Anticancer activity of two ruthenium(II) polypyridyl complexes toward Hepatocellular carcinoma HepG-2 cells

Article abstract of DOI:10.1016/j.poly.2019.05.017

Two novel ruthenium(II) polypyridyl complexes [Ru(bpy)₂(ETPIP)](ClO₄)₂ (Ru(II)-1) and [Ru(phen)₂(ETPIP)](ClO₄)₂ (Ru(II)-2) (bpy = 2,2′-bipyridine, ETPIP = 2-(4-(thiophen-2-ylethynyl)phenyl)-1H-imidazo[4,5-f][1,10]phenanthroline, phen = 1,10-phenanthroline) have been synthesized and characterized. The DNA binding behaviors were evaluated using electronic absorption titration, luminescence spectra and viscosity measurement, revealing an intercalative mode. The cytotoxicity of the ligand and Ru(II) complexes toward A549, HepG-2, SGC-7901 and Hela was assayed by MTT ((3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide)) method. Notably, ETPIP shows no anticancer activity against selected cell lines, and complexes Ru(II)-1 (IC₅₀ = 18.4 ± 2.1 μM) and Ru(II)-2 (IC₅₀ = 16.5 ± 1.7 μM) were found to be slightly more effective against HepG-2 cells than cisplatin (IC₅₀ = 26.4 ± 2.6 μM). Evaluation of cell invasion was performed with the Boyden chamber invasion assay. Additionally, the cell cycle distribution of HepG-2 cells was carried out by flow cytometry. Most importantly, the further anticancer mechanism of the Ru(II) complexes was explored by apoptosis, intracellular reactive oxygen species (ROS) levels and mitochondrial membrane potentials. These results reveal that complexes Ru(II)-1 and Ru(II)-2 could induce apoptosis in HepG-2 cells via a ROS-mediated mitochondrial dysfunction pathway.

Full text of DOI:10.1016/j.poly.2019.05.017

Polyhedron 169 (2019) 209–218
Contents lists available at ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
Anticancer activity of two ruthenium(II) polypyridyl complexes toward
Hepatocellular carcinoma HepG-2 cells
a,
⇑
b
b
b
b
b
b
Guang-Bin Jiang , Wen-Yao Zhang , Miao He , Yi-Ying Gu , Lan Bai , Yang-Jie Wang , Qiao-Yan Yi ,
Fan Du
a
b
Guangxi Key Laboratory of Electrochemical and Magneto-chemical Function Materials, College of Chemistry and Bioengineering, Guilin University of Technology, Guilin
5
41004, China
b
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two novel ruthenium(II) polypyridyl complexes [Ru(bpy) (ETPIP)](ClO ) (Ru(II)-1) and [Ru
2
4 2
Received 10 March 2019
Accepted 9 May 2019
Available online 18 May 2019
0
(phen)
2
(ETPIP)](ClO
4
)
2
(Ru(II)-2) (bpy = 2,2 -bipyridine, ETPIP = 2-(4-(thiophen-2-ylethynyl)phenyl)-
1H-imidazo[4,5-f][1,10]phenanthroline, phen = 1,10-phenanthroline) have been synthesized and charac-
terized. The DNA binding behaviors were evaluated using electronic absorption titration, luminescence
spectra and viscosity measurement, revealing an intercalative mode. The cytotoxicity of the ligand and
Ru(II) complexes toward A549, HepG-2, SGC-7901 and Hela was assayed by MTT ((3-(4,5-dimethylthi-
azo-2-yl)-2,5-diphenyltetrazolium bromide)) method. Notably, ETPIP shows no anticancer activity
Keywords:
Ruthenium(II) polypyridyl complex
DNA binding
Reactive oxygen species
Cell invasion
Mitochondrial membrane potentials
against selected cell lines, and complexes Ru(II)-1 (IC50 = 18.4 ± 2.1
l
M) and Ru(II)-2
M) were found to be slightly more effective against HepG-2 cells than cisplatin
M). Evaluation of cell invasion was performed with the Boyden chamber invasion
(IC50 = 16.5 ± 1.7
IC50 = 26.4 ± 2.6
l
l
(
assay. Additionally, the cell cycle distribution of HepG-2 cells was carried out by flow cytometry. Most
importantly, the further anticancer mechanism of the Ru(II) complexes was explored by apoptosis, intra-
cellular reactive oxygen species (ROS) levels and mitochondrial membrane potentials. These results
reveal that complexes Ru(II)-1 and Ru(II)-2 could induce apoptosis in HepG-2 cells via a ROS-mediated
mitochondrial dysfunction pathway.
Ó 2019 Elsevier Ltd. All rights reserved.
1
. Introduction
nium polypyridyl complexes on the anticancer field have made sig-
nificant progress. It has been reported that [Ru(phen)
2
(MHPIP)]
The discovery of cisplatin as anticancer drug by Rosenberg et al.
(ClO (phen = 1,10-phenanthroline) can effectively inhibit the
4 2
)
opens the new pathway for cancer chemotherapy [1,2]. To date,
cisplatin and its analogues are some of the more effective
chemotherapeutics drugs in clinical use [3–5]. However, even
though the treatment of various malignant cancers with cisplatin
is quite successful, this comes at the price of serious side effects
such as nephrotoxicity, myelotoxicity, neural damage, and ototox-
icity which limit its clinical applicability [6,7]. Therefore, obtaining
other metal-based anticancer agents, which could broaden the
spectrum of anti-tumors, reduce the occurrence of side effects,
and overcome platinum resistance, has attracted widespread
attention [8,9]. It was believed that the complexes containing
ruthenium exhibit low toxicity, easily absorbed and rapidly
excreted by the body, and will become one of the most promising
anti-cancer drugs [10–23]. In recent years, the studies of the ruthe-
proliferation of HepG-2 cells by inducing apoptotic cell death
through ROS-mediated mitochondrial dysfunction pathways [24].
