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DNA-binding, molecular docking studies and biological activity studies of ruthenium(ii) polypyridyl complexes
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Cite this: RSC Adv., 2017, 7, 34945
DNA-binding, molecular docking studies and
biological activity studies of ruthenium(II)
polypyridyl complexes†
Bing Tang,a Fang Shen,b Dan Wan,a Bo-Hong Guo,a Yang-Jie Wang,a Qiao-Yan Yia
and Yun-Jun Liu *ac
Anticancer properties of chemically synthesized compounds have been evaluated for efficacy and
selectivity.
A
new
ligand
PTCP
(PTCP
¼
2-phenanthren-9-yl-1H-1,3,7,8-tetraazacyclopenta[l]
phenanthrene) and its three new ruthenium(II) polypyridyl complexes [Ru(N–N)2(PTCP)](ClO4)2 (N–N:
phen ¼ 1,10-phenanthroline 1; dmp ¼ 2,9-dimethyl-1,10-phenanthroline 2; ttbpy ¼ 4,40 -di-tert-butyl2,20 -bipyridine 3) were synthesized and characterized by elemental analysis, ESI-MS, IR, 1H NMR and 13C
NMR. In this report, we investigated the cytotoxicity in vitro of the complexes against several cancer cell
lines SGC-7901, HepG2, HeLa, SiHa and normal cell NIH3T3. The results show that complexes show
highly anti-proliferation activity toward SGC-7901 and low cytotoxic activity against normal cell NIH3T3.
The relationship between anti-proliferation and molecular interaction mechanism of the complexes was
also elucidated. The apoptosis was assayed by flow cytometry. The changes of mitochondrial membrane
potential and the ROS levels were measured by flow cytometry. The cell invasion, cell cycle arrest and
the expression of Bcl-2 family proteins were studied in detail. N-Acetylcysteine (NAC) was used in
several experiments to testify the effect of the complexes on apoptosis. The results demonstrate that the
Received 6th May 2017
Accepted 5th July 2017
complexes induce apoptosis in SGC-7901 cells through a ROS-mediated mitochondrial dysfunction
pathway, which was accompanied by the regulation of Bcl-2 family proteins. In addition, the interaction
DOI: 10.1039/c7ra05103d
of the complexes with calf thymus DNA (CT-DNA) shows that the complexes interact with DNA through
rsc.li/rsc-advances
partial intercalation mode.
1. Introduction
Cisplatin and its derivatives are still used in more than 50% of
the treatment drugs for cancer patients.1–4 Platinum-based
compounds are the most widely used clinically. However, the
clinical drawbacks of cisplatin are also obvious, including the
limited applicability, the acquired resistance, and the serious
side effects, such as neurotoxicity and nephrotoxicity.5,6 Thus,
a number of other metal-based drugs have been sought with the
hope of nding an inexpensive metal complex with fewer
adverse effects and improved therapeutic value. It is well known
that ruthenium compounds with special properties to mimic
iron by binding to biological molecules, reduce general toxicity
and strong affinity to cancer tissues than normal tissues, which
a
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, 510006, P.
R. China. E-mail: lyjche@163.com
b
Department of Applied Chemistry, South China Agricultural University, Guangzhou
510642, P. R. China
c
Guangdong Cosmetics Engineering & Technology Research Center, Guangzhou,
510006, P. R. China
† Electronic supplementary
10.1039/c7ra05103d
information
(ESI)
This journal is © The Royal Society of Chemistry 2017
available.
See
DOI:
make them be potential candidates as anticancer drugs.7–16 So
far, two ruthenium complexes have been proved to display
potential anticancer activities; KP1019 and KP1339 have
entered clinical trials.17,18 These complexes exhibit some
advantages over cisplatin, for instance, their activity against
cisplatin-resistant cancer cell lines and higher selectivity for
cancer cells over normal cells, which can reduce side effects.19–22
Several mechanisms have been proposed to elucidate the anticancer activities of ruthenium complexes, including the inhibition of metastasis,23,24 interaction with DNA and proteins,25,26
generation of ROS,27 induction of apoptosis.28 Based on our
previous work,29–33 we found that ruthenium(II) polypyridyl
complexes appear to show high activity against cancer cells. To
obtain more information on the anticancer activity of ruthenium complexes, in this study, a new ligand PTCP (PTCP ¼ 2phenanthren-9-yl-1H-1,3,7,8-tetraazacyclopenta[l]phenanthrene) and its three Ru(II) polypyridyl complexes: [Ru(N–N)2(PTCP)](ClO4)2 (N–N: phen ¼ 1,10-phenanthroline 1; dmp ¼ 2,9dimethyl-1,10-phenanthroline 2 and ttbpy ¼ 4,40 -di-tert-butyl2,20 -bipyridine 3, Scheme 1) were synthesized and characterized
by elemental analysis, ESI-MS, IR, 1HNMR, 13C NMR. The
cytotoxicity in vitro of the ligand and complexes toward SGC7901, HepG2, HeLa, SiHa and normal NIH3T3 cells was
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Scheme 1
The synthetic route of ligand and complexes.
evaluated by MTT method. The apoptosis in SGC-7901 cells
induced by the complexes was quantied with Annexin V/PI
staining method. The cell cycle arrest, reactive oxygen species
and mitochondrial membrane potential (MMP) in SGC-7901
cells were detected by ow cytometry. The matrigel invasion
assay was investigated to evaluate the capacity of the complexes
to inhibit the invasion of SGC-7901 cells. The expression of Bcl-2
family proteins was studied by western blot. Additionally, the
NAC was used in the cell viability, ROS levels and MMP assay to
compare with those incubated without NAC, which was used to
further explore the apoptotic-inducing mechanism. The DNAbinding of the complexes with CT-DNA was performed by
electronic absorption titration, luminescence spectra, viscosity
measurements and molecular docking.
2.
Paper
Results and discussion
2.1. Synthesis and characterization
The ligand PTCP was prepared through condensation of 1,10phenanthroline-5,6-dione with phenanthrene-9-carbaldehyde
using a similar method to that described by Steck and Day.34
The ruthenium(II) complexes were synthesized by the direct
reaction with the appropriate precursor complexes in ethylene
glycol. In the spectra of ESI-MS for the ruthenium(II) complexes,
the expected signal of [M 2ClO4]2+ was observed. The
measured molecular weights were consistent with the expected
values. In the 13C NMR spectra for complexes 2 and 3, the
signals in the range of 24–36 ppm are attributed to the methyl
group. The UV-vis and luminescence spectra of the complexes (5
mM) in PBS solution were detected. The UV-vis spectra of the
complexes consist of two well resolved bands in the range 200–
600 nm. The high-energy absorption bands below 300 nm are
attributed to an intraligand (IL) p–p* transition. The low-energy
absorption bands at 450–500 nm are assigned to a metal-toligand charge-transfer (MLCT) transition.
2.2. DNA-binding studies
2.2.1. Electronic absorption titration. The absorption
spectra of the complexes (5 mM) 1–3 in the absence or presence
of increasing amounts of CT-DNA are shown in Fig. 1. With
34946 | RSC Adv., 2017, 7, 34945–34958
increasing concentration of CT-DNA, a red shi of 2 nm for 1,
5 nm for 2 and 2 nm for 3 was observed at the MLCT bands. The
percentage hypochromicity at the MLCT band of complexes 1
(464 nm), 2 (473 nm) and 3 (469 nm) upon binding to DNA was
determined to be 17.7%, 33.3% and 28.2%, respectively. In
order to quantitatively compare the binding strength of the
complexes, the intrinsic binding constant Kb of the complexes
with CT-DNA was determined by monitoring the changes of
absorbance at the MLCT band with increasing concentration of
DNA. The values of Kb are 7.24 (0.21) 103 M1 for 1, 5.82
(0.15) 103 M1 for 2 and 3.63 (0.11) 103 M1 for 3,
respectively. The DNA-binding strength of the complexes
follows the order of 2 > 1 > 3. Substitution on the 2- and 9positions of the ancillary phen ligands may cause severe steric
constraints near the RuII core when the complex interacts into
the DNA base pairs. The methyl groups may come into close
proximity of base pairs at the interaction sites. These steric
clashes cause a diminution of the intrinsic constant. Thus,
complex 2 shows less DNA-binding constant than complex 1.
