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B. Kim et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
2. Materials and methods
OH
OH
O
2.1. Preparation of N-Nicotinoyl dopamine synthesis
Dicyclohexylcarbodiimide (Alfa Aesar, Karlsruhe, Germany) was
added to Pyridine (Sigma-Aldrich Co., St. Louis, MO, USA) solution
containing dopamine hydrochloride (Sigma-Aldrich) and Nicotinic
acid (Sigma-Aldrich). The reaction mixture was stirred at room
temperature for 12 h and filtered. The solvent was removed by
distillation in vacuum condition. The residue was rinsed with
water and purified by medium pressure liquid chromatography
(MPLC) (Biotage Isolera, Uppsala, Sweden) (Fig. 1). MPLC separa-
tions were carried out on a glass column packed with snap ultra-
silica gel at a flow rate of 25 ml/min. Chloroform-Methanol (30:1)
was used as mobile phase. The identification of NND was per-
formed by thin layer chromatography (TLC). NND were separated
by silica gel plate (Kieselgel 60, Merck) with chloroform: methanol
(10:1). To visualize resolved NND, the plates were detected under a
UV lamp (254 nm). NND components were achieved by scraping
the separated spots from the plate. After extraction with methanol,
the samples were concentrated and dried using a centrifugal
evaporator (ScanSpeed 40, Gyrozen, Seoul, Korea).
N
H
N
Fig. 1. The structure of Nicotinoyl dopamine.
Epilife medium (Invitrogen). After 3 days, cells were fixed in ice-
cold methanol for 10 min at ꢁ20 °C. The anti-cytokeratin antibody
(1:100) (Abcam, Cambridge, MA, USA) was applied for 2 h at room
temperature followed by a tetramethyl rhodamine isothiocyanate
(TRITC)-conjugated secondary antibody (1:100) (Santa Cruz Bio-
technology, Santa Cruz, CA, USA). The cell nuclei were counter
stained using DAPI-containing VectorShield mounting medium
(Vector Laboratories, Burlingame, CA, USA). The melanosomes
transferred from melanocytes to keratinocytes were detected by
CFDA positive signal in the keratinocytes using LSM 510 META
confocal microscope (Zeiss, Germany). The relative numbers of
melanosome transferred were estimated by counting CFDA posi-
tive spots within the recipient keratinocytes in five random mi-
croscopic fields per well at x 200 magnification in three in-
dependent experiments.
2.2. Cell culture
Normal human keratinocytes were purchased from Cascade
Biologics (Portland, OR, USA) and maintained in EPI-500 medium
containing human keratinocyte growth factor (Gibco-BRL/Life
Technologies, Grand Island, NY, USA). B16F10 murine melanoma
cells (ATCC, Manassas, VA, USA) were maintained in DMEM (Gibco,
Grand Island, NY, USA), supplemented with 10% (v/v) fetal bovine
serum (FBS) (Gibco-BRL/Life Technologies, Grand Island, NY, USA)
2.5. Flow cytometry
Human melanocytes were stained with the succinimidyl ester
of 2 M CFDA (Molecular Probes) in HBSS for 30 min. These mel-
μ
and 1% penicillin–streptomycin (10,000 U and 10,000 g/ml)
μ
anocytes were then co-cultured for 3 days with human keratino-
cytes at a ratio of 1:2, respectively, and maintained in Epilife
medium. On the third days, adherent cells were harvested and
washed twice with phosphate buffered saline (PBS). Cells were
detached with trypsin, washed in PBS containing 0.4% FBS and
0.2% sodium azide, and fixed with BD Cytofix/Cytoperm solution
(BD Biosciences, Rockville, MD, USA) for 20 min at 4 °C, and then
incubated with anti-pancytokeratin for 30 min (Invitrogen), fol-
lowed by R-phycoerythrin (R-PE) conjugated secondary antibody
at 4 °C for another 30 min. Cells were analyzed by flow cytometry
using FACS Calibur (BD Biosciences). Data were analyzed using
WinMDI 2.8 software (Scripps Institute, La Jolla, CA, USA). The
melanosome transfer efficacy was determined as the number of
cytokeratin-positive and CFDA-positive cells divided by the total
number of cytokeratin-positive cells.
(Gibco-BRL/Life Technologies, Grand Island, NY, USA) in 5% CO2 at
37 °C. Normal human melanocytes were maintained Medium 254
with human melanocyte growth supplement (Invitrogen, Carlsbad,
CA, USA) in 5% CO2 at 37 °C.
2.3. MTT assay
The MTT assay was used to evaluate the effects of WCE on cell
viability. RAW 264.7 cells were plated in 96-well plates at a den-
sity of 1 ꢀ 104 cells/well for 24 h. The cells were treated with
varying concentrations of NND for 24 h at 37 °C, followed by 50 L
μ
of 2 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT; Sigma-Aldrich, St. Louis, MO, USA) solution to each
well and further incubation for 4 h at 37 °C. The media were dis-
carded and 100 l of dimethyl sulfoxide (DMSO) was added to
μ
each well to solubilize the formazan. Optical density was mea-
sured at 540 nm on a SpectraMax 190 microplate reader (Mole-
cular Devices, Sunnyvale, CA, USA).
2.6. Immunocytochemistry
Cells were fixed with 4% paraformaldehyde in PBS for 15 min at
room temperature and washed with PBS. For permeabilization,
cells were treated with 0.1% Triton X-100 for 15 min at room
temperature. And cells were blocked with 5% BSA and 1% normal
goat serum in PBS for 30 min at room temperature and succes-
2.4. Immunofluorescence staining and measurement of melanosome
transfer
Normal human epidermal keratinocytes were maintained in
Epilife medium with human keratinocyte growth supplement
(Invitrogen, Carlsbad, CA, USA), and maintained in Medium 254
with human melanocyte growth supplement (Invitrogen). The
cells were maintained by changing with fresh medium at every 2–
3 days. Human melanocytes were stained with the succinimidyl
ester of carboxy fluorescein diacetate (CFDA) (Molecular Probes,
sively incubated with mouse anti- -catenin (1:100, BD transduc-
β
tion laboratory, Lexington, KY, USA) and anti-tyrosinase antibody
(1:200, Abcam, Cambridge, UK) overnight at 4 °C. Cells were rinsed
with PBS and then incubated with Alexa Fluor 488-conjugated
goat anti-mouse antibody (Molecular Probes, Eugene, OR, 1:400)
for 1 h at room temperature, counterstained with 4′-6-Diamidino-
2-phenylindole (DAPI) (Boehringer Mannheim, Mannheim, Ger-
many, 1:5000), and examined under confocal microscope, LSM510
META (Carl Zeiss, Gottingen, Germany).
Eugene, OR, USA), at 2 M in Hank's balanced salt solution (HBSS)
μ
for 30 min. These melanocytes were then co-cultured for 3 days
with human keratinocytes at a ratio of 1:2 on to Lab-Teks
Chamber slide (Nunc Inc., Naperville, IL, USA), and maintained in
Please cite this article as: Kim, B., et al., N-Nicotinoyl dopamine inhibits skin pigmentation by suppressing of melanosome transfer. Eur J
Kim, Bora
