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Bifunctional ruthenium(ii) polypyridyl complexes of curcumin as potential anticancer agents.
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Zhu, J. Zhao and S. Gou, Dalton Trans., 2020, DOI: 10.1039/D0DT01040E.
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Bifunctional Ruthenium(II) Polypyridyl Complexes of Curcumin as
Potential Anticancer Agents
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Shuang Li,† a Gang Xu,† a Yuhua Zhu,b Jian Zhao,*a Shaohua Gou*a
Ru(II)-polypyridyl complexes have been widely studied and well established for their antitumor properties. Modifications
of the coordination environment around the Ru atom through proper choice of the ligand can lead to different modes of
action and result in greatly improved anticancer efficacy. Herein, two Ru(II)-polypyridyl complexes of curcumin were
synthesized and characterized as potential anticancer agents. In vitro tests indicated that complexes 1 and 2 displayed
excellent antiproliferative activity against the tested cancer cell lines, especially complex 2, which exhibited superior
cytotoxicity compared to curcumin and cisplatin. Further biological evaluations demonstrated that complexes 1 and 2 can
cause cell apoptosis via DNA interaction and MEK/ERK signaling pathway, which is the first example of a Ru(II)-polypyridyl
complex to inhibit MEK/ERK signaling pathway as well as DNA intercalation. Overall, this work suggests that coordination
with bioactive agents may endow Ru(II)-polypyridyl complexes with improved pharmaceutical properties and synergistic
effects
for
cancer
therapy.
INTRODUCTION
Platinum-based drugs, as outstanding representatives of
chemotherapeutic drugs, have been successfully applied in the
clinical treatment of various solid tumors.1 However, their clinical
applications are obstructed on account of severe side effects and
insurmountable drug resistance.2 Under such circumstances, many
other metal complexes, especially Ru-based complexes, have been
designed and explored as alternative agents for cancer therapy. For
example, three notable Ru-based complexes (NAMI-A, NKP-1339
and TLD1433) have entered clinical trials, which has spurred
attempts to develop novel Ru-based agents with different modes of
action for cancer therapy.3 Over the last two decades, Ru(II)polypyridyl complexes have been showing great potential in
biomedical applications due to their unique biological and
physiochemical features,4 For example, Ru(II)-polypyridyl complexes
have the ability to bind with different biological targets upon the
modification of the ligands.5 As a result, Ru(II)-polypyridyl
complexes have been extensively developed and evaluated for their
antitumor activities and some of which have shown great potential
as anticancer candidates.6
Curcumin, a β-diketone polyphenol originated from the
rhizome of the herbaceous turmeric (Curcuma longa), is a
a. Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research and
Pharmaceutical Research Center, School of Chemistry and Chemical Engineering,
Southeast University, Nanjing 211189, P. R. China.
b. State Key Laboratory of Analytical Chemistry for Life Science, Nanjing University,
Nanjing 210093, P. R. China.
† These authors contributed equally to this work.
Electronic
Supplementary
Information
(ESI)
available:
See
DOI: 10.1039/x0xx00000x
promising candidate for cancer therapy. Although the
underlying anticancer mechanisms of curcumin has not been
thoroughly explored, its tumor selectivity and low side effects
has sparked a large number of clinical trials.7 However, the
clinical application of curcumin is still stagnant due to its poor
water solubility, little tissue accumulation and instability under
physiological conditions.8 Thus, improving the hydrolytic
stability of curcumin is crucial for promoting its clinical
treatment. Benefiting from the β-diketone structure of
curcumin, it can form thermodynamically stable compounds
with many metal complexes cations, enabling them to reach
biological targets without decomposition,9 and some of the
complexes showed potent anticancer efficacy both in vitro and
in vivo.10 Remarkably, arene-Ru(II) complexes of curcumin
have been widely studied as potential anticancer agents. For
example, the complex [(p-cymene)Ru(cur)Cl] has been
developed and reported by Caruso and co-workers, which
exhibited superior cytotoxicity in the HCT116 cells.11
Subsequently, the same group found that the anticancer
activity of arene-ruthenium(II) curcuminoid complexes may be
correlated with increase of curcuminoid lipophilicity.12 Besides,
Dyson and co-workers have developed several RAPTA-type
Ru(II)-(arene)-curcumin complexes with good aqueous
solubility and excellent anticancer efficacy.13
It is worth noting that modifications of the coordination
environments of Ru(II)-polypyridyl complexes through proper
choice of the ligand can lead to different modes of action and
greatly improved anticancer efficacy.14 To date, limited efforts
have been made in designing Ru(II)-polypyridyl complexes with
curcumin ligand for anticancer study. Based on the studies
above, we intended to utilize [Ru(bpy)3]Cl2 as a model complex
and take the place of its one bipyridine by curcumin to get
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2+
Ru
N
2+
2Cl-
N
N
N
Ru
N
N
N
2Cl-
N
N
N
O O
[Ru(bpy)3]Cl2
[Ru(bpy)2(acac)]Cl2
+
Cl-
N
N
N
Ru
HO
1
N
N
N
N
O O
O
Cl-
N
+
O
O
OH
HO
N
Ru
N
O O
O
OH
2
Fig. 1 Ru(II)-polypyridyl complexes studied in this work.
