Hypopigmentary action of dihydropyranocoumarin D2, a decursin derivative, as a MITF-degrading agent

Article abstract of DOI:10.1021/np900455j

In this study, the decursin derivative dihydropyranocoumarin D2 (1) was selected for its effects on melanogenesis using a spontaneously immortalized mouse melanocyte cell line (Mel-Ab). The results showed that 1 effectively inhibited melanin synthesis in

Full text of DOI:10.1021/np900455j

J. Nat. Prod. 2010, 73, 797–800
797
Hypopigmentary Action of Dihydropyranocoumarin D2, a Decursin Derivative, as a
MITF-Degrading Agent
Dong-Seok Kim,† So-Hee Park,‡ Hyun-Kyung Lee,‡ Soo-Jin Choo,§ Jee Hyun Lee,⊥ Gyu Yong Song,⊥ Ick-Dong Yoo,§
Sun-Bang Kwon,‡ Jung-Im Na,‡ and Kyoung-Chan Park*,‡
Department of Biochemistry, Chung-Ang UniVersity College of Medicine, 221 Heukseok-Dong Dongjak-Gu, Seoul 156-756, Korea, Department of
Dermatology, Seoul National UniVersity Bundang Hospital, 300 Gumi-Dong, Bundang-Gu, Seongnam-Si, Gyeonggi-do, 463-707, Korea, National
Cosmeceutical Research Center, Korea Research Institute of Bioscience and Biotechnology, 111 Gwahangno, Yuseong-Gu, Daejeon 305-806, Korea,
and College of Pharmacy, Chungnam National UniVersity, 220 Gung-Dong, Yuseong-Gu, Daejeon 305-764, Korea
ReceiVed July 27, 2009
In this study, the decursin derivative dihydropyranocoumarin D2 (1) was selected for its effects on melanogenesis using
a spontaneously immortalized mouse melanocyte cell line (Mel-Ab). The results showed that 1 effectively inhibited
melanin synthesis in a concentration-dependent manner, but that it did not inhibit tyrosinase in a cell-free system. In
addition, the changes in ERK, Akt, and microphthalmia-associated transcription factor (MITF) in response to treatment
with 1 were assessed. The results revealed that ERK was dramatically up-regulated and MITF was down-regulated in
response to treatment with 1, but that Akt was unchanged. Therefore, the effects of 1 on melanogenesis were examined
in the absence or presence of PD98059 (a specific inhibitor of the ERK pathway). PD98059 restored hypopigmentation
and the down-regulation of MITF induced by 1. Finally, MITF down-regulation by 1 was clearly restored by both
chloroquine, a lysosomal proteolysis inhibitor, and MG132, a proteasome inhibitor.
Angelica gigas Nakai (Umbelliferae) is a monocarpous biennial
or short-lived perennial plant found in forests, in grasslands, and
along stream banks in mainland China, Japan, and Korea. The roots
of A. gigas are used in traditional Chinese medicine. In addition, it
has been reported that decursin from A. gigas has antitumor and
antibacterial activities.1,2 Furthermore, decursin exhibits protective
effects against glutamate-induced neurotoxicity, which suggests that
it has antioxidative stress activity.3
melanin synthesis at the transcription level.8-10 Furthermore, it has
been reported that activation of the extracellular signal-regulated
kinase (ERK) pathway leads to MITF degradation and subsequent
inhibition of melanin synthesis.11-13 In the present study, the effects
of 1 on melanin synthesis and tyrosinase activity, including the
signaling pathways of MITF degradation, were examined in a
spontaneously immortalized mouse melanocyte cell line (Mel-Ab).
The production and distribution of melanin pigment is a major
determinant of skin and hair color. Tyrosinase is a major melano-
somal enzyme involved in melanogenesis that catalyzes the rate-
limiting reaction of the melanogenic process.4,5 Therefore, melanin
production depends primarily on the expression and activation of
tyrosinase.6
It was recently reported that an ethanol extract of A. gigas roots
effectively inhibited isobutylmethylxanthine-induced melanogenesis
in B16 melanoma cells.7 However, to the best of our knowledge,
there have been no studies conducted to evaluate the effects of
decursin on melanogenesis, even though it is one of the primary
constituents of A. gigas. Therefore, decursin derivatives were
screened for their inhibitory effects on tyrosinase, and dihydropy-
ranocoumarin D2 (1) was selected as a promising lead compound.