Xu and co-workers first reported that [Ru(N–N)
2
-(1-Py-bC)](PF
6
)
2
0
(N–N = 2,2 -bipyridine (bpy); 1,10-phenanthroline (phen), 4,7-
diphenyl-1,10-phenanthroline (DIP); 1-Py-bC = 1-(2-pyridyl)-b-
carboline) can induce apoptosis and autophagy simultaneously in
Hela cells through a ROS mediated mechanism [25]. Very recently,
2
+
Ru(II) complex [(piq)Ru(bpy)
2
]
(piq = phenylisoquinolinate,
0
bpy = 2,2 -bipyridine) shows high inhibition of cell growth toward
MDA-MB-231 cells [26]. It is worth to mention that two ruthe-
nium-based complexes, NAMI-A and KP1019 have entered clinical
trials [27,28].
The incorporation of heterocycle can partially modify the
biological properties of the parent molecules, thus heterocycle
containing compounds are significant oriented targets in medici-
nal chemistry. In this report, a thiophene-containing ligand
⇑
Corresponding author. Fax: +86 773 899 1304.
E-mail address: jianggb@glut.edu.cn (G.-B. Jiang).
ETPIP
(ETPIP = 2-(4-(thiophen-2-ylethynyl)phenyl)-1H-imidazo
https://doi.org/10.1016/j.poly.2019.05.017
277-5387/Ó 2019 Elsevier Ltd. All rights reserved.
0

2
10
G.-B. Jiang et al. / Polyhedron 169 (2019) 209–218
[
4,5-f][1,10]phenanthroline) and its two ruthenium(II) polypyridyl
complexes [Ru(bpy) (ETPIP)](ClO (Ru(II)-1) and [Ru(phen)
ETPIP)](ClO (Ru(II)-2) (Scheme 1) were synthesized and char-
2.2. Synthesis of ligand and complexes
2
4
)
2
2
(
4
)
2
2.2.1. Synthesis of 4-(thiophen-2-ylethynyl)benzaldehyde
To a resealable Schlenk tube or alternatively, a screw-cap pres-
sure tube, were added 4-bromobenzaldehyde (1.0 mmol,
1
13
acterized by H NMR, C NMR, HRMS and IR. To the best of our
knowledge, this form of ETPIP-containing Ru(II) complexes is used
for the first time as anticancer agents. The interaction of the two
ruthenium(II) polypyridyl complexes with calf thymus (CT DNA)
was investigated via electronic absorption titration, viscosity mea-
surements and luminescence spectra. The cytotoxicity of the ligand
and Ru(II) complexes toward A549, HepG-2, SGC-7901 and Hela
were detected by MTT ((3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl-
tetrazolium bromide)) method. In addition, the possible anticancer
mechanism of the ruthenium(II) polypyridyl complexes was
explored by apoptosis, intracellular reactive oxygen species (ROS)
levels, cell invasion assay, cell cycle arrest, mitochondrial mem-
brane potentials and location in mitochondria. All these results
point out that these thiophene-containing Ru(II) complexes pos-
sess excellent anticancer activity that make them attractive as a
potentially promising candidate for the development of anticancer
agents.
3 2 2
1.0 equiv), CuI (10 mol %), Pd(PPh ) Cl (5 mol%), 2-ethynylthio-
phene (1.3 mmol, 1.3 equiv), Et N (8 mL) and a stir bar. The reac-
3
tion vessel was fitted with a rubber septum, and was evacuated
and back-filled with nitrogen. The reaction tube was sealed and
immersed in a preheated oil bath at 40 °C for 12 h and the solution
was stirred with the aid of a magnetic stirrer. After attaining ambi-
ent temperature, the reaction mixture was diluted with ethyl acet-
ate and filtered through a plug of silica gel. The filtrate was
concentrated and the resulting residue was purified by column
chromatography (silica gel, petroleum ether/EtOAc) to give the
desired substrates.
2.2.2. Synthesis of ligand (ETPIP)
A
mixture of 1,10-phenanthroline-5,6-dione (100 mg,
0
0
.500 mmol), 4-(thiophen-2-ylethynyl)benzaldehyde (106 mg,
.500 mmol), ammonium acetate (15 mmol, 1156.2 mg) and acetic
acid (30 mL) was refluxed with stirring for 4 h. The cooled solution
was diluted with water and neutralized with concentrated aqueous
ammonia. The brown precipitate was collected and purified by col-
umn chromatography on silica gel (60 ꢁ 100 mesh) with ethanol as
2
. Experimental
2.1. Materials and methods
eluent to give the compound as a brown yellow powder. Yield:
All reagents and solvents were purchased commercially and
1
3
38.0 mg, 84%. H NMR (400 MHz, DMSO-d
6
) d 9.02 (s, 2H), 8.91
used without further purification unless otherwise noted. Calf thy-
mus DNA (CT DNA) was obtained from the Sino American Biotech-
nology Company. Ltd. Ultrapure MilliQ water was used in all
experiments. DMSO and RPMI 1640 were purchased from Sigma.
Cell lines of HeLa (Human cervical cancer cell line), SGC-7901
(d, J = 7.7 Hz, 2H), 8.33 (d, J = 7.7 Hz, 2H), 7.95 (s, 1H), 7.80 (dd,
J = 7.1, 3.9 Hz, 2H), 7.76–7.66 (m, 3H), 7.32 (d, J = 4.7 Hz, 1H); IR:
m
1
C
= 3099, 2202, 1887, 1692, 1603, 1555, 1470, 1440, 1400, 1357,
ꢂ1
194, 1120, 943, 835, 733, 616 cm ; HRMS (ESI) m/z: calcd for
+
H
25 15
N
4
S [M+H] , 403.1012; found 403.1017.
(
human gastric carcinoma cells), HepG-2 (Hepatocellular carci-
noma cells) and A549 (Human lung carcinoma cells) were pur-
chased from the American Type Culture Collection. RuCl
3
ꢀ3H
2
O
2 4 2
2.2.3. Synthesis of [Ru(bpy) (ETPIP)](ClO ) (Ru(II)-1)
was obtained from the Kunming Institution of Precious Metals.
