An Isoquinolin-1(2H)-Imine Derivative Induces Cell Death via Generation of Reactive Oxygen Species and Activation of JNK in Human A549 Cancer Cells

Article abstract of DOI:10.1002/jcb.26093

Compound 11-benzoyl-10-chloro-7,9-difluoro-6-imino-2,3,4,6-tetrahydro-1H-pyrimido[1,2-b]isoquinoline-8-carbonitrile (HC6h) is a novel polyhalo 1,3-diazaheterocycle fused isoquinolin-1(2H)-imines derivative, which displays good anticancer activity and low

Full text of DOI:10.1002/jcb.26093

Article
An isoquinolin-1(2H)-imine derivative induces cell death via
generation of reactive oxygen species and activation of JNK in
human A549 cancer cells†
Jing Liu1# , Tongyang Liu1#, Hanchuan Mou1, Shuting Jia1, Chao Huang2, Shengjiao
Yan2, Jun Lin2*, Ying Luo1*, Jihong zhang1*
1
Laboratory of Molecular Genetics of Aging and Tumor, Faculty of Medicine，
Kunming University of Science and Technology, Kunming 650500, P.R. China
2 Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry Education,
School of Chemical Science and Technology, Yunnan University, Kunming, 650091,
P.R. China
*Correspondence to Jihong Zhang, email zhjihong2000@126.com. Lab of Molecular
Genetics of Aging and Tumor, Faculty of Medicine, Kunming University of Science
and Technology, 727 Jing Ming Nan Road, Chenggong County, Kunming 650500,
Yunnan Province, China.
Telephone: 0086-871-65920753
Fax: 0086-871-65920753
# The authors contribute to the paper equally
†This article has been accepted for publication and undergone full peer review but has
not been through the copyediting, typesetting, pagination and proofreading process,
which may lead to differences between this version and the Version of Record. Please
cite this article as doi: [10.1002/jcb.26093]
Received 20 March 2017; Revised 25 April 2017; Accepted 25 April 2017
Journal of Cellular Biochemistry
This article is protected by copyright. All rights reserved
DOI 10.1002/jcb.26093
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1

Abstract
Background:Compound11-benzoyl-10-chloro-7,9-difluoro-6-imino-2,3,4,6-tetrahydr
o-1H-pyrimido[1,2-b]isoquinoline-8-carbonitrile (HC6h) is a novel polyhalo
1,3-diazaheterocycle fused isoquinolin-1(2H)-imines derivative, which displays good
anticancer activity and low toxicity in vivo. However, the underlying anticancer
mechanisms have not previously been identified.
Methods: The proliferation of A549 was assessed by MTT assay. The reactive oxygen
species (ROS) level was assessed in A549 with a H2DCFDA probe. Mitochondrial
membrane potential was measured using the JC-1 staining. Apoptosis were measured
by annexin-V/PI assay and autophagy by acridine orange staining and GFP-LC3
fluorescence assay. The expression of autophagic and apoptotic proteins was
determined by Western blot.
Results: The compound HC6h increased accumulation of vesicles, acridine
orange-stained cells and LC3-II in A549 cells. Inhibition of compound HC6h-induced
autophagy by bafilomycin A1 increased apoptosis. Compound HC6h enhanced
activation of caspase-3, caspase-9 and PARP cleavage in A549 cells. Compound
HC6h leads to the rapid generation of intracellular ROS. Moreover, compound HC6h
induced phosphorylation of JNK and was conferred by the increased ROS levels.
Furthermore, down-regulation of JNK attenuated autophagic and apoptotic effect in
response to HC6h.
Conclusion: The induction of ROS upon HC6h treatment leads to the activation of
JNK that mediates autophagy and apoptosis in human A549 cancer cells. This article is
protected by copyright. All rights reserved
Key words: isoquinolin; autophagy; apoptosis; ROS
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Introduction
Lung cancer is the most common cancer in both women and men, and also the leading
cause of cancer death. Although multiple diagnostic procedures and therapeutic
options are developed recently, the overall outlook has not greatly improved for most
patients，with the overall 5-year survival having only slightly increased over the last
decade from 15.7% to 17.4%. It is well known that resisting cell death is a hallmark of
cancer which contributes to tumor progress and resistance to conventional anti-tumor
therapies [1]. Therefore, targeting cancer cell death pathways is an efficient strategy to
kill cancer.
