molecules

Article
DDX3 Upregulates Hydrogen Peroxide-Induced Melanogenesis
in Sk-Mel-2 Human Melanoma Cells
Sanung Eom 1,† , Shinhui Lee 1,† , Jiwon Lee 1 , Hye Duck Yeom 2 , Seong-Gene Lee 1, * and Junho Lee 1, *

1 Department of Biotechnology, Chonnam National University, Gwangju 61886, Korea

2 GoPath Laboratories, Buffalo Grove, IL 60089, USA
* Correspondence: sglee3@chonnam.ac.kr (S.-G.L.); leejunho@chonnam.ac.kr (J.L.);
Tel.: +82-62-530-2160 (S.-G.L.); +82-62-530-2164 (J.L.)
† These authors contributed equally to this work.

Abstract: DDX3 is a DEAD-box RNA helicase with diverse biological functions through multicellular
pathways. The objective of this study was to investigate the role of DDX3 in regulating melanogenesis
by the exploring signaling pathways involved. Various concentrations of hydrogen peroxide were
used to induce melanogenesis in SK-Mel-2 human melanoma cells. Melanin content assays, tyrosinase
activity analysis, and Western blot analysis were performed to determine how DDX3 was involved
in melanogenesis. Transient transfection was performed to overexpress or silence DDX3 genes.
Immunoprecipitation was performed using an antityrosinase antibody. Based on the results of the cell
viability test, melanin content, and activity of tyrosinase, a key melanogenesis enzyme, in SK-Mel-2
human melanoma cells, hydrogen peroxide at 0.1 mM was chosen to induce melanogenesis. Treatment
with H2 O2 notably increased the promoter activity of DDX3. After treatment with hydroperoxide
for 4 h, melanin content and tyrosinase activity peaked in DDX3-transfected cells. Overexpression
of DDX3 increased melanin content and tyrosinase expression under oxidative stress induced by
H2 O2 . DDX3 co-immunoprecipitated with tyrosinase, a melanogenesis enzyme. The interaction
between DDX3 and tyrosinase was strongly increased under oxidative stress. DDX3 could increase
Citation: Eom, S.; Lee, S.; Lee, J.; melanogenesis under the H2 O2 -treated condition. Thus, targeting DDX3 could be a novel strategy to
Yeom, H.D.; Lee, S.-G.; Lee, J. DDX3
develop molecular therapy for skin diseases.
Upregulates Hydrogen
Peroxide-Induced Melanogenesis in
Keywords: DDX3; hydroperoxide; melanogenesis; SK-Mel-2 human cancer melanoma cell
Sk-Mel-2 Human Melanoma Cells.
Molecules 2022, 27, 7010. https://
doi.org/10.3390/molecules27207010

Academic Editor: Dong Wook Shin 1. Introduction

Received: 23 September 2022 Melanocytes are melanin-producing cells located in the bottom layer (the stratum
Accepted: 14 October 2022 basale) of the skin’s epidermis. Melanin [1], a pigment in the melanosome, is primarily re-
Published: 18 October 2022 sponsible for skin color. Besides defining an important human phenotypic trait, melanin has
a critical role in photoprotection due to its ability to absorb ultraviolet radiation (UVR) [2,3].
Publisher’s Note: MDPI stays neutral
The Fitzpatrick system is the most commonly used system to distinguish different skin
with regard to jurisdictional claims in
pigmentation phenotypes. It characterizes six phototypes (I-VI) by grading erythema and
published maps and institutional affil-
iations.
acquired pigmentation after exposure to UVR. Constitutive skin pigmentation as a geneti-
cally determined color in the absence of any external factor such as sun exposure can be
affected by various regulatory factors [4].
Melanin is synthesized by tyrosinase, a copper-containing metalloglycoprotein and
Copyright: © 2022 by the authors. a rate-limiting enzyme that is capable of utilizing L-tyrosine, dihydroxyphenylalanine
Licensee MDPI, Basel, Switzerland. (L-DOPA), and 5,6-dihydroxyindole as substrates. Other enzymes, including the tyrosinase-
This article is an open access article related proteins (TRP-1) and dopachrome tautomerase, also known as TRP-2, are also
distributed under the terms and responsible for melanogenesis. Melanogenesis occurs via enzymatic conversion of amino
conditions of the Creative Commons acid tyrosine to melanin pigments through a series of intermediates [5]. Firstly, L-tyrosine
Attribution (CC BY) license (https:// is hydroxylated to form L-DOPA. Subsequently, L-DOPA is oxidized to L-dopaquinone,
creativecommons.org/licenses/by/ which is further processed into either eumelanin (black or brown pigment) or pheomelanin
4.0/).

