-1, and TRP-2, we performed Western blottingusing the cell lysate of B16 melanoma cells treated withCEFV in the presence or absence of �-MSH. As shownin Fig. 5A, �-MSH treatment markedly increased theprotein levels of tyrosinase, and induction was signifi-cantly inhibited by CEFV, in a dose-dependent manner.However, the expression of two other melanogenicproteins, TRP-1 and TRP-2, showed no significantincrease on stimulation with �-MSH alone, and theirlevels were unchanged on CEFV treatment. To examine

A

B

C

Fig. 2. Effect of CEFV on Melanogenesis in B16 Melanoma Cells.Cells were treated with various concentrations of arbutin and

CEFV (10, 50, 100 mg/mL) in the presence of �-MSH (200 nM).Tyrosinase activity (A) and melanin contents (B) from cellularlysates were determined as described in ‘‘Materials and Methods.’’For visual observation, control and treated cells were collected andphotographed (C). Data were expressed as the means� SD for threeindependent experiments. Values not sharing the same letter aresignificantly different (p < 0:05).

Fig. 3. Intracellular Tyrosinase Activity by DOPA Staining.Cells were treated with CEFV (50, 100 mg/mL) in the presence of

�-MSH (200 nM) for 3 d and stained with DOPA, as described inMaterials and Methods. Control: not treated with �-MSH, �-MSH;treated with �-MSH 200 nM alone, �-MSH + CEFV50; �-MSH + CEFV 50 mg/mL, �-MSH + CE100; �-MSH + CEFV100 mg/mL, (�200).

Fig. 4. Effect of CEFV on Intracellular Active Tyrosinase Amountson Zymography.

Cellular lysates were resolved on polyacrylamide gel by electro-phoresis. The gel was soaked with 0.1M sodium phosphate buffer(pH 6.8) containing 5mM L-DOPA. Data were expressed as themeans� SD of three independent experiments. #p < 0:001 vs.control, not treated with �-MSH. ��p < 0:01 vs. the group treatedwith �-MSH alone.

844 Y.-J. KWAK et al.

whether the inhibition of tyrosinase protein expressionby CEFV was due to a decreased level of transcription,we performed both RT-PCR and quantitative real-timePCR using specific primers. As shown in Fig. 5B and C,the mRNA level of tyrosinase was markedly decreasedby treatment with CEFV in a dose-dependent manner,whereas the mRNA levels of TRP-1 and TRP-2showed no significant change in the CEFV-treated B16melanoma cells. These results clearly indicate thatCEFV inhibits the expression of tyrosinase, which playsa pivotal role in melanogenesis, at the transcriptionaland translational levels.

Effects of CEFV on the signaling pathway involved inmelanogenesis

It is well known that MITF expression is inducedthrough phosphorylation of CREB in �-MSH-treatedB16 cells.11,18) Hence we investigated to determinewhether CEFV affects MITF expression and CREBphosphorylation. As shown in Fig. 6, �-MSH-inducedMITF expression was decreased by CEFV in a dose-dependent manner at the transcriptional and translationallevels. On the other hand, phosphorylated CREB washardly detectable in �-MSH-untreated B16 cells, butwas markedly increased by �-MSH treatment. CEFVremarkably decreased �-MSH-induced CREB phospho-rylation in a dose-dependent manner. This indicates thatCEFV downregulated MITF expression through inhib-ition of CREB phosphorylation.

It has been reported that the ERK pathway isintimately involved in melanogenesis, and that activa-tion of the ERK pathway induces MITF degradation,which subsequently decreases melanin synthesis.14,15,35)

Hence we investigated to determine whether CEFVinfluences ERK activation. As shown in Fig. 7A, thelevel of phosphorylated ERK by �-MSH stimulationpeaked at 5min, and diminished almost to zero at60min, whereas ERK phosphorylation by CEFV treat-ment was strongly activated at 5min and this activation

was sustained for at least 60min. Next we investigatedto determine whether ERK activation by CEFV inducesdownregulation of MITF, which plays a pivotal role inmelanogenesis. �-MSH-treated B16 melanoma cellswere pretreated with U0126, a specific inhibitor of theERK pathway, to inhibit ERK phosphorylation, and

A

C

B

Fig. 5. Effects of CEFV on Protein and mRNA Expression of Melanogenic Enzymes.A, Whole-cell lysates were subjected to Western blot analysis with antibodies against tyrosinase, TRP-1 and TRP-2. B, RT-PCR analysis of

tyrosine, TRP-1, and TRP-2 mRNA levels. C, mRNA expression of tyrosine, TRP-1, and TRP-2 was analyzed by quantitative real-time PCR.Results are shown as relative gene expression (normalized to �-actin), where the �-MSH-treated expression levels were set to 1. Experimentswere repeated 3 times to confirm reproducibility of results. Data were expressed as the means� SD for three independent experiments.�p < 0:05, ��p < 0:01 vs. control group treated with �-MSH.