In the present study, we have investigated the hypopigmentary effects
and the possible mechanisms by which 1 inhibits melanogenesis.
Results and Discussion
Cytotoxicity of Dihydropyranocoumarin D2 (1) in Mel-
Ab Cells. The effects of several decursin derivatives on melano-
genesis were tested to identify new skin-whitening agents, and 1
was found to induce a strong hypopigmentary effect. To determine
the toxicity of 1, Mel-Ab cells were treated with increasing doses
of this compound (1, 10, 50, 100 µM), after which the cell viability
was measured using a crystal violet assay. The results revealed that
1 has no cytotoxic effect on Mel-Ab cells at the concentrations
tested (data not shown).
Dihydropyranocoumarin D2 (1) Decreases Melanogenesis
in Mel-Ab Cells. To investigate the hypopigmentary effect of 1,
Mel-Ab cells were treated with 1 (1-100 µM) for four days, after
which the cells were photographed under a phase-contrast micro-
scope. The results revealed that treatment with 1 strongly reduced
melanin synthesis (Figure 1A). In addition, the melanin content
was measured. Consistent with the microscopic observations, 1
significantly inhibited melanogenesis in a dose-dependent manner
(Figure 1B).
Tyrosinase plays a major role in regulating the rate-limiting step
involved in melanin synthesis;14 therefore, mushroom and plant
tyrosinases have been used to identify potent tyrosinase inhibitors
in many studies.15,16 However, some substances inhibit melano-
genesis by regulating the transcription of tyrosinase without
inhibiting tyrosinase activity directly.12,13,17 Therefore, the tyrosi-
nase activity of cells treated with 1 was measured. The results
showed that the tyrosinase activity decreased as a function of the
concentration of 1 (Figure 2A). These findings demonstrated that
1 regulated tyrosinase and subsequently suppressed melanin
synthesis in Mel-Ab cells. To determine if 1 inhibits tyrosinase
activity directly, its effects on mushroom tyrosinase were measured
in a cell-free system. The results revealed that 1 did not affect
Interestingly, 1 did not inhibit tyrosinase activity in an in vitro
assay. Therefore, the major signaling pathways related to melano-
genesis were investigated. Microphthalmia-associated transcription
factor (MITF) is known to regulate tyrosinase expression and
* To whom correspondence should be addressed. Tel: 82-31-787-7311.
Fax: 82-31-787-4058. E-mail: gcpark@snu.ac.kr.
† Chung-Ang University College of Medicine.
‡ Seoul National University Bundang Hospital.
§ National Cosmeceutical Research Center.
⊥ Chungnam National University.
10.1021/np900455j  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 04/14/2010

798 Journal of Natural Products, 2010, Vol. 73, No. 5
Kim et al.
Figure 3. Effects of dihydropyranocoumarin D2 (1) on ERK, Akt,
and MITF. (A) Following 24 h of serum starvation, Mel-Ab cells
were treated with 50 µM 1 for the indicated times. Whole cell
lysates were then subjected to western blot analysis using antibodies
against phospho-specific ERK (p-ERK), phospho-specific Akt (p-
Akt), MITF, and tyrosinase. (B) Cells were cultured with 50 µM 1
for 24-72 h, and the whole cell lysates were then subjected to
western blot analysis with antibodies against MITF and tyrosinase.
Equal protein loadings were confirmed using anti-actin antibody.
that ERK and Akt activation play important roles in the regulation
of melanin synthesis.18-20 Additionally, ERK activation has been
shown to reduce tyrosinase gene transcription.11 Moreover, we have
confirmed that ERK pathway activation is involved in decreased
melanin production in human and mouse melanocytes.12,13,17,21
Thus, 1 was evaluated to determine if it influenced the ERK and
Akt signaling pathway. As shown in Figure 3A, 1 activated ERK
in a time-dependent manner, while Akt was not affected. Interest-
ingly, ERK activation was not transient, but was sustained for at
least 360 min. In accordance with the ERK activation, the MITF
level was down-regulated by 1 at 60-360 min. These findings
suggested that the reduced levels of melanogenic protein were
responsible for decreased melanogenesis. MITF is a major tran-
scription factor responsible for regulation of the expression of
melanogenic enzymes such as tyrosinase, TRP-1, and TRP-2.22-24
Since 1 did not affect the tyrosinase activity directly in a cell-free
system, the MITF and tyrosinase protein levels were evaluated
following treatment with 1. The results showed that 1 (50 µM)
reduced both the MITF and tyrosinase protein levels (Figure 3B).
