Phenanthroimidazole derivatives act as potent inducer of autophagy by activating DNA damage pathway

Article abstract of DOI:10.1016/j.bioorg.2019.102940

A series of imidazo[4,5f][1,10]phenanthroline derivatives (1–6) have been synthesized in this study, and their inhibitory activity was evaluated by MTT assay. Results showed that all of these compounds demonstrate a promising inhibitory activity against a panel of human cancer cell lines. The 6, the most effective compound with IC₅₀ of approximately 2.3 ± 0.1 μM, was against the growth and could induce autophagy of HepG2 cells. This condition was confirmed by abundant autophagic vacuoles appearing in cells and evident ultrastructural changes observed under transmission electron microscopy. The autophage induced by 6 has also been demonstrated by up-regulating LC3-II and Beclin1. The apoptosis and G2/M phase cell cycle arrest through DSB damage have also been confirmed after the HepG2 cells were treated by 6. These multiple effects, especially induction apoptosis and autophagy, indicate the potential of 6 for development as a novel anticancer drug.

Full text of DOI:10.1016/j.bioorg.2019.102940

Accepted Manuscript
Phenanthroimidazole Derivatives Act as PotentInducer of Autophagy by Acti-
vating DNA Damage Pathway
Hao Zhang, Yue Song, Li Li, Shuang-Yan Zhang, Qiong Wu, Wen-Jie Mei,
Hui-Min Liu, Xi-Cheng Wang
PII:
S0045-2068(18)31569-4
https://doi.org/10.1016/j.bioorg.2019.102940
102940
DOI:
Article Number:
Reference:
YBIOO 102940
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
31 December 2018
21 March 2019
18 April 2019
Please cite this article as: H. Zhang, Y. Song, L. Li, S-Y. Zhang, Q. Wu, W-J. Mei, H-M. Liu, X-C. Wang,
Phenanthroimidazole Derivatives Act as PotentInducer of Autophagy by Activating DNA Damage Pathway,
Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.102940
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Phenanthroimidazole Derivatives Act as Potent Inducer
of Autophagy by Activating DNA Damage Pathway
Hao Zhanga, #, Yue Songa, #, Li Lib, c, Shuang-Yan Zhangb, c, Qiong Wub, c, *, Wen-Jie
Meib, c, d*, Hui-Min Liua, Xi-Cheng Wanga, *
aThe First Affiliated Hospital of Guangdong Pharmaceutical University, Guangzhou
510006, PR China
bSchool of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR
China
cGuangdong Province Engineering Technology Centre for Molecular Probe and
Biomedicine Imagining, Guangzhou 510006, PR China
dGuangzhou key laboratory of construction and application of new drug screening
model systems, Guangdong Pharmaceutical University, Guangzhou 510006, China
#These authors contributed equally to the work.

Abstract: A series of imidazo[4,5f][1,10]phenanthroline derivatives (1–6) have been
synthesized in this study, and their inhibitory activity was evaluated by MTT assay.
Results showed that all of these compounds demonstrate a promising inhibitory
activity against a panel of human cancer cell lines. The 6, the most effective compound
with IC50 of approximately 2.3  0.1 µM, was against the growth and could induce
autophagy of HepG2 cells. This condition was confirmed by abundant autophagic
vacuoles appearing in cells and evident ultrastructural changes observed under
transmission electron microscopy. The autophage induced by 6 has also been
demonstrated by up-regulating LC3-II and Beclin1. The apoptosis and G2/M phase
cell cycle arrest through DSB damage have also been confirmed after the HepG2 cells
were treated by 6. These multiple effects, especially induction apoptosis and
autophagy, indicate the potential of 6 for development as a novel anticancer drug.
Keywords: Hepatocellular carcinoma, autophagy, apoptosis Inducer, phenanthroline
derivative，DNA damage，Zebrafish，

