MHY440, a novel topoisomerase I inhibitor, induces cell cycle arrest and apoptosis via a ROS-dependent DNA damage signaling pathway in AGS human gastric cancer cells

Article abstract of DOI:10.3390/molecules24010096

We investigated the antitumor activity and action mechanism of MHY440 in AGS human gastric cancer cells. MHY440 inhibited topoisomerase (Topo) I activity and was associated with a DNA damage response signaling pathway. It exhibited a stronger anti-proliferative effect on AGS cells relative to Hs27 human foreskin fibroblast cells, and this effect was both time- and concentration-dependent. MHY440 also increased cell arrest in the G2/M phase by decreasing cyclin B1, Cdc2, and Cdc25c, and upregulating p53 and p73. MHY440 induced AGS cell apoptosis through the upregulation of Fas-L, Fas, and Bax as well as the proteolysis of BH3 interacting-domain death agonist and poly(ADP-ribose) polymerase. It also contributed to the loss of mitochondrial membrane potential. The apoptotic cell death induced by MHY440 was inhibited by pretreatment with Z-VAD-FMK, a pan-caspase inhibitor, indicating that apoptosis was caspase-dependent. Moreover, the apoptotic effect of MHY440 was reactive oxygen species (ROS)-dependent, as evidenced by the inhibition of MHY440-induced PARP cleavage and ROS generation via N-acetylcysteine-induced ROS scavenging. Taken together, MHY440 showed anticancer effects by inhibiting Topo I, regulating the cell cycle, inducing apoptosis through caspase activation, and generating ROS, suggesting that MHY440 has considerable potential as a therapeutic agent for human gastric cancer.

