Microwave-assisted synthesis of arene ruthenium(II) complexes that induce S-phase arrest in cancer cells by DNA damage-mediated p53 phosphorylation

Article abstract of DOI:10.1016/j.ejmech.2013.01.037

A series of arene ruthenium(II) complexes coordinated by phenanthroimidazole derivates, [(C₆H₆)Ru(L)Cl] Cl·2H₂O (1b L = IP, 2b L = p-NMe₂PIP, 3b L = p-MeOPIP, 4b L = p-HOPIP, 5b L = p-COOHPIP, 6b L = p-CF_3<

Full text of DOI:10.1016/j.ejmech.2013.01.037

European Journal of Medicinal Chemistry 63 (2013) 57e63
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Microwave-assisted synthesis of arene ruthenium(II) complexes that induce
S-phase arrest in cancer cells by DNA damage-mediated p53 phosphorylation
,
,
,
b
a,
b,
c
Qiong Wu a 1, Cundong Fan b 1, Tianfeng Chen b , Chaoran Liu , Wenjie Mei
**
**
, Sidong Chen ,
*
Baoguo Wang c, Yunyun Chen d, Wenjie Zheng b
,
a School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, China
b Department of Chemistry, Jinan University, Guangzhou, China
c Key Laboratory of Molecular Epidemiology, Guangdong Pharmaceutical University, Guangzhou, China
d School of Pharmacy, Sun Yat-sen University, Guangzhou, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of arene ruthenium(II) complexes coordinated by phenanthroimidazole derivates, [(C6H6)Ru(L)
Cl]Cl$2H2O (1b L ¼ IP, 2b L ¼ p-NMe2PIP, 3b L ¼ p-MeOPIP, 4b L ¼ p-HOPIP, 5b L ¼ p-COOHPIP, 6b L ¼ p-
CF3PIP, 7b L ¼ p-BrPIP) have been synthesized in yields of 89e92% under microwave irradiation in
30 min, and the crystal structure of 1b by XRD gives a typical “piano stool” conformation. The antitumor
activity of these complexes against various tumor cells have been evaluated by MTT assay, and the results
show that this type of arene Ru(II) complexes exhibit acceptable inhibitory effect against all of these
tumor cells, especially osteosarcoma MG-63 cells, but with low toxicity toward HK-2 human normal cells.
Studies on the mechanism revealed that cell cycle arrest at S-phase in MG-63 cells induced by the arene
Ru(II) complex 2b, which was confirmed by the increase in the percentage of cells at S-phase and down-
regulator of cyclin A. The further studies by Comet assay at single cell level indicated that DNA damage in
MG-63 cells was triggered by 2b, following with the up-regulation of phosphorylated p53 and histone.
The studies by spectroscopy in vitro also indicate that 2b bind to DNA molecule by intercalative mode to
disturb the bio-function of tumor cells. In conclusion, the synthetic arene Ru(II) complexes could serve as
novel p53 activator with potential application in cancer chemotherapy.
Received 11 October 2012
Received in revised form
25 January 2013
Accepted 30 January 2013
Available online 8 February 2013
Keywords:
Arene ruthenium complexes
Microwave assisted synthesis
S-Phase arrest
DNA damage
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
arene Ru(II) complex [(h
6-arene)RuII(en)Cl]þ, which exhibit great
inhibitory activity against various human tumor cells, can unwind
the double strand helix of DNA by forming monofunctional adducts
with DNA in intercalative mode [13,14].