Introduction
Hepatocellular carcinoma (HCC) is the major histological subtype of liver cancer
and is the fifth most prevalent malignancy worldwide [1, 2]. Despite recent
therapeutic advancement, HCC has a poor prognosis with a high rate of mortality due
to rapid recurrence and metastasis [3]. In China, HCC is the second most frequent
cause of cancer-related mortality in males and the third in females [4, 5]. Liver
resection currently remains the most effective intervention modality with localized
stage HCC. However, the majority of HCC patients are diagnosed at advanced stages
with underlying liver dysfunction, thereby making the cure with resection treatment
impossible [6-8]. In addition, treatment options among patients with unresectable
HCC may include other palliative treatments, such as percutaneous ablation, TACE,
cytotoxic chemotherapy, and three-dimensional conformal radiotherapy[9]. However,
the effectiveness of these therapies needs further investigation [10-13]. Therefore,
most patients rely on chemotherapy to prolong their lifespan. However, chemotherapy
has shown low efficacy and unbearable side effects, thereby resulting in poor
prognosis [14-17].
Autophagy, a type II programmed cell death, is a tightly-regulated catabolic
process that involves the degradation of cytoplasm and cellular organelles via
lysosomes [18-20]. In a tumor microenvironment, autophagy is a two-edged sword
given that it is involved in cell survival and death [21-24]. Moreover, autophagy plays
a major role in cell survival, growth, and homeostasis under hypoxic or metabolic
stresses, and it becomes a cell survival pathway. Cellular stress can also lead to death

itself once it is continuous or progressive. Therefore, autophagy could be positive or
negative for cancer treatment depending on the environment [25-28]. The therapeutic
target of the autophagic signaling pathway might be a novel molecular approach to
kill cancer cells considering that autophagy is a fundamental process [29].
In recent decades, phenanthroline derivatives have attracted much attention because
of their wide applications as duplex DNA-intercalating agents, molecular ‘light
switch’, and antitumor chemotherapy drugs because of their unique capabilities as
chelating agents [30-32]. In our previous studies, imidazo[4,5f][1,10]phenanthroline
derivatives exhibited anticancer activity and induced tumor cell apoptosis via the
NF-κB pathway [33]. Additionally, phenanthroimidazole derivatives have been
further confirmed to act as potential inhibitors against various tumor cells by
selectively targeting telomeric G-quadruplexes [34].
In this study, seven novel imidazo[4,5f][1,10]phenanthroline derivatives (Fig. 1)
have been synthesized with a yield of 87% under microwave irradiation [35]. In
addition, the derivatives' biological activities were further elucidated. During the
investigation process, we found that 6 efficiently resists the growth of human
hepatoma G2 cells. A surprise discovery, namely, autophagy, played a major role in
the regulation of cell death. Thus, 6 is a promising agent as an autophagy inducer in
the treatment of HCC. However, the potential molecular mechanism of the inhibiting
cancer function of 6 remains obscure. Therefore, we designed a series of studies to
explore the antitumor effect of 6 in human hepatoma G2 cells and to further elucidate
its molecular mechanism.

Results and discussion
Biological activity
The antiproliferative activities of compounds 1–6 were evaluated using the MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and the
inhibitory activities (IC50) of these compounds and cisplatin after 72-hour treatment
are demonstrated in Table 1. The tested cancer cells, especially the HepG2 cell lines,
exhibited a significant cytotoxic effect on the complexes. The IC50 of 6 against
HepG2 cells is 2.33 μM, which is much less than that of cisplatin (93.09 μM).
Treatment of HepG2 cells with 6 remarkably showed a downward trend of the cell
viability in a dosage-dependent manner (Fig. 2B). The changes in cell morphology
were first examined under in the phase-contrast observation to clarify the manner by
which 6 affected HepG2 cell growth (Fig. 2A). After cells were treated with 6 for 24 h,
we observed evident morphological changes, such as shrinking of considerable cells,
a decrease of adherent cells, a gradual increase of detached cells, and an emergence of
many suspended cells along with cell debris. The decrease of cell viability and the
change in cell morphology proved the growth-suppressing effect of 6 on HepG2 cells.
In vivo inhibitory effect of 6 on zebrafish xenotransplanted liver cancer model
In decades, the zebrafish (Danio rerio) has been developed as a prominent disease
model in various disease treatments before clinical research, especially in the field of
tumor therapy [36, 37]. Moreover, zebrafish is also permeable to many small
molecules and thus potentially useful for drug screening. This study aims to further
appraise the cancer-preventing effect of the most active compound 6 against HepG2

