Figure
1.
HCP
exhibits
low
toxicity
to
PIG1
and
MelanA
cells.
(A)
After
incubation
with
various
concentrations
(0-100
µg/ml)
of
HCP
for
24h,
cell
viability
was
determined
by
MTS
assay.
(B)
After
incubation
with
various
concentrations
(0-90nM)
of
ET-1
for
24h,
cell
viability
was
determined
by
MTS
assay.
(C)
After
pre-treating
with
ET1
for
24h,
cells
were
then
treated
with
HCP
for
the
indicated
concentration
for
another
24h
before
harvesting.
Then
cell
cycle
of
PIG1
and
MelanA
were
detected
by
flow
cytometry.
Data
represent
as
mean
±
SD,
*P
0.05,
**P
0.01,
and
***P
0.001
vs
control.
Ns
represents
no
significate
difference.
162 JOURNAL OF COSMETIC SCIENCE

163 Hydrolyzed Conchiolin Protein Inhibits Melanin
caused no marked cytotoxicity in PIG1 cells, while 0-90 nM ET-1 had no toxic effects on
MelanA cells. Therefore, 20 nM ET-1 was selected for further experiments.
HCP INHIBITS MELANIN TRANSFER OF PIG1 AND MELANA CELLS
It is reported that the formation and growth of melanocyte dendrites are the prerequisites
for the transfer of melanosomes. To assess the potential impact of HCP on melanin transfer
in melanocytes, we examined alterations in melanocyte morphology following HCP
treatments. As shown in Figure 2A, 20 nM ET-1 significantly increased the number of cell
dendrites when compared to the control. However, when 1 µg/ml or 10 µg/ml HCP were
applied, the number of dendrites significantly decreased. After treating the cells with ET-1,
there was a significant increase in the proportion of cells with dendrites equal to or greater
than three. However, after 24 hours of HCP treatment, this ratio significantly decreased
in a concentration-dependent manner (Figure 2B). A similar trend was also observed in
MelanA cells (Figures 2C and 2D). These results demonstrated that HCP can greatly inhibit
ET-1-induced increase in the number and length of dendrites in MelanA and PIG1 cells,
indicating its role in inhibiting melanin transfer. To further confirm the impact of HCP on
melanin transfer, we assessed melanin secretion from cultured MelanA cells into a phenol
red-free medium. As expected, 20 nM ET-1 markedly increased melanin content in the
medium, while HCP treatment effectively reversed the ET-1-induced increase of melanin
in the medium and fully returned it to the normal level (Figures 2E and 2F). Together, all
the data show that HCP possesses a new role of inhibiting melanin transfer in melanocytes.
HCP INHIBITS MELANOSOME TRANSPORT-RELATED GENE EXPRESSION
Rac1 is one of the key factors in the transport of melanosomes from melanocytes.12 Rab27a
has been found to be anchored to melanosomes and promotes the movement of melanosomes
along actin filaments.13 Thus, we performed Western blot experiments to detect whether
HCP influenced the expression of these two key factors involving in melanosome transport.
As shown in Figures 3A to 3C, ET-1 elevated the mRNA and protein levels of Rac1
Table I
The Percentage of Cells in Each Phase of the Cell Cycle in Melana and PIG1 Cells With
Different Treatments.