A novel quinazoline-based analog induces G2/M cell cycle arrest and apoptosis in human A549 lung cancer cells via a ROS-dependent mechanism

Article abstract of DOI:10.1016/j.bbrc.2017.03.034

6-amino-4-(4-phenoxyphenylethylamino)quinazoline (QNZ) is an excellent quinazoline-containing NF-κB inhibitor also acting as a novel anticancer agent. Considering both the medicinal significance of quinazoline scaffold and the tunable functionality of Mic

Full text of DOI:10.1016/j.bbrc.2017.03.034

Accepted Manuscript
A novel quinazoline-based analog induces G2/M cell cycle arrest and apoptosis in
human A549 lung cancer cells via a ROS-dependent mechanism
Hailong Shi, Yan Li, Xiaorong Ren, Yaohong Zhang, Zhen Yang, Chenze Qi
PII:
S0006-291X(17)30491-6
10.1016/j.bbrc.2017.03.034
YBBRC 37421
DOI:
Reference:
To appear in: Biochemical and Biophysical Research Communications
Received Date: 23 February 2017
Accepted Date: 11 March 2017
Please cite this article as: H. Shi, Y. Li, X. Ren, Y. Zhang, Z. Yang, C. Qi, A novel quinazoline-based
analog induces G2/M cell cycle arrest and apoptosis in human A549 lung cancer cells via a ROS-
dependent mechanism, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/
j.bbrc.2017.03.034.
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A novel quinazoline-based analog induces G2/M cell cycle arrest and
apoptosis in human A549 lung cancer cells via a ROS-dependent
mechanism
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Hailong Shi,1 Yan Li,1 Xiaorong Ren, Yaohong Zhang, Zhen Yang, Chenze Qi*
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Zhejiang Key Laboratory of Alternative Technologies for Fine Chemicals Process,
School of Chemistry and Chemical Engineering, Shaoxing University, Shaoxing,
Zhejiang Province 312000, China
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∗Corresponding author. Fax: +86 575 88345682
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E-mail: qichenze@usx.edu.cn
1 These authors contributed equally to this work.
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24 Abstract
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6-amino-4-(4-phenoxyphenylethylamino)quinazoline (QNZ) is an excellent
quinazoline-containing NF-κB inhibitor also acting as a novel anticancer agent.
Considering both the medicinal significance of quinazoline scaffold and the tunable
functionality
of
Michael
acceptor-centric
pharmacophores
in
the
electrophilicity-based prooxidant strategy, we designed a novel QNZ-inspired
electrophilic molecule QNZ-A by introducing a Michael acceptor unit at position-6 of
quinazoline ring in QNZ. Our results identified QNZ-A as a promising selective
cytotoxic agent against A549 cells. QNZ-A, by virtue of its Michael acceptor unit,
induced reactive oxygen species (ROS) accumulation associated with collapse of the
redox buffering system in A549 cells. This caused up-regulation of p53-inducible p21
and down-regulation of redox sensitive Cdc25C along with Cyclin B1/Cdk1, leading
to a G2/M cell cycle arrest and final cell apoptosis. By contrast, QNZ-B, a reduction
product of QNZ-A lacking the Michael acceptor unit failed to induce ROS generation
and all these cell cycle-related events. In conclusion, this work provided a successful
example of designing QNZ-directed anticancer agent by a ROS-promoting strategy
and identified QNZ-A as a selective anticancer agent against A549 cells through
G2/M cell cycle arrest and apoptosis via a ROS-dependent mechanism
.
