Dinuclear Organoruthenium Complexes Exhibiting Antiproliferative Activity through DNA Damage and a Reactive-Oxygen-Species-Mediated Endoplasmic Reticulum Stress Pathway

Article abstract of DOI:10.1021/acs.inorgchem.8b03447

Subtle ligand modifications on ruthenium arene complexes can lead to different mechanisms of action and result in significant changes in the anticancer efficacy. Herein, four novel dinuclear ruthenium(II) arene complexes were designed and prepared. In vit

Full text of DOI:10.1021/acs.inorgchem.8b03447

Article
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
pubs.acs.org/IC
Dinuclear Organoruthenium Complexes Exhibiting Antiproliferative
Activity through DNA Damage and a Reactive-Oxygen-Species-
Mediated Endoplasmic Reticulum Stress Pathway
†
,‡
†
†
†,‡
,†,‡
Jian Zhao, Shuang Li, Xinyi Wang, Gang Xu, and Shaohua Gou*
†
‡
Pharmaceutical Research Center and School of Chemistry and Chemical Engineering and Jiangsu Province Hi-Tech Key
Laboratory for Biomedical Research, Southeast University, Nanjing 211189, China
*
S Supporting Information
ABSTRACT: Subtle ligand modifications on ruthenium arene complexes can lead
to different mechanisms of action and result in significant changes in the anticancer
efficacy. Herein, four novel dinuclear ruthenium(II) arene complexes were designed
and prepared. In vitro tests indicated that complexes 1−3 displayed moderate
antiproliferative activity against the tested cancer cells, while the cytotoxicity of
complex 4 is superior or comparable to that of cisplatin. Further studies indicated
that complexes 1−4 induce cell death through DNA interaction and a reactive-
oxygen-species-mediated endoplasmic reticulum (ER) stress pathway, which is the
first example of an organometallic ruthenium(II) arene complex to induce ER stress
as well as DNA interaction. This kind of dinuclear ruthenium(II) arene complex
has unique biological characteristics and is a promising model for new anticancer
drug development.
INTRODUCTION
■
Platinum-based anticancer drugs, including cisplatin, carbopla-
tin, and oxaliplatin, have achieved tremendous successes in
cancer therapy, which are still used in more than 50% of all
1
−3
chemotherapeutic regimens. However, the severe side effects
and inevitable drug resistance have limited their clinical
application. Ruthenium anticancer agents with relatively lower
toxicity are regarded as promising anticancer drug candidates,
which may have the potential to overcome the limitations of the
4
−12
platinum drugs.
So far, two ruthenium(III) complexes
(
NAMI-A, KP1019, and its sodium salt KP1339) have entered
13−16
clinical trials (Figure 1).
In recent years, ruthenium(II) arene complexes have attracted
much attention because several compounds are close to clinical
trials, such as RM175 and RAPTA-C from the groups of Sadler
17−19
and Dyson, respectively.
It was initially anticipated that
ruthenium(II) arene complexes exert their anticancer effects by
interaction with DNA. However, different mechanisms of
2
0,21
action, such as inhibition of the activity of the proteins
catalytic hydride transfer reactions in cells,
or
Figure 1. Representative anticancer ruthenium(II) complexes.
22−24
were reported.
Moreover, subtle ligand modifications on ruthenium(II) arene
complexes can lead to different modes of action and result in
3
0−33
lower toxicities.
The notable examples are the trinuclear
25−29
platinum(II) compound BBR3464 evaluated in clinical trials
and dinuclear rhodium(II) carboxylate complexes with sig-
nificant in vitro and in vivo anticancer activities by inhibiting
nucleic acid and protein synthesis.
modify the action modes and improve the therapeutic properties
significant changes in the anticancer efficacy.
Therefore,
the arene rings and coordination ligands of the organo-
ruthenium complexes may provide more opportunities to design
anticancer agents with different anticancer mechanisms.
Polynuclear metal complexes provide a promising class of
compounds for anticancer agent design and discovery, which
may display additional advantages over their mononuclear
counterparts by offering improved anticancer efficacies and
2
,34
Thus, the strategy to
Received: December 11, 2018
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.inorgchem.8b03447
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Article
Figure 2. Ruthenium(II) complexes 1−4 studied in this work.
a
Scheme 1. Preparation of Ligands L1 and L2 and Complexes 1−4
a
Reagents and conditions: (a) MeOH, rt, 24 h; (b) [(arene)RuCl ] , DCM, rt, 12 h.
2
2
Table 1. log POW and Cytotoxicity Data for 1−4
IC50 values (μM)
a
b
c
d
e
compound
log POW
T24
MCF-7
A549
A549/Cis
RF
1
2
3
4
−1.73 ± 0.15
−1.32 ± 0.08
−1.21 ± 0.11
−0.96 ± 0.07
−2.03 ± 0.47
20.6 ± 1.9
28.0 ± 2.5
29.5 ± 1.7
8.4 ± 1.0
23.4 ± 2.1
27.7 ± 1.3
25.8 ± 1.5
8.6 ± 0.6
15.5 ± 1.3
19.6 ± 1.2
22.4 ± 2.1
7.5 ± 0.5
9.6 ± 0.3
15.7 ± 1.1
18.6 ± 3.6
24.0 ± 0.9
7.8 ± 0.6
1.01
0.95
1.07
1.04
4.58
f
cisplatin
13.5 ± 0.3
9.5 ± 0.6
c
44.0 ± 1.7
a
b
d
Human bladder carcinoma cell line. Human breast carcinoma cell line. Human non-small-cell lung cancer cell line. Cisplatin-resistant non-
small-cell lung cancer cell line. Resistant factor (RF) defined as IC in A549/Cis/IC50 in A549. Cited from ref 36.
e
f
5
0
of complexes by engineering polynuclear scaffolds is an effective
with electrospray ionization mass spectrometry (ESI-MS;
Figures S1−S12). The ESI-MS spectra of compounds 1−4
showed the highest isotope at 459.08, 509.04, 487.11, and
537.13, respectively, corresponding to the dication species of
35
way for anticancer drug discovery. Here, we report a series of
dinuclear ruthenium(II) arene complexes with two metal
centers bridged by a relatively rigid aromatic chain that are
expected to exert their activities by different anticancer
mechanisms.
