The compound 2-(naphthalene-2-thio)-5,8-dimethoxy-1,4-naphthoquinone induces apoptosis via reactive oxygen species-regulated mitogen-activated protein kinase, protein kinase B, and signal transducer and activator of transcription 3 signaling in human gastric cancer cells

Article abstract of DOI:10.1002/ddr.21442

(Table presented.). It is reported that 1,4-naphthoquinones and their derivatives have potent antitumor activity in various cancers, although their clinical application is limited by observed side effects. To improve the therapeutic efficacy of naphthoquinones in the treatment of cancer and to reduce side effects, we synthesized a novel naphthoquinone derivative, 2-(naphthalene-2-thio)-5,8-dimethoxy-1,4-naphthoquinone (NTDMNQ). In this study, we explored the effects of NTDMNQ on apoptosis in gastric cancer cells with a focus on reactive oxygen species (ROS) production. Our results demonstrated that NTDMNQ exhibited the cytotoxic effects on gastric cancer cells in a dose-dependent manner. NTDMNQ significantly induced mitochondrial-related apoptosis in AGS cells and increased the accumulation of ROS. However, pre-treatment with N-acetyl-L-cysteine (NAC), an ROS scavenger, inhibited the NTDMNQ-induced apoptosis. In addition, NTDMNQ increased the phosphorylation of p38 kinase and c-Jun N-terminal kinase (JNK) and decreased the phosphorylation of extracellular signal-regulated kinase (ERK), protein kinase B (Akt), and Signal Transducer and Activator of Transcription 3 (STAT3); these effects were blocked by mitogen-activated protein kinase (MAPK) inhibitor and NAC. Taken together, the present findings indicate that NTDMNQ-induced gastric cancer cell apoptosis via ROS-mediated regulation of the MAPK, Akt, and STAT3 signaling pathways. Therefore, NTDMNQ may be a potential treatment for gastric cancer as well as other tumor types.

Full text of DOI:10.1002/ddr.21442

Received: 9 May 2018
Revised: 12 June 2018
Accepted: 18 June 2018
DOI: 10.1002/ddr.21442
R E S E A R C H A R T I C L E
The compound 2-(naphthalene-2-thio)-5,8-dimethoxy-
1,4-naphthoquinone induces apoptosis via reactive oxygen
species-regulated mitogen-activated protein kinase, protein
kinase B, and signal transducer and activator of transcription
3 signaling in human gastric cancer cells
Jia-Ru Wang1 | Gui-Nan Shen1 | Ying-Hua Luo2 | Xian-Ji Piao3 | Meng Shen4 |
Chang Liu1 | Yue Wang1 | Ling-Qi Meng1 | Yi Zhang1 | Hao Wang1 | Jin-Qian Li1 |
Wan-Ting Xu1 | Yang Liu1 | Hu-Nan Sun1 | Ying-Hao Han1 | Mei-Hua Jin1 |
Long-Kui Cao4 | Cheng-Hao Jin1,4
Enabling Technologies
Strategy, Management & Health Policy
Hit, Lead &
Candidate Discovery
Preclinical Research
& Development
Clinical
Research
Post-Market
Research
1Department of Biochemistry and Molecular Biology, College of Life Science & Technology, Heilongjiang Bayi Agricultural University, Daqing, China
2College of Animal Science & Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, China
3Department of Gynaecology and Obstetrics, The Fifth Affiliated Hospital of Harbin Medical University, Daqing, China
4College of Food Science & Technology, Heilongjiang Bayi Agricultural University, Daqing, China
Correspondence
It is reported that 1,4-naphthoquinones and their derivatives have potent antitumor activity
Cheng-Hao Jin, College of Life Science and
in various cancers, although their clinical application is limited by observed side effects. To
improve the therapeutic efficacy of naphthoquinones in the treatment of cancer and to
reduce side effects, we synthesized a novel naphthoquinone derivative, 2-(naphthalene-
2-thio)-5,8-dimethoxy-1,4-naphthoquinone (NTDMNQ). In this study, we explored the
effects of NTDMNQ on apoptosis in gastric cancer cells with a focus on reactive oxygen spe-
cies (ROS) production. Our results demonstrated that NTDMNQ exhibited the cytotoxic
effects on gastric cancer cells in a dose-dependent manner. NTDMNQ significantly induced
mitochondrial-related apoptosis in AGS cells and increased the accumulation of ROS. How-
ever, pre-treatment with N-acetyl-L-cysteine (NAC), an ROS scavenger, inhibited the
NTDMNQ-induced apoptosis. In addition, NTDMNQ increased the phosphorylation of p38
kinase and c-Jun N-terminal kinase (JNK) and decreased the phosphorylation of extracellular
signal-regulated kinase (ERK), protein kinase B (Akt), and Signal Transducer and Activator of
Transcription 3 (STAT3); these effects were blocked by mitogen-activated protein kinase
(MAPK) inhibitor and NAC. Taken together, the present findings indicate that
NTDMNQ-induced gastric cancer cell apoptosis via ROS-mediated regulation of the MAPK,
Akt, and STAT3 signaling pathways. Therefore, NTDMNQ may be a potential treatment for
gastric cancer as well as other tumor types.
Technology, Heilongjiang Bayi Agricultural
University, Daqing 163319, China.
Email: jinchenghao3727@qq.com
and
Long-Kui Cao, College of Food Science &
Technology, Heilongjiang Bayi Agricultural
University, Daqing 163319, China.
Email: caolongkui2013@163.com
Funding information
Heilongjiang Bayi Agricultural University,
Grant/Award Number: XYB2012-14;
Postdoctoral Scientific Research Foundation of
Heilongjiang Province of China, Grant/Award
Number: LBH-Q13132; Natural Science
Foundation of Heilongjiang Province of China,
Grant/Award Number: LC2015036
Wang, Shen, and Luo contributed equally to this work.
Drug Dev Res. 2018;1–12.
wileyonlinelibrary.com/journal/ddr
© 2018 Wiley Periodicals, Inc.
1