liver cancer cells in vivo. An experimental liver cancer xenograft model was used in
this study to evaluate the inhibition on the growth of the HepG2 cells in vivo, and the
fluorescence-labeled tumor cells were microinjected into the blood circulation of
transgenic zebrafish (fil1:EGFP) embryos for 48 h. The hematogenous tumor cells
were immediately disseminated in the embryo after injection near the subintestinal
vessel (SIV) [38].
As shown in Fig. 2A, a certain amount of HepG2 cells were located near the SIV area
in the zebrafish which dealt without drug at 0 h. However, an increasing number of
tumor cells were distinctly observed after 48 h. HepG2 cells can infinitely proliferate
in the zebrafish model [39]. Then, the number of HepG2 cells was slightly increased
at 48 h over 0 h in the zebrafish after being treated with 6 (1 μM) (Fig. 2B).
Furthermore, the number of HepG2 cells almost remain constant with a slight increase
48 h after treatment with 6 (3 μM), thereby indicating that this class of
phenanthroimidazole derivatives, especially for 6, can effectively suppress the
proliferation of HepG2 cells in zebrafish. This compound can be developed as a
potential agent to inhibit liver cancer.
Autophage of HepG2 cells induced by 6
Observation of autophagic vacuolization in cytoplasm by inverted phase contrast
microscopy
The morphological characteristics of HepG2 cells treated with 6 were further
observed by using an inverted phase contrast microscope. The normal control group’s
cells excellently adhered to smooth surface membrane, have a good refractive index,

and show active nuclear fission. Remarkable morphological changes were observed
when the tumor cells were treated with 6 for 24 h (Fig. 3A). Moreover, abundant
autophagic vacuoles with varying sizes appeared, and cytoplasmic vacuoles
progressively became larger and denser with increasing concentration of 6. In addition,
considerable cells treated with 6 at 3 and 5 µM concentrations became round,
shrunken, and suspended at 24 h.
Western Blot
First, we examined the expression of LC3, the mammalian homolog to the yeast
Atg8 gene, via Western blot to further confirm the progression of autophagy. The LC3
protein can specifically label autophagosomal membranes, and the conversion of
LC3-I to LC3-II is generally accepted as an autophagy marker [40]. During autophagic
stress, the 16-kDa cytosolic form (LC3-I) is cleaved and post-translationally modified
(LC3-II, 14 kDa) and translocated to the autophagosome [41]. After culturing HepG2
cells with compound 6 at 0, 1, 3, and 5 μM for 24 h, a remarkable increase in the ratio of
LC3-II/LC3-I was observed compared with the vehicle-treated control. Additionally,
treatment with 6 increased the ratio of LC3-II/LC3-I in a concentration-dependent
manner (Fig. 3B). Beclin-1, one of the major autophagy-related gene (ATG) proteins, is
the mammalian homolog of the yeast protein ATG6 that correlates directly with
autophagic vesicle formation [42] and controls vital steps in the autophagic pathway.
The Western blot analysis revealed the relative upregulation of Beclin-1 levels in a
dose-dependent manner after 6 treatment. These results indicate that 6 can induce
autophagy in HepG2 cells.

Immunofluorescence microscopy
Cells were further analyzed by fluorescence microscopy. As shown in Fig. 3c,
treatment of HepG2 cells with 0, 1, and 5 μM compound 6 displayed an increase in
not only the number but also the size of MAP-LC3-positive points. This result
indicated that 6 treatment induced the formation of autophagosomes.
Transmission electron microscopy
Transmission electron microscopy (TEM) was applied to detect the ultrastructural
morphological changes of compound 6-treated HepG2 cells to further determine
whether the cell vacuolization induced by 6 is involved in autophagy. The HepG2
cells untreated with 6 exhibited normal ultrastructural morphology of cytoplasm,
organelles, and nuclei (Fig. 4A). The most prominent morphological change in
6-treated cells was the formation of abundant autophagic vacuoles sequestrating
cytoplasm and organelles, such as mitochondria and endoplasmic reticulum.
Double-membranes, giant autophagosomes filled with degraded organelles, and
autolysosomes were frequently observed (Fig. 4B). TEM, the standard method for
detecting autophagy, was performed to observe the formation of autophagosomes.
Compound 6 could induce HepG2 cells to generate autophagy, which was consistent
with the vacuolization obtained by inverted phase contrast microscopy.
Apoptosis of HepG2 cells induced by 6
A flow cytometric assay was used to distinguish the percentage of early and late
apoptosis and necrotic cells by Annexin-V-FITC and PI double staining. The HepG2
cells were treated for 24 h with compound 6. As shown in Fig. 5A, the percentages of