Cell death includes apoptosis, autophagy and necrosis. Apoptosis, type I
programmed cell death (PCD), plays an important role in chemotherapy-induced
tumor cell death in variety of cancers[2]. Moreover, apoptosis activation in cancer
therapy is irreversible and is commonly regarded as an efficient way to kill cancer
cells. Apoptosis can be classified into two distinct signaling pathways: the extrinsic
and intrinsic pathway [3]. In the extrinsic pathway, apoptosis is triggered by death
receptors and activated by death receptor ligands (FASL, TNFα and TRAIL). On the
other hand, the mitochondrial-initiated pathway (the intrinsic apoptosis) is usually
associated with mitochondrial outer membrane permeabilization (MOMP) and the
release of pro-apoptotic proteins such as Bax and activation of caspases [4].
Autophagy is a basic catabolic process, which is not only involved in energy
homeostasis, cellular components turnover and development but also in cancer cell
death. Autophagy is regarded as type-II PCD program that occurs in various cancer
cells in response to anticancer therapies[5]. In some cellular settings, autophagy
serves as a cell survival mechanism suppressing apoptosis, or in others it can lead to
cell fate, either being together with apoptosis or being an alternative mechanism when
the apoptosis is defective[6].
Reactive oxygen species（ROS）act as mediators of cell fate determined by the extent
of oxidative damage, under physiological conditions, low levels of ROS result in
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3

growth adaptation and survival[7]. In contrast, excessive ROS production induces
cellular damage and promotes apoptotic, necrotic and autophagic cell death[8].
Cancer cells have a higher metabolic rate than normal cells, leading to comparatively
higher intracellular ROS concentrations. Therefore, cancer cells are under increased
oxidative stress, causing them to be more vulnerable to ROS-mediated insults induced
by exogenous agents[9, 10]. It is well known that various ROS-inducing compounds
are effective in cancer therapy by causing mitochondrial damage and cell death[11]. In
addition, ROS are regulators of mitogen-activated protein kinase(MAPKs) signaling
transduction, which mediates intracellular signal transduction in response to different
physiological stimuli and stressing conditions[12]. C-Jun NH2-terminal kinase (JNK)
is one of the major MAPKs, JNK activation induce apoptosis. An increase in
intracellular ROS, as well as activation of MAPKs plays a key role in many cellular
events, including apoptosis and autophagy[13-15].
11-benzoyl-10-chloro-7,9-difluoro-6-imino-2,3,4,6-tetrahydro-1H-pyrimido[1,2-b]i
soquinoline-8-carbonitrile (HC6h) compound is polyhalo 1,3-diazaheterocycle fused
isoquinolin-1(2H)-imines derivative. It has been shown that isoquinolin-1(2H)-imine
derivatives exhibit anti-malarial, anti-inflammatory, anti-tumor and antiviral
properties[16]. We previously evaluated a series of polyhalo 1,3-diazaheterocycle
fused issoquinolin-1(2H)-imines derivatives to ascertain the cytotoxicity in a series of
cancer cell lines. We found that HC6h compound showed good anticancer activity in
vitro and in vivo. Also, the HC6h compound inhibited S180 and H22 mice tumor
growth and did not show any acute toxicity at the dosage of 5000mg/kg[16]. However,
the underlying anti-tumor mechanism has not been identified before.
