Journal of Inorganic Biochemistry 152 (2015) 19

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Journal of Inorganic Biochemistry

journal homepage: www.elsevier.com/locate/jinorgbio

Cytotoxic activity, DNA damage, cellular uptake, apoptosis and western

blot analysis of ruthenium(II) polypyridyl complex against human lung
decarcinoma A549 cell
Shang-Hai Lai a, Guang-Bin Jiang a, Jun-Hua Yao b, Wei Li a, Bing-Jie Han a, Cheng Zhang a,
Chuan-Chuan Zeng a, Yun-Jun Liu a,
a
b

School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China

Instrumentation Analysis and Research Center, Sun Yat-Sen University, Guangzhou 510275, PR China

a r t i c l e

i n f o

Article history:
Received 22 May 2015
Received in revised form 30 July 2015
Accepted 5 August 2015
Available online 12 August 2015
Keywords:
Ruthenium(II) complex
Apoptosis
Cell cycle arrest
Mitochondrial membrane potential
Western blot analysis

a b s t r a c t
A new ruthenium(II) polypyridyl complex [Ru(dmp)2(pddppn)](ClO4)2 Ru1 was synthesized and characterized.
The cytotoxic activity in vitro of the complex was evaluated by MTT method. Ru1 shows high effect on the inhibition of the cell growth against BEL-7402, HeLa, MG-63 and A549 cells with low IC50 values of 1.6 0.4, 9.0
0.8, 1.5 0.2 and 1.5 0.3 M, respectively. The cellular uptake indicates that Ru1 can enter into the cytoplasm
and accumulate in the cell nuclei. Ru1 can induce apoptosis in A549 cells and enhance the levels of reactive oxygen species (ROS) and induce the decrease of mitochondrial membrane potential. In addition, Ru1 can downregulate the levels of Bcl-2, Bcl-x, Bak, and Bim expression and up-regulate the expression of Bag-1 and Bad.
The complex induces apoptosis of A549 cells through an intrinsic ROS-mediated mitochondrial dysfunction pathway, which was accompanied by regulating the expression of caspases and Bcl-2 family proteins.
2015 Elsevier Inc. All rights reserved.

1. Introduction
Therapy with cytotoxic compounds or small molecule inhibitors is a
major strategy to treat human cancer at the disseminated stage. However, the occurrence of drug resistance and unwanted side-effects remains
a major obstacle for successful long-term treatment [1,2]. Cisplatin is
one of the most widely used anticancer drugs. Signicant side effects
and drug resistance have limited the clinical applications of cisplatin.
In recent years, the anticancer activity of the ruthenium complexes
has been paid great attention. Up to now, two ruthenium complexes
NAMI-A ([ImH][trans-RuCl4(DMSO)(Im)]) and KP1019 ([IndH][transRuCl4(Ind)2]) have entered clinical trials. NAMI-A has been used as an
antimetastatic drug and KP1019 has been employed as colon carcinomas drug [3,4]. A number of ruthenium(II) polypyridyl complexes display unique antitumor properties [518]. [Ru(phpy)(bpy)(dppn)]+ is
6 times more active than the platinum drug against HeLa cells, and it
is able to disrupt the mitochondria membrane potential [19]. [(6hexamethylbenzene)Ru(dmp)(-Cl)2Sn(CH3)2Cl] exhibits signicantly
good cytotoxicity on both HeLa (IC50 = 5.2 M) and HepG-2 (IC50 =
7.4 M) cells [20]. [Ru(dip)2(1-Py-C)]2+ shows high inhibition of cell

Corresponding author.
E-mail address: lyjche@163.com (Y.-J. Liu).

http://dx.doi.org/10.1016/j.jinorgbio.2015.08.012
0162-0134/ 2015 Elsevier Inc. All rights reserved.