Full text of DOI:10.3390/molecules24010096

molecules
Article
MHY440, a Novel Topoisomerase I Inhibitor, Induces
Cell Cycle Arrest and Apoptosis via a
ROS-Dependent DNA Damage Signaling Pathway in
AGS Human Gastric Cancer Cells
†
†
Jung Yoon Jang , Yong Jung Kang , Bokyung Sung, Min Jeong Kim, Chaeun Park,
Dongwan Kang, Hyung Ryong Moon , Hae Young Chung and Nam Deuk Kim *
College of Pharmacy, Molecular Inflammation Research Center for Aging Intervention (MRCA), Pusan National
University, Busan 46241, Korea; jungyoon486@hanmail.net (J.Y.J.); biogreen80@hotmail.com (Y.J.K.);
auvers1516@gmail.com (B.S.); nanum105@hanmail.net (M.J.K.); sauryyyy@pusan.ac.kr (C.P.);
3
*
†
607@pusan.ac.kr (D.K.); mhr108@pusan.ac.kr (H.R.M.); hyjung@pusan.ac.kr (H.Y.C.)
Correspondence: nadkim@pusan.ac.kr; Tel.: +82-51-510-2801; Fax: +82-51-513-6754
These authors contributed equally to this work.
Academic Editor: Tiziano Tuccinardi
Received: 12 December 2018; Accepted: 24 December 2018; Published: 28 December 2018
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: We investigated the antitumor activity and action mechanism of MHY440 in AGS human
gastric cancer cells. MHY440 inhibited topoisomerase (Topo) I activity and was associated with
a DNA damage response signaling pathway. It exhibited a stronger anti-proliferative effect on
AGS cells relative to Hs27 human foreskin fibroblast cells, and this effect was both time- and
concentration-dependent. MHY440 also increased cell arrest in the G2/M phase by decreasing
cyclin B1, Cdc2, and Cdc25c, and upregulating p53 and p73. MHY440 induced AGS cell apoptosis
through the upregulation of Fas-L, Fas, and Bax as well as the proteolysis of BH3 interacting-domain
death agonist and poly(ADP-ribose) polymerase. It also contributed to the loss of mitochondrial
membrane potential. The apoptotic cell death induced by MHY440 was inhibited by pretreatment with
Z-VAD-FMK, a pan-caspase inhibitor, indicating that apoptosis was caspase-dependent. Moreover,
the apoptotic effect of MHY440 was reactive oxygen species (ROS)-dependent, as evidenced by the
inhibition of MHY440-induced PARP cleavage and ROS generation via N-acetylcysteine-induced
ROS scavenging. Taken together, MHY440 showed anticancer effects by inhibiting Topo I, regulating
the cell cycle, inducing apoptosis through caspase activation, and generating ROS, suggesting that
MHY440 has considerable potential as a therapeutic agent for human gastric cancer.
Keywords: MHY440; topoisomerase inhibitor; cell cycle arrest; apoptosis; gastric cancer cells
1
. Introduction
Gastric cancer (GC) is the third leading cause of cancer death in both sexes worldwide, and it
is especially common in less developed countries [1]. In Asia, GC is the third-most common cancer
after breast cancer and lung cancer, and it is the second most frequent cause of cancer death after lung
cancer. Although the incidence and mortality of GC are declining in many Asian countries, including
South Korea, it still remains an important public health issue [2]. Therefore, the development of new
anticancer drugs and effective therapeutic strategies for patients with GC is needed to increase the
efficacy of treatment.
Topoisomerase (Topo) is a highly specialized nuclear enzyme involved in the correction of
topological DNA errors during the elimination, replication, transcription, recombination, and
chromosomal condensation of DNA [3,4]. Topo acts by sequentially breaking and recombining one or
Molecules 2019, 24, 96; doi:10.3390/molecules24010096
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two strands of DNA, depending on the type of Topo involved in the process [
5
,
6]. There are two types
of Topo in humans: topoisomerase type I (Topo I) and topoisomerase type II (Topo II). Topo I breaks
and recombines single strands of the double helix structure, while topo II cleaves and recombines both
strands of DNA [7]. In fact, Topo activity, particularly inhibition of Topo I, is a key mechanism for a
variety of anticancer agents. Inhibition of Topo I can lead to changes in DNA structure as well as DNA
damage and can ultimately result in the induction of apoptosis [8].
Apoptosis is an essential process of programmed cell death in multicellular organisms.
This cellular process prevents cancer by eliminating unwanted or unnecessary cells during development
or by neutralizing cells that are potentially deleterious to DNA damage [9]. Apoptosis is initiated
by various stresses, such as reactive oxygen species (ROS), DNA damage factors (e.g., radiation),
heat shock, serum deprivation, viral infection, and hypoxia. ROS are considered a toxic product of
cellular metabolism and can act as a signaling molecule that regulates many physiological processes [10].
Previous studies have shown that oxidative stress can lead to apoptosis through the extrinsic apoptotic
receptor pathway as well as the endogenous mitochondrial apoptotic pathway [11,12].
Camptothecin is an alkaloid isolated from the stem wood of the Chinese tree,
Camptotheca acuminata. This compound selectively inhibits the nuclear enzyme Topo I. However,
because of its low solubility, numerous derivatives and analogues were synthesized. Among them,
topotecan is approved by the U.S. FDA (Food and Drug Administration) for the treatment of ovarian
and lung cancer. Another camptothecin derivative irinotecan is approved for the treatment of colorectal
cancer. There are, however, certain clinical limitations of the camptothecin derivatives. These include:
(
1) spontaneous inactivation to the form of lactones in the blood, (2) resistance of cancer cells to
camptothecins by overexpressing membrane transporters, and (3) dose-limiting side effects such as
diarrhea and myelosuppression such as neutropenia [13 14]. To overcome these limitations, several
,
laboratories are trying to develop non-camptothecin Topo I inhibitors.
Psorospermin, a natural substance, showed topoisomerase II-induced DNA alkylation activity
and compound A showed DNA alkylation activity (Figure 1A) [15,16]. Psorospermin and compound
A each have a flat xanthone and benzo[b]acridinone template, and both compounds have an epoxy
functional group in common at the similar position. For the discovery of a new anticancer agent,
MHY440 with an epoxy group at the similar position and a flat acridinone template was designed
and synthesized.
This study was conducted to characterize MHY440 [1-hydroxy-3-((R/S)-oxiran-2-ylmethoxy)-10-
((R/S)-oxiran-2-ylmethyl) acridin-9(10H)-one] (Figure 1A) as a novel Topo I inhibitor, assess the
cytotoxic effect of MHY440 on GC cells, and define the underlying molecular mechanism.
2
. Results
2
.1. Effects of MHY440 on Topo I and DNA Damage Signaling Pathway in AGS Cells
To confirm whether MHY440 inhibits Topo, a cell-free system was used. As shown in Figure 1B,
MHY440 inhibited the activity of Topo I in a concentration-dependent manner. Camptothecin, a known
Topo I inhibitor, was used as the positive control. Both camptothecin and MHY440 inhibited human
Topo I and prevented the unwinding of the supercoiled DNA substrate. We confirmed that MHY440 is
an inhibitor of Topo I; however, MHY440 did not demonstrate inhibition of Topo IIα (data not shown).
We next examined the expression of DNA damage-related proteins after treatment with
MHY440. Ataxia telangiectasia mutated (ATM) is a well-known DNA damage sensor and regulator.
After exposure to oxidative stress or DNA damage stresses, such as Topo I and II inhibitors, ATM kinase
is activated by phosphorylation at Ser1981 and ataxia telangiectasia and Rad3-related (ATR) kinase
is activated by phosphorylation at Ser428 [17]. The activation of these proteins by phosphorylation
results in the phosphorylation of numerous downstream substrates, including Chk1, Chk2, p53, H2AX,
etc., ultimately resulting in cell cycle arrest and apoptosis [18
of AGS cells to MHY440 markedly increased the protein levels of p-ATM, p-ATR,
,
19]. As shown in Figure 1C, the exposure
-H2AX, p-Chk1,
γ

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p-Chk2, and p-p53 in a concentration-dependent manner. These results indicate that MHY440 is a
novel Topo I inhibitor and induced a DNA damage signaling pathway in AGS cells.
Figure 1. The chemical structure of the novel Topo I inhibitor, MHY440, and the effect of
MHY440 on the DNA damage response in AGS cells. (
A) The rationale for the design of MHY440
[1-hydroxy-3-((R/S)-oxiran-2-ylmethoxy)-10-((R/S)-oxiran-2-ylmethyl) acridin-9(10H)-one]. (B)
Supercoiled substrate pHOT1 DNA (0.25 µg) was incubated with various concentrations of MHY440
◦
(
0, 5, 50, 100, and 500
agarose gel containing EtBr. M, relaxed pHOT1 marker DNA; UT, supercoiled substrate pHOT1 DNA;
CPT, camptothecin. ( ) Western blot analysis of total cell lysates from cells treated with increasing
concentrations of MHY440 for 24 h. The blots were probed with p-ATM (Ser1981), p-ATR (Ser428),
-H2AX (Ser139), p-Chk1 (Ser345), p-Chk2 (Thr68), and p-p53 (Ser15). -actin was used as a loading
µM) and Topo I at 37 C for 30 min. The reaction product was separated on a 1%
C
γ
β
control. Representative results from three independent experiments are shown.
2
.2. Molecular Docking Simulation of Topo I with MHY440
To further test the effect of MHY440 on Topo I, a protein-ligand docking simulation was performed
using the AutoDock Vina and AutoDock 4 programs. The docking score between the ligand and
the receptor can be expressed in terms of different energy sources including electrostatic energy,
Van der Waals energy, and salvation energy. The predicted 3-D structure of Topo I, camptothecin
(
Figure 2A), and MHY440 (Figure 2B) are shown. The black lines indicate camptothecin and MHY440,
respectively (Figure 2A,B). The docking simulation was effective with a considerable score. The binding
energies of camptothecin and Topo I were 5.2 (Autodock Vina) and 5.73 (AutoDock 4) kcal/mol,
respectively. The binding energies of MHY440 and Topo I were 4.4 (Autodock Vina) and 4.88
AutoDock 4) kcal/mol, respectively (Table 1). Camptothecin interacted with the amino acid residue
−
−
−
−
(