Here we investigated the mechanism of HC6h induced cell death in human
adenocarcinoma lung cancer cells (A549). Exposure of A549 cells to HC6h resulted in
cell death with morphological and biochemical changes characteristic of autophagy
and apoptosis. Furthermore, we identified that the involvement of ROS production as
well as JNK activation as upstream of HC6h-induced autophagic and apoptotic
pathways. In addition, in combination with autophagy inhibitor, bafilomycin increased
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the apoptotic effect of HC6h in A549 cells, suggesting that autophagic effect induced
by HC6h acted as a cell repair mechanism to counteract the damaging effect caused
by HC6h-induced ROS. Thus our results demonstrated the cytotoxic activity of HC6h
by inducing both autophagy and apoptosis in A549 cells and its potential as a
promising chemotherapeutic agent.
Materials and Methods
Chemicals
HC6h compound was synthesized according to the literature[17] at Key Laboratory of
Medicinal Chemistry for Natural Resource (Yunnan University), Ministry Education,
School of Chemical Science and Technology, Yunnan University, Kunming, P.R.
China. In detail,
a
dry mortar was charged with HKAs
1
(1-phenyl-2-(tetrahydropyrimidin-2(1H)-ylidene) ethan-1-one,
1
mmol) and
polyhaloisophthalonitrile 4 (5-chloro-2,4,6-trifluoroisophthalonitrile, 1.1 mmol). The
mixture was mixed at room temperature by vigorously grinding with a pestle for a few
minutes (1-2 min). The mixture was placed in a microwave tube and irradiated in a
microwave reactor (Discover), with control of power and temperature by infrared
detection, at 120℃ for 12 min (maximum power 200 W). After it was cooled, the
resulting mixture was transferred to a 50 mL flask, and dissolved in 25 mL of dioxane,
and addition of t-BuOK (1.5 mmol). Stirring at room temperature, the reaction process
was monitored by TLC. After completion, the reaction mixture was poured into 60
mL of water and filtered to obtain the crude products, which were purified by column
chromatography (petrol/ethyl acetate ) 1:3, v/v) on silica-gel to give the desired
product HC6h. The structure of HC6h that was used in our study is shown in Fig.1.
Compounds were prepared as 100 mM stock solutions in DMSO and aliquots stored
at -20 ℃, protected from light. Reagents, unless specified otherwise, originated from
Sigma-Aldrich Ltd (China).
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Cell lines and culture conditions
Human colon cancer cell lines (HCT116, HT29), human ovarian cancer (SK-OV-3),
human breast cancer (MCF-7), human glioblastoma (U251), human lung cancer
(A549)and normal lung fibroblast (MRC-5) cell lines were obtained from the
American Type Culture Collection(Manassas, VA); HCT116, HT29, SK-OV-3 and
A549 cells were grown in RPMI1640 medium (Hyclone, Thermo Scientific)
supplemented with 10% fetal bovine serum (FBS), MRC-5, U251and MCF-7 cells
were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone,
Thermo Scientific) supplemented with 10% FBS. All cells were incubated at 37 °C
under 5% CO2. All cell lines were verified as mycoplasma free.
Growth Inhibitory Assays
Growth inhibition of human cancer cells by the synthetic polyhalo
1,3-diazaheterocycle fused iaoquinolin-1(2H)-imines derivative was assessed by the
MTT assay, along with DMSO as a control. Different human cancer cell lines were
treated with compound at various concentrations (0μM, 0.1μM, 1μM, 10μM, 50μM,
100μM).
After
a
72
h
incubation,
MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was added to the
wells (50 μl; 0.4 mg/ml) and incubated another 4h. Medium were aspirated and
DMSO (150 μl) was added to each well. Absorbance was measured at 490 nm using
2030 Multi-label Reader (Perkin-Elmer Victor X5, US). Compound concentrations
causing 50% growth inhibition (IC50) were calculated.
Measurement of intracellular ROS
Cellular ROS content was determined using the oxidation-sensitive fluorescent probe
dye, 2’,7’-dichlorofluorescein diacetate (H2DCFDA) according to the manufacturer’s
instructions. Briefly, cells were collected and washed once with PBS and labeled for
30 min at 37C in the dark with 10 µM H2DCFDA probe. The cells were then washed
three times with PBS, and ROS levels (fluorescence intensity) were determined by
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6

flow cytometry.