Results and discussion
+
Cl
Cl
Cl
Cl-
a)
Ru
N
Ru
b)
N
Ru
N
Cl
N
N
Cl
N
N
c)
N
O
HO
Ru
O O
2
0.8
0.6
0.4
0.0
400
500
600
700
Wavelength (nm)
Cl-
N
N
(b)
800
Curcumin
Complex 1
Complex 2
500
400
300
200
100
0
300
+
N
Curcumin
Complex 1
Complex 2
0.2
Cl
N
N
N
The stability of 1 and 2 was evaluated by observing the
electronic spectral changes at different times in hepes buffer
(pH = 7.2). As shown in Figure S7, minor changes of the
absorption spectra were observed for complexes 1 and 2
within 4 h. In contrast, the absorption bands of curcumin
decreased dramatically fast, indicating the degradation of
curcumin under the tested conditions. This study indicated
that coordination of curcumin to Ru(II)-polypyridyl moiety can
prevent the hydrolysis of curcumin under physiological
conditions.
The lipophilicity of the compounds is highly associated with
cellular uptake.14(c) Therefore, the log P (the octanol-water
partition coefficient) values of complexes 1-2 and
[Ru(bpy)2(acac)]Cl2 were measured using the shake-flask
method. As revealed in Table 1, The log P values of 1 and 2
were 0.75 and 1.06, respectively, both of which were higher
than those of [Ru(bpy)2(acac)]Cl2 (-1.2) and [Ru(bpy)3]Cl2 (0.41), implying that complexes 1-2 have higher lipophilicity
than [Ru(bpy)2(acac)]Cl2 and [Ru(bpy)3]Cl2.
1.0
Scheme 1 Chemical Structures and Synthetic Procedures of Complex 2.
Cl
Stability and lipophilicity
(a) 1.2
Synthesis and characterization
Ru
characterized by 1H NMR spectra (Figures S7). The
absorption
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and emission spectra of complexes 1 and
2 were
studied (Fig.
2). Notably, the photoluminescence intensity of the complexes
1 and 2 was extremely low in aerated solution. Therefore, the
emission spectra of complexes 1 and 2 were further measured
in deaerated methanol solution (Fig. 2b). The emission spectra
of complexes 1 and 2 are located at around 620 nm, which are
red-shifted compared to the emission of curcumin located at
around 550 nm. Overall, the emission spectrum may be
attributed to phosphorescence from 3MLCT (metal-to-ligand
charge transfer) transitions.
Fluorescence Intensity
complex 1 (Figure 1). Notably, complex 1 displayed potent
antimicrobial activity both in vitro and in vivo.15 It has been
reported that dppz-containing Ru(II) complexes (dppz =
dipyrido[3,2-a:2’, 3’-c]phenazine) can intercalate between DNA
base pairs and serve as DNA molecular light switches.16 The
dppn (benzo-[i]dipyrido[3,2-a:2’,3’-c]phenazine) is a derivative
of dppz, which may have the potential to intercalate into DNA
due to its excellent planar conjugated structure.17 Thus, to
further improve the DNA binding ability as well as in vitro
anticancer activity of complex 1, another bipyridine in complex
1 was replaced with dppn to obtain complex 2. These two
Ru(II)-polypyridyl complexes with curcumin as ligand were
designed as potential anticancer agents, with the goal of
increasing the hydrolytic stability of curcumin and improving
the pharmacological activity of the Ru(II)-polypyridyl
complexes. Herein, two ruthenium(II) polypyridyl complexes of
curcumin were synthesized, and their biological activities as
well as potential anticancer mechanisms were explored.
500
600
700
Wavelength (nm)
800
Fig. 2 UV-vis absorbance (a) and emission spectra (b) of curcumin and complexes 1 and
2 in MeOH (20 μM). Emission spectra of 1 (λex = 420 nm) and 2 (λex = 450 nm) were
measured in deaerated MeOH.
Electrostatic potential maps
O
OH
Reagents and conditions: a) MeOH, bpy, reflux, 12 h; b) dppn, LiCl, DMF, reflux, 4 h; c)
curcumin, LiCl, EtOH/H2O (3:1), reflux, 12 h.
Complex 1 was prepared according to previous literature,15
and complex 2 was synthesized by following the steps outlined
in Scheme 1. The composition and purity of complexes 1 and 2
were determined by 1H and 13C NMR spectra, ESI-MS
spectroscopy along with elemental analysis (Fig. S1-S6).