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WANG ET AL.
KEYWORDS
2-(naphthalene-2-thio)-5,8-dimethoxy-1,4-naphthoquinone, apoptosis, gastric cancer cells,
MAPK signaling pathway, reactive oxygen species
At present, some anticancer drugs have been shown to increase the level
of intracellular ROS, repressing cancer cell growth via modulation of the
1
| INTRODUCTION
Gastric cancer (GC) is a common malignant tumor of the digestive sys-
tem. According to a recent report, gastric cancer is the third leading
cause of cancer death and the fifth most frequent cancer worldwide,
particularly in Eastern Asia (Luo et al., 2016). Nearly two-thirds of new
diagnoses take place in developing countries, with 42% occurring in
China alone (Kong et al., 2016; Wu et al., 2017). At present, the most
widely used treatments for gastric cancer include surgery, chemother-
apy and radiotherapy (Zhang et al., 2016). Nevertheless, the overall
prognosis is frequently poor; the relative 5-year survival rate for gas-
tric cancer is lower than 30% (Zheng et al., 2014). A variety of chemo-
therapy drugs, including 5-fluorouracil (5-FU) and cisplatin have been
used to treat gastric cancer; however, these drugs not only affect can-
cer cells but also normal cells (Van Cutsem et al., 2006; Wang et al.,
2016). As a result, there is a need to identify novel, more effective
and less toxic antitumor agents.
MAPK and Akt signaling pathways, the Bcl-2 associated X (Bax)/B-cell
lymphoma-2 (Bcl-2) ratio, and cysteinyl aspartate-specific proteinase-3
(caspase-3) protein expression in clinical practice (Tsai et al., 2017; Yin
et al., 2017). Together, these finding indicate that ROS could adjust cell
growth and death via regulated apoptotic-related signaling pathways.
The compound 1,4-naphthoquinone is an organic compound that is
reported to exhibit a variety of pharmacological properties involving anti-
inflammatory, antimicrobial, antiviral and antitumor effects (Bhasin, Chet-
tiar, Etter, Mok, & Li, 2013; Coelho-Cerqueira, Netz, Canto, Pinto, &
Follmer, 2014; Hafeez et al., 2013). For example, 1,4-naphthoquinone
fused Gold (i) N-heterocyclic carbene complexes could enhance cancer
cell death and reduce tumor burden by perturb antioxidant homeostasis
(McCall et al., 2017). Although 1,4-naphthoquinone derivatives may
inhibit lung, breast and prostate cancer cell proliferation, they are not
widely used as anticancer drugs because of their high toxicity and side
effects (Bolton, Trush, Penning, Dryhurst, & Monks, 2000; O'Brien,
1991). Some studies have shown that naphthoquinone derivatives strong
toxicity may be because of the hydroxyl in their structure. The Dihydroxy
of C5 and C8 make quinones less electrophilic, but it seems to increase
the efficiency of redox cycling and result in greater toxicity (Ollinger &
Brunmark, 1991). Therefore, substitution of hydroxyl structure is an
effective way to reduce the toxic effect. Indeed, our previous
study showed that 1,4-naphthoquinone derivatives—2-butylsulfinyl-1,-
4-naphthoquinone (BSNQ), and 2-octylsulfinyl-1,4-naphthoquinone
(OSNQ) considerably decreased the cytotoxicity and increased antitumor
activity in HCC cells (Liu, Shen et al., 2017).
Apoptotic pathways are activated by various signals, including the
MAPK, protein kinase B (Akt), and STAT3 signaling pathways (Kim
et al., 2014; Sampson et al., 2015). The MAPK signaling pathways
involving ERK, p38, and JNK play crucial roles in cell growth, develop-
ment, differentiation, apoptosis, and cell growth arrest (Yu, Kim, Yu, &
Kim, 2013) Many studies have suggested that Akt is activated by
drugs, extracellular cytokines, pressure, and other factors, such as the
phosphatidylinositol 3-kinase (PI3K) signaling pathway (Yang, Guo,
Shao, & Gao, 2017). Activated Akt can induce tumor cell apoptosis via
modulation of downstream signaling (Costa et al., 2015; Porta,
Paglino, & Mosca, 2014). The STAT3 signaling pathway is constitu-
tively activated in many cancer cells, such as gastric and breast cancer
(Samsonov et al., 2013; Zhu, Wang, Guo, Liu, & Dong, 2016). There-
fore, findings have shown that the MAPK, Akt, and STAT3 signaling
pathways are viable therapeutic targets for cancer treatment.
In the present study, to better improve the antitumor activity and
reduce toxicity of 1,4-naphthoquinone derivatives, we substituted the
dihydroxy of C5 and C8 with methoxy, and then synthesized a
novel naphthoquinone derivative named 2-(naphthalene-2-thio)-
5,8-dimethoxy-1,4-naphthoquinone (NTDMNQ). Then, we investigated
the cytotoxic effects, apoptotic effects, ROS generation effects, and
underlying mechanisms of NTDMNQ in gastric cancer cells.
ROS are generated during the metabolism of oxygen, which gener-
ates free radicals, including hydroxyl radicals (OH−), superoxide anions
(O2−), singlet oxygen, and hydrogen peroxide (H2O2) (Günes-Bayir et al.,
2017; Luo et al., 2016). Previous studies have reported that the produc-
tion of intracellular ROS is vital for redox balance, cell signaling, and cell
growth. Under conditions of oxidative stress, increased ROS generation
may overcome cellular antioxidant defenses and induce cancer cell apo-
ptosis (Chen et al., 2017). Additionally, the accumulation of intracellular
ROS also initiates the destruction of the mitochondrial membrane, indi-
rectly bringing about the induction of apoptosis (Li, Zhang, & Wang,
2017). Therefore, the search for novel anticancer therapies may primarily
concentrate on intercellular apoptosis-stimulating agents that are medi-
ated by various pathways. One well-known molecular pathway that
induces apoptosis is the production of ROS (Ren, Yin, Yu, & Xiao, 2017).
2
| MATERIALS AND METHODS
2.1 | Chemicals and reagents
The chemicals used in compound synthesis were purchased from the
Sigma-Aldrich Chemical Company (St. Louis, MO). 5-FU (MedChem
Express; Princeton, NJ) and NTDMNQ were dissolved in 100%
dimethyl sulfoxide (DMSO, St. Louis, MO) to prepare a 20 mM stock
solution, and stored at −20 ꢀC. Other chemicals were analytical grade.

WANG ET AL.
3
Roswell Park Memorial Institute (RPMI) 1,640, dulbecco's modified
(40.3 mol) in CH3CN (450 ml) and CHCl3 (150 ml). The resulting mix-
ture was then stirred at room temperature for 1 hr. The organic layer
was separated, dried over sodium sulfate, and concentrated in vacuo.
The residue was recrystallized from MeOH to give the title compound
5,8-dimethoxy-1,4-naphthoquinone (DMNQ; Shen et al., 2009; Sun
et al., 2016).
eagle's medium (DMEM), fetal bovine serum (FBS), penicillin, and strepto-
mycin were purchased from Gibco (Waltham, MA). Annexin V apoptosis
detection kit, 20,70-dichlorofluorescein diacetate (DCFH-DA) and NAC
were obtained from the Beyotime Institute Biotechnology (Shanghai,
China). ECL was obtained from Thermo Fisher Scientific (Waltham, MA).
All antibodies were purchased from Santa Cruz Biotechnology Inc.
(Dallas, TX).
2.5 | Synthesis of 2-(naphthalene-2-thio)-
5,8-dimethoxy-1,4-naphthoquinone
2.2 | Synthesis of naphthazarin
The 1-naphthalenethiol (1.65 mmol) was added to a solution of DMNQ
(1.38 mmol) and MeOH (30 ml). The mixture was then stirred at room
temperaturefor 4 hr. A solution of sodium dichromate (Na2Cr2O7ꢁ2H2O,
0.76 mmol) and 98% H2SO4 (0.23 mmol) was then added and stirred for
2 min. The acidic solution was extracted with dichloromethane (CHCl2,
60 ml). The organic layer was washed with brine (40 ml), dried over
anhydrous sodium sulfate, filtered, and then concentrated under reduced
pressure. The residue was separated by chromatography (hexane:
EtOAc = 1:4) to yield the title compound 2-(naphthalene-2-thio)-5,-
8-dimethoxy-1,4-naphthoquinone (NTDMNQ).
AlCl3 (142 g) and NaCl (28.3 g) were melted at 150–155 ꢀC, and a
mixture of 1,4-dimethoxybenzene (0.12 mol) and maleic anhydride
(0.24 mol) was added to the melted mixture. The temperature was
sustained at 170–175 ꢀC for 1–2 min and the dark red melt was
allowed to cool. Next, the distilled water (1,400 ml) and 98% HCl
(100 ml) were added to the melt mixture and continuous mixed for
12 hr to generate naphthazarin (Chang et al., 2017).
2.3 | Synthesis of 1,4,5,8-tetramethoxynaphthalene
Nuclear magnetic resonance (NMR) spectra were recorded on a
JNM-AL 400 (400 MHz). Chemical shifts (d) are given in ppm down-
field from tetramethylsilane (TMS) as the internal standard. MS spec-
tra were collected with an AB SCIEX API 2000 LC/MS/MS (Applied
Biosystems, Inc., Carlsbad, CA) and LCMS-IT-TOF (Shimadzu Scientific
Inc., Beijing, China).
The mixture of naphthazarin (19.0 g), tetrahydrofuran (C4H8O,
200 ml), Na2S2O4 (10.6 g), distilled water (200 ml), and tetrabutylam-
monium bromide (C16H36BrN, 2.0 g) were mixed for 1 hr. Then, a
solution of C2H6O4S (25 ml), NaOH (40 ml), and Na2S2O4 (10.6 g)
were added to the mixture stirred for 22 hr at room temperature. The
1,4,5,8-tetramethoxynaphthalene was then recovered by filtration
and recrystallized from petroleum ether (b.p. 90–120 ꢀC) (Huot &
Brassard, 2011; Xu, Guo, Xu, & Li, 2006).
2.6 | Cell culture
The nine GC cells and two normal cells were purchased from American
Type Culture Collection (ATCC; Rockville, MD). Because the liver is the
important target organ for drug toxicity testing, and human lung fibro-
blasts (IMR-90) are extracted from embryonic cells, their primary proper-
ties has not transformed, which can directly reflect the toxicity effects in
the toxicity study, we chosen the QSG-7701 and IMR-90 as controls.
2.4 | Synthesis of 5,8-dimethoxy-1,4-
naphthoquinone
A solution of cerium ammonium nitrate (CAN, 54 g) in water was
added dropwise to a solution of 1,4,5,8-tetramethoxynaphthalene
FIGURE 1 The synthetic process and chemical structure identification of NTDMNQ