A mixture of cis-[Ru(bpy)
2
Cl O (158.7 mg, 0.3 mmol) and
2
]ꢀ2H
2
0
2
,2 -Bipyridine and 1,10-phenanthroline were obtained from the
ETPIP (120.6 mg, 0.3 mmol) in ethylene glycol (12 mL) was heated
at 150 °C under argon for 8 h to give a clear red solution. Upon
cooling, a red precipitate was obtained by dropwise addition of sat-
Guangzhou Chemical Reagent Factory.
Analytical thin layer chromatography was performed by using
commercially prepared 100–400 mesh silica gel plates (GF254
)
urated aqueous NaClO
column chromatography on neutral alumina with a mixture of
CH CN-toluene (1:1, v/v) as eluent. The red band was collected.
4
solution. The crude product was purified by
and visualization was effected at 254 nm. Mass spectra were
recorded on a Thermo Scientific ISQ gas chromatograph-mass
spectrometer. The data of HRMS was carried out on a high-resolu-
tion mass spectrometer (LCMS-IT-TOF). IR spectra were obtained
either as potassium bromide pellets or as liquid films between
two potassium bromide pellets with a Bruker TENSOR 27 spec-
3
The solvent was removed under reduced pressure and a red pow-
1
der was obtained. Yield: 224.5 mg, 73%. H NMR (400 MHz, DMSO-
6
d ) d 8.97 (d, J = 63.5 Hz, 6H), 8.20 (dd, J = 77.3, 35.0 Hz, 8H), 7.81
1
3
(d, J = 86.1 Hz, 12H), 7.36 (d, J = 41.7 Hz, 4H); C NMR (100 MHz,
DMSO-d ) d 157.3, 157.1, 151.9, 149.9, 149.9, 145.4, 138.4, 138.3,
1
trometer. H NMR spectra were recorded on a Varian-500 spec-
6
trometer with DMSO-d
as an internal standard at 400 MHz at room temperature.
6
as solvent and tetramethylsilane (TMS)
132.3, 130.9, 130.8, 130.8, 130.1, 128.4, 128.2, 127.6, 127.2,
126.6, 124.9, 124.9, 124.3, 123.9, 123.9, 121.5, 119.7, 89.0, 87.2;
Scheme 1. The synthetic route of ligand and ruthenium complexes.

G.-B. Jiang et al. / Polyhedron 169 (2019) 209–218
211
IR:
m
= 3074, 1602, 1512, 1477, 1463, 1446, 1271, 1244, 1199, 1093,
uble colored formazan product, which can be measured spec-
trophotometrically after dissolving in DMSO. Cells were seeded
in 96-well plates with 8000 cells/well and divided into control
and treatment group. The tested complexes were then added to
ꢂ1
8
43, 806, 766, 742, 729, 624 cm ; HRMS (ESI) m/z: calcd for C45
-
+
29 8 4
H N RuS [M-2ClO -H] , 815.1285; found 815.1291.
–6
the wells to achieve final concentrations ranging from 10 to
2
.2.4. Synthesis of [Ru(phen)
This complex was synthesized in an identical manner to that
described for complex Ru(II)-1, with cis-[Ru(phen) Cl
]ꢀ2H
23,29]. In place of cis-[Ru(bpy) Cl O. Yield: 254.3 mg, 79%.
]ꢀ2H
) d 8.97 (d, J = 7.5 Hz, 2H), 8.78 (d,
2 4 2
(ETPIP)](ClO ) (Ru(II)-2)
–
4
1
0
M. Control wells were prepared by addition of culture med-
ium (100 L). After 48 h incubation, culture medium was removed
and cells were washed using PBS. 5 mg/mL of MTT was diluted by
PBS (1 mL MTT stock add 10 ml PBS) and 100 L of it was added
l
2
2
2
O
[
2
2
2
1
l
H NMR (400 MHz, DMSO-d
6
into every well. Then, the plate was incubated for 4 h until for-
mazan was produced. The purple-blue formazan precipitate was
dissolved in 100 lL of DMSO and the absorbance values were
J = 7.8 Hz, 4H), 8.40 (s, 4H), 8.31 (d, J = 7.2 Hz, 2H), 8.18 (dd,
J = 26.7, 3.0 Hz, 4H), 8.00 (s, 2H), 7.90 (s, 1H), 7.84–7.70 (m, 6H),
.65 (d, J = 6.8 Hz, 3H), 7.28 (s, 1H); 1 C NMR (100 MHz, DMSO-
3
7
determined at 490 nm by a multi-well plate reader. Data obtained
from at least three separate experiments, while there were
untreated and DMSO treated cells as negative and positive con-
trols, respectively.
d
1
1
8
1
6
) d 153.9, 153.2, 153.1, 150.2, 147.8, 145.7, 137.3, 135.7, 134.0,
32.3, 132.2, 131.1, 131.0, 130.8, 130.7, 130.1, 128.5, 127.6,
27.1, 126.8, 126.8, 126.4, 126.3, 124.4, 124.4, 123.7, 121.5, 89.0,
7.2; IR:
m
= 3066, 1601, 1576, 1511, 1478, 1361, 1250, 1200,
ꢂ1
092, 843, 804, 740, 721, 697, 624 cm ; HRMS (ESI) m/z: calcd
+
29 8 4
for C49H N RuS [M-2ClO -H] , 863.1286; found 863.1289.
2.5. Apoptosis assessment by AO/EB staining
Caution: Perchlorate salts of metal compounds with organic
ligands are potentially explosive, and only small amounts of the
material should be prepared and handled with great care.
