Chiral ruthenium(ii) complex as potent radiosensitizer of 125I through DNA-damage-mediated apoptosis

Article abstract of DOI:10.1039/c8ra03383h

A chiral ruthenium(ii) complex, Λ-[Ru(bpy)₂(o-tFMPIP)] (ClO₄)₂ (o-tFMPIP = 2′-trifluoromethylphenyl) imidazo [4,5-f][1,10]phenanthroline, was prepared and evaluated for its enhancement of the radiosensitivity of ¹²⁵

Full text of DOI:10.1039/c8ra03383h

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Chiral ruthenium(II) complex as potent
1
25
radiosensitizer of
I through DNA-damage-
Cite this: RSC Adv., 2018, 8, 20612
mediated apoptosis†
a
a
b
b
a
a
Mingjun Bai,‡ Zhaolin Zeng,‡ Li Li, Qiong Wu, * Yanyang Zhang, Tao Pan,
Luwen Mu,
a
a
a
a
b
Duo Zhu, Shouhai Guan,* Qiang Xie* and Wenjie Mei
0
A chiral ruthenium(II) complex, L-[Ru(bpy)
2
(o-tFMPIP)] (ClO
4
)
2
(o-tFMPIP ¼ 2 -trifluoromethylphenyl)
imidazo [4,5-f][1,10]phenanthroline, was prepared and evaluated for its enhancement of the
1
25
radiosensitivity of
I seeds. The synthetic Ru(II) complex, LR042, effectively enhanced growth inhibition
125
against HepG2 human hepatocellular liver carcinoma cells induced by
I seeds and consequently
effectively promoted the apoptosis of tumor cells with increasing level of cleave-caspase-3.
Furthermore, the results of immunofluorescence indicated that LR042 enhanced the phosphorylation of
125
Received 19th April 2018
Accepted 15th May 2018
H2AX by
I seeds vigorously in response to damaged DNA. LR042 improved DNA damage induced by
125
I seeds, which resulted in apoptosis through the activation of the p53/AKT signal. In conclusion,
125
DOI: 10.1039/c8ra03383h
synthetic LR042 can be further developed as a potential radiosensitizer of
cancer therapy.
I seed radiotherapy for
rsc.li/rsc-advances
reduce the damage to the surrounding normal tissues and
1
. Introduction
1
25
medical personnel. When low-energy I seeds are implanted,
8
125
For decades, radiotherapy is one of the most common and the gamma rays can be focused on the target area. Thus,
I
effective tumor therapy techniques in clinical use. Generally, seeds have been applied to the treatment of head and neck
9,10
125
radiotherapy is commonly used as a clinical noninvasive means carcinoma, recurrent colorectal cancer.
However,
I seed
to treat tumors. High-energy X-rays or g-rays kill tumor cells by radiotherapy still cannot eradicate hypoxic tumors efficiently
1
1
inducing DNA damage or causing radical damage that in turn due to their insensitivity to radiation.
1
25
1,2
breaks down the DNA. However, the application of radio-
therapy technology to cancer treatment has many limitations. combination of chemotherapy and radiotherapy has become
To enhance the sensitivity to
I seed radiation, the
12
Radiotherapy has inefficient ability against cancer cells, a standard treatment option. It's reported that gold nano-
because of the strong resistance of cancer cells to external beam particles (GNPs) can enhance radiotherapeutic efficacy of
1
25
I
3
4
13
radiotherapy (EBRT), and high rate of local recurrence.
and use as tumor-targeted radiosensitizer in oncotherap.
Furthermore, adjacent normal tissues could be damaged in the Currently, ruthenium(II) complexes, which show excellent
5
process of radiation treatment. Therefore, exploring a new inhibitory activity against various tumors but low cytotoxicity
treatment modality for tumor patients is necessary to overcome toward human normal cells, have been widely studied for their
the effect of EBRT and reduce its side effects on the surrounding potential utility in chemotherapy. Increasing numbers of ruth-
normal tissue.
enium(II) complexes have been developed as potential anti-
1
25
I seeds have an average energy of 27.4–31.4 keV, and their cancer drugs. For instance, NAMI-A and KP1019 have been
6
14,15
valid radius is 1.7 cm in tissue. They have been widely applied successfully entered into clinical trials.