4
WANG ET AL.
FIGURE 2 Effects of NTDMNQ on cell viability in human gastric cancer cells. Nine human gastric cancer cell lines (AGS, MKN-28, MKN-45, NCI-
N87, SNU-216, SNU-484, SNU-668, YCC-1, YCC-6, and YCC-16) and two normal cells (IMR-90 and QSG-7701) were treated with different
concentrations (0.1, 0.3, 1.0, 3.0 and 30 μmol/L) of 5-FU or NTDMNQ for 24 hr. Error bars indicate means ꢂ SD of three independent
experiments (ap < 0.05, bp < 0.01, cp < 0.001 indicate significant differences)
AGS, MKN-28, MKN-45, SNU-216, SNU-484, IMR-90, and QSG-7701
cells were maintained in RPMI 1640 medium. And NCI-N87, YCC-1,
YCC-6, YCC-16 cells were grown in DMEM medium. Medium was sup-
plemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml strepto-
mycin. All cells were cultured in humidified atmosphere of 5% CO2 at
37 ꢀC, and cells were maintained at 80% confluence level.
Instruments Inc., Winooski, VT). Cell viability was expressed as the per-
centage change in absorbance compared to control values.
2.8 | Cell apoptosis analysis
Cell apoptosis was detected by the Annexin V apoptosis detection kit
and flow cytometry. AGS cells were seeded in 6-well plates (1 × 105
cells/well), and treated with 1 μmol/L 5-FU and NTDMNQ at 3, 6,
12, and 24 hr. Following treatment, the cells were collected and
2.7 | Cell viability assay
Cell viability was determined using the MTT assay. Gastric cancer cells
(AGS, MKN-28, MKN-45, NCI-N87, SNU-216, SNU-484, YCC-1, YCC-6,
and YCC-16) and normal human embryo lung IMR-90 cells and liver
QSG-7701 cells were collected during the logarithmic phase of growth.
All cells were seeded in 96-well plates at 1 × 104 cells/well, then treated
with various doses (0.1, 0.3, 1.0, 3.0, and 30 μmol/L) of 5-FU and
NTDMNQ for 24 hr. Adding the MTT solution (20 μl; 5 mg/ml) and cul-
tured for 2 hr at 37 ꢀC. Formazan crystals were lysed in 100 μl of DMSO,
and cells were shocked for 10 min. Absorbance was then measured at a
wavelength of 490 nm with an automatic micro-plate reader (BioTek
TABLE 1 IC50 value of 5-FU and NTDMNQ in gastric cancer
5-FU
NTDMNQ
Cell name
(μM)
(μM)
AGS
25.07 ꢂ 3.02
17.96 ꢂ 3.39
13.75 ꢂ 1.84
27.89 ꢂ 4.47
24.67 ꢂ 3.49
19.29 ꢂ 2.62
17.81 ꢂ 1.67
26.17 ꢂ 3.29
27.59 ꢂ 9.91
0.42 ꢂ 0.06
1.26 ꢂ 0.05
0.82 ꢂ 0.08
0.89 ꢂ 0.13
1.44 ꢂ 0.18
1.53 ꢂ 0.19
1.30 ꢂ 0.15
0.80 ꢂ 0.11
1.58 ꢂ 0.69
MKN-28
MKN-45
SNU-216
SNU-484
NCI-N87
YCC-1
YCC-6
YCC-16

WANG ET AL.
5
FIGURE 3 Induction of apoptosis caused by NTDMNQ in AGS cells. (a) AGS cells were treated with 1 μmol/L of NTDMNQ and 5-FU for
different time periods (3, 6, 12, and 24 hr), and stained with Annexin V-FITC/PI solution. The morphology and fluorescence intensity were
observed under a fluorescence microscope (magnification: ×200). (b) Quantification of fluorescent intensity. (c) AGS cells were treated with the
1 μmol/L NTDMNQ and 5-FU for 3, 6, 12, and 24 hr, and stained with Annexin V-FITC/PI solution and analyzed by flow cytometer.
(d) Quantification of the percentage of apoptotic cells. (e) Apoptosis-related proteins Bcl-xl, Bax, cleaved-PARP, and cleaved-caspase-3 expression
levels were measured by western blotting. (f ) Quantification of the band density was analyzed by imageJ software. Error bars indicate
means ꢂ SD of three independent experiments (ap < 0.05, bp < 0.01, cp < 0.001 indicate significant differences)
washed once in phosphate-buffered saline (PBS). Cells were resus-
pended in 195 μl Annexin V binding buffer, dual staining was per-
formed with 3 μl Annexin V-FITC and 2 μl PI, and incubated for
20 min at the room temperature (RT) in the dark. Apoptotic cells were
analyzed using the EVOS FL Auto Cell Imaging System (Thermo Fisher
Scientific, Inc.) and flow cytometer (Beckman Coulter, Inc., Brea, CA).
membranes. Membranes were blocked for 1 hr with 5% skim milk at RT,
and then incubated overnight with primary antibodies against mouse
monoclonal α-tubulin, Bax, Bcl-xl, Poly (ADP-ribose) polymerase-1
(PARP-1), cleaved caspase-3, p-ERK, p-JNK, JNK, p-p38, p-STAT3 (Tyr
705), and STAT3. Rabbit polyclonal ERK2, p38α/β, Akt, p-Akt at 4 ꢀC (all
from Santa Cruz Biotechnology, Inc., Dallas, TX). The membranes were
washed in TBST, and incubated with Goat Anti-Rabbit IgG and Goat
Anti-Mouse IgG secondary antibodies at rt for 2 hr. Following incuba-
tions, Pierce ECL was added, bands were detected using an Amersham
Imager 600 (GE, Fairfield, CT) and evaluated using ImageJ software. Rela-
tive protein expression in treated cells was compared to the control
group. The endogenous control was α-tubulin.
2.9 | Western blotting analysis
Cells were extracted in lysis buffer, and then centrifuged for 30 min at
4
ꢀC and 12,000 × g. The supernatants of 30 μg were electrophoresed
on 8%–12% SDS-PAGE gels and then transferred onto nitrocellulose