Molecules 2022, 27, 7010. https://doi.org/10.3390/molecules27207010 https://www.mdpi.com/journal/molecules

Molecules 2022, 27, 7010 2 of 11

(yellow or red pigment) [6]. Dopaquinone generally forms eumelanin through sponta-
neous reactions involving cyclization, decarboxylation, oxidation, and polymerization.
TRP-2 can generate 5,6-dihydroxyindole-2-carboxylic acid (DHICA) from dopachrome.
TRP-1 catalyzes the oxidation of DHICA to indole-5,6-quinone carboxylic acid. In the
absence of thiols, dopaquinone is immediately converted to dopachrome, leading to eu-
melanin production. However, when glutathione (GSH) and cysteine are present, they
can react with dopaquinone intermediates to divert melanin pigment synthesis from eu-
melanin to pheomelanin through cysteinyl DOPA [7]. Besides these enzymatic reactions,
the melanogenic pathway also involves non-enzymatic reactions involving o-quinones
generated enzymatically by the action of tyrosinase to produce several unstable intermedi-
ates that can polymerize to form melanin. A series of both enzymatic and nonenzymatic
reactions in the synthesis of eumelanin and pheomelanin subsequently result in H2 O2
formation [8]. Intrinsic factors such as the inflammatory, endocrine, and central nervous
systems, keratinocytes, and fibroblasts can interact with normal skin melanocytes. Extrinsic
factors such as drug and ultraviolet radiation also play an important role in regulating
its activity [6,9]. Once synthesized, melanin is contained in a special organelle called a
melanosome that moves along arm-like structures called dendrites to reach keratinocytes.
DEAD-box DDX3 (DDX3X) has all the properties of an RNA-helicase family, including
RNA unwinding activity, transition factor activity, adenosine triphosphate activity, or
presence of promoters [10]. DDX3 participates in various cell processes, including cycle
progression, cell division, innate immune response, viral replication, and tumor develop-
ment. DDX3 also has a variety of functions throughout the lifecycle of various viruses.
DDX3 mutation can prevent replication of human immunodeficiency virus (HIV), hepatitis
C virus (HCV), and other viruses [11,12]. DDX, on the other hand, exhibits antiviral effects
on dengue and hepatitis B viruses through interferon stimulation of beta generation. The
role of DDX3 in different types of cancer is somewhat controversial [13]. DDX3 acts as a
tumor gene in one type of cancer, but shows different forms of tumor suppressant prop-
erties [14–16]. Human DDX3 helicase is now considered a new attractive target for new
drug development.
The oxidative stress could interact with melanogenesis, probably because the photo-
generation of ROS reactive oxidative stress could be led by eumelanin and pheomelanin
induced by a potential source of H2 O2 [17,18]. H2 O2 and other reactive oxygen species also
play an important role in regulating many intracellular pathways, for example, hydroxyl
radicals and superoxide radical (O2 •−) in melanocytes [19,20]. Sarangarajana et al. have
presented that H2 O2 level is directly proportion to the synthesis of melanin in normal
melanocytes. Antioxidant or pro-oxidant abilities of H2 O2 depend on the redox state of
melanocytes [21]. H2 O2 or NO and oxidative damage stimulating the α-MSH/MC1R
or MITF signaling pathway and leading to melanogenesis could mediate the increase
in melanin production [22,23]. In melanin synthesis in mammalian skin, H2 O2 is a by-
product which is produced following UV irradiation, interacting with chromophores and
melanin [24,25]. In a previous study, we have analyzed effects of H2 O2 on melanogenesis
using human melanoma SK-Mel-2 cells and mouse melanoma B16F10 cells by measuring
melanin content and analyzing expression levels of melanogenesis-related proteins [26],
including cAMP-responsive element binding protein (CREB), microphthalmia-associated
transcription factor (MITF), tyrosinase, and phenylalanine hydroxylase (PAH). Results
of that study showed that H2 O2 could induce melanogenesis by upregulating PAH and
activating cAMP/p-CREB/MITF signaling by increasing intracellular cAMP levels through
the induction of ATP5B. Although DDX3 is related to a lot of cellular pathways, the bio-
logical function of DDX3 in melanogenesis remains unclear. We have previously reported
the molecular mechanism involved in the effect of DDX3 on intrinsic apoptosis in HeLa
cells. The objective of the present study was to investigate the novel function of DDX3 in
melanogenesis. SK-Mel-2 human melanoma cells were selected as a model and H2 O2 was
selected as a melanogenesis-inducing agent to study the biological function of DDX3 in
melanogenesis, focusing on signaling pathways.
Molecules 2022, 27, x FOR PEER REVIEW 3 of 12

selected as a melanogenesis-inducing agent to study the biological function of DDX3 in