Keywords: Reactive oxygen species; Cell cycle; G2/M arrest; Michael acceptor;
Quinazoline
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45 1. Introduction
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Reactive oxygen species (ROS) play important roles in cell growth by regulating the
major mediators in many cell signaling pathways [1]. Cells can balance the production
of ROS with their removal by an antioxidant defense system under normal
physiological conditions [1]. However, once the intracellular redox equilibrium
collapses, the excessive ROS may induce direct cellular damage, in turn causing cell
growth inhibition and apoptosis by activating specific redox-sensitive cell death
signaling pathways [2]. Cancer cells characterized by mitochondrial defects,
malignant proliferation and metastatic ability, exhibit greater ROS stress than normal
cells, thereby are more susceptible to further ROS production and easier to trigger the
critical “toxic threshold” [3, 4]. This intrinsic differences in the redox status between
cancer cells and normal cells favors an anticancer strategy that selectively kills cancer
cells by ROS-promoting and decreasing the antioxidant capacity of cancer cells,
whereas is harmless to the normal cells owing to their lower basal level of
endogenous ROS and stronger antioxidant capacity [3, 4].
Numerous promising cytotoxic drugs that kill cancer cells by the abrogation of
proliferative signals have been reported to possess ROS-generating ability, including
certain commonly used chemotherapeutic agents such as commercial anticancer drug
5-fluorouracil [5] and paclitaxel [6]. One of the most exciting findings regarding
ROS-based anticancer agents is that much of these drugs contain electrophilic
Michael acceptor pharmacophores which can be employed as tools to fine tune
biological activity depending on their multiple reactivities [7]. Michael acceptors can
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form covalent adducts with critical thiol residues in redox-sensitive proteins that
regulate celluar redox status such as glutathione and thioredoxin, thereby inducing
dramatic oxidative stress in target cancer cells [8, 9]. In this respect, for example,
natural products parthenolide [10] and piperlongumine [11]characterized by
α,β-unsaturated carbonyl moiety in their molecular structures can preferentially
induce apoptosis in human cancer cells via ROS generation over normal cells,
vigorously supporting the concept of ROS-based selective cancer killing [3, 4].
Quinazoline unit has been found as an important scaffold for many drugs with
broad spectrum of biological activities such as anti-inflammatory, antioxidant and
anticancer [12]. Recent drug optimization efforts have generated several novel
quinazoline-derived compounds exerting potent anti-cancer activity via ROS
generation
[13].
In
this
study,
our
interest
was
focused
on
a
6-amino-4-(4-phenoxyphenylethylamino)quinazoline
(QNZ),
quinazoline-containing compound, known for its excellent NF-κB inhibitory activity
[14]. Additionally, QNZ also exhibits as a novel anti-cancer reagent [15]. However,
the action mechanisms behind are still unclear, and there is as yet scarcely any
research related to designing QNZ-inspired anticancer agents based on the
ROS-promoting strategy. In this study, we tried to design a QNZ-based prooxidant by
introducing a Michael acceptor unit at position-6 of quinazoline ring in QNZ to
increase its electrophilicity. To probe the possibility of Michael acceptor-dependent
prooxidant mechanism, we synthesized two analogs: QNZ-A with a Michael acceptor
unit and its reduction product QNZ-B lacking the Michael acceptor unit. Our results
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confirm for the first time that QNZ-A as a novel electrophilic compound can
selectively kill human non-small cell lung cancer (A549) cells mainly by
ROS-mediated cell cycle arrest and apoptosis. It is worthy of further study for its
potentials in the investigation of Michael acceptor-dependent redox intervention
related molecular mechanisms and as a possible lead structure to develop new cancer
drug.
95 2. Materials and methods
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2.1 Materials
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RPMI medium 1640, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium
bromide (MTT), Propidium iodide (PI), RNAse, 2',7'-Dichlorofluorescin diacetate
(DCFH-DA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The
antibodies against p53, p21, Cyclin B1, Cdk1 and Cdc25C were from Cell Signaling
Technology (Beverly, MA, USA). QNZ, QNZ-A and QNZ-B were synthesized in our
lab (the details and spectra data shown in the Supplementary Materials).
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2.2. Electrophilicity assessment by NMR
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1H NMR spectra of QNZ-A (50 mM in d6-DMSO) before and after the incubation
with benzyl mercaptan (75 mM in d6-DMSO) at set intervals (3 h, 2 or 5 day) were
recorded using a Bruker AV 400 (Bruker Biospin Co. Ltd., Switzerland) spectrometer.
