← Back
Evaluation of anticancer effect in vitro and in vivo of iridium(III) complexes on gastric carcinoma SGC-7901 cells.
Accepted Manuscript
Evaluation of anticancer effect in vitro and in vivo of iridium(III) complexes on gastric
carcinoma SGC-7901�cells
Wen-Yao Zhang, Yang-Jie Wang, Fan Du, Miao He, Yi-Ying Gu, Lan Bai, Lin-Lin
Yang, Yun-Jun Liu
PII:
S0223-5234(19)30520-3
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.003
Reference:
EJMECH 11406
To appear in:
European Journal of Medicinal Chemistry
Received Date: 18 April 2019
Revised Date:
23 May 2019
Accepted Date: 2 June 2019
Please cite this article as: W.-Y. Zhang, Y.-J. Wang, F. Du, M. He, Y.-Y. Gu, L. Bai, L.-L. Yang, Y.-J.
Liu, Evaluation of anticancer effect in vitro and in vivo of iridium(III) complexes on gastric carcinoma
SGC-7901�cells,
European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.06.003.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Graphical abstract
Three new iridium(III) complexes were synthesized and characterized. The
cytotoxicity in vitro and in vivo was investigated. The complexes show high
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
anticancer activity in vitro and in vivo.
ACCEPTED MANUSCRIPT
Submitted to Eur J Med Chem.
Evaluation of anticancer effect in vitro and in vivo of iridium(III)
RI
PT
complexes on gastric carcinoma SGC-7901 cells
Wen-Yao Zhanga, Yang-Jie Wanga, Fan Dua, Miao Hea, Yi-Ying Gua, Lan Baia,
a
M
AN
U
SC
Lin-Lin Yangb,*, Yun-Jun Liua,c*
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, 510006,
P.R. China
Department of Pediatrics, Guangdong Women and Children Hospital, Guangzhou
510000, P.R. China
School of Chemistry and Chemical Engineering, Guangdong Pharmaceutical
EP
c
TE
D
b
AC
C
University, Zhongshan, 528458, P.R. China.
*Corresponding author. E-mail address: fy_yanglinlin@126.com (L.L. Yang);
1
ACCEPTED MANUSCRIPT
lyjche@gdpu.edu.cn (Y. J. Liu).
Abstract This work mainly introduces the synthesis and characterization of three
RI
PT
iridium(III) complexes [Ir(ppy)2(adppz)](PF6) (Ir-1), [Ir(bzq)2(addpz)](PF6) (Ir-2) and
[Ir(piq)2(adppz)](PF6) (Ir-3). The complexes are more cytotoxic than cisplatin against
tumor cell lines such as SGC-7901, A549, HeLa, Eca-109, HepG2 and BEL-7402.
SC
The toxicity test results indicated that complexes Ir-1, Ir-2 and Ir-3 can effectively
inhibit the cell growth of SGC-7901 cells, and the measured IC50 values are 1.8 ± 0.4,
M
AN
U
1.6 ± 0.3 and 0.8 ± 0.1 µM, respectively. AO/EB staining and flow apoptosis
confirmed that SGC-7901 cells were caused apoptosis after being treated with the
complexes. Along with the increase of endogenous ROS and Ca2+ levels,
TE
D
mitochondrial membrane potential collapse and massive release of cytochrome c, it is
fully demonstrated that these complexes induce apoptosis through ROS-mediated
mitochondrial pathway. At the same time, the complex Ir-3 is outstanding in the
EP
inhibition of tumor growth in vivo. Combined with the above results, it provides a
AC
C
favorable foundation for the future development of more effective anti-tumor drugs.
Keywords: Iridium(III) complexes; Cytotoxicity in vitro and vivo; Apoptosis; Cell
cycle arrest; western blot.
1. Introduction
Cancer is the most deadly disease in the world today, with liver, stomach, lung,
2
ACCEPTED MANUSCRIPT
ovary and rectal cancer being the most common cancers with high mortality [1-3].
The easy transfer of tumor cells and rapid and excessive proliferation are the main
challenges of cancer treatment [4-7]. Therefore, the design and development of
RI
PT
effective anti-tumor drugs with outstanding tumor cytotoxicity and apoptotic rate and
better inhibition of tumor spread have attracted much attention in the field of drug
research and development. At present, metal platinum drugs (cisplatin, carboplatin,
SC
oxaliplatin) can effectively treat various types of cancer and have been successfully
M
AN
U
applied clinically [8-13]. However, with the continuous use in the clinic, the serious
toxic and side effects of platinum drugs, drug resistance and selectivity are quickly
exposed to the public's field of vision [14-18]. This drawback has quickly stimulated
the exploration of other metal drugs by various scientific research groups around the
TE
D
world, and strived to find drugs that can reduce side effects, expand the spectrum of
sensitive tumors, and try to overcome resistance. Among many metal drugs,
organometallic iridium(III) complexes have been paid great attention by virtue of their
EP
rich functionality, multiple oxygen states and excellent photosensitivity [18-23]. Some
AC
C
iridium(III) complexes reveal high anticancer and antibacterial activity and can
effectively inhibit the cancer cell proliferation [24-40]. To obtain more information on
anticancer
activity
of
iridium(III)
complexes,
in
this
paper,
a
11-nitrodipyrido[3,2-a:2',3'-c]phenazine
(NDPPZ)
and
its
three
iridium(III)
complexes:
(ppy
[Ir(ppy)2(NDPPZ)](PF6)
=
2-phenylpyridine,
ligand
Ir-1),
[Ir(bzq)2(NDPPZ)](PF6) (bzq = benzo[h]quinolone, Ir-2) and [Ir(piq)2(NDPPZ)](PF6)
(piq = 1-phenylisoquinoline, Ir-3, Scheme 1) were synthesized and characterized by
3
ACCEPTED MANUSCRIPT
elemental analysis, ESI-MS, 1H NMR and 13C NMR. The cytotoxicity of the
complexes against cancer SGC-7901, A549, HepG2, BEL-7402, Eca-109 and normal
NIH3T3 cells was evaluated by 3-(4,5-dimethylthiazole)-2,5-diphenyltetraazolium
RI
PT
bromide (MTT) methods. The cellular morphological apoptosis, cell substructure
localization, Ca2+ level, cytochrome c content and cytoskeleton were detected and
analyzed by high-content cell imaging system. Apoptosis rate, cell cycle arrest, ROS
SC
level and mitochondrial membrane potential into the SGC-7901 cells were detected.
M
AN
U
In addition, we have conducted research on tumor invasion assay and molecular
protein expression in order to further understand the apoptotic mechanism. The results
of nude mice experiments further illustrate the inhibitory effect of iridium complexes
on tumor cell growth, and effectively support the possibility that iridium complexes
TE
D
will become clinical anti-tumor drugs in the future. Finally, we hope this work can
contribute to the development of anti-cancer drugs.
EP
2. Results and discussion
AC
C
2.1. In vitro growth inhibitory activity
The anticancer activities of complexes (Ir-1-Ir-3) were evaluated against six tumor
cell lines: SGC-7901 (human gastric cancer cell line), A549 (human pulmonary
adenocarcinoma cell), HeLa (human cervical carcinoma cell line), Eca-109 (human
esophageal cancer cell line), HepG2 (Human hepatocellular liver carcinoma cell line),
BEL-7402 (Human hepatocellular liver carcinoma cell line)) and normal cell NIH3T3
4
ACCEPTED MANUSCRIPT
(3T3-Swiss albino) using MTT methods [41-43]. Cisplatin was used as a positive
control. The IC50 values of the complexes toward the above cell lines are listed in
Table 1. As an expectation, ligand NDPPZ shows low or no cytotoxicity toward the
RI
PT
selected cancer cells. From table 1, all the complexes show strong toxicity against the
cancer cell lines, especially SGC-7901 cells with a low IC50 value of 1.8 ± 0.4 µM for
Ir-1, 1.6 ± 0.3 µM for Ir-2 and 0.8 ± 0.1 µM for Ir-3. In addition, the results also
SC
indicate that antitumor activity of the complexes follows the order of Ir-3 > Ir-2 >
M
AN
U
Ir-1 toward SGC-7901 cells. Complex 3 reveals the highest cytotoxicity in vitro
among the three complexes against SGC-7901 and A549 cells. According to our
previous work [41], we infer that this may be caused by large hydrophobicity of piq
compared with ppy or bzq, which makes it easy for complex 3 to enter into the cells.
TE
D
On the other hand, -NO2 as substituent group may increase the hydrophobicity of the
complexes. Hence, these complexes exhibit high anticancer activity toward the
selected cancer cells. However, for A549 cells, the anticancer activity of the
EP
complexes follows the order of Ir-3 > Ir-1 > Ir-2. Hence, we conclude that different
AC
C
complexes show different anticancer effect on the different cancer cells. In addition,
the complexes reveal higher cytotoxic activity than cisplatin against the selected
cancer cells under identical conditions. Because the complexes are sensitive to
SGC-7901 cell, therefore, this cell line was selected for undergoing the following
experiments. In order to observe whether the luminescence spectra of the complexes
perturb the green fluorescence in the all cell experiments, the luminescence spectra of
complexes Ir-1, Ir-2 and Ir-3 were determined in PBS solution at ambient
5
ACCEPTED MANUSCRIPT
temperature. As shown in Fig. S1 (supporting information), Ir-1, Ir-2 and Ir-3 can
emit at 599 nm, 595 nm and 605 nm, respectively. Whereas the emission wavelengths
of DCHF-DA for ROS, JC-1 for mitochondrial membrane potential, Fluo-3AM for
RI
PT
intracellular Ca2+ concentration and cyto c are 525 nm, 529 525 nm and 454 nm,
respectively. Hence, the emission data of the complexes do not perturb the cell
SC
experiments.
M
AN
U
2.2. Location and change of mitochondrial membrane potential (MMP)
The MTT data indicated that the Ir(III) complexes Ir-1, Ir-2 and Ir-3 have
significant antiproliferative activity. As one of the most important subcellular
structures, mitochondria participate in cellular energy production and apoptotic
TE
D
signaling pathways [44]. To affirm whether the mitochondria are a target, we used
tracer dye Mito Tracker ® Deep Red FM (Thermo Fisher, 100 nM) [45] to further
EP
explore whether the complexes locate at the mitochondria. As shown in Fig. 1A, the
mitochondria were stained red by Mito Tracker Red FM, complexes Ir-1, Ir-2 and
AC
C
Ir-3 emitted green fluorescence when SGC-7901 cells were treated with 2.0 µM of
Ir-1, Ir-2 and 1.0 µM of Ir-3 for 6 h. It is worth noting when we overlay the red and
green images, we will surprisingly find that the two images can be completely
integrated. In other words, these complexes have a certain degree of targeting to
mitochondria.