6
WANG ET AL.
NTDMNQ: 1H NMR (CDCl3, 400 MHz) δ 8.09 (s, 1H), 7.91 (m, 2H),
7.85 (d, J = 6.0 Hz, 1H), 7.58 (m, 2H), 7.50 (d, J = 6.8 Hz, 1H), 7.32 (d,
J = 7.2 Hz, 1H), 7.20 (d, J = 7.2 Hz, 1H), 5.97 (s, 1H), 3.99 (s, 3H), 3.91 (s,
3H); 13C NMR (CDCl3, 150 MHz) δ 182.00 (C-1), 181.75 (C-4), 154.37 (C-
5), 153.37 (C-8), 153.7 (C-2), 134.76 (C-90), 131.26 (C-10), 130.02 (C-100),
128.83 (C0-2), 127.96 (C0-8), 127.90 (C-50), 127.7 (C-3), 121.27 (C-7),
120.96 (C-6), 120.66 (C-10), 119.60 (C-9), 56.98 (OCH3), 56.89 (OCH3),
127.68 (C0-4), 126.96 (C0-3), 121.27 (C0-7), 121.08 (C0-6); MS: m/z
399.31 (M + Na)+
2.10 | Measurement of ROS levels
The accumulation of intracellular ROS was determined by using
DCFH-DA staining, and many studies have shown that DCFH-DA stain-
ing is one of the most widely used techniques for directly measuring the
redox state of cells (Afri, Frimer, & Cohen, 2004; Eruslanov & Kusmartsev,
2010). AGS cells were treated with NTDMNQ (1 μmol/L) for 3, 6, 12, and
24 hr, followed by centrifuged for 5 min at 6,000 × g. Cells were washed
once in PBS, and staining with 10 μmol/L DCFH-DA for an additional
30 min at 37 ꢀC in darkness, then analyzed by flow cytometer.
These results confirm the successful synthesis of NTDMNQ.
2.11 | Statistical analysis
3.2 | NTDMNQ inhibited the viability of gastric
cancer cells
Statistical analysis was performed using SPSS 19.0 software, and results
are represented as means ꢂ SD and analysis of variance (one-way
ANOVA). The value of p < .05 was considered statistically significant.
As shown in Figure 2a, NTDMNQ significantly inhibited the growth of nine
different types of gastric cancer cells in a dose-dependent manner; the IC50
values of 5-FU and NTDMNQ for each cell line show in Table 1. In addition,
there were no obvious cytotoxic effects of NTDMNQ compared with
5-FU-treated group on IMR-90 and QSG-7701 cells (Figure 2b). Because
the AGS cells showed higher sensitivity to NTDMNQ compared with other
gastric cancer cells, we chose the AGS cells for further study.
3
| RESULTS
3.1 | Identification of the compound's structure
To synthesize 1,4-naphthoquinone derivatives with improved antican-
cer activity, we performed a series of steps to modify its chemical
structure (Figure 1a). The residue was separated by chromatography
(hexane: EtOAc = 1:4) to isolate the compound NTDMNQ (Figure 1b).
By performing NMR at a wavelength of 400 MHz, we analyzed the H
and C spectra in the deuterated chloroform solvent and identified the
following structures:
3.3 | NTDMNQ-induced apoptosis in AGS cells
Compared to the control group, treatment with 5-FU and NTDMNQ for
24 hr caused morphological changes in the cells, specifically, a change
from an inerratic rhombus to a spherical particle (Figure 3a,b). Additionally,
the fluorescent intensity of the AGS cells was gradually enhanced in a
time-dependent manner. Compared with 5-FU-treated cells, the
FIGURE 4 Effects of NTDMNQ on MAPK, Akt, and STAT3 signaling pathways in AGS cells. (a) The expression levels of p-ERK, p-JNK, p-p38, p-Akt,
and p-STAT3 were measured by western blotting, α-tubulin was used as an internal control. (b) Quantification of the p-ERK, p-JNK, p-p38, p-Akt, and
p-STAT3 were analyzed by imageJ software. Error bars indicate means ꢂ SD of three independent experiments (ap < 0.05, bp < 0.01, cp < 0.001
indicate significant differences)

WANG ET AL.
7
fluorescent intensity was markedly increased in the NTDMNQ-treated
group. As shown in Figure 3c,d, there was a small percentage (7.46%) of
apoptotic cells at 0 hr, and treatment with NTDMNQ for 24 hr dramati-
cally increased apoptotic cells to 63.68%. This was significantly higher than
the 5-FU-treated group. In addition, NTDMNQ significantly increased the
protein expression levels of Bax, cleaved-caspase-3, and cleaved-PARP,
while decreasing the protein expression levels of Bcl-xl (Figure 3e,f ).
Taken together, these indicated that NTDMNQ-induced apoptosis in AGS
cells via regulating the mitochondrial apoptotic pathways.
and p-p38 in a time-dependent manner. Thus, NTDMNQ induced
apoptosis via the regulation of the MAPK, Akt, and STAT3 signaling
pathways in AGS cells.
3.5 | NTDMNQ induced apoptosis by accelerating
ROS generation in AGS cells
As shown in Figure 5a,b, NTDMNQ significantly increased intercel-
lular ROS accumulation in a time-dependent manner. However,
pretreatment with NAC apparently decreased NTDMNQ-induced
apoptosis (Figure 5c,d). In addition, NAC markedly increased the
expression levels of p-ERK, p-Akt, and p-STAT3, and decreased the
expression levels of p-JNK, p-p38, and cleaved-caspase-3
(Figure 5e,f ). These findings demonstrate that ROS generation is a
key regulator of NTDMNQ-induced apoptosis. In addition, the
3.4 | NTDMNQ regulated the MAPK, Akt, and
STAT3 signaling pathways
As shown in Figure 4, NTDMNQ significantly decreased the levels
of p-ERK, p-Akt, and p-STAT3, and increased the levels of p-JNK
FIGURE 5 The effects of NTDMNQ-induced ROS generation and apoptosis in AGS cells. (a,b) AGS cells were treated with NTDMNQ (1 μmol/L)
and intracellular ROS generation measured by flow cytometer. (c,d) Inhibition of NTDMNQ-induced apoptosis. AGS cells were pretreated with
NAC (5 μmol/L) for 30 min, then treated with NTDMNQ (1 μmol/L) for 24 hr. apoptosis was determined by flow cytometer. (e,f ) p-ERK, p-JNK,
p-p38, p-Akt, p-STAT3, cleaved-caspase-3, and cleaved-PARP protein expression levels were determined by western blotting. Error bars indicate
means ꢂ SD of three independent experiments (ap < 0.05, bp < 0.01, cp < 0.001 indicate significant differences)

8
WANG ET AL.
FR180204 (ERK inhibitor), SP600125 (JNK inhibitor), and
SB203580 (p38 inhibitor) were used to further investigate the role
of MAPK and STAT3 signaling pathways in cells apoptosis driven
by NTDMNQ. The protein expression level of p-STAT3 was
enhanced by ERK inhibitor, and this effect was inhibited by JNK
inhibitor or p38 inhibitor (Figure 6a–f ). These results suggested
that MAPK was involved in mediating the STAT3 signaling pathway
and induced AGS cells apoptosis.
4
| DISCUSSION
Naphthoquinone derivatives have been reported to have various
pharmacological activities, such as anti-inflammatory, antiviral, and
especially anticancer properties (Lee et al., 2011). In addition, sev-
eral naphthoquinone derivatives promote the apoptosis of cancer
cells via an ROS-dependent mechanism (Ong, Yong, Lim, & Ho,
2015). However, studies with naphthoquinone derivatives
FIGURE 6 The effects of NTDMNQ on MAPK and STAT3 signaling pathways in AGS cells. (a) AGS cells were pretreatment with 12.1 μmol/L of
FR180204 for 30 min, and then treated with NTDMNQ for 24 hr, the expression levels of p-ERK, p-STAT3, and cleaved-caspase-3 were
determined by western blotting. (b) Quantification of the p-ERK, p-STAT3, and cleaved-caspase-3 were analyzed by imageJ software. (c) AGS
cells were pretreatment with 12.1 μmol/L of SP600125 for 30 min, and then treated with NTDMNQ for 24 hr, the expression levels of p-JNK, p-
STAT3, and cleaved-caspase-3 were determined by western blotting. (d) Quantification of the p-JNK, p-STAT3, and cleaved-caspase-3 were
analyzed by imageJ software. (e) AGS cells were pretreatment with 12.1 μmol/L of SB203580 for 30 min, and then treated with NTDMNQ for
24 hr, the expression levels of p-p38, p-STAT3, and cleaved-caspase-3 were determined by western blotting. (f ) Quantification of the p-p38, p-
STAT3, and cleaved-caspase-3 were analyzed by imageJ software. Error bars indicate means ꢂ SD of three independent experiments (ap < 0.05,
bp < 0.01, cp < 0.001 indicate significant differences)