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GLU418 with a single hydrogen bond, and MHY440 interacted with two active site amino acid residues
ASN352 and TYR426 with two hydrogen bonds (Figure 2C,D and Table 1). The binding energy data
in Table 1 indicated that camptothecin exhibits greater binding affinity than MHY440. However,
MHY440 interacted by two hydrogen bonding with ASN352 and TYR426. In addition, TYR426 and
MET428 residues were involved in hydrophobic interactions with MHY440. Camptothecin was the
reported compound, interacted with two active-site amino acid residues, PHE361 and GLU418, with
one hydrogen bond. PHE361 residue was involved in hydrophobic interaction with camptothecin.
In general, it is known that the bond strength of hydrogen bonds is very strong, so the number of
hydrogen bonds often means strength of bond strength. Thus, it can be predicted that MHY440 is more
cohesive than camptothecin to Topo I.
Table 1. Topo I inhibitory activity of MHY440.
Binding Energy (kcal/mol) a
No. of b
H-Bond Interacting
Residues b
Van der Waals Bondb
Compounds
H-Bond
Interaction Residues
AutoDock Vina
AutoDock 4
Camptothecin
MHY440
−5.2
−5.73
1
2
GLU418
ASN352, TYR426
PHE361
TYR426, MET428
−4.4
−4.88
a
The binding energy represents the binding affinity and capacity for the active site of the Topo I enzyme.
The number of hydrogen bonds and all amino acid residues of the enzyme-inhibitor complex were determined
b
using the AutoDock Vina and AutoDock 4 programs.
Figure 2. Computational structure prediction for docking simulation between Topo I and MHY440.
(
A
) Predicted 3-D structure of Topo I. The black lines indicate the site of camptothecin. (
image of the binding site of MHY440 in Topo I shows predicted binding affinity of MHY440 with Topo I.
The binding sites of camptothecin ( ) and MHY440 ( ) with Topo I are shown. Pharmacophore results
obtained using LigandScout 4.1.5 showing hydrogen-bonding (green and red arrows) and hydrophobic
yellow) interactions.
B) Magnified
C
D
(

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2
.3. Effects of MHY440 on the Growth of AGS Cells
To investigate the effects of MHY440 on the viability of the human GC cell line (AGS) and the
human foreskin fibroblast cell line (Hs27), the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) assay was performed. Cell viability was reduced with MHY440 treatment in a
concentration- and time-dependent manner. As shown in Figure 3A, in AGS cells, the half-maximal
inhibitory concentration (IC ) of MHY440 at 24 and 48 h was 3.40 µM and 1.83 µM, respectively.
50
However, MHY440 exhibited decreased cytotoxic effects on Hs27 cells (Figure 3B), suggesting MHY440
exerts its inhibitory effects specifically on the proliferation of cancer cells. Therefore, only AGS cells
were used for further experiments.
Figure 3. Effect of MHY440 on the viability of AGS human gastric cancer cells and Hs27 human
foreskin fibroblast cells. (
and 48 h. The percentage of cell viability was then determined using the MTT assay. (
were treated with MHY440 for 24 and 48 h, and cell viability was determined by MTT assay. The results
are expressed as a percentage of vehicle-treated controls standard deviation (SD) in three separate
A) AGS cells were cultured with increasing concentrations of MHY440 for 24
B
) Hs27 cells
±
experiments. Significance was calculated by the Student’s t-test (* p < 0.05, ** p < 0.01, and *** p < 0.001
compared with vehicle-treated cells).
2
.4. Effects of MHY440 on the Cell Cycle in AGS Cells
To investigate whether MHY440 affects cell cycle distribution, AGS cells were treated with various
concentrations of MHY440 for 24 h and then analyzed for cell cycle progression using flow cytometry.
As shown in Figure 4A, MHY440 exposure resulted in an accumulation of cells at G2/M phase.
Flow cell analysis demonstrated that 45.58% of cells cultured with 1.25
phase compared to 28.54% of control cells. In addition, the sub-G1 population increased from 1.88%
in the control group to 39.87% in cells treated with 5.0 M MHY440 (Figure 4B). Next, we examined
µM MHY440 were in G2/M
µ
whether MHY440 regulates the expression of G2/M cell cycle regulators. Cells were treated with
various concentrations of MHY440 for 24 h and the level of G2/M cell cycle regulating proteins were
examined using western blot analysis. As shown in Figure 4C, MHY440 treatment markedly decreased
cyclin B1 in a concentration-dependent manner in AGS cells; Cdc2 and Cdc25c proteins were also
slightly decreased. The transcription factor p53 is induced by a number of stress signals. Cell cycle