It is well known that the earliest change in apoptosis is the decrease in
6
ACCEPTED MANUSCRIPT
mitochondrial membrane potential. To investigate whether these metal complexes
induce apoptosis in mitochondria-mediated manner, We measured the change of
mitochondrial membrane by loading SGC-7901 cells with fluorescent probe JC-1
RI
PT
(5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide) [46,47]. It
is convenient to detect the change of mitochondrial membrane potential according to
the change of fluorescent color. When the mitochondrial membrane potential is at a
SC
high potential, JC-I can accumulate in the mitochondrial matrix and emit red
M
AN
U
fluorescence. Conversely, if it is not able to accumulate in the mitochondrial matrix,
the JC-1 monomer will emit green fluorescence. As depicted in Fig. 1B, It is clear that
the red fluorescence was gradually replaced by green fluorescence on addition of Ir(III)
complexes into SGC-7901 cells. Therefore, the experimental results indicate that
TE
D
complexes Ir-1-Ir-3 can cause destruction of mitochondrial membrane integrity and
decrease in transmembrane potential in SGC-7901 cells. Thus the results confirmed
that mitochondria are involved in mediating apoptosis.
EP
To investigate whether there is a correlation between mitochondrial membrane
AC
C
potential and reactive oxygen species, the changes in the mitochondrial membrane
potential caused by the complexes in the presence of NAC [48] were further
quantified. The relative ratio of red/green fluorescence intensity after the complexes
treated SGC-7901 cells was determined by flow cytometry, which in turn reflected the
mitochondrial depolarization ratio. As shown in Fig. 1C, in the control (a) or in the
presence of NAC (b), the ratios of red/green are 11.97 and 19.41, respectively.
Treatment of SGC-7901 cells with 1.0 µM of Ir-1 (c), 2.0 µM of Ir-1 (d) and Ir-1 (2.0
7
ACCEPTED MANUSCRIPT
µM) + NAC (8 µM) (e), 1.0 µM of Ir-2 (f), 2.0 µM of Ir-2 (g) and Ir-2 (2.0 µM) +
NAC (8 µM) (h), 0.5 µM of Ir-3 (i), 1.0 µM of Ir-3 (g) and Ir-3 (1.0 µM) + NAC (8
µM) (k) for 24 h, the ratios of the red/green fluorescence are 1.24, 0.095 and 0.81 for
RI
PT
Ir-1, 0.91, 0.16 and 0.33 for Ir-2, 2.98, 0.38 and 1.68 for Ir-3, respectively. According
to the above experimental data, we can summarize two conclusions: (I) the complexes
(Ir-1, Ir-2 and Ir-3) exhibited dose tolerance to induce mitochondrial membrane
SC
potential collapse. (II) we can clearly see that in the presence of NAC, the red/green
M
AN
U
fluorescence ratio increases significantly compared with the complexes alone. The
results indicate that NAC has an inhibitory effect on the collapse of mitochondrial
membrane potential. Therefore, the accumulation of active oxygen will accelerate the
TE
D
decrease of mitochondrial membrane potential.
2.3. Intracellular ROS measurements
Endogenous reactive oxygen species (ROS) are considered to be key factors in
EP
cell signaling pathways, especially in terms of apoptosis and inflammation [49-51].
AC
C
Regulating the homeostasis of reactive oxygen species in the body has become an
important research direction for antitumor. In order to gain a deep understanding of
the anti-tumor mechanism of these complexes, we used the specific fluorescent probe
DCFH-DA to detect changes in the reactive oxygen species in SGC-7901 cells. As
shown in Fig. 2A, in the control (a), a weakly green fluorescence points could be
observed. However, SGC-7901 cells were incubated with Rosup (b, positive control),
2.0 µM of Ir-1 (c), 2.0 µM of Ir-2 (d) and 1.0 µM of Ir-3 (e) for 24 h, a number of
8
ACCEPTED MANUSCRIPT
green fluorescence points were found notably. Moreover, we further quantified the
fluorescence intensity of the treated cells by flow cytometry. As see from Fig. 2B,
SGC-7901 cells were exposed to Ir-1-Ir-3 for 24 h in the absence or presence of NAC,
RI
PT
the DCF fluorescent intensity increases 28.86, 16.81 and 19.55 times than that in the
control, and the effect of Ir-1-Ir-3 on the ROS levels were higher than Rosup group.
The data obtained from Fig.2A and 2B suggest that these complexes can significantly
SC
increase the level of cellular reactive oxygen species, and that the complex Ir-1
M
AN
U
induces the production of reactive oxygen species more strongly than other complexes.
The results demonstrate that NAC inhibits the production of cellular ROS.
Superoxide anion is one of the important components of intracellular ROS, which
can cause damage to DNA and RNA at high concentrations. The determination of
TE
D
intracellular superoxide anion content was performed using fluorescent probe
dihydroethidium (DHE) [52]. The probe DHE will be dehydrogenated by the
intracellular superoxide anion to form the ethidium. Ethidium produces clear, bright
EP
red fluorescence when combined with DNA or RNA. As shown in Fig. 2C, in the
AC
C
control (a), weak red fluorescence could be observed. However, SGC-7901 cells were
incubated with 2.0 µM of Ir-1 (b), 2.0 µM of Ir-2 (c) and 1.0 µM of Ir-3 (d) for 24 h,
a plentiful of red fluorescence points were observed clearly. To quantitatively
compare the effects of complexes on O2·‒ levels, the DHE fluorescent intensity was
determined by ImageXpress Micro XLS system (MD company, US) in the presence
or absence of NAC. As shown in Fig. 2D, SGC-7901 cells were exposed to Ir-1-Ir-3
for 24 h, the DHE fluorescent intensity increases 70.80, 62.17 and 89.09 times than
9
ACCEPTED MANUSCRIPT
that in the control. The obtained results indicate that complexes can significantly
increase the level of O2•– in SGC-7901 cells, while NAC can inhibit the production of
O2•– in cells.
RI
PT
As a reactive oxygen molecule, NO plays an important role in the anti-tumor
process. The endogenous NO level of cells can be conveniently detected using the
fluorescent probe 3-amino-4-aminomethyl-2',7'-difluorescein diacetate (DAF-FMDA).
SC
As indicated in Fig. 2E, treatment of SGC-7901 cells displays an obvious green
M
AN
U
fluorescence after incubation with complexes (Ir-1-Ir-3) in comparison with the
untreated cells. Then, we also quantitatively evaluate the endogenous NO levels. As
shown in Fig. 2F, SGC-7901 cells were exposed to Ir-1-Ir-3 for 24 h, the
DAF-FMDA fluorescent intensity increases 1.09, 1.81 and 2.24 times than the control.
TE
D
Combined with the data above, complexes can increase the intracellular ROS levels.
As a major member of the MAPK pathway, P38 MAPK plays an important role
in the regulation of various physiological signals such as cell stress, inflammation,
EP
apoptosis and cell cycle [53-57]. High levels of the intracellular ROS can activate the
AC
C
downstream protein P38 to induce apoptosis. Activation of P38 protein is essential for
inducing cell apoptosis. Therefore, we assayed the expression of P38 protein after the
treatment of SGC-7901 cells with complexes Ir-1-Ir-3 by western blotting. See from
Fig. 2G, we can clearly observe that the protein expression of P38 is significantly
up-regulated after 24 h of exposure of SGC-7901 cells to the complexes. Therefore,
these results showed that the complexes can increase the intracellular ROS level, and
then induced apoptosis of SGC-7901 cells through ROS-P38 signaling pathway.
10
ACCEPTED MANUSCRIPT
2.4. Apoptosis assay with AO/EB and Annex V/PI double staining methods
The MTT results indicated that the complexes have a significant effect on
RI
PT
reducing the proliferation of SGC-7901 cells. To further understand the effect on
apoptosis, the AO/EB and Annex V/PI double staining methods [58-60] were carried
out to assess the changes in nuclear morphology and the degree of apoptosis.
SC
SGC-7901 cells stained with acridine orange (AO) and ethidium bromide (EB) are
M
AN
U
shown in Fig. 3A. In the control (a), the living cells were stained bright green and
exhibited homogeneous nuclei staining in spots. After the treatment of SGC-7901
cells with 1.0 µM of Ir-1 (b), 1.0 µM Ir-2 (c), and 0.5 µM Ir-3 (d) for 24 h, the
apoptotic morphological features such as cell blebbing, nuclear shrinkage, chromatin
TE
D
condensation and fragmentation, as well as red necrotic cells, were observed. In order
to more accurately quantify the effect of apoptosis, flow cytometry was used to study
the proportion of apoptosis in cells treated with the complexes. As show in Fig. 3B,
EP
quantitative results showed that the early apoptosis rate of cells in SGC-7901 cells (a)
AC
C
treated with Ir-1 (b, 1.0 µM), Ir-2 (c, 1.0 µM) and Ir-3 (d, 0.5 µM) for 48 h was
8.36%, 13.50% and 11.20%, respectively. The results indicate that the complexes can
induce early apoptosis in SGC-7901 cells.
2.5. DNA damage assay
Elevated levels of intracellular ROS mediate DNA oxidative damage and
ultimately induce apoptosis [61-63]. Comet assay as a most commonly used means to
11
ACCEPTED MANUSCRIPT
assess genotoxicity was implemented in current study to evaluate the complexes
inducing DNA damage. As shown in Fig. 4, it is clearly to see no DNA damage in the
control (a). However, after SGC-7901 cells were incubated with 1.0 µM of Ir-1 (b),
RI
PT
1.0 µM of Ir-2 (c) and 0.5 µM of Ir-3 (d) for 24 h, obviously and clearly-formed
comets were observed. The results demonstrate that the complexes can induce DNA
SC
damage which further triggers apoptosis.
M
AN
U
2.6. Autophagy induced by the complexes
Recent studies have shown that there is a close relationship between autophagy
and the occurrence of tumor cells, especially when the cells are autophagic, which can
cause tumor cell apoptosis [64]. To further explore the mechanism of apoptosis
TE
D
induced by the complex, the assessment of intracellular autophagy in SGC-7901 was
detected by the specific dye monodansylcadaverine (MDC), and the intensity of
fluorescence reflected the degree of autophagy [65]. The autophagy image is shown in
EP
Fig. 5A, we could find the weak fluorescence in the control (a). After the treatment of
AC
C
SGC-7901 cells with Ir-1-Ir-3 for 24 h, bright green fluorescent cycles were observed,
which suggests that complexes Ir-1-Ir-3 can induce autophagy with the formation of
autophagic vacuoles. The process of autophagy is always accompanied by some key
protein expression such as LC3, Beclin-1 and p62. The proteins LC3-II and Beclin-1
are directly involved in the formation of autophagosomes, and the content of LC3-II is
directly proportional to the content of autophagic vesicles [66]. As a substrate for
autophagosome formation, the level of protein p62 will decrease when a large amount
12
ACCEPTED MANUSCRIPT
of autophagy occurs. Therefore, Western blotting was used to assess the expression of
individual proteins in SGC-7901 cells. As depicted in Fig. 5B, after SGC-7901 cells
were treated with the complexes, the expression level of p62 protein was significantly
RI
PT
down-regulated, whereas the expression levels of Beclin-1 and LC3-II showed an
increasing trend, and the LC3-II/LC3-I ratio increased significantly. The results of the
data indicate that the complexes can induce autophagy.