Mitochondrial Membrane Potential Assay
Mitochondrial membrane potential was measured by flow cytometry using the
lipophilic cation 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolyl-carbocyanine
iodide (JC-1;). In healthy cells, JC-1 stains the mitochondria red whereas in cells with
decreased mitochondrial membrane potential, JC-1 remains in the cytoplasm and
emits green fluorescence. A549 cells treated at indicated concentrations and times
were harvested and incubated with JC-1 at a final concentration of 1µg/mL for 30 min
at 37℃. After staining, cells were washed twice with ice-cold PBS and subjected to
flow cytometry analysis.
Western blot analysis
Treated cells were pelleted and lysed in the lysis buffer (2.5 mM EDTA, 2.5 mM
EGTA, 20 mMNaF, 1 mM Na3VO4, 100 Mm NaCl, 0.5% triton X-100) and freshly
added protease inhibitor. Protein concentration was determined by Bradford assay.
Cellular protein (50 μg) were separated by SDS polyacrylamide gel electrophoresis
and blotted onto PVDF membranes. Membranes were blocked and probed overnight
with the respective primary antibodies (LC3, p53, p-p53, PUMA, Bax, Bcl-2,
caspase-3, caspase-9, PARP, JNK, p-JNK, p62, VDAC and tubulin,) (Cell Signaling
Technology) followed by horseradish-peroxidase-coupled secondary antibodies.
Chemiluminescence was performed with ECL kit (Thermo).
Cytochrome C release assay
A549 cells treated with vehicle and HC6h compound for 24 h were harvested and
washed once with ice-cold PBS. The cells were then incubated with extraction buffer
(Mitochondria/Cytosol Fractionation Kit, Abcam) on ice for 10 min. The supernatant
containing the cytosol proteins and precipitations containing mitochondria were used
for western blot analysis.
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Annexin-V/PI assay
The Annexin-V/PI assay was performed with flow cytometry (BD, FACS Vantage SE)
using the FITC-labeled Annexin-V and PI kit (Santa Cruz) according to the
manufacturer’s instructions. Briefly, cells were pelleted following HC6h treatment at
indicated time, washed in PBS and resuspended in 100 μl of binding buffer containing
annexin V-FITC and propidium iodide. Another 400 μl of binding buffer were added
to the cells before flow cytometric analysis.
Quantification of acidic vesicular organelles with acridine orange
A549 cells were seeded into 6-well plates and allowed to attach overnight before
treatment with 24 μM HC6h and vehicle. Cells were stained with acridine orange
(final concentration 1 μg/ml) for 15 min. Media were removed and cells washed with
PBS, trypsinized, re-suspended in PBS and analyzed by flow cytometry; green
(510-530 nm, FL1-H channel) and red (> 650 nm, FL3-H channel). The bar for FL3-H
in the control sample was set so that the acidic vesicular organelle (AVO) positive
cells represented approximately 5% of the population. Compound-treated samples
were measured under the same conditions.
Detection of punctuate GFP-LC3 punctate
GFP-LC3 was kindly provided by Dr EngHui Chew (National University of
Singapore). A549 cells were seeded onto cover slips in 6-well plates at a density of
1x105 cells/well and left overnight. Cells were treated with 2, 6 12 and 24μM HC6h
and vehicle, then transfected with 4 μg GFP-LC3 plasmid using LipofectAMINE2000
(Invitrogen Corporation)) according to manufacturer’s instructions. After 72 h cells
were fixed with 4% paraformaldehyde in PBS at 4 ºC for 30 min, and GFP-LC3
fluorescence was examined under a fluorescence microscope. For each slide, 100 cells
were analyzed and mean numbers of punctate GFP-LC3 spots/cell were calculated.