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arrest and apoptosis are the most prominent results of p53 activation [20]. In addition, p73 is a protein
associated with p53, and it is considered a tumor suppressor because it is structurally similar to
p53. It is involved in cell cycle regulation and induction of apoptosis [21]. Therefore, we examined
the expression of p53 and p73 in AGS cells treated with MHY440. Our results show that MHY440
treatment increased the expression of both p53 and p73 in a concentration-dependent manner in
AGS cells (Figure 4C). In summary, these results indicate that MHY440 induced cell cycle arrest by
controlling the expression of key proteins involved in the regulation of G2/M phase in AGS cells.
Figure 4. The effect of MHY440 on cell cycle regulation in AGS cells. (A) Cells were treated with
MHY440 at indicated concentrations for 24 h, stained with propidium iodide (PI), and then subjected to
flow cytometry analysis to determine their distribution at each phase of the cell cycle. Representative
results from three independent experiments are shown. (
of four independent experiments. Significance was determined using Student’s t-test (* p < 0.05,
* p < 0.01, and *** p < 0.001 compared with vehicle-treated cells). ( ) After MHY440 treatment for 24 h,
cells were subjected to western blot analysis for the following proteins: cyclin B1, Cdc2, Cdc25c, p53,
and p73. -actin was used as a protein loading control. Representative results from three independent
experiments are shown.
B) Results are expressed as means
±
SD
*
C
β

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2
.5. Effects of MHY440 on the Induction of Apoptosis in AGS Cells
We investigated whether the MHY440-dependent growth inhibition in AGS cells is mediated
by apoptosis via analyzing the features of nuclear morphological changes. AGS cells treated
with MHY440 displayed cell shrinkage and rounding as well as a decrease in cell number in a
concentration-dependent manner compared with the untreated control group. Hoechst 33342 staining
confirmed the induction of apoptosis in AGS cells treated with MHY440 for 24 h. MHY440-treated
cells showed nuclear fragmentation, which is characteristic of chromatin condensation and apoptosis,
whereas control cells showed normal circular morphology of the nucleus (Figure 5A). To confirm that
MHY440-induced cell death was indeed apoptosis, we performed flow cytometry using Annexin V and
PI staining. As shown in Figure 5B, the ratio of late apoptotic cells (upper right quadrant, Annexin V/PI
positive) increased from 4.6% to 64.6% after 24 h of exposure to 5.0 µM MHY440. The results of flow
cytometry also indicated that MHY440-induced apoptosis was concentration-dependent (Figure 5C).
Treatment of AGS cells with MHY440 for 24 h resulted in a concentration-dependent internucleosomal
DNA fragmentation (Figure 5D). To investigate the molecular mechanism of apoptotic cell death by
MHY440 treatment, western blot analysis was conducted with the antibodies for apoptotic marker
proteins. As shown in Figure 5E, MHY440 upregulated the death receptor Fas and its ligand Fas-L
in a concentration-dependent manner. In addition, the expression of the pro-apoptotic protein Bax
by MHY440 treatment was increased compared to the control groups. Furthermore, the levels of
total BID expression were decreased with MHY440 treatment, but truncated Bid (tBid) expression
progressively increased depending on the concentration of MHY440 treatment. tBid localizes at the
mitochondrial membrane and can stimulate the release of cytochrome c and promote the progression of
apoptosis [11
of apoptosis. Cleavage of PARP was evident by the appearance of an 85 kDa fragment (uncleaved
PARP protein is 116 kDa) observed by western blot analysis in AGS cells treated with either 2.5 M or
.0 µM of MHY440. These results suggest that MHY440 induced apoptosis in AGS cells.
,12]. MHY440 treatment also caused proteolytic degradation of PARP, a molecular marker
µ
5

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Figure 5. The effect of MHY440 on apoptosis in AGS cells. (
treated cells. Nuclei of AGS cells stained with Hoechst 33342 to visualize DNA. The stained cells and
nuclei were then observed with a fluorescence microscope (original magnification, 400 ). Arrows
indicate apoptotic cells. ( ) The effect of MHY440 on cell death was determined via Annexin
V-FITC/PI analysis using flow cytometry. ( ) Results are expressed as the means SD of three
independent experiments. Significance was determined using Student’s t-test (* p < 0.05 compared with
vehicle-treated cells). ( ) Representative results of DNA analysis from three independent experiments
are shown. M, Marker. ( ) Western blot analysis of total cell lysates of cells treated with increasing
A) Morphological changes of MHY440-
×
B
C
±
D
E
concentrations of MHY440 for 24 h. The blots were probed with antibodies against Fas-L, Fas, Bax,
BID, and PARP. β-actin was used as a loading control. Representative results from three independent
experiments are shown.
2
.6. Effects of MHY440 on the Mitochondrial Membrane Potential (MMP)
We determined whether the death of these cells is related to the disruption of MMP. The effect
of MHY440 on the MMP in AGS cells was determined using flow cytometry after staining with
0 0 0 0
5
,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetra-ethylbenzimidazolylcarbocyanine iodide (JC-1), (a lipophilic cationic
dye selectively entering mitochondria). The frequency of cells with JC-1 monomers, which are
predominant in regions with low MMP (lower right panel of fluorescence cytogram), increased
in MHY440-treated AGS cells, suggesting that MHY440 destroyed the integrity of the mitochondria,
as measured by the concentration-dependent loss of MMP (Figure 6A). The average populations
of AGS cells with damaged membrane potentials from three individual experiments were 3.6%,
10.3%, 32.4%, and 66.8% at 0, 1.25, 2.5, and 5.0 µM MHY440 concentration, respectively (Figure 6B).
Overall, these results indicate that mitochondrial dysfunction may also contribute to MHY440-induced
apoptosis in AGS cells.

