Key words
melanogenesis - tara tannin - melanosome - Microphthalmia-Associated Transcription Factor (MITF)
Introduction
Skin pigmentation contributes significantly to our overall appearance. A change in
skin pigmentation, such what is observed in hypomelanosis, occurs when there is
decreased melanogenesis or the failure of the mature melanosome to transfer to the
dendritic tips of melanocytes [1]. Some of the
diseases that cause hypomelanosis are chemical leukoderma, pityriasis alba,
inflammatory diseases, and several infectious diseases [2]. Melanin is the biopolymer responsible for
pigmentation and has important functions that include protection of the skin from UV
radiation, neural cells from toxicants, and the inner ear from noise-induced
temporary hearing loss [3]
[4]. Moreover, melanin is strongly correlated
with the prevention of the accumulation of free radicals/reactive oxygen
species (ROS) generated by exposure of the skin to UV radiation [5]. Melanin is produced in the subcellular
organelle melanosomes of neuro crest-derived melanocytes in a series of
oxidation-reduction reactions catalyzed by melanogenic enzymes tyrosinase (TYR),
tyrosinase-related protein 1 (TRP1), and dopachrome tautomerase (DCT) [6]. Effective pigmentation depends largely on
the sorting and trafficking of melanosomes from melanocytes involving several
proteins such as RAB27A and MYOVA, the so-called melanosome-transport proteins [7]
[8] that, like the melanogenic enzymes, are
under the transcriptional regulation of the microphthalmia-associated transcription
factor (MITF) [9], the master regulator of
melanogenesis [10]. In turn, several signaling
pathways, including the cAMP-dependent pathway, regulate MITF [11].
In the past decade, there has been an increase in the reports on the efficacy of
natural products in promoting melanogenesis [12]
[13]
[14]. These natural products, contained in
plant extracts or as a pure compound, have been demonstrated to have a regulatory
effect on MITF and melanogenesis by regulating either TYR expression or activity
[15].
Tara tannin ([Fig. 1a]) is a natural product
isolated from pods of tara (Caesalpinia spinosa) that has been extensively
studied as a source of galloylquinic acids [16] and gallic acid [17]; except
for its antifouling effect [18]
[19], no other bioactivities of tara tannin
have been reported. In this study, the effect of a tara tannin sample, which also
includes other gallic acid derivatives and gallotannins ([Fig. 1b]), on melanogenesis was determined
using the murine pigment cell model B16F10 cells and human epidermal melanocytes. To
get an understanding of the signaling transduction or the mechanism underlying the
observed effect on melanogenesis, the effect of tara tannin on the global gene
expression in B16F10 cells was determined using DNA microarray.
Fig. 1 Chemical structure of tara tannin (benzoic acid,
3,4-dihydroxy-5-((3,4,5-trihydroxybenzoyl)oxy)-,
5-(((4-carboxy-4-hydroxy-2,6-bis((3,4,5-trihydroxybenzoyl)oxy)cyclohexyl)oxy)carbonyl)-2,3-dihydroxyphenyl
ester, (1S-(1-α,2-α,4-α,6-β)) from the pods
of Caesalpinia spinosa
a and HPLC analysis profile b.
Results
Before the effective concentration of tara tannin on melanogenesis was determined,
the assessment of its cytotoxicity on B16F10 cells was determined first using the
MTT assay. Results showed that treatment with 25, 50, and 75 µM tara
tannin for 24 and 48 h did not have any cytotoxic effect on B16F10 cells
([Fig. 2a]). At 25 µM,
cell proliferation actually increased significantly compared with the control group.
Based on this result, tara tannin at 25 µM concentration was used in
the succeeding experiments.
Fig. 2 Tara tannin has no cytotoxic effect and promotes melanin
biosynthesis in B16F10 cells. a B16F10 cells proliferation determined
using MTT assay. B16F10 cells (3 × 104 cells/well of
96-well plate) were treated with tara tannin or TT (0, 25, 50,
75 µM) for 24 and 48 h; b Intracellular
melanin content of B16F10 after 48, 72, and 96 h treatment with
25 µM tara tannin (TT) or 200 nM
α-MSH; c Intracellular melanin from B16F10 cells
treated with 25 µM tara tannin (TT) or 200 nM
α-MSH for 72 h treatment; d Cell culture plates
after 72 h of incubation with 25 µM tara tannin (TT)