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Small interfering RNA (siRNA) transfection
A549 cells were seeded into 6-well plate at a density of 1x106cells /well and allowed
to reach approximately 70% confluence on the day of transfection. The siRNA
constructs used were obtained as siRNA control and siRNA against JNK (Genechem
Biotech, China). Cells were transfected with 100nM siRNA using electroporation
method (Amaxa, 10700634 AAD-1001, Germany) according to manufacturer’s
instruction. At 24h after transfection, the cells were treated with compound HC6h for
24h and examined by western blot.
Statistical analysis
The results were expressed as mean ±standard deviation (SD). Statistical differences
were evaluated using the two-tailed Student’s t-test and analysis of variance (ANOVA)
followed by q- test, considered significant at *p <0.05, **p <0.01 or ***p <0.001.
Results
HC6h decreases cell viability in human cancer cells
To assess the effect of HC6h on the inhibition of cell growth, A549, HCT116, HT29,
MCF-7, SK-OV-3，U251 and MRC5 cells were exposed to various concentrations of
HC6h compound for 72 h. As shown in table 1, the IC50 values of HC6h were 7.64µM
for A549 cells, 13.87µM for HCT116, 13.65µM for HT29, 14.38µM for MCF-7,
17.06 µM for SK-OV-3, 15.02µM for U251 and 21.56µM for MRC-5 cells. As HC6h
showed relatively good anticancer activity in A549 cells, we therefore investigated the
mechanism of anticancer effect of HC6h in A549 cells.
HC6h rapidly induces the accumulation of intracellular ROS
It is reported that ROS can induce autophagy as well as apoptosis [15, 18]. Therefore,
ROS generation was analyzed in HC6h-treated A549 cells by performing
2’,7’-dichlorodihydrofluorescein diacetate(H2DCFDA) staining followed by flow
cytometry, H2O2 was included as a positive control. As shown in Figure2A, HC6h
induced ROS generation in a time dependent manner, as reflected by the increase of
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fluorescence intensity. The highest amount of ROS was generated after 6 h treatment
of 24 µM HC6h compound in A549 cells. In addition, the enhanced ROS levels were
significantly attenuated by treatment with N-acetylcysteine (NAC). Moreover,
combination of NAC and HC6h attenuated the apoptotic and autophagic effects, as
shown by diminished caspase-9 and caspase-3 activation, PARP cleavage, p53 and
PUMA enhancement, also a decrease in LC3-II levels (Figure2B).
HC6h induced apoptosis in A549 cells
The apoptotic effect of HC6h treatment in A549 cells was explored. Mitochondria are
known to play a pivotal role to apoptosis in response to many stresses and the loss of
mitochondrial membrane potential (MMP) is a sensitive marker of early
mitochondrial damage during apoptosis. We therefore used JC1 staining assay to
investigate the mitochondrial membrane potential. HC6h treatment of A549 cells for
3h results in 12.5% decrease in mitochondrial membrane potential. 17.8% decrease
was observed at 6h and 67.8% decrease in mitochondria membrane potential was
observed at 12h (Figure 3A). Thus, mitochondrial membrane potential was
significantly decreased after HC6h treatment in A549 cells, which indicated that
accumulation of damaged mitochondria. Cytochrome C localized in the mitochondrial
inner membrane and release into the cytosol upon apoptosis induction, exposure of
A549 cells to HC6h induced the release of cytochrome C from mitochondria to the
cytosol (Figure 3B). Bcl-2 family includes Bcl-2 and Bcl-2 related membranes such
as Bax plays a crucial role in regulation of apoptosis. HC6h treatment can
significantly reduce the expression level of anti-apoptotic Bcl-2 while induce the level
of pro-apoptotic Bax in a dose dependent manner, leading to an increase in Bax/Bcl-2
ratio (3.26 fold vs. control), which is in favor for apoptosis induction (Figure3C).
Bcl-2 family members located in the mitochondrial membranes and trigger the
activation of caspase[19].To gain further insight into the mechanism by which HC6h
induced cell death, we examined the effect of HC6h treatment on the apoptosis of
A549 cells. Flow cytometry analysis via Annexin V/PI staining was performed to
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10

investigate the pro-apoptotic effect on A549 cells. It was shown that HC6h treated
cells undergo apoptosis at significantly higher rate than control cells with a
concentration dependent manner (Figure 3D).