Acridine orange (AO) and ethidium bromide (EB) staining
method was carried out to evaluate morphological evidence of
5
apoptosis on the treated cells. Briefly, 2 ꢄ 10 HepG-2 cells were
2.3. Ruthenium(II) complexes-CT-DNA binding
seeded on chamber slides in a 12-well plate and allowed to attach
overnight and the cells were treated with ruthenium(II) complexes
The absorption spectra of target complexes were collected in
(
8.5 mM). After the treatment (24 h), the HepG-2 cells were coun-
the presence of increasing concentration of calf thymus DNA
CT DNA). Stock solutions of target complexes were made by dis-
terstained with acridine orange (AO) and ethidium bromide (EB)
(
ꢂ1
ꢂ1
(
AO: 100 mg mL , EB: 100 mg mL ) and incubated for 10 min.
solution of the complexes in dimethyl sulfoxide and diluting to a
required concentration using a buffer [5 mM Tris–HCl, 50 mM
NaCl, pH 7.0]. The concentration of DNA was calculated using
The unbound dye was removed by washing with PBS and the cells
were fixed with methanol and glacial acetic acid (3:1) for 1 h at
room temperature. The nuclear morphology of the cells was
observed and imaged with a fluorescence microscope (Nikon,
Yokohama, Japan).
ꢂ1
ꢂ1
the molar extinction coefficient of 6600 M cm
at 260 nm
[
30]. DNA was added to both the sample cuvette and the refer-
ence cuvette. After the DNA was allowed to equilibrate with the
complex solution for 5 min, the spectra were measured [31].
The absorption titrations of the complex in buffer were performed
using a fixed concentration (5.0 mM) for complex to which incre-
ments of the DNA stock solution were added. The intrinsic-bind-
2.6. Apoptosis assay by flow cytometry
Induction of apoptosis was studied by Annexin V-FITC and Pro-
ing constant
equation:
b
K was calculated according to the following
5
pidium iodide (PI) binding assay. HepG-2 cells (6 ꢄ 10 ) were trea-
ted with different concentration (8.5 lM and 16.5 lM) of
À
Á
À
Á
Â
À
ÁÃ
½
DNAꢃ=
e
a
ꢂ
e
f
¼ ½DNAꢃ=
e
b
ꢂ
e
f
þ 1= K
b
e
b
ꢂ
e
f
ruthenium(II) complexes at 37 °C for 24 h to quantify normal and
apoptosis HepG-2 cells. After treatment, cells were trypsinized
and resuspended in original media to include the dead cells and
washed with PBS. Cells were pelleted and resuspended in 1X media
binding buffer and then labelled with Annexin V-FITC and propid-
ium iodide (PI) according to manufacturer’s instructions followed
by incubation for 30 min in the dark. The fluorescence intensities
of the cells of each of the three batches were examined in tripli-
cates by FACS Caliber flow cytometer (Beckman Dickinson & Co.,
Franklin Lakes, NJ). A minimum of 10,000 cells were analyzed per
sample. A minimum of 10,000 cells were analyzed per sample.
The fluorescence of cell population and the acquisition was then
performed using FlowJo software.
In this equation, [DNA] is the concentration of DNA added,
a
f b
while e , e and e corresponds to the apparent molar absorptivity,
the molar absorptivity for the free complex [32], and the molar
absorptivity for the complex when saturated, respectively.
The viscosity of a DNA solution was measured in the presence of
increasing amounts of complexes Ru(II)-1 and Ru(II)-2. The flow
time was measured with a digital stopwatch, each sample was
measured at least five times, and then the average flow time was
calculated [33]. Relative viscosities for DNA in the presence and
absence of target complexes were calculated from the relation
0
0
g
= (t – t )/t , where t is the observed flow time of the DNA-con-
0
taining solution and t is the flow time of buffer alone [34,35].
1
/3
The change in the viscosity was presented as (
binding ratio [Ru]/[DNA] [36], where is the viscosity of DNA
is the viscosity of
g
/
g
0
)
versus
g
2.7. Reactive oxygen species (ROS) levels studies
solution in the presence of complexes and
g
0
DNA solution alone.
For measuring the total ROS level, HepG-2 cells were seeded in
a 12-well plate and allowed to attach overnight. Medium was
replaced with fresh medium, cells were treated with ruthenium
2.4. Cytotoxicity assay in vitro
(
II) complexes and allowed to incubate for 24 h. After treatment,
Cytotoxic effect of the complexes against selected tumor cell
lines were determined by a rapid colorimetric assay, using MTT
the cells were washed twice with cold PBS and subsequently pre-
stained with DCFH-DA (10 mM) and incubated at 37 °C in the dark
for 30 min. Finally, the cells were washed with 3ꢄ PBS, and then
imaged and quantitatively analyzed using a confocal fluorescence
microscope.
(
[
3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide)
37]. This assay is based on the metabolic reduction of soluble
MTT by mitochondrial enzyme activity of viable cells into an insol-

2
12
G.-B. Jiang et al. / Polyhedron 169 (2019) 209–218
2.8. The change of mitochondrial membrane potential assay
Table 1. As shown in Table 1, unsurprisingly, ligand ETPIP is found
to display no cytotoxic activity toward the selected tumor cell
The HepG-2 cells were seeded in 12-well plates at a density of
lines. To our delight, complex Ru(II)-1 displayed significant and
selective cytotoxicities with IC50 values lower than 20 lM against
HepG-2 cells. In addition, Ru(II)-2 exhibited excellent activity
5
2
ꢄ 10 cells/mL, and the IC50/2 doses of Ru(II)-1 and Ru(II)-2 were
added to cells. The cells were incubated in 5% CO
2
air-conditioned
atmosphere at 37 °C. After 24 h of incubation, the cells were
washed two times with PBS. Subsequently, JC-1 dye (1 g/mL)
against A549, HepG-2, SGC-7901 and Hela with IC50 values of
26.1 ± 2.6, 16.5 ± 1.7, 28.1 ± 2.6 and 14.9 ± 1.5 lM, respectively.
l
was added and incubated for 20 min in the dark at 37 °C. After
being washed with PBS, the cells were covered with PBS, and
observed under an ImageXpress Micro XLS system.