or 200 nM α-MSH; e Total melanin content
(intracellular and melanin in the growth media) after 72 h treatment
with 25 µM tara tannin (TT) or 200 nM
α-MSH. Data is expressed as mean±SD
(n=5). * Indicates significance at p≤0.05 while
** indicates significance at p≤0.01.
To determine the effect of tara tannin on melanin biosynthesis, the melanin content
of B16F10 cells treated with 25 µM tara tannin or alpha
melanocyte-stimulating hormone (α-MSH) for 48, 72, and 96 h
was quantified. A significant increase in the intracellular melanin content (6-fold
vs. control) was observed following treatment with tara tannin for
72 h ([Fig. 2b]). The increase in the
melanin content was also evident in the pelleted melanin from cells treated with
tara tannin for 72 h ([Fig. 2c]). It
was also noted that after the 72 h treatment, the growth medium in
25 µM tara tannin-treated cells turned dark in color compared to the
control and α-MSH plates ([Fig.
2d]) due to the melanosomes that were released from the cells. The total
melanin produced by the cells, which included both the intracellular melanin and the
melanin collected from the growth medium ([Fig.
2e]), was significantly increased by tara tannin.
To investigate the underlying reason for the increased melanogenesis, the effect of
tara tannin on the expression of TYR, TRP1, and DCT was determined. Treatment with
25 µM tara tannin for 48 h increased the TYR, TRP1, and DCT
expression, comparable to 200 nM α-MSH. ([Fig. 3a–c]). Results of the
quantification of the mRNA expression level of these melanogenic enzymes showed that
they were significantly upregulated, especially after 48 h of treatment
([Fig. 3]d–f). When the tara
tannin-treated cells’ growth medium color was observed to have turned darker
in color compared to the control, we hypothesized that it could be due to the
melanin released from the cells, which means that tara tannin has an effect on
melanosome transport. We then verified if our hypothesis was correct by determining
the expression of melanosome transport-associated proteins MYO5A and RAB27A. As
shown in [Fig. 3h] and [i], tara tannin increased MYO5A and RAB27A
expression after 48 h. Since the melanogenic enzymes TYR, TRP1, and DCT, as
well as the melanosome transport proteins MYO5A and RAB27A, are under the
transcriptional regulation of MITF, determination of the effect of tara tannin on
MITF expression was done, and the results showed that treatment with
25 µM tara tannin for 48 h increased the mRNA level of
Mitf ([Fig. 3g]) and the MITF
protein expression ([Fig. 3j]).
Fig. 3 Tara tannin increased the expression level of melanogenic
enzymes and melanosome transport proteins in B16F10 cells. a-c
Protein expression (western blot band intensities) of the TYR, TRP1, and DCT
protein expression after 48 H treatment with 25 µM tara
tannin (TT) or or 200 nM α-MSH. Real-time PCR analysis of the
expression of d
Tyr, e
Trp1,
f
Dct, and
g
Mitf mRNA in B1F10 cells treated with tara tannin for 24, 48, and 72
h. Western blot band intensities of h MYO5A and I RAB27A after
48 and 72 h treatment; j MITF protein expression after 48 h
treatment. Data is expressed as mean±SD (n=5).
*Indicates significance at p≤0.05; **
indicates significance at p≤0.01.
To find out if the observed effect of tara tannin on B16 cells will also be observed
on human cells, the effect of tara tannin on human epidermal melanocytes (HEM) was
established. First, noncytotoxic concentrations of tara tannin on HEM were
determined using MTT assay, and the results showed that tara tannin was not
cytotoxic at up to 10 µM. In contrast, there is a dose-dependent
decrease in cell proliferation in mitomycin C-treated cells (positive control) as
shown in [Fig. 4a]. HEM proliferation was
actually increased when treated with 5 µM tara tannin ([Fig. 4a]). Based on this result,
5 µM tara tannin was used in the succeeding experiments.
Quantification of the melanin content in HEM treated with 5 µM tara
tannin or 200 nM α-MSH (positive control) showed that
treatment with tara tannin for 72 h caused a 2-fold significant increase in
the melanin content ([Fig. 4b]). The melanin
content in [Fig. 4b] is the sum of the
melanin present in the culture medium and the intracellular melanin ([Fig. 4c] and [d], respectively).