These findings suggest that the down-regulation of both MITF and
tyrosinase is related to 1-induced hypopigmentary effects.
Figure 1. Effects of dihydropyranocoumarin D2 (1) on melano-
genesis. Mel-Ab cells were cultured with 1-100 µM 1 for four
days. (A) Phase-contrast photomicrographs were then taken using
a digital video camera. The results shown are an example of three
replicate experiments. (B) The melanin content was measured as
described in the Experimental Section. The results shown are the
average of three replicate experiments ( SD (**p < 0.01 when
compared to an untreated control).
tyrosinase activity directly (Figure 2B), although it strongly inhibited
melanogenesis (Figure 1B). Taken together, these findings dem-
onstrate that 1 inhibits melanogenesis, possibly through the down-
regulation of tyrosinase.
Dihydropyranocoumarin D2 (1) Activates ERK and
Down-regulates MITF and Tyrosinase. It has been reported
Effect of the ERK Pathway Inhibition on Melanin
Synthesis. Previous studies have shown that ERK is an important
kinase involved in melanin synthesis because ERK activation
induces the phosphorylation and subsequent degradation of
Figure 2. Effects of dihydropyranocoumarin D2 (1) on tyrosinase activity. (A) Tyrosinase activity in a cell-free system was measured as
described in the text. Mushroom tyrosinase was used for a tyrosinase activity assay in a cell-free system. (B) Mel-Ab cells were cultured
with 1-100 µM 1 for four days, and the cellular tyrosinase activity was then measured as described in the text. The results shown are the
average of three replicate experiments ( SD (**p < 0.01 when compared to an untreated control).

Dihydropyranocoumarin D2
Journal of Natural Products, 2010, Vol. 73, No. 5 799
Figure 4. Effects of dihydropyranocoumarin D2 (1) on melanogenesis in the presence of PD98059. Mel-Ab cells were pretreated with 20
µM PD98059 for 1 h, after which they were cultured with 50 µM 1 for four days. (A) Phase-contrast photographs were then taken using
a digital video camera. (B) The melanin content was measured as described in the Experimental Section. Each determination was made in
triplicate, and the data shown represent the mean ( SD.
MITF.18,19,25 Our group has also reported that sphingosine-1-
phosphate immediately activates ERK and causes MITF degradation
within 3 h of sphingosine-1-phosphate treatment.13 In the present
study, 1 also decreased the levels of MITF. These results suggest
that MITF and tyrosinase down-regulation is mediated by 1-induced
ERK activation. To investigate the relationship between 1-induced
hypopigmentation and the ERK pathway, Mel-Ab cells were
cultured for four days in the absence or presence of PD98059 (a
specific inhibitor of the ERK pathway), after which they were
photographed under a phase-contrast microscope (Figure 4A). The
results showed that PD98059 abrogated the hypopigmentary effect
induced by 1. In addition, the melanin synthesis was also measured,
and the results were consistent with the microscopic observations
(Figure 4B).
