The design of 1,4-naphthoquinone derivatives and mechanisms underlying apoptosis induction through ROS-dependent MAPK/Akt/STAT3 pathways in human lung cancer cells

Article abstract of DOI:10.1016/j.bmc.2019.03.002

The natural compound 1,4-naphthoquinone has potent anti-tumor activity. However, the clinical application of 1,4-naphthoquinone and its derivatives has been limited by their side effects. In this study, we attempted to reduce the toxicity of 1,4-naphthoquinone by synthesizing two derivatives: 2,3-dihydro-2,3-epoxy-2-propylsulfonyl-5,8-dimethoxy-1,4-naphthoquinone (EPDMNQ) and 2,3-dihydro-2,3-epoxy-2-nonylsulfonyl-5,8-dimethoxy-1,4-naphthoquinone (ENDMNQ). Then we evaluated the cytotoxicity and molecular mechanisms of these compounds in lung cancer cells. EPDMNQ and ENDMNQ significantly inhibited the viabilities of three lung cancer cell lines and induced A549 cell cycle arrest at the G1 phase. In addition, they induced the apoptosis of A549 lung cancer cells by increasing the phosphorylation of p38 and c-Jun N-terminal kinase (p-JNK), and decreasing the phosphorylation of extracellular signal-related kinase (p-ERK), protein kinase B (Akt), and signal transducer and activator of transcription 3 (STAT3). Furthermore, they increased reactive oxygen species (ROS) levels in A549 cells; however, pretreatment with the ROS inhibitor N-acetyl-L-cysteine significantly inhibited EPDMNQ- and ENDMNQ-mediated apoptosis and reversed apoptotic proteins expression. In conclusion, EPDMNQ and ENDMNQ induced G1 phase cell cycle arrest and apoptosis in A549 cells via the ROS-mediated activation of mitogen activated protein kinase (MAPK), Akt and STAT3 signaling pathways.

Full text of DOI:10.1016/j.bmc.2019.03.002

Accepted Manuscript
The design of 1,4-Naphthoquinone derivatives and mechanisms underlying
apoptosis induction through ROS-dependent MAPK/Akt/STAT3 pathways in
human lung cancer cells
Yi Zhang, Ying-Hua Luo, Xian-Ji Piao, Gui-Nan Shen, Jia-Ru Wang, Yu-Chao
Feng, Jin-Qian Li, Wan-Ting Xu, Yu Zhang, Tong Zhang, Chang-Yuan Wang,
Cheng-Hao Jin
PII:
S0968-0896(18)31939-4
https://doi.org/10.1016/j.bmc.2019.03.002
BMC 14794
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
15 November 2018
14 February 2019
1 March 2019
Please cite this article as: Zhang, Y., Luo, Y-H., Piao, X-J., Shen, G-N., Wang, J-R., Feng, Y-C., Li, J-Q., Xu, W-
T., Zhang, Y., Zhang, T., Wang, C-Y., Jin, C-H., The design of 1,4-Naphthoquinone derivatives and mechanisms
underlying apoptosis induction through ROS-dependent MAPK/Akt/STAT3 pathways in human lung cancer cells,
Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.03.002
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The design of 1,4-Naphthoquinone derivatives and mechanisms underlying
apoptosis induction through ROS-dependent MAPK/Akt/STAT3 pathways in
human lung cancer cells.
Yi Zhanga,1, Ying-Hua Luob,1, Xian-Ji Piaoc,1, Gui-Nan Shena, Jia-Ru Wanga, Yu-Chao Fengd,
Jin-Qian Lia, Wan-Ting Xua, Yu Zhanga, Tong Zhanga, Chang-Yuan Wangd,*, Cheng-Hao
Jina,d,*
a Department of Biochemistry and Molecular Biology, College of Life Science & Technology,
Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang, 163319, China.
b College of Animal Science & Veterinary Medicine, Heilongjiang Bayi Agricultural University,
Daqing, Heilongjiang, 163319, China.
c Department of Gynaecology and Obstetrics, the Fifth Affiliated Hospital of Harbin Medical
University, Daqing, Heilongjiang, 163316, China.
d College of Food Science, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang,
163319, China.
1 Contributed equally to this study as first authors.
* Corresponding authors.
E-mail addresses: jinchenghao3727@qq.com (CH.Jin), 406726021@qq.com (CY. Wang).

Abstract: The natural compound 1,4-naphthoquinone has potent anti-tumor activity. However,
the clinical application of 1,4-naphthoquinone and its derivatives has been limited by their side
effects. In this study, we attempted to reduce the toxicity of 1,4-naphthoquinone by synthesizing
two derivatives: 2,3-dihydro-2,3-epoxy-2-propylsulfonyl- 5,8-dimethoxy-1,4-naphthoquinone
(EPDMNQ) and 2,3-dihydro-2,3-epoxy-2- nonylsulfonyl-5,8-dimethoxy-1,4-naphthoquinone
(ENDMNQ). Then we evaluated the cytotoxicity and molecular mechanisms of these compounds in
lung cancer cells. EPDMNQ and ENDMNQ significantly inhibited the viabilities of three lung
cancer cell lines and induced A549 cell cycle arrest at the G1 phase. In addition, they induced the
apoptosis of A549 lung cancer cells by increasing the phosphorylation of p38 and c-Jun N-terminal
kinase (p-JNK), and decreasing the phosphorylation of extracellular signal-related kinase (p-ERK),
protein kinase B (Akt), and signal transducer and activator of transcription 3 (STAT3).
Furthermore, they increased reactive oxygen species (ROS) levels in A549 cells; however,
pretreatment with the ROS inhibitor N-acetyl-L-cysteine significantly inhibited EPDMNQ- and
ENDMNQ-mediated apoptosis and reversed apoptotic proteins expression. In conclusion,
EPDMNQ and ENDMNQ induced G1 phase cell cycle arrest and apoptosis in A549 cells via the
ROS-mediated activation of mitogen activated protein kinase (MAPK), Akt and STAT3 signaling
pathways.
Keywords: 1,4-naphthoquinone, Akt, apoptosis, lung cancer, MAPK, ROS, STAT3
Introduction
Lung cancer is a common malignant tumor with high morbidity and mortality. There are
230,000 new lung cancer cases worldwide each year.1 At present, chemotherapy is the standard
treatment for patients with advanced non-small cell lung cancer,2 however, chemotherapy drugs
have high toxicity and low selectivity. Therefore, the development of novel drugs with high efficacy
and low toxicity is necessary.