Gasser and
for permanent implantation in the treatment of cancers due to coworkers found that polypyridyl ruthenium(II) complexes can
7
125
16
their high precision and low complication rate.
I seeds can develop into potential apoptosis inducers of cancer cells.
Different ruthenium complexes have anticancer effects against
variety of cancer cells, especially against metastatic
a
17–19
a
Department of Vascular Interventional Radiology, The Third Affiliated Hospital, Sun cancers.
Our previous study found [Ru(phen)
2
(p-
Yat-sen University, 600 Tianhe Road, Guangzhou, Guangdong, China, 510630. E-mail: tFMPIP)](ClO
)
as a potential dual functional agent for the
4
2
xieqiangdoctorxie@126.com; Guanshouhai1148598268@qq.com
inhibition of the proliferation of tumor cells through stabilizing
c-myc G4 DNA. Therefore, ruthenium(II) complexes may also be
b
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, China,
20
5
†
1
‡
10006. E-mail: wuqiongniu.1113@163.com
Electronic supplementary information (ESI) available. See DOI:
0.1039/c8ra03383h
candidate agents for radiosensitization.
Inspired by these ndings, the present study synthesized
a chiral ruthenium(II) complex and found that such a complex can
Equally contribution.
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125
+ +
1
effectively enhance the
I-induced growth inhibition against DMSO, m/z): 365.7 ([M + H ] , calc. 365.1). H NMR (in DMSO-
human hepatocellular liver carcinoma (HepG2) cells through the d6, d/ppm): 9.03 (2H, d); 8.88 (2H, d); 7.99 (2H, d); 7.87 (4H, m).
induction of apoptosis by triggering DNA damage. The complex
also activated p53 signaling pathways to enhance the anticancer
2
.3 Synthesis of L-[Ru(bpy) (o-tFMPIP)] (ClO ) . (LR042)
2 4 2
efficacy of radiation. In summary (Scheme 1), synthetic LR042 is
a promising radiation sensitizer for I seed radiotherapy.
L-[Ru(bpy)
2
(o-tFMPIP)] (ClO
4
)
2
(LR042) was synthesized
125
24
following the literature procedure with some modications.
mixture
A
0
of
[Ru(bpy) (py) ][O,O -dibenzoyl-L-tartrate]$12H O
2
2
2
2
. Experimental
(520 mg, 0.4 mmol), o-tFMPIP (188.4 mg, 0.6 mmol), and ethylene
glycol (54 mL) was irradiated with microwaves for 25 min at
2.1 Materials and methods
ꢀ
1
30 C. The cooled reaction mixture was diluted with water. The
All reagents and solvents were purchased commercially and used
without purication unless noted. Distilled water was used in all
experiments. All aqueous solutions were prepared with double-
distilled water. Ruthenium(III) chloride hydrate was purchased
from Mitsuwa Chemical. cis-Ru(bpy) Cl $2H2O, [Ru(bpy) (py) ]
sodium perchlorate added to the ltered liquor produced an
orange suspended solid. The dark-red solid was collected,
washed with water and small amounts of ethanol, dried under
vacuum, and puried using column chromatography on
2
2
2
2
alumina. The solvent was removed under reduced pressure, and
0
Cl , and [Ru(bpy) (py) ][O,O -dibenzoyl-L-tartrate]$12H O were
prepared and characterized according to the literature.