Fig. 4 Tara tannin promoted melanin biosynthesis in human epidermal
melanocytes (HEM). a HEM proliferation determined using MTT assay.
HEM (3 × 104 cells/well of 96-well plate) were treated
with tara tannin or TT (0, 5, 10, 15 µM) for 48 and 72 h;
MTT assay was run together with mitomycin C (positive control); b
Total melanin content of HEM after 72 h of incubation with
5 µM tara tannin or 200 nM α-MSH; c
Cell culture plates after 72 h of incubation with 5 µM tara
tannin or 200 nM α-MSH; d Intracellular melanin from
HEM treated with 5 µM tara tannin (TT) or 200 nM
α-MSH for 72 h treatment; Data is expressed as
mean±SD (n=5). *Indicates significance at
p≤0.05 while ** indicates significance at
p≤0.01.
To investigate the underlying reason for the observed increase in melanogenesis in
HEM following treatment with 5 µM tara tannin for 24, 48, and
72 h, the mRNA expression level of the melanogenic enzymes and MITF
was quantified. Results showed that after 48 h treatment,
5 µM tara tannin significantly increased the TYR,
TRP1, and DCT mRNA level ([Fig. 5a–c]) at all time points. Moreover, Mitf mRNA
expression was also significantly upregulated by tara tannin treatment after
48 h (2.5-fold vs. control) and 72 h (1.5-fold vs.
control). As expected, treatment with the hormone α-MSH increased the
MITF expression by 1.5-fold (vs. control) ([Fig. 5d]). The blackish color of the cell culture medium was attributed
to the release of melanin from the cells. The effect of tara tannin on melanosome
transport was then evaluated by quantifying the mRNA level of MYO5A and RAB27A. Tara
tannin upregulated RAB27A and MYO5A genes’ expression, with the increase in
expression higher after 48 h of treatment than after 24 h and at its
highest after 72 h ([Fig. 5e] and
[f]).
Fig. 5 Tara tannin increased the mRNA expression level of melanogenic
enzymes and melanosome transport proteins in HEM. HEM were treated with
5 µM tara tannin or 200 nM α-MSH for 24 or
48 h after which total RNA were extracted and complementary DNAs
(cDNAs) were synthesized using reverse transcription PCR. The cDNAs were
used as template for real-time PCR analysis. a Tyrosinase
(TYR) gene expression; b Tyrosinase-related protein 1
(TRP1) gene expression. c Dopachrome tautomerase
(DCT) gene expression; d Microphthalmia-associated
transcription factor (MITF) gene expression; e Myosin VA
(MYOVA) gene expression. f
RAB27A gene expression. Data is expressed as mean±SD
(n=5). *Indicates significance at p≤0.05
while ** indicates significance at
p≤0.01.
To elucidate the molecular mechanisms affected by tara tannin treatment, the gene
expression profile of B16F10 cells treated with tara tannin was determined.
Differential expression analysis results showed that 1067 genes, out of which 720
genes were upregulated and 347 genes were downregulated, were modulated in B16F10
cells treated with tara tannin for 72 h. Significantly upregulated
(p≥0.05) genes include Cxcr7, Tmem204, Fdft1,
Cmpk2, Ankrd37, Mcm3, Adamts4, Narf,
Aldoc, Dhcr24, Shisa2, Esco2, Insig1,
Hmgcs1, Stard4, Ak4, Jarid2, Hspa1b,
Tet1, and Nrep (Suppl. [Table
1a]
). The genes relevant for melanogenesis that were significantly expressed
(fold-change ≥1.5 or ≤−1.5) were Kit, Prkca,
Camk2g, Mitf, Prkaca, Plcb4, Mapk3,
Adcy7, Fzd7, Ctnnb1, Map2k1, Adcy9,
Adcy3, Fzd2, Fzd3, and Pomc (Suppl. [Table 1b]). Visualization of the modulated
genes on Kegg Pathway, using DAVID, revealed that tara tannin-modulated genes that
were significant in signaling pathways that regulate melanogenesis such as the MAPK
and the cAMP signaling pathways ([Fig. 6]).
The other genes that were differentially expressed by tara tannin treatment were
significant in several pathways including the cell cycle, DNA replication, and
metabolic pathways ([Table 1]). The number of
genes that tara tannin modulated increased with the increase in treatment time
(24 h, to 48 h, and to 72 h). Significant effect on
melanogenesis-associated genes were observed after 72 h of treatment with
tara tannin ([Fig. 6]).