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2.3. Cell Culture
A549 cells, human hepatocellular carcinoma (HepG2) cells and human umbilical vein
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endothelial (HUVEC) cells (Shanghai Institute of Biochemistry and Cell Biology,
Chinese Academy of Sciences) were cultured in RPMI-1640 medium supplemented
with 10% (v/v) FBS, 2 g/L NaHCO3, 2 mM glutamine, 100 kU/L penicillin and 100
kU/L streptomycin at 37 °C in an atmosphere of 5% CO2.
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2.4. Cytotoxicity assay
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Cells (3 × 103/well) were treated with graded concentrations of test compounds for 48
h, and then incubated with MTT (0.5 mg/mL) for 4 h. When necessary, the cells were
pretreated with N-acetylcysteine (NAC, 10 mM), dithiothreitol (DTT, 500 µM) or
α-tocopherol (VE, 500 µM) for 1 h before adding QNZ-A. Then the medium was
substituted with DMSO for OD determination using a microplate reader (Model 550,
Bio-Rad, CA).
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2.5. Cell cycle and apoptosis analysis
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A549 cells (2 × 105 cells/well) were treated with test compounds for 24 h (in cell
cycle analysis) or 48 h (in cell apoptosis analysis). When necessary, the cells were
pretreated with NAC (10 mM) or VE (500 µM) for 1 h before QNZ-A was added.
Cells were harvested for cell cycle and apoptosis analysis using a flow cytometer
(Becton–Dickinson, San Jose, CA, USA) as described in our previous work [16].
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2.6. Intracellular ROS assay
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A549 cells (3 × 105 cells/well) treated with test compounds for 4 h or 8 h, with or
without pre-incubation with NAC or DTT or VE, were harvested, stained with
DCFH-DA and analyzed by flow cytometry (Becton–Dickinson, San Jose, CA, USA)
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[17]. The ROS level in HUVEC cells was also assayed after treated with QNZ-A (15
µM) for 8h.
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2.7. Intracellular glutathione assay
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A549 cells (3 × 105 cells/well) treated with compounds as desired were harvested,
lysed and centrifugated, the supernatant was used to determine the total glutathione
(GSH) and glutathione disulfide (GSSG) levels using a GSH and GSSG Assay Kit
(Beyotime Biotechnology, Jiangsu, China) by an Infinite M200 Pro microplate reader
(Tecan, USA). The GSH levels were calculated as previously described [17].
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2.8. Real-time quantitative PCR (RT-qPCR) analysis
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A549 cells (2 × 106 cells/dish) treated with QNZ-A (5, 15 µM) for 24 h with or
without pre-incubation with NAC were collected and total RNA was extracted using
Total RNA Extraction Kit (Tiangen Biotech Co., Ltd., Beijing, China). The reverse
transcription was performed at 42 °C for 1 h and 70 °C for 10 min. RT-qPCR was
performed using Brilliant II SYBR Green QPCR Master Mix (Agilent technologies,
Santa Clara, CA, USA) on a Stratagene Mx3005P QPCR System (Agilent
technologies, Santa Clara, CA, USA). The cycling conditions were: 10 min at 95 °C
followed by 40 cycles of amplification (30 s at 95 °C and 50 s at 60 °C ). All data
were analyzed by the ∆∆Ct method using GAPDH as the endogenous reference and
were normalized to the non-treated control. Primer sequences are shown in Table S1
in Supplementary materials.
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2.9. Western Blot analysis
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A549 cells (3 × 106 cells/dish) were treated with QNZ-A (5, 15 µM) or QNZ-B (15
µM) for 24 h with or without pre-incubation with NAC or VE. After cells lysed, the
protein lysates were harvested for western blots as previously described [17]. The
signals were finally detected using an enhanced ImageQuant chemiluminescence
system (GE Healthcare, Pittsburgh, PA, USA).