Ru(bpy)2(acac)]Cl2 was synthesized as control, which was
The electrostatic potential surfaces (ESP) of 1 and 2 were
obtained by density functional theory (DFT) calculations.18 As
shown in Fig. 3, the relatively green-colored region at the
curcumin moiety in complexes 1 and 2 defined a region of high
electron density. The corresponding blue area at bipyridine
moiety depicts a diminished electronic density. In general, a
similar profile was observed for complexes 1 and 2, except that
electron density at dppn moiety in complex 2 is higher than
bipyridine moiety in complex 1.
2 | J. Name., 2012, 00, 1-3
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Fig. 3 Electrostatic potential surfaces of complexes 1 (a) and 2 (b). ESP maps
(from -0.10 au in green, to +0.15 au in blue) drawn onto an electron density
isosurface (0.004 au) for the same compounds.
To further visually assess the cytotoxicity View
induced
by
Article Online
DOI: 10.1039/D0DT01040E
complexes 1 and 2 against cancer cells,
calcein AM and
propidium iodide (PI) co-staining assay was performed to dye
the living and dead cells. As shown in Fig. 4, A549 cells
maintained high viability for the control group. However, cells
treated with complexes 1 and 2 were effectively killed as
revealed by the intense red fluorescence, demonstrating the
considerable antitumor potency of 1 and 2. More effective
anticancer activity of complex 2 was observed from a lower
proportion of living cells as compared with 1, which was in
correspondence with the results of IC50 values.
In vitro cytotoxicity study
The antiproliferative properties of complexes 1 and 2 were
evaluated against A549 (human non-small-cell lung cancer),
MCF-7 (human breast adenocarcinoma) and SGC7901 (human
gastric cancer) cell lines together with curcumin, cisplatin,
[Ru(bpy)3]Cl2 and [Ru(bpy)2(acac)]Cl2 for comparison by using
the MTT assay. As shown in Table 1 and Figure S9, both
complexes 1 and 2 exhibited considerable cytotoxicity against
the tested cancer cell lines with IC50 values ranging from 2.1 to
5.8 μM. Comparing [Ru(bpy)2(acac)]Cl2 and complex 1, the IC50
values of [Ru(bpy)2(acac)]Cl2 against A549 (8.6 μM), MCF-7
(11.7 μM), and SGC7901 (10.3 μM) cells are much higher than
those of complex 1 (3.8, 5.2 and 5.8 μM, respectively), thus
indicating that replacing acetylacetone in [Ru(bpy)2(acac)]Cl2
with curcumin can greatly improve the cytotoxicity of complex
1. Importantly, complex 2 showed much superior activity to
those of reference compounds against the tested cancer cell
lines, which were > 17.0-, > 5.7- and > 4.8-fold as potent as
[Ru(bpy)3]Cl2,
curcumin
and
cisplatin,
respectively,
demonstrating that introduction of curcumin to the Ru(II)polypyridyl moieties is an effective way to improve the
cytotoxic activity of the Ru(II)-polypyridyl complexes.
Table 1 Log P values and cytotoxicity data for complexes 1-2, curcumin, Cisplatin,
[Ru(bpy)3]Cl2 and [Ru(bpy)2(acac)]Cl2
IC50 values (μM) a
Compound
logP
A549 b
MCF-7 c
SGC7901 d
1
3.8 ± 0.2
5.2 ± 0.4
5.8 ± 0.4
0.75
2
2.1 ± 0.2
2.3 ± 0.1
2.7 ± 0.2
1.06
Curcumin
13.1 ± 1.1
13.0 ± 0.9
15.4 ± 1.0
NDe
Cisplatin
13.8 ± 0.7
11.4 ± 0.3
13.0 ± 0.9
-2.03f
[Ru(bpy)3]Cl2
47.5 ± 1.9
39.2 ± 2.8
> 50.0
-0.41g
[Ru(bpy)2(acac)]Cl2
8.6 ± 0.3
11.7 ± 0.8
10.3 ± 0.5
-1.2
a The IC values were determined after 72 h of drug exposure; b human non-small-cell
50
lung cancer; c human breast adenocarcinoma cell line; d human gastric cancer cell line;
eNot determined; fCited from ref 19; gCited from ref 20.
Fig. 4 Fluorescence images of A549 cells co-stained with calcein AM and
propidium iodide (PI) after 24 h of treatment with complexes 1 and 2 at
concentration of 10 μM (size bar = 30 μm).
DNA interaction
DNA is the main target for Ru(II)-polypyridyl complexes, which
generally interact with DNA through electrostatic interaction,
intercalation and/or groove binding.5(b), 5(c) In order to
investigate the interaction pattern between 1, 2 and DNA,
competitive binding experiments were executed by monitoring
the emission intensity of DNA-bound ethidium bromide (EB)
upon the addition of 1 and 2. It was reported that the
fluorescence intensity can be reduced by DNA groove binders
or intercalators. Nevertheless, the reduction induced by
groove binders is moderate, while intercalators can reduce the
intensity significantly as a consequence of the replacement of
EB.21 As exhibited in Figure 5, the emission intensity of CTDNA-EB system (λex = 537 nm and λem = 597 nm) gradually
decreased with increasing the concentration of two complexes,
demonstrating complexes 1-2 can interact with DNA through
groove binding or intercalation. Furthermore, it was apparent
that the decrease of fluorescence intensity of CT-DNA-EB
complex caused by complex 2 was more severe than that of
complex 1. To futher illustrate the DNA binding affinity of 1-2,
the value of the apparent DNA binding constant (Kapp) were
calculated using the following equation.
