Synthesis, characterization, cellular uptake and apoptosis-inducing properties of two highly cytotoxic cyclometalated ruthenium(II) β-carboline complexes

Article abstract of DOI:10.1016/j.ejmech.2017.09.007

Two new cyclometalated Ru(II) complexes of the general formula [Ru(N-N)₂(1-Ph-βC)](PF₆), where N–N = 4,4′-dimethyl-2,2′-bipyridine (dmb, Ru1), 2,2′-bipyridine (bpy, Ru2), and 1-Ph-βC (1-phenyl-9H-pyrido[3,4-b]indole) is a β-carboline alkaloids derivatives, have been synthesized and characterized. The in vitro cytotoxicities, cellular uptake and localization, cell cycle arrest and apoptosis-inducing mechanisms of these complexes have been extensively explored by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, inductively coupled plasma mass spectrometry (ICP-MS), flow cytometry, comet assay, inverted fluorescence microscope as well as western blotting experimental techniques. Notably, Ru1 and Ru2 exhibit potent antiproliferative activities against selected human cancer cell lines with IC₅₀ values lower than those of cisplatin and other non-cyclometalated Ru(II) β-carboline complexes. The cellular uptake and localization exhibit that these complexes can accumulate in the cell nuclei. Further antitumor mechanism studies show that Ru1 and Ru2 can cause cell cycle arrest in the G0/G1 phase by regulating cell cycle relative proteins and induce apoptosis through mitochondrial dysfunction, reactive oxygen species (ROS) accumulation and ROS-mediated DNA damage.

Full text of DOI:10.1016/j.ejmech.2017.09.007

Accepted Manuscript
Synthesis, characterization, cellular uptake and apoptosis-inducing properties of two
highly cytotoxic cyclometalated ruthenium(II) β-carboline complexes
Jincan Chen, Fa Peng, Yao Zhang, Baojun Li, Ji She, Xinming Jie, Zhilin Zou, Man
Chen, Lanmei Chen
PII:
S0223-5234(17)30689-X
10.1016/j.ejmech.2017.09.007
EJMECH 9722
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 July 2017
Revised Date: 26 August 2017
Accepted Date: 4 September 2017
Please cite this article as: J. Chen, F. Peng, Y. Zhang, B. Li, J. She, X. Jie, Z. Zou, M. Chen, L. Chen,
Synthesis, characterization, cellular uptake and apoptosis-inducing properties of two highly cytotoxic
cyclometalated ruthenium(II) β-carboline complexes, European Journal of Medicinal Chemistry (2017),
doi: 10.1016/j.ejmech.2017.09.007.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Synthesis, characterization, cellular uptake and apoptosis-inducing
properties of two highly cytotoxic cyclometalated ruthenium(II)
β-carboline complexes
The cellular uptake, in vitro cytotoxicities, cell cycle arrest and apoptosis-inducing
mechanism of two new cyclometalated ruthenium(II) β-carboline complexes have
been extensively explored by ICP-MS, MTT assay, flow cytometry, inverted
fluorescence microscope as well as western blotting experimental techniques.

ACCEPTED MANUSCRIPT
Synthesis, characterization, cellular uptake and apoptosis-inducing
properties of two highly cytotoxic cyclometalated ruthenium(II)
β-carboline complexes
a,b
c
c
c
c
a
d
Jincan Chen , Fa Peng , Yao Zhang , Baojun Li , Ji She , Xinming Jie , Zhilin Zou ,
d
c*
Man Chen , Lanmei Chen
a
Analysis Centre of Guangdong Medical University, Zhanjiang 524023, China
b
Guangdong Key Laboratory for Research and Development of Nature Drugs, Guangdong
Medical University, Zhanjiang 524023, China
c
School of Pharmacy, Guangdong Medical University, Zhanjiang 524023, China
d
The First Clinical Medical College, Guangdong Medical University, Zhanjiang 524023, China
*
Corresponding author. Tel./fax: Tel: 86-759-2388-568
E-mail addresses: lanmeichen@126.com
Abstract
Two new cyclometalated Ru(II) complexes of the general formula
[
Ru(N-N) (1-Ph-βC)](PF ), where N–N = 4,4’-dimethyl-2,2’-bipyridine (dmb, Ru1),
2 6
2
,2’-bipyridine (bpy, Ru2), and 1-Ph-βC (1-phenyl-9H-pyrido[3,4-b]indole) is a
β-carboline alkaloids derivatives, have been synthesized and characterized. The in
vitro cytotoxicities, cellular uptake and localization, cell cycle arrest and
apoptosis-inducing mechanisms of these complexes have been extensively explored
by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay,
inductively coupled plasma mass spectrometry (ICP-MS), flow cytometry, comet
assay, inverted fluorescence microscope as well as western blotting experimental
techniques. Notably, Ru1 and Ru2 exhibit potent antiproliferative activities against
selected human cancer cell lines with IC values lower than those of cisplatin and
5
0
other non-cyclometalated Ru(II) β-carboline complexes. The cellular uptake and
localization exhibit that these complexes can accumulate in the cell nuclei. Further
antitumor mechanism studies show that Ru1 and Ru2 can cause cell cycle arrest in
1

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the G0/G1 phase by regulating cell cycle relative proteins and induce apoptosis
through mitochondrial dysfunction, reactive oxygen species (ROS) accumulation and
ROS-mediated DNA damage.
Key words: Cyclometalated Ru(II) complexes; β-carboline alkaloids; Cytotoxicity;
Apoptosis; Mitochondrial dysfunction
1
. Introduction
Cisplatin, which discovered in 1969, is one of the foremost and widely used metal
based anticancer drugs for cancer therapy [1,2]. However, significant side effects and
drug resistance limited its clinical applications and have also stimulated the
development of non-platinum metal-based therapeutics [3]. Ruthenium-based
complexes have been regarded as one of the most promising alternatives because of
their rich synthetic chemistry, variable oxidation states and potential use as
therapeutic anticancer agents with low toxicity to normal cells [4,5].
A number of ruthenium compounds show promising anticancer activity, and some
of them exhibit a cytotoxic potency similar to or better than that of cisplatin [6-11].
6
+
For example, the piano-stool complexes [(η -arene)RuCl(en)]
(en
=
1
,2-ethylenediamine), exhibit anticancer activities in vitro comparable to that of
cisplatin in a number of cancer cell lines [12,13]. The RAPTA complexes
6
[
(η -arene)-RuCl (pta)] (pta = 1,3,5-triaza-7-phosphatricyclo-[3.3.1.1]-decane), are
2
known to have selective activity on metastatic tumors both in vitro and in vivo similar
to that of NAMI-A [14,15]. Recently, it has been noted that the cyclometallated Ru(II)
complexes, in which one or more of the nitrogen donors of ruthenium complexes are
replaced by a carbon donor atom, exhibit promising cytotoxic properties, and some of
which are even more efficient than cisplatin [16-18]. Moreover, it has been found that
the cyclometalation of Ru(II) complexes can efficiently enhance the lipophilicity of
these complexes and significantly improve their cellular uptake [18-20], which are
important factors influencing their cytotoxicities.
Some previous works have showed that Ru(II) polypyridyl complexes with
2