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2.10. Statistical Analysis
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Data are expressed as mean ± SD of the results obtained from at least three
independent experiments. Significant differences (P < 0.05) between the means of two
groups were analyzed by Student's t-test using SPSS 17.0 (SPSS Inc., USA).
160 3. Results
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3.1. Synthesis and electrophilicity assay
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As is outlined in Fig. 1A, QNZ-A, a novel QNZ analog, was constructed via a
nucleophilic acyl substitution reaction between acryloyl chloride and QNZ. Its
reduction product QNZ-B, was obtained by catalytic hydrogenation of QNZ-A over
Pd/C. To clarify whether introduction of Michael acceptor-pharmacophore could
increase the electrophilicity of the parent QNZ, the 1H NMR spectroscopy changes of
QNZ-A in presence of the sulfydryl-containing reagent benzyl mercaptan were
monitored. After incubating a mixture of QNZ-A and benzyl mercaptan at a molar
ratio of 1:1.5 in d6-DMSO, the three groups of double doublets of the olefinic protons
at δ 5.81, δ 6.31 and δ 6.50 disappeared over time, associating with an appearance of
a strong multiplet at δ 2.70 indicative of the conjugate-addition (Fig. 1B). In
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comparision, the addition does not occur for QNZ and QNZ-B under the same
conditions (data not shown), implying that QNZ-A is more electrophilic by virtue of
its Michael acceptor moiety.
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3.2. Selective cytotoxicity toward cancer cells in a ROS-dependent fashion
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A series of dose-response curves shown in Fig. 1C allowed us to identify QNZ-A
(IC50 = 6.7 µM) as the most excellent anti-proliferative agent in A549 cells. In
contrast, its reduction product QNZ-B (IC50 = 71.1 µM ) with the Michael acceptor
unit completely abolished, was even less active than the leading QNZ (IC50 = 41.1
µM), suggesting that introduction of the electrophilic Michael acceptor was essential
for enhancing the cytotoxicity. The cytotoxicity of QNZ-A against HepG2 cancer
cells together with HUVEC normal cells was also tested. Minimal cytotoxicity was
observed in HUVEC normal cells, revealing that normal cells display greater
tolerance to QNZ-A compared to cancer cells, in which A549 cells was the most
sensitive one (Fig. 1D). Based on the excellent selectivity, all the further studies on
the cytotoxic mechanisms of QNZ-A were focused on A549 cells.
Considering ROS as one of the leading causes of growth inhibition and cell death,
three antioxidants including NAC, DTT and VE were employed to clarify the role of
ROS in the cytotoxicity induced by QNZ-A. As shown in Fig. 1E, pretreatment with
NAC or DTT, acting as both a ROS scavenger and a sulfhydryl-containing
nucleophile to preferentially react with the Michael donor, almost completely reversed
the cytotoxicity induced by QNZ-A. A significant reversion effect was also achieved
for VE as another important ROS scavenger but with no nucleophilic activity. The
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above results clearly indicate that ROS generation dramatically contributes to the
cytotoxicity of QNZ-A, and its Michael acceptor unit also plays a pivotal role.
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Fig. 1 here
3.3. ROS-dependent G2/M cell cycle arrest and apoptosis
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To investigate the possible mechanisms underlying the cytotoxicity, we further
analyzed the effects of QNZ-A on cell cycle distribution and apoptosis by flow
cytometry. As shown in Fig. 2A, 24 h of treatment with QNZ-A caused a remarkable
dose-dependent accumulation of cells in G2/M phase. Increasing concentration from 5
to 15 µM led to a successive increase of G2/M-phase cell population from 12.57%
(control) to 33.69% (15 µM). Additionally, after a longer duration (48 h) treatment,
apoptosis of A549 cells was also strikingly triggered by QNZ-A in a
concentration-dependent manner (Fig. 2B). In contrast, 15 µM of QNZ-B exhibited
an insignificant effect on both the cell cycle arrest and induction of apoptosis (Fig. 2A
and B). In addition, pretreatment with NAC or even VE noticeably reversed the cell
cycle arrest and apoptosis induced by QNZ-A (Fig. 2A and B), in line with the results
obtained by the cytotoxicity assay.