growth on HeLa with an low IC50 of 1.9 0.2 M [21], [Ru(dppz)2

(CppH)]2+ specically targeted mitochondria and showed a low IC50
value comparable to cisplatin in HeLa [22]. [Ru(bpy)2(addppn)]2+ exhibit very high cytotoxic effect and can effectively induce the apoptosis
of BEL-7402 cells [23]. [Ru(Hdpa)2(dppz)]2 + exhibits a cytotoxicity
against human cervical epidermoid carcinoma cell line (ME180) with
potency approximately 8 times more than cisplatin for 24 h incubation
[24]. [Ru(bpy)2(2,9-dimethyl-dpq)]2+ has no cytotoxicity toward A549
cells in the dark with an IC50 value of 250 (5) M, but on irradiation
with N450 nm light for 3 min, the complex shows high cytotoxicity
with an IC50 value of 1.2 ( 0.1) M [25]. [Ru(dmp)2(addpz)]2 +
shows very high inhibitory effect on SK-BR-3 cell growth with a low
IC50 value of 2.2 0.3 M [26]. Complex [Ru(Hdpa)2(7-F-dppz)]2 +
can effectively inhibit the growth in HeLa cells [27]. Schatzschneider
reported that the planarity of ligand of ruthenium complexes plays
important role in antitumor activity [28]. In order to obtain more insight
into anticancer activity of ruthenium complex, in this report, we designed a new ruthenium (II) complex [Ru(dmp)2(pddppn)](ClO4)2
(Scheme 1). The complex [Ru(dmp)2(pddppn)](ClO4)2 was synthesized
and characterized by UVvis spectra, elemental analysis, ESI-MS, 1H
NMR and 13C NMR. The cytotoxicity in vitro of the complex against
BEL-7402, HeLa, MG-63 and A549 cells was assayed by MTT method.
The morphological apoptosis and comet assay in A549 cells were investigated. The cellular uptake was observed under uorescent microscopy.

S.-H. Lai et al. / Journal of Inorganic Biochemistry 152 (2015) 19

Scheme 1. The synthetic route of complex Ru1.

The cell cycle arrest was analyzed by ow cytometry. The reactive oxygen species, mitochondrial membrane potential and western blot were
also investigated in detail.
2. Experimental sections
2.1. Materials and method
All reagents and solvents were purchased commercially and used
without further purication unless otherwise noted. Ultrapure MilliQ
water was used in all experiments. DMSO, phenanthrenequinone and
RPMI 1640 were purchased from Sigma. Cell lines of BEL-7402 (Hepatocellular), HeLa (human cervical cancer cell line), A549 (human lung
denocarcinoma cell line), MG-63 (human osteosarcoma) were purchased from the American Type Culture Collection. RuCl3 3H2O was
purchased from the Kunming Institution of Precious Metals. 1,10phenanthroline was obtained from the Guangzhou Chemical Reagent
Factory.
Microanalyses (C, H, and N) were obtained with a Perkin-Elmer
240Q elemental analyzer. Electrospray ionization mass spectra (ESI-MS)
were recorded on a LCQ system (Finnigan MAT, USA) using methanol
as mobile phase. The spray voltage, tube lens offset, capillary voltage
and capillary temperature were set at 4.50 KV, 30.00 V, 23.00 V and
200 C, respectively, and the quoted m/z values are for the major
peaks in the isotope distribution. 1 H NMR and 13 C NMR spectra
were recorded on a Varian-500 spectrometer with DMSO-d 6 as
solvent and tetramethylsilane (TMS) as an internal standard at
500 MHz at room temperature.

temperature, a red precipitate was obtained by dropwise addition of

saturated aqueous NaClO4 solution. The red precipitate was dried in
vacuo. The crude product was puried by column chromatography on
neutral alumina with a mixture of CH3CN-toluene (3:1, v/v) as eluent.
The red band was collected. The solvent was removed under reduced
pressure and a red powder was obtained. Yield: 70%. Anal. Calc for
C60H40N10Cl2O8Ru: C, 60.00; H, 3.36; N, 11.66%. Found: C, 60.22; H,
3.28; N, 11.48%. max nm (relative intensity) for the complex (20 M)
in DMF: 263 nm (0.886), 301 nm (0.154), 445 nm (0.080), in CH3CN:
269 nm (0.141), 304 nm (0.134), 440 nm (0.064), in ethanol: 270 nm
(0.113), 311 nm (0.128) and 446 nm (0.074). ESI-MS (CH3CN, m/z):
1001.3 [(M-2ClO4-H)]+, 501.4 [(M-2ClO4)]2 +. 1H NMR (DMSO-d6):
9.44 (d, 2H, J = 8.0 Hz), 9.39 (s, 2H), 9.33 (d, 2H, J = 7.5 Hz), 8.95
(d, 2H, J = 8.5 Hz), 8.75 (d, 2H, J = 8.0 Hz), 8.51 (d, 2H, J = 8.5 Hz),
8.46 (d, 2H, J = 8.5 Hz), 8.29 (d, 2H, J = 8.0 Hz), 8.00 (d, 2H, J =
8.5 Hz), 7.91 (t, 2H, J = 7.5 Hz), 7.87 (t, 2H, J = 7.5 Hz), 7.64 (dd, 2H,
J = 5.5, J = 5.5 Hz), 7.52 (d, 2H, J = 6.0 Hz), 7.48 (d, 2H, J = 8.0 Hz),
1.95 (s, 6H), 1.93 (s, 6H). 13C NMR (DMSO-d6): 168.08, 166.85, 154.19,
152.06, 148.79, 147.61, 144.74, 142.05, 140.59, 139.61, 138.33, 136.82,
132.31, 129.89, 129.62, 128.99, 128.71, 128.30, 127.61, 127.20, 126.86,
126.56, 123.75, 26.21, 24.57.
2.4. Cell culture
Cell was cultured in RPMI 1640 medium supplemented with heat
inactivated fetal bovine serum (FBS, 10%), penicillin (100 g/mL) and
streptomycin (100 g/mL). Cells were maintained at 37 C in a 5% CO2
incubator. Ru1 was dissolved in DMSO and the nal concentration of
DMSO is 0.05%.