A549 cells activated incomplete autophagy with HC6h treatment
Light microscopy observation of HC6h (24µM, 48 h) treated A549 cells revealed
several morphological changes, such as cytoplasmic vacuolation and cell shrinkage
(Figure 4A). These vacuoles might arise due to the cell autophagy. We therefore
postulated that autophagy would be triggered in A549 cells on HC6h treatment.
Formation of acidic vesicular organelles (AVOs) was observed in HC6h-treated cells
in time dependent manner when stained with the lysosome-tropic agent acridine
orange by FACS analysis, and formation of AVO was markedly suppressed in the
presence of 3-methyladenine (3-MA), an early stage of autophagosome inhibitor
(Figure 4B). LC3 is an autophagy marker which exists in two forms, an 18-KDa
cytosolic protein (LC3-I) and a processed 16-kDa form (LC3-II) that is
membrane-bound and increased during autophagy by conversion from LC3-I [20].
Therefore, we examined LC3 distribution in response to HC6h treatment in A549 cells.
As shown in Figure 4C, in control cells, GFP-LC3 was weakly expressed in the
cytoplasm, whereas, HC6h treatment remarkably increased punctuate structure of
GFP-LC3, also the percentage of puncta of GFP-LC3 was enhanced with the
increasing concentration of HC6h. Furthermore, induction of autophagy was further
supported by the conversion of light chain 3 LC3-1 to LC3-II in a concentration
dependent manner upon HC6h treatment (Figure 4D). To further identify whether the
autophagy induced by HC6h treatment was complete, we analyzed the expression of
LC3 and p62 by using autophagosome inhibitor 3-MA and CQ. Our data showed that
the expression level of autophagy substrate p62 had a little increasing and the
phenomenon was more significant after 3-MA or CQ treatment together. These results
suggest the induction of an incomplete after HC6h treatment.
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Inhibition of autophagy by HC6h induced apoptosis
Cell death could be augmented when autophagy is suppressed, suggesting a
pro-survival role of autophagy. To assess the role of autophagy in HC6h induced
cancer cell death, autophagy inhibitor bafilomycin A1was used in combination with
HC6h in A549 cells, apoptosis induction was examined in A549 cells by flow
cytometry following annexin-V/PI staining of cells exposed to HC6h in the presence
or absence of bafilomycin. Treatment with HC6h alone (24μM) induced apoptosis in
A549 cells with an early apoptotic percentage of 14.3%. Bafilomycin(100nM) per se
did not have any effect on apoptosis in A549 cells; combination of HC6h with
bafilomycin increased significant apoptosis (23.0%) compared with HC6h alone
treatment (14.3%)(Figure 5A). Characteristic morphological changes associated with
apoptosis induction were observed after simultaneous incubation with bafilomycin
and HC6h: cells rounded up, appeared smaller and detached from the plates (data not
shown). Moreover, cleavage of PARP, caspase-9 and caspase-3 was observed at low
concentration of HC6h (2μM) in combination with bafilomycin, compared with HC6h
alone treatment (12 μM) (Figure 5B). These data indicate that A549 cells underwent
increased apoptosis when exposure to HC6h compound combined with autophagy
inhibitor bafilomycin. The inhibition of HC6h-induced autophagy accelerated HC6h
induced apoptosis, these findings suggest that HC6h-induced autophagy may play
some role as a survival mechanism against apoptosis.
Activation of JNK
JNK，a member of MAPK family, plays essential role in cell death. The JNK pathway
is critical for the mediation of both autophagy and apoptosis. HC6h induced JNK
phosphorylation (p-JNK) in a concentration-dependent manner. Notably, the
inductions of p-JNK and p-53 were conferred by the increased ROS levels, as NAC
inhibited JNK activation, suggesting that intracellular ROS acts supstream of JNK to
mediate the actions of compound HC6h (Figure 6A). Furthermore, JNK serves as a
critical mediator of HC6h-induced apoptosis as evidenced by the rescue of PARP and
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12

caspase-3 cleavage and the LC3 conversion by JNK inhibitor SP600125 and
siRNA-mediated depletion of JNK (Figure 6B and 6C). Taken together, these results
indicate that the activation of JNK is required for HC6h-induced apoptosis in A549
cells.