25
2
2
2
2
red microcrystals were obtained. ESI-MS (in CH CN, m/z): 389.3
3
2
1,22
ꢁ 2+
1
(
[M–2ClO
4
] , calc. 389.1). H NMR (in DMSO-d
6
, d/ppm): 9.01
Microwave-assisted synthesis was performed using an Anton
Paar Monowave 300 microwave reactor. ESI-MS spectra were
obtained in methanol on an Agilent 1100 ESI-MS system operated
at room temperature. Cellular localization and immunouores-
cence experiments were performed with an LSM800 (Germany).
(
1
d, J ¼ 8.2 Hz, 2H), 8.87 (dd, J ¼ 22.5, 8.2 Hz, 4H), 8.23 (td, J ¼ 8.1,
.4 Hz, 2H), 8.12 (td, J ¼ 8.1, 1.4 Hz, 2H), 8.10–8.04 (m, 4H), 7.94
(
(
dt, J ¼ 8.2, 6.0 Hz, 4H), 7.87 (dd, J ¼ 10.8, 5.6 Hz, 4H), 7.67–7.64
13
m, 4H), 7.63–7.57 (m, 4H), 7.38–7.34 (m, 4H). C NMR (151
MHz, DMSO) d 155.14 (s), 154.89 (s), 149.93 (s), 149.76 (s), 148.20
(
(
s), 143.45 (s), 136.23 (s), 136.09 (s), 131.03 (s), 130.80 (s), 128.69
s), 126.24 (s), 126.13 (s), 124.77 (s), 122.76 (s).
0
2
[
.2 Synthesis of (2 -triuoromethylphenyl) imidazo [4,5-f]
1,10]phenanthroline. (o-tFMPIP)
o-tFMPIP was synthesized according to the literature proce- 2.4 Synthesis of D-[Ru(bpy) (o-tFMPIP)] (ClO ) (DR042)
2
4 2
2
3
dures
with some modication.
A
mixture of 1,10-
D-[Ru(bpy) (o-tFMPIP)] (ClO ) (DR042) was synthesized by
2
4 2
0
phenanthroline-5,6-dione (315.06 mg, 1.50 mmol), 2 -tri-
uoromethylphenylaldehyde (339.81 mg, 2.25 mmol), ammo-
nium acetate (4 g, 51.9 mmol), and glacial acetic acid (20 mL)
was heated at 100 C for 20 min under microwave radiation. pH
value was adjusted to 7.0 at room temperature. The solution was
ltered and dried in vacuum to obtain a yellow precipitate,
which was collected and washed with water and small amounts
of ethanol. The crude product dissolved in ethanol was puried
by ltration on a silica gel column (60–100 mesh). ESI-MS (in
0
above method, but [Ru(bpy) (py) ][O,O -dibenzoyl-L-tartrate]$
2
2

0
1
2H
dibenzoyl-D-tartrate]$12H
ꢁ +
4
CH CN, m/z): 876.6 ([M–ClO ] , calc. 877.1), 776.9 ([M–2ClO –
2
O (520 mg, 0.4 mmol) was replaced by [Ru(bpy)
2 2
(py) ][O,O -
2
O (520 mg, 0.4 mmol). ESI-MS (in
ꢀ
ꢁ
4
3
+ +
ꢁ
2+
1
H ] , calc. 777.1), 388.5 ([M–2ClO
4
]
, calc. 389.1). H NMR

(
500 MHz, DMSO) d 9.01 (d, J ¼ 8.2 Hz, 1H), 8.88 (dd, J ¼ 18.7,
8
1
6
.2 Hz, 2H), 8.23 (td, J ¼ 8.0, 1.4 Hz, 1H), 8.13 (td, J ¼ 8.0, 1.4 Hz,
H), 8.10–8.05 (m, 2H), 7.97–7.91 (m, 2H), 7.88 (dd, J ¼ 10.7,
.6 Hz, 2H), 7.66 (d, J ¼ 5.1 Hz, 1H), 7.63–7.58 (m, 1H), 7.36
1
3
(
ddd, J ¼ 7.2, 5.8, 1.1 Hz, 1H). C NMR (151 MHz, DMSO)
d 155.16 (s), 154.91 (s), 149.99 (s), 149.73 (s), 148.10 (s), 143.58
(
(
s), 136.36 (s), 136.10 (s), 131.06 (s), 130.87 (s), 128.63 (s), 126.25
s), 126.16 (s), 124.91 (s), 122.88 (s).