Owing to large steric hindrance of tertiary butyl, complex 3
displays the least DNA-binding constant among the three
complexes. The DNA-binding constants are less than those of
so-called DNA-intercalative Ru(II) complexes (1.1 104 to 4.8
104 M1).35,36 Thus, we consider that the complexes interact with
CT-DNA through partial intercalative mode.
2.2.2. Luminescence spectra. Complex 2 can not emit
luminescence. Complexes 1 and 3 can emit luminescence in
Tris buffer at ambient temperature, with a maximum appearing
at 583 and 601 nm, respectively. As shown in Fig. 2, upon
addition of CT-DNA, the emission intensity caused by 1 (a)
grows about 1.78 times larger than that in the absence of DNA.
This implies that complex 1 can interact with DNA and be
protected by DNA efficiently, since the hydrophobic environment inside the DNA helix reduces the accessibility of solvent
water molecules to the complex and the complex mobility is
restricted at the binding site, leading to a decrease of the
vibrational modes of relaxation. However, with the increasing
concentration of DNA, the emission intensity induced by
complex 3 (b) decreases about 1.73 times than that of the
original.
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Fig. 1 Absorption spectra of complex in Tris–HCl buffer upon addition of CT-DNA at room temperature in the presence of complexes 1 (a), 2 (b)
and 3 (c), [Ru] ¼ 5 mM, [DNA] ¼ 0–19.8 mM. Arrow shows the absorbance changing upon the increase of DNA concentration. Plots of (3a 3f)/(3b
3f) vs. [DNA] for the titration of DNA with Ru(II) complexes.
Fig. 2 Emission spectra of complexes 1 (a) and 3 (b) in Tris–HCl buffer in the absence and presence of CT-DNA. Arrow shows the intensity
change upon increasing DNA concentrations.
2.3. Viscosity measurements
Viscosity measurements that are sensitive to length change of
DNA are regarded as the least ambiguous and the most critical
tests of binding mode in solution in the absence of crystallographic structural data or NMR spectra.37,38 It is popularly
accepted that a classical intercalation mode results in lengthening the DNA helix, as base pairs are separated to accommodate the binding ligand, leading to the increase of DNA
viscosity. In contrast, a partial and/or non-classical intercalation of ligand could bend (or kink) the DNA helix, reduce its
effective length and, concomitantly, its viscosity.37,38 As shown
in Fig. 3, the relative viscosity of DNA increases at a low ratio of
[Ru]/[DNA]. However, on increasing concentration of complexes
1–3, the relative viscosity of DNA solution decreases. The
viscosity results demonstrate that the complexes interact with
DNA through partial intercalation.
with DNA through partial intercalation at the major groove. The
resulting relative binding energies of docked 1, 2 and 3 with
DNA are 37.18, 31.95 and 9.70 kJ mol1, respectively. The
more negative binding energy shows more potent DNA-binding
affinity. Thus, the DNA-binding affinities follow the order of 1 >
2 > 3. This is completely consistent with the results obtained
from the electronic absorption titration. In addition, the results
indicate that there are certain hydrogen-bonding interaction
between the complexes and DNA. The length of these hydrogen
2.4. Molecular docking with DNA
The molecular docking technique can contribute to rational
drug design and mechanistic studies by placing a small molecule into the binding site of the DNA target specic region
mainly in a non-covalent mode.39 To explore the most feasible
binding site, interaction mode and binding affinity docking
studies have been performed on complexes 1–3 with B-DNA
(PDB ID: 1BNA). As shown in Fig. 4, the complexes interact
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Effect of increasing amounts of complexes 1–3 on the relative
viscosity of CT-DNA at 25 (0.1) C, [DNA] ¼ 3.97 mM.
Fig. 3
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Paper
Fig. 4 Molecular docked models of complexes 1 (a), 2 (b) and 3 (c) with DNA (PDB 1D:1BNA). The hydrogen bonds between the complexes and
DNA are labeled using red dashed lines.
bonds are 2.058 Å (N (imidazole)/H6 (DA-5)) for 1, 2.523 Å (H
(imidazole N)/O4 (DT-19)) for 2 and 1.991 Å (N (imidazole)/
H7 (DA-18)) for 3, respectively.
2.6. Apoptosis assay by ow cytometry
2.5. Cytotoxic activity in vitro assay
The cytotoxic activity in vitro of the ligand and complexes
against cancer cell lines was determined by MTT method, and
cisplatin was used as a positive reference. The IC50 values of the
ligand and complexes are listed in Table 1. The ligand shows
high cytotoxicity toward HepG2, HeLa cancer cell lines and
normal NIH3T3 cells. However, when ligand bonded to metal to
form complexes, the cytotoxicity activity against normal cell line
has been reduced. As expectation, complexes 1, 2 and 3 display
high cytotoxicity activity toward SGC-7901 cells and low cytotoxicity toward normal cells (NIH3T3). Unexpectedly, all
complexes have no cytotoxicity on HepG2 cells and complexes 3
show high cytotoxic effect against NIH3T3 cells. All the
complexes exhibit lower cytotoxic activity than cisplatin toward
the selected cell lines, but comparable to that of [Ru(bpy)2
(TCPI)]2+ (IC50 ¼ 18.3 2.2 mM, TCPI ¼ 2-(3-1H-1,3,7,8-tetraazacyclopenta[l]phenanthren-2-yl)phenylbenzo[de]isoquinoline1,3-dione).40 Through comparison of the cytotoxic activity of the
same complex against different cancer cell lines or different
complexes against the same cancer cell line, we conclude that
(1) the same complex shows different cytotoxic effect on the
different cell growth, this may be related to the amount which
the complex enters into the cell. (2) Different complexes also
displays different cytotoxic activity against the same cancer cell,
this may be caused by different structures of the different
complexes. Comparing the IC50 values of complexes toward the
Table 1 The IC50 (mM) values of ligand and complexes toward the
selected cell lines
Complexes SGC-7901
HepG2
HeLa
SiHa
NIH3T3
PTCP
1
2
3
Cisplatin
0.8 0.3
>100
>100
>100
23.4 2.5
2.7 0.5
72.3 4.8
>100
>100
7.2 1.2
>100
61.3 5.7
>100
38.5 3.3
13.4 2.1
2.7 0.5
>100
>100
9.0 1.3
—
>100
14.5 1.8
12.5 0.6
18.2 1.7
3.4 0.4
34948 | RSC Adv., 2017, 7, 34945–34958
cancer cell lines, we found that SGC-7901 was more sensitive to
complexes than others. Thus this cell was selected for the
following experiments.
Apoptosis is a normal phenomenon in the process of development and growth in organisms,41 and many diseases
including cancer have been connected with the excessive
activation or inhibition of apoptotic signaling pathway.42,43 To
evaluate the effect of the complexes on apoptosis in SGC-7901
cell, we used ow cytometry analysis to determine the
percentage of apoptotic cells. As shown in Fig. 5, in the control
(a), the percentage of the apoptotic cells is 0.38%. SGC-7901
cells were treated with 12.5 and 25.0 mM of complexes 1 (b
and c), 2 (d and e) and 3 (f and g) for 24 h, the percentages of
apoptotic cells are 5.52% and 7.24% for 1, 1.61% and 1.74%
for 2, 5.33% and 28.5% for 3, respectively. Complex 3 shows
the highest apoptotic effect on SGC-7901 cells among these
complexes. Furthermore, the complexes induce apoptosis in
a concentration-dependent manner. The apoptotic effect of
the complexes against SGC-7901 cell follows the order of 3 > 1 >
2. This is not consistent with the order of cytotoxic activity of
2 > 1 > 3.