2.2. Synthesis of complex

2.5. In vitro cytotoxicity assays
1,10-Phenanthroline-5,6-dione [29], ligand dadppz [30] and [Ru(dmp)2
(dadppz)](ClO4)2 [31] were prepared according to the methods in the
literature.
2.3. Synthesis of [Ru(dmp)2(pddppn)](ClO4)2 (Ru1)
0.104 g of phenanthraquinone (0.5 mmol) in 30 mL of glacial acetic
acid was added to the solution of [Ru(dmp)2(dadppz)](ClO4)2 (0.515 g,
0.5 mmol) in 10 mL of acetonitrile. The reaction mixture was reuxed
under argon for 6 h to give a clear red solution and the solvent was removed to about 10 mL under reduced pressure, and then cooled to room

MTT assay procedures were used [32]. Cells were placed in 96-well
microassay culture plates (8 104 cells per well) and grown overnight
at 37 C in a 5% CO2 incubator. The tested compound was then added to
the wells to achieve nal concentrations ranging from 106 to 104 M.
Control wells were prepared by addition of culture medium (100 L).
The plates were incubated at 37 C in a 5% CO2 incubator for 48 h.
Upon completion of the incubation, stock MTT dye solution (20 L,
5 mg/mL) was added to each well. After 4 h, buffer (100 L) containing
N,N-dimethylformamide (50%) and sodium dodecyl sulfate (20%) was
added to solubilize the MTT formazan. The optical density of each well

S.-H. Lai et al. / Journal of Inorganic Biochemistry 152 (2015) 19

was then measured with a microplate spectrophotometer at a wavelength of 490 nm. The IC50 values were calculated by plotting the percentage viability versus concentration on a logarithmic graph and
reading off the concentration at which 50% of cells remained viable relative to the control. Each experiment was repeated at least three times
to obtain the mean values.

2.6. Apoptosis assay by AO/EB and Hoechst 33258 staining methods

A549 cells were seeded onto chamber slides in six-well plates at a
density of 2 105 cells per well and incubated for 24 h. The cells were
cultured in RPMI 1640 supplemented with 10% of FBS and incubated
at 37 C in a 5% CO2. The medium was removed and replaced with medium (nal DMSO concentration 0.05% v/v) containing Ru1 (0.5 M or
1.0 M) for 24 h. The medium was removed and the cells were washed
with ice-cold PBS, and xed with formalin (4%, w/v). Cell nuclei were
counterstained with AO/EB (100 g/mL AO, 100 g/mL EB) or Hoechst
33258 (10 g/mL in PBS) for 10 min, Then the cells were imaged by uorescence microscope (Nikon, Yokohama, Japan) with excitation at
350 nm and emission at 460 nm.