Molecules 2022, 27, 7010 melanogenesis, focusing on signaling pathways. 3 of 11

2. Results
2.1.2.HResults
2O2 Activates Melanogenesis and DDX3 Promoter Activity

2.1.It Hhas
2 O2been reported
Activates that H2O2and
Melanogenesis canDDX3
induce the expression
Promoter Activity of melanogenesis-related
genes including CREB, MITF, tyrosinase, and PAH
It has been reported that H2 O2 can induce the expression in SK-Mel-2 cells
ofand B16F10 cells [26].
melanogenesis-related
In genes
this study, effects of various concentrations of H 2O2 on cell viability of SK-Mel-2 human
including CREB, MITF, tyrosinase, and PAH in SK-Mel-2 cells and B16F10 cells [26].
melanoma
In this study,cells effects
were determined. Results are shown
of various concentrations of H2inO2Figure
on cell1A. BasedofonSK-Mel-2
viability these results,
human
0.1melanoma
mM H2O2 was cells were determined. Results are shown in Figure 1A. Based on pthese
chosen for performing subsequent experiments (F2,46 = 29.3, < 0.0001).
results,
Melanin expression level in human SK-Mel-2 cells was obviously
0.1 mM H2 O2 was chosen for performing subsequent experiments (F2,46 = 29.3, p < 0.0001).increased at 4 hrs after
treatment
Melaninwith 0.1 mMlevel
expression H2O2in(Figure
human1B). As confirmed
SK-Mel-2 cells was byobviously
a one-wayincreased
repeatedat measures
4 hrs after
comparisons
treatment with 0.1 mM H2 O2 (Figure 1B). As confirmed by a one-way repeated activity
post hoc test, exposure to 0.1 mM H 2 O2 for 4 h increased tyrosinase measures
bycomparisons
2.53-fold comparedpost hoctotest,
control (p < 0.0001).
exposure to 0.1 mM DDX3H2 Ois2 known to have antyrosinase
for 4 h increased importantactivity
role
in by
cell2.53-fold
cycle regulation [3,27]. In our previous study, we investigated
compared to control (p < 0.0001). DDX3 is known to have an important role the effect of DDX3
onin intrinsic
cell cycle apoptosis
regulation in sanguinarine-treated
[3,27]. In our previous HeLa cellswe
study, [28]. Here, the effect
investigated of DDX3
the effect on
of DDX3
melanogenesis induced in
on intrinsic apoptosis bysanguinarine-treated
H2O2 was evaluated.HeLa It hascells
been reported
[28]. thateffect
Here, the 0.1 mM H2O2on
of DDX3
canmelanogenesis
activate the melanogenesis
induced by H2 O process [29,30]. Thus,
2 was evaluated. It haswebeen
firstreported
assessedthatwhether
0.1 mM HH 2O2O
2 2
could induce DDX3 promoter activity. Cells were transfected with pGL2
can activate the melanogenesis process [29,30]. Thus, we first assessed whether H2 O2 could basic/DDX3 pro-
moter
induce for DDX3
24 h and then treated
promoter with
activity. H2O
Cells 2 transiently
were transfected(0.1with
mMpGL2for 4 h) before harvesting
basic/DDX3 promoter
cells
forfor
24 ha and
luciferase assay. with
then treated Results
H2 Oshowed that DDX3
2 transiently (0.1 mM promoter activityharvesting
for 4 h) before was gradually
cells for
increased
a luciferasein 0.1assay.
mM H 2O2-treated
Results showed cells in aDDX3
that time-dependent mannerwas
promoter activity for gradually
4 h (Figureincreased
1D) (p
< 0.0001).
in 0.1 mM These
H2 Oresults may
2 -treated suggest
cells an association manner
in a time-dependent betweenfor DDX3 and melanogenesis
4 h (Figure 1D) (p < 0.0001).
induced
These by H2Omay
results 2 in human
suggestmelanoma cells. between DDX3 and melanogenesis induced by
an association
H2 O2 in human melanoma cells.