early apoptotic were 18.80%, 22.42%, and 49.52% with 1, 5, and 10 µM treatment,
respectively, whereas the proportion of untreated cells is only 1.15%. The result
indicated that the 6 induced HepG2 cell apoptosis. Furthermore, the effect
considerably increases in a dose-dependent manner.
Induced G2/M phase cell cycle arrest in HepG2 cells
We monitor the changes of compound 6 on cell cycle perturbations considering the
possibility that the induction of cell growth inhibition might be mediated through the
regulation of the cell cycle. The cell cycle distribution was performed by flow
cytometric analysis, as shown in Fig. 5B. After a 24-hour treatment, the G2/M phase
population of the untreated cells was 2.2%, and the ratio of G2/M was considerably
increased (6.2%, 11.2%, and 14.9% cells at 1, 5, and 10 μM concentrations of 6,
respectively). The compound 6 treatment resulted in an appreciable arrest of HepG2
cells in the G2/M phase of the cell cycle compared with the control group.
Specifically, this cell cycle accumulation at the G2/M phase after 6 treatment and the
changes were concentration dependent.
The DNA damage induction is an effective mode of action of anticancer agents.
Anticancer agents act by producing sufficient DNA strand breaks in cancer cells to
evoke cell repair systems, cell cycle arrest, or cell death programs [43, 44].
DNA damage in HepG2 cells induced by 6
We used the DNA ladder analysis by agarose gel electrophoresis to directly
visualize the DSBs in tumor cells induced by 6. This analysis reportedly detects DSBs
induced by various DNA-damaging agents. DSBs in tumor cells were further tested.

The reaction samples were separated by 1% agarose gel electrophoresis and visualized
by UV light after standard ethidium bromide staining. As shown in Fig. 6A, the ladders
in cells treated with 6 were more remarkable than those in untreated cells. On the basis
of the above data, the 6 was determined as a novel DNA-damaging anticancer agent.
Furthermore, a formation of γH2AX foci showed a dose-dependent increase in
6-treated cells compared with control cells in the immunofluorescence assay (Fig. 6C).
Compared with the control, the increased expression and nuclear translocation of
γ-H2AX, which is a sensitive central marker for DNA damage, were further
confirmed by Western blot analysis [45, 46]. Moreover, the expression of 6-induced
γ-H2AX was markedly increased in a concentration-dependent manner at 24 hours
after treatment (Fig. 6B).
As previously reported, imidazo[4,5f][1,10]phenanthroline derivatives induce
apoptosis in cancer cells via suppression of NF-κB activity and down-regulation of
c-myc gene expression. In this study, the phenanthroline derivatives successfully
inhibited the cell proliferation of HepG2 cells, thereby activating autophagy and
apoptosis. Moreover, the phenanthroline derivatives stimulated the DNA
double-strand breaks in HepG2 cells, which play a critical role in mediating the G2/M
phase transition, thereby leading to the accumulation of unrepaired DSBs and cell
apoptosis. These increased DNA lesions might be due to the inhibition of DNA
damage repair. Abundant cytoplasmic vacuoles were observed in 6-treated HepG2
cells under an inverted phase contrast microscope. Moreover, the vacuoles were
confirmed as autophagosomes by transmission electron microscopy, which is the

“golden standard” for autophagy. According to the study, the treatment caused an
increase of Beclin-1 and LC3-II expressions at the molecular level and resulted in cell
apoptosis and autophagy (Fig. 7).
This study is the first to demonstrate that the capability of phenanthroline
derivatives in initiating not only apoptosis but also autophagic cell death further
promises an attractive strategy for developing advanced anticancer drugs. Moreover,
phenanthroline derivatives are effective in therapeutic approaches for treating cancers
to specifically target the autophagic cell death machine.
Conclusions
In conclusion, a series of imidazo[4,5f][1,10]phenanthroline derivatives exhibited
high anticancer activities against several cancer cell lines. Compound 6 is identified as
a potential antitumor agent and exerts its antineoplastic action by inhibiting cell
proliferation and inducing cell apoptosis and autophagy. Autophagic cell death induced
by 6 underlines its potential utilization as a new cancer treatment modality. In
particular, autophagy may provide leverage to treat chemo-resistant HCC on the basis
of ineffective apoptosis.
Experimental section
Materials and method
All solvents and buffer fractions were of analytical and biology grades and used
as received, respectively. Imidazo[4,5f][1,10]phenanthroline (Alfa Aesar), cisplatin
(Acros), MTT (Sigma), Annexin V-FITC/PI (TOYOBO), and Trizol (Invitrogen)
antibodies for detecting LC3B, Beclin-1, γ-H2AX, and β-actin were purchased from