Apoptosis, a form of programmed cell death, is regulated by multiple signaling pathways.3
Defects in apoptosis cause the unregulated proliferation of cancer cells, thus, inducing the apoptosis
of cancer cells through multiple signaling pathways is a feasible scheme for developing novel anti-
cancer agents.4 Mitogen-activated protein kinase (MAPK) plays an important role in regulating of
cell growth, differentiation, apoptosis and other important cellular physiological processes.5 In
addition, many chemotherapeutic drugs and natural compounds induce cancer cell apoptosis
through the activation of the MAPK signaling pathway.6 Therefore, this pathway may be a potential
target for cancer treatment.
The Akt pathway is closely related to the development of malignant tumors, it regulates the
cell cycle, apoptosis and cell proliferation.7 Activation of Akt pathways inhibits the phosphorylation
of cyclin D and E, which cannot be degraded and accumulates in cells, causing the cell to enter the
S phase from G1.8 In addition, activation of this signaling pathway also inhibits the activity of
cyclin-dependent kinases (CDK) inhibitors p21 and p27, thereby promoting cell proliferation.9,10
Signal transducer and activator of transcription 3 (STAT3) signaling is critical for cellular
processes, such as cell differentiation, growth and apoptosis, and its activation is closely related to
the occurrence of various tumors, this makes STAT3 an attractive therapeutic target for cancers.11
The persistently inhibited STAT3 down-regulates its downstream protein expression such as Bcl-2,
Bcl-XL and caspase-3, which contribute to controlled proliferation of cancer cells through blocking
cell cycle progression and promoting apoptosis.12
Reactive oxygen species (ROS) are formed during aerobic respiration of eukaryotic cells, and
participate in the regulation of cell proliferation, apoptosis and other metabolic activities.13,14 There
is evidence that Increasing intracellular ROS levels can suppress the growth of cancer cells and
induce cellular apoptosis by mediating MAPK and STAT3 signaling components.15,16 Akt is
representative ROS-responsive signaling pathways. When intracellular ROS continuously
accumulate, Akt signaling pathway is inhibited, which eventually leads to cell cycle arrest in lung
cancer cells.17

The natural small compound 1,4-naphthoquinone has many biological activities, such as anti-
tumor effects, and as such, has promise as a novel anti-cancer drug.18,19 However, the application of
1,4-naphthoquinone is limited by its side effects and unclear anti-tumor mechanisms.
We previously synthesized 1,4-naphthoquinone derivatives BSNQ and OSNQ, showing good
anti-tumor effects.18 In order to further reduce the side effects of 1,4-naphthoquinone and improve
its anti-tumor effects, we synthesized novel 1,4-naphthoquinone derivatives, including 2,3-dihydro-
2,3-epoxy-2-propylsulfonyl-5,8-dimethoxy- 1,4-naphthoquinone (EPDMNQ) and 2,3-dihydro-2,3-
epoxy-2-nonylsulfonyl-5,8- dimethoxy-1,4-naphthoquinone (ENDMNQ) after modification of its
chemical structure. We elucidated the effects of EPDMNQ and ENDMNQ on the proliferation, cell
cycle and ROS generation in human lung cancer A549 cells. In addition, we evaluated the
molecular mechanisms underlying the apoptosis induced by these compounds.
Materials and Methods
Chemicals and reagents
The chemicals used for compound synthesis were purchased from Sigma (St. Louis, MO,
USA). Solvents were of reagent grade and used without further purification. Fluorouracil (5-FU;
MedChem Express, Princeton, NJ, USA) was dissolved in DMSO (Sigma) and stored at -20°C.
Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, and
streptomycin were purchased from Gibco (Waltham, MA, USA). The 2´,7´-dichlorofluorescin
diacetate (DCFH-DA), the Annexin V-FITC Apoptosis Detection Kit, and N-acetyl-L-cysteine
(NAC) were purchased from Beyotime Biotechnology (Shanghai, China). All of the antibodies were
purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA).
Synthesis and identification of EPDMNQ and ENDMNQ
Maleic anhydride (0.12 M) and 1,4-dimethoxybenzene (0.24 M) produced 5,8-dihydroxy-1,4-
naphthoquinone under the catalysis of molten aluminum chloride (142 g) and sodium chloride (28.3

g). Then, 5,8-dihydroxy-1,4-naphthoquinone (19.0 g) reacted with sodium hyposulfite (10.6 g),
dimethyl sulfate (25 mL), and sodium hydroxide (40 mL) to generate 1,4,5,8-
tetramethoxynaphthalene, which produced 5,8-dimethoxy-1,4-naphthoquinone (DMNQ) under the
oxidation of ceric ammonium nitrate (54.0 g). Through the catalysis of concentrated sulfuric acid
(0.23 mM) and sodium dichromate (0.76 mM), DMNQ (1.38 mM) reacted with mercaptan (1.65
mM) to generate the mercaptoid compound, which was oxidized by m-chloroperoxybenzoic acid
(MCPBA, 0.44 mM) to form the final products, 1,4-naphthoquinone derivatives EPDMNQ and
ENDMNQ (Fig. 1A, B). EPDMNQ and ENDMNQ were validated by nuclear magnetic resonance
(NMR) and mass spectrometry (MS). NMR spectra were recorded on the JNM-AL 600 (600 MHz)
NMR spectrometer. Chemical shifts (d) are given in ppm downfield from tetramethylsilan as the
internal standard. MS spectra were collected with the AB SCIEX API 2000 LC/MS/MS (Applied
Biosystems, Inc., Foster City, CA, USA) and LCMS-IT-TOF (Shimadzu Scientific Inc., Beijing,
China) systems. Finally, the two compounds were dissolved in DMSO to a stock concentration of
20 mM for subsequent experiments.
Figure 1. Synthesis of the 1,4-naphthoquinone derivatives EPDMNQ and ENDMNQ. (A) Synthetic
route of EPDMNQ and ENDMNQ. (B) Structural formulas of EPDMNQ and ENDMNQ.