SC
It is well known that the relationship between autophagy and the proliferation of
M
AN
U
tumor cells is twofold. On one hand, autophagy can prompt cell proliferation; On the
other hand, autophagy can inhibit the cell proliferation. To understand the relationship
between autophagy and cell proliferation, the viability of SGC-7901 cells was
assessed by the MTT method in the presence of 3-MA or NAC. As shown in Fig. 6, in
TE
D
the presence of NAC (Ir-1-Ir-3 + NAC, yellow line) and 3-MA (Ir-1-Ir-3 + 3MA,
purple line), NAC induce an increase of the cell viability compared with Ir-1-Ir-3
(blue line), but 3-MA induces more cell death. All of the above data indicate that
EP
autophagy prompts cell proliferation, whereas high levels of reactive oxygen species
AC
C
(ROS) accelerate cell death.
2.7. Determination of intracellular Ca2+ levels
It is currently accepted that the transduction of intracellular related signals and the
implementation of the pre-apoptotic phase are inseparable from the involvement of
Ca2+ [67]. High levels of reactive oxygen species (ROS) will open the mitochondrial
permeability transition pores and will increase calcium uptake in the mitochondria.
13
ACCEPTED MANUSCRIPT
Once Ca2+ overload occurs in the mitochondria, cytochrome c is released to activate
caspase, and finally apoptosis will occur [68]. Therefore, we evaluate changes in
intracellular Ca2+ levels after the treatment of SGC-7901 cells with the complexes
RI
PT
using the specific fluorescent probe Fluo-3AM. As shown in Fig. 7A, the green
fluorescence of the experimental group (c for Ir-1, d for Ir-2 and e for Ir-3) was
significantly stronger than that of the control group (a). However, in the absence of
SC
Ca2+ free medium (b), the green fluorescence is similar to the control. Next, we
M
AN
U
further quantified the fluorescence intensity. It is apparent from Fig. 7B that the
fluorescence intensity of the control group is lower than that of the complexes-treated
groups, whereas in the absence Ca2+ free medium, the fluorescence intensity is slight
high compared with the control. The data confirmed that the complexes can
TE
D
significantly increase the intracellular Ca2+ level.
2.8. Release of cytochrome c
EP
As the main electron carrier in the mitochondrial respiratory chain, cytochrome c
AC
C
is a key factor in cell signal transduction [69]. Cytochrome c, a key factor in the
process of apoptosis, is released into the cytoplasm and can bind to apoptosis-related
factors and activate caspase-9, thereby triggering apoptosis [70]. It can be seen from
Fig. 8A that the green fluorescence after the treatment of SGC-7901 cells with the
complexes (Ir-1-Ir-3) was significantly stronger than that of the control group. The
results indicate that cytochrome c can be released from mitochondria after the
treatment of SGC-7901 cells with the complexes. In addition, the integrated
14
ACCEPTED MANUSCRIPT
fluorescent intensity/cell was determined by Multi Wavelength Cell Scoring module.
See from Fig. 8B, the green fluorescence increases by 4.37 times for Ir-1, 3.79 times
for Ir-2 and 4.58 times for Ir-3 than that in the control. The experimental results
RI
PT
demonstrate that the complexes can induce a release of cytochrome c.
2.9. Cell invasion assays
SC
One of the main reasons for the lack of effective methods for cancer treatment is
M
AN
U
that tumor cells are easy to metastasize and spread [71]. Therefore, it is essential to
evaluate the effects of the three complexes on inhibiting the metastatic spread of
SGC-7901 cells. Invasion assay results are shown in Fig. 9A and B, and we can
observe that these complexes have outstanding inhibitory effects on the invasion of
TE
D
SGC-7901 cells. Additionally, the percentage of inhibiting the cell invasion is 51.5%
for Ir-1, 49.3% for Ir-2 and 74.0% for Ir-3 after SGC-7901 cells were exposed to 2.0
µM of the complexes, respectively. It is interesting to note that all complexes exhibit
EP
excellent concentration-dependent manner for cell invasion inhibition. All the above
AC
C
data results further reveal that the obtained complexes are effective on inhibiting cell
migration.
2.10. Cell cycle analysis
The current research reveals that anticancer drugs show different degrees and
properties for the tumor cell cycle [72,73]. Through the evaluation of the degree of
cell cycle damage of SGC-7901 cells, a deep understanding of the mechanism of
15
ACCEPTED MANUSCRIPT
apoptosis induced by complexes. To investigate the effects of the complexes on the
cell cycle, flow cytometry analysis of SGC-7901 cells treated with dye iodized
pyridine was used. As shown in Fig.10, flow cytometry results showed that deal with
RI
PT
complexes for 24 h caused distinguished accumulation of cells in G0/G1 phase.
Compared with the control (43.95%, Fig. 10a), the percentage of SGC-7901 cells in
G0/G1 phase increased by 50.87% for Ir-1 (b), 48.71% for Ir-2 (c) and 54.24% for
SC
Ir-3 (d) after treated with complexes for 24 h. The data clearly display that these
2.11. Tubulin polymerization assay
M
AN
U
complexes cause the cell cycle to be arrested at the G0/G1 phase.
In large number of studies on the morphology of cytoskeleton, it is found that
TE
D
changes in cytoskeletal structure are essential for the morphological changes of
apoptotic cells [74]. Microtubules are not only the key in maintaining the
morphological structure of cells, but also essential for the transport of substances and
EP
the transduction of signals [75]. Therefore, we labeled microtubules and cytoskeleton
AC
C
by immunofluorescent methods to further explore the mechanism of inhibition of
SGC-7901 cell growth by complexes. As shown in Fig. 11, there was no change in the
morphology of the microtubules in the control cells (a), and a perfect fusiform shape
was exhibited. However, after treatment with the complexes Ir-1 (b), Ir-2 (c) and Ir-3
(d) for 10 h, the cell microtubule structure exhibited a circular shape. The results
confirmed that the complexes inhibited the microtubule polymerization and caused a
significant change in the cytoskeleton morphology, eventually leading to cell
16
ACCEPTED MANUSCRIPT
morphology collapse.
2.12. Assays of Bcl-2 family proteins by western blot
RI
PT
To further clarify the apoptotic mechanism induced by the complexes, we
examined the expression of related protein molecules by Western Blot assay [76,77].
The early key apoptotic factor caspase-3 mediates the entire apoptotic event of cells
SC
after being activated by substrate-cleaved PARP [78]. The P-p53 tumor-associated
M
AN
U
gene, a cell-mediated signal transduction pathway plays an important role in
regulating cell activities. As we all known, Bcl-2 and Bcl-xl inhibits apoptosis,
whereas Bax, Bid and Bak stimulate apoptosis. As shown in Fig. 12, the results
demonstrated that treatment of SGC-7901 cells with Ir-1-Ir-3 led to activate caspase
TE
D
3 and cleavage of PARP, which further confirms that the complexes can induce
apoptosis in SGC-7901 cells. The treatment of SGC-7901 cells induces a decrease in
the expression of phosphorylation of p53 (P-p53) and the antiapoptotic proteins Bcl-2
EP
and Bcl-xL. Besides, the expression levels of Bax, Bid and Bak were up-regulated.
AC
C
Although complex 3 exhibits the highest anticancer activity against SGC-7901 cells
among the three complexes, owing to different proteins with different structures,
therefore, different complexes cause different up-regulation or down-regulation
toward Bak or Bcl-xL. These results indicate that the complexes induce apoptosis
through regulating the expression of Bcl-2 family proteins.
2.13. In vivo antitumor effect
17
ACCEPTED MANUSCRIPT
The results of a series of in vitro experiments confirmed that the complexes
showed excellent inhibitory effects on the proliferation against the tested tumor cells.
The LD50 value measured by the 10-day acute toxicity test for Ir-3 is 8.4 mg/Kg. Next,
RI
PT
we implanted SGC-7901 cells into nude mice and grew corresponding transplanted
tumors to further evaluate the inhibitory effect of Ir-3 in vivo. The corresponding
tumor volume and weight were measured and recorded in the control group, cisplatin
SC
(positive group) and nude mice treated with 0.75 mg/kg and 1.50 mg/kg Ir-3 per day.
M
AN
U
As can be clearly seen from the data of Fig. 13A-D, after 7 days of treatment with
Ir-3, the tumor growth inhibition effect was significantly higher than that of the
control group. The inhibiting percentages of tumor growth induced by cisplatin, 0.75
mg/kg and 1.50 mg/kg are 30.71%, 37.97% and 43.93%, respectively (Fig. 13D).
TE
D
Complex Ir-3 displays obvious antitumor activity, and the tumor inhibition rate is
higher than cisplatin under identical condition. The results indicate that complex Ir-3
reveals a concentration-dependent manner to inhibit the tumor growth.
EP
Through the pathological analysis of tumors and tissues in Fig. 14, the following
AC
C
results can be obtained: in the blank control group, the lungs of the nude mice were
congested with ablation of the alveolar septum, emphysema and local alveolar plaque
hemorrhage. In the positive group, there were also symptoms such as pulmonary
congestion and emphysema, but there were also lace-like changes in the tracheal wall
and scattered small metastases. The lungs of nude mice treated with the complexes
showed extensive alveolar hemorrhage except for the same condition as the positive
group. In addition, there was no significant difference between the hearts of nude
18
ACCEPTED MANUSCRIPT
mice in each group, which showed local myocardial eosinophilic changes and edema
fractures. Similarly, we also observed that the liver tissue of each group showed
visible focal cancer metastasis, small piece of hepatocyte necrosis and small piece of
RI
PT
hemorrhage. The glomeruli of the three groups of nude mice showed congestion,
tubular edema and interstitial siltation. There was no difference in brain tissue of each
group of nude mice showing mild congestion and edema. At the same time, it can be
SC
observed that the tumor capsule is intact in the blank group, the growth is vigorous,
M
AN
U
the division phase is clearly visible, and the large-scale necrosis in the tumor. The
tumor performance of the positive group was basically the same as that of the blank
control, but the range of tumor necrosis was significantly reduced. The results after
treatment with the complexes were not significantly different from those of the
TE
D
positive groups. Therefore, complex Ir-3 may become a potent anti-tumor drug
candidate in the future.