Most importantly, the anticancer activities of Ru(II)-1 and Ru(II)-
2 are slightly more effective than that of cisplatin against HepG-2
cells under the identical conditions. The difference in cytotoxic
activity of the complexes Ru(II)-1 and Ru(II)-2 toward the
same tumor cell line may be caused by different ancillary
ligands, different ancillary ligands induce different function of
complexes [38].
2.9. Location assay of the complex in the mitochondria
5
HepG-2 cells were seeded in 12-well plates at 2 ꢄ 10 cells/well
and incubated for 24 h at 37 °C and 5% CO . After that, 1.0 mM of the
incubator
2
complexes were added to the wells at 37 °C in a 5% CO
2
3.2. Apoptosis assay with AO/EB and Annex V/PI double staining
methods
for 4 h and further co-incubated with MitoTracker Ò Deep Green
FM (150 nM) at 37 °C for 0.5 h. Upon completion of the incubation,
the wells were washed three times with ice-cold PBS. After
discarding the culture medium, the cells were imaged under an
ImageXpress Micro XLS system.
Disruption or inappropriate regulation of apoptotic and necrosis
processes can result in several diseases including cancer [24]. In
order to observe the morphological changes of HepG-2 cells, acri-
dine orange (AO)/ethidium bromide (EB) fluorescent staining assay
was conducted to distinguish the live, apoptotic and necrotic cells
2.10. Matrigel invasion assay
[
39]. In this method, AO, the vital dye, penetrates into both live and
dead cells and emits green fluorescence when it binds to double-s-
tandard nucleic acid. Ethidium bromide (EB) provides a red–orange
fluorescence by staining fragmented DNA of nuclear membrane
ruptured cells [40]. Therefore, the viable cells will be uniformly
stained green, apoptotic cells are stained green and contain apop-
totic characteristics such as cell blebbing, nuclear shrinkage and
chromatin condensation, necrotic cells are stained as red and can
be found by the AO/EB double staining [38]. After HepG-2 cells
For the cell invasion assays, Matrigel (BD Biosciences) was
thawed on ice at 4 °C overnight and diluted with serum-free med-
ium at a ratio of 1:3. Then, the Transwell chambers were coated
with 30
7 °C for 4 h. Subsequently, 1.5 ꢄ 10 HepG-2 cells in serum-free
RPMI-1640 containing the indicated concentration of ruthenium
II) complexes were seeded into the prepared Transwell chambers.
Afterward, 400 L or RPMI-1640 with 20% FBS and the indicated
lL diluted Matrigel in a 24-well plate and incubated at
5
3
(
l
were exposed to 8.5 lM of different Ru(II) complexes (Ru(II)-1
concentration of ruthenium(II) complexes were added to the lower
and Ru(II)-2) for 24 h, the observations are shown in Fig. 1. Control
cells (a) have shown uniform green cells, which confirm the pres-
ence of live cells. The treatment of HepG-2 cells with Ru(II)-1 (b)
and Ru(II)-2 (c), the apoptotic cells with apoptotic features such
as cell blebbing, nuclear shrinkage and chromatin condensation,
as well as red necrotic cells, were observed. The observations
demonstrated that the complexes Ru(II)-1 and Ru(II)-2 can induce
apoptosis in HepG-2 cells.
chamber. The 24-well plate was then incubated at 37 °C with 5%
CO for 24 h. The cells were fixed and stained as in the migration
2
assay. The membranes were photographed and the invading cells
were counted under a light microscope. The mean values from
three independent assays were calculated.
2.11. Cell cycle arrest by flow cytometry
5
In order to further quantitatively compare the apoptotic effect
of the ruthenium(II) complexes, Annex V/PI double staining was
employed to determine the percentage of apoptotic cells. As shown
in Fig. 2, HepG-2 cells treated with different concentrations of
complexes Ru(II)-1 (b, c) and Ru(II)-2 (d, e) for 24 h, the percent-
age of apoptotic (Q3) cells was increased to 14.6% for Ru(II)-1 and
HepG-2 cells were seeded in 6-well plates at 3 ꢄ 10 cells/well
and incubated overnight to allow attachment. The cells were trea-
ted with the indicated concentration of the target complexes. After
4 h of incubation, HepG-2 cells were trypsinised and washed with
PBS 3 times. The resulting pellet was fixed with 70% ethanol and
stored at ꢂ20 °C for one day. Fixed cells were washed with PBS, fol-
lowed by the addition of 50 mg/mL RNAse and staining with
2
1
5.1% for Ru(II)-2 in comparison with the control (a, 2.6%), indicat-
ing that the Ru(II)-1 and Ru(II)-2 could induce early apoptosis in
HepG-2 cells. In addition, the level of apoptosis shows a concentra-
tion-dependent manner.
5
0 mg/mL propidium iodide (PI) (Sigma-Aldrich, USA). PI binds
DNA as well as RNA. Thus, the addition of RNAse enzyme was
essential to allow PI to bind RNA directly to obtain an accurate
cell-cycle profile. The HepG-2 cells were incubated between
3
0 min and 60 min at 37 °C. Finally, the cell-cycle kinetics were
3.3. Location assay of the complexes and mitochondrial membrane
potential (MMP) analysis
analysed using a FACS Caliber flow cytometer.
Disruption and permanent dissipation of the inner mitochon-
drial membrane potential (DWm) is an event that is associated
3
. Results and discussion
3.1. In vitro cytotoxicity assay
Table 1
IC50 (lM) values of ligand and [Ru] complexes against the selected cancer cell lines.
The cytotoxicity of ETPIP and its two Ru(II) complexes (Ru(II)-1
and Ru(II)-2) in vitro were evaluated on four cancer cell lines
A549, HepG-2, SGC-7901 and Hela) using the MTT method assay.
The preliminary bioassay results were compared with cisplatin.