2
+
[(arene) Ru L] (L = L1 or L2). All of the spectral data were
2
2
compatible with the proposed molecular structures of complexes
−4. Notably, complexes 1−4 showed similar absorption
1
RESULTS AND DISCUSSION
spectra, except that the absorption peaks of complexes 2 and 4
at about 350 nm showed red shifts in comparison to complexes 1
and 3 (Figure S13). The stability of complexes 1−4 in an
aqueous solution was evaluated by UV−vis spectroscopy
(Figure S14). No detectable changes of the absorption bands
of complexes 1−4 were observed after 12 h, demonstrating that
■
2
Synthesis and Characterization. Complexes 1−4 (Figure
) with different arene and iminopyridyl ligands were prepared
by following the procedure shown in Scheme 1. The resulting
dinuclear ruthenium(II) complexes were characterized by
1
13
elemental analysis and H and C NMR spectroscopy along
B
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these complexes were stable to aquation. Moreover, the
the bond distances for complexes 1−4, except that the distances
of the Ru−Cl bonds of complexes 3 and 4 are longer than those
of complexes 1 and 2, which is due to the increased electron
lipophilicity of complexes 1−4 was determined using the
shake-flask method (Table 1). The log P values for complexes
OW
II
1
−4 range from −1.73 to −0.96, showing that all compounds
density at Ru from the aromatic ligand of complexes 3 and 4.
were relatively hydrophilic.
The EPSs exhibited that the Cl atoms are relatively electron-rich
sites of complexes 1−4 (Figure 3). However, the color
distributions in the EPSs of complexes 1−4 are rather flat,
indicating that these dinuclear ruthenium(II) complexes are
relatively nonpolar.
Electrostatic Potential Surface (EPS). Density functional
theory (DFT) calculations were applied to obtain the structural
and electronic information on complexes 1−4. The optimized
geometries depicted in Figure 3 show very minor differences in
In Vitro Cytotoxicity. The antiproliferative activities against
three human cancer cell lines (A549, MCF-7, and T24 cancer
cell lines) have been investigated for all of the complexes
together with cisplatin as a positive agent by the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(
MTT) method. IC50 values were determined from dose-
survival curves (Table 1 and Figure S15). According to the IC50
values, complexes 1−4 showed moderate antiproliferative
activities against the tested cancer cell lines. Notably, complex
4
exhibited considerable cytotoxicity against the cancer cell lines
with the IC50 range from 7.5 to 8.6 μM, which displayed
comparable or superior cytotoxicity to that of cisplatin (range
from 9.5 to 13.5 μM for cisplatin).
In terms of the strong antiproliferative effects on the tested
cancer cell lines, complexes 1−4 were further studied to
determine whether these dinuclear ruthenium(II) arene
complexes can overcome cisplatin resistance. As shown in
Table 1, the IC50 value of cisplatin against A549/Cis was
increased to 44.0 μM, whereas the cytotoxicity of complexes 1−
4
against cisplatin-sensitive A549 and cisplatin-resistant A549/
Cis cells was quite similar to that of almost the same resistance
factor values ranging from 0.95 to 1.07, indicating that these
ruthenium(II) complexes can overcome cisplatin resistance.
Significantly, complex 4 (7.8 μM) is 5.6-fold as potent as
cisplatin (44.0 μM) against A549/Cis cells.
Figure 3. Structures and EPSs for dinuclear ruthenium(II) complexes
1
−4. EPS maps (from −0.10 au in red to +0.20 au in blue) drawn onto
an electron density isosurface (0.004 au) for the same compounds.
Interaction with DNA. DNA is a critical therapeutic target
for metal-based anticancer agents, which may interact with the
DNA through covalent binding, intercalation, groove binding, or
Figure 4. UV−vis specta of complexes 1−4 (25 μM) in Tris-HCl with increasing concentrations of CT-DNA (0−120 μM) at room temperature. Inset:
Plot of A /(A − A ) versus 1/[DNA]: (a) complex 1; (b) complex 2; (c) complex 3; d) complex 4.
0
0
C
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Figure 5. Fluorescence (λexc = 540 nm) quenching curves of EtBr bound to DNA in the presence of complexes 1−4. [DNA] = 25 μM, [EtBr] = 20 μM,
and [complex] = 0−33 μM.
Figure 6. Molecular docked model of complexes 1−4 with DNA (PDB 1BNA): (a) 1; (b) 2; (c) 3; (d) 4.
electrostatic interactions. 7−43 Thus, the interaction of com-
plexes 1−4 with DNA was studied by UV−vis absorbance and
fluorescence spectroscopy.
3
remarkably by complexes 2 and 4 than by complexes 1 and 3,
probably attributed to the intercalation effect of the quinoline
ligand. This study hints that complex 1 may interact with DNA
mainly through groove binding, while complexes 2−4 can be
proposed to interact via the DNA groove binding in addition to
intercalation interactions.