EP
3. Conclusions
AC
C
In the current study, three new compounds [Ir(ppy)2(NDPPZ)](PF6) (Ir-1),
[Ir(bzq)2(NDPPZ)](PF6) (Ir-2) and [Ir(piq)2(NDPPZ)](PF6) (Ir-3) were synthesized,
characterized and evaluated for biological activity. The experimental data showed that
the three complexes have the most prominent inhibitory effect on SGC-7901 cell
proliferation. In vitro studies have shown that complexes can enhance endogenous
ROS and calcium levels, thereby mitigating mitochondrial mode potential and
inducing apoptosis in SGC-7901 cells. These complexes are excellent in inhibiting the
19
ACCEPTED MANUSCRIPT
migration of tumor cells and inhibiting their growth at the G0/G1 phase. At the same
time, the DNA damage of the cells is further explained by the results of the comet
assay and the molecular expression level of the cleavage PARP. In addition, the
RI
PT
complexes can also activate caspase-3 by promoting the release of cytochrome c in
mitochondria, thereby inducing the regulation of Bcl-2 family protein expression.
Finally, the in vivo activity evaluation results showed that the complex Ir-3 has a
SC
significant inhibitory effect on tumor growth. In summary, the results of the above
M
AN
U
data indicate that the complexes induce apoptosis in SGC-7901 cells through the
following three pathways (Fig. 15): (I) The complexes can significantly increase the
level of endogenous reactive oxygen species (ROS) in the cells, and the excessive
accumulation of ROS induces the production of autophagy, resulting in apoptosis; (II)
TE
D
Excessive reactive oxygen species cause Ca2+ loading after opening of the
mitochondrial membrane permeability transition pore. At the same time,
mitochondrial function is further destroyed, causing a large release of cytochrome c,
EP
leading to apoptosis; (III) The complexes damage cellular DNA by altering the
AC
C
expression of PARP protein, and block cell division in G0/G1 phase, promoting
apoptosis. Hence, this work will be beneficial to the future anti-tumor mechanism and
design of new Ir(III) complexes.
4. Experimental
4.1 Materials and methods
2-phenylpyridine, benzo[h]quinoline, 1-phenylisoquinoline, 1,10-phenanthroline,
20
ACCEPTED MANUSCRIPT
4-nitrobenzene-1,2-diamine were purchased from Sigma-Aldrich and used without
further purification unless otherwise noted. Ultrapure MilliQ water was used in all
experiments. FBS and RPMI 1640 were purchased from Gibco company. Fluorescent
RI
PT
dye kits and related consumables are sourced from Beyotime Biotechnology. The
cancer SGC-7901, A549, HeLa, Eca-109, HepG2, BEL-7402 and normal NIH3T3 cell
line were purchased from the American Type Culture Collection. IrCl3.3H2O was
SC
purchased from the Kunming Institution of Precious Metals.
M
AN
U
Microanalysis (C, H, and N) was carried out with a PerkinElmer 240Q elemental
analyzer. Electrospray ionization mass spectra (ESI-MS) were recorded on a LCQ
system (Finnigan MAT, USA) using acetonitrile as the mobile phase. The spray
voltage, tube lens offset, capillary voltage and capillary temperature were set at 4.50
TE
D
kV, 30.00 V, 23.00 V and 200 ℃, respectively, and the quoted m/z values are for the
major peaks in the isotope distribution. 1H NMR and 13C NMR spectra were recorded
on a Varian-500 spectrometer with DMSO-d6 as a solvent and tetramethylsilane
AC
C
EP
(TMS) as an internal standard at 500 MHz at room temperature.
4.2. Synthesis of complexes
4.2.1. Synthesis of complex [Ir(ppy)2(NDPPZ)]PF6 (Ir-1)
A mixture of cis-[Ir(ppy)2Cl]2 (0.28 g, 0.25 mmol) [79] and NDPPZ [80] (0.164
g, 0.50 mmol) were dissolved in dichloromethane and methanol mixed solution
(VCH2Cl2: VCH3OH = 2:1), then the mixture was refluxed under argon for 6 h to appear a
clear red brown solution. And then the solution was cooled to room temperature, a
21
ACCEPTED MANUSCRIPT
large amount of yellow precipitated product appeared after the dropwise addition of
ammonium hexafluorophosphate and stirring for 2 h. The crude complex was purified
by column chromatography on neutral alumina with dichloromethane-acetone (1:3,
RI
PT
v/v) as eluent. After removing the solvent under reduced pressure and further drying,
a bright yellow pure complex was obtained. Yield: 82%. Anal. Calc for
C40H25N7O2IrPF6: C, 49.38; H, 2.59; N, 10.08%. Found: C, 49.25; H, 2.69 N, 10.02%.
H NMR (DMSO-d6, 500 MHz) (Fig. S2a, supporting information): δ 9.72 (dd, 2H, J
SC
1
M
AN
U
= 5.5, J = 4.5 Hz), 9.23 (d, 1H, J = 2.5 Hz), 8.77 (d, 1H, J = 7.0 Hz), 8.69 (d, 1H, J =
9.5 Hz), 8.33 (d, 2H, J = 5.0 Hz), 8.28 (d, 2H, J = 8.0 Hz), 8.72-8.18 (m, 2H), 7.96 (d,
2H, J = 7.5 Hz), 7.91 (t, 2H, J = 8.0 Hz), 7.67 (t, 2H, J = 6.0 Hz), 7.09-7.04 (m, 4H),
6.97 (t, 2H, J = 7.5 Hz), 6.28 (d, 2H, J = 7.5 Hz). 13C NMR (DMSO-d6, 125 MHz)
TE
D
(Fig. S3a, supporting information): 166.85, 152.56, 152.41, 150.42, 150.14, 149.65,
149.51, 148.85, 144.08, 143.78, 142.66, 142.19, 140.58, 138.97, 135.69, 135.48,
131.56, 131.25, 130.47, 130.43, 128.94, 128.88, 125.57, 125.35, 125.22, 123.92,
EP
122.68, 120.12. ESI-MS (CH3CN): 828.0 ([M ‒ PF6]+). HRMS (CH3CN): 828.1678
AC
C
([M ‒ PF6]+) (Fig. S4a, supporting information).
4.2.2. Synthesis of complex [Ir(bzq)2(NDPPZ)]PF6 (Ir-2)
Obtaining this complex by a method similar to that described for the synthesis of
complex Ir-1, with [Ir(bzq)2Cl]2∙2H2O [79] in place of [Ir(ppy)2Cl]2∙2H2O. Yield:
76%. Anal. Calc for C44H25N7O2IrPF6: C, 51.77; H, 2.47; N, 9.60%. Found: C, 51.89;
H, 2.35; N, 9.71%.
1
H NMR (DMSO-d6, 500 MHz) (Fig. S2b, supporting
22
ACCEPTED MANUSCRIPT
information): δ 9.70 (d, 2H, J = 8.0 Hz), 9.23 (d, 1H, J = 2.5 Hz), 8.78 (d, 1H, J = 6.5
Hz), 8.68 (d, 1H, J = 9.0 Hz), 8.53 (d, 2H, J = 8.0 Hz), 8.30 (t, 2H, J = 5.0 Hz),
8.14-8.07 (m, 4H), 7.98 (d, 2H, J = 9.0 Hz), 7.87 (d, 2H, J = 9.0 Hz), 7.58 (d, 2H, J =
RI
PT
8.0 Hz), 7.52-7.49 (m, 2H), 7.22 (t, 2H, J = 7.5 Hz), 6.31 (d, 2H, J = 7.0 Hz). 13C
NMR (DMSO-d6, 125 MHz) (Fig. S3b, supporting information): 156.32, 153.09,
152.94, 150.85, 150.57, 149.38, 148.85, 146.29, 143.77, 142.67, 142.21, 140.57,
SC
140.35, 137.81, 135.71, 135.48, 133.86, 131.55, 130.43, 130.38, 129.85, 129.59,
M
AN
U
128.93, 128.87, 128.62, 126.81, 125.57, 125.35, 124.34, 122.81, 120.66. ESI-MS
(CH3CN): 876.0 ([M ‒ PF6]+). HRMS (CH3CN): 876.1687 ([M ‒ PF6]+) (Fig. S4b,
supporting information).
TE
D
4.2.3. Synthesis of complex [Ir(piq)2(NDPPZ)]PF6 (Ir-3)
The complex is obtained by the same method as described in the synthesis of
complex Ir-1, with [Ir(piq)2Cl]2∙2H2O [79] in place of [Ir(ppy)2Cl]2∙2H2O. Yield: 78%.
EP
Anal. Calc for C48H29N7O2IrPF6: C, 53.73; H, 2.72; N, 9.13%. Found: C, 53.82; H,
AC
C
2.79; N, 9.01%. 1H NMR (DMSO-d6, 500 MHz) (Fig. S2c, supporting information): δ
9.73 (d, 2H, J = 6.5 Hz), 9.20 (d, 1H, J = 2.5 Hz), 9.02 (d, 2H, J = 8.5 Hz), 8.76 (d,
1H, J = 6.5 Hz), 8.67 (d, 1H, J = 9.5 Hz), 8.40 (d, 2H, J = 8.0 Hz), 8.20-8.15 (m, 4H),
8.05 (t, 2H, J = 6.5 Hz), 7.88 (t, 4H, J = 7.5 Hz), 7.56 (t, 2H, J = 6.5 Hz), 7.50 (dd, 2H,
J = 6.0, J = 6.5 Hz), 7.18 (t, 2H, J = 7.5 Hz), 6.98 (t, 2H, J = 7.0 Hz), 6.28 (d, 2H, J =
7.5 Hz). 13C NMR (DMSO-d6, 125 MHz) (Fig. S3c, supporting information): 167.88,
152.87, 152.52, 152.36, 150.24, 149.95, 148.83, 145.43, 143.77, 142.65, 142.19,
23
ACCEPTED MANUSCRIPT
141.21, 140.57, 136.67, 135.76, 135.54, 132.22, 131.68, 131.55, 130.79, 130.73,
130.55, 130.49, 129.51, 128.97, 128.92, 127.76, 126.53, 125.63, 125.54, 125.34,
122.65, 122.21. ESI-MS (CH3CN): 928.07 ([M ‒ PF6]+). HRMS (CH3CN): 928.2034
RI
PT
([M ‒ PF6]+) (Fig. S4c, supporting information).
4.3. Cytotoxicity assay in vitro
SC
Cancer cells suspension were seeded in 96-well microarray plates (1 × 104 cells
M
AN
U
per well) and incubated overnight at 37 °C in a 5% CO2 incubator. All tested
complexes were dissolved in DMSO, and the final concentration of the complexes
ranged from 3.125-100 µM. The 96-well plates were incubated for 48 hours at 37 °C
in a 5% CO2 incubator. When the incubation is complete and the culture in the wells
TE
D
is removed, add 90 µL of the culture medium and 10 µL of the MTT dye solution (20
µL, 5 mg/mL) to each well. After 4 h, buffer (100 µL) containing dimethylformamide
(50%) and sodium dodecyl sulfate (20%) was added to solubilize the MTT formazan.