The activity was expressed as the concentration (IC50) that causes
0% inhibition of cancer cell proliferation and is summarized in
complex
A549
HepG-2
SGC-7901
Hela
(
ETPIP
>100
>100
>100
>100
Ru(II)-1
Ru(II)-2
Cisplatin
38.0 ± 3.5
26.1 ± 2.6
8.2 ± 1.4
18.4 ± 2.1
16.5 ± 1.7
26.4 ± 2.6
41.7 ± 4.3
28.1 ± 2.6
4.4 ± 1.3
21.0 ± 2.3
14.9 ± 1.5
8.3 ± 1.1
5

G.-B. Jiang et al. / Polyhedron 169 (2019) 209–218
213
Fig. 1. Apoptosis in HepG-2 cells (a) exposure to 8.5 mM of complexes Ru(II)-1 (b) and Ru(II)-2 (c) for 24 h and the cells were stained with AO/EB.
Fig. 2. Apoptosis was assayed with Annex V/PI staining HepG-2 cells (a) in the presence of Ru(II)-1 (8.5
16.5 M) (e) for 24 h.
lM) (b), Ru(II)-1 (16.5 lM) (c), Ru(II)-2 (8.5 lM) (d), and Ru(II)-2
(
l
with the intrinsic pathway of apoptosis [41]. Therefore, to further
clarify the possible mechanism of apoptosis. The location of the
ruthenium(II) polypyridyl complexes in the mitochondrial was
detected using Mito TrackerÒ Deep Green FM (ThermoFisher,
the red/green fluorescence of JC-1 by ImageXpress Micro XLS sys-
tem. JC-1 can be aggregated in a MMP-dependent manner in mito-
chondria, where green fluorescence indicates a decrease in MMP
and red fluorescence means high membrane potentials [43]. As
shown in Fig. 4, in the control (a), JC-1 emits red fluorescence cor-
responding to high MMP. HepG-2 cells were incubated with CCCP
(carbonylcyanide-m-chlorophenylhydrazone, b, positive control),
1
00 nM) as a green fluorescent dye [42]. As shown in Fig. 3, in
the control, the mitochondria were stained in green by Mito
Ò
Tracker Deep Green. The treatment of HepG-2 cells with 1.0 lM
of Ru(II)-1 and Ru(II)-2 for 4 h, the complexes emits red fluores-
cence. The merge of the green and red fluorescence indicates that
the ruthenium(II) polypyridyl complexes could arrive the cyto-
plasm through the cell membrane and accumulate in the mito-
chondria. To further evaluate the antitumor mechanism. The
effects of complexes Ru(II)-1 and Ru(II)-2 on the mitochondrial
membrane potential of HepG-2 cells was monitored by detecting
Ru(II)-1 (8.5 lM, c) and Ru(II)-2 (8.5 lM, d) for 24 h, JC-1 emits
green fluorescence corresponding to low MMP. Therefore, the
two Ru(II) complexes caused a remarkable decrease in MMP, as
evidenced by the fluorescence shift from red to green. Subse-
quently, the ratios of red/green fluorescent intensity were also
determined as shown in Fig. 5. In the control, the ratio of red/green
fluorescence is 0.74. Treatment of HepG-2 cells with 16.5 lM of

2
14
G.-B. Jiang et al. / Polyhedron 169 (2019) 209–218
Fig. 3. (A) Location of complexes in the mitochondria in HepG-2 cells exposure to 1.0 lM of complexes Ru(II)-1 and Ru(II)-2 for 4 h.
Fig. 4. The changes of mitochondrial membrane potential was studied after HepG-2 cells (a) were treated with CCCP (b), 8.5
4 h and the cells were imaged under a fluorescent microscope.
lM of complexes Ru(II)-1 (c) and Ru(II)-2 (d) for
2
MMP induced by Ru(II)-1 and Ru(II)-2 were clearly concentra-
tion-dependent in HepG-2 cells.
3.4. Intracellular reactive oxygen species levels determination
Dysfunction of mitochondrial functions, such as the reduction
of mitochondrial membrane potential (MMP), may result in over-
generation of intracellular reactive oxygen species (ROS) [44].
The ROS generated in cancer cells were monitored using the fluo-
0
0
rescent probe 2 ,7 -dichlorodihydrofluorescein diacetate (DCFH-
DA), which could primarily be hydrolyzed and penetrate the cell
membrane to form DCFH [45]. DCFH has non-fluorescence and
cannot pass through the cell membrane. Then, DCFH can be oxi-
dized by ROS into fluorescent DCF. Therefore, the fluorescence
intensity of DCF can reflect the content of intracellular ROS [46].
The DCF fluorescent intensity was tested by ImageXpress Micro
XLS system. As shown in Fig. 6, in the control (a), no obvious green
fluorescence image was detected. After HepG-2 cells were incu-
bated with Rosup (positive control, b), Ru(II)-1 (8.5
lM, c) and
Ru(II)-2 (8.5 M, d) for 24 h, the remarkable fluorescence images
l
Fig. 5. The ratio of the red/green fluorescent intensity was determined after HepG-
*
were observed. These results indicate that ROS in HepG-2 cells
can be generated and complexes (Ru(II)-1 and Ru(II)-2) can
increase the contents of ROS. To further quantitatively evaluate
the levels of the ROS induced by Ru(II)-1 and Ru(II)-2. The DCF flu-
orescent intensity was tested by ImageXpress Micro XLS system. As
shown in Fig. 7, in the control, the DCF fluorescent intensity is 8.6.