2.7. DNA damage studies
DNA damage was quantied through the single cell gel electrophoresis, using the comet assay.44 DNA fragmentation is
a hallmark of apoptosis, mitotic catastrophe or both.45 DNA
strand breaks were detected in SGC-7901 cells upon treatment
with the complexes in a cell-based alkaline comet assay,
cisplatin was used as a positive control. As shown in Fig. 6, in
the control (a), SGC-7901 cells fail to show any comet-like
appearance. Exposure of SGC-7901 cells to cisplatin (2.5 mM,
b) and 12.5 mM of complexes 1 (c), 2 (d) and 3 (e) for 24 h
induced considerable strand breakage on chromosomal DNA
and led to the appearance of an obscure “halo” around the
nucleus of the SGC-7901 cells. The statistically signicant and
well-formed comets were observed that indicated severe DNA
damage. The results of the comet assay reveal that the DNA of
a single cell underwent degradation as a consequence of direct
DNA damage or rapid apoptosis.46
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Fig. 5 The percentage of apoptotic cell was determined by flow cytometry. SGC-7901 cells (a) exposure to 12.5 and 25.0 mM of complexes 1 (b
and c), 2 (d and e) and 3 (f and g) for 24 h.
Comet assay of SGC-7901 cell (a) exposure to 2.5 mM of
cisplatin (b) and 12.5 mM of complexes 1 (c), 2 (d) and 3 (e) for 24 h.
Cisplatin was used as a positive control.
Fig. 6
2.8. Determination of the level of reactive oxygen species
(ROS)
It is well known that pharmacological ROS damage might be
a potential element to eliminate cancer cells, which suggests
that ROS can induce apoptosis in cancer cells.47 The literature reported that there is an inseparable relationship
between the apoptosis or senescence process in cancer cells
and the levels of ROS.48 Thus we speculated that the
apoptosis of SGC-7901 cells may be mediated by ROS. To
verify whether or not our speculation is correct, DCFH-DA
was used as a uorescent probe to determine the change of
the level of the ROS induced by the complexes. Inside the cell,
DCFH-DA is transferred to a uorescent product, namely,
dichlorouorescein (DCF).49,50 As shown in Fig. 7A, in the
control (a), no obvious green spots were found. Aer the
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treatment of SGC-7901 cells with 12.5 mM of complexes 1 (b),
2 (c) and 3 (d) for 24 h, the bright green spots were observed.
Comparing the green uorescent intensity, complex 2
induces weaker green uorescence than those induced by
complexes 1 and 3 under identical conditions. To quantitatively compare the effect of the complexes on ROS levels, the
DCF uorescent intensity is determined by ow cytometry. As
shown in Fig. 7B, in the control (a), the DCF uorescence
intensity is 10.3. When SGC-7901 cells were incubated with
25 mM of complexes 1 (c), 2 (e) and 3 (g), the DCF uorescent
intensity increases 45.4, 5.0 and 60.6 times than the original,
respectively. The effect of the complexes on ROS levels
follows the order of 3 > 1 > 2. This is consistent with the
apoptotic effect. Moreover, the ROS levels induced by the
complexes show a concentration-dependent manner. N-Acetylcysteine (NAC, 5 mM) is an inhibitor to inhibit the
production of ROS. In the presence of NAC, the effect of the
complexes on the ROS levels are shown in Fig. 7C, in the
control (a), the DCF uorescence intensity is 12.8. Aer SGC7901 cells were exposed to 12.5 mM of complexes 1 (b), 2 (c)
and 3 (d), the DCF uorescence intensity increases 11.2, 1.88
and 16.9 times than the original. The increasing degrees are
less than those in the absence of NAC (17.6 times for 1, 3.6
times for 2 and 24.4 times for 3). This demonstrates that the
addition of NAC into the complexes lead to a decrease in the
ROS levels. Although complex 2 shows higher cytotoxic
activity against SGC-7901 cell, this complex displays lower
effect on the production of ROS levels. Additionally, we also
investigated the relationship between cell viability and ROS
levels. As shown in Fig. 7D, in the presence of NAC, the cell
viability induced by the complexes increases, which indicates
that ROS levels can enhance the ability of the complexes to
kill cancer cells.
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Fig. 7 (A) Intracellular ROS was detected in SGC-7901 cells (a) exposure to 12.5 mM of complexes 1 (b), 2 (c) and 3 (d) for 24 h. (B) The DCF
fluorescence intensity was determined in SGC-7901 cells (a) exposure to 12.5 and 25.0 mM of complexes 1 (b and c), 2 (d and e) and 3 (f and g) for
24 h. (C) In the presence of NAC, the DCF fluorescence intensity was detected in SGC-7901 cells (a) exposed to 12.5 mM of complexes 1 (b), 2 (c)
and 3 (d) for 24 h. (D) The cell viability induced by the complexes for 24 h was assayed in the absence or presence of NAC.
34950 | RSC Adv., 2017, 7, 34945–34958
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2.9. Mitochondrial membrane potential detection
The inner mitochondrial membrane is a main position of
production of ROS produced as co-products of the mitochondrial
electron transport chain. Mitochondria are a critical organelle
involved in maintaining cellular homeostasis and producing
cellular energy (adenosine triphosphate, ATP) depending on
oxidative phosphorylation,51,52 which induces apoptosis as the
intrinsic apoptotic pathway and adjusts calcium homeostasis.53,54 It
is conrmed that the mitochondria membrane potential (MMP,
DJm) is a major symbol in the early apoptosis, and JC-1 was used
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as uorescent probe to measure the MMP.55–57 The above
mentioned ROS levels assay shows that apoptosis in SGC-7901 cells
treated with complexes 1–3 was induced by excessive release of
ROS. According to the mitochondrial membrane potential assay
kit, JC-1 accumulates in matrix to form JC-1 aggregates which can
emit red uorescence corresponding to high mitochondrial
membrane potential. On the contrast, JC-1 forms monomer to
emit green uorescence corresponding to low mitochondrial
membrane potential. The ratio of red/green uorescence intensity
was determined by ow cytometry. As shown in Fig. 8A, in the
Fig. 8 The ratio of red/green fluorescence was determined in the absence (A) or presence (B) of NAC while SGC-7901 cells (a) were treated with
12.5 mM of complexes 1 (b), 2 (c) and 3 (d) for 24 h.
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control (a, up and down), the ratio of the red/green uorescence is
6.24. The complexes (12.5 mM) treated SGC-7901 cells for 24 h, the
ratios of the red/green are 1.44, 0.53 and 0.42 for 1–3, respectively.
The reduction in the ratio indicates that the red uorescence
decreases and the green uorescence increases, which suggests
that the complexes can induce a reduction in the mitochondrial
membrane potential. To explore the inuence of NAC on the
changes in mitochondrial membrane potential, in the presence of
NAC, the ratios of the red/green uorescence are shown in Fig. 8B.
Comparing the ratio of the red/green uorescence in the presence
of NAC with those in the absence of NAC, the ratios in the presence
of NAC increase, which indicates that high ROS level induces more
changes in the mitochondrial membrane potential.
2.10.
Cell cycle arrest studies
DNA is the most highlight of the target for anticancer drugs to
exert toxic effects.58 Most anti-tumor drugs eliminate cancer
cells by inducing apoptosis,59,60 while others cause cell death via
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disturbing the mitotic process.61 In order to evaluate the effect
of the complexes on cell cycle distribution of SGC-7901, the cell
cycle arrest of SGC-7901 cells were investigated by ow cytometry. As shown in Fig. 9, in the control, the percentage in the
cells at G0/G1 is 55.60 (3.2)%. Aer the treatment of SGC-7901
cells with 12.5 mM of the complexes, an increase of 7.27% for 1,
2.69% for 2 and 9.66% for 3 in the percentage at G0/G1 phase
was observed, which was accompanied by the corresponding
reduction of 5.22% for 1, 4.53% for 2 and 2.52% for 3 in the
percentage of cells in the G2/M phase. The data demonstrates
that the complexes inhibit the cell growth in the G0/G1 phase.