KEB [EB]=Kapp [complex]
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1.0
EB+DNA
0.9
600
0.8
500
0.7
400
2
4
6
8
10 12 14
[complex] μM
300
200
100
0
EB alone
570 600 630 660 690 720 750 780
Wavelength (nm)
800
700
600
500
1.0
EB+DNA
0.8
I/I0
700
Fluorescence Intensity
(b)
800
I/I0
Fluorescence Intensity
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(a)
0.6
0.4
400
2
4
6
8
10
12
[complex] μM
14
300
200
100
0
EB alone
570 600 630 660 690 720 750 780
Wavelength (nm)
Fig. 5 Fluorescence emission spectra of CT-DNA-EtBr quenched by increasing the
concentrations of complexes 1 (a) and 2 (b). [CT-DNA] = 50 μM; [EB] = 25 μM;
[complex] = 0-14 μM.
Molecular docking studies of complexes 1 and 2 with a DNA
duplex structure (PDB ID: 4jd8) were carried out using
Autodock 4.2 package.23 As presented in Fig. 6, the predicted
optimal binding mode of complex 1 and DNA showed that the
curcumin skeleton of complex 1 fit into the groove of the DNA
in a parallel manner with respect to the DNA backbone.
However, a very different mode was observed for complex 2,
which showed that the dppn ligand intercalated between DNA
base pairs like dppz-containing Ru(II) complexes. The binding
energies of complexes 1 and 2 with DNA were -5.77 and -11.05
kcal/mol, respectively, indicating that complex 2 with dppn
ligand exhibited higher DNA-binding ability, which is in
accordance with the competitive binding experiments. Taken
together, DNA binding studies imply that complex 1 may
interact with DNA through groove binding, while complex 2 is
proposed to interact with DNA via intercalation.
Western Blot Study
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DOI: 10.1039/D0DT01040E
It was reported that curcumin plays an important role in cell
proliferation, differentiation, and apoptosis through the
extracellular signal-regulated kinase (ERK) signaling pathway.24
To further discover the modes of action of the resulting Ru(II)
complexes, western blot was performed to investigate the
expression of p-ERK1/2 and p-MEK1/2 in Ru(II)-treated A549
cells, since activation of MEK/ERK pathway is mediated
through their phosphorylation.25 As shown in Fig. 7, the
expression of p-ERK1/2 was decreased after treatment with 1
and 2, reflecting the potent efficacy of the Ru(II) complexes in
inhibiting enzymatic activity of MEK1 kinase. Moreover, the
expression of p-ERK1/2 after treatment with complex 2 was
relatively lower than that of complex 1 at concentration of 10
μM (Fig. 7b), demonstrating the stronger inhibitory effect of
complex 2. Besides, it is noted that the phosphorylation of
MEK1 was also inhibited by complexes 1 and 2, indicating that
these complexes could also block the phosphorylation of MEK1
by upstream kinase.26 Taken together, this study confirmed
that the Ru(II)-polypyridyl-curcuminato complexes can inhibit
the MEK/ERK signaling pathway.
Celllular uptake
Both cellular uptake and subcellular location are crucial factors
impacting the biological activities of compounds.4 Given weak
luminescence of complexes 1-2, inductively coupled plasma
mass spectrometry (ICP-MS) was used to measure cellular
uptake efficacy of 1, 2 and [Ru(bpy)2(acac)]Cl2 in A549 cells. As
revealed by Figure S10, the amount of cellular uptake for
complex 2 (65.4 pmol/106 cells) was approximately 1.2-time
larger than that of 1 (53.5 pmol/106 cells), which may be
attributed to the higher lipophilicity of 2. Moreover, the
accumulation of both complexes 1 and 2 was higher than that
of [Ru(bpy)2(acac)]Cl2 (41.9 pmol/106 cells). Notably, about
56.9% of Ru content of complex 2 was located in nucleus,
which was much higher than those of complex 1 (30.1%) and
[Ru(bpy)2(acac)]Cl2 (29.6%). Overall, the increased cellular
uptake and nuclear distribution of complex 2 may be
responsible for its high cytotoxicity.
Fig. 6 Molecular docked modes of complexes 1 (a) and 2 (b) with DNA duplex
(PDB ID: 4jd8);(c)Docked complex 2 (magenta) superimposed over co-crystallized
[Ru(phen)2(dppz)]2+ (cyan).