Figure 1. H2 O2 activates melanogenesis and DDX3 promoter activity. SK-Mel-2 cells were treated
Figure
with 1. H2O2 activates
various melanogenesis
concentrations and
of H2 O2 for 4 hDDX3 promoter
and then activity.
harvested (A) orSK-Mel-2 cellswith
were treated were0.1treated
mM H2 O2
with
forvarious
variousconcentrations ofthen
time courses and H2Oharvested
2 for 4 h and
forthen harvested
melanin content(A) or were
assay (B) ortreated withassay
tyrosinase 0.1 mM
(C). Cells
H2were
O2 fortransfected
various time courses
with pGL2 and then harvested
basic/DDX3 promoterforfor
melanin content by
24 h followed assay (B) or tyrosinase
treatment with 0.1 mM H2 O2
for 4 h before harvesting for luciferase assay (D). Data were analyzed using one-way ANOVA with
repeated measurements followed by multiple comparisons post hoc test. *, p < 0.05; **, p < 0.001;
***, p < 0.0001 compared to control.
 Molecules 2022, 27, 7010 4 of 11

2.2. DDX3 Induces Melanogenesis in H2 O2 -Treated Condition Cells

Tyrosinase is known to be a key enzyme in regulating the mammalian melanin synthe-
sis pathway [31]. Melanogenesis can be induced by diverse signaling pathways through
activation of pigment-related proteins such as microphthalmia-associated transcription
factor (MITF), tyrosinase (TYR), tyrosine-related protein-1 (TRP-1), and tyrosine-related
protein-2 (TRP-2). Among these proteins, only tyrosinase is essential for melanogenesis [32].
To verify the effect of DDX3 on melanogenesis in H2 O2 -treated cells, SK-Mel- 2 cells were
transiently transfected with shDDX or WTDDX3 for 48 h and then treated with 0.1 mM
for H2 O2 for 4 h. Melanin content (Figure 2A) was then measured after harvesting cells.
Tyrosinase assay results are shown in Figure 2B. Two-way ANOVA revealed a significant
effect (or a non-significant effect) of H2 O2 treatment on melanin content, a significant effect
of DDX3 (p < 0.0001 and p < 0.0001, respectively) and a significant interaction between
these factors (p < 0.0001). Further, two-way ANOVA revealed a significant effect of H2 O2
treatment on tyrosinase activity, a significant effect of DDX3 (p < 0.0001 and p < 0.0001,
respectively) and a significant interaction between these factors (p < 0.0001). Post hoc
analysis showed that overexpression of DDX3 increased melanin content (by 1.5 times) and
tyrosinase activity (by 1.6 times) after H2 O2 treatment compared to control whereas DDX3
knockdown slightly decreased melanin content and tyrosinase activity compared to the
control cells. However, SK-Mel-2 cells with DDX3 knockdown by si-DDX3 did not show
much difference in melanin content or tyrosinase activity compared to non-treated5 H
Molecules 2022, 27, x FOR PEER REVIEW O2
of2 12
cells. These results imply that there is an association between melanogenesis and DDX3 in
H2 O2 -treated cells (Figure 2A,B).

DDX3induces
Figure2.2.DDX3
Figure inducesmelanogenesis
melanogenesisininHH2O2O 2 -treated
2-treated
conditions.SK-Mel-2
conditions. SK-Mel-2cells
cellswere
weretransiently
transiently
transfected with shDDX or WTDDX3 for 48 h and then treated with 0.1 mM
transfected with shDDX or WTDDX3 for 48 hrs and then treated with 0.1 mM H2O22 for H O 2 4 h4before
for h before
harvestingfor
harvesting formelanin
melanincontent
contentassay
assay(A),
(A),tyrosinase
tyrosinaseassay
assay(B),
(B),and
andWestern
Westernblot
blot(C).
(C).The
Thehisto-
histogram
gram shows relative expression levels of tyrosinase in H2O2-treated SK-Mel-2 cells with or without
DDX (D). The data are presented as the mean ± SEM of 4–6 samples from each group. Data were
analyzed using two-way ANOVA followed by multiple comparisons post hoc test. * p < 0.05, ** p <
0.001, *** p < 0.0001 compared to untreated control; ## p < 0.001, ### p < 0.0001 indicate differences
compared with H2O2-treated cells after transfection of WTDDX3 or shDDX. $$ p < 0.001 indicates
differences compared with H2O2-treated cells after transfection of WTDDX3. & p < 0.05 indicates
differences compared with H2O2-treated cells after transfection of shDDX3.
 Molecules 2022, 27, 7010 5 of 11

shows relative expression levels of tyrosinase in H2 O2 -treated SK-Mel-2 cells with or without DDX
(D). The data are presented as the mean ± SEM of 4–6 samples from each group. Data were analyzed
using two-way ANOVA followed by multiple comparisons post hoc test. * p < 0.05, ** p < 0.001,
*** p < 0.0001 compared to untreated control; ## p < 0.001, ### p < 0.0001 indicate differences compared
with H2 O2 -treated cells after transfection of WTDDX3 or shDDX. $$ p < 0.001 indicates differences
compared with H2 O2 -treated cells after transfection of WTDDX3. & p < 0.05 indicates differences
compared with H2 O2 -treated cells after transfection of shDDX3.