Cell lines and cell cultures
The human lung cancer cell lines (A549, NCI-H23, and NCI-H460), human normal lung
fibroblasts cells IMR-90, human normal liver cells QSG-7701 and human embryonic kidney cells
HEK-293A were purchased from the American Type Culture Collection (Manassas, VA, USA).
NCI-H23, NCI-H460 and QSG-7701 cells were cultured in RPMI 1640 medium. A549, IMR-90
and HEK-293A cells were cultured in DMEM medium. Medium contained 10% FBS, 100 U/mL
penicillin, and 100 μg/mL streptomycin in a humidified 5% CO2 incubator at 37°C.
Cell viability assay
The cell survival rate was measured by the MTT cell viability assay. A549, NCI-H23 and NCI-
H460 cells in the logarithmic growth phase were seeded in 96-well plates culture at a density of 1 ×
104 cells/well. Then the cells were treated with different concentrations (1, 3, 10, 30 and 100 μM) of
5-FU, EPDMNQ or ENDMNQ for 24 h. Subsequently, 20 μL MTT (5 mg/mL) was added to each
well and the plate was further incubated for 2 h at 37°C. The formazan crystals were dissolved in
100 μL DMSO. The absorbance was measured at 490 nm using the Bio-Tek EXL808 microplate
reader (BioTek Instruments, Winooski, VT, USA).
Cell apoptosis analysis
Cell apoptosis was detected using the Annexin V Apoptosis Detection Kit. A549 cells were
cultured in 6-well plates (1 × 105 cells/well) and treated with 3 μM 5-FU, EPDMNQ or ENDMNQ
for different time points (3, 6, 12 and 24 h). Following treatment, the cells were incubated with 200
μL binding buffer and stained with Annexin V-FITC and PI in the dark for 20 min, after which
apoptotic cells were analyzed using fluorescence inverted microscope (Thermo Fisher Scientific
Inc., Waltham, MA, USA) and flow cytometer (Beckman Coulter, Brea, CA, USA).

Cell cycle analysis
A549 cells were cultured in 6-well plates (1 × 105 cells/well) and treated with 3 μM EPDMNQ
or ENDMNQ for different time points (3, 6, 12 and 24 h). Cells were fixed in 70% ethanol at 4°C
for 12 h and washed with PBS 2 times, then incubated with RNase and PI (Beyotime
Biotechnology, Shanghai, China) at 37°C for 30 min. The cellular DNA content was analyzed by
flow cytometry.
Western blot analysis
Equal amounts of protein (30 μg) from cell lysates were separated by 12% sodium dodecyl
sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The
membranes were blocked in 5% skim milk at 37°C for 2 h, and probed with different primary
antibodies against specific proteins at 4°C overnight. After washing with Tris-buffered saline and
Tween 20 (TBST), the membranes were incubated with horseradish peroxidase-conjugated anti-IgG
secondary antibody in TBST at room temperature for 1 h. Immunoreactive protein bands were
detected with an Amersham imager (AI600; GE, Fairfield, CT, USA). The blots were analyzed
using Image J 1.46r (National Institutes of Health, Bethesda, MD, USA). Apoptosis- and cell cycle-
related protein levels were normalized to the matching densitometry value of β-actin as the internal
control. the protein intensity of p-p38 (Thr 180 and Tyr 182), p-JNK (Thr 183 and Tyr 185), p-ERK
(Tyr 204), p-Akt (Ser 473) and p-STAT3 (Tyr 705) was normalized to the expression levels of p38,
JNK, ERK, Akt and STAT3, respectively.
Detection of intracellular ROS
A549 cells were pretreated with 10 mM DCFH-DA for 30 min and then incubated with 3 μM
5-FU, EPDMNQ or ENDMNQ for 3, 6, 12 and 24 h at 37°C. Next, the cells were washed twice
with phosphate-buffered saline, and intracellular ROS levels were measured by flow cytometry.