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Fig. 2 here
3.4. ROS accumulation and imbalance of cellular redox homeostasis
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Based on the above results, we further measured the intracellular ROS levels in A549
cells. As shown in Fig. 3A, treatment with QNZ-A caused an obvious intracellular
ROS accumulation in a dose- and time-dependent manner. The fluorescence intensity
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measured in cells treated with QNZ-A (15 µM) were increased by 4.6-fold relative to
the vehicle control at 8 h. In contrast, QNZ-B had almost no ROS-generating ability
under the same conditions. As expected, the ROS accumulation induced by QNZ-A
was almost completely abolished by pretreating the cells with any one of the
antioxidants including NAC, DTT and VE (Fig. 3B). Additionally, compared with the
ROS accumulation observed in A549 cells, QNZ-A (15 µM) only raised the ROS
level by no more than 15% in HUVEC normal cells at 8 h, highlighting a ROS-based
cancer cell selectivity.
We subsequently determined the ratio of GSH/GSSG in A549 cells to ascertain
whether the ROS accumulation induced by QNZ-A resulted in an imbalance of
intracellular redox state. After exposed to QNZ-A for 4 and 8 h, a dose- and
time-dependent decrease and increase for the GSH and GSSG levels in cells was
observed, respectively (Fig. 3D and E). Thus the GSH/GSSG ratios calculated based
on the measured GSH and GSSG levels were undoubtedly decreased by QNZ-A (Fig.
3F). In contrast, QNZ-B was inactive in changing the glutathione levels (Fig. 3D-F).
And pretreatment with NAC or VE also abolished these alterations induced by
QNZ-A (Fig. 3D-F), supporting that the falling apart of intracellular redox buffering
system was associated with ROS generation.
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Fig. 3 here
3.5. Molecular Mechanisms for ROS-Dependent G2/M Cell Cycle Arrest
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To further clarify the molecular mechanisms by which QNZ-A induced
ROS-dependent G2/M cell cycle arrest in A549 cells, the effect of QNZ-A on the
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redox sensitive G2/M checkpoint regulators was investigated by RT-qPCR and
Western blotting (Fig. 4A and B). Upon the treatment with QNZ-A (5 or 15 µM) for
24h, an obvious dose-dependent down-regulation of Cdc25C, Cyclin B1 and Cdk1
along with up-regulation of p53 and p21 was observed both on levels of mRNA and
protein (Fig. 4A and B). However, Fig. 4B shows that QNZ-B (15 µM) barely
affected the expression of the above proteins. In addition, pretreatment with NAC or
VE reversed all these protein expression changes induced by QNZ-A (Fig. 4B), which
is in line with the results from the cell cycle analysis, highlighting the pivotal role of
ROS in regulating expression of the above redox active cell-cycle-regulatory proteins.
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Fig. 4 here
247 4. Discussion
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In this work, QNZ-A, a QNZ inspired electrophilic molecule, was designed by
introducing a Michael acceptor unit (Fig. 1A and B), and was identified as a potential
selective anticancer agent in terms of its preferential cytotoxicity toward A549 and
HepG2 cancer cells over HUVEC normal cells (Fig. 1D). The cancer cell selectivity
was further supported by the significant difference in inducing the accumulation of
ROS (Fig. 3C) between A549 and HUVEC cells. Abrogating the cytotoxicity of
QNZ-A by antioxidants NAC, DTT and VE (Fig. 1E) indicated ROS were involved in
the cytotoxicity mechanism. It is worth noting that NAC and DTT acting as both
antioxidants and nucleophiles can reverse the cytotoxicity of QNZ-A more
thoroughly than VE, supporting that Michael acceptor unit also contributes to its
activity. In addition, the cytotoxicity of QNZ-A towards A549 cells is predominately
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mediated by G2/M cell cycle arrest and subsequent apoptosis, in which the Michael
acceptor-dependent ROS generation also plays a central role (Fig. 2A and B). This is
further supported by the fact that NAC and VE completely block the ROS
accumulation induced by QNZ-A, while QNZ-B as a reduction product of QNZ-A
with no Michael acceptor-pharmacophore is inactive in inducing ROS-generation (Fig.