2.7. Comet assay

DNA damage was investigated by means of comet assay. A549 cells
in culture medium were incubated with 1.0, 2.5 and 5.0 M of Ru1 at
37 C for 24 h. The cells were harvested by a trypsinization process at
24 h. A total of 100 L of 0.5% normal agarose in PBS was dropped gently
onto a fully frosted microslide, covered immediately with a coverslip,
and then placed at 4 C for 10 min. The coverslip was removed after
the gel has been xed. 50 L of the cell suspension (200 cells/L) was
mixed with 50 L of 1% low melting agarose preserved at 37 C. A total
of 100 L of this mixture was applied quickly on top of the gel, coated
over the microslide, covered immediately with a coverslip, and then
placed at 4 C for 10 min. The coverslip was again removed after the
gel has been xed. A third coating of 50 L of 0.5% low melting agarose
was placed on the gel and allowed to place at 4 C for 15 min. After solidication of the agarose, the coverslips were removed, and the slides
were immersed in an ice-cold lysis solution (2.5 M NaCl, 100 mM
EDTA, 10 mM Tris, 90 mM sodium sarcosinate, NaOH, pH 10, 1% Triton
X-100 and 10% DMSO) and placed in a refrigerator at 4 C for 2 h. All
of the above operations were performed under low lighting conditions
to avoid additional DNA damage. After the removal of the lysis solution,
the slides were placed horizontally in an electrophoresis chamber. The
reservoirs were lled with an electrophoresis buffer (300 mM NaOH,
1.2 mM EDTA) until the slides were just immersed in the buffer solution,
and the DNA was allowed to unwind for 30 min in the electrophoresis
solution. Then the electrophoresis was carried out at 25 V and 300 mA
for 20 min. After electrophoresis, the slides were removed, and washed
thrice in a neutralization buffer (400 mM Tris, HCl, pH 7.5). Nuclear DNA
was stained with 20 L of EtBr (20 g/mL) in the dark for 20 min. The
slides were washed in chilled distilled water for 10 min to neutralize
the excess alkali, air-dried and scored for comets by uorescence
microscopy.

2.9. Reactive oxygen species (ROS) levels studies

A549 cells were seeded into six-well plates (Costar, Corning Corp,
New York) at a density of 1 106 cells per well and incubated for 24 h.
The cells were cultured in RPMI 1640 medium supplemented with 10%
of FBS and incubated at 37 C and 5% CO2. The medium was removed
and replaced with medium (nal DMSO concentration 0.05% v/v) containing different concentrations of Ru1 for 24 h. The medium was removed again. The uorescent dye 2,7-dichlorodihydrouorescein
diacetate (DCFH-DA) was added to the medium with a nal concentration of 10 M to cover the cells. The treated cells were then washed
with cold PBS-EDTA twice, collected by trypsinization and centrifugation
at 1500 rpm for 5 min, and resuspended in PBS-EDTA. Fluorescence was
imaged with uorescent microscope at an excitation wavelength of
488 nm and emission at 525 nm.
2.10. Mitochondrial membrane potentials assay
A549 cells were treated with Ru1 for 24 h in 12-well plates and were
then washed three times with cold PBS. The cells were then detached
with trypsin-EDTA solution. The collected cells were incubated for
20 min with 1 g/mL of JC-1 in culture medium at 37 C in the dark.
The cells were immediately centrifuged to remove the supernatant.
Cell pellets were suspended in PBS and then imaged under uorescence
microscope.
2.11. Apoptosis assay by ow cytometry
After chemical treatment, 1 106 cells were harvested, washed with
PBS, xed with 70% ethanol, and nally maintained at 4 C for at least
12 h. The pellets were stained with a uorescent probe solution containing 50 g/mL propidium iodide and 1 mg/mL Annexin V in PBS on ice in
the dark for 15 min. The uorescence emission was measured at 530 nm
using 488 nm excitation with a FACS Calibur ow cytometry (Beckman
Dickinson & Co., Franklin Lakes, NJ). A minimum of 10,000 cells were
analyzed per sample.
2.12. Cell cycle arrest by ow cytometry
A549 or BEL-7402 or MG-63 cells were seeded into six-well plates
(Costar, Corning Corp, New York) at a density of 1 106 cells per well
and incubated for 24 h. The cells were cultured in RPMI 1640 supplemented with 10% of FBS and incubated at 37 C and 5% CO2. The medium
was removed and replaced with medium (nal DMSO concentration
0.05% v/v) containing Ru1 (1.0 M). After incubation for 24 h, the cell
layer was trypsinized and washed with cold PBS and xed with 70%
ethanol. Twenty microliters of RNAse (0.2 mg/mL) and 20 L of
propidium iodide (0.02 mg/mL) were added to the cell suspensions
and the mixtures were incubated at 37 C for 30 min. The samples
were then analyzed with a FACS Calibur ow cytometry. The number
of cells analyzed for each sample was 10,000 [33].
2.13. Western blot analysis
A549 cells were seeded in 3.5-cm dishes for 24 h and incubated with
different concentrations of complex in the presence of 10% FBS. Then the
cells were harvested in lysis buffer. After sonication, the samples were

2.8. Cellular uptake

A549 cells were placed in 24-well microassay culture plates
(4 104 cells per well) and grown overnight at 37 C in a 5% CO2 incubator. Different concentrations of Ru1 were then added to the wells. The
plates were incubated at 37 C in a 5% CO2 incubator for 24 h. Upon completion of the incubation, the wells were washed three times with PBS.
After discarding the culture medium, the cells were visualized by uorescent microscopy.