Statistical analysis
The results are expressed as the mean ± standard deviation, and all of the experiments were
performed three times. The continuous data were analyzed by one-way analysis of variance
followed by Tukey’s post hoc tests using SPSS 19.0 statistical software, and *p<0.05, **p<0.01
and ***p<0.001 were considered to be statistically significant.
Results
Synthesis of EPDMNQ and ENDMNQ
Through a series of chemical reactions, we modified the chemical structure of 1,4-
naphthoquinone and obtained two compounds, after which their hydrogen and carbon spectra were
1
obtained by NMR. EPDMNQ: H-NMR (CDCl3, 600 MHz): δ 7.14 (s, 2H), 4.5 (s, 1H), 3.99 (s,
13
6H), 3.60 (m, J = 6.5 Hz, 2H), 1.9 (m, 2H), 1.1 (m, 3H); C NMR (CDCl3, 150 MHz) δ 187.2 (C-
1), 184.6 (C-4), 153.4 (C-5), 152.8 (C-8), 152.7 (C-2), 130.3 (C-3), 128.3 (C-7), 120.7 (C-6), 120.3
(C-10), 120.1 (C-9), 56.8 (OCH3), 56.4 (OCH3), 56.9 (C-1’), 49.8 (C-2’), 14.9 (C-3’); IT-TOF/MS:
1
m/z 364.41 (M + Na)+. ENDMNQ: H-NMR (CDCl3, 600 MHz): δ 7.14 (s, 2H), 4.5 (s, 1H), 3.99
(s, 6H), 3.60 (m, J = 6.4 Hz, 2H), 1.9 (m, 14H), 1.1 (m, 3H); 13C NMR (CDCl3, 150 MHz): δ191.1
(C-1), 189.3 (C-4), 154.8 (C-5), 153.4 (C-8), 152.8 (C-2), 129.9 (C-3), 128.3 (C-7), 120.7 (C-6),
120.6 (C-10), 119.1 (C-9), 56.8 (OCH3), 56.5 (OCH3), 56.9 (C-1’), 45.3 (C-2’), 29.7 (C-3’), 25.9
(C-4’), 20.5 (C-5’), 15.2 (C-6’), 14.7 (C-6’), 14.7 (C-7’), 14.1 (C-8’) and 13.1 (C-9’); IT-TOF/MS:
m/z 447.56 (M + Na)+. These results confirmed that EPDMNQ and ENDMNQ had been
successfully synthesized.
EPDMNQ and ENDMNQ inhibit lung cancer cell proliferation
The MTT assay was used to determine the survival rate of three lung cancer cell lines (A549,
NCI-H23, and NCI-H460). As shown in Figure 2A, EPDMNQ and ENDMNQ had significant
(p<0.001) effects on cell viability, and EPDMNQ was most cytotoxic. A549 cells were more

sensitive to the treatments (EPDMNQ/IC50=2.59 μM, ENDMNQ/IC50=7.54 μM) than the other cell
lines, therefore these cells were used for subsequent experiments. Because the liver and kidney are
the important target organ for drug toxicity testing, and human lung fibroblasts (IMR-90) are
extracted from embryonic cells, their primary properties have not transformed, which can directly
reflect the toxic effects in the toxicity study, so we chose the IMR-90, QSG-7701 and HEK-293A as
controls. As shown in Figure 2B, compared with 5-FU, EPDMNQ and ENDMNQ had no
significant cytotoxic effects on normal cells.
Figure 2. Cytotoxic effects of EPDMNQ and ENDMNQ on lung cancer cells. (A) Human lung
cancer cell lines A549, NCI-H23, and NCI-H460 were treated with different concentrations (1, 3,
10, 30 or 100 μM) of 5-FU, EPDMNQ or ENDMNQ for 24 h. Cell viability was determined by the
MTT assay. (B) Human normal lung fibroblasts cells IMR-90, human normal liver cells QSG-7701
and human embryonic kidney cells HEK-293A were treated with different concentrations (1, 3, 10,

30 or 100 μM) of 5-FU, EPDMNQ or ENDMNQ for 24 h. Cell viability was determined by the
MTT assay. The data are expressed as the means ± SD of the results from three independent
experiments (* p<0.05, ** p <0.01, *** p <0.001 indicate significant differences).
EPDMNQ and ENDMNQ induce apoptosis in A549 cells
To verify that EPDMNQ and ENDMNQ induce apoptosis in lung cancer cells, we observed
the cell morphology by Annexin V-FITC/PI staining. As shown in Figure 3A, cell shrinkage and
nuclear condensation, typical apoptotic morphological features, were observed and the fluorescence
intensities increased with treatment time. EPDMNQ and ENDMNQ increased A549 cell apoptosis
in a time-dependent manner (Fig. 3B). To elucidate the molecular mechanisms underlying
EPDMNQ- and ENDMNQ-mediated apoptosis, we used western blotting to examine the expression
of apoptosis-related proteins. As shown in Figure 3C, there was a decreased expression of the anti-
apoptotic protein Bcl-2, and increased expression of the pro-apoptotic protein Bad. In addition, cle-
caspase-3 and cleaved poly (ADP-ribose) polymerase (cle-PARP), important biomarkers of
apoptosis, were activated by EPDMNQ and ENDMNQ in a time-dependent manner.

Figure 3. EPDMNQ and ENDMNQ induce apoptosis in A549 lung cancer cells. (A) A549 cells
were treated with 3 μM 5-FU, EPDMNQ or ENDMNQ for different time points (3, 6, 12 or 24 h)
and stained with Annexin V-FITC/PI. (B) Apoptosis distribution was determined by flow cytometry
after treatment with 3 μM 5-FU, EPDMNQ or ENDMNQ for different time points (3, 6, 12 or 24
h). (C) Apoptosis-related protein Bcl-2, Bad, cle-caspase-3, and cle-PARP was measured by
western blotting. The data are expressed as the means ± SD of the results from three independent
experiments (* p<0.05, ** p <0.01, *** p <0.001 indicate significant differences).
EPDMNQ and ENDMNQ induce cell cycle arrest at G1 phase
To investigate whether EPDMNQ and ENDMNQ-induced proliferation inhibition and
apoptosis are caused by cell cycle arrest, cell cycle was analyzed with flow cytometry and the
expression levels of cell cycle associated proteins were detected by western blotting. As shown in
Figure 4A, after treatment with EPDMNQ or ENDMNQ, G1 phase arrest occurred in a time-
dependent manner and cell numbers in S and G2 phase decreased. In addition, western blot results
showed that the expression levels of CDK4/6 and cyclinD1/E were repressed, and p-Akt, p21 and
p27 were increased in a time-dependent manner (Fig. 4B).