EP
The microplate reader was used to measure the absorbance of each well at a
AC
C
wavelength of 490 nm. The IC50 values were determined by plotting the percentage of
viable cells versus concentration on a logarithmic graph and reading off the
concentration at which 50% of cells remain viable relative to the control. Each set of
experiments must be repeated at least three times to calculate the average.
4.4. Localization assay of the complex in the mitochondria
SGC-7901 cells were placed in 12-well microassay culture plates (1 × 105 cells
24
ACCEPTED MANUSCRIPT
per well) and grown overnight at 37 oC in a 5% CO2 incubator. The compounds of the
corresponding concentration were added to the wells at 37 oC in a 5% CO2 incubator
for 9 h and the cells were stained with MitoTracker Deep Red FM (150 nM) at 37 oC
RI
PT
for 30 min. Upon completion of the incubation, the wells were washed three times
with ice-cold PBS. After discarding the culture medium, the cells were imaged under
M
AN
U
4.5. Mitochondrial membrane potential (MMP) assay
SC
ImageXpress Micro XLS system (MD company, US).
SGC-7901 cells in 12-well plates were treated with complexes Ir-1-Ir-3 for 24 h,
and then washed three times with 500 µL of cold PBS per well. The cancer cells are
separated
by
using
trypsin-EDTA
solution.
1
µg/mL
of
JC-1
TE
D
(5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide) was used to dye
the cells at room temperature for 20 minutes in the dark. The cells were immediately
centrifuged to remove the supernatant. Then the cell pellets were suspended in PBS
EP
and imaged under ImageXpress Micro XLS system (MD company, US), and the ratio
AC
C
of red/green fluorescence intensity was determined by flow cytometry.
4.6. Reactive oxygen species (ROS) detection
4.6.1. Intracellular ROS levels detection
The specific fluorescent probe 2,7-dichloro-dihydrofluorescein diacetate
(DCFH-DA) was used to investigate the changes of intracellular ROS levels.
SGC-7901 cells were placed in a 12-well plate at a density of 1.5 × 105 per well. After
25
ACCEPTED MANUSCRIPT
24 h, the medium in the wells was replaced with medium containing the
corresponding concentration of Ir-1-Ir-3 and incubation for 24 h. Finally, the cells
pellets were suspended in PBS and imaged under ImageXpress Micro XLS system
4.6.2. Intracellular superoxide anion detection
SC
cytometry.
RI
PT
(MD company, US), and the DCF fluorescence intensity was calculated by flow
M
AN
U
The level of superoxide anion in cells is detected by DHE (Dihydroethidium) as
a fluorescent probe. SGC-7901 cells were placed in a 12-well plate at a density of 1.5
× 105 per well. After 24 h, the medium in the wells was replaced with medium
containing the corresponding concentration of Ir-1-Ir-3 and incubation for 24 h. The
TE
D
cells were then treated in the dark for 30 min with PBS solution containing 10 µM
DHE dye. Finally, the cells are imaged and the respective fluorescence intensities are
EP
calculated by using the ImageXpress Micro XLS system (MD company, US).
AC
C
4.6.3. Intracellular nitric oxide level detection
DAF-FM DA (3-amino-4-aminomethyl-2',7'-difluorescein diacetate was used as
fluorescence probe to determine the intracellular nitric oxide levels. SGC-7901 cells
with a density of 1.5 × 105 per well were seeded in 12 wells and incubated overnight
in an incubator. Next, replace the medium in the 12-well plate with the medium
containing the corresponding concentration of Ir-1-Ir-3 and continue to be incubated
for 24 h. Finally, the cells are imaged and the respective fluorescence intensities are
26
ACCEPTED MANUSCRIPT
calculated by using the ImageXpress Micro XLS system (MD company, US).
4.7. Apoptosis assay by AO/EB staining methods
RI
PT
Acridine orange and ethidium bromide (AO/EB) staining was used to detect the
apoptotic morphology of SGC-7901 cells. SGC-7901 cells were placed in a 12-well
plate at a density of 1.5 × 105 per well. After 24 h, the medium in the wells was
SC
replaced with medium containing the corresponding concentration of Ir-1-Ir-3 and
M
AN
U
incubation for 24 h. Then the cells in the wells were washed with cold PBS, the cells
were dyed by acridine range (AO) and ethidium bromide (EB) (AO: 100 µg/mL, EB:
100 µg/mL) for 10 min in the dark. The cells were imaged by ImageXpress Micro
TE
D
XLS system (MD company, US).
4.8. Apoptosis assay by flow cytometry
SGC-7901 cells in the 6-well plate were treated with Ir-1-Ir-3 and collected by
EP
using a trypsin-EDTA solution. After the supernatant was removed and the cells were
AC
C
washed and stained with PBS solution containing 500 mg/mL pyridine iodide (PI) and
1 mg/mL annexin V-FITC for 20 min in a dark. The fluorescence was measured at 530
nm using 488 nm excitation with an FACS Calibur flow cytometer (Beckman
Dickinson & Co., Franklin Lakes, NJ). A minimum of 10,000 cells were analyzed per
sample.
4.9. DNA damage assay
27
ACCEPTED MANUSCRIPT
DNA damage was investigated by means of comet assay. SGC-7901 cells in
culture medium were incubated with complexes Ir-1, Ir-2 and Ir-3 at 37 °C for 24 h.
The cells were harvested by a trypsinization process at 24 h. A total of 100 µL of 0.5%
RI
PT
normal agarose in PBS was dropped gently onto a fully frosted microslide, covered
immediately with a coverslip, and then placed at 4 °C for 10 min. The coverslip was
removed after the gel has been fixed. 50 µL of the cell suspension (200 cells/µL) was
SC
mixed with 50 µL of 1% low melting agarose preserved at 37 °C. A total of 100 µL of
M
AN
U
this mixture was applied quickly on top of the gel, coated over the microslide, covered
immediately with a coverslip, and then placed at 4 °C for 10 min. The coverslip was
again removed after the gel has been fixed. A third coating of 50 µL of 0.5% low
melting agarose was placed on the gel and allowed to place at 4 °C for 15 min. After
TE
D
solidification of the agarose, the coverslips were removed, and the slides were
immersed in an ice-cold lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 90
mM sodium sarcosinate, NaOH, pH 10, 1% Triton X-100 and 10% DMSO) and
EP
placed in a refrigerator at 4 °C for 2 h. All of the above operations were performed
AC
C
under low lighting conditions to avoid additional DNA damage. After the removal of
the lysis solution, the slides were placed horizontally in an electrophoresis chamber.
The reservoirs were filled with an electrophoresis buffer (300 mM NaOH, 1.2 mM
EDTA) until the slides were just immersed in the buffer solution, and the DNA was
allowed to unwind for 30 min in the electrophoresis solution. Then the electrophoresis
was carried out at 25 V and 300 mA for 20 min. After electrophoresis, the slides were
removed, and washed thrice in a neutralization buffer (400 mM Tris, HCl, pH 7.5).
28
ACCEPTED MANUSCRIPT
Nuclear DNA was stained with 20 µL of EtBr (20 µg/mL) in the dark for 20 min. The
slides were washed in chilled distilled water for 10 min to neutralize the excess alkali,
air-dried and scored for comets by fluorescence microscopy. A total of 10 comets on
4.10. Autophagy induced by the complexes
RI
PT
each gel were scored.
SC
SGC-7901 cells were placed into a 12-well plate and incubate overnight. Replace
M
AN
U
the medium in the plate with a new medium containing the corresponding
concentration of Ir-1-Ir-3 and incubate for 24 h at 37 °C in a 5% CO2 incubator. After
the incubation was completed, the medium in the wells was removed and the cells
were washed three times with cold PBS. The plated cells were then stained with MDC
TE
D
(monodansylcadaverine) solution (50 mM) for 20 minutes at 37 °C. Finally, the cells
were imaged using the ImageXpress Micro XLS system (MD, US).
EP
4.11. Detection of Ca2+ levels
AC
C
SGC-7901 cells in 12-well plates were incubated with the fluorescent probe
Fluo-3AM for 24 h after treatment with Ir-1-Ir-3 to detect changes in intracellular
Ca2+ levels. Fluo-3MA is easily cleaved by esterase to form Fluo-3, which will
produce strong fluorescence when combined with Ca2+. Fluorescent dyes in the wells
that are not bound to the cells are washed three times with cold PBS. Finally,
SGC-7901 cells were imaged by ImageXpress Micro XLS system (MD, US) and the
fluorescence intensity of each well was calculated.
29
ACCEPTED MANUSCRIPT
4.12. Release of cytochrome c
SGC-7901 cells were seeded in a 12-well plate and incubated overnight. Then
RI
PT
cells were treated with different concentrations of the complexes for 24 h.
Subsequently, the cells were fixed with ice-cold immunol staining fix solution for 30
min at room temperature. After blocking cells with immunol staining blocking buffer
SC
for 1 h, the cells were treated with the primary antibody against cytochrome c (1:50
M
AN
U
dilution) overnight at 4 oC. Next, the plate was washed with immunol staining wash
buffer three times and probed with Alexa Fluor 488-Labeled Goat Anti-Mouse IgG
(1:500 dilution) in the dark for 1 h at room temperature. Finally, the cells were
washed with immunol staining wash buffer three times and the cell nuclei were
TE
D
stained with DAPI. The images were obtained using ImageXpress Micro XLS system,
and Multi Wavelength Cell Scoring module was used to analyze the data. The
integrated intensity/cell which represents the fluorescence intensity of each cell was
EP
used to measure the release of cyto-c. The fluorescence intensity of each cell was
AC
C
calculated as the total fluorescence intensity divided by the number of cells.
4.13. Matrigel invasion assay
The BD Matrigel invasion chamber was used to investigate the ability of
complexes to inhibit tumor cell invasion. After the matrix glue was placed in the
invading chamber and solidified at 37 oC, the serum-free medium containing
SGC-7901 cells and the corresponding concentration of Ir-1-Ir-3 was placed in the
30
ACCEPTED MANUSCRIPT
upper chamber. After incubation for 24 h, the cells were fixed with 4%
paraformaldehyde and stained with 0.1% crystal violet. After removing the uninvaded
cells in the upper chamber, the invading cells were photographed by optical
RI
PT
microscope and the number was recorded. The experimental data were taken from the
average of three independent experiments.
SC
4.14. Cell cycle arrest studies
M
AN
U
SGC-7901 cells with a density of 5 × 105 per well were plated in 6-well plates
(Costar, Corning Corp, New York) and incubated until tightly attached. After the
incubation, RPMI 1640 medium containing 10% FBS in the wells was removed and
replaced with a new medium containing the corresponding concentration of Ir-1-Ir-3.