Fig. 6 Tara tannin modulated genes relevant to melanogenesis
regulation. KEGG pathway showing the signaling pathways that regulate
melanogenesis (Source: DAVID, david.ncifcrf.gov). Tara tannin-upregulated
genes are in red boxes while tara tannin-downregulated genes are in green
boxes. RNA used for DNA microarray analysis was extracted from B16F10 cells
after treatment with 25 µM tara tannin for 72 h. The
data was obtained by running the data in the Affymetrix Transcription
Console Software.
Table 1 Signaling pathways modulated by 25 µM
tara tannin (72 h) in B16F10 cells (p≤0.05).1
|
Signaling pathways
|
P-value
|
|
Cell cycle
|
2.7×10–13+
|
|
DNA replication
|
7.1×10–11
|
|
Metabolic pathways
|
2.5×10–10
|
|
Progesterone-mediated oocyte maturation
|
2.4×10–9
|
|
Biosynthesis of antibiotics
|
8.8×10–9
|
|
Oocyte meiosis
|
3.3×10–7
|
|
Mismatch repair
|
1.0×10–6
|
|
Steroid biosynthesis
|
1.3×10–6
|
|
VEGF signaling pathway
|
1.6×10–2
|
|
MAPK signaling pathway
|
2.0×10–2
|
|
Melanogenesis
|
3.0×10–2
|
1Analyzed using Transcriptome Analysis Console Software
(Affymetrix).
Discussion
Skin and hair pigmentation are the most obvious human phenotypes, and a change in the
color of some parts of the skin or hair causes severe psychological stress [1]
[20]. We have previously reported that certain
plant oil and extracts can promote melanogenesis [12]
[14]. Here, we demonstrated how a
plant-derived extract, tara tannin, promotes pigmentation. Tara tannin increased
melanin biosynthesis as shown by the preliminary test results using murine pigment
cell model B16F10 ([Figs. 2] and [3]). This melanogenesis promotion effect in
murine cell model was also observed in HEM ([Fig.
4]), wherein the expression of melanogenic enzymes TYR, TRP1, and DCT as
well as their transcription factor, MITF, were increased. Other natural products,
such as quercetin, lupenone, and fisetin, have been reported to promote melanin
biosynthesis by increasing the TYR activity [21] via the MAPK pathway [12] or by
constitutive activation of CREB [22].
Similarly, tara tannin promotes melanogenesis but by increasing the melanogenesis
enzyme TYR’s expression at the transcriptional level. At present, there are
no reports on the effect of tara tannin on melanogenesis.
Tara tannin ([Fig. 1a]) is a main component of
tara powder obtained from tara pods (Caesalpinia spinosa). It has a
gallotannin structure [23]
[24] and can thus be hydrolyzed enzymatically
[25]. Tara powder has been reported to
contain free gallic acid (2.6%) [26].
Even more gallic acid and ellagic acid may be released upon hydrolysis during, for
example, prolonged storage). The tara powder used in this study also contained
2–3% gallic acid. Gallic acid and ellagic acid have inhibitory
effect on melanogenesis [27]
[28]. To evaluate the effect of gallic acid,
B16 cells were treated with free gallic acid, and the results demonstrated that
gallic acid also inhibits melanogenesis in our experimental setup (data not
shown).
In this study, tara tannin-treated B16F10 or HEM cells promoted melanogenesis at up
to 72 h of treatment ([Fig. 2b],
[d], and [e]; [Fig.
4]b–[]d). It is also noteworthy
to mention that tara tannin at lower concentration (5 μM) was effective in
promoting melanogenesis in HEM while a higher dosage is required for murine cell
model B16F10 wherein higher concentration appears to decrease the cell proliferation
([Fig. 4a]). Tara tannin modulates the
signaling pathways ([Fig. 6]) that serve to
regulate the expression of the genes of the enzymes catalyzing melanogenesis in
B16F10 cells and in HEM ([Fig.
3]a–[]d; [Fig. 5]a–[]c) as well as upregulates MITF, the master
regulator of melanogenesis ([Fig. 3e]; [Fig. 5d]). The expression of the melanogenic
enzyme proteins B16F10 is not directly proportional to their observed mRNA
expression, and this could be due to post-translational events upon which tara
tannin may or may not have a direct effect.
