159 Hydrolyzed Conchiolin Protein Inhibits Melanin
have been employed for skin whitening by inhibiting melanin production, there is still a
lack of drugs that specifically target melanin transfer. Current whitening compounds, such
as hydroquinone and alpha arbutin, have been reported to be carcinogenic and can cause
redness or irritation when used in large amounts.9 Therefore, there has been an increasing
emphasis on the development of safer whitening products.
Pearl extract is rich in minerals and amino acids. Pearl extracts can enhance skin moisture, skin
repair, and UV protection without any unwanted side effects. It has been demonstrated that a
soluble pearl extract possesses the ability to inhibit melanin synthesis in B16 melanoma cells.10
In vitro studies have also shown that HCP exhibits anti–inflammatory and anti–apoptotic
properties.11 HCP is a hydrolyzing derivative of conchiolin protein extracted from pearls. As
a significant active component of pearl extract, HCP has been found to be biocompatible, but
the specific effects of HCP on melanin synthesis and transport remain unknown.
In this study, we aimed to investigate the effects of HCP on melanocyte morphology and
melanosome transport, and to further elucidate the underlying mechanism. We observed
that HCP effectively suppressed melanocyte dendrite extension induced by ET-1 and
downregulated the expression of genes involved in melanosome transport, including MITF,
Rab27A, and Rac1, suggesting a novel role for HCP in regulating melanosome transport.
Additionally, HCP was found to negatively regulate the activity of the Wnt/β-catenin
signaling pathway, leading to the decrease of pigmentation in zebrafish. Therefore, it seems
that HCP can inhibit melanin transfer by suppressing the activity of Wnt/β-Catenin
signaling pathway in melanocytes.
MATERIALS AND METHODS
REAGENTS
HCP was obtained from OSM Biology Co., Ltd. (Zhejiang, China). ET-1 was purchased
from MedChemExpress Co., Ltd. (Shanghai, China).
CELL CULTURE
PIG1 and MelanA cells were purchased from American Type Culture Collection (ATTC,
Virginia, USA) and cultured at 37°C with 5% CO
2 atmosphere in Dulbecco’s Modified
Eagle Medium (DMEM) containing 10% fetal bovine serum, 100 U/ml penicillin, and
100 µg/ml streptomycin.
CELL VIABILITY ASSAY
PIG1 and MelanA cells were cultured in 96-well plates at a density of 1.5 × 105/ml. Cells
were treated with HCP and ET-1 at various concentrations for 24 hours. For PIG1 cells, we
selected HCP treatment concentrations of 0, 0.01, 0.1, 5, 10, 20, 50, 80, and 100 µg/ml.
For MelanA cells, we selected HCP treatment concentrations of 0, 0.1, 1, 2, 4, 8, 10,
20, 40, 80 µg/ml. After a brief wash with medium, 3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) (0.5 mg/ml in DMEM)
was used for the quantification of metabolically active cells, and samples were measured
photometrically at 490 nm.

160 JOURNAL OF COSMETIC SCIENCE
FLOW CYTOMETRIC ANALYSIS
PIG1 and MelanA cells (1 × 106) were trypsinized and washed in ice-cold phosphate
buffered saline (PBS). Cells were then fixed with 70% ethanol for 12 hours at 4°C. After
washing, cells were resuspended and incubated at 37°C for 30 minutes in 0.5 ml of PBS
containing 10 mg/ml propidium iodine (Sigma) and 5 mg/ml RNase A (Sigma). Proportions
of cells in either G1 or S phase of cell cycle were analyzed by flow cytometry using a BD
FACSCalibur™ (Becton-Dickinson, New Jersey, USA).
IMMUNOFLUORESCENCE ANALYSIS
PIG1 and MelanA cells were fixed with 4% paraformaldehyde for 10 minutes, washed
with PBS 2 times, and permeabilized with 0.1% Triton X-100 for 10 minutes. After
blocking with 0.2% BSA for 30 minutes, cells were incubated with the prepared FITC-
labeled phalloidin working solution or antibody of anti-β-Catenin at room temperature,
for 30 minutes in the dark. Fluorescent images were obtained by an Olympus fluorescence
microscope (Olympus Corporation, Tokyo, Japan) and the number of melanocyte dendrites
was counted.
RNA ISOLATION AND REAL-TIME QUANTITATIVE PCR (RT-QPCR)
Cell RNAs were isolated using the Invitrogen TRIzol® reagent (Thermo Fisher Scientific,
Massachusetts, USA). cDNAs were synthesized by using the M-MLV reverse transcriptase
kit (Promega Corporation, Wisconsin, USA) and used as the templates for quantitative
polymerase chain reaction (qPCR) (MxPro QPCR Software, Agilent Technologies, USA)
according to manufactural instructions. The primers were as followed: homo glyceraldehyde
3-phosphate dehydrogenase (GAPDH): forward, 5’-ACCCAGAAGACTGTGGATGG-3’,
reverse, 5’-TTCAGCTCAGGGATGACCTT-3’ homo Rac1: forward, 5’-
GAGACGGAGCTGTTGGTAAAA-3’, reverse, 5’- ATAGGCCCAGATTCACTGGTT-3’
homo Rab27a: forward, 5’-ACAACAGTGGGCATTGATTTCA-3’, reverse,
5’-AAGCTACGAAACCTCTCCTGC-3’ mus GAPDH: forward, 5’-ACCCAGAAGAC
TGTGGATGG-3’, reverse, 5’-TTCAGCTCAGGGATGACCTT-3’ mus Rac1: forward,
5’-TCCGCAAACAGATGTGTTCTTA-3’, Reverse, 5’-CGCACCTCAGGATACCACT
TT-3’ mus Rab27a: forward, 5’-TCGGATGGAGATTACGATTACCT-3’, reverse,
5’-TTTTCCCTGAAATCAATGCCCA-3’. qRT-PCR was performed to detect the
expression of related genes according to the following conditions: 95°C for 15 seconds,
55°C for 30 seconds, and 72°C for 30 seconds (40 cycles).
WESTERN BLOT ANALYSIS
Cells were seeded into a 6-well plate at a density of 2 × 105/ml. 20 nM ET-1 was added
to treat cells for 24 hours. Subsequently, cells were treated with different concentrations of
HCP. After 24 hours, radio–immunoprecipitation was used for total protein extraction. The
extracted proteins were then separated by 10% SDS-polyacrylamide gel and transferred to
nitrocellulose filter membranes. After blocking with 5% BSA for 1 hour, membranes were