TE
D
After incubating for 24 h, the cell strain was obtained by trypsinization, and the cells
were washed with cold PBS. After being fixed with 75% alcohol, 20 µL of RNAse
(0.2 mg / mL) and 20 µL of propidium iodide (0.02 mg / mL) were suspended from
AC
C
EP
the cell pellet and incubated for 30 min at room temperature.
4.15. Effects of the complexes on microtubule network
SGC-7901 cells in 12-well plates were treated with Ir-1-Ir-3 for 24 h, and the
cells were fixed with immunostaining fixative solution for 30 min. After removing the
fixative solution, the cells were washed three times with cold PBS and incubated with
the immunostaining blocking solution for 1 h at room temperature. After incubation
and washing, SGC-7901 cells were incubated with anti-rabbit monoclonal
31
ACCEPTED MANUSCRIPT
anti-α-tubulin antibody (1:100 dilution) overnight at 4 °C. After washing the cells
three times with PBST, the cells were incubated with anti-rabbit FITC-conjugated IgG
antibody (1:500 dilution) for 1 h, and then the nuclei were stained with Hoechst
RI
PT
33258. The cells was imaged and analyzed by ImageXpress Micro XLS system (MD,
US).
SC
4.16. Western blotting assay
M
AN
U
When the SGC-7901 cells in the 6-well plate were attached and grown at a high
density, the medium containing the corresponding concentration of Ir-1-Ir-3 was
replaced with the original medium and incubation was continued for 24 h. Rapidly
obtain cell protein suspension by cell lysis buffer on ice bath, then perform low
TE
D
temperature high speed centrifugation for 15 min. After obtaining the supernatant, the
concentration of the corresponding protein was determined by BCA (bicinchoninic
acid) working solution. Add these processed proteins to the same amount of protein
EP
for sodium dodecyl sulfate-polyacrylamide gel electrophoresis using a micro syringe.
AC
C
The separation gel was transferred to a PVDF membrane, which was then blocked
with TBST buffer containing 5% skim milk powder for 4 h. The PVDF membrane
was washed four times with TBST buffer after overnight at 4 °C with the specific
antibody selected for specificity. The labeled secondary antibody was incubated with
the PDVF membrane bound to the primary antibody for 1 h at room temperature. The
blots were visualized using Amersham ECL Plus western blotting detection reagents
according to the manufacturer's instructions. To assess the presence of comparable
32
ACCEPTED MANUSCRIPT
amounts of protein in each lane, the membranes were stripped finally to detect the
β-actin.
RI
PT
4.17. Acute toxicity assay
Healthy Kunming (KM) mice (6-8 weeks old) weighing 18-22 g were housed in
rooms in which the temperature was approximately 22-24 oC, with a relative humidity
SC
of 40-50%, and in a 12 h light-dark cycle was used. Sterile food and water were
M
AN
U
provided according to institutional guidelines. All animals were provided by the
Laboratory Animal Center of Guangdong Pharmaceutical University. All animal
procedures were approved by the Animal Ethical Committee of Guangdong
Pharmaceutical University. Prior to each experiment, the mice were fasted overnight
TE
D
and allowed free access to water. Various doses of Ir-3 ranging from 1 to 10 mg/kg
dissolved in a 0.5% DMSO were administered intraperitoneally to different groups of
healthy KM mice; each group contained 6 mice. After the compound had been
EP
administered, the mice were observed continuously for the first 2 h for any gross
AC
C
behavioral changes or death, intermittently for the next 24 h and thereafter for 10 days
to determine the onset of any delayed effects. All animals were sacrificed at the 10
days after drug administration and checked macroscopically for possible damage to
the heart, liver, and kidneys. Mice that died immediately following drug
administration were also examined for any possible organ damage.
4.18. Antitumor activity assay
33
ACCEPTED MANUSCRIPT
Mice with human tumor xenografts (HOS) were provided by the Laboratory
Animal Center of Sun Yat-Sen University. Different doses of 0.75 and 1.50 mg/kg of
Ir-3 were injected intraperitoneally into mice of different group (each group contained
RI
PT
6 mice) once a day for seven consecutive days beginning 24 h after inoculation. This
dose was the maximum tolerated dose based on our preliminary studies. Cisplatin (2
mg/kg) was used as a positive control. Control mice were injected with the vehicle.
SC
Compounds were administered by exact body weight, with the injection volume of
M
AN
U
200 µL. The weights of the animals were recorded every day. All animals were
sacrificed on the eighth days after tumor inoculation, and the tumors were excised and
weighed. The inhibition rate was calculated as follow:
[(C - T)/C] × 100%
TE
D
T is the average tumor weight of the treated group and C is the average tumor weight
of the negative control group [81].
EP
4.19. Ir-3 on histopathological study
AC
C
In order to evaluate the degree of damage of Ir-3 to tumor tissues and other
normal organs in nude mice, when the animals were sacrificed, the tumor, brain, heart,
lung, liver and kidney of nude mice were taken and fixed with 4% paraformaldehyde
for 24 hours. After fixation, each tissue was embedded in paraffin and cut into 5 µm
sections. The sections were then stained with hematoxylin and eosin staining kits
purchased from the Beyotime Biotechnology Institute.
34
ACCEPTED MANUSCRIPT
4.20. Data analysis
All data was expressed as means ± SD. Statistical significance was evaluated by
a t-test. Differences were considered to be significant when a *P value was less than
RI
PT
0.05.
Acknowledgements
the
Natural
Science
Foundation
of
M
AN
U
(21877018),
SC
This work was supported by the National Natural Science Foundation of China
Guangdong
Province
(2016A030313728) and Special Funds of Key Disciplines Construction from
AC
C
EP
TE
D
Guangdong and Zhongshan cooperating.
35
RI
PT
ACCEPTED MANUSCRIPT
References
[1] G. Rohini, J. Haribabu, K.N. Aneesrahman, N.S.P. Bhuvanesh, K. Ramaiah, R.
SC
Karvembu, A. Sreekanth, Polyhedron. 152 (2018) 147-154.
M
AN
U
[2] A. Kumar, A. Kumar, R.K. Gupta, R.P. Paitandi, K.B. Singh, S.K. Trigun, M.S.
Hundal, D.S. Pandey, J. Organometallic. Chem. 801 (2016) 68-79.
[3] A.Z. El-Sonbati, A.F. Shoair, A.A. El-Bindary, M.A. Diab, A.S. Mohamed, J. Mol.
Liq. 209 (2015) 635-647.
(2015) 522-531.
TE
D
[4] P.Y. Zhang, H.Y. Huang, Y. Chen, J.Q. Wang, L.N. Ji, H. Chao, Biomaterials. 53
[5] X. Chen, L.L. Sun, Y. Chen, X.L. Cheng, W.J Wu, L.N. J, H. Chao, Biomaterials.
EP
58 (2015) 72-81.
AC
C
[6] Y. Chen, L.P. Qiao, L.N. Ji, H. Chao, Biomaterials. 35 (2014) 2-13.
[7] Q. Du, LH. Guo, M. Tian, X. X. Ge, Y.L. Yang, X.Y. Jian, Z.S. Xu, Z.Z. Tian, Z.
Liu, Organometallics. 17 (2018) 2880-2889
[8] T. Tanaka, M. Kasai, S. Kobayashi, Exp. Cell. Res. 370 (2018)454-460.
[9] G.Y. Li, Q. Lin, L.L. Sun, C.S. Feng, P.Y. Zhang, B.L. Yu, Y. Chen, H. Wang, L.N.
Ji, H. Chao, Biomaterials. 53 (2015) 285-295.
[10] L. He, J.J. Cao, D.Y. Zhang, L. Hao, M.F. Zhang, C.P. Tan, L.N. Ji, Z.W. Mao,
36
ACCEPTED MANUSCRIPT
Sensor Actuat B-Chem, 262 (2018) 313–325.
[11] J.S. Nam, M.G. Kang, J. Kang, S.Y. Park, S.J. Lee, H.T. Kim, J.K. Seo, O.H.
Kwon, M.H. Lim, H.W. Rhee, T.H. Kwon, J. Am. Chem. Soc. 138(2016)
RI
PT
10968-10977.
[12] J.J. Cao, Y. Zheng, X.W. Wu, C.P. Tan, M.H. Chen, N. Wu, L.N. Ji, Z.W. Mao, J.
Med. Chem. 62 (2019) 3311–3322.
SC
[13] P.C.C. James, E.B. Hannah, J.I. Song, A.S. Nichola, P.E.B. Nicolas, I.B.P.J.
M
AN
U
Sadler, R.C. Isolda, J. Med. Chem. 61 (2018) 9246–9255.
[14] H.J. Zhong, W.H. Wang, T.S. Kang, H. Yan, Y.L. Yang, L.P. Xu, Y. Q. Wang, D.L.
Ma, C.H. Leung, J. Med. Chem. 60 (2017) 497–503.
[15] Z.W. Mao, M.Q. Chen, X.S. Tan, J. Liu, W.X. Tang, Inorg. Chem. 34 (1995)
TE
D
2889-2893.
[16] F.X. Wang, M.H. Chen, Y.N. Lin, H. Zhang, C.P. Tan, L.N. Ji, Z.W. Mao, ACS
Appl. Mater. Inter. 9 (2017) 42471–42481.
EP
[17] D.Y. Zhang, Y. Zheng, C.P. Tan, J.H. Sun, W. Zhang, L.N. Ji, Z.W. Mao, ACS
AC
C
Appl. Mater. Inter. 9 (2017) 6761-6771.
[18] W. Zhang, J. Shen, H. Su, G. Mu, J H. Sun, C.P. Tan, L.N. Ji, Z.W. Mao, ACS
Appl. Mater. Inter. 8 (2016) 13332–13340.
[19] M. H. Chen, F. X. Wang, J. J. Cao, C. P. Tan, L. N. Ji, Z. W. Mao, ACS Appl.
Mater. Inter. 9 (2017) 13304-13314.
[20] C. Gossens, I. Tavernelli, U. Rothlisberger, J. Am. Chem. Soc. 130 (2008)
10921-10928.
37
ACCEPTED MANUSCRIPT
[21] F. Wang, J. Xu, A. Habtemariam, J. Bella, P. J. Sadler, J. Am. Chem. Soc. 127
(2014) 17734-17743.
[22] C. Haimei, J.A. Parkinson, P. Simon, R.A. Coxall, R.O. Gould, P.J. Sadler, J. Am.
RI
PT
Chem. Soc. 124 (2002) 3064-3082.
[23] W. Bao, X.W. Liu, Y.L. Lv, G.H. Lu, F. Li, F. Zhang, B. Liu, D. Li, W. Wei, Y. Li,
ACS. Nano. 13 (2019) 260–273.
M
AN
U
Liu, J. Biol. Inorg. Chem. 24 (2019) 151-169.
SC
[24] Q.Y. Yi, W.Y. Zhang, M. He, F. Du, X.Z. Wang, Y.J. Wang, Y.Y. Gu, L. Bai, Y.J.