2.5 Cell culture
The cell lines used in this study, including the human hepato-
cellular liver carcinoma (HepG2) cell line, human colon cancer
(
SW480) cell line, and human lung adenocarcinoma (A549) cell
line, were purchased from the American Type Culture Collection
ATCC, Manassas, VA). All cell lines were maintained in a Roswell
(
Park Memorial Institute medium: Dulbecco's modied Eagle
medium supplemented with bovine serum albumin (BSA; 10%),
penicillin (100 units per mL), and streptomycin (50 units per mL)
ꢀ
at 37 C in a CO
2
incubator (95% relative humidity and 5% CO
2
).
Scheme 1 (A) Chemical structure of the target ruthenium(II) poly-
pyridyl complex of LR042. (B) Application diagram of the model of the
125
I seed brachytherapy source. (C) Schematic diagram of the exper-
2
.6 MTT assay
1
25
imental setup for the measurement of radial dose function for
I
125
Cell viability was determined by measuring the ability of cells to
transform MTT to a purple formazan dye. Cells were seeded in
seeds. The I seeds were arranged regularly on the top of the 96 well
plate.
26
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6-well tissue culture plates for 24 h. The antiproliferative were incubated with primary antibodies at 1 : 1000 dilutions in
1
25
ꢀ
effects of ruthenium(II) complex and I
were assessed using 5% nonfat milk overnight at 4 C, and then secondary anti-
MTT assay. The cells were incubated with different concentra- bodies were conjugated with horseradish peroxidase at 1 : 2000
tions of ruthenium(II) complex (0–20 mM) with and without the dilution for 1 h at room temperature.
1
25
125
treatment of I
seed radiation. The
I seeds were arranged
regularly on the top of the 96 well plate, which keep a certain
distance (0.9 cm) between two seeds. Then, the 96 well plate
2
.10 Immunouorescence
4
HepG2 cells in complete growth medium at 5 ꢂ 10 cells per mL
were incubated with LR042 (5 mM) for 24 h, unless otherwise
stated. The cells were washed once in PBS, xed, permeabilized
simultaneously using 4% paraformaldehyde with 1% Triton X-
1
25
containing cancer cells covered on the I seeds, which cancer
cells were radiated in a distance about 1 mm (the thickness of
1
25
9
6 well plate) with I seeds, and every well was located in the
center of four seeds, which keep the well with same radiation
100 in PBS, quenched with 0.1 M glycine in PBS, and blocked
energy. Thereaer, the cells were cultured for 72 h. At the end of
ꢀ
overnight at 4 C with 3% (w/v) BSA. The xed cells were stained
with primary antibodies as indicated. Cell morphology was
observed using a laser confocal microscope.
ꢁ
1
incubation, 20 mL per well of MTT solution (5 mg mL in
phosphate-buffered saline (PBS)) was added and incubated for
30
27
5
h. The medium was aspirated and replaced with 150 mL per
well of DMSO to dissolve the formazan salt formed. The color
intensity was measured at 570 nm using a microplate spectro- 3. Results and discussion
photometer (Versa Max). The cell viability of the treatment
groups was expressed as the percentage of the control.