WANG ET AL.
9
similar to the compound which we synthesized (Ollinger & Brunmark,
1991; Sidlauskas, Sidlauskiene, Li, & Liobikas, 2017; Zhang, Hirsch,
Mohsen, & Samuni, 1994). On the basis of the above, we speculate
that NTDMNQ could interfere with the mitochondrial electron trans-
fer chain and induced apoptosis via regulating the mitochondrial apo-
ptotic pathways.
Recent studies have shown that ROS can induce apoptosis in vari-
ous cell systems (Ma et al., 2014). The ROS plays a central role in cellular
survival and function, although cell death was caused by excessive gener-
ation of ROS-induced oxidative stress (Chien et al., 2014). Some studies
show that the highly redox active naphthoquinones undergo one-
electron reduction when the organs with a sufficient blood supply and in
an aerobic conditions, and generate free-radical intermediates, resulting
in the accumulation of ROS (Qiu et al., 2018; Wellington, 2015). Our
study indicated that NTDMNQ induced apoptosis through increasing
intracellular ROS levels (Figure 5a,b). NAC is an antioxidant with ROS
scavenging that have a potentially benefits effect for the treatment of
diseases (Aldini et al., 2018). Because of the NAC that have well-
described antioxidant and radical scavenging activity, it has also been
used as an antioxidant in several in many studies (Dodd, Dean, Copolov,
Malhi, & Berk, 2008). Such as, NAC could inhibit the quinalizarin-induced
ROS generation in lung cancer A549 cells and blocked cryptotanshinone-
induced apoptosis in gastric cancer cells (Liu et al., 2017; Meng et al.,
2018). In this study, to determine the relationship of NTDMNQ-induced
apoptosis and ROS, the NAC was used to pretreat the AGS cells. And
the result shows that NAC significantly inhibited NTDMNQ-induced
apoptosis by decreasing ROS levels (Figure 5c,d). These findings clearly
demonstrate our hypothesis that NTDMNQ-induced apoptosis in AGS
cells via up-regulating the generation of ROS. In addition, ROS have been
demonstrated to induce apoptosis in malignant tumor cells by activating
the caspase, MAPK, PI3K/Akt, and STAT3 signaling pathways (Miao, Yu,
Ren, & Yang, 2017; Zhong et al., 2016). MAPK is a family of serine/thre-
onine kinases. It was reported that the activation of p38 and JNK
induces apoptosis via the phosphorylation of various pro-apoptotic
downstream effectors, while the ERK pathway is primarily associated
with cell survival (Lohberger et al., 2015; Shin et al., 2012). Studies have
shown that naphthoquinone derivatives activate the ERK, JNK and p38
signaling pathways by stimulating ROS generation (Ong et al., 2015). The
activation of STAT3 and Akt may be a key mechanism in the treatment
of a variety of cancers (Wu et al., 2013). In addition, Akt may regulate
apoptosis via modulation of Bcl-2 family members (Zhang et al., 2012).
The STAT3 signaling pathway is also a key anti-apoptotic pathway in
cancer cells because it is associated with cell proliferation, immune eva-
sion, and apoptotic arrest (Liu et al., 2015; Wang, Tang, & Zhang, 2017).
These effects are most likely mediated through ROS-induced activation
of the STAT3 pathway (Miao, Yu, Ren, & Yang, 2017). Therefore, we
investigated the cellular signaling pathways involved in NTDMNQ-
induced apoptosis. Our results showed that p38 and JNK phosphoryla-
tion was markedly increased after NTDMNQ-treated, and ERK, Akt, and
STAT3 phosphorylation was significantly decreased (Figure 4). Further-
more, NAC inhibited MAPK, Akt, and STAT3 activation, which indicates
that NTDMNQ-induced apoptosis in AGS cells by the ROS-mediated
MAPK, Akt, and STAT3 signaling pathways (Figure 5e,f ). Additionally, to
determine whether STAT3 was regulated by NTDMNQ-induced MAPK
activation, the MAPK inhibitor (including FR180204, SP600125, and
FIGURE 7 Schematic illustration of the underlying mechanism of
NTDMNQ effects on signaling pathways and the induction of
apoptosis in gastric cancer cells. The mechanism of NTDMNQ
anticancer activity as related to the MAPK, Akt, and STAT3 signal
pathways
indicated that these compounds exhibit significant toxicity, possibly
due to the hydroxyl group in their structure (Ollinger & Brunmark,
1991). To reduce their toxicity and improve the antitumor activity
of compound, we substituted the dihydroxy groups at C5 and C8
with methoxy groups and then synthesized a novel naphthoqui-
none derivative called NTDMNQ (Figure 1). Our results showed
that NTDMNQ-treated gastric cancer cells exhibited considerably
higher rates of apoptosis than control treated cells (Figure 3b,c).
These results were equivalent to the MTT assay results, which
showed that NTDMNQ suppressed the proliferation of nine kinds
of gastric cancer cells in a dose-dependent manner (Figure 2).
Therefore, we hypothesized that the loss of viability in gastric cells
treated with NTDMNQ is likely because of the induction of
apoptosis.
Apoptosis as a cell defense mechanism that plays an important
role in inhibiting tumor development (Zhu et al., 2012). Apoptosis has
two primary pathways: The extrinsic and the intrinsic mitochondrial
pathways, and among them, the Bcl-2 family members are known to
mediate the apoptotic process (Yu, Kim, Yu, & Kim, 2013). It was
reported that Ramentaceone, a naphthoquinone derivative, induced
apoptosis in breast cancer cells by increasing the expression levels of
the pro-apoptotic proteins Bax, cleaved-caspase-3, and cleaved-PARP,
and decreasing the expression level of the anti-apoptotic protein Bcl-
xl via inhibition of the PI3K/Akt signaling pathway (Kawiak & Loj-
kowska, 2016). Similarly, our results suggested that NTDMNQ
induced apoptosis via decreasing the protein expression level of Bcl-xl
and increasing the expression levels of pro-apoptotic protein Bax,
cleaved-caspase-3, and cleaved-PARP (Figure 3e,f ). In addition, it has
been reported that 5-hydroxy-1,4-naphthalenedione (juglone) which
is 1,4-naphthoquinione derivative may interact with the mitochondrial
electron transfer chain (ETC) in mitochondria. The intracellular free
radical production by quinones is thought to be a consequence of O2
reduction by semiquinone intermediates. The semiquinone is formed
via one-electron reduction of the corresponding quinone catalyzed by
NADPH-cytochrome P450 reductase. The structures of juglone are