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bioactive alkaloids as ligands offers new opportunities for the design of novel
anticancer drugs with enhanced activity [21-23]. The β-carboline alkaloids are a class
of synthetic and naturally occurring compounds that possess promising biological
activity and pharmacological functions including sedative, antiviral, antimicrobial and
anticancer activities [24]. It has been reported that β-carboline alkaloids can exert
antitumor activities through multiple mechanisms, such as interfering with DNA
synthesis [25], inhibiting DNA topoisomerases I and II [26, 27] etc. Some
ruthenium(II)-β-carboline complexes with [Ru(tpy)(N-N)(L)] type reported by Chen
et al. show anticancer activity against various cancer cells [28]. Another ruthenium(II)
2
+
β-carboline complex [Ru(tpy)(Nh)3] (Nh=Norharman) also synthesized by this
research group exhibits high and selective cytotoxicity induced p53-mediated
apoptosis [29]. A series of ruthenium(II) complexes containing a β-carboline alkaloid
as ligand reported by Tan et al. can simultaneously induce autophagy and apoptosis in
tumor cells, and among of which, the most active drug, complex
[
Ru(DIP) (1-Py-βC)](PF6) (DIP= 4,7-diphenyl-1,10-phenanthroline), exhibits higher
2 2
cytotoxic potency than cisplatin [30].
These findings encouraged us to further optimized these Ru(II) β-carboline
complexes by improving their cytotoxicity and their water solubility. We focus on the
cycloruthenated Ru(II) complex of 1-phenyl-9H-pyrido[3,4-b]indole (1-Ph-βC). So in
+
this work, two new cyclometalated Ru(II) complexes [Ru(dmb) (1-Ph-βC)] (Ru1)
2
+
and [Ru(bpy) (1-Ph-βC)] (Ru2) contained the bioactive β-carboline derivatives as
2
1
ligands were designed, synthesized and characterized by ESI-MS, H-NMR and
UV-Vis. For elucidating the anticancer functions and molecular mechanisms of Ru1
and Ru2, the cytotoxicity, cellular uptake and localization, apoptosis, cell cycle arrest,
mitochondrial membrane potential, reactive oxygen species and DNA damage in
HeLa cells have been investigated.
3

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Fig.1. The structures of 1-Ph-βC, dmb, bpy, Ru1 and Ru2.
2
. Results and discussion
2
.1. Synthesis and characterization
The ligand 1-phenyl-9H-pyrido[3,4-b]indole (1-Ph-βC) was synthesized
according to the literature method [31]. Ru1 and Ru2 were prepared by the reaction
of cis-[Ru(N-N) Cl ] (N-N = dmb, bpy) with 1-Ph-βC in ethyl alcohol at room
2
2
temperature for 12 h. The synthetic route of the ligand 1-Ph-βC and title complexes
Ru1 and Ru2 was presented in Supporting Information (Fig. S1). The desired Ru(II)
complexes were isolated as the chloride salt, and purified by column chromatography.
The ligand and the formation of the complexes were confirmed by elemental analysis,
1
ESI-MS and H NMR (Fig. S2-S5). In the ESI-MS spectra for the Ru(II) complexes,
+
all of the expected signals [M-PF ] were observed. The measured molecular weights
6
were in accordance with expected values.
2
.2. UV-visible spectral studies
The stability of Ru1 and Ru2 in PBS solutions at 298 K was analyzed by UV-Vis
4

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absorption spectroscopy. As shown in Fig. S6, there is no obvious change in the
UV-Vis absorption spectra of Ru(II) complexes during 24 h at 298 K. From Fig. S6
we can see that, both two complexes display several absorption bands in the UV and
visible spectral regions. The intense bands occurring between 250 nm and 330 nm are
attributed to an intraligand (IL) π–π* transition, and the bands at the range of 330–400
nm are attributed to π–π* transitions. The bands occurring in the visible region can be
assigned to metal-to-ligand charge transfer transitions (MLCT) [32]. In Ru1, two
intense absorption bands are observed at 449 nm and 509 nm. The latter is attributed
2
+
2+
to Ru → dmb transitions, and the band centered at 449 nm is assigned to Ru →
-Ph-βC metal-to-ligand transition [32]. Similar observations are obtained for the Ru2
complex in the visible region. In Ru2, two intense bands are centered at 436 nm and
1
4
96 nm.
Electronic absorption spectroscopy is the commonest means to study the interaction
of metal complexes with DNA [33]. The spectral profile of the complexes Ru1 and
Ru2 in the increasing concentration of CT DNA is shown in Fig. S7. As the DNA
concentration increased, the metal-to-ligand charge transfer (MLCT) bands of Ru1 at
4
49 nm, Ru2 at 436 nm exhibit hypochromism of about 14.0% and 23.4%,
respectively. These spectral characteristics obviously suggest that these complexes
interact with DNA most likely through a mode that involves a stacking interaction
between the aromatic chromophore and base pairs of DNA. To further evaluate the
binding strength of the complexes with DNA, the intrinsic binding constant K was
b
determined by monitoring the changes in the absorbance at the MLCT band. The Kb
3
−1
4
−1
values of Ru1 and Ru2 are 4.6×10 M and 3.1×10 M , respectively. These values
+
5
−1
are smaller than that of [Ru(bpy)(phpy)(dppz)] (8.5×10
M ) [17],
2
+
6
−1
[
Ru(bpy) (1-Py-βC)]
(1.08×10 M )
[30] and ruthenium methylimidazole
2
2
+
5
−1
complex [Ru(MeIm) (p-cpip)] (5.5×10 M ) [34]. Moreover, it is suggest that
4
complex Ru1 shows the less binding strength with CT-DNA than complex Ru2
because of the steric hindrance caused by the methyl groups present at the 4 and 4′
positions of the ancillary ligand dmb [35].
5