Figure 4. EPDMNQ and ENDMNQ induced G1 cycle arrest and altered the cell cycle regulatory
proteins in A549 lung cancer cells. (A) Cell cycle distribution of A549 cells analysed by flow
cytometer after treatment with 3 μM EPDMNQ or ENDMNQ for different time points (3, 6, 12 or
24 h). (B) The expression levels of G1 cell cycle associated proteins p-Akt, CDK4/6, cyclin D1/E,
p21 and p27 were detected by western blotting after treated with 3 μM EPDMNQ or ENDMNQ for
different time points (3, 6, 12 or 24 h). β-actin was used as the internal control. The data are

expressed as the means ± SD of the results from three independent experiments (* p<0.05, ** p
<0.01, *** p <0.001 indicate significant differences).
EPDMNQ and ENDMNQ regulate the MAPK and STAT3 signaling pathways
To determine the signaling pathways via which EPDMNQ and ENDMNQ mediate apoptosis,
we assessed the protein expression of signaling pathways that regulate apoptosis. As shown in
Figure 5A and 5B, EPDMNQ and ENDMNQ treatment significantly (p<0.001) decreased the
phosphorylation of ERK and STAT3, and significantly (p<0.001) increased the phosphorylation of
p38 and JNK. These results indicate that EPDMNQ and ENDMNQ induce apoptosis by activating
the MAPK and STAT3 signaling pathways. SB203580 (p38 inhibitor), SP600125 (JNK inhibitor),
FR180204 (ERK inhibitor) and LY10249 (Akt inhibitor) were applied to confirm the role of
MAPK, Akt and STAT3 signaling pathways in the EPDMNQ or ENDMNQ -induced apoptosis and
cell cycle arrest on lung cancer cells. A549 cells were pre-treated with 12.1 μM of SB203580,
SP600125, FR180204 or LY10249 for 30 min followed by treatment with EPDMNQ or ENDMNQ
for 24ꢀh. The decreased protein expression levels of p-STAT3 induced by EPDMNQ or ENDMNQ
were suppressed by adding the p38 inhibitor and JNK inhibitor, enhanced by adding the ERK
inhibitor, but the change of cle-caspase-3 protein expression levels was completely opposite to p-
STAT3 (Fig. 6A-6C). These results showed that MAPK was involved in regulating the STAT3
signaling pathway and induced apoptosis in lung cancer A549 cells. In addition, the inhibitory
effect of the EPDMNQ or ENDMNQ on CDK6 and Cyclin D1 was intensified by adding the Akt
inhibitor, but p27 protein expression levels were increased (Fig. 6D). These results showed that Akt
signaling pathway regulates the cell cycle of lung cancer A549 cells.

Figure 5. EPDMNQ and ENDMNQ regulate MAPK and STAT3 signaling pathway-related
proteins in A549 lung cancer cells. A549 cells were treated with 3 μM EPDMNQ (A) or ENDMNQ
(B) for different time points (3, 6, 12 or 24 h). The expression levels of MAPK and STAT3
pathway-related proteins were detected by western blotting. β-actin was used as the internal control.
The data are expressed as the means ± SD of the results from three independent experiments (*
p<0.05, ** p <0.01, *** p <0.001 indicate significant differences).

Figure 6. Effect of EPDMNQ and ENDMNQ on MAPK, Akt and STAT3 signaling pathways in
A549 cells. (A) Expression of p-p38, p-STAT3, and cle-caspase-3 in the EPDMNQ or ENDMNQ
and p38 inhibitor SB203580-treated A549 cells. (C) Expressions of p-JNK, p-STAT3, and cle-
caspase-3 in the EPDMNQ or ENDMNQ and JNK inhibitor SP600125-treated A549 cells. (E)
Expressions of p-ERK, p-STAT3, and cleaved caspase-3 in the EPDMNQ or ENDMNQ and ERK
inhibitor FR180204-treated A549 cells. (E) Expressions of p-Akt, CDK6, Cyclin D1 and p27 in the
EPDMNQ or ENDMNQ and Akt inhibitor HY10249-treated A549 cells. The data are expressed as

the means ± SD of the results from three independent experiments (* p <0.05, ** p <0.01, *** p
<0.001 indicate significant differences).
EPDMNQ and ENDMNQ induce ROS-mediated apoptosis in A549 cells
To investigate the correlation of ROS with EPDMNQ and ENDMNQ-mediated apoptosis in
A549 cells, we used flow cytometry to detect ROS levels. EPDMNQ and ENDMNQ significantly
(p<0.001) increased ROS levels in A549 cells in a time-dependent manner (Fig. 7A). However,
after the addition of NAC, an ROS scavenger, apoptosis was significantly (p<0.001) suppressed
(Fig. 7B). In addition, NAC reversed the effects of EPDMNQ and ENDMNQ by increasing p-ERK,
p-Akt, and p-STAT3 levels and decreasing p-p38, p-JNK, cle-caspase-3, and cle-PARP levels (Fig.
7C). These data showed that EPDMNQ and ENDMNQ caused apoptosis in A549 cells by inducing
ROS-mediated activation of MAPK, Akt and STAT3 signaling.

Figure 7. EPDMNQ and ENDMNQ induce ROS accumulation in A549 lung cancer cells. (A)
A549 cells were treated with 3 μM 5-FU, EPDMNQ or ENDMNQ for different time points (3, 6, 12