A

B

C

Fig. 6. Effects of CEFV on CREB Phosphorylation and MITFExpression.

After serum starvation for 16 h, cells were pretreated with CEFVfor 30min at the concentrations indicated and stimulated with�-MSH for 30min. A, Whole-cell lysates were then subjected toWestern blot analysis using antibodies against MITF and phospho-specific CREB (p-CREB). Equal protein loadings were confirmedusing anti-GAPDH and anti-CREB (CREB) antibodies. B, RT-PCRanalysis of MITF mRNA levels. C, mRNA expressions of MITF wasanalyzed by quantitative real-time PCR. Results are shown asrelative gene expression (normalized to �-actin), where the �-MSH-treated expression levels were set to 1. Experiments were repeated 3times to ascertain reproducibility of results. Data were expressed asthe means� SD for three independent experiments. �p < 0:05,��p < 0:01 vs. control group treated with �-MSH.

Viola mandshurica Inhibits Melanogenesis 845

were identified both ERK and MITF protein levels byWestern blot analysis. As shown in Fig. 7B, CEFVinduced ERK activation, whereas U0126 markedlyinhibited ERK activation and abrogated MITF degrada-tion. However, CEFV restored ERK activation inhibitedby U0126 treatment, which resulted in MITF reduction.Since a diminished level of MITF protein can beinduced by decreased MITF gene expression, weexamined whether CEFV has an effect on MITFtranscription by ERK activation. As shown in Fig. 7C,MITF mRNA level did not change significantly byCEFV-induced ERK activation, suggesting that MITFprotein reduction by CEFV is due to MITF degradation,not to suppressed MITF gene expression.

Taken together, these results suggest a dual role ofCEFV involving the downregulation of MITF expres-sion through a reduction of phosphorylated CREB levelsand MITF degradation via activation of the ERKpathway.

Discussion

To our knowledge, this is the first study to report thatchloroform extract from fermented Viola mandshurica(CEFV) has potent inhibitory effects on melanogenesisin �-MSH-induced B16 melanoma cells. The inhibitoryeffects were dose-dependent without significant cyto-toxicity. CEFV-induced melanin reduction was accom-panied by a corresponding decrease in tyrosinaseactivity, suggesting a possible mechanism of CEFVaction. It is especially notable that the depigmentingeffects of CEFV on melanogenesis were stronger thanthose of arbutin, used widely as an ingredient inwhitening cosmetics, suggesting that CEFV from aplant can be used as a safe skin-whitening agent.

Melanogenesis is known to be regulated by thetyrosinase gene family, including tyrosinase, TRP-1,and TRP-2.2–5) In melanocytes and melanoma cells,melanin production is controlled mainly by the expres-sion and activation of tyrosinase, which catalyzes therate-limiting step of the melanogenic process. Thus, thedownregulation of tyrosinase has been proposed to beresponsible for decreased melanin production.3) CEFVstrongly inhibits intracellular tyrosinase activity in �-MSH-stimulated B16 melanoma cells, as demonstratedby cellular tyrosinase and DOPA oxidation zymographyassays, suggesting that the decrease in tyrosinaseactivity by CEFV was not due to inhibition of enzymeactivity. In addition, CEFV inhibited tyrosinase expres-sion in a dose-dependent manner, as indicated by thereduced mRNA and protein levels of tyrosinase in theCEFV-treated cells, but CEFV had no significant effectson mRNA and protein expressions of TRP-1 and TRP-2,which act downstream to tyrosinase in the melaninbiosynthetic pathway.3–5) These results are in accord-ance with a recent report36) that demonstrated thatpyrroloquinoline quinone (PQQ) inhibited tyrosinaseexpression in �-MSH-stimulated B16 melanoma cells ina dose-dependent manner, but not TRP-1 and TRP-2expressions. Thus, the present study clearly indicatesthat CEFV inhibits tyrosinase expression at the tran-scription level, which results in downregulation ofmelanin production.On the basis of these results, next we investigated