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2
.3. Cytotoxicity assay in vitro
Table 1
IC values (µM) of Ru1 and Ru2 against the selected human cancer cell lines .
a
50
IC (µM)
5
0
Complex
A549
>200
>200
HeLa
>161 ± 5.5
>200
NCl-H460
>200
HepG-2
>168± 6.2
>181 ± 7.2
102.2 ± 2.8
10.3 ± 1.2
15.8 ± 2.1
86.2 ± 12.3
30.1 ± 2.0
MCF-7
>200
BEAS-2B
--
cis-[Ru(dmb) Cl ]
2
2
cis-[Ru(bpy) Cl ]
>200
>200
--
2
2
1-Ph-βC
79.5 ± 2.6
2.2 ± 0.2
3.6 ± 0.3
--
45.8 ± 3.8
1.9 ± 0.4
3.4 ± 0.3
61.2 ± 3.9
18.2± 1.2
75.2 ± 3.2
3.8 ± 0.5
4.9 ± 0.8
--
99.7± 3.8
17.2 ± 2.2
18.4 ± 1.2
102.2 ± 14.5
16.2 ± 0.9
>100
Ru1
Ru2
10.6 ± 0.6
11.2 ± 0.5
--
b
Ru-Py [30]
Cisplatin
27.2± 1.4
33.2±2.2
13.2± 0.7
a
Cell viability was determined by MTT assay after treatment for 48 h.
b
Ru-Py = [Ru(bpy) (1-Py-βC)](PF ) , 1-Py-βC = 1-(2-pyridyl)-β-carboline.
2
6 2
The ligand 1-Ph-βC, two Ru(II) synthetic precursors cis-[Ru(dmb) Cl ] and
2
2
cis-[Ru(bpy) Cl ], and corresponding cyclometalated Ru(II) complexes Ru1 and Ru2
2
2
were evaluated against five selected human cancer cell lines (lung adenocarcinoma
cell A549, human lung cancer NCl-H460, hepatocellular carcinoma HepG2, breast
cancer MCF-7 and cervical cancer HeLa). For comparison, the toxicity of cisplatin
was also evaluated. As a control, the toxicity of these complexes was also tested
against the normal human cell line (immortalized human bronchial epithelial cells
BEAS-2B). Table 1 shows the IC values of the different complexes as determined
5
0
by an MTT assay after a 48 h incubation. Significant differences are observed that
exceeded expectations with Ru1 and Ru2 exhibiting much higher activities (IC50
values ranged from 1.9 to 18.4 µM) than cis-[Ru(dmb) Cl ] and cis-[Ru(bpy) Cl ],
2
2
2
2
which are almost inactive against all the cell lines tested (IC > 200 µM). The ligand
5
0
1
-Ph-βC presents moderate activities (IC50 values ranged from 45.8 to102.2 µM).
6

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Notably, against the human cervical cancer cell line HeLa, Ru1 and Ru2 display an
IC values (1.9 µM and 3.4µM, respectively) that are over 32- and 18-fold lower than
5
0
that of non-cyclometalated Ru(II) β-carboline complex Ru-Py (61.2 µM) [30].
Moreover, the activities of Ru1 and Ru2 are about 9-fold and 5-fold higher than that
of cisplatin against HeLa cells under the same conditions.
Therefore, our results clearly show that the coordination of the cyclometalated
ligand 1-Ph-βC to polypyridyl-Ru(II) centers is a significant strategy to results in
more cytotoxic complexes. Interestingly, Ru1 is more potent than Ru2 against all the
cancer cell lines screened under identical condition at 48 h treatment, especially HeLa
cells. Thus, their cytotoxic effect against HeLa cells was further evaluated. Data
obtained are revealed in Fig. S8, Ru1 and Ru2 at indicated concentrations resulted in
marked reduction of the viability of HeLa cells in a concentration- and
time-dependent manner. Moreover, we noticed that the order of DNA binding affinity
is K (Ru1) < K (Ru2), which is not consistent with their antiproliferative activities.
b
b
So we deduced that DNA may be not the main target of these ruthenium complexes.
2
.4. The cellular uptake and localization of Ru(II) complexes
7

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Fig.2. (A) The logPo/w values of Ru1 and Ru2. (B) Cellular ruthenium concentrations determined
in HeLa cells after 1, 3 and 6 h incubated with Ru1 and Ru2 at 2, 4, 6, 8 µM, respectively. (C)
Intracellular uptake of Ru1 in HeLa cells under different endocytosis-inhibited conditions. Before
6
h incubation of Ru1 (4 µM), cells were incubated with specific endocytosis inhibitors, see the
experimental section for details. (D) Subcellular distribution of Ru1 in HeLa cells after incubated
with 4 µM of Ru1 for different times.
It is well known that lipophilicity of an anticancer agent has a vital influence on
its cytotoxicity. For some ruthenium [30,36,37] and platinum based compounds
[
38,39], increasing lipophilicities enhances the cellular uptake and, consequently, the
cytotoxic activities. Oil–water partition coefficient (logP ) provides a measure of
o/w
drug lipophilicity, which indicates the ability of the molecule to pass through cell
8

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membranes. “shake-flask” method was used to evaluate the logPo/w values of Ru1 and
Ru2 by using ICP-MS. As shown in Fig. 2A, both Ru1 and Ru2 exhibit positive
logPo/w values, showing them to be lipophilic in nature. Moreover, Ru1 (logPo/w
=
1
.38 ± 0.06) displays higher logPo/w value than Ru2 (logPo/w = 1.22 ± 0.04). Such
result is in accordance with the reported that a positive correlation between
hydrophobicity and cytotoxic activity for several classes of metal-based anticancer
complexes [30,40-42].
The cellular uptake characteristics of transition metal-based drugs are considered
to be one important factor influencing their cytotoxicity [43-45]. To investigate a
possible relationship between the dose of cellular uptake and cytotoxic activities of
title complexes, the quantitative determination of the ruthenium level inside the HeLa
cells has been performed by ICP-MS, and the results were represented as ng of Ru per
6
1
0 cells. As shown in Fig. 2B, the cellular uptake of Ru1 and Ru2 exhibit a
significantly dose- and time-dependent manner. In additon, Ru1 displays higher
cellular uptake than that of Ru2 in all of the test times and drug concentrations, which
is positively correlated to their cytotoxicity and lipophilicity.
The approaches of drugs entry into cells have important effect on their
bioactivities. To elucidate the anticancer mechanism of title complexes, we investigate
the routes of the cellular entry. Normally, small drug molecules enter into cells mainly
through two pathways, namely, energy-dependent (such as endocytosis and active
transport) and non-energy-dependent (facilitated diffusion) [46,47]. To get more
insight into the internalization pathway of Ru1, we treated HeLa cells with Ru1 at
low temperature (4 °C) or pretreated with oligomycin in combination with DOG,
which can block adenosine 5′-triphosphate (ATP) production to discriminate between
passive and active mechanisms. As shown in Fig. 2C, low temperature or oligomycin
in combination with DOG can strongly inhibit the cellular uptake of Ru1, indicating
that the energy-dependent active transport is involved in the cellular uptake of Ru1.
Endocytosis is the most common energy-dependent method. Next, we further
analyzed the internalization pathway of Ru1 in the presence of two endocytosis
inhibitors nystatin (clathrin-mediated endocytosis) and sucrose (lipid raft-mediated
9