As shown in [Fig. 2b], an increase in the
melanin content was the highest (6-fold) in B16F10 cells treated with tara tannin
for 72 h. This means that after 72 h, the melanin was
“transported” or released from the cells (into the growth medium),
and this could be the reason why the intracellular melanin content of the cells
after the 96 h treatment is the same as the melanin content of the cells
after 48 h ([Fig. 2e]). It is
important to note that tara tannin can also promote melanosome transport since
constitutive pigmentation is not just dependent on the quantity of melanin but also
on the transfer and distribution from the melanocytes into the neighboring
keratinocytes [7]. Moreover, the cellular
organelle melanosome serves as the site of melanin synthesis, storage, and transport
[8], and its uniform distribution in the
epidermis characterizes normal pigmentation. Melanosomes are transported to the tips
of the melanocyte dendrites via several melanosome transport proteins including
MYOVA, RAB27A, and MLPH that bind to the melanosome [29]. Among these proteins, MYOVA and RAB27A play an important role in
melanosome transport while melanophilin (MLPH) regulates the activity of MYOVA and
dynein [30]
[31]. Other reports also identify RAB27A and
MLPH) as an organelle-associated receptor for MYOVA [29]
[32]. In this study, tara tannin was shown to
have a positive effect on melanosome transport, and this was supported by the data
on the expression of the melanosome transport proteins MYOVA and RAB27 that were
both increased by tara tannin ([Fig. 3]a,
[]b, [5]e and [f]) and demonstrated by the
cell culture media becoming darker in color due to the presence of melanin ([Fig. 2d] and [Fig. 4c]). MITF is not just the master regulator of melanogenesis [33], but it is also the transcription factor of
genes involved in melanocyte survival and several cellular events including
melanosome transport [34]
[35].
Using DNA microarray, tara tannin was found to modulate genes that are associated
with melanogenesis (Suppl. [Table 1b]).
Several signaling pathways that were also regulated by tara tannin as shown in [Table 1] (cell cycle, DNA replication,
vascular endothelial growth factor) may not play a direct role in regulating
melanogenesis, as illustrated in [Fig. 6],
but may have contributed to the overall effect of tara tannin. The highly
upregulated genes (Suppl. [Table 1a])
Cxcr7 regulates normal human epidermal melanocyte migration [36] while MCM3 is involved in the initiation of
eukaryotic genome replication and shares a DNA replication factor with MITF [37]
An understanding of the genetic determinants of human pigmentation could help
identify the molecular mechanisms of pigmentation-associated conditions including
tanning response and skin cancers [38].
The benefits from using natural products in the stimulation of melanogenesis have
long been recognized. Plants are a rich source of compounds that can promote
melanogenesis [12]
[14]
[39]
[40] and are one of the most widely used
sources of pharmaceuticals. In this study, we have demonstrated that tara tannin can
effectively increase the melanin production in B16F10 cells. This is the first
report on the effect of tara tannin on melanogenesis and the underlying mechanism
involved in this effect.
Materials and Methods
Cells and cell culture
B16F10 murine melanoma cells were purchased from the Riken Cell Bank in Tsukuba
and cultured in RPMI1640 (Thermo Fisher Scientific) supplemented with
10% FBS (Sigma). Moderately pigmented neonatal HEM cells (Gibco) were
cultured in Medium 254 (Thermo Fisher Scientific) supplemented with human
melanocyte growth supplement with or without phorbol 12-meristate 13-acetate or
PMA (HMGS) [S-016–5, (Thermo Fisher Scientific)]. Cells were incubated
at 37°C in an incubator with 5% CO2. Photographs of
the cell cultures were taken using Leica DMIL light microscope camera (DFC290
HD).
Tara tannin sample preparation and composition analysis
The tara tannin sample used in this study was extracted by Nano Innovation
Laboratories, Ltd. from powdered Peruvian tara pods purchased from Kawamura
Tsusho Co. Ltd. Briefly, a liter of ethanol was added to 0.33 kg tara
powder, and the mixture was continuously stirred for 1 h at 60°C
after which the mixture was cooled to 20°C and filtered in vacuo.
Distilled water and ethanol were added and stirred (40°C). Finally, the
solution was filtered in vacuo and stored in −20°C until use.