10
WANG ET AL.
Bioorganic
org/10.1016/j.bmc.2013.05.017
&
Medicinal Chemistry, 21(15), 4662–4669. https://doi.
SB203580) were used to investigate the relation between MAPK and
STAT3. These data suggested that the reactivation of the MAPK inhibitor
via NTDMNQ-inhibited the STAT3 signaling pathway, demonstrating
that STAT3 was regulated through the MAPK signaling pathway
(Figure 6).
Bolton, J. L., Trush, M. A., Penning, T. M., Dryhurst, G., & Monks, T. J.
(2000). Role of quinones in toxicology. Chemical Research in Toxicology,
13(3), 135–160.
Chang, G., Zhou, Y., Zhu, R. Z., Liu, Z. H., Cao, Y. B., & Cao, R. B. (2017).
Method for synthesizing 5,8-dihydroxy-1,4-naphthoquinone with mol-
ten salt. China, Patent NO. 201710669130.8.
Chen, X., Dai, X., Zou, P., Chen, W., Rajamanickam, V., Feng, C., … Liang, G.
(2017). Curcuminoid EF24 enhances the anti-tumour activity of Akt inhibi-
tor MK-2206 through ROS-mediated endoplasmic reticulum stress and
mitochondrial dysfunction in gastric cancer. British Journal of Pharmacology,
174(10), 1131–1146. https://doi.org/10.1111/bph.13765
5
| CONCLUSIONS
In summary, we synthesized
a novel naphthoquinone derivative,
2-(naphthalene-2-thio)-5,8-dimethoxy-1,4-naphthoquinone (NTDMNQ),
and the anticancer molecular mechanisms of NTDMNQ were investi-
gated. NTDMNQ induce apoptosis in human GC cells through ROS-
mediated MAPK/Akt/STAT3 signaling pathways (Figure 7). Based on our
observations, NTDMNQ may be a candidate for the development and
exploitation of antigastric cancer drugs.
Chien, C. C., Wu, M. S., Shen, S. C., Ko, C. H., Chen, C. H., Yang, L. L., …
Chen, Y. C. (2014). Activation of JNK contributes to
evodiamine-induced apoptosis and G2/M arrest in human colorectal
carcinoma cells: A structure-activity study of evodiamine. PLoS One,
9(6), e99729. https://doi.org/10.1371/journal.pone.0099729
Coelho-Cerqueira, E., Netz, P. A., Canto, V. P. D., Pinto, A. C., & Follmer, C.
(2014). Beyond topoisomerase inhibition: Antitumor 1,4-naphthoquinones
as potential inhibitors of human monoamine oxidase. Chemical Biology &
Drug Design, 83(4), 401–410. https://doi.org/10.1111/cbdd.12255
Costa, C., Pereira, S., Lima, L., Peixoto, A., Fernandes, E., Neves, D., …
Santos, L. L. (2015). Abnormal protein glycosylation and activated
PI3K/Akt/mTOR pathway: Role in bladder cancer prognosis and tar-
geted therapeutics. PLoS One, 10(11), e0141253. https://doi.org/10.
1371/journal.pone.0141253
Dodd, S., Dean, O., Copolov, D. L., Malhi, G. S., & Berk, M. (2008).
N-acetylcysteine for antioxidant therapy: Pharmacology and clinical
utility. Expert Opinion on Biological Therapy, 8(12), 1955–1962. https://
doi.org/10.1517/14728220802517901
Eruslanov, E., & Kusmartsev, S. (2010). Identification of ROS using oxidized
DCFDA and flow-cytometry. Methods in Molecular Biology, 594,
57–72. https://doi.org/10.1007/978-1-60761-411-1_4
ACKNOWLEDGMENTS
This work was funded by the Natural Science Foundation of Heilongjiang
Province of China (LC2015036), the Postdoctoral Scientific Research
Foundation of Heilongjiang Province of China (LBH-Q13132), the
Research Project of Heilongjiang Bayi Agricultural University
(XYB2012-14), and the Scientific Research Foundation of Heilongjiang
Provincial Education Department of China (1252HQ007).
Günes-Bayir, A., Kiziltan, H. S., Kocyigit, A., Güler, E. M., Karatas¸, E., &
Toprak, A. (2017). Effects of natural phenolic compound carvacrol on the
human gastric adenocarcinoma (AGS) cells in vitro. Anti-Cancer Drugs,
28(5), 522–530. https://doi.org/10.1097/CAD.0000000000000491
Hafeez, B. B., Zhong, W., Fischer, J. W., Mustafa, A., Shi, X., Meske, L., …
CONFLICT OF INTEREST
The authors declare they have no conflict of interest.
Verma, A. K. (2013). Plumbagin,
a medicinal plant (Plumbago
zeylanica)-derived 1,4-naphthoquinone, inhibits growth and metastasis
of human prostate cancer PC-3M-luciferase cells in an orthotopic
xenograft mouse model. Molecular Oncology, 7(3), 428–439. https://
doi.org/10.1016/j.molonc.2012.12.001
AUTHORS' CONTRIBUTIONS
C.-H. J and D.-J. Z.: conceived and designed the experiments; J.-R. W.,
G.-N. S., and Y.-H. L.: wrote the article and participated in the experi-
ment; X.-J. P., C. L., and L.-Q. M.: assessed the cytotoxic effects; Y. W.
and Y. Z.: performed the apoptotic analysis; H. W., W.-T. X., J.-Q. L.,
and Y. L.: performed western blotting analysis; H.-N. S., Y.-H. H., and
M.-H. J. performed the signaling analysis; All authors read and
approved the final version of the manuscript.
Huot, R., & Brassard, P. (2011). Friedel–crafts condensations with maleic
anhydrides III. The synthesis of polyhydroxylated naphthoquinones.
Canadian Journal of Chemistry, 52(5), 838–842.
Kawiak, A., & Lojkowska, E. (2016). Ramentaceone, a Naphthoquinone
Derived from Drosera sp., Induces Apoptosis by Suppressing PI3K/Akt
Signaling in Breast Cancer Cells. PLoS One, 11(2), e0147718. https://
doi.org/10.1371/journal.pone.0147718.
Kim, M. C., Lee, H. J., Lim, B., Ha, K. T., Kim, S. Y., So, I., & Kim, B. J. (2014).
Quercetin induces apoptosis by inhibiting MAPKs and TRPM7 chan-
nels in AGS cells. International Journal of Molecular Medicine, 33(6),
1657–1663. https://doi.org/10.3892/ijmm.2014.1704
ORCID
Cheng-Hao Jin
http://orcid.org/0000-0003-4431-2623
Kong, G. M., Tao, W. H., Diao, Y. L., Fang, P. H., Wang, J. J., Bo, P., &
Qian, F. (2016). Melittin induces human gastric cancer cell apoptosis
via activation of mitochondrial pathway. World Journal of Gastroenterol-
ogy, 22(11), 3186–3195. https://doi.org/10.3748/wjg.v22.i11.3186
Lee, J. J., Zhang, W. Y., Yi, H., Kim, Y., Kim, I. S., Shen, G. N., …
Myung, C. S. (2011). Anti-proliferative actions of 2-decylami-
no-5,8-dimethoxy-1,4-naphthoquinone in vascular smooth muscle
cells. Biochemical and Biophysical Research Communications, 411(1),
213–218. https://doi.org/10.1016/j.bbrc.2011.06.145
Li, H., Zhang, X., & Wang, W. (2017). Anticancer activity of 5, 7--
dimethoxyflavone against liver cancer cell line HEPG2 involves apo-
ptosis, ROS generation and cell cycle arrest. African Journal of
Traditional, Complementary, and Alternative Medicines, 14(4), 213–220.
https://doi.org/10.21010/ajtcam.v14i4.24
REFERENCES
Afri, M., Frimer, A. A., & Cohen, Y. (2004). Active oxygen chemistry
within the liposomal bilayer. Part IV: Locating 20,70-dichlorofluorescein
(DCF),
20,70-dichlorodihydrofluorescein
(DCFH)
and
20,70-
dichlorodihydrofluorescein diacetate (DCFH-DA) in the lipid bilayer.
Chemistry and Physics of Lipids, 131(1), 123–133. https://doi.org/10.
1016/j.chemphyslip.2004.04.006
Aldini, G., Altomare, A., Baron, G., Vistoli, G., Carini, M., Borsani, L., &
Sergio, F. (2018). N-acetylcysteine as an antioxidant and disulphide
breaking agent: The reasons why. Free Radical Research, 9, 1–12.
https://doi.org/10.1080/10715762.2018.1468564
Bhasin, D., Chettiar, S. N., Etter, J. P., Mok, M., & Li, P. K. (2013). Antican-
cer activity and SAR studies of substituted 1,4-naphthoquinones.
Liu, C., Shen, G. N., Luo, Y. H., Piao, X. J., Jiang, X. Y., Meng, L. Q., …
Jin, C. H. (2017). Novel 1,4-naphthoquinone derivatives induce