or 24 h) and stained with DCFH-DA (10 μM). Intracellular ROS levels were determined by flow
cytometry. (B) A549 cells were treated with NAC for 30 min and then incubated with 3 μM
EPDMNQ or ENDMNQ for 24 h. Cell apoptosis were determined by flow cytometry. (C) A549
cells were treated with 10 mM NAC for 30 min before treatment with EPDMNQ or ENDMNQ for
24 h. The apoptotic protein expression was measured by western blotting. The data are expressed as
the means ± SD of the results from three independent experiments (* p <0.05, ** p <0.01, *** p
<0.001 indicate significant differences).
Discussion
The compound 1,4-naphthoquinone is commonly found in nature, and its structure exists in a
variety of drugs.20 Research has shown that 1,4-naphthoquinone has good biological activity,21 but
its application has been limited by its potent cytotoxicity, so we used 1,4-naphthquinone as the core
structure to develop novel compounds.
The structural design of 1,4-naphthquinone includes several main features. First, the
bioactivity of naphthone depends on the electronegativity of the quinone ring,22 and the strong
toxicity of naphthone may be due to the hydroxyl in its structure.23 Therefore, we introduced the
methoxy group into the C5 and C8 positions to increase the electrophilicity of the quinonoid ring
and reduce cytotoxicity. Second, when the branch chain of naphthquinone is introduced into sulfur
atoms, the bond dissociation enthalpy significantly decreases, and the dissociation activity of the
compounds significantly increases.24 Therefore, we introduced sulfonyl at the C2 position to
enhance its bioactivity. Third, by changing the length and spatial structure of the alkane chain on
the C2 position, we aimed to increase the fat solubility of the compounds and study the effects of
carbon chain length and volume on anti-cancer activity.25 Based on the above principles of
structural design, we ultimately synthesized EPDMNQ and ENDMNQ.
Eliminating abnormal cells through apoptosis, is an important way to maintain organism
homeostasis.26 Thus, induction of cancer cell apoptosis may be a good strategy for cancer

treatment.27 Mitochondria play a critical role in the regulation of apoptosis, and the mitochondrial
apoptotic pathway is a common target for anti-cancer drugs.28 Naphthquinones, such as shikonin,
juglone and β-Lapachone, can induce mitochondrial apoptosis by regulating the expression of Bcl-
2, Bcl-XL and Bax, resulting in activation of the caspase cascade, leading to eventual cell death.29-31
Our experiments led to a similar conclusion, as EPDMNQ and ENDMNQ enhanced expression of
the pro-apoptotic protein Bad, and inhibited the expression of Bcl-2, leading to mitochondrial-
mediated apoptosis of A549 lung cancer cells.
Inhibiting the proliferation of cancer cells by regulating the cell cycle is another important
strategy for cancer treatment.32 CDK and association with regulatory cyclins regulate cell cycle by
forming specialized cyclin/CDK complexes, and inhibitory proteins further regulate activity of
cyclin/CDK complexes. Such as p27, p21 and p57 block the interaction of CDK4 and CDK6 with
their respective targets.33,34 Indeed, our results indicated that EPDMNQ and ENDMNQ increased
the expression of p21 and p27, decreased the expression of CDK4/6 and cyclin D1/E, and
eventually led to A549 cell cycle arrest at the G1 phase.
ROS are chemically reactive radicals or non-radical molecules derived from molecular
oxygen.35 ROS directly interacts with critical signaling molecules to initiate signaling in a broad
variety of cellular processes, such as ROS homeostasis, target gene regulation, mitochondrial
oxidative stress and apoptosis.36-38 By inducing cells to produce a quantity of ROS, cancer cells will
undergo oxidative stress, triggering mitochondrial dependent apoptosis.39 Our results were in
accordance with those studies, after EPDMNQ and ENDMNQ treatment, ROS accumulated in the
cells and further activated p38 and JNK signaling pathways, inhibiting ERK and STAT3 signaling
pathway, ultimately causing mitochondrial-mediated apoptosis of A549 lung cancer cells. However,
after the addition of the ROS scavenger, NAC, these effects were prevented, the expression of pro-
apoptotic proteins (p-p38 and p-JNK) decreased and the expression of anti-apoptotic proteins (p-
ERK and p-STAT3) increased, the apoptosis induced by EPDMNQ and ENDMNQ was
significantly inhibited. In addition, the results showed that the reactivation of the p38 inhibitor, JNK

inhibitor or ERK inhibitor by EPDMNQ and ENDMNQ-inhibited the STAT3 signaling pathway,
indicating that STAT3 was regulated by the MAPK signaling pathway. In other words, when the
MAPK signaling pathway is activated, it can regulate the STAT3 signaling pathway and further
induce apoptosis.
On the other hand, accumulating evidence has indicated that ROS serve as critical signaling
molecules for cell proliferation and survival.40 The accumulation of ROS activates the Akt, p53 and
NF-κB signaling pathways, which in turn causes cell cycle arrest and eventually regulates the
proliferation of cancer cells.41,42 Our experimental results showed that ROS accumulation induced
by EPDMNQ and ENDMNQ inhibited Akt signaling pathway, and further regulated the expression
of CDK and cyclin, finally causing G1 phase arrest of A549 lung cancer cells. The above results
showed that apoptosis regulated by MAPK and STAT3 signaling pathways and cycle arrest
regulated by Akt signaling pathways were closely related to ROS generation.
The reason why EPDMNQ and ENDMNQ had low cytotoxicity in normal cells may be due to
the fact that these cells have a strong antioxidant defense system and can resist oxidative stress
damage. However, cancer cells have high impaired antioxidant defense mechanisms43, which lead
to ROS accumulation and oxidative stress. In addition, many studies have found that most
naphthraquinones cause a metabolic imbalance in intracellular ROS levels.44,45 Therefore, we
conclude that naphthalene compounds have the same or similar targets in the process of ROS
metabolism, although future studies are needed for the identification of specific targets.
In this study, we found that EPDMNQ had better anti-cancer effect than ENDMNQ, and we
speculated that this difference was caused by two reasons. First, two compounds have low polarity
and high lipid solubility, so EPDMNQ with a lower molecular mass is more likely to cross the cell
membrane and enter the cell to perform biological functions. Second, ENDMNQ with longer
carbon chain has good film-forming property, so its own effector group is not easy to be exposed
and binding with the corresponding target molecule.