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Figure 6. The effect of MHY440 on the mitochondrial membrane potential (∆Ψm) in AGS cells. (A) To
analyze ∆Ψm, AGS cells were incubated with the indicated concentrations of MHY440 for 24 h, stained
with JC-1 dye, and analyzed using flow cytometry. The data shown represent three independent
experiments with similar results. (
B
) Quantitative data are provided by means of green fluorescence
SD of three
(depolarized ∆Ψm) in the monomeric form of JC-1. The results are expressed as means
±
individual experiments. Significance was determined using Student’s t-test (** p < 0.01 compared with
vehicle-treated cells).
2
.7. Effects of MHY440 on the Caspase Activation
Pro-caspases are precursors of caspases, which are the executors of the apoptotic process [22].
However, a decrease in the level of a pro-caspase may not accurately reflect the activation of caspase.
We, therefore, evaluated the effect of MHY440 on caspase activation using specific substrates. The
histograms in Figure 7A show that caspase-3 activation was increased almost three-fold in AGS cells
treated with 5.0
µM MHY440, while caspase-8 and caspase-9 did not increase more than two-fold
(
Figure 7A). To identify the relevance of caspase activation in MHY440-induced apoptosis, AGS cells
were cultured in the presence and absence of the broad-spectrum caspase inhibitor Z-VAD-FMK and
analyzed using flow cytometry and western blot analysis. As shown in Figure 7B, pretreatment of
cells with Z-VAD-FMK partially decreased the accumulation of sub-G1 fractions induced by MHY440.
To further demonstrate this result, western blot analysis for PARP cleavage was conducted using the
same experimental conditions. Consistent with the cell death measured by flow cytometry, western
blot analysis of PARP showed that pretreatment with Z-VAD-FMK significantly inhibited the cleavage
of MHY440-induced PARP (Figure 7C). These results suggest that activation of caspases contributed to
the MHY440-induced apoptosis in AGS cells.

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Figure 7. The effect of caspases on MHY440-induced apoptosis in AGS cells. (A) MHY440-treated cell
lysates were assayed for caspase-3, -8, and -9 activities using DEVD-pNA, IETD-pNA and LEHD-pNA
substrates, respectively. The emitted fluorescent products were measured. Data are expressed as
the means
) Cells were pretreated with 40
for 24 h. Cells were stained with PI and analyzed using flow cytometry. The results are expressed
as means SD of three individual experiments. Significance was determined using Student’s t-test
* p < 0.05, ** p < 0.01 and *** p < 0.001 compared with vehicle-treated cells). ( ) Total cell lysates were
prepared and immunoblotted for PARP. -actin was used as a loading control. Representative results
±
SD of triplicate samples. The results represent one of three independent experiments.
(
B
µ
M Z-VAD-FMK for 30 min and then treated with 2.5 M MHY440
µ
±
(
C
β
from three independent experiments are shown.
2
.8. Effects of MHY440 on the ROS Generation
We examined the production of ROS in AGS cells after treatment with MHY440 and determined
whether this was the mechanism for the induction of apoptosis. Intracellular ROS levels were quantified
0
0
using the fluorescent probe 2 ,7 -dichlorofluorescein diacetate (DCF-DA). AGS cells were treated with
.0 M MHY440 for various times. As shown in Figure 8A, the maximum intracellular level of ROS
was at 1 h after exposure to 5.0 M MHY440. When AGS cells were treated for 1 h with increasing
concentrations of MHY440, ROS generation was most abundant in 5.0 M MHY440 treatment
Figure 8B). AGS cells were then pretreated with N-acetyl-L-cysteine (NAC), a well-known ROS
5
µ
µ
µ
(
scavenger, and then exposed to 5.0 µM MHY440 for 1 h. At the end of the incubation period, AGS cells
were analyzed using fluorescence microscopy and the levels of intracellular ROS were measured. As
shown in Figure 8C, when AGS cells were treated with 5.0 M MHY440, ROS was generated, and
µ
a green color appeared in the cells. However, pretreatment of cells with NAC considerably blocked
the generation of ROS in MHY440-treated AGS cells, as evidenced by a decrease in green color
(Figure 8C,D).

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Figure 8. The role of ROS on MHY440-induced apoptosis in AGS cells. (
.0 M MHY440 for several different times and stained with DCF-DA. Intracellular ROS levels were
measured using flow cytometry. Data are means SD of three separate experiments. Significance
was determined using Student’s t-test (*** p < 0.001 compared with vehicle-treated cells). ( ) Cells
were treated at various concentrations for 1 h. Data are expressed as the means SD of three
separate experiments. Significance was determined using Student’s t-test (** p < 0.01 compared with
vehicle-treated cells). ( ) Cells were pretreated with or without 5 mM NAC for 1 h and then treated
with 5.0 M MHY440 for 1 h. Intracellular ROS levels were measured using fluorescence microscopy.
Representative results from three independent experiments are shown. ( ) Cells were treated with
.0 M MHY440 for 1 h after pretreatment with or without 5 mM NAC for 1 h. Data are means SD of
A) Cells were treated with
5
µ
±
B
±
C
µ
D
5
µ
±
three separate experiments. Significance was determined using Student’s t-test (* p < 0.05 compared
with vehicle-treated cells; # p < 0.05 compared with 5.0
µ
M MHY440-treated cells). (
E) The expression
of apoptosis in cells pretreated with 5 mM NAC and 2.5
µM MHY440 was determined using PI staining
and flow cytometry analysis. Data are means
determined using Student’s t-test (** p < 0.01 compared with vehicle-treated cells; p < 0.05 compared
±
SD of three separate experiments. Significance was
#
with 5.0
after pretreatment with or without 5 mM NAC were analyzed using western blot analysis for the
expression level of PARP. -actin was used as a loading control. Representative results from three
independent experiments are shown. ( ) Total cell lysates from cells treated with 2.5 M MHY440
alone or pretreated with 5.0 mM NAC for 24 h were analyzed using western blot analysis for the
following antibodies: p-ATM (Ser1981), p-ATR (Ser428), -H2AX (Ser139), p-Chk1 (Ser345), p-Chk2
Thr68), and p-p53 (Ser15). -actin was used as a loading control. Representative results from three
independent experiments are shown.
µM MHY440-treated cells). (F) Total cell lysates of cells treated with or without 2.5 µM MHY440
β
G
µ
γ
(
β