Table 1
IC50 values of Ru1 toward BEL-7402, HeLa, MG-63 and A549 cell lines.
Complex

Ru1
pddppn
Cisplatin

IC50 (M)
BEL-7402

HeLa

MG-63

A549

1.6 0.4
N200
11.4 1.8

9.0 0.8
N200
7.3 1.4

1.5 0.2
N200
6.5 0.5

1.5 0.3
N200
6.6 1.1

S.-H. Lai et al. / Journal of Inorganic Biochemistry 152 (2015) 19

Fig. 1. The cell viability of Ru1 on BEL-7402, A549 (a) and MG-63 (b) cell proliferation in vitro. Each point is the mean standard, error obtained from three independent experiments.

centrifuged for 20 min at 13,000 g. The protein concentration of the

supernatant was determined by BCA assay. Sodium dodecyl sulfate
polyacrylamide gel electrophoresis was done loading equal amount of
proteins per lane. Gels were then transferred to poly (vinylidene
diuoride) membranes (Millipore) and blocked with 5% non-fat milk
in TBST (20 mM TrisHCl, 150 mM NaCl, 0.05% Tween 20, pH 8.0) buffer
for 1 h. The membranes were incubated with primary antibodies at
1:5000 dilutions in 5% non-fat milk overnight at 4 C, and after washed
for four times with TBST for a total of 30 min, then the secondary antibodies conjugated with horseradish peroxidase at 1:5000 dilution for
1 h at room temperature and washed for four times with TBST. The
blots were visualized with the Amersham ECL Plus western blotting
detection reagents according to the manufacturer's instructions. To
assess the presence of comparable amount of proteins in each lane,
the membranes were stripped nally to detect the -actin.

3. Results and discussion

3.1. Chemistry
The complex [Ru(dmp)2(pddppn)](ClO4)2 was synthesized by the
direct reaction of parent complex [Ru(dmp)2(dadppz)](ClO4)2 with
phenanthraquinone in glacial acetic acid. The desired ruthenium(II)
complex was isolated as the perchlorate and puried by column chromatography. The synthesized ruthenium(II) complex was characterized
by UVvis, elemental analysis, ESI-MS, 1H NMR and 13C NMR. In the ESIMS spectra of the complex, all of the expected signals of [M-2ClO4-H]+
and [M-2ClO4]2 + were observed. The measured molecular weights
were consistent with the expected values. The complex can be dissolved
in DMSO, DMF, CH3CN and ethanol, and low solubility in water.
3.2. Cytotoxicity in vitro assay by MTT method

2.14. Data analysis

All data was expressed as means SD. Statistical signicance was
evaluated by a t-test. Differences were considered to be signicant
when a P value was less than 0.05.

In the studies on cytotoxicity, the culture medium and cisplatin were

used as negative and positive control, respectively. The cytotoxic activity after 48 h of incubation of cell lines with Ru1 was expressed as IC50
(drug concentration reducing the number of living cells by 50%). The

Fig. 2. A549 cells were stained with AO/EB (a, b, c) and Hoechst 33258 (d, e, f) exposed to different concentrations of Ru1 for 24 h. Liv, Nec and Apop stand for living, necrotic and apoptotic
cells.

S.-H. Lai et al. / Journal of Inorganic Biochemistry 152 (2015) 19

Fig. 3. Images of A549 cell exposure to 2.5 M of Ru1 and DAPI-stained at 37 C for 24 h.

IC50 values of Ru1 against BEL-7402, HeLa, MG-63 and A549 cells are
listed in Table 1, and the cell viability of BEL-7402, A549 and MG-63 is
depicted in Fig. 1. As expectation, ligand pddppn is found to exhibit no
cytotoxic activity against the above cell lines. Ru1 shows high cytotoxic
activity with low IC50 values of 1.6 0.4, 9.0 0.8, 1.5 0.2 and 1.5
0.3 M toward BEL-7402, HeLa, MG-63 and A549 cells. Comparing the
IC50 values of Ru1 with cisplatin, Ru1 is found to show higher cytotoxicity than cisplatin and its parent complex [Ru(dmp)2(dadppz)]2 +
(IC50 values are 16.4 1.5, 11.0 1.1 and 25.7 2.4 M) [31] toward
BEL-7402, MG-63 and A549 cells under the same conditions. Additionally, Fig. 1 indicates that the cell viability is concentration-dependent,
and the cell viability decreases with increasing concentrations of Ru1.
Also, Table 1 shows when ligand pddppn bonded metal to form complex, the cytotoxic activity is greatly enhanced. Since the complex
displays the same cytotoxic effect on MG-63 and A549 cells, in this report, we selected A549 cell line for further investigation of the underlying mechanisms accounting for the action of ruthenium complex.