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endocytosis) [48]. After treatment with both endocytosis inhibitors, we found that
cellular uptake level of Ru1 was significantly reduced in the nystain group and
moderately decreased in the sucrose group. These results suggested Ru1 can be
transported into the cells mainly through clathrin-mediated endocytosis pathway.
It is widely considered that TfR, a transmembrane glycoprotein, is overexpressed
on the membrane of many cancer cells. TfR-directed targeting cellular uptake has
been regarded as an efficient way for delivery of metal-based agents to malignant
tissues [49,50]. Guo et al find that Tf/TfR serves as a mediator enhanced the delivery
of organometallic Ru(II) complexes into tumor cells [50]. Thus, we speculate that the
intracellular uptake of Ru1 in HeLa cancer cell lines may associate with its transferrin
receptor (TfR) expression profiles. The results in Fig. 2C demonstrate that the
intracellular uptake of Ru1 is barely blocked by pretreatment of anti-TfR, indicating
TfR-mediated endocytosis is not involved in the uptake of Ru1 in HeLa cells.
In addition, the subcellular distribution of Ru1 in HeLa cells was further
determined by ICP-MS. Fig. 2D showed that Ru1 was predominantly accumulated in
the nuclei and only a small fraction of complex was found in mitochondria and
cytoplasm, which implied the localization of Ru1 in the nucleus. At a dose of 4 µM,
treatment with Ru1 for 6 h, nearly 80% of Ru1 within the HeLa cells was localized in
the nuclei. These results suggest that the cyclometalated complex Ru1 is a
nuclei-targeting Ru(II) complex, like cyclometalated Ru(II) complex,
+
[
Ru(bpy)(phpy)(dppz)] [17].
2
.5. Ru(II) complexes induce cell cycle arrest by regulating cell cycle regulatory
proteins
1
0

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Fig.3. Ru(II) complexes induced cell cycle arrest in HeLa cells. (A) Cell cycle distribution was
performed by PI staining after co-incubated with Ru1 and Ru2 for 24 h. (B) The effects of Ru1 on
the expression levels of cell cycle relative proteins were performed by western blotting, β-actin
was used as internal control. HeLa cells were treated with various concentrations of Ru1 for 24 h.
It is reported that inhibition of cancer cell proliferation by cytotoxic drugs could be
the result of cell cycle arrest, apoptosis or their combination [51]. To investigate
whether the anti-proliferative effect of Ru1 and Ru2 on HeLa cells was triggered by
cell cycle arrest, the cell cycle phase ratio was measured by flow cytometry with
propidium iodide (PI) staining. As shown in Fig. 3A, for both Ru1 and Ru2 treatment
groups, the percentage of cells is increased at G0/G1 phase and decreased at S and
11

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G2/M phases in a concentration-dependent manner. In additon, Fig. 3A also displays
that exposure of the HeLa cells to title complexes results in marked dose-dependent
increase in the proportion of apoptotic cells as reflected by the subdiploid peak
(sub-G1). The value of sub-G1 of Ru1 treatment group varies from 13.63% (1.0 µM)
to 40.99% (4.0 µM) with increasing treated concentration. For Ru2 treatment group,
this value varies from 6.88% (1.0 µM) to 30.07% (4.0 µM). Meanwhile, Fig. S9
shows that the cells in the sub-G1 phase in these Ru1-treated groups also significantly
increase in a time-dependent manner, when compared with DMSO treated control.
Obviously, these results demonstrate that the antiproliferative effect induced by Ru1
and Ru2 on HeLa cells occurs in G0/G1 phase.
To investigate the molecular basis by which Ru(II) complexes inhibited the G0/G1
transition in cancel cells, we treated cells with Ru1 and then analyzed the expression
of proteins involved in cell cycle regulation. We found that Ru1 treatment inhibited
Cyclin D1/D3 and cyclin E expression and reduced the expression of CDK4 and
CDK6; in contrast, p27, p21 and p18 were increased in HeLa cells (Fig. 3B). Cyclin
D and cyclin E (along with CDK2, CDK4, and CDK6) play important roles in the
progression of cells through the G1 phase of the cell cycle [52]. The Cip/Kip family
proteins p27, p21 and p18 are well-known CDK inhibitors, up-regulation the
expression levels of those can block G1-S transition [53]. The expression level of cell
division cycle 25A (CDC25A), which acts as an upstream regulator of the
CDK/cyclin complex [54], was significantly inhibited by Ru1. Ru1 treatment also
increased the expression of p53 and phosphorylated p53 at ser15 site (p-p53ser15),
which is tumor suppressor proteins, up-regulation the expression levels of those can
promote the cell cycle arrest and induce cell apoptosis [55,56].
2
.6. Ru(II) complexes induce HeLa cell apoptosis
1
2

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Fig.4. Ru(II) complexes induce HeLa cells apoptosis. (A) HeLa cells stained with Hoechst 33342
after treatment of Ru(II) complexes at indicated concentrations for 24 h. (B) HeLa cells apoptosis
was detected by annexin V/PI assay after co-incubation with various concentrations of Ru(II)
complexes for 24 h. (C) Caspase activities were measured by using specific fluorescent substrates
for caspase-3/8/9. (D) The expression levels of caspase-3, PARP and cleaved caspase-3/8/9 and
PARP were evaluated in a dose-dependent manner with Ru1 treatment for 24 h. β-actin was used
as internal control.
1
3