2
cells were treated with 8.5 and 16.5 lM of Ru(II) complexes for 24 h P < 0.05
represents significant differences compared with control. (Color online.)
complexes Ru(II)-1 and Ru(II)-2, the ratios of red/green fluores-
cence are 0.51 and 0.54, respectively. The decreases of the ratio
indicate that the green fluorescent intensities increase and the
red fluorescent intensities reduce. In addition, the impairment in
However, HepG-2 cells were treated with 16.5 lM of complexes

G.-B. Jiang et al. / Polyhedron 169 (2019) 209–218
215
Fig. 6. Intracellular ROS was detected in HepG-2 cells (a) exposure to Rosup (b, positive control), 8.5 lM of Ru(II)-1 (c) and Ru(II)-2 (d) for 24 h.
cells were exposed to 8.5
lM of Ru(II)-1 (b) and Ru(II)-2 (c) for
2
4 hour, obvious red fluorescence was observed, indicating that
the complexes can enhance intracellular superoxide anion levels.
To compare the effect of the Ru(II)-1 and Ru(II)-2 on superoxide
anion levels, the fluorescent intensity was tested by ImageXpress
Micro XLS system. As shown in Fig. 9, the red fluorescence inten-
sity follows the order of Ru(II)-2 > Ru(II)-1.
Subsequently, DAF-FM DA was used as a fluorescent indicator of
intracellular nitric oxide (NO). After entering into the cells, this cell
membrane permeable probe was hydrolyzed and then was able to
react with NO to generate strong green fluorescence.[48] As show
in Fig. 10, treatment of HepG-2 cells (a) with 8.5 lM of Ru(II)-1 (b)
Fig. 7. The DCF fluorescent intensity was determined after HepG-2 cells treated
with different concentration of the complexes for 24 h.
Ru(II)-1 and Ru(II)-2, the fluorescent intensities are 40.8 and 70.3,
respectively. Compared the complexes with the control, the fluo-
rescent intensities of DCF grow 4.74 and 8.17 times than the orig-
inal. Unsurprisingly, the level of ROS shows a concentration-
dependent manner.
We also measured intracellular superoxide anion OÅ -
levels of
2
complexes treated HepG-2 cells using a fluorescent probe, dihy-
droethidium (DHE). The non-fluorescent DHE could penetrate cells
Å -
freely and interact with O
2
to form the membrane-impermeant
ethidium cation, which becomes fluorescent upon intercalating
DNA, thus the fluorescence intensity of ethidium-DNA can demon-
Å -
strate the level of intracellular O
2
[47]. As shown in Fig. 8, in the
Fig. 9. The DHE fluorescent intensity was determined after HepG-2 cells treated
with different concentration of the complexes for 24 h.
control (a), feeble red fluorescence was detected. After HepG-2
Fig. 8. The superoxide anion level was assayed after 24 h of HepG-2 cells (a) with 8.5 lM of Ru(II)-1 (b) and Ru(II)-2 (c) and the cells were stained with DHE.

2
16
G.-B. Jiang et al. / Polyhedron 169 (2019) 209–218
Fig. 10. The intracellular NO levels were detected after HepG-2 cells (a) were exposed to 8.5 lM of Ru(II)-1 (b) and Ru(II)-2 (c) for 24 h.
and Ru(II)-2 (c) for 24 h resulted in an increase of green fluores-
cence. To quantitatively the effect of Ru(II)-1 and Ru(II)-2 on intra-
cellular NO levels, the fluorescent intensity was tested by
ImageXpress Micro XLS system, As shown in Fig. 11, in the control,
grow 9.86 and 12.51 times than the control. Furthermore, the level
of NO shows a concentration-dependent manner.
3.5. Transwell cell migration and invasion assay
the fluorescent intensity is 3.7. Compared Ru(II)-1 (16.5
lM) and
Ru(II)-2 (16.5 M) with the control, the fluorescent intensities
l
The migration and invasion are the significant factors in the
process of tumor metastasis [49]. Ru(II)-1 and Ru(II)-2 can induce
apoptosis and effectively inhibit the HepG-2 cells proliferation,
which stimulates us to explore the effects of the complexes on
inhibiting cell invasion [50]. Consequently, cellular migration was
detected by determining the ability of cells to migrate through a
transwell membrane using the Boyden chamber invasion assay
[
8
51]. As shown in Fig. 12, HepG-2 cells (a) were treated with
.5 M of Ru(II)-1 (b) and Ru(II)-2 (c) for 24 h, the number of cell
l
invasion decreases. In addition, the percentage of Ru(II) complexes
inhibiting the cell invasion is calculated in Fig. 13. HepG-2 cells
treated with different concentrations of Ru(II)-1 and Ru(II)-2
showed reduced cell migration from 37.9% to 75.2% as compared
to cells treated with vehicle control. These observations indicated
that Ru(II)-1 and Ru(II)-2 played a crucial role in the inhibition
of HepG-2 migration.
3.6. Cell cycle arrest studies
The proliferation inhibition or death of cells is the result of
apoptosis, cell cycle arrest or a combined action of both [52].
Therefore, the ability of the ruthenium(II) complexes to arrest cell
cycle was studied. As shown in Fig. 14, in the control (HepG-2), the
percentage in the cell cycle at S phase is 22.11%. The treatment of
HepG-2 cells with 8.5 lM of Ru(II)-1 and Ru(II)-2 for 24 h resulted
in an increase of 20.31% and 20.83% in the cells at S phase, respec-
tively, at the same time, a reduction of 26.96% and 25.44% in the
cell at G2/M phase compared with the control were discovered.
The results revealed a cell cycle arrest induced by the complexes
at the S phase in HepG-2 cells.
Fig. 11. The DAF-FMDA fluorescent intensity induced by the complexes was
determined by ImageXpress Micro XLS system. P < 0.05 represents significant
differences compared with control.
*
Fig. 12. Microscope images of invading HepG-2 cells (a) induced by 8.5 lM of Ru(II)-1 (b) and Ru(II)-2 (c) for 24 h.

G.-B. Jiang et al. / Polyhedron 169 (2019) 209–218
217
Fig. 15. The effect of increasing the amounts of the Ru(II)-1 and Ru(II)-2 on the
relative viscosity of CT DNA at 25 (±0.1) °C. [DNA] = 0.25 mM.