Discussion
Chemotherapy remains the mainstream of tumor treatments, and the ultimate goal of
successful chemotherapy is to induce robust apoptosis with simultaneous inhibition of
survival signaling in cancer cells with minimal toxicity to normal cells. Autophagy as
well as apoptosis is an emerging target as anticancer therapy. A variety of evidence
has represented the complex interplay between apoptosis and autophagy and which
can cooperate, antagonize or assist each other[6, 21]. In several studies, autophagy
induction has been shown to enhance tumor cell survival, thereby counteracting or
restricting the efficacy of apoptosis induction by chemotherapeutic agents[22]. One
previous study reported that HC6h showed good anti-cancer effect in various cancer
cell lines and also inhibited S180 and H22 mice tumor growth and did not show any
acute toxicity at the dosage of 5000mg/kg[16]. In our study we found that HC6h
compound possess good anticancer activity in A549 cancer cells (IC50=7.64μM)
compared with normal fibroblast MRC-5 cells (IC50=21.56μM) (Table 1). It is well
documented that cancer cells frequently have increased burden of oxidative stress and
therefore are likely to be more sensitive to the damage promoted by further ROS
insults[23]. Regardless of how ROS are generated, ROS rise has two potential
consequences: damage to cellular components and triggering various signaling
pathways[10, 24]. A wide arrange of evidence shows a link between ROS and
autophagy and apoptosis[7, 25, 26]. Here we found that HC6h induces apoptosis and
autophagy in cancer cells through the induction of oxidative stress. Treatment of
cancer cells with HC6h in vitro leads to the rapid generation of ROS. Scavenging of
ROS by antioxidant NAC completely reversed the HC6h-induced apoptosis and
autophagy (Figure 2).
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13

Apoptosis is a process essential for development and maintenance of tissue
homeostasis, and also a major process to eradicate cancer cells. It is conceivable that
PUMA (p53 upregulated modulator of apoptosis) plays an important role in
p53-mediated apoptosis and its over-expression is associated with the translocation of
Bax to the mitochondria that in turn result in the loss of MMP and subsequently the
release of cytochrome C. The release of cytochrome C initiates the formation of
apoptosome through interaction with Apaf-1 (apoptotic protease-activating factor)
which is a key regulator of programmed cell death [4, 27]. We revealed that
mitochondria dysfunction was observed after exposure of A549 cells to HC6h, which
was displayed by some alterations at mitochondria-related events, such as loss of
mitochondrial membrane potential, cytochrome c release and changes of Bax and
Bcl-2 expressions (Figure 3). HC6h also leads to PUMA and p53 up-regulation,
caspase-9, caspase-3 and PARP cleavage. Therefore efficient intrinsic apoptosis
pathway is triggered by HC6h treatment in A549 cells.
In addition, we found that autophagy was induced by HC6h as evidenced by
accumulation of AO-staining acidic vesicles, formation of autophagosomes observed
with increased number of puncta LC3 and up-regulation of LC3-II. However, we
found that the expression level of autophagy substrate p62 increased with
concentration dependent. Furthermore, the increasing was more significant with 3-MA
and CQ treatment. 3-MA is selective PI3K inhibitors which can inhibit the shift of
LC3-1 to LC3-II and block the formation of autophagosome. Another small molecules
CQ inhibits autophagy by destroying the lysosome function. Therefore, the autophagy
induced by HC6h was an incomplete autophagy in A549 cells. It is well known that
autophagy could limit the effects of cytotoxic anticancer agents through its ability to
clear damaged organelles and proteins, which may help cancer cells to survive
chemotherapy-induced stress. In these scenarios, suppressing autophagy could be
beneficial to treatment outcome. We revealed that autophagy inhibitor bafilomycinA1
significantly augmented cell death on HC6h treatment in A549 cells (Figure 4).