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Cell cycle arrest triggered by various stimulating factors may result in the blockage
of cell division and apoptosis. To determine whether or not Ru1 and Ru2 could
induce chromatin condensation and fragmentation, both of which were recognized as
morphological features of apoptosis, the Hoechst 33342 staining technique for
fluorescence microscopy was performed. HeLa cells were treated with different
concentrations of Ru(II) complexes for 24 h, and then photographed using an inverted
fluorescence microscope. As shown in Fig. 4A, the cells with apoptotic features such
as nuclear fragmentation, chromatin condensation and plasma membrane blebbing are
observed. In addition, the apoptosis assay was also performed by acridine
orange/ethidium bromide (AO/EB) dual-staining method. AO is a vital dye and can
stain both live and dead cells and shows green fluorescence. EB stains only apoptotic
or necrotic cells that have lost their membrane integrity and appear red fluorescence.
Fig. S10 displays that, in control cells, the normal morphology and homogeneous
green fluorescence are obtained. When cells are pre-incubated with Ru1 and Ru2, the
orange or red fluorescence, that are the clearly morphological features of apoptosis,
are observed. These preliminary results indicate that both Ru1 and Ru2 can induce
apoptosis of HeLa cells.
To further confirm the nature of cell death induced by Ru1 and Ru2, annexin
V-FITC/PI staining was performed, and the results were analyzied by using flow
cytometry. Fig. 4B shows that pre-incubation of HeLa cells with different
concentrations of Ru1 or Ru2 for 24 h enhances the percentage of apoptotic cells.
Comparing the apoptotic effect, we can find that Ru1 displays more effective
apoptotic activity than Ru2 under identical conditions, which is consistent with their
cytotoxic activity.
A large number of evidence has indicated that caspases play vital roles for initiation
and performance of apoptosis [57-59]. Apoptotic pathways can be divided into the
extrinsic and intrinsic pathways, which are dependent on the cleavage of caspases
[
60]. Caspase-3 is a vital regulatory protein of cell apoptosis, the activation of which
lead to the cleavage of poly ADP-ribose polymerase (PARP), thus to induce the cells
1
4

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apoptosis [61]. To further clarify the mechanism of cells apoptosis induced by Ru1,
the activities of caspase-3, -8 and -9 were examined by using specific fluorescent
substrates. As shown in Fig. 4C, pre-incubation of HeLa cells with different
concentration of Ru1 exhibits dramatic enhancement in caspase-3, -8 and -9 activities.
Besides, the results of western blot assay in Fig. 4D illustrate that the expression
levels of cleaved-PARP, cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9
increase in a dose- dependent manner, and the expression level of total caspase-3
decrease in a dose- dependent manner, which suggesting both extrinsic and intrinsic
apoptosis pathways are involved in Ru1-induced apoptosis in HeLa cells.
2
.7. Ru(II) complexes induce mitochondrial dysfunction
1
5

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Fig.5. Ru1 and Ru2 induce mitochondrial dysfunction. (A) Fluorescence microscope analysis of
cellular MMP level by JC-1 staining after 2 µM of Ru1 or Ru2 treatment for 24 h. (B) Flow
cytometry analysis of cellular MMP level after 1, 2 and 4 µM of Ru1 or Ru2 treatment for 24 h.
(C) Real-time imaging the same cells treated with 2 µM of Ru1 for different times. Cell
morphology was captured by differential reflection (DIC) microscope. Mitochondria and
nucleuses were visualized by red and blue fluorescence, respectively. The bottom panel is DIC
images. (D) The expression levels of Bcl-2 family protein were performed in a dose-dependent
1
6

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manner. β-actin was used as internal control. (E) The expression levels of cytochrome c in cytosol
and in mitochondria, respectively, were performed after 1, 2, 4 µM of Ru1 treatment for 24 h.
Mitochondrion, which plays an important role in apoptosis, can release
pro-apoptotic factors such as cytochrome c and other apoptosis inducing factors.
[
17,62]. Mitochondrial dysfunction is involved in apoptotic cell. Generally, the
mitochondrial dysfunction is determined by measuring changes in the loss of
mitochondrial membrane potential (MMP) using an inverted fluorescence microscope
and
,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidalylcarbo cyanine iodide (JC-1) as
fluorescent probe. As shown in Fig. 5A, compared with control, Ru1 (2.0 µM) or Ru2
2.0 µM) treatment cause a red to green color shift in most of the treated cells,
flow
cytometry
after
staining
live
cells
with
5
(
indicating loss of MMP. The changes of MMP were also detected quantificationally
by determining the percentage of the red and green fluorescent intensity using flow
cytometry (Fig. 5B). As shown in Fig. 5B, after 6 h of incubation with Ru(II)
complexes, the green fluorescence of the JC-1 monomers increase from 2.7% to
7
4.5% and 48.4% for Ru1 (4 µM) and Ru2 (4 µM), respectively.
To determine whether or not, mitochondria are pivotal in controlling cell growth
and death, real-time living cell experiment was carried out. We observed the changes
of mitochondria (stained by Mito-Tracker; nuclei stained by DAPI) in HeLa cells
treated with 2.0 µM of Ru1 for different time intervals. As shown in Fig. 5C, obvious
mitochondrial fragmentation, nuclear condensation and cytoplasmic shrinkage can be
observed after treatment for 3 h, further indicating the mitochondrial dysfunction.
Additionally, western blot analysis was performed to confirm the mitochondrial
pathways in apoptosis which induced by Ru1. The results in Fig. 5D indicate that
treatment with Ru1 dose dependently suppresses the expression of the anti-apoptotic
protein Bcl-xl and Bcl-2. As a result of these changes, the ratios of Bcl-2/Bax and
Bcl-xl/Bad decrease, and thus leading to the release of cytochrome c (an apoptosis
inducing factor) into the cytosol. Actually, the experimental results suggest that
cytochrome c of cytosol increase in a dose-dependent manner, while those in
1
7

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mitochondria decrease (Fig. 5E). This result further confirms that Ru1 induces
mitochondrial dysfunction and the mitochondrial pathway is involved in Ru1-induced
apoptosis in Hela cells.
2
.8. Ru(II) complexes induce intracellular ROS accumulation
Fig.6. (A) ROS generation was determined by flow cytometry, HeLa cells stained with DCFH-DA
after Ru(II) complexes treatment for 6 h. (B) The cellular ROS level was detected by amicroplate
reader after exposure to Ru1 for different times in HeLa cells. (C) Flow cytometry analysis of
1
8