Cell Cycle Distribution
The effects of complexes 1 and 2 on cell cycle distribution of
A549 cells were determined by flow cytometry with propidium
iodide (PI) staining. As shown in Fig. 8, complexes 1 and 2 can
block the cell cycle at the G0/G1 phase in A549 cells compared
to the untreated group (control), which is different from the
most of the anticancer agents (eg. cisplatin) that block the cell
cycle in the S or G2/M phases, implying the different modes of
action of the resulting Ru(II) complexes.27 Besides, the
percentages of cells arrested at the G0/G1 phase by complex 2
4 | J. Name., 2012, 00, 1-3
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Where KEB (4.94 × 105 M-1) is the average DNA binding
constant of EthBr,22 [EB] is the concentration of EB (25 μM),
[complex] is the concentration of the complex at 50%
fluorescence intensity from the plots of the relative intensities
I/I0 against the concentrations of complexes 1-2. The Kapp
values for 1 and 2 are 4.79 × 105 M-1 and 1.14 × 106 M-1
respectively, implying that complex 2 with dppn ligand showed
higher DNA-binding ability relative to complex 1 with
bipyridine ligand.
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were higher than those of complex 1 at concentrations of 1
and 5 μM, respectively, demonstrating that treatment with
complex 2 affected the G0/G1 populations more than
treatment with complex 1.
Fig. 7 (a) The expression of p-MEK and p-ERK proteins in A549 cells was analyzed
by western blot after being treated with complexes 1 and 2 for 24 h. The data
represents three parallel experiments with similar results. (b) The relative
expression content of p-MEK and p-ERK proteins was measured through
densitometric analysis and normalized with GAPDH. The data was representative
of the density of protein band/density of GAPDH band.
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DOI: 10.1039/D0DT01040E
Fig. 9 Apoptosis analysis of A549 cells determined by flow cytometry after
treatment with complees 1 and 2 for 24 h. Data are expressed as the mean (± SD)
for three independent experiments.
Experimental Section
Materials and Measurements
Fig. 8 Cell cycle distribution of A549 cancer cells after being incubated with
complexes 1 and 2 at concentrations of 1 μM and 5 μM for 24 h. Data are quoted
as mean ± SD of three replicates.
Apoptosis studies
Apoptosis is a means of programmed cell death in multicellular
organisms, which generally confers advantages during an
organism’s life cycle. The potential of complexes 1 and 2 to
induce cell death in A549 cells was determined by Annexin
V/PI double-labeling assay. As shown in Fig. 9, treatment with
1 and 2 increased the apoptotic populations (early and late
apoptotic cells) of A549 cells compared with untreated cells
(control). Moreover, the apoptotic rates of the A549 cells were
increased with increasing concentrations of complexes 1-2,
suggesting that the resulting Ru(II) complexes could induce
cancer cell death in a concentration-dependent manner.
Overall, this study indicated that complexes 1 and 2 can induce
cancer cell death through an apoptotic pathway. Moreover,
Hoechst 33358 staining assay was conducted to confirm the
cell apoptosis induced by 1 and 2. As shown in Fig. S9, A549
cells treated with complexes 1 and 2 exhibited cytoplasm
shrinkage and fragmentation, accompanied by intensely bright
blue fluorescence in nucleus, whereas the control group
showed negligible fluorescence intensity, implying that 1 and 2
could induce cancer cell death through an apoptotic pathway.
All reagents are analytically pure and used without additional
purification. cis-[Ru(bpy)2]Cl2 and cis-[Ru(bpy)(dppn)]Cl2 are
synthesized according to previously published literature.28
Cancer cell lines were obtained from Jiangsu KeyGEN BioTECH
company (China), and pBR322 plasmid DNA was purchased
from Thermofisher, China. 1H and 13C NMR spectra were
performed on a Bruker Avance III-HD 600 MHz spectrometer.
Mass spectroscopy was measured on an Agilent 6224 ESI/TOF
MS instrument, and elemental analysis of C, H, and N was
performed on a Vario MICRO CHNOS elemental analyzer
(Elemental). UV-vis spectra were measured on Shimadzu
UV2600 instrument. DNA competitive binding experiments
were carried out using a SHIMADZU RF-600 fluorometer. Cell
cycle distribution and apoptosis experiments were conducted
using flow cytometry (FAC Scan, Becton Dickenson). The cell
accumulation of the complexes was conducted on a
PerkinElmer NexION® 1000G ICP Mass spectrometer.
General Synthetic Procedure and Characterization of Complexes 1
and 2
cis-[Ru(bpy)2Cl2] or cis-[Ru(bpy)(dppn)Cl2] (0.5 mmol),
curcumin (368.4 mg, 1.0 mmol) and LiCl (42.0 mg, 1.0 mmol)
was adequately mixed in EtOH/H2O (3:1) and stirred at refluxing
temperature for 12 h. A black hybrid solution was obtained,
and the solvent was evaporated under vacuum. The final
product was purified by using dichloromethane/methanol
(50:1, v/v) through preparative column chromatography (basic
Al2O3).