Santa Cruz. Microanalysis (C, H, and N) was performed with a vario EL elemental
1
analyzer. The H-NMR spectra were recorded on a Varian INOVA 500NB NMR
spectrometer with (CD3)2SO as solvent at room temperature and TMS as the internal
standard. Electrospray mass spectra were registered with a Thermo Finnigan LCQ
1
13
DECA XP ion trap mass spectrometer, equipped with an ESI source. The H and C
NMR spectra were recorded on a Varian-300 spectrometer.
Synthesis and characterization
Phenanthroimidazole derivatives were synthesized in accordance with the
literature procedure with some modifications. Generally,
a
mixture of
1,10-phenanthroline-5,6-dione (1.50 mmol), benzaldehyde (2.25 mmol), ammonium
acetate (51.9 mmol), and glacial acetic acid was heated at 100 °C for 20 min under
microwave irradiation (Fig. 1). Then, 20 mL of water was added, and the pH value
was adjusted to 7.0 at room temperature [47-49]. The solution was filtered and dried
in a vacuum to obtain a yellow precipitate. The product was purified in a silica gel
column by using ethanol as eluent. The target compounds were characterized by
ESI-MS and 1H- and 13C-NMR spectroscopy.
Cell culture
The A549 (lung adenocarcinoma), MDA-MB-231 (human breast cancer), MCF-7
(human breast cancer), EC-1 (esophageal cancer), L-02 (human normal liver), C6 (rat
glioma cell), and HepG2 (human liver HCC cells) were obtained from the National
Key Lab of Molecular Oncology, Cancer Institute and Cancer Hospital, Chinese
Academy of Medical Science. All cell lines were cultured in DMEM media (Gibco,

Gaithersburg, MD, USA) supplemented with FBS (10%), penicillin (100 units/ml),
and streptomycin (50 units/ml) at 37 °C in a CO2 incubator (95% relative humidity, 5%
CO2).
MTT assay
Cell viability was determined by the ability of cells to transform
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl. The dehydrogenase in the mitochondria of
living cells can restore the yellow MTT to blue-violet [50]. Cells were seeded in
96-well culture plates (Costar) (5×103 cells/well) for 24 h. After incubation with the
cell gradient concentration compound for 72 h, 20 μL of MTT solution per well was
added to continue incubation for 4 h. A microplate reader (SpectroAmaxt 250)
measures the absorbance intensity of cell growth conditions at a wavelength of
490 nm. The percent cell survival was calculated using the formula: (A490 of treatment
group/A490 of untreated group)×100%, and the IC50 values were procured using SPSS
statistics 17.0.
The establishment of zebrafish embryo liver cancer model.
The HepG2 cells (~107 cells/mL) were labeled red fluorescence by DiI, which the
suspended HepG2-DiI cells were injected 10-50 nL into the perivitelline space near
the subintestinal vessels (SIVs) of the transgenic zebrafish (fil1:EGFP) embryos at 48
hpf by using WPI microinjector. Juvenile zebra fish of liver cancer model (48 h old)
were incubated in 6-well plates (10 fishes in every well) with 2 mL solutions without
or with 6 (0, 1 and 3 μM) in aquaculture water. The effect of 6 in liver cancer zebra
fish were observed every 24 h with a fluorescence microscopy. The relevant ethical