The composition of this tara tannin sample was determined using HPLC with UV
detector set at 254 nm. Samples were injected onto a TSKgel ODS-80Ts
column (4.6φ × 150) (Tosoh Corporation) maintained at
40°C. A dual-gradient using acetonitrile/0.1% formic
acid (solvent A) and water/0.1% formic acid (solvent B) was used
for elution at a flow rate of 1 ml/min over 33 mins as
follows: 95% B for 0.01 min; gradient to 65% B for
25 min; 5% B for 50 s min; gradient to 5% B for
1 min 10 s; 95% B for 2 min; and 95% for
3 min to re-equilibrate the column. For the bioassays, stock solution of
tara tannin sample (>95% purity) was prepared by dissolving it
in 70% ethanol (70% ethyl alcohol and 30% milli-Q water)
and, prior to use, was filter-sterilized using a 0.22 μm filter
(Merck Millipore) and stored at −20°C until use. Treatment with
tara tannin was prepared by mixing the tara tannin stock solution in the growth
medium for human epidermal melanocytes or B16F10 cells. Tara powder is rich in
gallotannins and also contains small quantities of catechin derivatives. A
characteristic compound is tara tannin (benzoic acid,
3,4-dihydroxy-5-((3,4,5-trihydroxybenzoyl)oxy)-,
5-(((4-carboxy-4-hydroxy-2,6-bis((3,4,5-trihydroxybenzoyl)oxy)cyclohexyl)oxy)carbonyl)-2,3-dihydroxyphenyl
ester,
(1S-(1-α,2-α,4-α,6-β))-)
([Fig. 1a]). HPLC-MS analysis
revealed the presence of more than 40 individual gallotannins in tara pod
extracts [41]. The tara powder sample used
in this study showed a tannin fingerprint similar to that presented in ([Fig. 1b]) [41].
Cell viability assay
Briefly, B16F10 or HEM (3 × 103 cells/well) were
seeded onto 96-well plates (Falcon) and incubated at 37oC in an
incubator with 5% CO2. After overnight incubation, the growth
medium was replaced by fresh growth medium with or without tara tannin (5, 10,
15, 25, 50, or 75 μM) then incubated for 24 and 48 h at
37oC in an incubator with 5% CO2. After
specified incubation period, MTT solution at concentration
5 mg/ml (Wako) was added, and the cells incubated further for
6−8 h. To completely dissolve the formazan crystals, 10%
sodium dodecyl sulfate (SDS) (Wako) was added, and incubated overnight. The cell
viability was calculated based on the absorbances obtained at 570 nm
using a microplate reader (Powerscan HT; Dainippon Pharmaceuticals USA
Corporation). Blanks containing only medium, MTT, and SDS were subtracted from
the average values of the absorbances. Mitomycin C (>95% purity;
Merck) was used a positive control.
Melanin content determination
B16F10 cells or HEM were seeded at a density of 5 × 105 cells
onto 10-cm petri dishes (Falcon) and cultured as described above. After
24 h incubation, the growth medium was replaced by fresh growth medium
with or without 5, 10, 15, and 25 µM tara tannin or
200 nM α-MSH (Sigma;>95% purity), the
positive control, and then incubated further for 48, 72, and 96 h. The
cells were harvested by trypsination (0.25% trypsin⁄
0.02% EDTA in PBS; Gibco) and solubilized by sonication after addition
of 1% Triton X-100 (Sigma). Melanin was purified and precipitated in
10% trichloroacetic acid. Melanin in the spent growth medium was also
collected for quantification. The precipitated melanin from both the cells and
the growth medium was washed with 70% ethanol and then solubilized in
8 N NaOH with incubation at 80 °C for
2 h. The melanin content was then quantified spectrophotometrically
(410 nm) and by comparing to a standard curve of synthetic melanin
(Sigma) and expressed as melanin content per cell. The cell counts and cell
viability were evaluated flow cytometrically as reported previously [14].