The cellular uptake characteristics of a small molecule are critical to

its application as a therapeutic or diagnostic agent [34]. In order to testify whether or not Ru1 can be transported into the cellular interior, the
cellular uptake and localization was investigated using uorescence microscope. After A549 cells exposed to 2.5 M of Ru1 for 24 h, the cells
were stained with DAPI. As shown in Fig. 3, the blue channel shows
DAPI-stained nuclei, the red channel displays the luminescence of Ru1
with an excitation wavelength of 460 nm, and the overlay represents
cellular association of the complex. The complete overlay of blue channel and red channel suggests that Ru1 can be successfully taken up by
A549 cells and the complex can be transported into the cellular interior
and accumulates in the cell nuclei.

3.3. Apoptosis assay by AO/EB staining method

3.5. The detection of ROS levels

Apoptosis was investigated with AO/EB staining method. According

to the difference in membrane integrity between necrotic and apoptosis, AO can pass through cell membrane, but EB cannot. After the treatment of A549 cells with 0.5 and 1.0 M of Ru1 for 24 h, the apoptotic
effect is shown in Fig. 2a. In the control, the living cells of A549 were
stained bright green in spots. A549 cell exposure to 0.5 and 1.0 M of
Ru1 (Fig. 2b and c) for 24 h, green or orange apoptotic cells containing
apoptotic bodies, as well as red necrotic cells, were observed. Additionally, A549 cells (Fig. 2d) were treated with 0.5 (Fig. 2e) and 1.0 M
(Fig. 2f) of Ru1 for 24 h and stained with Hoechst 33258, apoptotic features such as nuclear shrinkage and chromatin condensation were also

Reactive oxygen species (ROS) play an important role in cancer cell

death and apoptosis. In the assay of ROS, 2,7-dichlorodihydrouorescein
diacetate (DCFH-DA) was used as uorescence probe. DCFH-DA is
cleaved by intracellular esterases into its non-uorescent form (DCFH).
Then the non-uorescent substrate is oxidized by intracellular free radicals to produce a uorescent product DCF [35,36]. The changes of the
levels of ROS generation can be evaluated according to the uorescent
intensity of DCF. The uorescent intensity of DCF is proportional to the
amount of peroxide (ROS) produced by the cells [37]. As shown in
Fig. 4A, in the control, little uorescent image was observed. After the
treatment of A549 cells with Rosup (positive control, b) and 1.0 (c) and

observed. These results suggest that Ru1 can induce apoptosis in A549
cells.
3.4. Cellular uptake studies

Fig. 4. (A) Intracellular ROS was detected in A549 cell (a) exposure to Rosup (b) and 1.0 (c) and 5.0 (d) M of Ru1 for 24 h. (B) DCF uorescence intensity on ROS generation in A549 cells
exposed to Rosup and 1.0 and 5.0 M of Ru1 for 24 h. Rosup was used as a positive control.

S.-H. Lai et al. / Journal of Inorganic Biochemistry 152 (2015) 19

Fig. 5. Comet assay of EB-stained control (a) and 1 M (b), 2.5 M (c), and 5.0 M (d) of Ru1 treated A549 cancer cells at 24 h incubation.

5.0 M (d) of Ru1, the uorescent images were found. Additionally, ROS
accumulation was quantied by determining the DCF uorescent intensity with microplate analyzer (Fig. 4B). Treatment of A549 cells with Rosup
(positive control) and 1.0 and 5.0 M of Ru1 resulted in a 3.12, 1.52 and
1.95-fold increase in the uorescent intensity compared with the control.
Obviously, treatments of A549 cells with the complex led to the enhancement of cellular uorescence, which indicated the overproduction of
superoxide. Similar results were observed in other ruthenium(II)
complexes [38,39].
3.6. DNA damage
Single cell gel electrophoresis (comet assay) in an agarose gel matrix
was used to study DNA fragmentation. As shown in Fig. 5a, in the control, A549 cells fail to show a comet like appearance. Treatment of