Molecular docking studies were made to elucidate the DNA
binding mode of complexes 1−4. The optimized structures of
these complexes were docked with a B-DNA structure (PDB
1BNA) using AutoDock 4.2. The most stable binding
conformations of these dinuclear ruthenium(II) complexes
and DNA show that complexes 1−4 fit into the minor groove of
the DNA in a parallel manner with respect to the DNA backbone
(Figure 6), suggesting that complexes 1−4 interact with the
minor groove of DNA.
The electronic absorption titration was carried out with
results shown in Figure 4. Obviously, the hypochromisms at
2
07−221 nm and bathochromic shifts (10−14 nm) were
observed for all of the complexes with increasing concentrations
of CT-DNA (Table S1), probably because of the groove binding
as well as intercalation of the dinuclear ruthenium(II) complexes
4
4,45
with CT-DNA.
The binding constant K was calculated
b
using the Benesi−Hildebrand equation (Table S1). These
4
−1
ruthenium(II) complexes have affinity constants of ∼10 M
for DNA. Among them, complexes 2 and 4 with iminoquinoline
ligands showed enhanced binding efficacy compared with
complexes 1 and 3 with an iminopyridine ligand.
In order to study the DNA binding mode further, a
competitive binding experiment was carried out by monitoring
the emission intensity of DNA-bound ethidium bromide (EtBr)
upon the addition of complexes 1−4. Both DNA groove binders
and intercalators reduce the fluorescence intensity, but the
intercalators can reduce the intensity significantly because of the
replacement of EtBr, while the intensity reduction is moderate
Because atomic force microscopy (AFM) is a useful tool to
study the interaction of DNA with small molecules and offer an
intuitive and direct way to observe the morphological changes of
DNA, it was applied to monitor the interaction of complexes 1−
4 with plasmid DNA (pBR322). As shown in Figure S16, DNA
condensation was observed after 24 h of incubation of the
ruthenium(II) complex with pBR322 plasmid DNA, which is
mainly attributed to the insertion of the dinuclear ruthenium(II)
complex into DNA chains.
46
for groove binders. As shown in Figure 5, the observed
quenching upon the addition of complexes 1−4 is 22.1%, 50.5%,
4
3.5%, and 84.5%, respectively. Notably, the fluorescence
With the purpose of confirming the binding mode of these
intensity of the DNA−EtBr complex was reduced more
complexes with DNA, agarose gel electrophoresis was applied to
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Figure 7. Gel electrophoretic mobility pattern of pBR322 plasmid DNA incubated with various concentrations of ruthenium(II) complexes. Lanes 1−
(0, 10, 20, 40, 80, 160, 320, 640, and 1280 μM) + DNA: (a) 1; (b) 2; (c) 3; (d) 4.
9
study the interaction of complexes 1−4 with pBR322 plasmid
DNA (Figure 7). A decrease in the rate of migration for closed-
circular DNA (form I) was obviously observed for complexes 1
and 2, indicating the covalent binding of the complexes with
4
7
pBR322 DNA. However, no migration was observed for
complexes 3 and 4. Moreover, the plasmid DNA gradually
disappeared with increasing concentrations of complexes 1−4,
indicating that these complexes can inhibit the intercalation of
EtBr in plasmid DNA at high concentrations, especially
complexes 2 and 4, which is in accordance with the results of
an EtBr competitive binding experiment. This study indicates
that complexes 1 and 2 can covalently bind to DNA, while
complexes 3 and 4 interact with DNA through intercalation
interactions.
Figure 8. Quantification of the flow cytometric results in Figure S17
showing the percentage of cells with increased intracellular DCF
oxidation compared to that of control cells. The data are representative
of three independent experiments. The results are mean ± SD (n = 3).
In summary, the interactions of complexes 1−4 with DNA
have been investigated by means of UV−vis, EtBr competition,
computational docking, AFM, and agarose gel electrophoresis.
The results show that complex 1 may interact with DNA
through the minor groove binding and covalent binding, while
complex 2 binds covalently and intercalatively to DNA,
simultaneously. Complexes 3 and 4 interact with DNA through
the minor groove binding and intercalation interactions.
ROS Generation. Ruthenium(II) arene complexes have
been reported to induce ROS generation in cancer cells, which is
(
**) p < 0.01 compared with the value of the control.
localized largely on ligand L1 or L2. The HOMO−LUMO
energy gaps of these complexes vary as 1 (1.91 eV) > 3 (1.79 eV)
≈
2 (1.75 eV) > 4 (1.62 eV), suggesting that electrons in
complex 4 are easier to excite from the HOMO to LUMO than
in complexes 1−3. The lower HOMO−LUMO energy gap in
complex 4 may result in easier electron transfer and ROS
generation, which is in accordance with the data of ROS
generation. The study implicates that electron transfer of these
complexes can be responsible for ROS generation.
48
responsible for the observed cytotoxicity. Thus, the levels of
ROS in A549 cells induced by complexes 1−4 were determined
by flow cytometry. As shown in Figure S17, the dinuclear
ruthenium(II) complexes markedly increased the ROS levels in
A549 cells, especially complex 4 (Figure 8). The relative order of
the ROS levels induced by these complexes is 4 ≫ 3 > 2 > 1.