Due to the unavoidable high toxicity, drug resistance and severe
side effects of platinum-based anticancer drugs [1,2], ruthenium(II)
complexes, especially arene ruthenium(II) complexes have been
considered as one of the most promising alternatives owing to their
low toxicity, high antitumor activity and strong DNA-binding af-
finity [3e5]. DNA has long been considered as a common target for
antitumor drugs, such as cisplatin, doxorubicin, 5-fluorouracil and
paclitaxel which exhibit their therapeutic effects by bonding to
DNA molecules [6,7], and there are a large number of evidence
indicated that arene Ru(II) complexes can bind to disturb the rep-
lication of DNA [8e12]. For example, Sadler et al. indicated that
In recently, Dyson et al. elucidated that RAPTA-C-induced DNA
damage was observed for EAC cells, following the arrest of cell cycle
in G2/M phase, as the results the cells proliferation was suppressed
[15]. It was worth to indicate that the half-life and transcriptional
activity of p53, which are in the central region of the protein
responsible for DNA binding [16], are increased in response to the
treatment of RAPTA-C [17e20]. It’s also reported that up-regulate
p53 expression was also observed in apoptosis induced by DW1/2
against melanoma cells [21]. Furthermore, cell cycle arrest in G1
phase through p53-depended and p53-independed mechanism
also reported for arene ruthenium-derived compounds (RDCs) [22].
However, there is still little information focused on the S-phase cell
cycle arrest through DNA damage-mediated p53 phosphorylation
induced by arene ruthenium complexes.
* Corresponding author. School of Pharmacy, Guangdong Pharmaceutical Uni-
versity, Guangzhou, China. Tel./fax: þ86 20 39352129.
** Corresponding authors.
In the present study, a series of arene Ru(II) complexes coordi-
nated by phenanthroimidazole derivatives (Scheme 1) have been
E-mail address: wenjiemei@126.com (W. Mei).
These authors contributed equally to the work.
1
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.037

58
Q. Wu et al. / European Journal of Medicinal Chemistry 63 (2013) 57e63
+
H
N
N
N
H
N
Cl
Cl
N
N
Cl-
Ru
Ru
R
Ru
MW
2
Cl
Cl
R
+ 2
N
Cl
N
60 °C,30min
1b-7b
1a-7a
1a, 1b R=H
2a, 2b R=p-NMe2C6H4 3a, 3b R=p-MeOC6H4 4a, 4b R=p-HOC6H4
7a, 7b R=p-BrC6H4
5a, 5b R=p-COOHC6H4 6a, 6b R=p-CF3C6H4
Scheme 1. Microwave-assisted synthesis route for arene Ru (II) complexes.
prepared in high yield under the irradiation of microwave, and the
cell cycle arrest in the S-phase through triggering DNA damage by
this type of arene ruthenium(II) complexes was investigated.
reduction in cell number, cell shrinkage and loss of cell-to-cell
contact. The reduction of cell viability and the change in cell mor-
phology both proved the growth inhibitory effect of Ru(II) complex
2b on MG-63 cells. Besides this potency, all Ru complexes showed
low cytotoxicity toward HK-2 human normal cells, suggesting that
the synthetic Ru(II) complexes possess the great selectivity be-
tween human cancer and normal cells and display potential
application in caner chemotherapy.
2. Result and discussion
2.1. Synthesis
The application of microwave irradiation [23] has significantly
increased the yield for most of the complexes to about 90%, which
were much higher than those of conventional synthesis method
(Table 1). Interestingly, complex 5b that could not be synthesized
by liquid phase synthesis method has been produced with yield of
47.5% under the microwave irradiation. Complex 1b was crystal-
lized and characterized by X-ray diffraction, and the results showed
that complex 1b displayed a typical “piano stool” structure (Fig. 1).
As shown in Table 2, the Ru atom was bonded with benzene ring
and ligand with an average RueC and RueN distance of 2.188 (5)
2.3. Mechanism studies
Growth inhibition in cancer cells by anticancer drugs could be
the result of induction of apoptosis or cell cycle arrest or a combi-
nation of these two modes. Therefore, we performed propidium
iodide (PI)-flow cytometric analysis to determine action modes of
complex 2b. The results reveal that complex 2b-induced growth
inhibition was mainly caused by cell cycle arrest at S-phase
(Fig. 2C). For instance, exposure of MG-63 cells to 5
mg/ml of
ꢀ
and 2.094 (4) A, respectively. The clear characterization of the
chemical structure of the complexes facilitates the understanding
of their anticancer mechanisms [24].
complex 2b for 24 h triggered a significant increase of cell pro-
portion at S-phase, accompanied by decrease in the percentage of
cells at G2/M phase (Fig. 2D), implying that p53 initiated a S-phase
arrest for DNA repair to achieve growth inhibition. Furthermore,
MG-63 cells exposed to complex 2b for 24 h showed dose-
dependent decrease in the expression levels of cyclin A (Fig. 2E),
which can form a complex with CDK to regulate the progression of
cells [25,26]. The down-regulation of cyclin A may disturb the
progression of cells at S-phase. Taken together, induction of
S-phase arrest by down-regulation of cyclin A could be the major
mechanism for cell death induced by complex 2b.