protocols used for the in vivo study for zebrafish were followed by the relevant laws.
Observation of cell ultrastructure under transmission electron microscope
Cells were harvested, precipitated, fixed with 2.5% glutaraldehyde in 0.1 mol/L
PBS (pH 7.4) for 90 min, washed with cacodylate buffer, and fixed in 2% osmium
tetroxide for 1 h. Then, the samples were rinsed with PBS and then gradually
dehydrated in a 10% gradient series of 50%–100% ethanol and propylene oxide and
embedded in Epon 812 resins. A microtome was used to create ultrathin sections,
which were stained with uranyl acetate and lead citrate. Finally, the ultrastructure of
the cells was observed under a transmission electron microscope.
Cell cycle and apoptosis analysis
The distribution of cells in the cell cycle phases was measured in accordance with
the cellular DNA content as measured by flow cytometry with a flow cytometer (BD
Biosciences). The HepG2 cells were seeded in six-well plates for 24 h and then
treated with different concentrations of compound 6. After 24 h, the floating and
attached cells were collected and centrifuged before being rinsed with PBS and then
fixed overnight at 4 °C with 70% ethanol. The cell cycle arrest was analyzed with an
Epics XL-MCL flow cytometer (Beckman Coulter, Miami, FL, USA). The suspension
was incubated with 5 μL Annexin-V-FITC and 10 μL PI for 10 min at room
temperature in the dark and analyzed with an Epics XL-MCL flow cytometer
(Beckman Coulter).All experiments were conducted in triplicate.
Immunofluorescence microscopy studies49
The HepG2 cells seeded in six-well plates grown on coverslips were treated with

drugs for 48 h and coated with poly-L-lysine. The coverslips were washed with PBS.
In addition, the cells were fixed with 4% formaldehyde, permeabilized in 0.1% Triton
X-100, and blocked with 4% goat serum in PBS. For LC3 staining, cells were
permeabilized with 0.1% saponin. The coverslips were incubated with the primary
antibody for 1 h, rinsed with PBS, and incubated with the secondary antibody for
30 min. The coverslip was washed again with PBS. After 10 min of dark-proof DAPI
staining, the cells were observed under a fluorescence microscope.
Transmission electron microscopy
The HepG2 cells were collected, pelleted, and fixed in 2.5% glutaraldehyde in
0.1%/L PBS (pH 7.4) for 90 min. The specimen was fixed with 1% osmium tetroxide
for 30 min after being washed with PBS. Then, the specimen was rinsed with PBS
and gradually dehydrated in a 10% gradient series of 50%–100% ethanol and
propylene oxide. The sample was embedded in Epon 812 resin and cured in an oven
at 60 °C. Ultrathin sections were obtained with a microtome. The sections were
stained with saturated uranyl acetate and lead citrate. The ultrastructure of the cells
was photographed with a transmission electron microscope (JEM-1230, JEOL,
Japan).
DNA fragmentation analysis by agarose gel electrophoresis
The high molecular weight fragments separate by pulsed-field gel electrophoresis,
and the nucleosome-sized fragments demonstrate a ladder pattern when separated on a
conventional agarose gel. The DNA was prepared using a DNA extraction kit
(Beyotime, Hangzhou, China). A total of 500 μL of lysis buffer containing protease

was added to the cell pellet after harvesting the cells by centrifugation and then, the
cells were incubated overnight at 50 °C. After digestion, the DNA was extracted by
standard phenol/chloroform extraction and mixed vigorously to separate the DNA
[51]. After adding ethanol and ammonium acetate and a short spin centrifugation
(1 min, 12,000 rpm), the DNA was precipitated. The obtained DNA was resuspended
with nuclease free water for further use. A total of 20 μL of extracted DNA was mixed
with a 5 μL loading buffer (Orange G, glycerol, TAE). The samples were resolved on
a 1.0% agarose gel (MP agarose, Boehringer Mannheim), and ethylene bromide
staining was performed. The gel images were captured with Tanon 2500 imaging
system (Shanghai, China).
Western blotting
The entire cell proteins were extracted by cell lysis buffer obtained from Cell
Signaling Technology. The protein concentration was calculated by BCA assay. The
proteins (20-80 μg) were separated by SDS-PAGE gel and transferred to a
fixed-PVDF transfer membrane by electroblotting [52]. After blocking with 5% skim
milk in TBST buffer for 1 h, the membrane was incubated with a 1:1000 diluted
o
primary antibody in 5% skim milk powder at 4 C overnight for subsequent washing,
followed by secondary antibody incubation. Moreover, the membrane was conjugated
with horseradish peroxidase for 1 h at room temperature at a 1:2000 dilution. Protein
bands contain an enhanced chemiluminescence system (Tanon 4200).
Acknowledgment
This work was supported by the National Natural Science Foundation of China (Grant