In conclusion, as shown in Figure 8, our study demonstrated that EPDMNQ and ENDMNQ
induced G1 phase cell cycle arrest and apoptosis in A549 cells via the ROS-mediated activation of
MAPK, Akt and STAT3 signaling pathways. These results provide a basis for the development of
1,4-naphthoquinone derivatives and their application as potential anti-tumor agents for lung cancer.
Figure 8. Overview of the pathways for EPDMNQ and ENDMNQ that induce apoptosis in A549
human lung cancer cells.
Acknowledgment
All the authors contributed to this research. YZ, YHL, XJP, and GNS took part in the analysis
and data interpretation, and critical revision of the manuscript. JRW, YCF, JQL and WTX took part
in evaluating the MTT assay, western blot analysis, and data interpretation. YZ and TZ conducted
the fluorometric analysis. CHJ and CYW contributed to all of the experimental designs. All of the
authors contributed to the critical reading and comments. This work was funded by the Multigrain

Production and Processing Characteristic Discipline Construction Project, the Postdoctoral
Scientific Research Foundation of Heilongjiang Province of China (LBH-Q13132) and National
key research and development plan “Regulation and activity retention technology and application of
coarse cereals active components in processing” (2017YFD0401203). In addition, we thank LetPub
(www.letpub.com) for its linguistic assistance during the preparation of this manuscript.
Conflict of interest
The authors declare they have no conflict of interest.
References
1 Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin. 2018; 68:7-30.
2 Xu K, Liu B, Liu Y. Impact of Brachyury on epithelial-mesenchymal transitions and
chemosensitivity in non-small cell lung cancer. Molecular Medicine Reports. 2015; 12:995–
1001.
3 Pandurangan AK, Divya T, Kumar K, et al. Colorectal carcinogenesis: Insights into the cell
death and signal transduction pathways: A review. World Journal of Gastrointestinal Oncology.
2018; 10:244–259.
4 Mehta RG, Murillo G, Naithani R, Peng X. Cancer chemoprevention by natural products: how
far have we come? Pharmaceutical Research. 2010; 27:950-61.
5 Luo Z, Zhu W, Guo Q, et al. Weaning Induced Hepatic Oxidative Stress, Apoptosis, and
Aminotransferases through MAPK Signaling Pathways in Piglets. Oxidative Medicine and
Cellular Longevity. 2016; 2016:4768541.
6 Beccafico S, Morozzi G, Marchetti MC, et al. Artesunate induces ROS-and p38 MAPK-
mediated apoptosis and counteracts tumor growth in vivo in embryonal rhabdomyosarcoma
cells. Carcinogenesis. 2015; 36:1071-83.

7 Nitulescu GM, Margina D, Juzenas P, et al. Akt inhibitors in cancer treatment: The long journey
from drug discovery to clinical use (Review). Intern0ational Journal of Oncology. 2016;
48:869–885.
8 Markowska A, Pawałowska M, Lubin J, Markowska J. Signalling pathways in endometrial
cancer. Contemp Oncol (Pozn). 2014;18(3):143-8.
9 Héron-Milhavet L, Franckhauser C, Rana V, et al. Only Akt1 is required for proliferation, while
Akt2 promotes cell cycle exit through p21 binding. Mol Cell Biol. 2006; 26(22):8267-80.
10 Shin I, Yakes FM, Rojo F, Shin NY, Bakin AV, Baselga J, Arteaga CL. PKB/Akt mediates cell-
cycle progression by phosphorylation of p27(Kip1) at threonine 157 and modulation of its
cellular localization. Nat Med. 2002; 8(10):1145–52.
11 Subramaniam A, Shanmugam MK, Perumal E, et al. Potential role of signal transducer and
activator of transcription (STAT)3 signaling pathway in inflammation, survival, proliferation
and invasion of hepatocellular carcinoma. Biochimica et Biophysica Acta. 2013; 1835:46-60.
12 Laudisi F, Cherubini F, Monteleone G, Stolfi C. STAT3 Interactors as Potential Therapeutic
Targets for Cancer Treatment. Int J Mol Sci. 2018; 19(6):1787.
13 Wang HM, Chuang SM, Su YC, et al. Downregulation of tumor-associated NADH oxidase,
Tnox (ENOX2), enhances capsaicin-induced inhibition of gastric cancer cell growth. Cell
Biochemistry and Biophysics. 2011; 61:355-66.
14 Di Meo S, Reed TT, Venditti P, et al. Role of ROS and RNS Sources in Physiological and
Pathological Conditions. Oxidative Medicine and Cellular Longevity. 2016; 2016:1245049.
15 Guo J, Dong F, Ding L, et al. A novel drug-free strategy of nano-pulse stimulation sequence
(NPSS) in oral cancer therapy: In vitro and in vivo study. Bioelectrochemistry. 2018; 123:26-
33.
16 Ong JY, Yong PV, Lim YM, et al. 2-Methoxy-1,4-naphthoquinone (MNQ) induces apoptosis of
A549 lung adenocarcinoma cells via oxidation-triggered JNK and p38 MAPK signaling
pathways. Life Sciences. 2015; 135:158-64.