2.2. Biological activity
The in vitro anticancer activities of the Ru complexes were
screened against a series of human cancer cell lines by MTT assay
after a 72 h treatment (Table 3). The results indicate that the anti-
proliferative effects of the complexes were cell-line specific. The
most active complex, 2b, with ep-NMe2C6H4 group on R position,
displayed a broad spectrum growth inhibition against several
cancer cells, with IC50 value lower than those of other Ru com-
plexes. MG-63 human osteosarcoma cells were sensitive to com-
plex 2b with IC50 value at 36.1 mg/ml. Treatment of MG-63 cells
with complex 2b significantly decreased the cell viability in a dose-
dependent manner (Fig. 2A). In the phase-contrast observation
(Fig. 2B), the cells treated with complex 2b for 72 h displayed
Table 1
Yields of Ru complexes by microwave-assisted and conventional synthesis methods.
Complexes
Microwave-assisted
Conventional
Temp./ꢀC
Time/h
Yield/%
Temp./ꢀC
Time/h
Yield/%
1b
2b
3b
4b
5b
6b
7b
60
60
60
60
60
60
60
0.5
0.5
0.5
0.5
0.5
0.5
0.5
91.2
90.3
91.4
90.8
47.5
91.3
89.9
60
60
60
60
60
60
60
4
4
4
4
4
4
4
63.5
71.4
68.8
58.7
/
69.4
56.2
Fig. 1. The ORTEP drawing of arene Ru(II) complex 1b.

Q. Wu et al. / European Journal of Medicinal Chemistry 63 (2013) 57e63
59
Table 2
intensity of the positive CD signal at 275 nm, and increase in the
relative viscosity of CT-DNA exposed to complex 2b.
Cytotoxic effects of arene Ru(II) complexes on human cancer cell lines.
Complexes IC50
(mg/ml)
3. Conclusions
MG-63 Hela
Neuro-2a MCF-7 HepG2 LNcap HK-2
1b
2b
3b
4b
5b
6b
7b
51.5
36.1
247.2
88.5
82.6
68.1
77.9
94.3
99.0
>320
>320
114.3
110.8
>320
>320
175.1
295.3
>320
79.5
78.6
191.7
82.8
In conclusion, a series of arene Ru(II) complexes coordinated by
phenanthroimidazole derivatives have been synthesized under
irradiation of microwave with high yield in 30 min. The synthetic
complexes displayed acceptable inhibitory activity against various
tumor cells, especially MG-63 cells. Further investigation on the
mechanisms revealed that this type of arene Ru(II) complexes could
induce S-phase arrest in tumor cells through DNA damage-
mediated p53 phosphorylation. Taken together, the synthetic ar-
ene Ru(II) complexes could serve as a novel p53 activator with
potential application in cancer chemotherapy.
157.2
138.6
157.3
>320
>320
186.1
176.9
>320
>320
>300
162.5
204.4
>320
>320
>320
118.7
91.1
125.6
112.0
169.0
178.0
104.0
126.8
106.4
158.0
232.0
129.8
2.4. DNA-binding studies
It is generally accepted that DNA is the target of Ru complexes.