Complex 1. Yield: 52.0%. Black-brown powder. Anal. Calcd (%)
for C41H35ClN4O6Ru: C 60.33, H 4.32, N 6.86, Found: C 60.18, H
4.37, N 6.97; ESI-MS: [M - Cl]+ = 781.1587; 1H NMR (600 MHz,
DMSO-d6) δ 3.75 (s, 6H), 5.96 (s, 1H), 6.58-6.61 (d, 2H, J = 15.8
Hz), 6.79-6.81 (d, 2H, J = 8.2 Hz), 6.88-6.90 (m, 2H), 6.95-6.97
(d, 2H, J = 15.7 Hz), 7.11-7.12 (m, 2H), 7.29-7.32 (t, 2H, J = 6.6
Hz), 7.76-7.80 (m, 4H), 7.91-7.93 (t, 2H, J = 7.6 Hz), 8.16-8.19 (t,
2H, J = 7.6 Hz), 8.64-8.65 (d, 2H, J = 5.3 Hz), 8.74-8.76 (d, 2H, J
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Journal Name
= 8.0 Hz), 8.85-8.86 (d, 2H, J = 8.1 Hz); 13C NMR (150 MHz,
DMSO-d6) δ 56.02, 101.87, 110.68, 116.08, 122.42, 123.97,
124.02, 126.26, 126.39, 126.93, 127.31, 135.44, 136.47, 137.04,
148.39, 148.77, 149.81, 153.23, 157.88, 159.24, 178.20 ppm.
cancer cell line) were incubated in culture medium
Viewcontaining
Article Online
DOI:
10% fetal bovine serum (FBS), 100 mg/mL
of 10.1039/D0DT01040E
streptomycin and
100 mg/mL of penicillin in a sterile incubator of 5% CO2 and 95%
air at 37 °C.
Complex 2. Yield: 40.5%. Black-brown powder. Anal. Calcd (%)
for C53H39ClN6O6Ru: C 64.14, H 3.96, N 8.47. Found: C 63.98, H
4.13, N 8.69; ESI-MS: m/z [M - Cl]+ = 957.2062; 1H NMR (600
MHz, DMSO-d6) δ 3.54 (s, 3H), 3.79 (s, 3H), 6.09 (s, 1H), 6.606.61 (d, 1H, J = 8.3 Hz), 6.69-6.71 (d, 2H, J = 15.9 Hz), 6.78-6.82
(m, 2H), 6.94-6.95 (m, 1H), 6.99 (m, 1H), 7.06-7.09 (d, 1H, J =
15.8 Hz), 7.15-7.17 (m, 2H), 7.21-7.23 (t, 1H, J = 6.6 Hz), 7.63
(m, 2H), 7.71-7.72 (m, 1H), 7.83-7.84 (d, 1H, J = 5.6 Hz), 7.887.93 (m, 2H), 8.09-8.10 (m, 1H), 8.23-8.29 (m, 4H), 8.75-8.77 (d,
1H, J = 8.2 Hz), 8.84-8.90 (m, 3H), 8.96 (m, 1H), 9.03-9.04 (d,
2H, J = 5.2 Hz), 9.26 (m, 1H), 9.43 (s, 1H), 9.57 (s, 1H); 13C NMR
(150 MHz, DMSO-d6) δ 55.70, 56.10, 102.66, 110.86, 116.06,
116.19, 122.56, 126.15, 126.35, 127.29, 127.36, 127.76, 129.63,
137.45, 140.55, 148.19, 148.47, 148.71, 148.93, 150.01, 151.81,
152.01, 153.32, 153.88, 155.38, 158.09, 159.21, 178.16, 178.84
ppm.
In Vitro Cytotoxicity
Preparation of [Ru(bpy)2(acac)]Cl2. [Ru(bpy)2(acac)]Cl2 was
prepared according to previous report.15 Yield: 72.4%. reddish
brown powder. 1H NMR (600 MHz, DMSO-d6) δ 1.80 (s, 6H),
5.37 (s, 1H), 7.24-7.26 (t, 2H, J = 7.2 Hz), 7.70-7.71 (d, 2H, J =
5.4 Hz), 7.75-7.80 (m, 2H), 7.84-7.89 (m, 2H), 8.18-8.21 (t, 2H, J
= 7.9 Hz), 8.66-8.67 (d, 2H, J = 5.3 Hz), 8.71-8.72 (d, 2H, J = 8.0
Hz), 8.84-8.85 (d, 2H, J = 8.0 Hz).