2.11.
Cell invasion is a process associated with cancer metastasis,
which refers to the cell migration through an extracellular
matrix.62 To investigate the efficiency of complexes 1, 2 and 3 on
inhibiting the cell invasion, the Matrigel invasion assay was
performed. As shown in Fig. 10A and B, the percentage of
inhibiting the cell invasion is 41.4% for 1, 44.7% for 2 and
28.9% for 3 aer SGC-7901 cells were exposed to 25.0 mM of the
complexes, respectively. It is clear that the inhibiting effects of
the complexes display a concentration-dependent manner. The
effect of the complexes at 25.0 mM on the cell invasion follows
the order of 2 > 1 > 3. This order is consistent with the cytotoxic
activity of the complexes toward SGC-7901 cells.
2.12.
Fig. 9 The cell cycle distribution of SGC-7901 cells (control) exposure
to 12.5 mM of complexes 1, 2 and 3 for 24 h.
Cell invasion studies
Autophagy induced by the complexes
Autophagy is an evolutionarily conserved process degrading
cellular proteins and cytoplasmic organelles, in which the
phagophores surround and pack organelles to form autophagosomes.63 The effect of the complexes on autophagy was
studied using monodansylcadaverine (MDC) as uorescent
probe. MDC is a specic, in vivo marker for autophagic vacuoles,
Fig. 10 (A) Microscope images of invading SGC-7901 cells (a) that have migrated through the Matrigel induced by 12.5 mM of the complexes 1 (b),
2 (c) and 3 (d) for 24 h. (B) The percentage of invading SGC-7901 cells induced by different concentration of the complexes 1 (red), 2 (green) and 3
(blue) for 24 h. *p < 0.05 represents significant differences compared with control.
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and MDC incorporation is an indicator of autophagic activity.64
As shown in Fig. 11A, in the control (a), no obvious spots are
found. The treatment of SGC-7901 cells with 12.5 mM of
complexes 1 and 2 for 24 h, no uorescence spots are observed
(data not present), which indicates that complexes 1 and 2 can
not induce autophagy. However, SGC-7901 cells were treated
with complex 3 (b), the MDC labeling green uorescent points
in the cytoplasm were discovered, which suggests that the
autophagic vacuoles were formed. The MDC uorescence
intensity was determined by ow cytometry. As shown in
Fig. 11B, in the control (a), the uorescence intensity is 2.72.
Aer SGC-7901 cells were exposed to 12.5 mM of complex 3 for
24 h, the uorescence intensity is 8.16. The MDC uorescence
intensity increases 3 times than the original, which also indicates that complex 3 can induce autophagy.
2.13.
Western blot analysis
The transduction of apoptotic signals requires the activation of
a cascade of cysteine proteases and caspases. Caspase 3, in
particular, plays a central role in the execution of apoptosis.65
Bcl-2 family proteins maintain the balance of cell death and
survival, which plays a key role in the regulation of apoptosis via
the control of mitochondrial membrane.66 NAC was used to
investigate whether ROS inuence the expression of those
proteins or not. Aer treatment of SGC-7901 cells with 12.5 mM
of complexes 1–3, the results are shown in Fig. 12. Complexes 1–
Western blot analysis of caspase 3, Bcl-2, Bcl-x, Bax and Bid in
SGC-7901 cells treated with 12.5 mM of complexes 1–3 in the absence
and presence of NAC for 24 h. SGC-7901 (a), SGC-7901 + NAC (b), 1
(c), 1 + NAC (d), 2 (e), 2 + NAC (f), 3 (g) and 3 + NAC (h). b-actin was
used as internal control.
Fig. 12
3 result in an increase in the expression of caspase-3, Bax and
Bid compared to the control. In the presence of NAC, the
expression of caspase 3, Bcl-2 and Bcl-x down-regulates,
whereas the expression of Bax and Bid up-regulates. This
shows that NAC can regulate the expression of Bcl-2 family
proteins.
Fig. 11 (A) Autophagy was assayed with MDC staining in SGC-7901 cells (a) induced by 12.5 mM of complex 3 (b) for 24 h. (B) The MDC green
fluorescence intensity in SGC-7901 cells (a) exposed to 12.5 mM of complex 3 (b) for 24 h.
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Fig. 13
The molecular mechanism of the complexes inducing apoptosis in SGC-7901 cell.
3.
Conclusions
In summary, we have synthesized three ruthenium(II )
complexes and investigate DNA-binding and the probable
anticancer mechanism in vitro of the complexes against SGC7901. These complexes interact with CT-DNA through partial
intercalative mode at DNA major groove. The complexes
show high cytotoxic activity toward SGC-7901 cells. They can
increase the ROS levels and induce a decrease in the mitochondrial membrane potential. The complexes inhibit the
cell growth at G0/G1 phase, and down-regulate the expression of Bcl-2 and Bcl-x, and up-regulate the expression of Bax
and Bid. Take above together, we consider that the
complexes induce apoptosis in SGC-7901 cell through the
following two pathways (Fig. 13): (1) rstly, the complexes
enhance ROS levels, and then the increase of ROS levels
induces a decrease in the mitochondrial membrane potential. Secondly, the changes of mitochondrial membrane
potential activate the expression of caspase 3. Thirdly, the
activation of caspase stimulates the cell apoptosis. (2) The
complexes can cause DNA damage, and then the complexes
inhibit the cell growth at G0/G1 phase. Finally, the
complexes induce cell apoptosis. This work will be helpful
for design and synthesis of new ruthenium( II) complexes as
potent anticancer drugs.
4. Experimental
4.1. Materials and methods
The reagents and solvents used in the experiments were
purchased commercially and used without further purication
unless special statement. Ultrapure water was used in all
experiments Calf Thymus DNA (CT-DNA) was obtained from the
Sino-American Biotechnology Company. pBR 322 DNA was obtained from Shanghai Sangon Biological Engineering & Services
Co., Ltd. Doubly distilled water was used to prepare buffers
(5 mM Tris(hydroxymethylaminomethane)–HCl, 50 mM NaCl,
pH ¼ 7.2). A solution of calf thymus DNA in the buffer gave
a ratio of UV absorbance at 260 and 280 nm of ca. 1.8–1.9 : 1,
indicating that the DNA was sufficiently free of protein.67 The
DNA concentration per nucleotide was determined by absorption spectroscopy using the molar absorption coefficient (6600
M1 cm1) at 260 nm.68
34954 | RSC Adv., 2017, 7, 34945–34958
DMSO and RPMI 1640 (Roswell Park Memorial Institute)
were purchased from GIBCO. Cell lines of SGC-7901, HepG2,
HeLa, SiHa and NIH3T3 were purchased from the American
Type Culture Collection. RuCl3$3H2O was purchased from the
Kunming Institution of Precious Metals. 1,10-Phenanthroline
was obtained from the Guangzhou Chemical Reagent factory.
Microanalyses (C, H, and N) were investigated with a PerkinElmer 240Q elemental analyzer. Electrospray ionization mass
spectra (ESI-MS) were recorded on a LCQ system (Finnigan
MAT, USA) using acetonitrile as mobile phase. The spray
voltage, tube lens offset, capillary voltage, and capillary
temperature were set at 4.50 kV, 30.00 V, 23.00 V, and 200 C,
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 solvent
and tetramethylsilane as an internal standard at 500 MHz at
room temperature.