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cellular ROS level by DCFH-DA staining after 2.0 µM of Ru1 treatment for 6 h. (D) Cells
apoptosis were detected by flow cytometry, HeLa cells were treated with Ru1 for 24 h with or
without pretreatment of NAC and GSH.
ROS, which regard as mediators of apoptosis, are important for the induction of
apoptosis in many cancer cells [63,64], and could enter the nucleus to cause DNA
damage [65]. To investigate whether Ru(II) complexes treatment could increase the
ROS level in HeLa cells, the cellular ROS level was detected by using an inverted
fluorescence microscope and flow cytometry after cells stained with a DCFH-DA
fluorescent dye. Fig. S11A displays that, compared with DMSO-treated control, HeLa
cells exhibite obvious green fluorescence when treated with 2.0 µM of Ru1 or Ru2
for 6 h, which indicates an increase in the cellular ROS level. A similar result is also
obtained by flow cytometry. As shown in Fig. 6A, at a dose of 4 µM, treatment with
Ru1 or Ru2 for 6 h results in marked increase of the mean fluorescent intensity (MFI)
by approximate 4.3- and 2.8- fold than control group, respectively.
Data obtain from Fig. 6B suggest that Ru1 significantly enhances cellular ROS
level in a dose- and time-dependent manner, and the ROS is detected as early as 5 min,
indicating ROS as an early event in respond to Ru1-induced apoptosis. To inquire into
the contribution of ROS in Ru1-induced HeLa cells apoptosis, two antioxidants
N-acetyl-L-cysteine (NAC) and glutathione (GSH) were used. As shown in Fig. 6C,
both NAC and GSH can substantially suppress the intracellular ROS levels. The
percentage of apoptotic cells was detected by Annexin V staining assay after Ru1
treatment with or without antioxidants NAC or GSH. From Fig. 6D, we can see that
the percentage of apoptosis cells induced by Ru1 obviously reduces when ROS is
inhibited by antioxidants. Meanwhile, Fig. S11B presents that both NAC and GSH
obviously increase cell viability. Taken together, these results suggest that activation
of ROS-generating by Ru(II) complexes plays an important role in inducing apoptotic
in HeLa cells.
1
9

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2
.9. Ru(II) complexes trigger DNA damage
Fig.7. DNA damage was examined by comet assay and western blotting experiment. (A) HeLa
cells were treated with various concentration of Ru1 or Ru2 for 24 h, DNA fragmentation was
examined by comet assay. (B) Quantification of DNA tails in the comet assay. The length of DNA
tails in microscopy images was quantified by ImageJ. (C) The expression levels of phosphorylated
proteins ATM、ATR、Chk1、Chk2、H2A.X were performed by western blotting. (D) Western
blotting analysis of expression levels of DNA damage proteins in HeLa cells in the presence of
GSH, β-actin was used as internal control.
A number of Ru(II) complexes can trigger DNA damage, also some Ru(II)
compouds exhibit photo-induced DNA cleavage activity [34,66,67]. DNA damage is
regarded as a hallmark of apoptosis [68,69]. It has been reported that excess
intracellular ROS can attack DNA, resulting in DNA damage and activation of
various damage sensor proteins such as ATM, Histone proteins, and the tumor
2
0

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suppressor gene p53 [70]. In this work, to study whether or not Ru1 can induce DNA
damage and activate DNA damage sensor proteins, such as Histone protein, which is a
marker of DNA double-strand breaks, single cell gel electrophoresis (comet assay)
and western blotting assay were performed. Comet assay is a simple and rapid method
for assessing DNA damage generated by DNA-targeting agents in single cells, and the
length of the comet tail represents the level of DNA damage. As shown in Fig. 7A, in
the control, HeLa cells fail to show a comet like appearance. After incubation with
Ru1 (1.0 µM) and Ru2 (1.0 µM) for 24 h, HeLa cells exhibite well-formed comet
tails, indicating that severe DNA damage has occurred. With extension of the
concentration to 4.0 µM, statistically significant comet tails appear, suggesting that
more DNA damage has occurred. The quantification of comet tails in the comet assay
is shown in Fig. 7B. The length of comet tails in microscopy images was quantified
by ImageJ. Differences. Fig. 7B suggests that Ru1 induces more DNA damage than
that of Ru2 induces.
In addition, Ru1-induced DNA damage was further confirmed by western blotting
assay (Fig. 7C), as evidenced by the up-regulation of the phosphorylation levels of
1
39
1981
428
H2A.X (Ser ), ATM (Ser ) and ATR (Ser ), which are the DNA damage
markers. Meanwhile, Chk1 and Chk2 are activated, as evidenced by the increase of
the phosphorylation levels of Chk1 and Chk2. Interestingly, when an antioxidant GSH
1
39
is added to the cells which incubated with Ru1, the levels of p-H2A.X (Ser ), p-p53
1
5
1981
428
(
Ser ), p-ATM (Ser ) and p-ATR(Ser ) are down-regulate (Fig. 7D). These
results demonstrate that Ru1 induces DNA damage through ROS overproduction, like
2
+
ruthenium (II) imidazole complex [Ru(MeIm) (p-cpip)] [34].
4
2
1

ACCEPTED MANUSCRIPT
Scheme 1: Proposed apoptosis pathways induced by Ru1 in HeLa cells.
3
. Conclusions
In this work, we synthesized two new cyclometalated Ru(II) complexes
+
+
[
Ru(dmb) (1-Ph-βC)] (Ru1) and [Ru(bpy) (1-Ph-βC)] (Ru2). Both Ru1 and Ru2
2 2
display IC values much lower than those of cisplatin and other non-cyclometalated
5
0
Ru(II)-β-carboline complexes [Ru(N-N) (1-Py-βC)](PF ) (N-N = bpy, phen). Notably,
2
6 2
the antiproliferative activities of Ru1 and Ru2 are much higher than those of
precursors cis-[Ru(bpy) Cl ], cis-[Ru(dmb) Cl ] and 1-Ph-βC, suggesting that the
2
2
2
2
significantly higher anticancer activity has been achieved by complexation of Ru(II)
polypyridyl moieties with 1-Ph-βC. The hydrophobicity, cellular uptake efficiency
and cytotoxic effect on tumor cells of Ru1 and Ru2 are significantly correlated. The
2
2

ACCEPTED MANUSCRIPT
higher lipophilic of Ru1, shows higher cellular uptake efficiency and is more activity
than Ru2.
The cellular uptake and localization exhibit that Ru(II) complexes can be
transported into the cells mainly through clathrin-mediated endocytosis pathway, and
these complexes can accumulate in the cell nuclei. Hoechst 33342 staining and
AO/EB dual-staining demonstrate that both Ru1 and Ru2 can effectively induce
apoptosis of HeLa cells. In addition, these complexes enhance the level of
intracellular ROS and induce a decrease of MMP. And further studies show that ROS
play vital roles in Ru1-induced apoptosis and cell viability. Western blotting analysis
show that Ru1 activates Caspase-3/8/9, increases the level of the pro-apoptotic
proteins Bad and Bax, decreases the levels of the anti-apoptotic protein Bcl-xl and
Bcl-2, thus leads to the release of cytochrome c in HeLa cells. Moreover, comet assay
and western blotting analysis indicate that Ru1 can induce DNA damage as evidenced
by the up-regulation of the phosphorylation levels of H2A.X, ATR and ATM, and the
latter two further phosphorylates the downstream effectors ChK1/2. Next, the
phosphorylated effectors downregulate the expression of CDC25A, thereby inhibiting
the CDK2/4 kinases and subsequently leading to cell cycle arrest at the G0/G1 phase.
Taking all these results together, we conclude that Ru1 and Ru2 can induce cell
apoptosis in HeLa cells mainly through mitochondrial dysfunction, intracellular ROS
accumulation and ROS-mediated DNA damage. The schematic diagram of this
proposed apoptosis pathway induced by Ru1 in HeLa cells is illustrated in Scheme 1.
These results will be helpful for design and synthesis of new cyclometalated Ru(II)
complexes as potent anticancer drugs.
4
4
. Experimental section
.1 Materials and general methods
All reagents and solvents were purchased commercially and used without further
purification unless specifically noted, and all aqueous solutions were prepared with
doubly distilled water. 1-phenyl-9H-pyrido[3,4-b]indole (1-Ph-βC) [31] and
2
3