Nos. 81572926, 81703349). The China Postdoctoral Science Foundation (Grant No.
2017M610576). The Provincial Major Scientific Research Projects in Universities of
Guangdong Province (Grant No. 2014KZDXM053), The Science and Technology
Project of Guangdong Province (Grant No. 2014A020212312),The projects of
Guangzhou key laboratory of construction and application of new drug screening
model systems(No.201805010006) and Key Laboratory of New Drug Discovery and
Evaluation of ordinary universities of Guangdong province (No. 2017KSYS002).
Conflicts of interest
The authors declare that they have no conflict of interest to disclose.
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Figures and Tables
Scheme 1. The synthesis route of phenanthroimidazole derivatives under microwave
irradiation.
Table 1 The inhibitory activity (IC50/μM) of the target compounds against different
cancer cells
Figure 1. (A) The morphological change of HepG2 cells in the absence and presence
of 6 (B) The cell viability of HepG2 cells induced by different concentration of 6.
Figure 2. (A) The growth inhibition of HepG2 cells induced by 6 in zebrafish
xenografts model. Dil-labeled HepG2 cells (red) were microinjected into zebrafish
embryos, and treatment with 6 (0, 1, 3 and 5 µM) for 48 h. (B) The proliferation
multiple of tumor cells were analyzed through the fluorescence intensity of tumor
cells at 48 h. The proliferation of the xenografts of HepG2 cells were imaged under a
fluorescence microscope (n=12/group).
Figure 3. Transmission electron microscopy showing the utophage process of HepG2
cells induced by 6. (A) The ultramicroscopic structure of HepG2 cell without drug
treatment. (B) The typical autophagic cells with many autophagic vacuoles (arrows)
of HepG2 cells treated with compound 6 for 24h.
Figure 4. (A) Fluorescence images of HepG2 cells showing endogenous MAP-LC3
levels examined by immunofluorescence staining. Nuclei (blue) were labeled by
DAPI. (B) HepG2 cells were exposed 6 for 24 h. beclin1, LC3B, and β-actin levels
were determined by Western Blot.
Figure 5. (A) Flow cytometric analysis of HepG2 cells treated with 6 (0, 1, 5 and 10
µM) for 24 h. (B) Flow cytometric analysis for apoptosis after treatment by Annexin
V-FITC and PI staining on HepG2 cells. After treatment with different doses of 6 (0,
1, 5 and 10 µM) for 24 h, apoptosis induction was observed. (C) Quantitative analysis
of cell cycle distribution induced by different concentration of 6. (D) The change of
apoptosis induced by different concentration of 6.
Figure 6. (A) Cells were treated with compound 6 for 0, 1 and 5 μM, and
translocation of γ-H2AX was detected by immunofluorescence staining. Cell nucleus

was located by DAPI staining assay. (B) HepG2 cells treated with or without 6 for 24
h were visually detected DSBs using the DNA ladder assay; (C) HepG2 cells were
exposed compound 6 for 24 h. γ-H2AX and β-actin levels were determined by
Western blot.
Figure 7. A schematic depicting the role of compound 6 induced autophagy and
apoptosis in HepG2 cells through DNA damage

1 R=-OH
4 R=-COOH
2 R=-F
5 R=-SO2CH3
3 R=-N(CH3)2
6 R=-CH3
Scheme 1.

Inhibitory activity IC50/μM
Comp.
MDA-MB-231
57.0 ± 0.2
89.0 ± 0.2
＞100
HepG2
8.6 ± 0.7
3.8±0.9
3.5±0.9
EC-1
C6
MCF-7
A549
LO2
0.6 ± 0.8
16.3 ± 0.4 56.7±0.9
6.3±0.9
1.4±0.9
1.3±0.8
1
2
3
7.3±0.2
1.1±0.02
1.3±0.3
68.0 ± 0.9 46.0 ± 0.3
4.4±0.6
3.2±0.9
20 .0± 0.3
/
57.5 ± 0.2
71.5±0.9
7.2±0.9
4
＞100
＞100
＞100
9.3±0.6
65.6 ± 0.1
＞100
56.3±0.8 22.0 ± 0.3
2.5 ± 0.3 42.2±0.9 24.0±0.8
2.4±0.9 13.0±0.7
5
13.7±0.4
2.0±0.07
2.3±0.9
20 .0± 0.8
/
/
6
cis-platin
17.8±0.9
93.1± 0.9
/
76.9±0.9 57.3±0.7 13.5±0.5
Table 1

Figure 1

Figure 2.