2-(3,5-Dibromo-4-hydroxyphenyl)imidazo [4,5-f][1,10] phenanthrolinoruthenium(II) complexes: Synthesis, characterization, cytotoxicity, apoptosis, DNA-binding and antioxidant activity

Article abstract of DOI:10.1007/s10534-010-9340-2

A new ligand DBHIP and its two ruthenium(II) complexes [Ru(dmb) ₂(DBHIP)](ClO₄)₂ (1) and [Ru(dmp) ₂(DBHIP)](ClO₄)₂ (2) have been synthesized and characterized. The cytotoxicity of DBHIP and complexes 1 and 2 has been assessed by MTT assay. The apoptosis studies were carried out with acridine orange/ethidium bromide (AO/EB) staining methods. The binding behaviors of these complexes to calf thymus DNA (CT-DNA) were studied by absorption titration, viscosity measurements, thermal denaturation and photoactivated cleavage. The DNA-binding constants of complexes 1 and 2 were determined to be 8.64 ± 0.16 × 10 (s = 1.34) and 2.79 ± 0.21 × 10 (s = 2.17) M ^-1. The results suggest that these complexes interact with DNA through intercalative mode. The studies on the mechanism of photocleavage demonstrate that superoxide anion radical (O₂^?-) and singlet oxygen (¹O₂) may play an important role in the DNA cleavage. The experiments on antioxidant activity show that these compounds also exhibit good antioxidant activity against hydroxyl radical (OH ^?). Springer Science+Business Media, LLC. 2010.