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To further confirm the relationship between ROS generation and apoptosis, the effect of NAC
was evaluated in cells treated with MHY440. As shown in Figure 8E, after exposure to with MHY440
with or without NAC pretreatment, the presence of cells with sub-G1 DNA content was assessed
using flow cytometry to quantify the onset of apoptosis. Cells pretreated with NAC significantly
inhibited apoptosis in MHY440-treated cells. Consistent with these observations, sequestration of ROS
by NAC effectively inhibited MHY440-induced PARP cleavage in AGS cells (Figure 8F). In addition, to
investigate the effect of ROS generation on the DNA damage response, we examined the effects that
treatment of MHY440 with or without NAC had on the expression of DNA damage response proteins.
We found that the inhibition of ROS by NAC effectively down-regulated the levels of MHY440-induced
DNA damage response proteins, including p-ATM, p-ATR, γ-H2AX, p-Chk1, p-Chk2, and p-p53, all of
which were increased after MHY440 treatment alone (Figure 8G). These results demonstrate that ROS
generation played an important role in the MHY440-mediated apoptotic pathways as well as the DNA
damage response pathways in AGS cells.
3
. Discussion
DNA Topo I controls the topological state of DNA in many cell processes, including DNA replication
and transcription [ ]. Compounds that inhibit Topo I activity have been widely used as anticancer
8
agents because of their ability to block DNA damage, trigger cell cycle arrest, and subsequently initiate
apoptosis [23]. FDA-approved Topo I inhibitors camptothecin derivatives topotecan and irinotecan are
currently used in the treatment of ovarian and colon cancer, respectively [24]. Based on these reports,
we examined the effect of MHY440 on HCT116 human colon cancer cells and AGS human gastric
cancer cells. After 24 h of MHY440 treatment, the IC50 of HCT116 cells and AGS cells was 5.24
.40 M, respectively. Based on these preliminary results, whole experiments were conducted using
AGS human gastric cancer cell line.
µM and
3
µ
Induction of DNA damage is a key mechanism of Topo inhibitors [25]. Suppression of Topo
activity and induction of DNA damage stimulates DNA repair enzymes [26]. DNA damage pathways
include damage sensors, signal transducers, and effectors. DNA damage causes activation of DNA
damage response elements, such as ATM and ATR. Activation of ATR is generally associated with
single-stranded DNA damage or arrest of DNA replication forks, whereas ATM activation is associated
with the initiation of signaling pathways involved with double-strand DNA breaks [26]. During
the inhibition of Topo activity, activated ATM and ATR directly influence the downstream proteins
BRCA1, H2AX, Chk1, and Chk2 through either direct or sequential steps, resulting in the inhibition of
downstream factors involved in cell cycle progression and cell survival [27]. Phosphorylated H2AX
and BRCA1 are involved in DNA repair and the activation of other repair factors, but phosphorylated
Chk1 and Chk2 activate cell cycle arrest and apoptosis-related factors [28].
It is well known that the progression of the cell cycle is tightly regulated by the interaction between
the activators of the cell cycle and inhibitors of the cell cycle. The progression of the eukaryotic cell
cycle is controlled by the coordinated activity of the Cdk-cyclin complex [29]. G2/M transitions are
primarily dependent on cyclin B1/Cdk1 activity. The activity of cyclin B1/Cdk1 can be activated by
Waf1/Cip1
Kip1
Cdc25c or inhibited by p53, p21
, and p27
[30]. Cdc25c is a key protein controlling cell
cycle G2/M transition and is an essential component of the checkpoint pathway that can be activated
in response to DNA damage. Activation of ATM by DNA damage mediates the induction of Chk1 and
Chk2, inhibition and degradation of Cdc25c, and activation of Cdk1 via Cdc25c, all of which result in
cell cycle blockade at G2/M [31]. In our study, exposure of AGS cells to MHY440 significantly induced
the activation of ATM, ATR, Chk1, and Chk2 via phosphorylation. Activation of the ATM/ATR and
Chk1/2 signaling axes inhibited Cdc25c, which in turn, inhibited cyclin B1/Cdk1 kinase activity and
induced cell cycle arrest in the G2/M phase (Figure 1C; Figure 4B,C).
The tumor suppressor protein, p53, is an important component of the cell machinery,
which regulates a variety of signaling pathways, including carcinogenesis, cell cycle, apoptosis, and
DNA damage responses under a variety of conditions. In response to Topo suppression or chemically-