To further elucidate the type of ROS induced by complex 4,
some ROS scavengers, such as superoxide dismutase (SOD),
tert-butyl alcohol (TBA), and N-acetyl-L-cysteine (NAC), were
cotreated with complex 4. As shown in Figures S18 and S19,
SOD and TBA treatment inhibited the ROS levels in the cells,
Western Blot. To further investigate the impact of ROS on
the cancer cells, we applied a Western blot technique to study
the expression of the transcription factor Nrf2, which can
49
regulate the expression of some antioxidant genes. As shown in
Figure 10, the levels of Nrf2 protein were increased after the
treatment of complexes 1−4. Because some reported ROS
induced by chemotherapeutic agents can trigger endoplasmic
49
reticulum (ER) stress via the IRE1/XPB-1s pathway, the
expression of two crucial ER stress markers, XBP-1s and CHOP,
was examined as well. It was observed that complexes 1−4
increased the accumulation of XBP-1s and CHOP, indicating
that these complexes can induce ER stress in cancer cells.
Cell Cycle Distribution. The influence of complex 4 on the
cell cycle progression of A549 and A549/Cis cells was evaluated
in the presence and absence of NAC by flow cytometry. As
shown in Figure S20, complex 4 induced the cell cycle at the S
phase in both A549 and A549/Cis cells with respect to the
untreated control. Moreover, the addition of NAC had a
negligible influence on the cell cycle distribution in A549/Cis
•
−
•
indicating that both superoxide ( O ) and hydroxyl ( OH)
2
radicals were generated. Moreover, the addition of NAC
(
nonspecific antioxidant) could greatly reduce ROS generation.
•
−
•
Taken together, this study suggests that both O2 and OH
radicals may be generated in A549 cells treated with complex 4.
Frontier Molecular Orbital. The highest occupied
molecular orbitals (HOMOs) and lowest unoccupied molecular
orbitals (LUMOs) of complexes 1−4 were calculated (Figure
−
9
). The HOMOs are localized largely on one Ru atom, Cl ions,
and arene groups in complexes 1−4, while the LUMOs are
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Figure 9. Frontier molecular orbital diagram and energy profiles for the HOMOs and LUMOs of complexes 1−4.
Figure 10. (a) Western blot analysis of the ER stress protein markers in A549 cells after treatment with complexes 1−4 at 20 μM. The data are
representative of three independent experiments. (b) Densitometric analysis of the expression of proteins normalized with GAPDH. The relative
expression of each protein was represented by the density of the protein band/density of the GAPDH band. (**) p < 0.01 compared with the value of
control.
Figure 11. Flow cytometry analysis for apoptosis of A549 and A549/Cis cells induced by complex 4 and cisplatin.
cells. As for A549 cells, the percentage of the cells arrested at the
S phase was slightly decreased after the addition of NAC. Taken
together, complex 4 inhibited the cell cycle in the S phase in
A549 and A549/Cis cells compared with the control cells, and
the addition of NAC had little effect on the cell cycle
distribution.
Apoptosis Study. Apoptosis is a programmed cell death
process in multicellular organisms, which is the main cell death
pathway induced by metal-based anticancer agents. With the
purpose of investigating the cause of the different cytotoxicities
of complex 4 and cisplatin, equimolar concentrations (20 μM)
were chosen for a comparison to elucidate the underlying
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mechanisms. Besides, an extra concentration (10 μM) was
evaluated for complex 4. As shown in Figure 11, both complex 4
and cisplatin induced an improved incidence of early-to-late-
stage apoptosis in A549 cells compared with untreated cells
obtained from Jiangsu KeyGEN BioTECH Co. (China). Cell apoptosis
and ROS experiments were measured by flow cytometry (FAC Scan,
Becton Dickenson).
General Procedure for the Synthesis of Complexes 1−4. A
methanol (MeOH) solution (50 mL) of 4,4′-methylenedianiline (1.2
mmol) and 2-quinolinecarbaldehyde or 2-pyridinecarbaldehyde (2.4
mmol) was stirred at room temperature for 24 h. Then RuL Cl (1.0
(
control). Moreover, the effect of complex 4 (43.5%) on the
cancer cell apoptosis is stronger than that of cisplatin (30.6%) at
0 μM in A549 cells. As for A549/Cis cells, the population of
2
2
2
mM) was added, and the resulting mixture was stirred at room
temperature for 12 h. The solvent was then removed by evaporation
under reduced pressure. The crude product was collected and
recrystallized from MeOH.
apoptotic cells induced by cisplatin (20.6%) decreased, while
complex 4 produced a population of apoptotic cells (42.4%)
similar to that in the A549 cells (43.5%) at 20 μM, which is in
accordance with the cytotoxicity results. Besides, the apoptotic
rates of the A549 cells were increased with increasing
concentration of complex 4, suggesting that complex 4 could
induce cancer cell death through an apoptotic pathway in a
concentration-dependent manner.
Complex 1. Yield: 0.45 g (45.5%); yellow-brown powder. Anal.
Calcd for C H Cl N Ru : C, 54.66; H, 4.89; N, 5.67. Found: C,
45 48
4
4
2
+
1
5
(
(
4.63; H, 4.79; N, 5.58; ESI-MS: m/z 459.08 ([M/2 − Cl] ). H NMR
600 MHz, CD OD): δ 1.08−1.09 (m, 12H), 2.25 (s, 6H), 2.58−2.62
3
m, 2H), 4.28 (s, 2H), 5.52−5.53 (d, 2H, J = 5.8 Hz), 5.62−5.63 (d, 2H,
J = 6.1 Hz), 5.70−5.71 (d, 2H, J = 6.1 Hz), 6.01−6.02 (d, 2H, J = 6.1
Hz), 7.56−7.58 (m, 4H), 7.80−7.82 (d, 4H, J = 8.2 Hz), 7.84−7.86 (m,
To explore the role of ROS in apoptosis induced by complex
, the A549 and A549/Cis cells were preincubated with the
1
3
4
2
H), 8.25−8.30 (m, 4H), 8.81−8.82 (m, 2H), 9.53−9.55 (m, 2H). C
antioxidant NAC before exposure to complex 4. As shown in
Figure S21, NAC preincubation caused an approximate 12% and
NMR (150 MHz, CD OD): δ 17.58, 20.90, 21.01, 31.02, 40.48, 85.39,
3
85.56, 86.31, 86.80, 103.99, 106.28, 122.64, 128.86, 129.81, 129.92,
1
39.70, 143.13, 150.60, 154.93, 155.84, 166.77.