WANG ET AL.
11
apoptosis via ROS-mediated p38/MAPK, Akt and STAT3 signaling in
insertion of a luciferase reporter in the cyclin D1 gene provides a modified
A549 cell line to screen for selective STAT3 inhibitors. PLoS One, 8(7),
e68391. https://doi.org/10.1371/journal.pone.0068391
human hepatoma Hep3B cells. International Journal of Biochemistry and
Cell Biology, 96, 9–19. https://doi.org/10.1016/j.biocel.2018.01.004
Liu, C., Sun, H. N., Luo, Y. H., Piao, X. J., Wu, D. D., Meng, L. Q., …
Jin, C. H. (2017). Cryptotanshinone induces ROS-mediated apoptosis
in human gastric cancer cells. Oncotarget, 8(70), 115398–115412.
https://doi.org/10.18632/oncotarget.23267
Liu, Y. F., Lu, Y. M., Qu, G. Q., Liu, Y., Chen, W. X., Liao, X. H., & Kong, W. M.
(2015). Ponicidin induces apoptosis via JAK2 and STAT3 signaling path-
ways in gastric carcinoma. International Journal of Molecular Sciences,
16(1), 1576–1589. https://doi.org/10.3390/ijms16011576
Lohberger, B., Leithner, A., Stuendl, N., Kaltenegger, H., Kullich, W., &
Steineckerfrohnwieser, B. (2015). Diacerein retards cell growth of
chondrosarcoma cells at the G2/M cell cycle checkpoint via cyclin
B1/CDK1 and CDK2 downregulation. BMC Cancer, 15, 891–901.
https://doi.org/10.1186/s12885-015-1915-4
Luo, D., Ni, Q., Ji, A., Gu, W., Wu, J., & Jiang, C. (2016). Dehydroabietic
acid derivative QC4 induces gastric cancer cell death via Oncosis and
apoptosis. BioMed Research International, 2016(3), 1–10. https://doi.
org/10.1155/2016/2581061
Luo, D., Xu, Z., Hu, X., Zhang, F., Bian, H., Li, N., … Gu, J. (2016). URI pre-
vents potassium dichromate-induced oxidative stress and cell death in
gastric cancer cells. American Journal of Translational Research, 8(12),
5399–5409.
Ma, W. D., Zou, Y. P., Wang, P., Yao, X. H., Sun, Y., Duan, M. H., … Yu, B.
(2014). Chimaphilin induces apoptosis in human breast cancer MCF-7
cells through a ROS-mediated mitochondrial pathway. Food and Chemi-
cal Toxicology, 70, 1–8. https://doi.org/10.1016/j.fct.2014.04.014
McCall, R., Miles, M., Lascuna, P., Burney, B., Patel, Z., Sidoran, K. J., …
Arambula, J. F. (2017). Dual targeting of the cancer antioxidant net-
work with 1,4-naphthoquinone fused gold(i) N-heterocyclic carbene
complexes. Chemical Science, 8(9), 5918–5929. https://doi.org/10.
1039/c7sc02153d
Meng, L. Q., Liu, C., Luo, Y. H., Piao, X. J., Wang, Y., Zhang, Y., … Jin, C. H.
(2018). Quinalizarin exerts an anti-tumour effect on lung cancer A549
cells by modulating the Akt, MAPK, STAT3 and p53 signalling path-
ways. Molecular Medicine Reports, 17(2), 2626–2634. https://doi.
org/10.3892/mmr.2017.8110
Miao, Z., Yu, F., Ren, Y., & Yang, J. (2017). D,l-Sulforaphane induces
ROS-dependent apoptosis in human Gliomablastoma cells by inactivat-
ing STAT3 signaling pathway. International Journal of Molecular Sci-
ences, 18(1), 72–85. https://doi.org/10.3390/ijms18010072
O'Brien, P. J. (1991). Molecular mechanisms of quinone cytotoxicity.
Chemico-Biological Interactions, 80(1), 1–41.
Shen, G. N., Choi, J. H., Gajulapati, K., Lee, J. H., Kim, Y. K., Rho, M. C., …
Choi, Y. (2009). 2-Substitiuted Thio- and Amino-5,8-dimethoxy-1,4-
naphthoquinones as a novel class of acyl-CoA: Cholestrol acyltransferase
inhibitors. Bulletin of the Korean Chemical Society, 30(5), 1088–1092.
Shin, D. Y., Kim, G. Y., Lee, J. H., Choi, B. T., Yoo, Y. H., & Choi, Y. H.
(2012). Apoptosis induction of human prostate carcinoma DU145 cells
by Diallyl disulfide via modulation of JNK and PI3K/AKT signaling
pathways. International Journal of Molecular Sciences, 13(11),
14158–14171. https://doi.org/10.3390/ijms131114158
Sidlauskas, K., Sidlauskiene, R., Li, N.,
&
Liobikas, J. (2017).
5-Hydroxy-1,4-naphthalenedione exerts anticancer effects on glioma
cells through interaction with the mitochondrial electron transport
chain. Neuroscience Letters, 639, 207–214. https://doi.org/10.1016/j.
neulet.2017.01.007
Sun, H. N., Shen, G. N., Jin, Y. Z., Jin, Y., Han, Y. H., Feng, L., … Jin, C. H.
(2016). 2-cyclohexylamino-5,8-dimethoxy-1,4-naphthoquinone inhibits
LPS- induced BV2 microglial activation through MAPK/NF-kB signal-
ing pathways. Heliyon, 2(7), e00132. https://doi.org/10.1016/j.heliyon.
2016.e00132
Tsai, M. H., Liu, J. F., Chiang, Y. C., Hu, S. C., Hsu, L. F., Lin, Y. C., …
Lee, C. W. (2017). Artocarpin, an isoprenyl flavonoid, induces
p53-dependent or independent apoptosis via ROS-mediated MAPKs
and Akt activation in non-small cell lung cancer cells. Oncotarget, 8(17),
28342–28358. https://doi.org/10.18632/oncotarget.16058
Van Cutsem, E., Moiseyenko, V. M., Tjulandin, S., Majlis, A., Constenla, M.,
Boni, C., & V325, Study. Group. (2006). Phase III study of docetaxel
and cisplatin plus fluorouracil compared with cisplatin and fluorouracil
as first-line therapy for advanced gastric cancer: A report of the V325
study group. Journal of Clinical Oncology, 24(31), 4991–4997. https://
doi.org/10.1200/JCO.2006.06.8429
Wang, D., Tang, S., & Zhang, Q. (2017). Maslinic acid suppresses the
growth of human gastric cells by inducing apoptosis via inhibition of
the interleukin-6 mediated Janus kinase/signal transducer and activa-
tor of transcription
3 signaling pathway. Oncology Letters, 13(6),
4875–4881. https://doi.org/10.3892/ol.2017.6073
Wang, J., Xu, R., Li, J., Bai, Y., Liu, T., Jiao, S., … Shen, L. (2016). Randomized
multicenter phase III study of a modified docetaxel and cisplatin plus fluo-
rouracil regimen compared with cisplatin and fluorouracil as first-line ther-
apy for advanced or locally recurrent gastric cancer. Gastric Cancer, 19(1),
234–244. https://doi.org/10.1007/s10120-015-0457-4
Zhong, W. F., Wang, X. H., Pan, B., Li, F., Kuang, L., & Su, Z. X. (2016).
Eupatilin induces human renal cancer cell apoptosis via ROS-mediated
MAPK and PI3K/AKT signaling pathways. Oncology Letters, 12(4),
2894–2899. https://doi.org/10.3892/ol.2016.4989
Ollinger, K., & Brunmark, A. (1991). Effect of hydroxy substituent position
on 1,4-naphthoquinone toxicity to rat hepatocytes. The Journal of Bio-
logical Chemistry, 266(32), 21496–21503.
Ong, J. Y., Yong, P. V., Lim, Y. M.,
&
Ho, A. S. (2015).
Wellington, K. W. (2015). Understanding cancer and the anticancer activi-
ties of naphthoquinones-a review. RSC Advances, 46(20),
20309–20355. https://doi.org/10.1039/C4RA13547D
Wu, H., Liu, S., Gong, J., Liu, J., Zhang, Q., Leng, X., … Li, Y. (2017). VCPA, a
novel synthetic derivative of α-tocopheryl succinate, sensitizes human
gastric cancer to doxorubicin-induced apoptosis via ROS-dependent
mitochondrial dysfunction. Cancer Letters, 393, 22–32. https://doi.
org/10.1016/j.canlet.2017.02.007
Wu, K., Chang, Q., Lu, Y., Qiu, P., Chen, B., Thakur, C., … Chen, F. (2013).
Gefitinib resistance due to STAT3-mediated Akt activation in lung can-
cer cells. Oncotarget, 4(12), 2430–2438. https://doi.org/10.18632/
oncotarget.1431
2-Methoxy-1,4-naphthoquinone (MNQ) induces apoptosis of A549
lung adenocarcinoma cells via oxidation-triggered JNK and p38 MAPK
signaling pathways. Life Sciences, 135, 158–164. https://doi.org/10.
1016/j.lfs.2015.03.019
Porta, C., Paglino, C., & Mosca, A. (2014). Targeting PI3K/Akt/mTOR sig-
naling in cancer. Frontiers in Oncology, 4(4), 64–74. https://doi.org/10.
3389/fonc.2014.00064
Qiu, H. Y., Wang, P. F., Lin, H. Y., Tang, C. Y., Zhu, H. L., & Yang, Y. H.
(2018). Naphthoquinones: A continuing source for discovery of thera-
peutic antineoplastic agents. Chemical Biology & Drug Design, 91(3),
681–690. https://doi.org/10.1111/cbdd.13141
Ren, H. P., Yin, X. Y., Yu, H. Y., & Xiao, H. F. (2017). Stevioside induced
cytotoxicity in colon cancer cells via reactive oxygen species and
mitogen-activated protein kinase signaling pathways-mediated apopto-
sis. Oncology Letters, 13(4), 2337–2343. https://doi.org/10.3892/ol.
2017.5744
Sampson, V. B., Vetter, N. S., Kamara, D. F., Collier, A. B., Gresh, R. C., &
Kolb, E. A. (2015). Vorinostat enhances cytotoxicity of SN-38 and
Temozolomide in Ewing sarcoma cells and activates STAT3/AKT/-
MAPK pathways. PLoS One, 10(11), e0142704. https://doi.org/10.
1371/journal.pone.0142704
Xu, D. F., Guo, X. J., Xu, Z. H., & Li, S. S. (2006). A novel Total synthesis of
(ꢂ) Shikonin. Chinese Chemical Letters, 17(7), 871–873.
Yang, Y., Guo, J. X., Shao, Z. Q., & Gao, J. P. (2017). Matrine inhibits bladder
cancer cell growth and invasion in vitro through PI3K/AKT signaling path-
way: An experimental study. Asian Pacific Journal of Tropical Medicine,
10(5), 515–519. https://doi.org/10.1016/j.apjtm.2017.05.009
Yin, Z., Zhao, X., Yang, D., Cao, X., Yu, Y., Jiang, H., … Guo, Q. (2017).
LFG-500, a newly synthesized flavonoid, induces apoptosis in human
ovarian carcinoma SKOV3 cells with involvement of the reactive oxy-
gen species-mitochondria pathway. Experimental and Therapeutic Medi-
cine, 13(6), 2819–2827. https://doi.org/10.3892/etm.2017.4343
Samsonov, A., Zenser, N., Zhang, F., Zhang, H., Fetter, J., & Malkov, D. (2013).
Tagging of genomic STAT3 and STAT1 with fluorescent proteins and