ACCEPTED MANUSCRIPT
cis-[Ru(L) Cl ]·2H O (L = dmb, bpy) were prepared according to the literature
2
2
2
TM
methods [71]. Annexin V-FITC Apoptosis Detection Kit, QuantiPro BCA Assay Kit,
TM
ECL Start Western Blotting Detection Reagent, DMSO, MTT, phosphate buffered
saline solution (PBS), DNA (CT-DNA), PI, JC-1, GSH, NAC, DCFH-DA were
purchased from Sigma-Aldrich (St. Louis, MO, USA). Cisplatin was purchased from
Acros. Cell Mitochondria Isolation Kit and specific caspase substrates were purchased
from Beyotime (Shanghai, China). Ruthenium standard solution was purchased from
Aladdin Chemistry Co. (Shanghai, China). Primary and secondary antibodies were
purchased form Cell Signaling Technology Company. Anti-Transferrin Receptor
antibody (anti-TfR antibody) was purchased from Abcam (Shanghai, China). Comet
assay reagent kit was purchased from Trevigen (Gaithersburg, MD, USA). Protein
TM
bands were visualized using ChemiDoc XRS+ Imaging System (Bio-Rad, USA).
Flow cytometry was performed by EPICS XL-MCL (BECKMAN COULTER, USA)
and microscopic observation was performed by Ti-E (Nikon, Japan). Microanalyses
(C, H, and N) were carried out with a Perkin–Elmer 240Q elemental analyser.
Electrospray ionization mass spectrometry (ESI-MS) was recorded on an Agilent
1
LC-MS6430B Spectrometer using CH CN as mobile phase. H NMR spectra was
3
recorded on a Bruker AVANCE 400 spectrometer (400 MHz) with (CD ) SO as
3
2
solvent for the complexes at room temperature. All chemical shifts relative to
tetramethylsilane (TMS) were given.
4
4
.2. Synthesis and characteristics
.2.1. Synthesis of [Ru(dmb) (1-Ph-βC)] (PF ) (Ru1)
2
6
A mixture of cis-[Ru(dmb) Cl ]·2H O (0.27 g, 0.5 mmol), 1-Ph-βC (0.12 g, 0.5
2
2
2
mmol), AgNO (0.17 g, 1.0 mmol) and tetramethylammonium hydroxide (0.092 g, 0.5
3
mmol) were dissolved in anhydrous ethanol (15 mL), and then the mixture was heated
at reflux under nitrogen at 25 °C for 24 h to give a clear red solution. Upon
completion of the reaction, the red precipitate was obtained by a dropwise addition of
saturated aqueous KPF solution. Finally, the red precipitate was dried under vacuum
6
and purified by chromatography over alumina (200 mesh), using
2
4

ACCEPTED MANUSCRIPT
acetonitrile/toluene-(1:2, v/v) as an eluent. Yield: 81.8 %. Anal. calc. for
C H F N PRu：C 57.41%，H 4.11%，N 9.80%；found: C 57.21%，H 4.09%，N
41
35
6
6
+
1
9
.81%. ESI-MS (MeCN): m/z = 713.2 ([M−PF ] ). H NMR (400 MHz, DMSO-d6) δ
6
11.77 (s, 1H), 8.64 (d, J = 1.7 Hz, 1H), 8.56 – 8.37 (m, 4H), 8.14 (d, J = 7.9 Hz, 1H),
7
.84 – 7.72 (m, 3H), 7.69 – 7.53 (m, 2H), 7.50 (d, J = 5.8 Hz, 1H), 7.44 – 7.33 (m,
H), 7.33 – 7.17 (m, 2H), 7.16 – 7.06 (m, 2H), 6.99 (td, J = 7.5, 1.4 Hz, 1H), 6.82 (td,
3
J = 7.3, 1.1 Hz, 1H), 6.51 (dd, J = 7.4, 1.3 Hz, 1H), 2.51 (s, 3H), 2.44 (s, 3H), 2.37 (s,
6
H).
4
.2.2. Synthesis of [Ru(bpy) (1-Ph-βC)] (PF ) (Ru2)
2
6
Ru2 was synthesized by a similar procedure to Ru1. Cis-[Ru(bpy) Cl ]·2H O
2
2
2
was used instead of cis-[Ru(dmb) Cl ]·2H O. Yield: 82.1%. Anal. calc. for
2
2
2
C H F N PRu：C 55.43%，H 3.39%，N 10.48%；found: C 55.37%，H 3.37%，
37
27
6
6
+
1
N 10.53%. ESI-MS (MeCN): m/z = 657.2 ([M−PF ] ). H NMR (400 MHz,
6
DMSO-d ) δ 11.82 (s, 1H), 8.81 (dt, J = 8.4, 1.1 Hz, 1H), 8.70 (dt, J = 8.2, 1.1 Hz,
6
1H), 8.64 (ddt, J = 10.5, 8.2, 1.1 Hz, 2H), 8.47–8.40 (m, 1H), 8.19–8.09 (m, 2H),
8.01–7.96 (m, 1H), 7.96–7.76 (m, 6H), 7.70–7.67 (m, 1H), 7.63–7.55 (m, 3H),
7.41–7.27 (m, 5H), 7.03 (td, J = 8.0, 7.6, 1.4 Hz, 1H), 6.85 (td, J = 7.3, 1.1 Hz, 1H),
6.47 (dd, J = 7.4, 1.3 Hz, 1H).
4
.3. Cytotoxicity assay in vitro
Five different tumor cell lines (lung adenocarcinoma cell A549, human lung cancer
NCl-H460, cervical cancer HeLa, breast cancer MCF-7, hepatocellular carcinoma
HepG2, and cervical cancer HeLa) and one normal cell lines (immortalized human
bronchial epithelial cells BEAS-2B) were purchased from American Type Culture
Collection (ATCC, Manassas, VA). All cell lines were cultured in Roswell Park
Memorial Institute 1640 (RPMI 1640) culture media supplemented with 10 % fetal
bovine serum (FBS) and incubated in incubator with 5 % CO at 37 °C unless
2
otherwise noted. The IC values were determined by MTT assay.
5
0
4
.4. Lipophilicity measurements
2
5