Complex 2. Yield: 0.41 g (37.7%); yellow-brown powder. Anal.
1
5% decrease in the percentage of apoptotic cells in A549 and
A549/Cis cells, respectively, indicating that ROS plays a crucial
role in cell apoptosis.
Calcd for C H Cl N Ru : C, 58.46; H, 4.81; N, 5.15. Found: C,
5
3
52
4
4
2
+
1
5
(
8.60; H, 4.74; N, 5.23. ESI-MS: m/z 509.04 ([M/2 − Cl] ). H NMR
600 MHz, CD OD): δ 0.86−0.87 (d, 6H, J = 6.8 Hz), 1.01−1.02 (d,
3
CONCLUSION
■
6H, J = 6.9 Hz), 2.31−2.32 (d, 6H, J = 2.9 Hz), 2.38−2.45 (m, 2H),
4.33−4.34 (d, 2H, J = 2.1 Hz), 5.36−5.37 (d, 2H, J = 6.1 Hz), 5.77−
5.78 (d, 2H, J = 6.1 Hz), 5.88−5.89 (d, 2H, J = 6.2 Hz), 6.04−6.05 (d,
In summary, four novel dinuclear ruthenium(II) arene
complexes were designed and synthesized. In vitro tests
indicated that the resulting ruthenium(II) complexes displayed
moderate antiproliferative activity against the tested cancer cells,
while the cytotoxicity of complex 4 is superior or comparable to
that of cisplatin. Further experimental and computational
studies showed that the ruthenium(II) complexes have affinity
2
(
8
H, J = 6.2 Hz), 7.64−7.66 (m, 4H), 8.00−8.02 (m, 6H), 8.20−8.23
m, 2H), 8.26−8.28 (d, 2H, J = 8.2 Hz), 8.34−8.35 (m, 2H, J = 7.3 Hz),
13
.82−8.84 (m, 4H), 9.08−9.09 (d, 2H, J = 3.7 Hz). C NMR (150
MHz, CD OD): δ 17.59, 20.47, 21.22, 31.06, 40.55, 85.30, 85.93, 86.70,
3
8
1
6.87, 105.08, 106.22, 122.63, 129.02, 129.14, 129.78, 130.04, 130.31,
33.18, 140.78, 143.47, 149.04, 150.93, 155.90, 167.39, 167.41.
Complex 3. Yield: 0.52 g (49.8%); yellow-brown powder. Anal.
4
−1
constants of ∼10 M for DNA by interacting with DNA mainly
through minor groove binding and/or intercalation interactions.
The agarose gel electrophoresis study indicates that complexes 1
and 2 can covalently bind to DNA, while complexes 3 and 4
interact with DNA through intercalation interactions. Moreover,
complexes 1−4 can generate significant levels of ROS and
further induce ER stress. Hence, we proposed that these
dinuclear ruthenium(II) complexes induce cell death through
DNA binding and a ROS-mediated ER stress pathway, which is
the first example of an organometallic ruthenium(II) arene
complex to induce ER stress as well as DNA interaction. The
findings of the present study help to unravel the mechanisms of
action of ruthenium(II) arene complexes. It is noteworthy that
complexes 1−4 have no cross-resistance with cisplatin due to the
different mechanisms of action. Overall, this kind of dinuclear
ruthenium(II) arene complex has unique biological character-
istics and is a promising model for new anticancer drug
development.
Calcd for C H Cl N Ru : C, 56.32; H, 5.40; N, 5.36. Found: C,
4
9
56
4
4
2
+
1
56.43; H, 5.29; N, 5.31. ESI-MS: m/z 487.11 ([M/2 − Cl] ). H NMR
(600 MHz, CD
OD): δ 1.93−1.94 (m, 36H), 4.23 (s, 2H), 7.53−7.55
3
(
(
m, 4H), 7.74−7.75 (d, 2H, J = 8.3 Hz), 7.84−7.87 (m, 2H), 8.19−8.24
m, 4H), 8.66−8.67 (d, 4H, J = 3.4 Hz), 9.00−9.01 (d, 2H, J = 5.5 Hz).
1
3
C NMR (150 MHz, CD OD): δ 14.19, 40.36, 96.64, 122.63, 128.67,
3
1
1
29.49, 129.85, 129.88, 139.19, 143.20,143.23, 149.26, 153.23, 155.04,
67.20.
Complex 4. Yield: 0.48 g (42.0%), yellow-brown powder. Anal.