the signaling pathway responsible for transcriptionaldownregulation of the tyrosinase gene by CEFV in�-MSH-stimulated B16 melanoma cells. CEFV wasfound to inhibit mRNA and protein levels of MITF in adose-dependent manner, MITF is known as a majortranscription factor in the regulation of tyrosinaseexpression, and thus plays a critical role in melaninbiosynthesis.8–11) Our results are consistent with thefindings of a recent report37) that xanthohumol (XH)significantly decreased mRNA and protein levels ofMITF, suggesting that XH inhibits MITF transcription.It has been reported that CREB phosphorylation by

cAMP pathway induces MITF expression and subse-quently upregulates tyrosinase expression, leading toincreased melanin synthesis. �-MSH, a cAMP elevatingagent, is well known to induce CREB phosphorylationand MITF expression.17,18) In addition, a previous studyfound that the effects of �-MSH on melanogenesis aremediated through activation of the cAMP pathway andPKA.38) A recent report has shown that piperlongumi-nine decreased CREB phosphorylation and MITFproduction in a dose-dependent manner in �-MSH-stimulated B16 melanoma cells, which resulted inreduced tyrosinase and melanin levels.39) In accordancewith these findings, our results indicate that CEFV alsoinhibits CREB phosphorylation and MITF expression in�-MSH-stimulated B16 melanoma cells.Previous studies have suggested that the ERK signal-

ing pathway is involved in the regulation of melano-genesis.13–15) Several studies have found that ERKactivation induces the phosphorylation of MITF atserine 73, which leads to ubiquitination of it followedby proteasome-mediated degradation.14–16) It has reportedthat sustained activation of ERK by sphingosine-1-phosphate (S1P)35) and C2-ceramide40) induced MITF

A

B

C

Fig. 7. Effects of CEFV on MITF Degradation via the ERKSignaling Pathway.A, After serum starvation for 16 h, cells were pretreated with

CEFV (100mg/mL) for the times indicated and stimulated with�-MSH for 30min. Whole-cell lysates were then subjected toWestern blot analysis using an antibody against phospho-specificERK1/2 (p-ERK1/2). Equal protein loadings were confirmed usingthe anti-ERK1/2 antibody. After serum starvation for 16 h, cellswere co-stimulated with �-MSH and CEFV (100 mg/mL) for 30minin the absence or presence of 10 mM of U0126, which was pre-incubated for 1 h to inhibit completely ERK pathway. B, Whole-celllysates were then subjected to Western blot analysis using antibodiesagainst MITF and p-ERK1/2. C, MITF mRNA level was analyzedby RT-PCR using total RNA isolated from the cells.

846 Y.-J. KWAK et al.

phosphorylation and subsequent degradation, resultingin reduced tyrosinase and melanin levels. In consistentlythese reports, our results indicate that CEFV inducedMITF degradation through sustained ERK activation.Furthermore, our results indicate that the reduction ofthe MITF protein level by CEFV is not due to decreasedlevels of MITF mRNA, suggesting that CEFV inducesMITF degradation, but does not suppress MITF geneexpression, like S1P.35)

In conclusion, we found here for the first time thatCEFV induces downregulation of melanogenesisthrough decreased CREB phosphorylation and ERKactivation, leading to a reduction in MITF expressionand consequently decreased tyrosinase expression andmelanin production in �-MSH-stimulated B16 melanomacells.

Acknowledgments

This study was financially supported by a Grant(no. 610003-03-1-SB210) from the Technology Devel-opment Program for Agriculture and Forestry Food andFisheries of the Ministry for Food, Agriculture, Forestry,and Fisheries of the Republic of Korea.

References

1) Hearing VJ, The Pigmentary System: ‘‘Physiology and Patho-

physiology,’’ eds. Nordlund JJ, Boissy RE, Hearing VJ, King

RA, and Ortonne JP, Oxford University Press, Inc., New York,

pp. 423–438 (1998).

2) Korner A and Pawelek J, Science, 217, 1163–1165 (1982).

3) Hearing VJ and Tsukamoto K, FASEB J., 5, 2902–2909 (1991).

4) Kameyama K, Takemura T, Hamada Y, Sakai C, Kondoh S,

Nishiyama S, Urabe K, and Hearing VJ, J. Invest. Dermatol.,

100, 126–131 (1993).

5) Tsukamoto K, Jackson IJ, Urabe K, Montague PM, and Hearing

VJ, EMBO J., 11, 519–526 (1992).

6) Hodgkinson CA, Moore KJ, Nakayama A, Steingrimsson E,

Copeland NG, Jenkins NA, and Aruheiter H, Cell, 74, 395–404

(1993).

7) Levy C, Khaled M, and Fisher DE, Trends Mol. Med., 12, 406–

414 (2006).

8) Yasumoto K, Yokoyama K, Shibata K, Tomita Y, and

Shibahara S, Mol. Cell. Biol., 14, 8058–8070 (1994).