3A and B).
The decrease in the intracellular GSH/GSSG ratio induced by QNZ-A in A549
cells indicates the collapse of intracellular redox buffering system. However, it is hard
to draw firm conclusion about whether the ROS accumulation is the trigger of this
change. QNZ-A which has proven to be an electrophile reactive to
sulfydryl-containing molecules (Fig. 1B), may react directly with millimolar
concentrations of intracellular GSH in all probability, causing the decrease of
GSH/GSSG ratio. From another standpoint, despite the slight decrease of the GSH
levels in A549 cells, the alteration is much more obviously in the GSSG levels than in
the GSH levels (Fig. 3D and E), indicating that the decline of GSH/GSSG ratio is
more dependent on the dramatic increase of intracellular GSSG levels. In addition,
pretreatment with antioxidant VE reversed all the alterations of intracellular GSH,
GSSG and GSH/GSSG ratio induced by QNZ-A (Fig. 3D-F), reflecting that ROS do
contribute to the collapse of redox homeostasis. It can be inferred from the above
results that the ROS accumulation would act as both a trigger and an effector of the
drop in GSH levels, leading to a vicious cycle and ensuing redox imbalance in A549
cells.
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In further research, we investigated the underlying molecular mechanisms for the
ROS-dependent G2/M-phase arrest induced by QNZ-A. An extensive number of
reports focused on redox regulation of cell growth and death, support the view that
ROS as key signaling intermediates participate in cell-cycle progression [18-20]. The
accumulation of ROS disturb the redox control of cell cycle progression via
phosphorylation and ubiquitination of cell cycle regulatory proteins such as cyclins,
Cdks and Cdk inhibitors, leading to aberrant cell proliferation and apoptosis [18-20].
Our results revealed that the mRNA and protein expression levels of G2/M checkpoint
regulators Cyclin B1 and Cdk1 were down-regulated by QNZ-A in a ROS-dependent
manner in A549 cells (Fig. 4A and B). The Cyclin B1/Cdk1 complex can be activated
by a redox sensitive cell-cycle-regulatory protein Cdc25C phosphatase through
phosphorylation of Thr161 and dephosphorylation of pThr14 and pTyr15 on Cdk1
[21]. What coincides is that a down-regulation of Cdc25C expression was also
observed in A549 cells treated with QNZ-A dose-dependently (Fig. 4A and B). In
addition, this down-regulation can be rescued by ROS-scavenger (Fig. 4A and B),
which is supported by the reported hypothesis that ROS can inactivate Cdc25C
through oxidization of cysteine 330 and 377 at the enzyme active site to form
intramolecular disulfide [21]. On the other hand, the activation of Cyclin B1/Cdk1
complex is also negatively regulated by p21, a downstream mediator of the tumor
suppressor p53 in a stressed situation [22, 23]. Our results showed that QNZ-A
increased both the mRNA and protein expression levels of p53 and p21 in a
ROS-dependent manner (Fig. 4A and B). It is well established that p53 is an important
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sensor of cellular stress conditions (such as excess ROS), playing important roles in
cell cycle arrest through up-regulating p21 and in apoptosis [22, 23]. So the
up-regulation of p53 may contribute not only to the G2/M phase arrest but also the
apoptosis induced by QNZ-A in A549 cells. QNZ-B, by contrast, failed to affect
expression of the above redox-sensitive target proteins under the same conditions (Fig.
4B), clearly indicating that electrophilic (Michael acceptor) moiety also at least
partially contributes to the collapse the cell-cycle-regulatory system in A549 cells.
In summary, a novel QNZ analog, QNZ-A, was first identified as a selective
cytotoxic agent toward cancer cells via a Michael acceptor-dependent prooxidant
mechanism. A possible model depicting the actions of QNZ-A is presented in Fig. 4C.