3
.1 Distribution model of the 125I seed brachytherapy
source
Either intracavitary or implantation brachytherapy needs an
accurate localization of the position of radiation source to
calculate the dose distribution. The dosimetric properties of
brachytherapy sources can be obtained by calculating the dosi-
2
.7 Cellular localization
4
HepG2 cells in a complete growth medium at 5 ꢂ 10 cells
ꢀ
per mL were incubated with LR042 (5 mM) for 24 h at 37 C unless
otherwise stated. Cells were washed three times with PBS, xed,
permeabilized simultaneously by using 4% p-formaldehyde, and
stained with DAPI (0.5 mg mL ) for 15 min. The cell morphology
metric parameters. The optimization of dose distribution is
125
important in brachytherapy. As shown in Scheme 1B, the
seeds were arranged uniformly on the 96-well plate. Four
I
I
ꢁ1
125
was observed using a confocal laser microscope.
seeds were positioned in one well with a xed distance (0.9 cm)
from one another. According to the literature, the radiation dose
of every well on the 96-well plate was calculated for 0.57 cm. The
2.8 Flow cytometric analysis
1
25
ꢁ1 ꢁ1
The cell cycle distribution and the apoptosis rate were analyzed radiation energy of every I seed was 0.992 ꢃ 0.025 cGy h
U .
28
125
using ow cytometry as previously described. Aer incubating Aer treatment with I seed for 72 h, the accumulated radiation
with different concentrations of LR042 (0, 5, and 10 mM), LR042 dose of the tumor cells reach 285.7 ꢃ 7.2 cGy U
ꢁ
1
31
.
1
25
(0, 5, and 10 mM), and I for 72 h, the cells were trypsinized,
ꢀ
125
washed with PBS, and xed with 70% ethanol overnight at 4 C. 3.2 Radiotherapy sensitization of LR042 to enhance I-
The xed cells were washed with PBS and stained with propidium seed-induced growth inhibition
iodide (PI) for 15 min in the dark. Then, the cell cycle arrest was
The antiproliferative activities of LR042 and DR042 were
analyzed using an Epics XL-MCL ow cytometer (Beckman
screened using MTT assay against human hepatocellular liver
Coulter, Miami, FL, USA). The treated and untreated cells were
carcinoma HepG2 cells, human colon cancer SW480 cells, and
trypsinized, washed with PBS, and costained with Annexin V and
human lung adenocarcinoma A549 cells. The inhibitory activi-
PI for 10 min, respectively. The apoptosis of cells was analyzed
ties (IC50) of the complex against different tumor cells are listed
using an Epics XL-MCL ow cytometer (Beckman Coulter).
in Table 1. It is found that laevo-isomer LR042 displayed great
growth inhibition against HepG2 cells (IC50 ¼ 9.63 mM) and
2
.9 Western blot analysis
The effects of HepG2 treated with LR042 and/or radiation on the
expression levels of proteins associated with different signaling cells after 72 h of treatment
125
Table 1 Inhibitory effect (IC50, mM) of LR042 and I on human cancer
29
pathways were examined using Western blot analysis. The
total cellular proteins were extracted by incubating the cells in
a lysis buffer obtained from Cell Signaling Technology. Protein
concentrations were determined using BCA assay. P53, Bax and
Bcl-2 caspase-3 and AKT were purchased from Abcam, Cell
Signaling Technology, and Proteintech. SDS-PAGE was per-
formed in 10% tricine gels, and equal amounts of protein were
loaded per lane. The procedure was conducted as described
previously. Aer electrophoresis, the separated proteins were
transferred to nitrocellulose membranes and blocked with 5%
nonfat milk in TBST buffer for 1 h. Thereaer, the membranes
IC50 (mM)
Comp.
HepG2
SW480
A549
HaCaT
LR042
LR042 with
9.63 ꢃ 1.49
1.81 ꢃ 0.50
1.13 ꢃ 0.32
0.79 ꢃ 0.90
>20
>20
9.65 ꢃ 0.21
10.61 ꢃ
125
I
0.38
Radiosensitivity
5.32
1.43
—
0.91
a
index
a
125
Radiosensitivity index ¼ IC50 (LR042)/IC50 (LR042 with
different cells.