To confirm the role of DDX3 in melanogenesis under H2 O2 treatment, tyrosinase

expression was checked by Western blot assay. The results were analyzed using a two-way
ANOVA, which showed a significant effect (or a non-significant effect) of H2 O2 treatment
on melanin content, a significant effect of DDX3 (p < 0.0001 and p < 0.0001, respectively)
and a significant interaction between these factors (p < 0.0001). Similarly, as revealed
by post hoc analysis, tyrosinase expression was enhanced in H2 O2 -treated transfected
cells (Figure 2C,D). DDX3 was increased 1.2 ± 0.6-fold compared to control in WTDDX3-
transfected cells and 0.4 ± 0.6-fold in shDDX3-treated cells. However, expression levels of
tyrosinase in WTDDX3 under the H2 O2 -treated condition were significantly higher than
those in shDDX3-treated cells. These findings suggest that overexpression of DDX3 can
induce melanogenesis in H2 O2 -treated SK-Mel-2 cells.

2.3. DDX3 Binds to Tyrosinase in H2 O2 -Treated Condition

The previous experiment did not clarify the mechanism of DDX3 involved in the
melanogenesis induced by H2 O2 in SK-Mel-2 cells. We hypothesized that DDX could bind
to tyrosinase, the key enzyme of melanogenesis. To test this hypothesis, SK-Mel-2 cells
were transiently transfected with WTDDX3 for 48 h and then treated with 0.1 mM H2 O2
for 4 h before harvesting for tyrosinase immune precipitation and Western blotting. Results
showed that DDX3 co-immunoprecipitated with tyrosine, the melanogenesis enzyme. Two-
way ANOVA revealed a significant effect of H2 O2 treatment on TYR, a significant effect of
DDX3 (p < 0.0001 and p < 0.0001, respectively), and a significant interaction between these
factors (p < 0.0001). A post hoc analysis confirmed that the impact of DDX3 on TYR was
Molecules 2022, 27, x FOR PEER REVIEW
strongly increased under oxidative stress as shown in Figure 3A,B. These results6suggest
of 12
that DDX3 might upregulate melanogenesis in SK-Mel-2 human melanoma cells by binding
to tyrosinase after H2 O2 treatment. Thus, we proposed a model for the effect of DDX3 on
H2 O2 -treated HeLa cells (Figure 4).

Figure 3. DDX3 binds to tyrosinase in H2 O2 -treated conditions. SK-Mel-2 cells were transiently
Figure 3. DDX3
transfected binds
with to tyrosinase
WTDDX3 for 48inh H 2O2then
and -treated conditions.
treated with 0.1SK-Mel-2
mM H2 O cells were transiently
2 for 4 h before harvesting
transfected with WTDDX3 for 48 h and then treated with 0.1 mM H 2O2 for 4 h before harvesting
for tyrosinase immunoprecipitation and Western blot experiment (A). The histogram shows relative
for tyrosinase immunoprecipitation and Western blot experiment (A). The histogram shows rela-
expression levels of tyrosinase in H2 O2 -treated SK-Mel-2 cells with or without DDX (B). Data were
tive expression levels of tyrosinase in H2O2-treated SK-Mel-2 cells with or without DDX (B). Data
analyzed using two-way ANOVA followed by multiple comparisons post hoc test. ** p < 0.001,
were analyzed using two-way ANOVA followed by multiple comparisons post hoc test. ** p <
0.001,p***
*** < 0.0001 compared
p < 0.0001 to control
compared cell. ##
to control p <##0.001
cell. indicates
p < 0.001 differences
indicates compared
differences with H
compared 2 O2 -treated
with
cells after transfection of WTDDX3.
H2O2-treated cells after transfection of WTDDX3.
 Figure 3. DDX3 binds to tyrosinase in H2O2-treated conditions. SK-Mel-2 cells were transiently
transfected with WTDDX3 for 48 h and then treated with 0.1 mM H2O2 for 4 h before harvesting
for tyrosinase immunoprecipitation and Western blot experiment (A). The histogram shows rela-
tive expression levels of tyrosinase in H2O2-treated SK-Mel-2 cells with or without DDX (B). Data
were analyzed using two-way ANOVA followed by multiple comparisons post hoc test. ** p <
Molecules 2022, 27, 7010 6 of 11
0.001, *** p < 0.0001 compared to control cell. ## p < 0.001 indicates differences compared with
H2O2-treated cells after transfection of WTDDX3.