To examine synthetic Ru complexes whether induce DNA damage,
Western blotting was employed to detect the changes of p53 and
histone phosphorylation, two of DNA damage marker, which can be
activated in response to DNA damage. As shown in Fig. 3A, MG-63
4. Experimental
4.1. Generals
cells exposed to 5e20
mg/ml of complex 2b for 24 h showed
All the chemicals including solvents were obtained from com-
mercial vendors and used as received. The arene Ru(II) complexes
were synthesized by Anton Paar GmbH monowave 300. The elec-
tronic absorption spectra were recorded on a Shimadzu UV-2550
Spectrophotometer and steady-state emission spectra were recor-
ded on a RF-5301 Fluorescence Spectrophotometer. The 1H NMR
spectra were recorded in DMSO-d6 on BrukerDRX2500 spec-
trometer and ESI-MS spectra were obtained in methanol on Agilent
1100 ESI-MS system, and the CD spectra were recorded on Jasco
J810 Circular Dichroism (CD) spectrophotometer. The X-ray in-
tensity data (0.40 mm ꢁ 0.20 mm ꢁ 0.10 mm) were collected at 293
(2) K on a Bruker SMART APEX 2K CCD-based X-ray diffractometer
a noticeable elevation of phosphorylated p53 at Ser15 in a dose-
dependent manner, implying that DNA damage was triggered in
cells exposed to complex 2b. Ser139eHistone H2AX, as another
DNA damage marker, was also up-regulated in MG-63 cells treated
with complex 2b in a dose-dependent manner. Complex 2b-
induced DNA damage was further confirmed by Comet assay at the
single-cell level, which is based on the ability of denatured or
cleaved DNA fragments to migrate out of the cell under the influ-
ence of an electric field [27]. As shown in Fig. 3B, complex 2b-
induced a remarkable DNA damage in a dose-dependent manner,
as evidenced by the increase in tail DNA. Taken together, our results
suggest that arene Ru complexes induce S-phase arrest in cancer
cells by triggering DNA damage-mediated p53 phosphorylation.
The interaction between the complexes and DNA was confirmed
by spectroscopic and viscosity analysis [28]. As shown in Fig. 3C,
upon the addition of calf thymus DNA (CT-DNA), obviously hypo-
chromism (18%) and red shift (Dl ¼ 8 nm) at LMCT (ligand-to-metal
charge transition) absorption at 350 nm were observed in the
spectra of complex 2b, with the intrinsic binding constant (K)
calculated according to the decay of LMCT absorption to be about
equipped with
a graphite-monochromated MoKa radiation
ꢀ
(l
¼ 0.71073 A). The collected frames were processed with the
software SHELXTL with 2001 Bruker Analytical X-ray Solutions.
4.2. Synthesis and characterization
4.2.1. Synthesis of 1a, 2a, 3a, 4a, 5a, 6a and 7a
A solution containing 1,10-phenanthroline-5,6-dione (1.6 mmol,
347 mg), substituted benzaldehyde (1.6 mmol), 20 ml of HAc and
NH4Ac (33 mmol, 2.53 g), was heated at 110 ꢀC under reflux for 4 h.
Then, 20 ml of water was added and the pH value was adjusted to
7.0 at room temperature. The solution was filtered and dried in
vacuum to obtain a yellow precipitate. The product was purified in
a silica gel column by using ethanol as eluent. 1a: yield 78.4%; mp.
217e219 ꢀC, ESI-MS (in MeOH): m/z: 219.1, ([M þ H]þ), 438.1,
([M þ 2H]2þ). 2a: yield 79.5%; mp. 226e228 ꢀC, ESI-MS (in MeOH):
m/z: 339.15, ([M þ H]þ), 678.1, ([M þ 2H]2þ). 3a: yield 64.4%; mp.
262e265 ꢀC, ESI-MS (in MeOH): m/z: 325.1, ([M þ H]þ), 650.3,
([M þ 2H]2þ). 4a: yiþeld 75.7%; mp. 234e236 ꢀC, ESI-MS (in MeOH):
m/z: 311.1, ([M þ H] ). 5a: yield 67.1%; mp. 232e235 ꢀC, ESI-MS (in
MeOH): m/z: 339.1, ([M þ H]þ). 6a: yield 67.7%; mp. 280e283 ꢀC,
ESI-MS (in MeOH): m/z: 363.1, ([M þ H]þ), 726.1, ([M þ 2H]2þ).