Calcd for C H Cl N Ru : C, 59.79; H, 5.28; N, 4.89. Found: C,
5
7
60
4
4
2
+
1
5
9.68; H, 5.37; N, 4.97. ESI-MS: m/z = 537.13 ([M/2 − Cl] ). H
NMR (600 MHz, CD OD): δ 1.81 (s, 36H), 4.22−4.24 (m, 2H),
3
7.55−7.56 (d, 4H, J = 7.6 Hz), 7.89−7.93 (m, 2H), 7.98−8.00 (m, 4H),
8.07−8.09 (m, 2H), 8.15−8.18 (m, 2H), 8.23−8.27 (m, 2H), 8.48−
8
2
1
1
.50 (d, 2H, J = 8.8 Hz), 8.66−8.69 (d, 2H, J = 8.5 Hz), 8.88−8.91 (m,
1
3
H). C NMR (150 MHz, CD OD): δ 14.37, 40.42, 96.95, 123.24,
3
24.29, 128.84, 128.85, 129.01, 129.99, 130.02, 130.26, 132.24, 140.03,
40.05, 143.71, 143.76, 148.93, 167.35, 167.40.
log POW Determination. The log POW determination of complexes
1
−4 was conducted using the shake-flask method. Excess 1−4 were
EXPERIMENTAL SECTION
Materials and Measurements. All chemicals and solvents were of
analytical reagent grade and were used without further purification.
■
dissolved in double-distilled water presaturated with n-octanol for 24 h
at 37 °C. The solution was filtered to remove undissolved ruthenium
complexes. Subsequently, the solution was added to an equal volume of
n-octanol (presaturated with water). The heterogeneous mixture was
shaken vigorously for 2 h before centrifuging for 15 min to achieve
phase separation. The initial and final concentrations of compounds in
an aqueous phase were determined by the UV−vis spectrum method,
50
1
Ligands L1 and L2 were prepared according to previous reports.
H
and 13C NMR spectra were measured on a Bruker Avance III-HD 600
MHz spectrometer. UV−vis spectra and kinetic traces were recorded
on a Shimadzu UV2600 instrument. The concentration of DNA in the
base pairs was determined by UV−vis absorbance at 260 nm taking
and the water−octanol partition coefficients (log P ) were calculated.
OW
1
−1
ε
as 13100 M− cm . Fluorescence measurements were
DFT Calculation. All calculations were performed using the
2
60 nm
51
performed using a FluoroMax-4 fluorometer. Scans were run at room
temperature with excitation and emission slit widths of 2.5 nm. Mass
spectrometry was measured by an Agilent 6224 ESI/TOF MS
instrument. Elemental analysis of C, H, and N used a Vario MICRO
CHNOS elemental analyzer (Elementar). Human cancer cells were
Gaussian 09 program package. Full geometry optimizations were
carried out for complexes 1−4 at the M06-L/6-31G*//LanL2DZ level
in the gas phase at 310.15 K and 1 atm.
52,53
Docking Study. Docking studies were carried out using AutoDock
54
4.2. The crystal structure of B-DNA (PDB 1BNA) has been taken
G
DOI: 10.1021/acs.inorgchem.8b03447
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
5
5
from the protein data bank. Visualization of the docked molecules has
been made by the software PyMOL. The docking simulation was
performed with the Lamarckian genetic algorithm for as much as 150
docking runs. Each run of the docking operation was terminated after a
maximum of 2500000 energy evaluations. During docking studies, the
DNA structure was kept rigid. Rotation in the complexes 1−4 was
permitted about all single bonds.
by centrifugation (5 min, 25 °C, 2000 rpm). Then, the cells were
washed twice with cold water and resuspended in a binding buffer (10
mM HEPES, 140 mM NaCl, 2.5 mM CaCl , pH 7.4). The cells were
2
stained with 5 μL of Annexin V-FITC and then with 5 μL of propidium
iodide (PI; 20 μg/mL) for 15 min in the dark at room temperature. The
fluorescence of the cells was detected by an Annexin V-FITC apoptosis
detection kit (Roche) according to the manufacturer’s protocol, and the
cells were quantified by system software (Cell Quest; BD Biosciences).
MTT Assay. The cytotoxicities of complexes 1−4 and cisplatin
(positive control) against A549, MCF-7, T24, and A549/Cis cells were
Electronic Absorption Titration Studies. Absorption spectral
titration experiments were performed by electronic spectroscopy under
physiological conditions (5 mM Tris-HCl/10 mM NaCl buffer
solution, pH 7.2) with 5 min of equilibration time. Spectra were
collected from 195 to 600 nm after the successive addition of CT-DNA
5
determined by means of MTT assay. Cells (10 /well) with better
vitality were seeded in 96-well plates. The compounds were dissolved
by DMF (40 mM) and diluted with a medium to various concentrations
(0.16, 0.63, 2.5, 10, 40, and 160 μM; the final concentration of DMF
was less than 0.4%). Cisplatin was diluted to the tested concentration
with a medium (0.16, 0.63, 2.5, 10, 40, and 160 μM). As for the negative
control, 0.4% DMF was added. The cells were incubated in the dark for
72 h. After that, the cells were stained with MTT (5 mg/mL) for
another 5 h, and then the medium was thrown away and replaced by
150 mL of dimethyl sulfoxide. The inhibition of cell growth induced by
the tested complexes was detected by measuring the absorbance of each
well at 570/630 nm using an enzyme-labeling instrument. The IC50
values were calculated by SPSS software after three parallel experiments.
Cell Cycle Measurement. A549 and A549/Cis cells were
(
0−120 μM) into a 3 mL solution of the complex (25 μM). Because the
electronic absorption of N,N-dimethylformamide (DMF) in 200−300
nm may overlap with the absorbance of compounds 1−4, they were
initially dissolved in MeOH (5 mM) and diluted with buffer to the
required concentration (the final concentration of MeOH was 0.5%).
Fluorescence Emission Spectrometry. The experiments were
carried out by the addition of compounds 1−4 to samples containing 25
μM CT-DNA (nucleotide) and 20 μM EtBr in a Tris buffer solution (5
mM Tris-HCl/10 mM NaCl buffer solution, pH 7.2). The stock
solutions of the complexes were prepared using MeOH (5 mM), and
they were further diluted to the required concentration using a Tris
buffer solution (the final concentration of MeOH was less than 2%).