Figure 4.
Figure Aproposed
4. A proposedmodel
modelof ofDDX3
DDX3for
forupregulating
upregulatingmelanogenesis
melanogenesisvia
viabinding
bindingtototyrosinase
tyrosinaseinin
H22OO2-treated
H 2 -treatedSK-Mel-2
SK-Mel-2cells.
cells.

3. Discussion
3. Discussion
DDX3 helicase can be a tumor suppressor in many different cancers, including breast,
DDX3 helicase can be a tumor suppressor in many different cancers, including breast,
lung, cervical, colorectal, and pancreatic cancers [10,33–35]. Moreover, DDX3 might play
lung, cervical, colorectal, and pancreatic cancers [10,33–35]. Moreover, DDX3 might play
different roles in the same type of cancer. For example, a decreased level of DDX3 has
different roles in the same type of cancer. For example, a decreased level of DDX3 has
been found in hepatocellular carcinoma (HCC) caused by hepatitis B virus (HBV), but
been found in hepatocellular carcinoma (HCC) caused by hepatitis B virus (HBV), but not
not in HCC caused by HCV [36]. DDX3 also plays dual roles in breast cancer [37] and
in HCC caused
colorectal bypatients
cancer HCV [36]. DDX3
[38]. also
Up to plays
now, dual
there roles
has beeninno
breast
exactcancer [37] andabout
explanation colorec-
the
tal cancer patients [38]. Up to now, there has been no exact explanation about the dual
dual role of DDX in a variety of cancers. DDX3 is involved in the cell signaling pathway role
of Wnt/β-catenin. It can affect the Wnt regulation cascade, which is crucial for DDX30 s
functions in cancer development [39]. DDX3 also modulates cell adhesion and represses
E-cadherin expression, resulting in increased cell migration and thus promoting tumor
progression [40]. Different roles of DDX helicases might be associated with mutations
in the DDX helicase (as can be exemplified by [41]) or virus infections, particularly HCV
or HBV. DDX3 knockdown with short interfering RNA (shRNA) or small molecules can
suppress cell motility and reduce the metastatic potential in cancer cells and a mouse
model [34]. The localization of DDX3 within the cell might also lead to different DDX3
functions. Usually, DDX3 accumulates in the cytoplasm of the cell. However, DDX3 can
also be exported from the nucleus to the cytoplasm during tumor progression. DDX3
helicase is a nucleo-cytoplasmic shuttling protein predominantly localized in the cytoplasm
of non-malignant cells. It has been suggested that its localization is altered during cell
transformation and that such alteration could even contribute to malignancy [42,43].
Hydrogen peroxide (H2 O2 ) production due to oxidative stress is associated with apop-
tosis and melanogenesis in melanocytes [26]. It has been reported that some suppressors
such as N-feruloyl serotonin can inhibit H2 O2 -induced melanogenesis and apoptosis [44].
DDX3 helicase can be a tumor suppressor in many different cancer types. However, the
role of DDX3 in melanogenesis has not been reported yet. This is the first report about
the involvement of DDX3 in melanogenesis. Results of this study showed that DDX3
upregulated melanogenesis in H2 O2 -treated SK-Mel-2 human melanoma cells. Our results
also suggested that DDX3 might promote melanogenesis in H2 O2 -treated SK-Mel-2 human
Molecules 2022, 27, 7010 7 of 11

melanoma cells by binding to tyrosinase. The identification of DDX3 binding partners