A549 cells with 1.0 (b), 2.5 (c) and 5.0 M (d) of Ru1 shows statistically
signicant and well-formed comets, and the length of the comet tail
represents the extent of DNA damage. The DNA damage may be caused
by superoxide produced by ROS. These results indicate that Ru1 can
induce DNA fragmentation, which is further evidence of apoptosis.
3.7. The changes in the mitochondrial membrane potential
Mitochondria act as a point of integration for apoptotic signals originating from both extrinsic and intrinsic apoptotic pathways [40,41].
5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolcarbocyanine iodide
(JC-1) was used as a uorescence probe in detecting the changes of
mitochondrial membrane potential induced by Ru1. It is well known
that JC-1 forms aggregates and shows red uorescence emission peak
corresponding to high mitochondrial membrane potential; JC-1 exists

Fig. 6. (A) Assay of A549 cell mitochondrial membrane potential with JC-1 as uorescence probe staining method. A549 cells (a) exposed to cccp (b) and 0.5 (c) and 1.0 (d) M of Ru1 for
24 h. cccp was used as a positive control. (B) The ratios of red/green uorescent intensity were determined after A549 cell exposure to 0.5 and 1.0 M of Ru1 for 24 h. (For interpretation of
the references to color in this gure legend, the reader is referred to the web version of this article.)

S.-H. Lai et al. / Journal of Inorganic Biochemistry 152 (2015) 19

Fig. 7. Cell cycle distribution of BEL-7402 (a), Ru1 + BEL-7402 (b), A549 (c), Ru1 + A549 (d) and MG-63 (e), Ru1 + MG-63 (f) for 24 h. Ru1 = 1.0 M.

as a monomer that emits green uorescence corresponding to low mitochondrial membrane potential. As shown in Fig. 6A, in the control, JC-1
emits red uorescence. A549 cells were exposed to cccp (positive control,
b) and 0.5 (c) and 1.0 M (d) of Ru1 for 24 h, JC-1 displays a green with
little red uorescence (JC-1 monomers). The changes from red to green
uorescence indicate the decrease of mitochondrial membrane potential.
The ratio of red/green uorescent intensity was assayed by ow cytometry. As shown in Fig. 6B, in the control, the ratio of red/green is 37.9 4.3,
while A549 cells were treated with 0.5 and 1.0 M of Ru1 for 24 h, the ratios of uorescent intensities of red/green are 33.0 3.4 and 15.6 2.2,
respectively. The reduction in the ratio of red/green indicates that the
red uorescence decreases and the green uorescence increases. These
results suggest that Ru1 can induce the decrease of mitochondrial membrane potential.

these. The status of cell cycle for cells treated with Ru1 (1.0 M) for
24 h was analyzed by ow cytometry. As shown in Fig. 7, BEL-7402
cells (a) were exposed to 1.0 M of Ru1 (b) and MG-63 cells (e) were
exposed to 1.0 M Ru1 (f) for 24 h, a large increase of 9.22% and 4.80%
in the percentage of the cells at G2/M phase was observed, accompanied
by a corresponding reduction of 8.28% and 5.30% in the percentage of
the cells in the G0/G1 phase, respectively. The results indicate that
Ru1 inhibits the cell growth of BEL-7402 and MG-63 cells in the G2/M
phase. Whereas the treatment of A549 cells (c) with 1.0 M of Ru1
(d) for 24 h, an obvious increase of 7.89% in the G0/G1 phase and corresponding decrease of 6.83% in the S phase were found, which suggests
that Ru1 inhibits the cell growth of A549 cells in the G0/G1 phase.
These observations show that Ru1 exhibits different anti-proliferative
mechanism against different cancer cells.

3.8. Cell cycle arrest assay

3.9. The expression of caspases and Bcl-2 family proteins

Inhibition of cancer cell proliferation by cytotoxic drugs could be the

result of induction of apoptosis or cell cycle arrest or a combination of

Apoptosis is primarily executed by a family of proteases known as

the caspases (cysteinyl, aspartate-specic proteases) [42]. During the

Fig. 8. Western blot analysis of caspases (a) and Bcl-2 family proteins (b) in A549 cells treated with different concentrations of Ru1 for 24 h. -Actin was used as internal control.