Molecules 2019, 24, 96
13 of 18
induced DNA damage, activated ATM or Chk2 directly activates p53 via phosphorylation, which
inhibits its interaction with the negative regulator murine double minute2 (MDM2) [32]. Activated
p53 induces Bax expression, which leads to an imbalance in the Bax/Bcl-2 ratio, resulting in the release
of cytochrome c from the mitochondria, disruption of the mitochondrial membrane potential, and the
induction of apoptosis. In our study, MHY440-treated AGS cells showed increased expression of p53
and Bax in addition to increased proteolysis of the BID protein (Figures 4C and 5E). MHY440 also
caused loss of mitochondrial membrane potential in AGS cells (Figure 6A,B).
In biological systems, ROS are constantly generated and removed. ROS also play an important
role in both homeostasis and disease. The excessive production of ROS in the mitochondria is known to
play an important role in the regulation of apoptosis [33]. The downregulation of survivin, a member
of inhibitor of apoptosis and an antagonist of apoptosis, was associated with ROS production in
cancer cell apoptosis [34–36]. Some anticancer agents, such as cisplatin, doxorubicin, mitomycin C,
and etoposide, are at least partially effective through the induction of ROS [37]. Oxidative stress
induced by ROS can damage cellular components, including DNA and proteins [38]. The continued
failure of cells to repair DNA lesions with the appropriate repair mechanisms can eventually translate
into double-strand DNA breaks, which ultimately lead to cell cycle arrest and cell death. Several
studies have shown that ROS can affect cell cycle progression and cell death by activating intracellular
signaling pathways sensitive to various oxidative stresses, such as ATM/ATR, Chk1/2, and c-Jun
N-terminal kinases (JNK) [39].
In conclusion, MHY440, as a novel Topo I inhibitor, inhibited the growth of AGS cells by inducing
a DNA damage response, arresting cell cycle at G2/M phase, and initiating apoptosis through the
activation of a caspase cascade and ROS generation. Overall, our results demonstrate that MHY440
has the potential to be used as a therapeutic agent for treating GC.
4
. Materials and Methods
4
.1. Chemicals and Reagents
The simplified code name and structure of MHY440 used in this study are shown in Figure 1A.
This compound was provided by Professor Hyung Ryong Moon (Pusan National University, Busan,
Korea). MHY440 was synthesized via condensation of methyl anthranilate with phloroglucinol
in the presence of p-toluenesulfonic acid followed by alkylation with epichlorohydrin in the
presence of potassium carbonate as shown in Supplementary material (Scheme 1). Compound
was obtained in a yield of 58% and alkylation of compound with epichlorohydrin afforded the
desired product MHY440 and a monoalkylated product, compound , at a yield of 16 and 20%,
1
1
2
respectively. MHY440 was dissolved in sterile dimethyl sulfoxide (DMSO) to make a 10 mM stock
◦
solution and stored at
−
20 C until use. Subsequent dilutions were performed in RPMI-1640 (GE
Healthcare Life Sciences, Logan, UT, USA). The maximum concentration of DMSO did not exceed
.1% (v/v), which did not affect cell growth. DMSO and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) were obtained from Amresco Inc. (Solon, OH, USA). Camptothecin,
propidium iodide (PI), N-acetyl-L-cysteine (NAC), and the monoclonal antibody against -actin were
0
β
purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies specific for phosphorylated (p-)
ataxia telangiectasia-mutated kinase (ATM, Ser1981), p-ataxia telangiectasia and rad3-related kinase
(
ATR, Ser428),
and BH3-interacting domain death agonist (BID) were purchased from Cell Signaling Technology
Danvers, MA, USA). Cyclin B1, cell division cycle protein 2 (Cdc2), Cdc25c, p53, p73, Fas-L, Fas,
γ-H2AX (Ser139), p-checkpoint kinase 1 (Chk1, Ser345), p-Chk2 (Thr68), p-p53 (Ser15),
(
Bcl-2-associated X protein (Bax), poly(ADP-ribose) polymerases (PARP), and Z-VAD-FMK were
obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Molecules 2019, 24, 96
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4
.2. Determination of Topo I Activity
A commercially available Topo I drug screening kit (TopoGEN, Inc., Buena Vista, CO, USA)
was used in this study, per the manufacturer’s instructions. Briefly, supercoiled substrate pHOT1
DNA substrate (0.25
µ
g) was incubated with Topo I reaction buffer (2
µ
L), MHY440, and four units
L)
g/mL)
◦
of human DNA Topo I at 37 C for 30 min. After the incubation, 10% sodium dodecyl sulfate (2
µ
was added, and then the reaction was terminated via decomposition with proteinase K (50
µ
◦
at 37 C for 15 min. The DNA was resolved on a 1% agarose gel in a gel electrophoresis system for
90 min. Ethidium bromide (EtBr) was added to the gel to aid in visualizing the DNA. The gel images
were determined via BioSpectrum AC imaging system (UVP, Upland, CA, USA).
4
.3. Docking Simulation of Topo I and MHY440
Because of automated docking capability, AutoDock Vina and AutoDock 4 were used for the
in silico protein–ligand docking simulation. The 3-D structure of Topo I was used in the crystal
structure of human Topo I (PDB ID: 1K4T). As a docking pocket, predefined binding site of Topo I
was used [40]. Docking simulations were performed between Topo I and MHY440 or camptothecin.
To prepare compounds for docking simulation, (1) 2-D structures were converted into 3-D structures,
(
(
2) charges were calculated, and (3) hydrogen atoms were added using the ChemSketch program
http://www.acdlabs.com/resources/freeware/chemsketch). The prediction of possible hydrogen
bonding residues between compounds and Topo I and generation of pharmacophores were analyzed
with LigandScout 4.1.5 program (Inte:Ligand, Vienna, Austria).
4
.4. Cell Culture and Cell Viability Analysis
The human GC cell line (AGS) and the human foreskin fibroblast cell line (Hs27) were
purchased from the American Type Culture Collection (Manassas, VA, USA) and grown in RPMI-1640
supplemented with 10% fetal bovine serum (GE Healthcare Life Sciences), 100 U/mL penicillin,
◦
and 100
µ
g/streptomycin (GE Healthcare Life Sciences) and incubated at 37 C with 5% CO .
2
Cell viability was measured using an MTT assay. For MTT analysis, cells were incubated on 24-well
culture plates and cultured for 24 or 48 h and then treated with or without various reagents at the
◦
indicated concentrations of MHY440. Cells were incubated with 0.5 mg/mL MTT in the dark at 37 C
for 2 h. The formazan produced by live cells were dissolved in DMSO, and the absorbance at 540 nm
was monitored with a multi-wall reader (Thermo Fisher Scientific, Waltham, MA, USA).
4
.5. Cell Cycle Analysis
Cells were treated for 24 h at the appropriate conditions and then were treated with trypsin.
The cells were then washed one time with cold phosphate-buffered saline (PBS) and then stored in
◦
7
0% ethanol overnight at
−
20 C. Fixed cells were pelleted and stained in cold PI solution (50
µg/mL
in PBS) in the dark for 30 min at room temperature. The stained DNA contents of the cells were
then analyzed using flow cytometry on a Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes,
NJ, USA).
4
.6. Western Blot Analysis
Cells were harvested, lysed, and an equivalent amount of protein was subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride
membranes for immunoblotting. The membranes were blocked with 5% nonfat dry milk in
Tris-buffered saline for 1 h with a Tween-20 buffer (TBS-T; 20 mM Tris, 100 mM NaCl, pH 7.5,
and 0.1% Tween-20) at room temperature. Membranes were then incubated with primary antibodies
◦
overnight at 4 C. The membranes were washed with a TBS-T buffer and incubated with horseradish
peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) for 1 h. The membranes