ACCEPTED MANUSCRIPT
Lipophilicity was determined by liquid–liquid extraction between n-octanol (oil)
and NaCl (0.9% w/v) using the flask-shaking method as previously described [34].
Briefly, the ruthenium complex in NaCl (0.9% w/v) was added to an equal volume of
n-octanol (oil), and the mixture was shaken for 48 h at 200 rpm at 25 ºC to allow
partitioning. After centrifuging the sample at 3000 rpm for 10 min, the aqueous layer
was used for ruthenium analysis, and the Ru content in the aqueous layer was then
measured by ICP-MS and used to calculate the logPo/w values according to the
equation logP =Log ([Ru] /[Ru] ).
o/w
o
w
4
.5. Cellular uptake
5
HeLa cells at a density of 5.0 × 10 cells were seeded in 60 mm tissue culture
dishes and allowed to reach 70% confluence, then the cells were incubated with the
different concentration of Ru(II) complexes for different times. After treatment, the
cells were treated with trypsin and collected in centrifugal tubes, and then washed
three times with cold PBS. The pellets were completely digested in 3 mL mixture (2
mL, 60% HNO and 1mL, H O ) for 24 h, and then diluted to 4 mL with ultrapure
3
2
2
water. Finally, the amount of Ru taken up by HeLa cells was determined by ICP-MS
NEXION-300X, PerkinElmer, USA) with a 100 ng/mL ruthenium standard solution
(
for drawing a standard line. Ruthenium standard solutions were freshly prepared
before each experiment. The water used for ICP-MS analysis was doubly deionized.
The uptake of Ru was calculated from the standard curve and expressed as the amount
6
of Ru (ng) taken up per 10 cells.
4
.6. The mechanism of cellular uptake
HeLa cells were seeded into 60 mm tissue culture dishes for 24 h, and then the
cells were pretreated with endocytosis inhibitors oligomycin
5
µM,
2
1
6
-deoxy-D-glucose (DOG) 50 µM, sucrose 0.25 mM, nystatin 10 µg/mL and anti-TfR
µg/mL) for 2 h or at 4 °C for 4 h, respectively, before treatment of 4 µM of Ru1 for
h. The control sample was just exposed to 4 µM of Ru1 at 37 °C for 6 h. Then the
2
6

ACCEPTED MANUSCRIPT
cells were trypsinized and collected. Finally, the intracellular uptake of Ru1 were
determined using ICP-MS.
4
.7. Subcellular distribution of Ru(II) complexes
The subcellular distribution of Ru1 was detected using Cell Mitochondria
Isolation Kit according to manufacturer's recommendations. Briefly, after incubation
with 4.0 M of Ru1 for different times, cells were harvested and washed twice with
µ
cold-PBS and re-suspended in the mitochondria isolation buffer for 15 min under low
temperature conditions. The suspension was homogenized using a Dounce
homogenizer, and then the homogenate was centrifuged at 600× g for 10 min at 4 ºC.
The pellets (nuclear fraction) were completely digested in 4 mL mixture (3 mL, 95%
HNO and 1 mL, H O ), and the supernatant was centrifuged at 11 000 × g for 10 min
3
2
2
at 4 °C. The resulting supernatants (cytoplasmic fractions) and pellets (mitochondrial
fractions) were also digested in 4 mL mixture, respectively. Finally, ruthenium
element was determined by ICP-MS with a 100 ng/mL ruthenium standard solution
for drawing a standard line.
4
.8. Cell cycle arrest analysis
Cell cycle distribution and apoptosis were analyzed by flow cytometry analysis
as described previously [67,72]. HeLa cells were plated in 6-well plate and incubated
for 24 h. Different concentrations of Ru(II) complexes were then added into the wells
and incubated for 24 h. After incubation, cells were collected and fixed in 1.5 mL
aqueous ethanol (75%, v/v) at -20 ̊C overnight, and then stained with PI (50 µg/mL)
in the presence of RNAase A (100 µg/mL) for 30 min at 37 ºC in the dark. The stained
cells were then analyzed using the flow cytometer.
4
.9. Apoptosis assay by Hoechst 33342 staining
5
HeLa cells (3×10 cells per well in 6-well plate) were incubated for 24 h and then
exposed to different concentrations of Ru1 or Ru2 complex for 24 h, respectively.
After treatment, cells were stained with 5 µg/mL Hoechst 33342, washed twice with
2
7

ACCEPTED MANUSCRIPT
PBS, and then photographed using an inverted fluorescence microscope.
4
.10. AO/EB staining method
HeLa cells were seeded in 6-well plates and treated in the absence or presence of
Ru(II) complexes for 24 h. After incubation, cells were washed twice with ice-cold
PBS and stained with 100 µg/mL concentration of AO/EB solutions, and then
photographed using an inverted fluorescence microscope.
4
.11. Apoptosis assay by annexin V/PI double staining
Different stages of apoptosis were distinguished using annexin V-FITC
Apoptosis Detection Kit. After incubation with various concentrations of complexes
for 24 h, cells were harvested and washed twice with PBS, and then resuspended in
5
00 µL binding buffer. The suspension were stained with 5 µL Annexin V-FITC and
0 µL PI at room temperature for 15 min in the dark, and then analyzed using the flow
1
cytometer.
4
.12. Caspase activity assay
According to the manufacturer’s protocol, HeLa cells were collected and
suspended in cell lysis buffer, and then incubated on ice for 15 min. After
centrifugation at 15,000 g for 15 minutes, the supernatants were collected and seeded
into 96-well plates. After then, specific caspase substrates, including Ac-DEVD- pNA
for caspase-3, Ac-IETD- pNA for caspase-8, and Ac-LEHD-pNA for caspase-9, were
added. Plates were incubated at 37°C for 2 h and caspase activity was determined by a
Microplate reader. Relative caspase activity was expressed as the percentage of
control (as 100%).
4
.13. Reactive oxygen species (ROS) assay
ROS level was detected after HeLa cells had been stained with 2,7-Dichlorodi
-hydrofluorescein diacetate (DCFH-DA). For flow cytometry analysis or microplate
analysis, collected cells were trypsinized and washed three times with PBS, and then
2
8