Dihydropyranocoumarin D2 (1) Decreased the MITF
Figure 5. Effect of the inhibition of protein degradation or the ERK
Protein Levels via Both Lysosomal and Proteosomal
pathway on MITF expression. Following serum starvation, Mel-
Degradation. Previously, we suggested that MITF degradation and
Ab cells were pretreated with 25 µg/mL of cyclohexamide for 1 h
subsequent tyrosinase down-regulation were related to ERK
as indicated. The cells were also pretreated with 25 µM MG132,
activation.13,26 However, the mechanism by which ERK regulates
100 µM chloroquine, or 20 µM PD98059 for 1 h, after which they
were treated with 50 µM 1 for 3 h. Whole cell lysates were then
MITF degradation and expression has not been completely eluci-
dated. It has been reported that MITF can be down-regulated via
ubiquitin-dependent proteasome degradation and that MITF deg-
radation results in decreased melanin synthesis.19,25 Thus, we
verified the down-regulation of MITF by 1 to determine if this
resulted from MITF degradation. To accomplish this, a proteasome
inhibitor, MG132, and/or the lysosomal proteolysis inhibitor
chloroquine was used. Specifically, cycloheximide was added to
Mel-Ab cells to inhibit protein synthesis, after which the cells were
pretreated with MG132 and/or chloroquine for 1 h followed by
3 h of treatment with 1. The MITF levels were then analyzed by
western blot analysis. As shown in Figure 5, the decrease in MITF
levels in response to treatment with 1 was restored by pretreatment
with MG132 or chloroquine. In addition, co-treatment of MG132
and chloroquine almost completely blocked the MITF down-
regulation. On the basis of these results, it may be suggested that
1 leads to MITF degradation via both a ubiquitin-proteasome
pathway and a lysosomal proteolysis pathway. In addition, PD98059
blocked 1-induced ERK activation and MITF degradation. These
results suggest that prolonged ERK activation in response to 1
contributes to the down-regulation of melanin synthesis via MITF
degradation.
subjected to western blot analysis using antibodies against phospho-
specific ERK or MITF. Equal protein loadings were confirmed using
anti-actin antibody.
degradation of MITF and down-regulation of tyrosinase at the
protein level, which contributes to the inhibition of melanin
synthesis. Thus, the results obtained suggest that compound 1 can
be considered as a potential skin-whitening agent by acting as an
MITF suppressor.
Experimental Section
General Experimental Procedures. Compound 1 and (S)-(+)-
decursinol27 were synthesized at Chungnam National University.
Cholera toxin (CT), 12-O-tetradecanoylphorbol 13-acetate (TPA),
synthetic melanin, L-DOPA, chloroquine, and mushroom tyrosinase
were obtained from Sigma (St Louis, MO), MG132 and cycloheximide
were obtained from Calbiochem (San Diego, CA), and PD098059 was
obtained from Cell Signaling Technology (Beverly, MA). Antibodies
recognizing phospho-specific ERK1/2 (Thr202/Tyr204, number 9101S)
and phospho-specific Akt (Ser473, #9271S) were purchased from Cell
Signaling Technology, microphthalmia Ab-3 (C5+D5, MS-773-P0) was
obtained from NeoMarkers (Fremont, CA), and tyrosinase (C-19) and
actin (I-19) were purchased from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA).
In summary, the results of the present study demonstrate that 1
activates the ERK pathway in a prolonged manner and leads to

800 Journal of Natural Products, 2010, Vol. 73, No. 5
Kim et al.
Synthesis of Dihydropyranocoumarin D2 (1). Two drops of N,N-
dimethyl formamide and thionyl chloride (4.44 mL, 60.9 mM) were
added to a solution of trans-cinnamic acid (1.81 g, 12.2 mM) in 20
mL of anhydrous benzene. After refluxing for 5 h at 70-80 °C, the
reaction solution was cooled to room temperature and concentrated
under reduced pressure to obtain cinnamoyl chloride. (S)-(+)-Decursinol
(2 g, 8.12 mM) was added to a solution of cinnamoyl chloride and
pyridine (1.97 mL, 24.4 mM) in anhydrous dichloromethane at room
temperature. After stirring for 2 h, the reaction mixture was concentrated
under reduced pressure. The resulting residue was then purified by silica
gel column chromatography (5-30% EtOAC in n-hexane) to obtain
(S)-(+)-3-phenylacrylic acid 2,2-dimethyl-8-oxo-3,4-dihydro-2H,8H-
pyrano[3,2-g]chromen-3-yl ester (1) (1.5 g, 49.3%) as a white solid:
[R]2D5 +42.0 (c 3, CHCl3); mp 136-137 °C; ESIMS m/z 377 (M +
H)+; Rf ) 0.40 (50% EtOAc/hexane).28 The purity (>99.5%) of 1 was
determined by chiral stationary phase HPLC analysis [HPLC (Shimadzu
LC-6AD, Kyoto, Japan), column (Chiralcel OD-H 0.46 cm # × 25
cm, Daicel Chemical Industries, Ltd., Co., Osaka, Japan), solvent (2-
propanol/n-hexane, 1:6), flow rate 1 mL].