Molecules 2019, 24, 96
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were washed with TBS-T buffer. Antigen-antibody complexes were detected using an enhanced
chemiluminescence detection system (GE Healthcare, Chicago, IL, USA).
4
.7. Nuclear Staining with Hoechst 33342
Cells were stained with 4
USA) at 37 C for 10 min. Cells were examined by a Nikon Eclipse TE 2000-U microscope (Nikon,
Tokyo, Japan).
µ
g/mL Hoechst 33342 (Life Technologies Corp., Grand Island, NY,
◦
4
.8. Annexin V Staining
The proportion of cells that actively progressed to apoptosis was quantitatively determined
using an Annexin V-FITC apoptosis detection kit (BD Biosciences). Cells were treated for 24 h at
the appropriate conditions and then harvested, treated with trypsin, washed once in cold PBS, and
resuspended in 1X Binding Buffer. The aggregated cells were stained in the dark for 15 min at room
temperature in PI and Annexin V-FITC solution. The stained cells were analyzed by an Accuri C6
flow cytometer.
4
.9. DNA Fragmentation Assay
Cells were lysed on ice for 30 min in a buffer containing 5 mM Tris-HCl (pH 7.5), 5 mM
EDTA, and 0.5% Triton X-100. The lysate was vortexed and then centrifuged at 27,000
×
g for 20
min. The fragmented DNA in the supernatant was RNase-treated and then treated with proteinase K
to degrade any exogenous proteins. The DNA was then extracted with a phenol/chloroform/isoamyl
alcohol mixture (25:24:1, v/v/v) and precipitated with isopropanol. DNA was separated from 1.6%
agarose gel, stained with 0.1 µg/mL EtBr, and visualized with a UV light source.
4
.10. Measurement of Mitochondrial Membrane Potential (MMP, ∆Ψm)
0
0
MMP was measured using a flow cytometer and a lipophilic cationic dye, 5,5 ,6,6 -tetrachloro-
0
0
1
,1 ,3,3 -tetra-ethylbenzimidazolylcarbocyanine iodide (JC-1; Calbiochem, San Diego, CA, USA).
JC-1 is a dye that stains the mitochondria of living cells in a membrane potential-dependent manner.
Cells were treated with various concentrations of MHY440, harvested, and washed with cold PBS.
◦
Cells were stained with 10
µ
M JC-1 for 20 min at 37 C in the dark. Cells were then washed with cold
PBS and analyzed using an Accuri C6 flow cytometer.
4
.11. Measurement of Caspase Activity
Cells were harvested, washed with cold PBS, and incubated with a lysis buffer (R&D Systems,
Inc., Minneapolis, MN, USA) for 10 min on ice. The lysed cells were centrifuged at 10,000
×
g for
1
min, and 100 g of protein was added to the reaction mixture containing 2 reaction buffer and
µ
×
substrates of colorimetric tetrapeptides, including DEVD-pNA for caspase-3, IETD-pNA for caspase-8,
◦
and LEHD-pNA for caspase-9. The reaction mixture was incubated at 37 C for 2 h, and then enzymatic
release of p-nitroaniline was quantitated at 405 nm using a multi-wall reader (Thermo Fisher Scientific).
4
.12. Measurement of Intracellular ROS Accumulation
The intracellular accumulation of ROS was monitored using the fluorescent probe 2 ,7 -
0
0
dichlorofluorescin diacetate (DCF-DA). A solution of 10
µM DCF-DA was added to the cells.
◦
After incubation at 37 C for 30 min, the intracellular accumulation of ROS was determined by a Nikon
Eclipse TE 2000-U microscope set at 488 nm for excitation and 530 nm for emission. Alternatively,
cells were rinsed with PBS, treated with trypsin, washed with PBS, and then analyzed by an Accuri C6
flow cytometer.

Molecules 2019, 24, 96
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4
.13. Statistical Analysis
Data are presented as means
analyzed via Student’s t-test. The mean was considered significantly different if * p < 0.05, ** p < 0.01,
and *** p < 0.001.
±
standard deviations (SD) of three separate experiments and
Supplementary Materials: The following are available online.
Author Contributions: J.Y.J. and Y.J.K. wrote the manuscript and performed the experiments. B.S. and M.J.K.
interpreted the data. C.P., D.K., and H.R.M. synthesized the compounds. H.Y.C. and N.D.K. coordinated the study
and interpretation of the data. All authors read and approved the final manuscript.
Funding: The present study was supported by a National Research Foundation of Korea (NRF) grant funded by
the Korea government (MSIP, no. 2009-0083538) and the Basic Research Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Education (2018R1D1A1B07044648).
Acknowledgments: We would like to thank the Aging Tissue Bank for providing research information.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).