4.2. Synthesis and characterization of ligand and complexes
4.2.1. Synthesis of ligand PTCP. A mixture of 1,10phenanthroline-5,6-dione (0.315 g, 1.5 mmol),69 phenanthrene9-carbaldehyde (0.309 g, 1.5 mmol) and ammonium acetate
(2.31 g, 30 mmol) in glacial acetic acid (30 mL) was reuxed with
stirring for 3 h. Aer completion of the reaction, the pink
precipitate was collected and washed with water and dried in
vacuo. Yield: 86%. Anal. calcd for C27H16N4: C, 81.80; H, 4.07; N,
14.13%. Found: C, 81.66; H, 4.02; N, 14.22%. ESI-MS: m/z ¼ 397
[M + H]. IR (KBr, cm1): 3057m, 1943w, 1689s, 1607m, 1545m,
1494s, 1427m, 1397m, 1246w, 1196w, 1072s, 1032m, 1012m,
894s, 804s, 738s, 681m, 624w.
4.2.2. Synthesis of [Ru(phen)2(PTCP)](ClO4)2 (1). A mixture
of [Ru(phen)2Cl2]$2H2O70 (0.284 g, 0.5 mmol) and PTCP
(0.198 g, 0.5 mmol) in ethylene glycol (30 mL) was reuxed
under argon for 8 h to give a clear red solution. Aer cooling to
room temperature, a red precipitate was obtained by the addition of an excess of saturated aqueous NaClO4 solution. The
crude product was puried by column chromatography on
neutral alumina with a mixture of CH3CN–toluene (3 : 1, v/v) as
eluent. The red band was collected. The solvent was removed
under reduced pressure and a red powder was obtained. Yield:
70%. Anal. calcd for C51H32N8Cl2O8Ru: C, 57.96; H, 3.05; N,
10.60%. Found: C, 58.03; H, 3.21; N, 10.68%. IR (KBr, cm1):
3056m, 1970m, 1681w, 1601m, 1577w, 1542m, 1492s, 1445w,
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1408s, 1366s, 1315w, 1245w, 1199s, 1144m, 1087s, 899w, 843s,
807m, 768m, 721s, 623s. 1H NMR (DMSO-d6): d 9.14 (d, 2H, J ¼
8.0 Hz), 9.04 (d, 1H, J ¼ 8.0 Hz), 8.98 (d, 2H, J ¼ 7.0 Hz), 8.80 (dd,
2H, J ¼ 4.5, J ¼ 4.5 Hz), 8.78 (dd, 2H, J ¼ 4.5, J ¼ 6.5 Hz), 8.48 (s,
1H), 8.41 (s, 4H), 8.22 (d, 1H, J ¼ 7.0 Hz), 8.18 (dd, 2H, J ¼ 5.0, J
¼ 5.5 Hz), 8.12 (dd, 2H, J ¼ 5.5, J ¼ 5.0 Hz), 8.07 (dd, 2H, J ¼ 5.5,
J ¼ 5.0 Hz), 7.86 (d, 4H, J ¼ 6.0 Hz), 7.81–7.77 (m, 6H). 13C NMR
(DMSO-d6, 125 MHz): 152.85, 152.68, 152.39, 150.45, 147.27,
147.18, 145.59, 136.82, 130.48, 130.42, 130.38, 129.86, 129.80,
129.27, 128.98, 128.53, 128.37, 128.08, 127.71, 127.66, 127.53,
126.86, 126.74, 126.67, 126.51, 126.36, 126.31, 126.04, 124.29,
124.25, 123.52, 123.23, 123.15, 122.70. ESI-MS (CH3CN): m/z
429.2 ([M 2ClO4]2+).
4.2.3. Synthesis of [Ru(dmp)2(PTCP)](ClO4)2 (2). This
complex was synthesized in a manner identical to that
described for 1, with [Ru(dmp)2Cl2]$2H2O71 in place of
[Ru(phen)2Cl2]$2H2O. Yield: 72%. Anal. calcd for C55H40N8Cl2O8Ru: C, 59.36; H, 3.62; N, 10.07%. Found: C, 59.25; H, 3.81; N,
10.15%. IR (KBr, cm1): 3058w, 2966w, 2926w, 1964m, 1625w,
1604w, 1589w, 1542m, 1508s, 1443s, 1403m, 1367s, 1349w,
1305m, 1246w, 1199m, 1088s, 903w, 856s, 810s, 771w, 727s,
623s. 1H NMR (DMSO-d6): d 9.00 (d, 3H, J ¼ 8.5 Hz), 8.94 (d, 4H,
J ¼ 6.0 Hz), 8.48 (s, 1H), 8.45 (dd, 4H, J ¼ 8.5, J ¼ 9.0 Hz), 8.26 (d,
2H, J ¼ 8.5 Hz), 8.16 (d, 1H, J ¼ 7.0 Hz), 8.01 (d, 2H, J ¼ 8.5 Hz),
7.84–7.73 (m, 4H), 7.54 (dd, 2H, J ¼ 5.5, J ¼ 5.5 Hz), 7.42 (d, 4H, J
¼ 8.0 Hz), 1.96 (s, 6H), 1.75 (s, 6H). 13C NMR (DMSO-d6, 125
MHz): 167.95, 166.40, 152.63, 150.74, 148.89, 147.81, 145.78,
138.13, 136.68, 130.82, 130.37, 130.32, 130.27, 129.76, 129.54,
129.23, 128.88, 128.18, 127.62, 127.55, 127.50, 127.43, 127.12,
126.87, 126.75, 126.59, 126.52, 126.07, 125.29, 123.45, 123.11,
25.61, 24.53. ESI-MS (CH3CN): m/z 457.0 ([M 2ClO4]2+).
4.2.4. Synthesis of [Ru(ttbpy)2(pttcp)](ClO4)2 (3). This
complex was synthesized in a manner identical to that
described for 1, with [Ru(ttbpy)2Cl2]$2H2O70 in place of
[Ru(phen)2Cl2]$2H2O. Yield: 70%. Anal. calcd for C63H64N8Cl2O8Ru: C, 61.36; H, 5.23; N, 9.09%. Found: C, 60.51; H, 5.62; N,
9.01%. IR (KBr, cm1): 2965s, 2871w, 1964m, 1614s, 1541m,
1481s, 1445m, 1413s, 1368s, 1316w, 1251m, 1201m, 1090s,
898m, 839m, 809m, 769m, 726s, 624s. 1H NMR (DMSO-d6):
d 9.13 (dd, 2H, J ¼ 8.5, J ¼ 8.5 Hz), 9.02 (dd, 2H, J ¼ 8.5, J ¼ 8.5
Hz), 8.95 (d, 1H, J ¼ 8.5 Hz), 8.88 (dd, 4H, J ¼ 7.0, J ¼ 6.5 Hz),
8.49 (s, 1H), 8.20 (d, 1H, J ¼ 8.0 Hz), 8.03 (d, 2H, J ¼ 5.0 Hz), 7.97
(dd, 2H, J ¼ 5.5, J ¼ 5.5 Hz), 7.85–7.81 (m, 2H), 7.77 (d, 2H, J ¼
6.0 Hz), 7.71 (d, 2H, J ¼ 6.0 Hz), 7.64 (dd, 2H, J ¼ 6.0, J ¼ 6.0 Hz),
7.52 (d, 2H, J ¼ 6.5 Hz), 7.36 (dd, 2H, J ¼ 6.0, J ¼ 7.0 Hz), 1.44 (s,
18H), 1.36 (s, 18H). 13C NMR (DMSO-d6, 125 MHz): 161.89,
161.71, 156.57, 156.41, 153.27, 150.85, 150.76, 149.46, 145.19,
130.39, 130.36, 130.23, 129.65, 129.25, 129.14, 128.89, 128.38,
128.19, 127.60, 127.44, 126.87, 126.61, 126.32, 124.86, 124.51,
123.46, 123.11, 121.85, 121.79, 35.55, 35.42, 30.11, 30.01. ESI-MS
(CH3CN): m/z 517.0 ([M 2ClO4]2+).
4.3. DNA-binding studies
The absorption titrations of ruthenium(II) complex in buffer
were performed by using a xed ruthenium complex concentration (20 mM) to which the DNA stock solution were added.