Cell Culture of Mel-Ab Cells. The Mel-Ab cell line is a mouse-
derived spontaneously immortalized melanocyte cell line that produces
large amounts of melanin.29 Mel-Ab cells were cultured in DMEM
supplemented with 10% fetal bovine serum, 100 nM TPA, 1 nM CT,
50 µg mL-1 streptomycin, and 50 U mL-1 penicillin at 37 °C under
5% CO2.
Cell Viability. Cell viability was determined using a crystal violet
assay.29 Briefly, the cells were incubated with 1 for 24 h, after which
the culture medium was removed and the cells were stained with 0.1%
crystal violet in 10% ethanol for 5 min at room temperature and then
rinsed four times with distilled water. The crystal violet retained by
the adherent cells was then extracted with 95% ethanol. Finally, the
absorbance was determined at 590 nm using an ELISA reader (Tecan,
Salzburg, Austria).
Measurement of Melanin Content and Microscopy. The melanin
content was measured using a slight modification of the method
described by Tsuboi et al.30 Briefly, the cells were treated with various
concentrations of 1 for four days. The cell pellets were then dissolved
in 1 mL of 1 N NaOH at 100 °C for 30 min and centrifuged for 20
min at 16000g, after which the optical density of the supernatants was
measured at 400 nm using an ELISA reader. Before measuring the
melanin content, the cells were observed under a phase-contrast
microscope (Olympus Optical Co., Tokyo, Japan) and photographed
using a CoolSNAPcf digital video camera system (Roper Scientific, Inc.,
Tucson, AZ) supported by the RS Image software (Roper Scientific).
Tyrosinase Activity. The tyrosinase activity was assessed using a
slight modification of the method described by Busca et al.31 Briefly,
Mel-Ab cells were cultured in six-well plates and then incubated with
1 at various concentrations for four days. The cells were then washed
with ice-cold phosphate-buffered saline and lysed with 0.1 M phosphate
buffer (pH 6.8) containing 1% Triton X-100. The cells were then
disrupted by freezing and thawing, after which the lysates were clarified
by centrifugation at 10000g for 5 min. After quantifying the protein
levels and adjusting the concentrations with lysis buffer, 90 µL of each
lysate containing the same amount of protein was placed in each well
of a 96-well plate, and 10 µL of 10 mM L-DOPA was then added to
each well. The control wells contained 90 µL of 0.1 M phosphate buffer
(pH 6.8) and 10 µL of 10 mM L-DOPA. The plates were incubated at
37 °C, and the absorbance at 475 nm was measured every 10 min for
at least 1 h using an ELISA plate reader. A cell-free assay system was
also used to examine the direct effects of 1 on tyrosinase activity.
Phosphate buffer (70 µL) containing various concentrations of 1 was
combined with 20 µL of 10 µg mL-1 mushroom tyrosinase, and 10 µL
of 10 mM L-DOPA was then added to each well. After incubation at
37 °C, the absorbance was measured at 475 nm.
5% dried milk in Tris-buffered saline containing 0.4% Tween 20. Next,
the blots were incubated with the appropriate primary antibodies at a
dilution of 1:1000 and then further incubated with horseradish peroxi-
dase-conjugated secondary antibody. The bound antibodies were
detected using an enhanced Chemiluminescence Plus kit (Amersham
International, Little Chalfont, UK).
Statistical Analysis. Differences among treatments were assessed
by analysis of variance (ANOVA) followed by Dunnett’s test. A p value
of <0.01 was taken to indicate statistical significance.
Acknowledgment. This study was supported by a grant (A050432)
from the Korea Health 21 R&D Project, Ministry of Health and Welfare,
Republic of Korea, and by the RIC program of MKE (Ministry of
Knowledge Economy) in Daejeon University.
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Western Blot Analysis. Cells were lysed in cell lysis buffer [62.5
mM Tris-HCl (pH 6.8), 2% SDS, 5% ꢀ-mercaptoethanol, 2 mM
phenylmethylsulfonyl fluoride, protease inhibitors (Complete, Roche,
Mannheim, Germany), 1 mM Na3VO4, 50 mM NaF, and 10 mM
EDTA], after which 10 µg of protein per lane was separated by SDS-
polyacrylamide gel electrophoresis. The separated proteins were then
blotted onto PVDF membranes, which were subsequently saturated with
NP900455J