Various apoptotic stimuli can rapidly activate MAPKs, which include JNK. So far,
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14

increasing evidences indicated that JNK is primarily activated by various stresses
including UV radiation, heat shock, oxidative stress, chemotherapeutic agents and
protein synthesis inhibitors, especially, oxidative stress is important [28]. We found
that A549 cells with HC6h treatment in vitro leads to the rapid generation of JNK
phosphorylation. Furthermore, the JNK inhibitor SP600125 and ShRNA JNK
significantly attenuate the promoting autophagy effects. In addition, JNK
phosphorylation was potently abolished by NAC suggested that HC6h induced
apoptosis and autophagy through ROS-dependent JNK pathway.
In conclusion, this study showed that isoquinolin-1(2H)-imine derivative HC6h
compound inhibited the proliferation of A549, HCT116 and HT29 cells. HC6h
induced apoptosis and autophagy via the ROS-dependent JNK signaling pathway in
A549 cells. Cancer cells activated incomplete autophagy with HC6h treatment and
inhibition of autophagy by autophagy inhibitor significantly augmented cell death on
HC6h treatment (Figure 7). This compelling evidence suggested that HC6h might be a
potential candidate for the development of new chemotherapy agents for the treatment
of non-small cell lung cancer. However, the potential of this molecule in the treatment
of other cancer is still unknown, further study need to be evaluated in other cancers.
Conflict of Interest
The authors declare no conflict of interest.
Acknowledgements
This study was supported by grants from the National Natural Science Foundation of
China (No. 81560601, U12022036, 81460457) and the Nature Science Foundation of
Yunnan Province (No. 2014FD011).
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15

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Figure Legends
Figure 1 chemical structure of HC6h
Figure 2 Induction of ROS accumulations in A549 cells. (A) Effect of HC6h on
ROS production. (B) Effects of HC6h and NAC on the expressions of apoptotic
protein.
Figure 3 Induction of apoptosis in A549 cells. (A)Loss of mitochondria membrane
potential was induced by HC6h treatment. (B) HC6h induced cytochrome c release.
(C) HC6h induced the ratio of Bax/Bcl-2 increased (D) Induction of apoptosis was
analyzed by flow cytometry.
Figure 4 A549 cells activated incomplete autophagy with HC6h treatment. (A)
Cell morphological change and double membrane vesicles were formed after HC6h
compound treatments. (B) Formation of AVOs (acidic vesicular organelles) was
determined in A549 cells treated with HC6h. (C) GFP-LC3 punctan induced by HC6h
over concentration course (D) Expression level of p62 and LC3-II with 3-MA and CQ
treatment.
Figure 5 Effects of autophagy inhibitor bafilomycin on apoptosis induced by
HC6h. (A) Apoptotic effects in combination with bafilomycin were analyzed by flow
cytometry with Annexin V/PI staining. (B) Apoptotic protein expressions in
combination with bafilomycin.
Figure 6 ROS generation induced by HC6h is necessary for activation JNK. (A)
Effects of NAC on the JNK and p-p53 activation in A549 cells. (B) Effects of
apoptosis and autophagy with JNK inhibitor SP600125. (C) Effects of apoptosis and
autophagy with silencing of JNK by siRNAs.
Figure 7 Proposed models representing the mechanism of action of HC6h in
A549 cells.
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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Figure 6
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Figure 7
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Table 1 IC50 values of HC6h on the growth inhibition of cancer cells
Cell lines IC50（μM）
Compound
MRC5
A549
HCT116
HT29
13.65±0.59
MCF-7
SK-OV-3
U251
HC6h
21.56±0.51
7.64±0.38
13.87±1.60
14.38±1.52
17.06±1.63
15.02±0.13
The growth inhibitory activity, determined as IC50 values for each cell line by 72 h MTT assay. Data represent the mean values of three independent
experiments.
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