indicated the function of DDX3 in melanogenesis and is notably novel for the mechanism
regulating pigmentation. DDX3 has been reported to be involved in various biogenesis
processes as a potential target for cancer treatment or regulation of apoptosis. Another
study has reported that cirsimaritin could stimulate melanogenesis in B16F10 cells and
SK-Mel-2 human epidermal melanocytes by upregulating MITF and tyrosinase expression
and CREB phosphorylation [45].
This study suggests a novel idea of using DDX3 molecular therapy for skin diseases
such as skin cancer or vitiligo disease. However, this study did not clearly elucidate the
molecular mechanism. In addition, our results were limited to cell lines and melanogenesis-
inducing agents. Along with binding proteins, DDX might promote other functions due to
stabilization of cell signaling. DDX3 has a variety of complex functions in gene expression
systems, viral transmission, and cancer diseases. It is known that DDX3 determines various
and contrasting types of cancer [34]. Correlations between DDX3 protein expression levels
and various cancer cells have been reported in several studies. In patients with severe lung
cancer, the expression level of DDX3 protein is low. A lower expression level of DDX3
has also been reported in patients with smoking history [34]. In contrast, the expression
level of DDX3 protein is high in patients with brain tumors, suggesting that it might be
a pathological cause of brain cancer. In liver cancer, the expression of DDX protein is
more common in men than in women. At the cellular level, DDX is mainly present in
the cytoplasm of cancer cells [46]. With regard to cancer diseases, DDX3 has been found
to have a variety of functions when it is combined with other proteins. Its main binding
proteins include p53, beta-catenin, and WNT, resulting in various signaling mechanisms.
p53 is known as a powerful cancer inhibitory protein that can directly activate DDX3
protein [47,48]. This phenomenon is known to control cell proliferation and stabilization
of genetic expression [49,50]. DDX3 can act on CK1-alpha in vertebrates and bind to beta-
catenin, thus promoting cancer and activating beta-catenin through multiple signaling
processes [38,40,51]. Furthermore, abnormal and non-expression of DDX3 can cause WNT
to form polymers, leading to cancer [52,53]. Further studies are needed to determine the
biological function of DDX3 in melanogenesis as well as the mechanism involved in the
effect of DDX3 in promoting melanogenesis under oxidative stress.
In this study, 0.1 mM hydrogen peroxide was used to induce melanogenesis in SK-
Mel-2 human cancer melanoma cells by increasing melanin content and the expression of
the melanin-synthesizing gene tyrosinase. Interestingly, H2 O2 treatment also increased the
promoter activity of DDX3. Under oxidative stress induced by H2 O2 , overexpression of
DDX3 increased melanin content and tyrosinase expression. By immunoprecipitation with
an antityrosinase antibody, it was confirmed that DDX3 could interact with tyrosinase and
that such interaction was increased under oxidative stress. A proposed model for DDX3
upregulation of melanogenesis under the H2 O2 -treated conditions was presented. This is
the first report about the effect of DDX3 on melanogenesis and its potential for developing
a molecular therapy to treat skin diseases. Further studies are needed to fully understand
the biological effect of DDX3 on the melanogenesis pathway.

4. Materials and Methods

4.1. Chemicals and Antibodies
Hydrogen peroxide (Cat. no. 216763, Sigma-Aldrich, St. Louis, MO, USA), L-DOPA
(Cat. no. 333786, Sigma-Aldrich, St. Louis, MO, USA), and protease inhibitor cocktail
were purchased from Merck KGaA (Darmstadt, Germany). Dulbecco’s modified Eagle’s
medium (DMEM), fetal bovine serum (FBS), and penicillin–streptomycin were obtained
from Gibco BRL (Eggenstein, Germany). The following antibodies were used to detect
gene expression: mouse polyclonal IgG anti-DDX3 (produced in rabbits and confirmed
by commercial anti-DDX3 antibodies purchased from Novus Biologicals, Littleton, CO,
USA; size 73 kDa), mouse monoclonal IgG anti-tyrosinase (SC56505, size 17 and 35 kDa),
mouse monoclonal IgG anti-β-actin (Biovision 3598-100, size 44 kDa), mouse monoclonal
Molecules 2022, 27, 7010 8 of 11

IgG anti-Myc tag (Millipore 05–724), and mouse monoclonal IgG anti-C-myc (SPM237)
(SC7277, size 67 kDa).

4.2. Cell Culture

SK-Mel-2 cells were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea)
and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10%
fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 ◦ C in a humidified at-
mosphere with 5% CO2 . Cells suspended in culture medium containing 10% FBS were
transferred into a flat-bottom 96-well plate. The number of cells/well was calculated to
reach the same confluence for all cell culture dishes: 9 × 103 cells/well for 96-well plates,
2.5 × 105 cells/well for 6-well plates, 7.5 × 105 cells/well for 60 mm dishes, and 2 × 106
for 100 mm dishes. Cells passaged fewer than 10 times were used at about 70–80% con-
fluence. Cells were treated with H2 O2 for 1 h to induce melanogenesis. Cell viability
was determined with an established colorimetric method using 2,3-bis(2-methoxy-4-nitro-
5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) obtained from WelGene
(Seoul, Korea).