7a: yield 63.4%; mp. 269e273 ꢀC, ESI-MS (in MeOH): m/z: 375.2,
([M þ H]þ), 750, ([M þ 2H]2þ), 772.8, ([M þ H þ Na]2þ).
7.78 ꢁ 105
M
ꢂ1. The interaction between complex 2b and CT-DNA
was also confirmed by the fluorescence quenching experiment in
EBeDNA system (Fig. 3D). The studies of CD (Fig. 3E) and viscosity
analysis (Fig. 3F) showed that complex 2b may bind to DNA in an
intercalating mode, as evidenced by a noticeable decrease in the
Table 3
Selected crystallographic data for 1b.
Identification code
1b
Empirical formula
Formula weight (K)
Temperature/K
Crystal system
Space group
C
23H21N4O2RuCl2
557.41
293(2)
Monoclinic
C2/c
21.2331(4)
14.6117(3)
13.6415(3)
90.00
106.843(2)
90.00
4050.74(14)
8
ꢀ
a (A)
ꢀ
b (A)
ꢀ
c (A)
a
b
g
(ꢀ)
(ꢀ)
(ꢀ)
4.2.2. Synthesis of 1b, 2b, 3b, 4b, 5b, 6b and 7b
A mixture of [(C6H6)RuCl2]2 (0.1 mmol, 50 mg) and 1a, 2a, 3a, 4a,
5a, 6a and 7a (0.2 mmol) in 20 ml of dichloromethane was heated
in microwave reactor at 60 ꢀC for 30 min. The solvent was removed
by rotary evaporation and the residue was dissolved in minimum
amounts of methanol and then filtered to remove unreacted ligand
to obtain a yellow crude product after recrystallization. 1b: yield:
91.2%. ESI-MS (in MeOH): m/z 435.1, ([M ꢂ Cl]þ). UVevisible in
3
ꢀ
V (A )
Z
rcalc mg/mm3
m/mmꢂ1
F(0000)
1.828
8.959
2248
8573
Reflections collected

60
Q. Wu et al. / European Journal of Medicinal Chemistry 63 (2013) 57e63
Fig. 2. Complex 2b-induced S-phase arrest in MG-63 cells. Cells were treated with complex 2b for 24 h. (A) Cytotoxic effects of complex 2b on MG-63 cells. (B) Morphological
change (magnification, 200ꢁ). (C, D) Change in cell cycle distribution. (E) Down-regulation of cyclin A by complex 2b. All data were obtained from three independent experiments
and presented as the means ꢃ SD. *P < 0.05 vs untreated control.
MeOH [lmax, nm (ε/Mꢂ1 cmꢂ1)]: 249 (29,585), 295 (20,095). IR (in
KBr, nmax/cmꢂ1): 3855.9, 3390.64, 3050.41, 1621.98, 1603.62,
1536.61, 1478.49, 1252.77, 1198.21, 1084.36, 848.13, 811.34,
d
162.61 (s), 155.41 (s), 144.84 (s), 134.43 (s), 130.27 (s), 127.64 (s),
116.29 (s), 88.63 (s), 57.30 (s). 4b: yield: 90.8%. ESI-MS (in MeOH):
m/z 527.07, ([M
ꢂ
Cl]þ). UVevisible in MeOH
[lmax, nm
724.29 cmꢂ1. 1H NMR (DMSO-d6,
d
ppm)
9.22 (d, J ¼ 7.9 Hz, 2H), 8.60 (d, J ¼ 8.1 Hz, 2H), 8.09 (dd, J ¼ 8.1,
5.3 Hz, 1H), 6.31 (s, 6H). 13C NMR (126 MHz, DMSO)
155.93 (s),
d