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RSC Advances
Ruthenium–DNA solutions were allowed to incubate for 5 min
before the absorption spectra were recorded. In order to further
illustrate the binding strength of the complex, the intrinsic
binding constant K with CT-DNA was obtained by monitored
the change in the absorbance at metal-to-ligand transfer
(MLCT), with increasing concentration of DNA, the following
equation was applied.72
[DNA]/(3a 3f) ¼ [DNA]/(3b 3f) + 1/[Kb(3b 3f)]
(1)
where [DNA] is the concentration of DNA in base pairs, 3a, 3f and
3b correspond to the apparent absorption coefficient Aobsd/[Ru],
the extinction coefficient for the free ruthenium complex and
the extinction coefficient for the ruthenium complex in the fully
bound form, respectively. In plots of [DNA]/(3a 3f) versus
[DNA], Kb is given by the ratio of slope to the intercept.
Viscosity measurements were carried out using an Ubbelodhe viscometer maintained at a constant temperature at 25.0
(0.1) C in a thermostatic bath. DNA samples approximately
200 base pairs in average length were prepared by sonicating in
order to minimize complexities arising from DNA exibility.73
Flow time was measured with a digital stopwatch, and each
sample was measured three times, and an average ow time was
calculated. Relative viscosities for DNA in the presence and
absence of complexes were calculated from the relation h ¼ (t
t0)/t0, where t is the observed ow time of the DNA-containing
solution and t0 is the ow time of buffer alone.74,75 Data were
presented as (h/h0)1/3 versus binding ratio,76 where h is the
viscosity of DNA in the presence of complex and h0 is the
viscosity of DNA alone.
4.4. Molecular docking
The optimized structures of the complexes were performed
using density function theory (DFT) B3LYP. The crystal data of
the B-DNA dodecamer d(CGCGAATTCGCG)2 (PDB 1D:1BNA)
were downloaded from the Protein Data Bank.77 The water
molecules and the ligands were removed from the 1BNA, and
Gasteiger charges were added to the complexes by Autodock 4.2
Tools (ADT) before performing docking calculations. The
binding site was centered on the DNA molecule and a grid box
was created with 60 60 60 points in which almost involved
the entire DNA molecule. The rigid docking protocol and 100
runs of the Lamarckian genetic algorithm for searching ligand
conformations were performed.
4.5. Cytotoxicity in vitro assay
Cell viability was determined by MTT assay.78 Cancer cells (8
103 cells per well) were seeded in 96-well for 24 h. Cells was
incubated with the tested compounds to achieve nal concentrations ranging from 106 to 104 M. Control wells were
prepared by addition of culture medium (100 mL) and cisplatin
was used as a positive control. Aer 48 h incubation, 10 mL of
MTT dye solution (5 mg mL1) was added to each well. Aer
incubation at 37 C for 4 h, buffer (100 mL) containing dimethylformamide (50%) and sodium dodecyl sulfate (20%) was
added to transform MTT to a purple formazan dye. The optical
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density of each well was then measured for three times to obtain
the mean values. The IC50 values were analyzed by soware of
SPSS.
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4.6. DNA damage assay
DNA damage was investigated by means of comet assay. SGC7901 cells in culture medium were incubated with 12.5 mM of
the complex for 24 h at 37 C. The cells were harvested by
a trypsinization process at 24 h. A total of 100 mL of 0.5% 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 aer the gel had
been set. A mixture of 50 mL of the cell suspension (200 cells per
mL) mixed with 50 mL of 1% low melting agarose was preserved
at 37 C. A total of 100 mL of 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 aer the gel had been set. A third
coating of 50 mL of 0.5% low melting agarose was placed on the
gel and allowed to place at 4 C for 15 min. Aer solidication 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 placed in a refrigerator at
4 C for 2 h. All of the above operations were performed under
low lighting conditions to avoid additional DNA damage. The
slides, aer removal from the lysis solution, were placed horizontally in an electrophoresis chamber. The reservoirs were
lled with an electrophoresis buffer (300 mM NaOH, 1.2 mM
EDTA) until the slides were just immersed in it, and the DNA
was allowed to unwind for 30 min in electrophoresis solution.
Then the electrophoresis was carried out at 25 V and 300 mA for
20 min. Aer electrophoresis, the slides were removed, washed
thrice in a neutralization buffer (400 mM Tris, HCl, pH 7.5).
Cells were stained with 20 mL of EB (20 mg mL1) 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 uorescent microscopy.
4.7. Annexin V-FITC apoptosis detection
SGC-7901 cells were seeded into 6-well plates at a density of 1
106 cells per well and incubated for 24 h. The different
concentration of compounds were added into the above well for
24 h, cells were collected and washed with PBS twice, and then
stained with uorescein isothiocyanate (FITC)-conjugated
Annexin V and then PI. Cells were quantied by a FACS Calibur ow cytometry (Beckman Dickinson & Co., Franklin Lakes,
NJ).
4.8. Measurement of mitochondrial membrane potential
(MMP, DJm)
SGC-7901 cells (2 105 per well) were treated using compounds
for 24 h. JC-1 (1 mg mL1) as uorescence probe for determination of MMP was added to stain cells at 37 C for 30 min.
Then the cells were washed twice with PBS and measured by
a ow cytometer. Data was analyzed by FlowJo V10.2 soware.
34956 | RSC Adv., 2017, 7, 34945–34958
Paper
4.9. Reactive oxygen species (ROS) detection
Intracellular ROS levels were measured with a uorescent dye
20 ,70 -dichlorodihydrouorescein diacetate (DCFH-DA). SGC7901 cells were seeded into 6-well plates at a density of 1
106 cells per well. Aer incubation for 24 h, the medium was
replaced with medium containing different concentrations of
compounds for 24 h. Then the cells were stained with 20 mM
DCFH-DA in PBS for 30 min in the dark. Finally, the cells were
harvested and washed twice with PBS, then the data were
determined by ow cytometry and analyzed with FlowJo V10.2
soware.
4.10.
Cell cycle arrest studies
SGC-7901 cells were seeded into six-well plates (Costar, Corning
Corp., New York) at a density of 1 106 cells per well and
incubated for 24 h. The cells were cultured in RPMI 1640 supplemented with 10% of FBS, and incubated at 37 C and 5%
CO2. The medium was removed and replaced with medium
(nal DMSO concentration 0.05% v/v) containing 12.5 mM
complexes 1, 2 and 3. Aer incubation for 24 h, the cell layer was
trypsinized and washed with cold PBS and xed with 70%
ethanol. Twenty mL of RNAse (0.2 mg mL1) and 20 mL of propidium iodide (0.02 mg mL1) were added to the cell suspensions and the mixtures were incubated at 37 C for 30 min. The
samples were then analyzed with a FACS Calibur ow cytometry.
The number of cells analyzed for each sample was 10 000.
4.11.
Anti-metastasis effect
The BD BioCoat™ Matrigel™ invasion chamber (BD Biosciences) was used according to the manufacturer's instructions.
Compounds were dissolved in cell media at the desired
concentration and added into Matrigel. Twenty-ve thousands
of SGC-7901 cells in serum free media were then seeded in the
top chamber of the two chamber Matrigel system. To the low
compartment, RPMI and 5% FBS were added as chemoattractant. Cells were allowed to invade for 24 h. Aer incubation, non-invading cells were removed from the upper surface
and cells on the lower surface were xed with 4% paraformaldehyde and stained with 0.1% of crystal violet.
Membranes were photographed and the invading cells were
counted under a light microscope. Mean values from three
independent assays were calculated.
4.12.
Autophagy studies
SGC-7901 cells were seeded onto chamber slides in 12-well
plates and incubated for 24 h. The cells were cultured in RPMI
1640 supplemented with 10% of FBS and incubated at 37 C in
5% CO2. The medium was removed and replaced with medium
(nal DMSO concentration, 0.05% v/v) containing the complex
for 24 h. The medium was removed again, and the cells were
washed with ice-cold PBS twice. Then the cells were stained with
MDC (monodansylcadaverine) solution (50 mM) for 10 min and
washed with PBS twice. The cells were observed and imaged
under uorescence microscope.