12
WANG ET AL.
Yu, S. M., Kim, S. J., Yu, S.-M., & Kim, S.-J. (2013). Thymoquinone-induced
reactive oxygen species causes apoptosis of chondrocytes via
PI3K/Akt and p38kinase pathway. Experimental Biology and Medicine,
238(7), 811–820. https://doi.org/10.1177/1535370213492685
Zhang, D. M., Liu, J. S., Tang, M. K., Yiu, A., Cao, H. H., Jiang, L., …
Ye, W. C. (2012). Bufotalin from Venenum Bufonis inhibits growth of
multidrug resistant HepG2 cells through G2/M cell cycle arrest and
apoptosis. European Journal of Pharmacology, 692(1–3), 19–28. https://
doi.org/10.1016/j.ejphar.2012.06.045
Zhu, W. Q., Wang, J., Guo, X. F., Liu, Z., & Dong, W. G. (2016). Thymoqui-
none inhibits proliferation in gastric cancer via the STAT3 pathway
in vivo and in vitro. World Journal of Gastroenterology, 22(16),
4149–4159. https://doi.org/10.3748/wjg.v22.i16.4149
Zhu, Z., Sun, H., Ma, G., Wang, Z., Li, E., Liu, Y., & Liu, Y. (2012). Bufalin
induces lung cancer cell apoptosis via the inhibition of PI3K/Akt path-
way. International Journal of Molecular Sciences, 13(2), 2025–2035.
https://doi.org/10.3390/ijms13022025
Zhang, R., Hirsch, O., Mohsen, M., & Samuni, A. (1994). Effects of nitroxide
stable radicals on juglone cytotoxicity. Archives of Biochemistry and Bio-
physics, 1312(2), 385–391.
Zhang, Y., Xu, W., Ni, P., Li, A., Zhou, J., & Xu, S. (2016). MiR-99a and
MiR-491 regulate cisplatin resistance in human gastric cancer cells by
targeting CAPNS1. International Journal of Biological Sciences, 12(12),
1437–1447. https://doi.org/10.7150/ijbs.16529
Zheng, L., Wu, C., Xi, P., Zhu, M., Zhang, L., Chen, S., … Zheng, Y. (2014).
The survival and the long-term trends of patients with gastric cancer in
Shanghai, China. BMC Cancer, 14(1), 300–311. https://doi.org/10.
1186/1471-2407-14-300
How to cite this article: Wang J-R, Shen G-N, Luo Y-H, et al.
The compound 2-(naphthalene-2-thio)-5,8-dimethoxy-1,4-
naphthoquinone induces apoptosis via reactive oxygen
species-regulated mitogen-activated protein kinase, protein
kinase B, and signal transducer and activator of transcription 3
signaling in human gastric cancer cells. Drug Dev Res. 2018;
1–12. https://doi.org/10.1002/ddr.21442