6
ꢂ1
Fig. 13. Percentage of inhibiting invasion of HepG-2 cells induced by different
concentration of Ru(II)-1 and Ru(II)-2 for 24 h. P < 0.05 represents significant
differences compared with control.
classical intercalator EB (1.4 ꢄ 10 M ) [55], but were higher than
that of complexes [Ru(dmp) (maip)](ClO (maip = 2-(3-amino-
phenyl)-imizado[4,5-f][1,10]phenanthroline, 3.23 ꢄ 10 M ) [56],
*
2
4 2
)
4
ꢂ1
2
+
and [Ru(dmb)
4,5-f][1,10]phenanthroline, 3.2 ꢄ 10 M ) [57].
The emission intensities of the Ru(II)-1 and Ru(II)-2 from their
2
(BFIP)] (BFIP = 2-benzo[b]furan-2-yl-1H-imidazo
4
ꢂ1
[
MLCT excited states upon excitation at 468 and 466 nm were
found to depend on CT DNA concentration [58]. The luminescence
spectra of complexes Ru(II)-1 and Ru(II)-2 in the presence of
increasing amounts of CT DNA in Tris buffer are shown in Fig. 2S
(
see the Supporting Information for details). With the addition of
DNA, The emission intensities of desired complexes Ru(II)-1 and
Ru(II)-2 grow to 1.42 and 1.43 times larger than the original,
respectively. This indicates that Ru(II)-1 and Ru(II)-2 can strongly
interact with CT DNA, and the complexes can be protected effi-
ciently by the hydrophobic environment inside the CT DNA helix.
In the past few decades considerable progress has been made to
evaluate transition metal complexes as structural probes of DNA. A
series of analytical measures developed to evidence metal com-
plexes-DNA interactions [59]. To further verify the modes of bind-
ing of complexes Ru(II)-1 and Ru(II)-2 to CT DNA, viscosity
measurements of DNA solutions were carried out in the presence
and absence of these complexes. The viscosity of DNA is sensitive
to length changes and is considered as the least equivocal and
the most significant clues of the DNA binding mode in solution
Fig. 14. The cell cycle arrest in HepG-2 cells exposed to 8.5 lM of complexes Ru(II)-
1
and Ru(II)-2 for 24 h.
3.7. Complexes-CT DNA binding studies
[
60]. The changes in the viscosity of CT DNA solution induced by
Electronic absorption spectroscopy is the effective method to
complexes Ru(II)-1 and Ru(II)-2 are shown in Fig. 15. Upon
increasing the concentration of complexes Ru(II)-1 and Ru(II)-2,
the relative viscosity of CT DNA increased obviously. These results
suggest that the target complexes bind CT DNA through a classical
intercalation model. The increased extent of viscosity depending
on its DNA binding strength follow the order of Ru(II)-2 > Ru(II)-
study the interaction of metal complexes with DNA [38]. The
absorption spectra of complexes Ru(II)-1 and Ru(II)-2 consist of
two or three resolved bands in the range 200–600 nm [53]. Absorp-
tion bands below 300 nm are caused by intraligand (IL) transition.
At 320 ? 350 nm (high energy), the spectra display a large change
band, corresponding to ETPIP ?
spectra show a strong MLCT bond at 466–468 nm contributed to
overlap the Ru(d ) ? ETPIP( *) and Ru(d ) ? bpy( *) or phen
*) [54]. Fig. S1 shows spectra obtained at increasing amounts
of CT DNA to Ru(II)-1 (5.0 M) and Ru(II)-2 (5.0 M) (see the Sup-
p* and p ? p* transitions. The
1
, which is consistent with that detected in electronic absorption
titration and luminescence spectra.
p
p
p
p
(p
l
l
4. Conclusions
porting Information for details). With increasing the concentration
of CT DNA, the MLCT transition bands of Ru(II)-1 at 468 nm and Ru
2
Two ruthenium(II) polypyridyl complexes [Ru(bpy) (ETPIP)]
(
II)-2 at 466 nm exhibit obvious hypochromism of about 24.3% and
4 2 2 4 2
(ClO ) (Ru(II)-1) and [Ru(phen) (ETPIP)](ClO ) (Ru(II)-2) were
2
of K
5.7% and bathochromism of 5 and 4 nm, respectively. The values
synthesized and well characterized. The complexes-CT DNA bind-
ing studies imply that the complexes Ru(II)-1 and Ru(II)-2 interact
with CT DNA via intercalative mode. Moreover, all the complexes
4
ꢂ1
5
ꢂ1
b
are 6.83 ꢄ 10 M and 1.00 ꢄ 10 M for the complexes Ru
(
II)-1 and Ru(II)-2, respectively, which were less than that of the

2
18
G.-B. Jiang et al. / Polyhedron 169 (2019) 209–218
[20] X. He, L. Jin, L. Tan, Spectrochim. Acta, Part A 135 (2015) 101.
displayed excellent anticancer activity toward HepG-2 cells. AO/EB
and Annex V/PI double staining studies showed that the complexes
could induce the apoptosis of HepG-2 cells. The location studies
demonstrate that the desired ruthenium(II) polypyridyl complexes
could arrive the cytoplasm through the cell membrane and accu-
mulate in the mitochondria. Additionally, these Ru(II) complexes
can obviously inhibit cell invasion in HepG-2 cells. The cell cycle
arrest studies reveal that complexes Ru(II)-1 and Ru(II)-2 induce
cell cycle arrest of HepG-2 cells at S phase. Further anticancer
mechanistic studies found that both the decrease of the mitochon-
drial membrane potential and increase of ROS contents were all
related to the apoptosis of HepG-2 cells. Consequently, the com-
plexes Ru(II)-1 and Ru(II)-2 induced apoptosis of the HepG-2 cells
through a ROS-mediated mitochondrial dysfunction pathway. This
work will be of helpful for further understanding the DNA binding
and synthesizing novel ruthenium(II) polypyridyl complexes as
potent antitumor agents.
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This work was supported by the Ph. D. Scientific Research Foun-
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