Western blot analysis
B16F10 cells (5 × 104 cells/ petri dishes) were seeded
and incubated at 37°C in an incubator with 5%
CO2. After 24 h incubation, the growth medium was
replaced with fresh growth medium with or without 25 μM tara
tannin and 200 nM α-MSH and incubated further for 24 and
48 h. After the specified incubation time, the protein samples were
extracted using radio immunoprecipitation assay lysis buffer (Sigma) with
0.1% protease inhibitor cocktail (Sigma), loaded into 10%
SDS-polyacrylamide gel, and subjected to electrophoresis (SDS-PAGE). The
proteins were transferred onto PVDF membrane and incubated in specific primary
antibodies against MITF (#110512; Assay Biotech); TYR (sc-7833; Santa Cruz
Biotechnology; 1:200) goat polyclonal; TRP1 (sc-166857; Santa Cruz
Biotechnology; 1:200) mouse monoclonal; DCT (sc-271356; Santa Cruz
Biotechnology; 1:200) mouse monoclonal; RAB27A (sc-22756; Santa Cruz
Biotechnology; 1:200) rabbit polyclonal; Myosin Va (sc-17706; Santa Cruz
Biotechnology; 1:200) goat polyclonal; and GAPDH (sc-32233; Santa Cruz
Biotechnology; 1:100) mouse monoclonal overnight at 4oC. Membranes
were washed with PBS with Tween-20 (PBST) before incubation with goat antimouse
IRDye 680LT or goat antiRabbit IRDye 800CW (LI-COR) secondary antibodies at room
temperature. Detection was carried out using OdysseyFc Imaging System (LI-COR
Inc.).
RNA extraction
B16F10 cells or HEM (5 × 104 cells/mL) were cultured
as described above and the RNA extracted using ISOGEN (Nippon Gene) as
previously reported [14]. The resulting
RNA solution was quantified using Nanodrop 2000 spectrophotometer (Thermo Fisher
Scientific).
Real-time PCR
RNA samples (1μg) were reverse transcribed using the SuperScript III
Reverse Transcription Kit (Invitrogen). The resulting cDNA was used as templates
for real-time PCR (rt-PCR) using TaqMan Gene Expression Master Mix (Applied
Biosystems), and specific primers for MITF (Hs01117294_m1), TYR (Hs00165976_m1),
TRP1 (Hs00167051_m1), DCT (Hs0198278_m1), MYOVA (Hs00165309_m1), RAB27A
(Hs00608302_m1), and GAPDH (Hs02786624_g1) (as an internal control) were used.
Rt-PCR was performed using 7500 Fast Real-time PCR System with 7500 software
version 2.0.5 (Applied Biosystems).
DNA microarray hybridization, imaging, and data analysis
DNA microarray was performed to determine the global gene expression changes in
B16F10 cells in relation to the observed melanogenesis promotion effect of tara
tannin. Single stranded cDNA was prepared from 200 ng of total RNA
following the manufacturer’s instructions for Affymetrix Gene Chip 30
IVT Express Kit (Affymetrix). Total RNA was reverse transcribed into
double-stranded cDNA, and biotin-labeled aRNA was generated using the 30 IVT
Express Labeling Kit (Affymetrix). Biotin-labeled aRNA was hybridized to the
Affymetrix mouse 430 PM Array strips (Affymetrix) for 16 h at 45°C at
the Hybridization Station (Affymetrix). Hybridized arrays were washed and
stained using the hybridization, wash, and stain Kit (Affymetrix) performed in
Affymetrix GeneAtlasTM Fluidics Station. The arrays were scanned
using the Affymetrix GeneAtlasTM Imaging Station.
The Affymetrix Expression Console Software was used to analyze the data by
running comparisons of gene expression in treated and control cells based on
mathematical algorithms. Results were based on the analysis of significance
(control vs. treatment) using 1-way between-subject ANOVA (paired) (p
value ≤0.05) and fold-change (linear) ≤ −2 or
≥2. The generated data was then analyzed using the Transcription
Analysis Console Software. Gene ontology and functional annotation chart were
derived using DAVID (database for annotation, visualization, and integrated
discovery) (david.ncifcrf.gov). The DNA microarray data comply with MIAME
guidelines and have been deposited in the ArrayExpress database at EMBL-EBI
(www.ebi.ac.uk/arrayexpress).
Statistical analysis
Statistical analysis was carried out using the using Student’s t-test
when 2 value sets were compared (control vs. sample). Mean
values±standard error deviations (SD) were calculated, and a value of
p≤0.05 was considered to be statistically significant.
For DNA microarray, the Affymetrix Expression Console Software was used to
analyze the data by running comparisons of gene expression in treated and
control cells based on mathematical algorithms. Results were based on the
analysis of significance (control vs. treatment) using 1-way
between-subject ANOVA (paired) (p value ≤0.05) and fold-change (linear)
≤−2 or ≥2.