: 9.84 (d, J ¼ 5.0 Hz, 2H),
(ε/Mꢂ1 cmꢂ1)]: 287 (46,030). IR (in KBr, nmax/cmꢂ1): 3865.69,
3400.23, 3068.44, 1611.12, 1481.83, 1276.56, 1175.75, 842.83,
d
808.44, 721.33. 1H NMR (in DMSO-d6,
d
/ppm): 9.70 (d, J ¼ 5.2 Hz,
145.19 (s), 144.96 (s), 134.34 (s), 128.00 (s), 88.67 (s). 2b: yield:
90.3%. ESI-MS (in MeOH): m/z 554.1, ([M ꢂ Cl]þ). UVevisible in
MeOH [lmax, nm (ε/Mꢂ1 cmꢂ1)]: 290 (43,780), 342 (38,460). IR (in
KBr, nmax/cmꢂ1): 3859.28, 3389.91, 3057.66, 1608.17, 1529.30,
2H), 9.07 (d, J ¼ 7.2 Hz, 2H), 8.76 (d, J ¼ 7.9 Hz, 2H), 8.11 (d,
J ¼ 10.6 Hz, 2H), 7.96 (dd, J ¼ 8.2, 5.2 Hz, 2H), 7.37 (s, 2H), 6.28 (s,
6H). 13C NMR (126 MHz, DMSO)
d 161.47 (s), 155.15 (s), 135.98 (s),
129.69 (s), 117.71 (s), 88.65 (s). 5b: yield: 45.7%. ESI-MS (in MeOH):
1486.91, 1202.82, 823.99, 812.25, 722.79. 1H NMR (in DMSO-d6,
d/
m/z 555.13, ([M
ꢂ
Cl]þ). UVevisible in MeOH
[lmax, nm
ppm): 10.20e9.76 (2H, m), 9.60 (2H, t, J ¼ 22.3 Hz), 9.40 (2H, d,
J ¼ 8.2 Hz), 9.11 (2H, dd, J ¼ 17.3, 8.0 Hz), 8.57e8.29 (2H, m), 8.28e
8.07 (2H, m), 6.91 (2H, t, J ¼ 16.9 Hz), 6.45e6.19 (6H, m). 13C NMR
(ε/Mꢂ1 cmꢂ1)]: 280 (35,385). IR (in KBr, nmax/cmꢂ1): 3860.42,
3414.54, 3080.94, 1612.23, 1548.55, 1481.39, 1253.06, 1182.67,
1072.72, 838.57, 806.33, 738.21. 1H NMR (DMSO-d6,
d/ppm): 9.96 (d,
(126 MHz, DMSO)
d
190.31 (s), 151.87 (s), 128.49 (s), 126.17 (s),
J ¼ 4.7 Hz, 2H), 9.20 (dd, J ¼ 31.3, 7.5 Hz, 2H), 8.70 (s, 2H), 8.32e8.06
116.98 (s), 112.21 (s), 111.50 (s), 87.19 (s). 3b: yield: 91.4%. ESI-MS (in
MeOH): m/z 541.13, ([M ꢂ Cl]þ). UVevisible in MeOH [lmax, nm
(ε/Mꢂ1 cmꢂ1)]: 289 (41,520). IR (in KBr, nmax/cmꢂ1): 3862.15,
3423.39, 2987.91, 1610.97, 1524.46, 1483.18, 1255.81, 1181.77,
(m, 2H), 7.37 (s, 2H), 6.32 (s, 6H). 13C NMR (126 MHz, DMSO)
d
156.04 (s), 153.65 (s), 145.34 (s), 131.84 (s), 128.64 (s), 127.96 (s),
88.70 (s). 6b: yield: 91.3%. ESI-MS (in MeOH): m/z 579.13,
([M ꢂ Cl]þ). UVevisible in MeOH [lmax, nm (ε/Mꢂ1 cmꢂ1)]: 290
(41,885). IR (in KBr, nmax/cmꢂ1): 66.14, 3401.22, 3059.80, 1607.30,
1620.53, 1551.47, 1454.98, 1167.3, 851.02, 810.46, 722.48. 1H NMR
1066.11, 839.63, 810.66, 722.07. 1H NMR (DMSO-d6,
d/ppm): 9.96
(dd, J ¼ 5.3, 1.1 Hz, 2H), 8.42 (d, J ¼ 8.6 Hz, 2H), 8.21 (s, 2H), 7.73 (d,
J ¼ 8.7 Hz, 2H), 7.37 (s, 2H), 6.33 (s, 6H). 13C NMR (126 MHz, DMSO)
(DMSO-d6,
d
ppm): 9.91 (d, J ¼ 4.7 Hz, 2H), 8.31 (t, J ¼ 11.1 Hz, 2H),

Q. Wu et al. / European Journal of Medicinal Chemistry 63 (2013) 57e63