4.3. Melanin Content Measurement

SK-Mel-2 cells were seeded into 6-wells plates and cultured for more than 24 h. After
reaching 50–70% confluence, cells were treated H2 O2 at different concentrations with a
time course. Cells were then washed with PBS and harvested by trypsinization. Cell pellets
were solubilized with 2 N NaOH at 80 ◦ C for 2 h and centrifuged at 12,000 rpm for 10 min
at 4 ◦ C. The optical density (OD) of the sample was then measured at 420 nm with an
ELISA VersaMax Microplate Reader (Versa Max, Remington). The amount of melanin was
normalized to the total protein amount in each sample.

4.4. Tyrosinase Activity Assay

The tyrosinase activity assay was carried out as previously described. Briefly, SK-Mel-2
cells were seeded into 6-well plates and cultured for more than 24 h to reach 50–70%
confluence before treatment. The number of cells/well was calculated to reach the same
confluence of 2.5 × 105 cells/well for 6-well plates. After preparing cell culture, cells were
harvested, washed with cold PBS, and then lysed with 450 µL of 50 mM PBS (pH 6.8)
containing 1% Triton X-100. The mixture was then frozen in dry ice methanol. A volume
of 50 µL 10 mM DOPA was added to each sample followed by incubation at 37 ◦ C for 4 h.
Cells were treated with H2 O2 for 1 h to induce melanogenesis. The optical density (OD)
of the sample was then measured at 475 nm with an ELISA VersaMax Microplate Reader
(Versa Max, Remington).

4.5. Luciferase Reporter Assay

To measure reporter gene activation (promoter activity), SK-Mel-2 cells were seeded
into 12-well plates at a density of 2 × 105 cells/well and transiently transfected with 0.5 µg
of a different expression vector for 24 h (for SK-Mel-2). Cells were co-transfected with 20 ng
renilla luciferase expression vector (pRL-TK) to normalize the transfection efficiency. After
incubation, cells were lysed with 250 µL reporter lysis buffer (Promega, Madison, WI, USA).
Luciferase activity from 50/100 µL lysate was measured using a SpectraMax Luminometer
(Molecular Devices, Sunnyvale, CA, USA).

4.6. Immunoprecipitation (IP)

After seeding into 100 mm dishes, SK-Mel-2 cells were cultured for more than 48 h
to reach a confluence at 70% or more before they were transfected with 15 µg pcDNA3-
myc/DDX3 vector for 24 h. Cells then were treated with 0.1 mM H2 O2 for 4 h or 8 h and
harvested to perform IP. Cell pellets were obtained after washing with PBS and centrifuging.
Cell lysate was then used to quantify protein concentration. Each sample (1.5 mg of protein
lysate) was mixed with antityrosinase antibody in a total volume of 500 µL lysis buffer,
Molecules 2022, 27, 7010 9 of 11

a complete protease inhibitor cocktail, and 50 µL Pierce™ Protein A/G Sepharose beads
(Calbiochem, San Diego, CA, USA). Immunoprecipitated proteins were then subjected to
SDS-PAGE followed by immunoblotting using specific antibodies.

4.7. Statistical Analysis

For melanogenesis activation experiments, statistically significant differences were
identified using one-way ANOVA for repeated measures followed by a multiple com-
parisons post hoc test. Experiments assessing melanin content, tyrosinase activity, and
expression were analyzed using a two-way ANOVA with a between-subject factor of
the H2 O2 treatment and a between subject factor of DDX3. Statistical analyses were per-
formed using Origin (Ver 8.5; Origin Lab, Northampton, MA, USA). Data represent the
mean ± SEM of at least three individual experiments. Differences between the group
means were considered significant when p < 0.05.

Author Contributions: Conceived of the presented idea and developed the theory and performed the
computations, S.E. and S.L.; verified the analytical methods, H.D.Y. and J.L. (Jiwon Lee); supervised
the findings of this work, S.-G.L. and J.L. (Junho Lee). All authors have read and agreed to the
published version of the manuscript.
Funding: This research was supported by the BK21 Fostering Outstanding Universities for Research
(FOUR) funded by the Ministry of Education (MOE, Korea) and National Research Foundation of
Korea (NRF).
Institutional Review Board Statement: The study was approved by the Institutional Review Board
of Chonnam National University (CNU IACUC-YB-201607).
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are contained within the article.
Conflicts of Interest: The authors declare no conflict of interest.

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