[25] B. Tang, D. Wan, Y.J. Wang, Q.Y. Yi, B.H. Guo, Y.J. Liu, Eur. J. Med. Chem. 145
(2018) 302-314.
[26] Q.Y. Yi, D. Wan, B. Tang, Y.Y. Wang, W.Y. Zhang, F. Du, M. He, Y.J. Liu, 145
TE
D
(2018) 338-349.
[27] Y.Y. Wang, Q.Y. Yi, W.Y. Zhang, F. Du, M. He, Y.J. Liu, Polyhedron 156 (2018)
320-331.
EP
[28] C. Zhang, S.H. Lai, H.H. Yang, D.G. Xing, C.C. Zeng, B. Tang, D. Wan, Y.J. Liu,
AC
C
RSC Adv. 7 (2017) 17752-17762.
[29] R.L. Guan, Y. Chen, L.L. Zeng, T.W. Rees, C.Z. Jin, J.J. Huang, Z.S. Chen, L.N.
Ji, H. Chao, Chem. Sci. 9 (2018) 5183-5190.
[30] Y.M. Ou, L.L. Zeng, H.Y. Huang, C.Z. jin, Y. Chen, L.N. Ji, H. Chao, Dalton
Trans. 46 (2017) 6734-6744.
[31] Y.L. Yang, L.H. Guo, X.X. Ge, S.P. Shi, Y.T. Gong, Z.S. Xu, X.F. Zheng, Z. Liu,
J. Inorg. Biochem. 191 (2019) 1-7
38
ACCEPTED MANUSCRIPT
[32] H.L. Hao, X.C. Liu, X.X. Ge, Y. Zhao, X. Tian, T. Ren, Y. Wang, C.F. Zhao, Z.
Liu, J. Inorg. Biochem. 192 (2019) 52-61.
[33] C. Pérez-Arnaiz, M. I. Acuña,
N. Busto, I. Echevarría,
M. Martínez-Alonso,
RI
PT
G. Espino, B. García, F. Domínguez, Eur. J. Med. Chem. 157 (2018) 279-293.
[34] C. Zhang, S.H. Lai, C.C. Zeng, B. Tang, D. Wan, J. Biol. Inorg. Chem. 21 (2016)
1047-1060.
SC
[35] X.D. Song, Y. Qian, R. Ben, X. Lu, H.L. Zhu, H. Chao, J. Zhao, J. Med. Chem.
M
AN
U
56 (2013) 6531-6535.
[36] V. Venkatesh, R. Berrocal-Martin, C.J. Wedge, I. Romero-Canelón, C.
Sanchez-Cano, J.I. Song, J.P.C. Coverdale, P.Y. Zhang, G.J. Clarkson, A.
Habtemariam, S.W. Magennis, R.J. Deeth, P.J. Sadler, Chem. Sci. 8 (2017)
TE
D
8271-8278.
[37] T.S. Kang, Z.F. Mao, C.T. Ng, M. Wang, W.H. Wang, C.M. Wang, S.M.Y.
Lee, Y.T. Wang, C.H. Leung, D.L. Ma, J. Med. Chem. 59 (2016) 4026-4031.
F.
Chen, J.
Moat, D.
EP
[38]
McFeely, G.
Clarkson, I.J.
Hands-Portman, J.P.
AC
C
Furner-Pardoe, F. Harrison, C.G. Dowson, P.J. Sadler, J. Med. Chem. 61 (2018)
7330-7344.
[39] J.J. Cao, Y. Zheng, X.W. Wu, C.P. Tan, M.H. Chen, N.Wu, L.N. Ji, Z.W. Mao, J.
Med. Chem. 62 (2019) 3311-3322.
[40] S. Lin, L.H. Lu, T.S. Kang, J.L. Mergny, C.H. Leung, D.L. Ma, Annal. Chem. 88
(2016) 10290-10295.
[41] H. Wang, L. Hu, W. Du, X. Tian, Q. Zhang, Z.J. Hu, L. Luo, H.P. Zhou, J.Y. Wu,
39
ACCEPTED MANUSCRIPT
Y.P. Tian, ACS Biomater. Sci. Eng. 3 (2017) 836-842.
[42] D. Solairaj, P. Rameshthangam, G. Arunachalam, INT. J. Biol. Macromol. 105
(2017) 608-619
RI
PT
[43] S.D. Guggilapu, L. Guntuku, T.S. Reddy, A. Nagarsenkar, D.K. Sigalapalli,
V.G.M. Naidu, S.K. Bhargava, B.N. Babu, Eur. J. Med. Chem. 138 (2017) 83-95.
[44] H.S. Jung, J. Han, J.H. Lee, J.H. Lee, J.M. Choi, H.S. Kweon, J.H. Han, J.H.
SC
Kim, K.M. Byun, J.H. Jung, C. Kang, J.S. Kim, J. Am. Chem. Soc. 137 (2015)
M
AN
U
3017-3023.
[45] A. Mallick, P. More, M.M.K. Syed, S. Basu, Appl. Mater. Inter. 8 (2016)
13218-13231.
[46] J.H. Cui, X. Zhang, G. Huang, Q.J. Zhang, J.Y. Dong, G.G. Sun, Q.Q. Meng, S.S.
TE
D
Li, Mol. Pharmaceut. 16 (2019) 409-421.
[47] M.D. Altıntop, B. Sever, G.A. Çiftçi, T.Z. Gülhan, Z. A. Kaplancıklı, A. Özdemir,
Eur. J. Med. Chem. 155 (2018) 905-924.
EP
[48] W.Y. Zhang, Q.Y. Yi, Y.J. Wang, F. Du, M. He, B. Tang, D. Wan, Y.J. Liu, H.L.
AC
C
Huang, Eur. J. Med. Chem. 151 (2018) 568-584.
[49] M. Zhu, J.B. Wang, J.W. Xie, L.P. Chen, X.Y. Wei, X. Jiang, M. Bao, Y.Y. Qiu, Q.
Chen, W.L. Li, C.X. Jiang, X.O. Zhou, L.P. Jiang, P.H. Qiu, J.Z. Wu, Eur. J. Med.
Chem. 157 (2018) 1395-1405.
[50] D.S. Kalinowski, P.C. Sharpe, P.V. Bernhardt, D.R. Richardson, J. Med. Chem.
50 (2007) 6212-25.
[51] F. Dai, W.J. Yan, X. Fu, Y.L. Zheng, Y.T. Du, X.Z. Bao, Y.F. Kang, X.L. Jin, B.
40
ACCEPTED MANUSCRIPT
Zhou, Eur. J. Med. Chem. 159 (2018) 317-323.
[52] J.J. Chen, Z.D. Luo, Z.N. Zhao, L.N. Xie, W.J. Zheng, T.F. Chen. Data In Brief. 8
(2016) 670-686.
Leung, Chem-An Asian J. 13 (2018) 275-279.
RI
PT
[53] K.J. Wu, H.J. Zhong, G.J. Yang, C. Wu, J.M. Huang, G.D. Li, D.L. Ma, C.H.
[54] C.C. Hsu, J.C. Lien, C.W. Chang, C.H. Chang, S.C. Kuo, T.F. Huang,
SC
Biochemical Pharmacology, 85 (2013) 385-395.
[55] K.J. Wu, H.J. Zhong, G.D. Li, C.F. Liu, H.M.D. Wang, D.L. Ma, C.H. Leung,
M
AN
U
Eur. J. Med. Chem. 143 (2018) 1021-1027.
[56] Q.C. Huang, L. Zhan, H.Y. Cao, J.B. Li, Y.H. Lyu, X. Guo, J. Zhang, L.L. Ji,
T.T. Ren, J.Z. An, B.R. Liu, Y.Z. Nie, J.L. Xing, Autophagy, 2016, 12 (2016)
999-1014.
TE
D
[57] R.P. Nishanth, R.G. Jyotsna, J.J. Schlager, S.M. Hussain, P. Reddanna,
Nanotoxicology, 5 (2011) 502-516.
EP
[58] J.H. Gao, G.L. Liang, B. Zhang, Y. Kuang, X.X. Zhang, B. Xu, J. Am. Chem.
Soc. 16 (2019) 409-421.
AC
C
[59] P. Naveen, F. Dallemer, R.J. Butcher, R. Prabhakaran, Inorg. Chim. Acta. 471
(2018) 724-734.
[60] S. Sampath, V. Veeramani, G.S. Krishnakumar, U. Sivalingam, S.L. Madurai, R.
Chellan, Biomed. Pharmacotcher 93 (2017) 296-307.
[61] C. Bartel, A.K. Bytzek, Y.Y. Scaffididomianello, G. Grabmann, M.A. Jakupec,
C.G. Hartinger, M. Galanski, B.K. Keppler, J. Biol. Inorg. Chem. 17 (2012)
41
ACCEPTED MANUSCRIPT
465-474.
[62] P. Jost, H. Svobodova, R. Stetina, Chem-Biol. Interact. 237 (2015) 31-37.
[63] M. Novak, B. Žegura, B. Modica, E. Heathd, M. Filipič, Sci. Total. Environ.
RI
PT
601-602 (2017) 293-300.
[64] X.D. Xu, Y. Zhao, M. Zhang, R.Z. He, X.H. Shi, X.J. Guo, C.J. Shi, F. Peng, M.
Wang, M. Shen, X. Wang, X. Li, R.Y. Qin, Int. J. Mol. Sci. 18 (2017) 370.
SC
[65] V. Pooladanda, S. Bandi, S.R. Mondi, K.M. Gottumukkala, C. Godugu, Toxicol.
M
AN
U
In. Vitor. 51 (2018) 114-128.
[66] Y.L. Li, P.H. Luo, J.C. Wang, J.B. Dai, X.C. Yang, H.H. Wu, B. Yang, Q.J. He,
Toxicol. Appl. Pharm. 274 (2014) 319-327.
[67] J. Das, S. Das, A. Paul, A. Samadder, A.R. Khuda-Bukhsh, J. Acupunct. Meridian.
TE
D
Stud. 7 (2014) 140-150.
[68] Y. Yu, Q. Xie, W. Liu, Y. Guo, N. Xu, L. Xu, S.B. Liu, S.Y. Li, Y. Xu, L.K. Sun,
Biomed. Pharmacother. 86 (2017) 8-15.
EP
[69] L.Y. Jin, L. Changhee, Virus. Res. 253 (2018) 112-123.
Lin, C.C. Wang, T.R. Jan, FREE. Radical. Bio.
AC
C
[70] H.Y. Wu, C.H. Huang, Y.H.
Med. 124 (2018) 311-318.
[71] S.P. Yang, Y.G. Zhang, Y. Luo, B.C. Xu, Y.Q. Yao, Y.L. Deng, F.F. Yang, T.H. Ye,
G. Wang, Z.Q. Cheng, Y. Zheng, Y.M. Xie, Biomed. Pharmacother. 103
(2018)101-110.