I) for
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SW480 cells (IC50 ¼ 1.31 mM) aer 72 h of treatment. However, indicated HepG2 cell death, in a special incubation of LR042 (10
1
25
dextro-isomer DR042 exhibited little suppression on different mM) and I (Fig. 1D). The evident reduction of cell viability and
tumor cells. These data suggested that LR042 displayed much marked cell morphology change indicated that LR042 can
1
25
more promising inhibitory effect against a variety of tumors effectively enhance the inhibitory activity of
cells than DR042 (Table S1 in ESI†). However, dextro-isomer the growth of tumors cells.
DR042 exhibited little suppression on different tumor cells.
I seeds against
These data suggested that LR042 displayed much more prom- 3.3 Drug distribution and localization
ising inhibitory effect against a variety of tumor cells than
Moreover, the cellular localization of LR042 in HepG2 cells
without and with the radiation of I were further investigated,
DR042.
However, with continuous and low-dose radiation with
125
1
25
I
as shown in Fig. 2. Accordingly, the ability of LR042 to
membrane-permeabilized cells was characterized by employing
confocal laser scanning microscopy (CLSM). As shown in Fig. 2,
the nucleus of HepG2 cells was highlighted to blue by the DNA
seeds for 72 h, the inhibitory activities of LR042 against HepG2
and SW480 cells decreased markedly to 1.81 and 0.79 mM,
respectively. As shown in Fig. 1, for HepG2 cells, LR042
inhibited the growth of HepG2 cells with increasing concen-
0
dye DAPI (4 ,6-diamidino-2-phenylindole). Without the drug,
1
25
tration. Then, the inhibitory effect of
I radiation effectively
the nucleus was round and plump. Aer treatment, LR042
emitted red uorescence that distributed in the whole cell,
increased. However, for the SW480 and A549 cells, little
1
25
improvement by
I radiation was observed. Synthetic LR042
mainly enriched in nucleus and a little distributed in the cyto-
1
25
enhanced the radiosensitivity of HepG2 cells to I seeds. The
IC50 values of the combined treatment against HepG2 cells
125
plasm. Moreover, combined with
I radiation, LR042 mainly
distributed in the whole cell with strong red uorescence, and
the cell nucleus notably shrank and condensed into smaller
balls. These results suggested that LR042 may cause cell death
1
25
dramatically decreased from 9.63 mM ( I seed radiation alone)
to 1.81 mM. Whereas for DR042, no obvious enhanced radio-
sensitivity was observed against various tumor cells (Table S1 in
ESI†). The radiosensitivity index was 5.32, which suggested
LR042 can induce tumor cell death at a low concentration under
32
by inducing DNA damage.
3.4 Biochemical mechanism studies
1
25
I radiation (Table 1). On the basis of its promising in vitro
1
25
3
.4.1 LR042 enhanced apoptosis induced by I radiation.
activity, LR042 was selected as an early lead for a preliminary
evaluation in further study.
Furthermore, ow cytometry was performed to examine the
inhibitory activity of LR042 and the combined treatment of
Furthermore, under the phase-contrast observation of the
1
25
1
25
LR042– I against the growth of HepG2 cells. Specically, the
suppression of cancer cell proliferation resulted from
apoptosis, cell cycle arrest, or the joint action of both modes
HepG2 cells, I seed radiation alone exerted little inuence on
the cell viability and morphology. Nevertheless, with the treat-
ment of LR042 at the concentration of 5 and 10 mM for 72 h, the
HepG2 cells displayed loss of cell-to-cell contact, dose-
dependent cell shrinkage, and reduction in cell number.