Stability Studies
The stability of complexes 1, 2 and curcumin were recorded on
a Shimadzu UV2600 UV-vis spectrophotometer equipped with
a thermostatically controlled cell holder. The stock solution of
the tested complexes were dissolved in MeOH, and then
diluted to 20 μM with Hepes buffer (10 mM, pH 7.2). The UVvis spectra were recorded every 10 min at 298 K.
log P Determination
Excess complexes 1, 2 and [Ru(bpy)2(acac)]Cl2 were dissolved
in n-octanol (presaturated with water) and then combined
with an equal volume of deionized water (presaturated with
octanol). The mixed solution was strongly shaken for 2 h and
then centrifuged for 15 min to remove undissolved complexes
and completely achieve phases separation. The concentrations
of complexes in two phases were determined through
standard working equation using UV-Vis spectroscopy. The log
P values were measured using this formula:
log P = log (Coctanol/Cwater)
DFT Calculation
The structures of complexes 1 and 2 were optimized at the
M06-L/6-31G*//LanL2DZ level in the gas phase at 310.15 K and
1 atm.29 All calculations were carried out using the Gaussian 09
program package.30
Cell Culture
A549 (human non-small-cell lung cancer), MCF-7 (human
breast adenocarcinoma cell line) and SGC7901 (human gastric
The cytotoxicity of complexes 1, 2, cisplatin, [Ru(bpy)3]Cl2, and
[Ru(bpy)2(acac)]Cl2 against three cancer cell lines was
evaluated by means of MTT assay. Cells with excellent viability
were dispersed into 96-well plates at a density of 104/well and
then incubated for 24 h at 37 °C in a 5% CO2 humidified air
incubator. The test compounds were dissolved with DMF and
then diluted with culture medium to the six required
concentrations (1.56, 3.13, 6.25, 12.5, 25, 50 μM). The final
concentration of DMF was less than 0.4%. Cisplatin was
dissolved and diluted with culture medium to same
concentrations. As for the negative control, 0.4% DMF was
added. Then these diluted solutions were added to test wells,
and incubated with cells at 37 °C for 72 h. After that, the cells
were treated with MTT (5 mg/mL) for additional 5 h. Then the
culture medium involving MTT was removed and 150 μL of
DMSO was added. Finally, the activity of complexes were
measured by an enzyme-labeling analyzer at UV absorption
intensity of 570/630 nm. The final experimental data was
analyzed by SPSS software through three parallel experiments.
Calcein AM and Propidium Iodide (PI) Co-Staining
For Calcein AM and propidium iodide (PI) co-staining assay,
A549 cells (105 per well) were seeded and cultured in confocal
dishes overnight at 37 °C. Then complexes 1 and 2 were added
to the cells with the final concentration of 10 μM. After 24 h
incubation, the cells were washed with PBS and stained with
Calcein AM/PI Double Stain Kit according to the instruction
manual. Fluorescence images of the stained cells were then
taken using a confocal microscope. For PI channel, the
excitation wavelength was 561 nm and the emission
wavelength range was 600-640 nm. For calcein AM channel,
the excitation wavelength was 488 nm and the emission was
detected at 500-540 nm.
DNA Competitive Binding Experiment
The experiments were performed by combining stock solutions
of 1 or 2 in MeOH to the mixed solution of 100 μM CT-DNA
and 50 μM EtBr under physiological conditions (5 mM TrisHCl/10 mM NaCl buffer solution, pH 7.2). The changes of the
fluorescence emission spectra with an excitation at 537 nm
were recorded after each successive addition of the tested
complexes (0-14 μM) and incubation at room temperature for
5 min to complete the interaction.
Molecular Docking
Molecular docking studies were performed using an autodock
4.2 package. The crystal structure of the DNA duplex structure
(PDB: 4jd8) were obtained from the protein data bank. The
docking simulation process was conducted for 200 rounds with
the lamarckian genetic algorithm search method. Each round
of docking operations required 2500000 energy optimizations.
6 | J. Name., 2012, 00, 1-3
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During the docking operation, the structure of DNA remained
fixed, and binding energy optimizations were performed by
rotation of the complexes. The final docking results of the
complexes and DNA were output through PyMOL software.
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Cell Uptake
A549 and MCF-7 cells with good viability were transferred into
6-well plates at 37 °C. After 12 h cultivation in medium, cells
were co-incubated with complexes 1, 2 and [Ru(bpy)2(acac)]Cl2
(5 μM) for 12 h. After the supernatants were removed, cells
were collected and washed 3 times with ice-cold PBS, then
centrifuged for 10 minutes, and resuspended in PBS (1 mL).
The nuclei component for measurement were extracted using
commercial nuclear extraction kit (Solarbio). The remaining
samples were then digested with HNO3 (65%, 200 μL) for 24 h
at room temperature. After three parallel experiments, the Ru
content were measured by ICP-MS.
Western Blot Assay
A549 cells with good viability were transferredView
to Article
a 6-well
Online
DOI: 10.1039/D0DT01040E
plate and cultured overnight at 37 °C. Complexes
1 and 2 were
dissolved with DMF and diluted to 1 μM and 5 μM by culture
medium. The final concentration of DMF was less than 0.4%.
For the negative control, 0.4% DMF was added. The diluted
complexes were added to these wells, and incubated for 24 h.