17 Xu H, Li, X, Ding W, et al. Deguelin induces the apoptosis of lung cancer cells through
regulating a ROS driven Akt pathway. Cancer Cell International. 2015; 15:25.
18 Liu C, Shen GN, Luo YH, et al. Novel 1,4-naphthoquinone derivatives induce apoptosis via
ROS-mediated p38/MAPK, Akt and STAT3 signaling in human hepatoma Hep3B cells. The
International Journal of Biochemistry & Cell Biology. 2018; 96:9-19.
19 Lee EB, Cheon MG, Cui J, et al. The quinone-based derivative, HMNQ induces apoptotic and
autophagic cell death by modulating reactive oxygen species in cancer cells. Oncotarget. 2017;
8:99637–99648.
20 Zhang RX, Zhang T, Chen K, et al. Sample Extraction and Simultaneous Chromatographic
Quantitation of Doxorubicin and Mitomycin C Following Drug Combination Delivery in
Nanoparticles to Tumor-bearing Mice. Journal of Visualized Experiments: JoVE. 2017; 128.
21 Im YS, Chung Y, Won DY, et al. Apoptotic effect of Naphthoquinone derivatives on HCT116
colon cancer cells. Genes & Genomics. 2010; 32:592–598.
22 Tu T, Giblin D, Gross ML. A Structural Determinant of Chemical Reactivity and Potential
Health Effects of Quinones from Natural Products. Chemical Research in Toxicology. 2011;
24:1527–1539.
23 Ollinger K, Brunmark A. Effect of hydroxy substituent position on 1,4-naphthoquinone toxicity
to rat hepatocytes. The Journal of Biological Chemistry. 1991; 266:21496-503.
24 Abiko Y, Shinkai Y, Unoki T, et al. Polysulfide Na2S4 regulates the activation of
PTEN/Akt/CREB signaling and cytotoxicity mediated by 1,4-naphthoquinone through
formation of sulfur adducts. Scientific Reports. 2017; 7:4814.
25 Suhara Y, Watanabe M, Motoyoshi S, et al. Synthesis of new vitamin K analogues as steroid
and xenobiotic receptor (SXR) agonists: insights into the biological role of the side chain part of
vitamin K. Journal of Medicinal Chemistry. 2011; 54:4918-22.
26 Hirose T, Horvitz HR. An Sp1 transcription factor coordinates caspase-dependent and -
independent apoptotic pathways. Nature. 2013; 500:354–358.

27 Mohammad RM, Muqbil I, Lowe L, et al. Broad targeting of resistance to apoptosis in cancer.
Seminars in Cancer Biology. 2015; 35:S78–S103.
28 Raza H, John A, Benedict S. Acetylsalicylic acid-induced oxidative stress, cell cycle arrest,
apoptosis and mitochondrial dysfunction in human hepatoma HepG2 cells. European Journal of
Pharmacology. 2011; 668:15-24.
29 Gara RK, Srivastava VK, Duggal S, et al. Shikonin selectively induces apoptosis in human
prostate cancer cells through the endoplasmic reticulum stress and mitochondrial apoptotic
pathway. Journal of Biomedical Science. 2015; 22:26.
30 Ji YB, Qu ZY, Zou X. Juglone-induced apoptosis in human gastric cancer SGC-7901 cells via
the mitochondrial pathway. Experimental and Toxicologic Pathologyꢀ: Official Journal of the
Gesellschaft Fur Toxikologische Pathologie. 2011; 63:69-78.
31 Kee JY, Han YH, Kim DS, et al. β-Lapachone suppresses the lung metastasis of melanoma via
the MAPK signaling pathway. PLoS ONE. 2017; 12:e0176937.
32 Wu X, Wang Y, Wang H, Wang Q, Wang L, Miao J, Cui F, Wang J. Quinacrine inhibits cell
growth and induces apoptosis in human gastric cancer cell line SGC-7901.Curr Ther Res Clin
Exp. 2012; 73:52–64.
33 Malumbres M. Cyclin-dependent kinases. Genome Biol. 2014; 15:122
34 Bendris N, Lemmers B, Blanchard JM. Cell cycle, cytoskeleton dynamics and beyond: the
many functions of cyclins and CDK inhibitors. Cell Cycle. 2015; 14:1786–98.
35 Dickinson BC, Chang CJ. Chemistry and biology of reactive oxygen species in signaling or
stress responses. Nature Chemical Biology. 2011; 7:504–511.
36 Holmström KM, Finkel T. Cellular mechanisms and physiological consequences of redox-
dependent signalling. Nature Reviews. Molecular Cell Biology. 2014; 15:411-21.
37 Valavanidis A, Vlachogianni T, Fiotakis K, Loridas S. Pulmonary Oxidative Stress,
Inflammation and Cancer: Respirable Particulate Matter, Fibrous Dusts and Ozone as Major

Causes of Lung Carcinogenesis through Reactive Oxygen Species Mechanisms. International
Journal of Environmental Research and Public Health. 2013; 10:3886–3907.
38 Xu C, Wang X, Zhu Y, et al. Rapamycin ameliorates cadmium-induced activation of MAPK
pathway and neuronal apoptosis by preventing mitochondrial ROS inactivation of PP2A.
Neuropharmacology. 2016; 105:270–284.
39 Gupta SC, Hevia D, Patchva S, et al. Upsides and Downsides of Reactive Oxygen Species for
Cancer: The Roles of Reactive Oxygen Species in Tumorigenesis, Prevention, and Therapy.
Antioxidants & Redox Signaling. 2012; 16:1295–1322.
40 Ray PD, Huang BW, Tsuji Y. Reactive oxygen species (ROS) homeostasis and redox regulation
in cellular signaling. Cellular Signalling. 2012; 24:981–990.
41 Guo CL, Wang LJ, Zhao Y, et al. A Novel Bromophenol Derivative BOS-102 Induces Cell
Cycle Arrest and Apoptosis in Human A549 Lung Cancer Cells via ROS-Mediated PI3K/Akt
and the MAPK Signaling Pathway. Mar Drugs. 2018;16(2):43.
42 Zhan L, Cao H, Wang G, et al. Drp1-mediated mitochondrial fission promotes cell proliferation
through crosstalk of p53 and NF-κB pathways in hepatocellular carcinoma. Oncotarget.
2016;7(40):65001-65011.
43 Oh B, Figtree G, Costa D, et al. Oxidative stress in prostate cancer patients: A systematic review
of case control studies. Prostate Int. 2016; 4(3):71-87.
44 Ghosh SK, Ganta A, Spanjaard RA. Discovery and cellular stress pathway analysis of 1,4-
naphthoquinone derivatives with novel, highly potent broad-spectrum anticancer activity.
Journal of Biomedical Science. 2018; 25:12.
45 Qiu HY, Wang PF, Lin HY, et al. Naphthoquinones: A continuing source for discovery of
therapeutic antineoplastic agents. Chemical Biology & Drug Design. 2017; 91:681–690.