61
2b
( g/ml)
μ
A
Control
5
10
20
p-p53 (Ser15)
53 KD
p-Histone H2A.X
(Ser139)
15 KD
42 KD
-actin
β
μg/ml
(
2b
)
B
Control
10
20
C
E
D
1.0
0.8
0.6
0.4
0.2
0.0
250
200
150
100
50
0.20
0.15
0.10
0.05
0.00
0.9
0.6
0.3
0.0
0
4
8
12 16
0
1
2
3
0
250
300
350
400
450
500
550
600
700
Wavelength/nm
Wavelength/6n5m0
F
10
5
1.6
1.4
1.2
1.0
0
-5
-10
-15
0.00
0.02
0.04
0.06
0.08
0.10
240
260
280
300
320
Wavelength/nm
[Ru] / [DNA]
Fig. 3. (A) Effects of complex 2b on the expression level of phosphorylated p53 and Histone H2A X. (B) DNA damage induced by complex 2b as examined by Comet assay. Cells were
treated for 24 h and the length of tail reflects DNA damage in the cells. (CeF) DNA-binding behavior of complex 2b with CT-DNA. (C) Absorption spectra of complex 2b in TriseHCl
buffer upon addition of increasing concentrations of CT-DNA. [Ru] ¼ 20
[DNA] ¼ 100 M. (E) CD spectra of CT-DNA with addition of complex 2b. [DNA] ¼ 300
viscosity of CT-DNA.
m
M. (D) Emission spectra of EB and CT-DNA in TriseHCl buffer with addition of complex 2b. [EB] ¼ 16
mM,
m
m
M, [Ru] ¼ 2 and 6
m
M. (F) Effects of complex 2b (-) and [Ru(bpy)3]þ (C) on the relative
8.13 (dd, J ¼ 23.8, 15.5 Hz, 2H), 7.37 (s, 2H), 6.98 (d, J ¼ 8.7 Hz, 2H),
6.32 (s, 6H). 13C NMR (126 MHz, DMSO)
156.03 (s), 145.39 (s),
129.24 (s), 127.95 (s), 88.70 (s). 7b: yield: 89.8%. ESI-MS (in MeOH):
4.3. Biochemical approach
4.3.1. MTT assay
d
m/z 590.9, ([M
ꢂ
Cl]þ). UVevisible in MeOH
[
lmax
,
nm
Human cancer cell lines, including melanoma A375, hepato-
cellular carcinoma HepG2 and colorectal adenocarcinoma SW620,
were purchased from American Type Culture Collection (ATCC,
Manassas, VA). The normal fibroblast Hs68 and kidney HK-2 cells
were also obtained from ATCC. All cell lines were maintained in
either RPMI-1640 or DMEM media supplemented with fetal bovine
serum (10%), penicillin (100 units/ml) and streptomycin (50 units/
ml) at 37 ꢀC in CO2 incubator (95% relative humidity, 5% CO2). Cell
(ε/Mꢂ1 cmꢂ1)]: 283 (46,320). IR (in KBr, nmax/cmꢂ1): 3869.75,
3369.99, 3042.42, 1625.14, 1603.67, 1543.71, 1459.87, 1275.97,
1186.24, 1070.50, 722.92. 1H NMR (DMSO-d6,
d ppm) d: 9.93 (dd,
J ¼ 5.3, 1.1 Hz, 2H), 8.31 (d, J ¼ 8.6 Hz, 2H), 8.18 (dd, J ¼ 8.1, 5.5 Hz,
2H), 7.84 (d, J ¼ 8.6 Hz, 2H), 7.34 (s, 2H), 6.30 (s, 6H). 13C NMR
(126 MHz, DMSO)
d 150.45 (s), 133.93 (s), 131.31 (s), 130.17 (s),
127.35 (s), 124.51 (s), 88.59 (s).