[72] Q. Zhou, C.Q. You, C. Zheng, Y.W. Gu, H.C. Gu, R. Zhang, H.S. Wu, B.W. Sun,
Life. Sci 206 (2018) 1-9.
42
ACCEPTED MANUSCRIPT
[73] X. Cai, L. Guo, F. Pei, X. Chang, R. Zhang, Arch. Biochem. Biophys. 644 (2018)
93-99.
[74] X. Tian, J. Zhang, F. Zhang, M. Zhao, X. Mei, Colloid. Surface. B. 165
RI
PT
(2018).278-285.
[75] S. Cheriyamundath, T. Mahaddalkar, S.N. Save, S. Choudhary, R.V. Hosur, M.
Lopus, Biomed. Pharmacother. 98 (2018) 76-81.
SC
[76] M. Koronkiewicz, Z. Chilmonczyk, Z. Kazimerczuk, A. Orzeszko, Eur. J.
M
AN
U
Pharmacol. 820 (2018) 146-155.
[77] J. Zhan, F. Feng, W. Qu, C.l. Wang, W.
Kitdamrongtham, A. Manosroi, J.
Manosroi, H. Tokuda, M. Abe, T. Akihisa, J. Ethnopharmacol. 214 (2018) 37-46.
[78] S.H. Lai, G.B. Jiang, J.H. Yao, W. Li, B.J. Han, C. Zhang, C.C. Zeng, Y.J. Liu, J.
TE
D
Inorg. Biochem.152 (2015) 1-9.
[79] S. Sprouse, K.A. King, P.J. Spellane, R.J. Watts, J. Am. Chem. Soc. 106 (1984)
6647-6653.
EP
[80] G. Ding, F. Liu, T. Yang, Y. Jiang, H. Fu, Y. Zhao, Bioorg. Med. Chem. 14 (2006)
AC
C
3766-3774.
[81] R.H. Cao, Q. Chen, X.R. Hou, H.S. Chen, H.J. Guan, Y. Ma, W.L. Peng, A.L. Xu,
Bioorg. Med. Chem. 12 (2004) 4613-4623.
43
Captions for Schemes and Figures
RI
PT
ACCEPTED MANUSCRIPT
Table 1 IC50 (µM) values of complexes toward selected cell lines.
SC
Scheme 1 Synthetic route of complexes Ir-1, Ir-2 and Ir-3
M
AN
U
Fig. 1 (A) Location assays of the complexes in the mitochondria. (B) The change in
the mitochondrial membrane potential after SGC-7901 cells (a) were treated
with CCCP (b, positive control), 2.0 µM Ir-1 (c) and 1.0 µM Ir-2 (d) and Ir-3
(e) for 24 h. (C) The ratio of red/green fluorescence intensity was determined
TE
D
after SGC-7901 cells (a), SGC-7901 + NAC (b) were incubated with
complexes Ir-1 (c, 1.0 µM), Ir-1 (d, 2.0 µM), Ir-1 (2.0 µM) + NAC (e); Ir-2
EP
(f, 1.0 µM), Ir-2 (g, 2.0 µM), Ir-2 (2.0 µM) + NAC (h) and Ir-3 (i, 1.0 µM),
Ir-2 (j, 2.0 µM), Ir-2 (2.0 µM) + NAC (k) for 24 h.
AC
C
Fig. 2 Intracellular ROS was detected in SGC-7901 cells (a) exposure to ROSUP (b,
positive control), 2.0 µM of Ir-1 (c), Ir-2 (d) and 1.0 µM of Ir-3 (e) for 24 h.
(B) the DCF fluorescence intensity was determined after SGC-7901 cells were
exposed to ROSUP and 2.0 µM of Ir-1, Ir-2 and 1.0 µM of Ir-3 in the
absence or presence of NAC for 24 h. (C) O2•‒ level was detected in
SGC-7901 cells (a) exposure to 2.0 µM of Ir-1 (b), Ir-2 (c) and 1.0 µM of
Ir-3 (d) for 24 h. (D) the DHE fluorescence intensity was determined after
44
ACCEPTED MANUSCRIPT
SGC-7901 cells were exposed to 2.0 µM of Ir-1, Ir-2 and 1.0 µM of Ir-3 in
the absence or presence of NAC for 24 h. (E) NO level was detected in
SGC-7901 cells (a) exposure to 2.0 µM of Ir-1 (b), Ir-2 (c) and 1.0 µM of
RI
PT
Ir-3 (d) for 24 h. (F) the DAF-FMDA fluorescence intensity was determined
after SGC-7901 cells were exposed to 2.0 µM of Ir-1, Ir-2 and 1.0 µM of Ir-3
in the absence or presence of NAC for 24 h. (G) The expression of p38MAPK
SC
induced by 2.0 µM of Ir-1, Ir-2 and 1.0 µM of Ir-3 for 24 h. *P < 0.05
M
AN
U
represents significant differences compared with control.
Fig. 3 (A) Apoptosis assays of SGC-7901 cells (a) exposed to 2.0 µM of Ir-1 (b), Ir-2
(c) and 1.0 µM of Ir-3 (d) for 24 h and the cell nuclei were stained with
AO/EB. (B) The apoptotic percentage of SGC-7901 cells (a) were treated with
TE
D
2.0 µM of Ir-1 (b), Ir-2 (c) and 1.0 µM Ir-3 (d) for 48 h.
Fig. 4 Comet assays of SGC-7901 (a) exposure to 2.0 µM of Ir-1 (b), Ir-2 (c) and 1.0
µM of Ir-3 (d) for 24 h.
EP
Fig. 5 (A) Autophagy was assayed after SGC-7901 cell (a) was treated with 2.0 µM
AC
C
of Ir-1 (b), Ir-2 (c) and 1.0 µM of Ir-3 (d) for 24 h. (B) The expression of p62,
Beclin-1 and LC3 while SGC-7901 cells were exposed to 2.0 µM of Ir-1 (b),
Ir-2 (c) and 1.0 µM of Ir-3 (d) for 24 h.
Fig. 6 Relationship between cell viability and ROS or autophagy after SGC-7901
cells were incubated with 2.0 µM of Ir-1 (b), Ir-2 (c) and 1.0 µM of Ir-3 (d) in
the absence or presence of NAC or 3-MA for 24 h.
Fig. 7 (A) Intracellular Ca2+ levels were assayed after SGC-7901 cells (a) were
45
ACCEPTED MANUSCRIPT
exposed to Ca2+ free medium (b), 2.0 µM of Ir-1 (c), Ir-2 (d) and 1.0 µM of Ir-3
(e) for 24 h. (B) Fluo-3 fluorescent intensity compared with the control was
determined after SGC-7901 cells were treated with Ca2+ free medium, 2.0 µM of
RI
PT
Ir-1, Ir-2 and 1.0 µM of Ir-3 for 24 h.
Fig. 8 (A) The release of cytochrome c was assayed after SGC-7901 cells (a) were
SC
exposed to 2.0 µM of Ir-1, Ir-2 and 1.0 µM of Ir-3 for 24 h. (B) The integrated
M
AN
U
fluorescent intensity/cell compared with control was determined after the
treatment of SGC-7901 cells with 2.0 µM of Ir-1, Ir-2 and 1.0 µM of Ir-3 for 24
h. *P < 0.05 represents significant differences compared with control.
Fig. 9 (A) Cell invasion of SGC-7901 cells (a) that have migrated through the
TE
D
Matrigel induced by 2.0 µM of Ir-1 (b), Ir-2 (c) and 1.0 µM of Ir-3 (d) for 24 h.
(B) Inhibiting percentage of cell invasion induced by different concentration of
complexes Ir-1, Ir-2 and Ir-3 for 24 h. *P < 0.05 represents significant
EP
differences compared with control.
AC
C
Fig. 10 Cell cycle distribution of SGC-7901 cells (a) exposure to 2.0 µM of Ir-1 (b),
Ir-2 (c) and 1.0 µM of Ir-3 (d) for 24 h.
Fig. 11 Assays of microtubules network of SGC-7901 cells (a) induced by 2.0 µM of
Ir-1 (b), Ir-2 (c) and 1.0 µM of Ir-3 (d) for 24 h.
Fig. 12 (A) Western blot analysis of p53, Cleaved PARP, caspase 3, Bcl-2 family
protein in SGC-7901 cells treated with 2.0 µM of Ir-1, Ir-2 and 1.0 µM of
Ir-3 for 24 h. β-actin was used as internal control.
46
ACCEPTED MANUSCRIPT
Fig. 13 The in vivo antitumor activity of complex Ir-3 against SGC-7901 xenograft
model (A) Relative volume of tumor. (B) Photographs of tumor from
treatment groups and vehicle group. (C) Tumor weight (Mean ± SD) g after
RI
PT
the tumor was treated with the complex Ir-3 for 7 days. (D) Inhibiting
percentage of tumor growth induced by cisplatin and different concentrations
of complex Ir-3. *P < 0.05 represents significant differences compared with
SC
control.
M
AN
U
Fig. 14 Effect of cisplatin and complex Ir-3 on the tissue: Lung, heart, liver, brain and
tumor compared to the control group.
AC
C
EP
TE
D
Fig. 15 The apoptotic mechanism induced by the complexes Ir-1, Ir-2 and Ir-3.
47
RI
PT
ACCEPTED MANUSCRIPT
Table 1 IC50 values of the complexes toward the selected cancer cell lines
A549
HeLa
Eca-109
NDPPZ
Ir-1
Ir-2
Ir-3
Cisplatin
82.1 ± 4.3
1.8 ± 0.4
1.6 ± 0.3
0.8 ± 0.1
3.6 ± 0.5
> 100
3.6 ± 0.2
4.2 ± 0.8
3.4 ± 0.2
7.5 ± 1.3
82.8 ± 3.5
3.6 ± 0.6
4.1 ± 1.1
4.1 ± 0.2
7.0 ± 1.0
81.6 ± 4.4
4.9 ± 0.5
5.7 ± 0.5
4.5 ± 1.2
11.2 ± 0.6
TE
D
EP
AC
C
HepG2
BEL-7402
SC
SGC-7901
> 200
5.2 ± 0.5
6.3 ± 1.4
5.7 ± 1.3
12.5 ± 1.5
M
AN
U
Complex
> 200
5.7 ± 0.7
5.5 ± 0.8
4.4 ± 1.2
10.8 ± 1.6
NIH 3T3
> 200
6.4 ± 1.0
6.1 ± 0.9
8.3 ± 1.7
ND
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
Highlights
The iridium(III) complexes were synthesized and characterized.
The cytotoxicity in vitro was studied by MTT method
RI
PT
The apoptosis, cell cycle arrest and intracellular ROS levels were assayed
AC
C
EP
TE
D
M
AN
U
SC
The effect of the complex on the tumor growth was evaluated