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4.13.
RSC Advances
Western blot analysis
SGC-7901 cells were seeded in 3.5 cm dishes for 24 h and
incubated with 12.5 mM of complexes 1, 2 and 3 in the presence
of 10% FBS. Then cells were harvested in lysis buffer. Aer
sonication, the samples were centrifuged for 20 min at 13 000g.
The protein concentration of the supernatant was determined
by BCA assay. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was done loading equal amount of proteins per
lane. Gels were then transferred to poly(vinylidene diuoride)
membranes (Millipore) and blocked with 5% non-fat milk in
TBST (20 mM Tris–HCl, 150 mM NaCl, 0.05% Tween 20, pH 8.0)
buffer for 1 h. The membranes were incubated with primary
antibodies at 1 : 5000 dilution in 5% non-fat milk overnight at
4 C, and aer washed four times with TBST for a total of
30 min, then the secondary antibodies conjugated with horseradish peroxidase at 1 : 5000 dilution for 1 h at room temperature and washed four times with TBST. The blots were
visualized with the Amersham ECL Plus western blotting
detection reagents according to the manufacturer's instructions. To assess the presence of comparable amount of proteins
in each lane, the membranes were stripped nally to detect the
b-actin.
5.
Data analysis
All data was expressed as means SD. Statistical signicance
was evaluated by a t-test. Differences were considered to be
signicant when a *p value was less than 0.05.
Acknowledgements
This work was supported by the National Nature Science
Foundation of China (No. 81403111), the Natural Science
Foundation of Guangdong Province (No. 2016A030313728) and
Project of Innovation for Enhancing Guangdong Pharmaceutical University, Provincial Experimental Teaching Demonstration Center of Chemistry & Chemical Engineering.
References
1 B. Rosenberg, L. Vancamp and T. Krigas, Nature, 1965, 205,
698–699.
2 B. Rosenberg, L. Vancamp, J. E. Trosko and V. H. Mansour,
Nature, 1969, 222, 385–386.
3 M. A. Jakupec, M. Galanski, V. B. Arion, C. G. Hartinger and
B. K. Keppler, Dalton Trans., 2008, 2, 183–194.
4 P. J. Dyson and G. Sava, Dalton Trans., 2006, 16, 1929–1933.
5 M. Markman, Expert Opin. Drug Saf., 2003, 2, 597–607.
6 P. C. Bruijnincx and P. J. Sadler, Curr. Opin. Chem. Biol.,
2008, 12, 197–206.
7 Y. K. Yan, M. Melchart, A. Habtemariam and P. J. Sadler,
Chem. Commun., 2005, 38, 4764–4776.
8 G. Süss-Fink, Dalton Trans., 2010, 39, 1673–1688.
9 A. K. Singha, D. S. Pandeyb, Q. Xua and P. Braunstein, Coord.
Chem. Rev., 2014, 270, 31–56.
This journal is © The Royal Society of Chemistry 2017
10 A. Bergamo, C. Gaiddon, J. H. M. Schellens, J. H. Beijnen and
G. J. Sava, J. Inorg. Biochem., 2012, 106, 90–99.
11 T. Khamrang, R. Kartikeyan, M. Velusamy, V. Rajendiran,
R. Dhivya, B. Perumalsamy, M. A. Akbarsha and
M. Palaniandavar, RSC Adv., 2016, 6, 114143–114258.
12 P. C. Hu, Y. Wang, Y. Zhang, H. Song, F. F. Gao, H. Y. Lin,
Z. H. Wang, L. Wei and F. Yang, RSC Adv., 2016, 6, 29963–
29976.
13 T. S. Kamatchi, P. Kalaivani, F. R. Fronczek, K. Natarajan and
R. Prabhakaran, RSC Adv., 2017, 7, 46531–46547.
14 L. M. Chen, F. Peng, G. D. Li, X. M. Jie, K. R. Cai, C. Cai,
Y. Zhong, H. Zeng, W. Li, Z. Zhang and J. C. Chen, J. Inorg.
Biochem., 2016, 156, 64–74.
15 W. X. Chen, X. D. Song, S. F. He, J. Sun, J. X. Chen, T. Wu and
Z. W. Mao, J. Inorg. Biochem., 2016, 164, 91–98.
16 C. Zhang, B. J. Han, C. C. Zeng, S. H. Lai, W. Li, B. Tang,
D. Wan, G. B. Jiang and Y. J. Liu, J. Inorg. Biochem., 2016,
157, 62–72.
17 I. Bratsos, S. Jedner, T. Gianferrara and E. Alessio, Chimia,
2007, 61, 692–697.
18 X. Meng, M. L. Leyva, M. Jenny, I. Gross, S. Benosman,
B. Fricker, S. Harlepp, P. H. Ebraud, A. Boos, P. Wlosik,
P. Bischoff, C. Sirlin, M. Pfeffer, J. P. Loeffler and
C. Gaiddon, Cancer Res., 2009, 69, 5458–5466.
19 G. Gupta, P. Nowak-Sliwinska, N. Herrero, P. J. Dyson and
B. Therrien, Inorg. Chim. Acta, 2014, 423, 524–529.
20 G. Sava, A. Bergamo and P. J. Dyson, Dalton Trans., 2011, 40,
9069–9075.
21 C. G. Hartinger, N. Metzler-Nolte and P. J. Dyson,
Organometallics, 2012, 31, 5677–5685.
22 K. J. Kilpin, S. Crot, T. Riedel, J. A. Kitchen and P. J. Dyson,
Dalton Trans., 2014, 43, 1443–1448.
23 G. Sava, S. Zorzet, C. Turrin, F. Vita, M. Soranzo, G. Zabucchi,
M. Cocchietto, A. Bergamo, S. Giovine, G. Pezzoni, L. Sartor
and S. Garbisa, Cancer Res., 2003, 91, 1898–1905.
24 A. Bergamo, A. Masi, P. J. Dyson and G. Sava, Internet J.
Oncol., 2008, 33, 1281–1289.
25 V. Moreno, M. Font-Bardia, T. Calvet, J. Lorenzo, F. X. Aviles,
M. H. Garcia, T. S. Morais, A. Valente and M. P. J. Robalo, J.
Inorg. Biochem., 2011, 105, 241–249.
26 A. Martinez, J. Suarez, T. Shand, R. S. Magliozzo and
R. A. J. Sanchez-Delgado, J. Inorg. Biochem., 2011, 105, 39–45.
27 S. Kapitza, M. A. Jakupec, M. Uhl, B. K. Keppler and
B. Marian, Cancer Lett., 2005, 226, 115–121.
28 L. Li, Y. Wong, T. Chen and W. Zheng, Dalton Trans., 2012,
41, 1138–1141.
29 S. H. Lai, W. Li, X. Z. Wang, C. Zhang, C. C. Zeng and
Y. J. Liu, RSC Adv., 2016, 6, 63143–63155.
30 W. Li, B. J. Han, J. H. Yao, G. B. Jiang, G. J. Lin, Y. Y. Xie,
H. L. Huang and Y. J. Liu, Spectrochim. Acta, Part A, 2015,
150, 127–134.
31 C. C. Zeng, S. H. Lai, J. H. Yao, C. Zhang, H. Yin, W. Li and
B. J. Han, Eur. J. Med. Chem., 2016, 122, 118–126.
32 B. J. Han, G. B. Jiang, J. H. Yao, W. Li, J. Wang, H. L. Huang
and Y. J. Liu, Spectrochim. Acta, Part A, 2015, 135, 840–849.
33 W. Li, B. J. Han, J. H. Yao, G. B. Jiang and Y. J. Liu, RSC Adv.,
2015, 5, 24534–24543.
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RSC Advances
34 E. A. Steck and A. R. Day, J. Am. Chem. Soc., 1943, 65, 452–
456.