The cells were then collected with trypsin and washed with
cold PBS. Then, cells were centrifugated (5 min, 2000 rpm),
then washed twice with cold water and resuspended in
binding buffer (10 mM Heps, 140 mM NaCl, 2.5 mM CaCl2, pH
7.4). Afterwards, cells were stained with 3 μL of Annexin VFITC (100 ng/mL) and 3 μL of PI (2 μg/mL). Cellular
fluorescence was quantified by flow cytometry (FAC Scan,
Becton Dickenson) using the Annexin V-FITC Apoptosis
Detection Kit (Roche) and analyzed by Cell Quest software.
Hoechst 33358 Staining for Apoptosis Studies
A549 cells were seeded in a 6-well plate and incubated
overnight in a 37 °C incubator. Then the cells were co-cultured
with complexes 1-2 (2.5 μM) for 24 h. Afterwards, cells were
washed three times with PBS buffer solution, and stained with
Hoechst 33358 for 10 min at 37 °C. After removing the staining
solution, the apoptosis cells were detected using fluorescence
microscopy with excitation wavelength of 346 nm.
A549 cells were transferred to a 6-well plate and incubated
overnight at 37 °C. Complexes 1 and 2 were diluted to the
required concentrations (0.1, 0.5, 2.5, 10 μM, the final
concentration of DMF was less than 0.4%) with culture
medium, and added to the cells for 24 h incubation at 37 °C.
For the negative control, 0.4% DMF was added. Afterwards,
the proteins extracted from the lysate were detached by 8-12%
SDA-PAGE (sodium dodecyl sulfate-polyacrylamide gel Conclusions
electrophoresis) and transferred to the PVDF (polyvinylidene
difluoride) membrane. Then the protein membrane was rinsed In summary, two curcumin-based Ru(II)-polypyridyl complexes were
for 1-2 minutes, and the blocking solution TBST (Tris buffered prepared and characterized. In vitro tests indicated that the
saline plus 0.1% Tween 20) was added. After that, this resulting Ru(II)-polypyridyl complexes showed much superior
membrane was incubated with the preliminary antibody activity to those of reference compounds curcumin, cisplatin,
(MEK1) overnight at 4 °C. A suitable dilution of horseradish [Ru(bpy)3]Cl2 and [Ru(bpy)2(acac)]Cl2 against the tested cancer cell
peroxidase (HRP)-labeled secondary antibody was further lines, especially complex 2, which were 17.0-, 5.7- and 4.8-fold as
added and incubated for 1 h at room temperature. Western potent as [Ru(bpy)3]Cl2, curcumin and cisplatin, respectively,
blotting can be detected using Ingen's Enlight and other demonstrating that the Ru(II)-polypyridyl moieties and curcumin
sensitive ECL luminescent solutions. High-sensitivity ECL have a positive cooperative effect on cancer cells. DNA binding
luminescent solution was used to detect proteins. The loading study revealed that complex 1 may interact with DNA through
control used Glyceraldehyde 3-phosphate dehydrogenase groove binding, while complex 2 is proposed to interact with DNA
(GAPDH).
via intercalation. Further results of western blot assay showed that
the resulting Ru(II) complexes have the potential to prevent the
phosphorylation of MEK1 and ERK1 in A549 cancer cells. Hence, we
Cell Cycle Distribution
proposed that the resulting Ru(II)-polypyridyl-curcuminato
A549 cells were seeded at the density of 2 × 105 per well in a 6- complexes induces apoptosis in cancer cells through DNA
well plate and incubated for 24 h in a 37 °C incubator. The intercalation and by inhibiting the MEK/ERK signaling pathway,
complexes were dissolved with DMF and diluted with medium which is the first example of a Ru(II)-polypyridyl complex to affect
to concentrations of 1 μM and 5 μM. The final concentration ERK signaling pathway as well as DNA interaction. However,
of DMF was less than 0.4%. As for the negative control, 0.4% considering the complexities of the cellular system, there may be
DMF was added. After 24 h incubation, the cells were washed existence of other targets for the greatly enhanced cytotoxicity of 1
with PBS and fixed with 70% ethanol for 24 h at 4 °C. After and 2. Thus, exploring Ru(II)-polypyridyl complexes with multiple
centrifugation to remove ethanol from the sample, the cells targets and different modes of action is an effective approach for
were stained by adding PI (50 μg/mL) and RNase Staining antitumor drug development.
Buffer (100 μg/mL) and incubated for 20 min. Flow cytometry
(FAC Scan, Becton Dickenson) was used to detect the number
of cells in each phase.
Conflicts of interest
Apoptosis studies by Flow Cytometry
There are no conflicts to declare.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
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We are grateful to the National Natural Science Foundation of
China (Grant 21601034)，Jiangsu Province Natural Science
Foundation (Grant BK20160664) and Fundamental Research
Funds for the Central Universities (2242020K40031) for
financial aid to this work.
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Ru(II)-polypyridyl-curcuminato complex induces cancer cells
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DOI: 10.1039/D0DT01040E