9) Hunt G, Todd C, Cresswell JE, and Thody AJ, J. Cell Sci., 107,

205–211 (1994).

10) Englaro W, Rezzonico R, Durand-Cle’ment M, Lallemand D,

Ortonne JP, and Ballotti R, J. Biol. Chem., 270, 24315–24320

(1995).

11) Busca R and Ballotti R, Pigment Cell Res., 13, 60–69 (2000).

12) Marshall CJ, Cell, 80, 179–185 (1995).

13) Englaro W, Bertolotto C, Busca R, Brunet A, Pages G, Ortonne

JP, and Ballotti R, J. Biol. Chem., 273, 9966–9970 (1998).

14) Hemesath TJ, Price ER, Takemoto C, Badalian T, and Fisher

DE, Nature, 391, 298–301 (1998).

15) Wu M, Hemesath TJ, Takemoto CM, Horstmann MA, Wells

AG, Price ER, Fisher DZ, and Fisher DE, Genes Dev., 14, 301–

312 (2000).

16) Xu W, Gong L, Haddad MM, Bischof O, Campisi J, Yeh ET,

and Medrano EE, Exp. Cell Res., 255, 135–143 (2000).

17) Steingrimsson E, Copeland NG, and Jenkins NA, Annu. Rev.

Genet., 38, 365–411 (2004).

18) Bertolotto C, Abbe P, Hemesath TJ, Bille K, Fisher DE,

Ortonne JP, and Ballotti R, J. Cell Biol., 142, 827–835 (1998).

19) Cayce KA, McMichael AJ, and Feldman SR, Dermatol. Nurs.,

16, 401–406 (2004).

20) Briganti S, Camera E, and Picardo M, Pigment Cell Res., 16,

101–110 (2003).

21) Kahn V, Pigment Cell Res., 8, 234–240 (1995).

22) Maeda K and Fukuda M, J. Pharmacol. Exp. Ther., 276, 765–

769 (1996).

23) Kubo I and Kinst-Hori I, J. Agric. Food Chem., 47, 4121–4125

(1999).

24) Chen QX and Kubo I, J. Agric. Food Chem., 50, 4108–4112

(2002).

25) Choi MY, Song HS, Hur HS, and Sim SS, Arch. Pharm. Res.,

31, 1166–1171 (2008).

26) Svangard E, Goransson U, Smith D, Verma C, Backlund A,

Bohlin L, and Claeson P, Phytochemistry, 64, 135–142 (2003).

27) Goransson U, Svangard E, Claeson P, and Bohlin L, Curr.

Protein Pept. Sci., 5, 317–329 (2004).

28) Carnat AP, Carnet A, Fraisse D, and Lamaison JL, J. Nat. Prod.,

61, 272–274 (1998).

29) Xie C, Veitch NC, Houghton PJ, and Simmonds MS, Chem.

Pharm. Bull., 51, 1204–1207 (2003).

30) Moon HI, Lee J, Kwak JH, Zee OP, and Chung JH, Biol. Pharm.

Bull., 28, 925–928 (2005).

31) Moon HI, Chung JH, Lee JK, and Zee OP, Arch. Pharm. Res.,

27, 730–733 (2004).

32) Takahashi H and Parsons PG, J. Invest. Dermatol., 98, 481–487

(1992).

33) Tsuboi T, Kondoh H, Hiratsuka J, and Mishima Y, Pigment Cell

Res., 11, 275–282 (1998).

34) Laskin JD and Piccinini LA, J. Biol. Chem., 261, 16626–16635

(1986).

35) Kim DS, Hwang ES, Lee JE, Kim SY, Kwon SB, and Park KC,

J. Cell Sci., 116, 1699–1706 (2003).

36) Sato K and Toriyama M, J. Dermatol. Sci., 53, 140–145 (2009).

37) Koo JH, Kim HT, Yoon HY, Kwon KB, Choi IW, Jung SH,

Kim HU, Park BH, and Park JW, Exp. Mol. Med., 40, 313–319

(2008).

38) Bertolotto C, Busca R, Abbe P, Bille K, Aberdam E, Ortonne

JP, and Ballotti R, Mol. Cell. Biol., 18, 694–702 (1998).

39) Kim KS, Kim JA, Eom SY, Lee SH, Min KR, and Kim Y,

Pigment Cell Res., 19, 90–98 (2006).

40) Kim DS, Kim SY, Chung JH, Kim KH, Eun HC, and Park KC,

Cell. Signal., 14, 779–785 (2002).

Viola mandshurica Inhibits Melanogenesis 847