35 Q. X. Zhen, B. H. Ye, Q. L. Zhang, J. G. Liu, H. Li, L. N. Ji and
L. Wang, J. Inorg. Biochem., 1999, 76, 47–53.
36 A. M. Pyle, J. P. Rehmann, R. Meshoyrer, C. V. Kumar,
N. J. Turro and J. K. Barton, J. Am. Chem. Soc., 1989, 111,
3051–3058.
37 C. V. Kumar and E. H. Asuncion, J. Am. Chem. Soc., 1993, 115,
8547–8553.
38 S. Satyanarayana, J. C. Dabroniak and J. B. Chaires,
Biochemistry, 1992, 31, 9319–9324.
39 R. Rohs, I. Bloch, H. Sklenar and Z. Shakked, Nucleic Acids
Res., 2005, 33, 7048–7057.
40 C. C. Zeng, C. Zhang, S. H. Lai, H. Yin, B. Tang, D. Wan and
Y. J. Liu, Inorg. Chem. Commun., 2016, 70, 210–218.
41 S. Elmore, Toxicol. Pathol., 2007, 35, 495–516.
42 C. Tse, A. R. Shoemaker, J. Adickes, M. G. Anderson, J. Chen,
S. Jin, E. F. Johnson, K. C. Marsh, M. J. Mitten, P. Nimmer,
L. Roberts, S. K. Tahir, Y. Xiao, X. Yang, H. Zhang, S. Fesik,
S. H. Rosenberg and S. W. Elmore, Cancer Res., 2008, 68,
3421–3428.
43 A. J. Souers, J. D. Leverson, E. R. Boghaert, S. L. Ackler,
N. D. Catron, J. Chen, B. D. Dayton, H. Ding,
S. H. Enschede, W. J. Fairbrother, D. C. Huang,
S. G. Hymowitz, S. Jin, S. L. Khaw, P. J. Kovar, L. T. Lam,
J. Lee, H. L. Maecker, K. C. Marsh, K. D. Mason,
M. J. Mitten, P. M. Nimmer, A. Oleksijew, C. H. Park,
C. M. Park, D. C. Phillips, A. W. Roberts, D. Sampath,
J. F. Seymour, M. L. Smith, G. M. Sullivan, S. K. Tahir,
C. Tse, M. D. Wendt, Y. Xiao, J. C. Xue, H. Zhang,
R. A. Humerickhouse, S. H. Rosenberg and S. W. Elmore,
Nat. Med., 2013, 19, 202–208.
44 R. R. Tice, E. Agurell, D. Anderson, B. Burlinson,
A. Hartmann, H. Kobayashi, Y. Miyamae, E. Rojas,
J. C. Ryu and Y. F. Sasaki, Environ. Mol. Mutagen., 2000, 35,
206–221.
45 C. Alapetite, T. Wachter, E. Sage and E. Moustacchi, Int. J.
Radiat. Biol., 1996, 69, 359–369.
46 P. Y. Zhang, J. Q. Wang, H. Y. Huang, L. P. Qiao, L. N. Ji and
H. Chao, Dalton Trans., 2013, 42, 8907–8917.
47 D. Trachootham, J. Alexandre and P. Huang, Nat. Rev. Drug
Discovery, 2009, 8, 579–591.
48 T. Ozben, J. Pharm. Sci., 2007, 96, 2181–2196.
49 A. Kawiak, J. Zawacka-Pankau, A. Wasilewska, G. Stasilojc,
J. Bigda and E. Lojkowska, J. Nat. Prod., 2012, 75, 9–14.
50 L. R. Silveira, L. Pereira-Da-Silva, C. Juel and Y. Hellsten, Free
Radical Biol. Med., 2003, 35, 455–464.
51 A. Heller, G. Brockhoff and A. Goepferich, Eur. J. Pharm.
Biopharm., 2012, 82, 1–18.
34958 | RSC Adv., 2017, 7, 34945–34958
Paper
52 S. E. Abu-Gosh, N. Kolvazon, B. Tirosh, I. Ringel and
E. Yavin, Mol. Pharmaceutics, 2009, 6, 1138–1144.
53 S. Fulda, L. Galluzzi and G. Kroemer, Nat. Rev. Drug
Discovery, 2010, 9, 447–464.
54 J. S. Armstrong, Br. J. Pharmacol., 2007, 151, 1154–1165.
55 L. Wang, K. Y. Lu, H. H. Hao, X. Y. Li, J. Wang, K. Wang,
J. Wang, Z. Yan, S. L. Zhang, Y. H. Du and H. R. Liu, PLoS
One, 2013, 8, e81296.
56 E. Cavalieri, A. Rigo, M. Bonifacio, A. C. D. Prati,
E. Guardalben, C. Bergamini, R. Fato, G. Pizzolo, H. Suzuki
and F. Vinante, J. Transl. Med., 2011, 9, 1–13.
57 Y. S. Shin, H. A. Shin, S. U. Kang, J. H. Kim, Y. T. Oh,
K. H. Park and C. H. Kim, PLoS One, 2013, 8, e6915.
58 F. Kopper, C. Bierwirth, M. Schon, M. Kunze, I. Elvers and
D. Kranz, Proc. Natl. Acad. Sci. U. S. A., 2013, 110, 16856–
16861.
59 L. Gamet-Payrastre, P. Li, S. Lumeau, G. Cassar,
M. A. Dupont, S. Chevolleau, N. Gasc, J. Tulliez and
F. Terce, Cancer Res., 2000, 60, 1426–1433.
60 J. Klucar and M. Al-Rubeai, FEBS Lett., 1997, 400, 127–130.
61 V. M. Dirsch, D. S. Antlsperger, H. Hentze and A. M. Vollmar,
Leukemia, 2002, 16, 74–83.
62 R. Horwitz and D. Webb, Curr. Biol., 2003, 13, R756–R759.
63 F. Zhao, Y. Zhao, Y. Liu, X. L. Chang, C. Y. Chen and
Y. L. Zhao, Small, 2011, 7, 1322–1377.
64 A. Biederbick, H. F. Kern and H. P. Elsasser, Eur. J. Cell Biol.,
1995, 66, 3–14.
65 A. Kawiak, J. Zawacka-Pankau, A. Wasilewska, G. Stasilojc,
J. Bigda and E. Lojkowska, J. Nat. Prod., 2012, 75, 9–14.
66 M. Degli Espost and C. Dive, Biochem. Biophys. Res.
Commun., 2003, 304, 455–461.
67 J. Marmur, J. Mol. Biol., 1961, 3, 208–218.
68 M. E. Reichmann, S. A. Rice, C. A. Thomas and P. Doty, J. Am.
Chem. Soc., 1954, 76, 3047–3053.
69 W. Paw and R. Eisenberg, Inorg. Chem., 1997, 36, 2287–2293.
70 B. P. Sullivan, D. J. Salmon and T. J. Meyer, Inorg. Chem.,
1978, 17, 3334–3341.
71 J. P. Collin and J. P. Sauvage, Inorg. Chem., 1986, 25, 135–141.
72 A. Wolfe, G. H. Shimer and T. Meehan, Biochemistry, 1987,
26, 6392–6396.
73 J. B. Chaires, N. Dattagupta and D. M. Crothers, Biochemistry,
1982, 21, 3933–3940.
74 S. Satyanarayana, J. C. Dabrowiak and J. B. Chaires,
Biochemistry, 1993, 32, 2573–2584.
75 S. Satyanaryana, J. C. Dabrowial and J. B. Chaires,
Biochemistry, 1982, 31, 9319–9324.
76 G. Cohen and H. Eisenberg, Biopolymers, 1969, 8, 45–55.
77 S. Tabassum, W. M. Al-Asbahy, M. Afzal, F. Arjmanda and
V. Bagchib, Dalton Trans., 2012, 41, 4955–4964.
78 T. Mosmann, J. Immunol. Methods, 1983, 65, 55–63.
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