S.-H. Lai et al. / Journal of Inorganic Biochemistry 152 (2015) 19

apoptosis process, the activation of caspase-3,-7,-8 and -9 is regarded as

one of the most obvious characteristics in many cell types [43]. The
activation of caspase-3, -7, -8, -9, procaspase-3, -7 and cleavage of
caspases-3, -7 and -8 were assayed by western blot analysis. As shown
in Fig. 8a, after the treatment of A549 cells with different concentrations
of Ru1, the levels of caspase-3, -7, -8, procaspase-3 and procaspase-7
expression decreased, whereas that of caspase-9, cleavage of caspase3, -7 and -8 increased. Anti-apoptotic Bcl-2 proteins, Bcl-x have also
been strongly pursued as therapeutic targets for apoptosis-inducing
anti-cancer strategies due to evidence of their overexpression in many
cancers and their association with poor prognosis and chemoresistance
in some of these cancers. The effect of Ru1 on the expression of
antiapoptotic proteins Bcl-2, Bcl-x, Bag-1 and proapoptotic proteins
Bad, Bak and Bim were investigated. As shown in Fig. 8b, A549 cells
were exposed to 0.5, 1.0 and 2.0 M of Ru1, the levels of the antiapoptotic proteins Bcl-2 and Bcl-x expression were down-regulated,
whereas the expression of Bag-1 was up-regulated. The level of
proapoptotic protein Bad increased, whereas the levels of Bak and Bim
expression were down-regulated. Additionally, the expression of p53
protein was also determined, with increasing the concentration of
Ru1, the expression of p53 increased. These data show that Ru1 can
activate caspases and regulate the expression of Bcl-2 family proteins.
4. Conclusions
In conclusion, we synthesized a new ruthenium(II) polypyridyl complex [Ru(dmp)2(pddppn)](ClO4)2. The complex shows very high cytotoxic activity toward BEL-7402, HeLa, MG-63 and A549 cells. Complex
Ru1 can induce apoptosis of A549 cells and can enter into the cytoplasm
and accumulate in the cell nuclei. Ru1 can enhance the level of ROS and
induce the decrease of mitochondrial membrane potential. Ru1 inhibits
the cell growth at G2/M phase in BEL-7402 and MG-63 cells and at G0/
G1 phase in A549 cells. The complex can down-regulate the expression
of Bcl-2, Bcl-x, Bak, Bim and up-regulate the levels of caspase-9, cleavage of caspase-3, -7 and -8, Bag-1 and Bad expression. These results suggest that Ru1 induces apoptosis through an intrinsic ROS-mediated
mitochondrial dysfunction pathway, which is accompanied by the regulation of Bcl-2 family proteins. The results will be of value for designing
new ruthenium(II) complexes as potent antitumor agents.
Abbreviations
Im
DMSO
Ind
phpy
bpy
dmp
dppn
CppH
dip
1-Py-C
dppz
addppn

imidazole
dimethylsulfoxide
indazole
2-phenylpyridine
2,2-bipyridine
2,9-dimethyl-1,10-phenthroline
benzo[i]dipyrido[3,2-a:2,3-c]phenazine
2-(2-pyridyl)pyrimidine-4-carboxylic acid
4,7-diphenyl-1,10-phenanthroline
1-(2-pyridyl)--carboline
dipyrido[3,2-a:2,3-c]phenazine
acenaphtheno[1,2-b]-1,4-diazabenzo[i]dipyrido[3,2-a:2,3c]phenazine
pddpn
phenantheno[1,2-b]-1,4-diazabenzo[i]dipyrido[3,2-a:2,3c]phenazine
Hdpa
2,2-dipyridylamine
2,9-dimethyl-dpq 2,9-dimethyl-dipyrido[3,2-f:2,3-h]quinoxaline
addpz
7-aminodipyrido[3,2-a:2,3-c]phenazine
7-F-dppz 7-uorodipyrido[3,2-a:2,3-c]phenazine
UVvis UVvisible
PBS
phosphate buffer saline
MTT
3-(4,5-dimethylthiazole)-2,5-diphenyltetraazolium bromide
RPMI
Roswell Park Memorial Institute
FBS
fetal bovine serum

DCHF
DCF
BCA
Tris
Tween
AO
EB
cccp

2,7-dichloro-3,6-uorandiol
dichlorouorescein
bicinchoninic acid
tris(hydroxymethylaminomethane)
polyoxyethy-lene monolaurate sorbaitan
acridine orange
ethidium bromide
carbonylcyanide-m-chloropenylhydrazone

Acknowledgments
This work was supported by the High-Level Personnel Project of
Guangdong Province in 2013 and the Joint Nature Science Fund of the
Department of Science and Technology and the First Afliated Hospital
of Guangdong Pharmaceutical University (No. GYFYLH201315).
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