QNZ-A, by virtue of its Michael acceptor unit, can induce ROS accumulation
associated with the collapse of the redox buffering system. The ROS burst triggers
redox-sensitive signaling pathways: up-regulation of p53 and p21, and
down-regulation of Cdc25C and Cyclin B1/Cdk1, leading to a final G2/M cell cycle
arrest and apoptosis in A549 cells. The above results represent a clear advantage of
QNZ-A as a lead compound for ROS-based cancer therapeutic purposes.
319 Acknowledgements
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This work was supported by Zhejiang Provincial Natural Science Foundation of China
(Grant No. LQ16C050001), Zhejiang Provincial Natural Science Foundation of China
(Grant No. LQ16H090004) and National Natural Science Foundation of China (Grant
No. 21302129).
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Fig.1. Synthesis, electrophilicity and cytotoxicity of QNZ analogs. (A) Synthetic
1
route of QNZ and its analogs QNZ-A and QNZ-B. (B) H NMR spectra of QNZ-A
in d6-DMSO before and after adding benzyl mercaptan. (C) Cytotoxicity of QNZ and
its analogs QNZ-A and QNZ-B against A549 cells. (D) Cytotoxicity of QNZ-A
against A549, HepG2 and HUVEC cells. (E) Cytotoxicity of QNZ-A against A549
cells in the absence or presence of pretreatment with NAC, DTT or VE.
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Fig.2. ROS-dependent G2/M cell cycle arrest (A) and apoptosis (B) induced by
QNZ-A in A549 cells. Cells were treated with the test compounds with indicated
concentrations for 24 h (in cell cycle analysis) or 48 h (in cell apoptosis analysis) in
the absence or presence of pretreatment with NAC or VE. Data are representative of
three independent experiments.
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Fig. 3. ROS accumulation and imbalance of cellular redox homeostasis induced by
QNZ-A. (A) Fold change of ROS stimulated by QNZ-A and QNZ-B with the
indicated concentrations after 4 h or 8 h of treatment in A549 cells. (B) Effect of ROS
scavengers NAC, DTT or VE on the ROS accumulation induced by QNZ-A in A549
cells. (C) A comparison of ROS accumulation in A549 cells and in HUVEC normal
cells treated with QNZ-A for 8 h. (D-F) Alterations of GSH levels (D), GSSG levels
(E), and GSH/GSSG ratios (F) in A549 cells treated by QNZ-A and QNZ-B with the
indicated concentrations for 4 h or 8 h, and effect of NAC or VE on these alterations.
Data are expressed as mean ± SD; n = 3, * P<0.05, ** P<0.01, *** P<0.001, vs.
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control; # P<0.05, ## P<0.01, ###P<0.001.
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Fig. 4. Molecular mechanisms for QNZ-A-induced G2/M cell cycle arrest. (A)
RT-qPCR analysis of mRNA expression levels of G2/M checkpoint regulators in
A549 cells treated with QNZ-A for 24 h with or without NAC pretreatment. Data are
expressed as mean ± SD; n = 3, * P<0.05, ** P<0.01, *** P<0.001, vs. control; #
P<0.05, ## P<0.01. (B) Western blot analysis of G2/M checkpoint proteins in A549
cells treated with QNZ-A and QNZ-B for 24 h in the absence or presence of
pretreatment with NAC or VE. Data are representative of three independent
experiments. (B) Proposed mechanisms underlying the ROS-mediated cytotoxicity of
QNZ-A in A549 cells.
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Highlights
•
•
•
•
•
Electrophilic molecule QNZ-A selectively kills A549 cancer cells by
ROS-promoting.
The cytotoxic mechanism relies on ROS-dependent G2/M-phase arrest and
apoptosis.
QNZ-A up regulates mRNA and protein levels of p53 and p21 in a
ROS-dependent way.
QNZ-A induces ROS-dependent G2/M arrest through Cdc25C/cyclin B1/Cdk1
pathway.
Michael acceptor-dependent prooxidant anticancer strategy is supported.