The influence of the addition of complexes 1−4 (0−33 μM) to the
EtBr−DNA mixture was measured by recording variations of the
fluorescence emission spectra with an excitation at 540 nm and an
emission between 550 and 720 nm.
5
transferred into 6-well plates, with a density of 10 /well, and cultured
overnight at 37 °C. Then, complex 4 was dissolved by DMF (10 mM)
and diluted with a medium to 20 μM (the final concentration of DMF
was 0.2%). As for the negative control, the same volume of DMF was
added. For NAC experiments, the cells were preincubated with a NAC
solution (2 mM in a growth medium) for 30 min. All adherent and
floating cells were collected and washed twice with phosphate-buffered
saline. Then, the cells were fixed with 70% ethanol at 4 °C for 24 h. After
being centrifuged, the cells were stained with a 50 μg/mL PI solution
containing 100 μg/mL RNase for 0.5 h at 37 °C. The samples were
measured by flow cytometry (FAC Scan, Becton Dickenson) using Cell
Quest software and recording PI in the FL2 channel.
AFM. The pBR322 plasmid DNA (200 ng/μL) was diluted to 5 ng/
μL with a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HEPES) buffer (40 mM HEPES, 5 mM MgCl , pH 7.2). Open-
circular and linear plasmid molecules were obtained after incubation of
a solution of supercoiled pBR322 plasmid DNA (5 ng/μL) at 333 K for
0 min. Complexes 1−4 were first dissolved in DMF (10 mM) and then
diluted to 0.5 mM with a HEPES buffer. The solutions of complexes 1−
(25 μL) and pBR322 (25 μL) were mixed together (the final
(
2
3
4
concentration of DMF was 2.5%). As for the negative control, 2.5%
DMF was added. After 24 h of incubation, the mixture was dropped
onto freshly cleaved mica. Then the samples were rinsed for 10 s with
deionized water and dried with nitrogen gas. The images were obtained
ROS Detection. A549 cells were cultured in black 96-well culture
plates (20000 cells/well) for 24 h. For a ROS quencher experiment, the
cells were preincubated with SOD (1500 units/mL), TBA (10 mM),
and NAC (10 mM) for 30 min. Thereafter, complexes 1−4 were
dissolved by DMF (10 mM) and diluted with a medium to 20 μM (the
final concentration of DMF was 0.2%). The ROS levels induced by
arene−ruthenium complexes increased initially but decreased with
time. Hence, 6 h of incubation was selected for the study. As for the
negative control, the same volume of DMF was added. Concentrated
hydrogen peroxide was diluted to 20 μM in a RPMI-1640 medium with
10% FBS and was added to the cells as a positive control. As for the
negative control, only a RPMI-1640 medium was added. Then, the cells
2
in air at room temperature on areas of 1.0 × 1.0 μm .
Gel Electrophoresis Study. DNA binding properties of complexes
1
−4 were investigated by agarose gel electrophoresis, and pBR322
26
plasmid DNA (50 ng/mL) was used as the target. Complexes 1−4 were
first dissolved in DMF (10 mM) and then diluted to the desired
concentrations with Tris-H PO (100 mm) buffer. pBR322 DNA (5
3
4
μL) and complexes 1−4 (5 μL) were added to each tube, and the
mixtures of ruthenium complexes and pBR322 plasmid DNA were then
incubated at 37 °C for 24 h. As for the negative control, the same
volume of DMF was added. Subsequently, the agarose gel [made up to
were incubated with a ROS probe, carboxy-H DCFDA (20 μM), for 30
2
min at 37 °C in the dark. The fluorescence was measured with a
microplate reader using an excitation at 485 nm and an emission at 535
nm. All experiments were done in triplicate, and data are depicted as
mean ± standard deviation (SD).
1
7
% (w/v)] was prepared with a TA buffer (50 mm Tris-acetate, pH
.4). The mixtures with a loading buffer (1 mL) were submitted to
electrophoresis in agarose gel in a TA buffer at 100 V for 90 min.
Agarose gels were then dyed with EtBr (0.5 mg/L) for 20 min. Bands
were imaged by using a Molecular Imager (Bio-Rad, USA) under UV
light.
Western Blot. A549 cells were grown in a 6-well plate at a density of
5
2 × 10 cells/well and cultured until the cell density reached 80%.
Complexes 1−4 were dissolved by DMF (10 mM) and diluted in media
to give the required concentration (20 μM) for the addition to the cells
(the final concentration of DMF was 0.2%), and the cells were cultured
for 24 h at 37 °C. As for the negative control, 0.2% DMF was added.
A549 cells were lysed in a cell lysis buffer and collected by centrifugation
at 13 000 rpm for 20 min at 4 °C. Proteins from cell lysates were
separated by 8−12% sodium dodecyl sulfate−polyacrylamide gel
electrophoresis (SDS−PAGE) and transferred onto a poly(vinylidine
difluoride) membrane (Amersham Biosciences). The membrane was
blocked with PBS with Tween 20 containing 5% nonfat dry milk for 1 h
and further incubated with primary antibodies (Nrf-2, XBP-1s, and
CHOP) overnight at 4 °C under gentle shaking. After that, the
membrane was incubated with the secondary antibody (1:2000) for 1 h
at room temperature (25 °C). Protein blots were detected with
chemiluminescence reagent (Thermo Fischer Scientifics Ltd.).