33
(
Fig. 3). As shown in Fig. 3, the exposure of HepG2 cells to 0, 5,
and 10 mM LR042 for 24 h exerted no evident inuence to the
1
25
1
25
cell cycle. Under I seed radiation, the number of cells under
Then, when LR042 was combined with I seed radiation, the
S-phase arrest signicantly increased. However, the combined
LR042– I treatment, did not exert evident changes to the cell
cell morphology profoundly shrank to a round shape, which
1
25
125
Fig. 1 LR042 enhanced I radiation-induced HepG2 cell (A), SW480
cell (B), and A549 cell (C) growth inhibition. These tumor cells were Fig. 2 Cellular localization and distribution of LR042 in the HepG2
125
pretreated with various concentrations of LR042 (0–20 mM) for 72 h cells. The cells were treated with LR042 and LR042– I for 24 h at
1
25
ꢀ
and treated with and without
I. (D) Changes in the morphology of 37 C: blue, DAPI; red, ruthenium(II) complexes, [LR042] ¼ 5 mM.
125
cells exposed to LR042 with and without I radiation were captured Fluorescent images were viewed using a confocal laser scanning
using fluorescence microscopy (ꢂ40 magnification).
microscope.
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Fig. 3 Cell cycle distribution of HepG2 cells incubated with LR042
125
with and without I radiation by using flow cytometric analysis. The
cells were treated with LR042 (0, 5, 10 and 20 mM) and cotreated with
125
and without I radiation for 72 h.
cycle. These results indicated that LR042 and the combination
1
25
of LR042– I did not cause tumor cells death by interfering the
34,35
cell cycle.
To further conrm the potential mechanism of the combi-
1
25
nation of LR042– I seeds, ow cytometry was also performed
to analyze cell apoptosis by staining the cells with Annexin V
and PI. As indicated in Fig. 4, increasing concentrations of free
LR042 slightly increased both the early (Q4 region) and late (Q2
region) stages of apoptosis. At 10 mM concentration of LR042,
1
.1% of the cells were in the early apoptotic stages and 4.4% in
1
25
the late stages. However, with the addition of I seed radiation
at a concentration of 10 mM LR042, 28.18% of the cells were in
early apoptosis and 17.15% in late apoptosis. LR042 may
1
25
effectively enhance
I-induced inhibitory activity against the
125
growth of HepG2 cells via apoptosis.
Fig. 4 (A) LR042 enhanced
I-induced HepG2 cell apoptosis. The
To investigate the underlying mechanisms for the cells were incubated with LR042 (0, 5, and 10 mM) and cotreated with
125
125
and without
I radiation for 72 h. (B) LR042 enhanced the
I-
cotreatment-induced apoptosis, the activation of Bcl-2 and Bax
were conrmed using Western blot analysis. As shown in
Fig. 4B, some certain up-regulate of Bax and down-regulate of
induced apoptosis of HepG2 cells and the related signaling pathways,
which were examined using Western blot analysis. The cells were
treated with LR042 (0, 10, and 20 mM) and cotreated with and without
1
25
125
Bcl-2 with the radiation of
I were observed. Moreover, with
I radiation for 72 h.
1
25
the addition of LR042 combined with
I, the up-regulate of
Bax and down-regulate of Bcl-2 were enhanced notably. It is well
known that Bax is a key pro-apoptotic protein which the
increasing expression followed with apoptosis, and Bcl-2 is an
important inhibitory apoptosis protein which the decreasing
expression followed with apoptosis. As a marker of apoptosis,
the induction of DNA double-strand breaks (DSBs) in HepG2
cells were investigated using confocal immunouorescence
assays (IFs) and staining with gH2AX (an early marker of DNA
38
damage response). HepG2 cells showed little activation of
gH2AX in response to LR042 treatment. However, the combi-
36
caspase-3 can be cleaved and activated during apoptosis. As
shown in Fig. 4B, cotreatment with LR042 induced the activa-
tion of caspase-3 in HepG2 cells aer 72 h of treatment as evi-
denced by the appearance of cleaved and increased levels of
caspase-3 (17 kDa) in comparison with the single treatment of
125
nation of LR042– I seeds (Fig. 5A) induced an increasing
number of gH2AX-positive cells. The enhancement of DNA
125
damage contributed to the synergistic effects of LR042 and
I
radiation in HepG2 cells.