62
Q. Wu et al. / European Journal of Medicinal Chemistry 63 (2013) 57e63
viability was determined by measuring the ability of cells to
transform MTT to a purple formazan dye [29]. Cells were seeded in
96-well tissue culture plates for 24 h. After incubation, 20 ml/well of
MTT solution (5 mg/ml phosphate buffered saline) was added and
incubated for 5 h. The color intensity of the formazan solution,
which reflects the cell growth condition, was measured at 570 nm
using a microplate spectrophotometer (SpectroAmaxÔ 250).
4.4.3. CD spectrum
CD spectral characteristics were compared for CT-DNA in the
absence and in the presence of complex 2b, respectively. Complex
2b has no intrinsic CD signals, as it is achiral so that any CD signal
above 300 nm can be attributed to the interaction of complex with
DNA. This increase of decrease was similar to that observed if DNA
was under identical conditions modified by cisplatin of ineffective
transplatin, respectively [33].
4.3.2. Flow cytometric analysis
The cell cycle distribution was analyzed by flow cytometry as
previously described [30]. Treated or untreated cells were trypsi-
nized, washed with PBS and fixed with 75% ethanol overnight
at ꢂ20 ꢀC. The fixed cells were washed with PBS and stained with
4.4.4. Viscosity
Viscosity test is one of the most effective methods to judge the
interaction mode of complexes with DNA. Prepare different con-
centrations of fixed solution of complex 2b and DNA in Tris/HCl
buffer media, which is [2b]/[DNA] ¼ 0, 0.02, 0.04, 0.06, 0.08, 0.1.
Before testing, keep them (30 ꢃ 0.1 ꢀC) 1 h in thermostatic water
propidium iodide (PI) (1.21 mg/ml Tris, 700 U/ml RNase, 50.1 mg/ml
PI, pH8.0) for 4 h in darkness. Cell cycle distribution was analyzed
using MultiCycle software (Phoenix Flow Systems, San Diego, CA).
Apoptotic cells with hypodiploid DNA content were measured by
quantifying the sub-G1 peak in the cell cycle pattern.
bath. The formula calculated viscosity is:
curves were obtained by a picture with (h/h0)
r(r ¼ [2b]/[DNA]) as X-axis.
h
¼ (t ꢂ t0)/t0. Viscosity
1/3 as Y-axis and with
4.3.3. Western blot analysis
Acknowledgments
Total cellular proteins were extracted by incubating cells in lysis
buffer obtained from Cell Signaling Technology and protein con-
centrations were determined by BCA assay. SDS-PAGE was done in
10% tricine gels loading equal amount of proteins per lane. After
electrophoresis, separated proteins were transferred to nitro-
cellulose membrane and blocked with 5% non-fat milk in TBST
buffer for 1 h. After then, the membranes were incubated with
primary antibodies at 1:1000 dilutions in 5% non-fat milk overnight
at 4 ꢀC, and then secondary antibodies conjugated with horseradish
peroxidase at 1:2000 dilution for 1 h at room temperature. Protein
bands were visualized on X-ray film using an enhanced chem-
iluminescence system (Kodak).
This work was supported by Natural Science Foundation of
China and Guangdong Province, Key Project of Science and Tech-
nology Department of Guangdong Province, Science and Technol-
ogy Planning Project of Guangdong Province, the Fundamental
Research Funds for the Central Universities, Program for New
Century Excellent Talents in University and the Doctoral Founda-
tion of Ministry of Education of China.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.01.037.
4.3.4. Comet assay
Single-cell gel electrophoresis for detection of DNA damage was
performed using the Comet assay reagent kit purchased from Tre-
vigen according to the manufacturer’s instructions. DNA was
stained with SYBR Green I (Trevigen) and visualized under a fluo-
rescence microscope (Nikon, Eclipse E-600). Fifty cells per slide
were selected randomly and their olive tail moments were deter-
mined using an image analysis system (Komet 3.1, Kinetics Imaging
Ltd., Liverpool) linked to a CCD camera.
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