Apoptosis Analysis by Flow Cytometry. On the basis of the IC50
values of our compounds, 10 and 20 μM were selected for the apoptosis
studies, which are 1.3−2.7-fold of the IC50 values of complex 4 against
A549 and A549/Cis cells. A549 and A549/Cis cells were cultured in a
RPMI-1640 medium with 10% fetal bovine serum (FBS). All media
were also supplemented with 100 mg/mL penicillin and 100 mg/mL
5
streptomycin. Cells were grown in a 6-well plate at a density of 2 × 10
cells/well and cultured overnight. Complex 4 was dissolved in DMF (10
mM) and diluted with a medium to the desired concentrations (the
final concentration of DMF was 0.1% for 10 μM and 0.2% for 20 μM).
Cisplatin was diluted to 20 μM with a medium. As for the negative
control, the same volume of DMF was added. For NAC experiments,
cells were preincubated with a NAC solution (2 mM in a growth
medium) for 30 min. After incubation for 24 h, the cells were collected
H
DOI: 10.1021/acs.inorgchem.8b03447
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as
the loading control.
Statistics. The student’s t test was applied to evaluate the
significance of the differences measured. Results were expressed as
the mean ± SD and considered to be significant when p < 0.05. The data
are representative of three independent experiments.
(9) Zhao, J.; Li, W.; Gou, S.; Li, S.; Lin, S.; Wei, Q.; Xu, G. Hypoxia-
Targeting Organometallic Ru (II)−Arene Complexes with Enhanced
Anticancer Activity in Hypoxic Cancer Cells. Inorg. Chem. 2018, 57,
8396−8403.
(10) Poynton, F. E.; Bright, S. A.; Blasco, S.; Williams, D. C.; Kelly, J.
M.; Gunnlaugsson, T. The development of ruthenium (II) polypyridyl
complexes and conjugates for in vitro cellular and in vivo applications.
Chem. Soc. Rev. 2017, 46, 7706−7756.
ASSOCIATED CONTENT
Supporting Information
■
(11) Li, J.; Tian, M.; Tian, Z.; Zhang, S.; Yan, C.; Shao, C.; Liu, Z.
*
S
Half-Sandwich Iridium (III) and Ruthenium (II) Complexes
Containing P∧P-Chelating Ligands: A New Class of Potent Anticancer
Agents with Unusual Redox Features. Inorg. Chem. 2018, 57, 1705−
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.8b03447.
1716.
1H and 13C NMR, ESI-MS, and UV−vis spectra of
complexes 1−4, dose-dependent cell viability curves,
AFM images, analysis of the ROS levels by flow
cytometry, cell cycle distribution, Flow cytometry
analysis, and absorption spectroscopic properties (PDF)
(
12) Zhao, J.; Zhang, D.; Hua, W.; Li, W.; Xu, G.; Gou, S. Anticancer
Activity of Bifunctional Organometallic Ru (II) Arene Complexes
Containing a 7-Hydroxycoumarin Group. Organometallics 2018, 37,
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and realities of the emerging metal-based drugs. Dalton Trans. 2011, 40,
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7
AUTHOR INFORMATION
Corresponding Author
E-mail: sgou@seu.edu.cn.
ORCID
(14) Hartinger, C. G.; Jakupec, M. A.; Zorbas-Seifried, S.; Groessl, M.;
Egger, A.; Berger, W.; Zorbas, H.; Dyson, P. J.; Keppler, B. K. KP1019, a
new redox-active anticancer agent-preclinical development and results
of a clinical phase I study in tumor patients. Chem. Biodiversity 2008, 5,
2140−2155.
■
*
Shaohua Gou: 0000-0003-0284-5480
Notes
The authors declare no competing financial interest.
(15) Riedl, C. A.; Flocke, L. S.; Hejl, M.; Roller, A.; Klose, M. H.;
Jakupec, M. A.; Kandioller, W.; Keppler, B. K. Introducing the 4-
Phenyl-1, 2, 3-Triazole Moiety as a Versatile Scaffold for the
Development of Cytotoxic Ruthenium (II) and Osmium (II) Arene
Cyclometalates. Inorg. Chem. 2017, 56, 528−541.
ACKNOWLEDGMENTS
■
(16) Trondl, R.; Heffeter, P.; Kowol, C. R.; Jakupec, M. A.; Berger, W.;
Keppler, B. K. NKP-1339, the first ruthenium-based anticancer drug on
the edge to clinical application. Chem. Sci. 2014, 5, 2925−2932.
We are grateful to the National Natural Science Foundation of
China (Grant 21601034) and Jiangsu Province Natural Science
Foundation (Grant BK20160664) for financial aid to this work.
Fundamental Research Funds for the Central Universities
(17) Guichard, S.; Else, R.; Reid, E.; Zeitlin, B.; Aird, R.; Muir, M.;
Dodds, M.; Fiebig, H.; Sadler, P.; Jodrell, D. Anti-tumour activity in
non-small cell lung cancer models and toxicity profiles for novel
ruthenium (II) based organo-metallic compounds. Biochem. Pharmacol.
(
Projects 2242016K30020 and 2242017K41025) and Priority
Academic Program Development of Jiangsu Higher Education
Institutions for the construction of fundamental facilities are also
appreciated.
2
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006, 71, 408−415.
18) Scolaro, C.; Bergamo, A.; Brescacin, L.; Delfino, R.; Cocchietto,
M.; Laurenczy, G.; Geldbach, T. J.; Sava, G.; Dyson, P. J. In vitro and in
vivo evaluation of ruthenium(II)-arene PTA complexes. J. Med. Chem.
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