Moreover, P53, a classic DNA damage response marker, is
1
25
I radiation. No changes were detected in the expression of
39
activated by AKT phosphorylation. AKT, also called protein
total caspase-3. These results indicated that the enhancement
of the HepG2 cell apoptosis contributed to the synergistic
kinase B, is described as the critical upstream mediator of wild-
type p53 and is known to suppress DNA replication. The pro-
apoptotic ability of p53 is activated through the expression of
AKT. Given the role of the AKT–p53 axis in triggering DNA-
damage-induced apoptosis, this study examined the protein
levels of AKT and p53 in response to treatment with free LR042
1
25
effects of LR042 and I radiation.
1
25
3.4.2 LR042 enhanced
I-induced DNA damage and
related signaling pathways. DNA is the most important and
sensitive target molecule for radiation biological effects. DNA
damage as the main mechanism for the combined treatments
125
and the combination of LR042– I seeds. Cellular extracts from
37
of radiotherapy and chemotherapy is reported. In this study,
40
the HepG2 cells revealed an increase in AKT–p53 protein.
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Fig.
6
Proposed signaling pathways of apoptosis induced by
125
LR042– I cotreatment. LR042 enhanced the radiosensitivity of cells
1
25
to
I-induced DNA damage. The activation of the p53 pathway
further activated AKT and caspase-3 and resulted in the apoptosis of
HepG2 cells.
4. Conclusions
In this study, it is found that laevo-isomer of chiral ruth-
enium(II) polypyridyl complex (LR042) exhibited much more
better inhibition than dextro-isomer (DR042). Moreover, LR042
1
25
could enhance the I -induced suppression on HepG2 cells
through the induction of apoptosis by accelerating DNA
damage. Further study indicated the occurrence of DNA damage
1
25
Fig. 5 (A) LR042 enhanced the
I-induced DNA damage of the
125
HepG2 cells. The cells were treated with LR042 and LR042– I for and activated downstream signaling pathways, including up-
4 h at 37 C. The confocal immunofluorescence assays (IF) of HepG2 regulation of the p53 and AKT, thereby nally resulting in an
ꢀ
2
cells were dealed with antibodies specific to DSBs biomarker gH2AX,
increase of radiation sensitivity and inhibition of tumor cells
125
[
LR042] ¼ 5 mM. (B) LR042 enhanced the
I-induced apoptosis of
proliferation. The synthetic LR042 can be further developed as
HepG2 cells and related signaling pathway. The DNA damage and
related signaling pathways were examined using Western blot analysis.
1
25
radiosensitizer of
I by inducing DNA-damage-mediated
The cells were treated with LR042 (0, 10, and 20 mM) and cotreated apoptosis for cancer therapy.
125
with and without I radiation for 72 h.
Conflicts of interest
As shown in Fig. 5B, aer treatment with free LR042, the
The authors declare no competing nancial interests.
expression level of p53 increased evidently, whereas little
1
25
signicant change was observed with free
I radiation.
Furthermore, the up-regulation of p53 was notably observed Acknowledgements
1
25
aer the combined treatments (LR042– I radiation). LR042
1
25
This work was supported by the authors acknowledge the
National Nature Science Foundation of China (81572926,
can enhance the I -induced DNA damage, up-regulate the
expression of p53, and further induce the apoptosis of HepG2
cells. The protein serine/threonine kinase AKT plays essential
81703349); the Provincial Major Scientic Research Projects in
41
Universities of Guangdong Province (2014KZDXM053); the
Science and Technology Project of Guangdong Province
(2014A020212312); the Innovation Projects in Universities of
Guangdong Province (2015cxqx151).
roles on the regulation of cell proliferation and survival.
Activated p53 up-regulation of AKT may lead to an irreversible
42
commitment to apoptotic cell death. As shown in Fig. 5B,
alone LR042 can decrease the expression level of AKT, but the
1
25
combination of LR042 with
I seed radiation can markedly
increase the expression of AKT. LR042 can serve as a potential
radiosensitizer to induce apoptosis of HepG2 cells through the
DNA-damage-activated AKT–p53 pathway (Fig. 6).
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