Full text of DOI:10.1007/s10534-010-9340-2

Biometals (2010) 23:739–752
DOI 10.1007/s10534-010-9340-2
2-(3,5-Dibromo-4-hydroxyphenyl)imidazo
[4,5-f][1,10]phenanthrolinoruthenium(II) complexes:
synthesis, characterization, cytotoxicity, apoptosis,
DNA-binding and antioxidant activity
•
Yun-Jun Liu Zhen-Hua Liang Zheng-Zheng Li
•
•
•
•
•
Cheng-Hui Zeng Jun-Hua Yao Hong-Liang Huang
Fu-Hai Wu
Received: 19 December 2009 / Accepted: 13 April 2010 / Published online: 27 April 2010
ꢀ Springer Science+Business Media, LLC. 2010
Abstract A new ligand DBHIP and its two ruthe-
nium(II) complexes [Ru(dmb)2(DBHIP)](ClO4)2 (1)
and [Ru(dmp)2(DBHIP)](ClO4)2 (2) have been syn-
thesized and characterized. The cytotoxicity of DBHIP
and complexes 1 and 2 has been assessed by MTT
assay. The apoptosis studies were carried out with
acridine orange/ethidium bromide (AO/EB) staining
methods. The binding behaviors of these complexes to
calf thymus DNA (CT-DNA) were studied by absorp-
tion titration, viscosity measurements, thermal denatur-
ation and photoactivated cleavage. The DNA-binding
constants of complexes 1 and 2 were determined to be
8.64 0.16 9 104 (s = 1.34) and 2.79 0.21 9 104
(s = 2.17) M-1. The results suggest that these com-
plexes interact with DNA through intercalative mode.
The studies on the mechanism of photocleavage dem-
onstrate that superoxide anion radical (O2•–) and singlet
oxygen (1O2) may play an important role in the DNA
cleavage. The experiments on antioxidant activity show
that these compounds also exhibit good antioxidant
activity against hydroxyl radical (OH•).
Keywords Ruthenium(II) complexes ꢀ DNA ꢀ
Cytotoxicity ꢀ Apoptosis ꢀ Antioxidant activity
Abbreviations
DBHIP
Y.-J. Liu (&) ꢀ Z.-H. Liang ꢀ Z.-Z. Li ꢀ C.-H. Zeng
School of Pharmacy, Guangdong Pharmaceutical
University, Guangzhou 510006,
People’s Republic of China
e-mail: lyjche@163.com
2-(3,5-Dibromo-4-
hydroxyphenyl)imidazo[4,5-
f][1,10]phenanthroline
dmb
dmp
MTT
4,40-Dimethyl-2,20-bipyridine
2,9-Dimethyl-1,10-phenanthroline
3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide
Acridine orange/ethidium bromide
Dimethyl sulfoxide
J.-H. Yao
Instrumentation Analysis and Research Center,
Sun Yat-Sen University, Guangzhou 510275,
People’s Republic of China
AO/EB
DMSO
RPMI
DMF
H.-L. Huang (&)
School of Life Science and Biopharmacology, Guangdong
Pharmaceutical University, Guangzhou 510006,
People’s Republic of China
Roswell Park Memorial Institute
N,N-Dimethylformamide
CT-DNA Calf thymus DNA
e-mail: hhongliang2004@yahoo.com.cn
MLCT
Tris
Metal to ligand charge transfer
F.-H. Wu (&)
Tris(hydroxymethyl)aminomethane
Electrospray mass spectroscopy
School of Public Health, Guangdong Pharmaceutical
University, Guangzhou 510006,
People’s Republic of China
ES–MS
NAMI-A [ImH][trans-RuCl4(DMSO)(Im)]
KP1019 [IndH][trans-RuCl4(Ind)2]
e-mail: fuhaiwu@163.com
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Biometals (2010) 23:739–752
(DBHIP)](ClO4)2 2 (Scheme 1). Thermal denaturation
and viscosity measurements show that the two complexes
interact with DNA by intercalative mode. The studies on
the mechanism of photocleavage demonstrate that
superoxide anion radical (O2•–) and singlet oxygen
(1O2) may play an important role in the DNA cleavage.
The cytotoxicity of these compounds has been evaluated
by MTT assay. The studies on the cytotoxicity show these
compounds exhibit high activity against BEL-7402
(hepatocellular), C-6 (Rat glioma), HepG-2 (hepatocel-
lular) and MCF-7 (breast cancer) cells in a dose-
dependent manner. The results of apoptosis assay suggest
that these complexes 1 and 2 can induce the apoptosis of
BEL-7402 cells. The experiments on antioxidant activity
show that these compounds exhibit good antioxidant
activity against hydroxyl radical (OH•).
Introduction
Due to rich photochemical and photophysical properties,
polypyridyl ruthenium(II) complexes have received
considerable attention as DNA-binding substrates. They
are photochemical stable in their ground and excited
state. The rich optical properties of these complexes
facilitate assessments of their DNA binding capabilities
as binding to DNA can be probed through changes in
absorption and emission spectra. Many octahedral
ruthenium(II) complexes have been proved to bind with
DNA through intercalative mode (Erkkila et al. 1999;
Metcalfe and Thomas 2003; Zeglis et al. 2007; Ghosh
et al. 2009;Liuetal.2009,2010;Sunetal.2009; Tan et al.
2009; Arockiasamy et al. 2009; Yu et al. 2009; Friedman
et al. 1990). These ruthenium complexes show interesting
properties. Complex [Ru(bpy)2(dppz)]2? (dppz = dipyr-
ido[3,2-a:20,30-c]phenazine) may act as ‘‘molecular light
switch’’ (Friedman et al. 1990) and exhibits cytotoxic
activity at low micromolar IC50 values (Schatzschneider
et al. 2008). Organometallic arene ruthenium compounds
of the general formula [Ru(g6-arene)(L)(L0)(L00)]Xn, in
which at least some of the ligands L are labile, have also
receiveda lot of attention recently, with structure–activity
studies against a variety of cancer cells lines (Yan et al.
2005; Melchart and Sadler 2006; Dyson 2007; Dougan
Materials and methods
Calf thymus DNA (CT-DNA) was obtained from the
Sino-American Biotechnology Company. pBR 322
DNA was obtained from Shanghai Sangon Biological
Engineering & Services Co., Ltd. Dimethyl sulfoxide
(DMSO) and RPMI 1640 were purchased from Sigma.
Cell lines of BEL-7402 (hepatocellular), C-6 (Rat
glioma), HepG-2 (hepatocellular) and MCF-7 (breast
cancer) were purchased from American Type Culture
Collection, agarose and ethidium bromide were
obtained from Aldrich. RuCl3ꢀxH2O was purchased
from Kunming Institution of Precious Metals. Doubly
distilled water was used to prepare buffers (5 mM
Tris(hydroxymethylaminomethane)–HCl, 50 mM NaCl,
pH = 7.2). A solution of calf thymus DNA (CT-DNA)
in the buffer gave a ratio of UV absorbance at 260 and
280 nm of ca. 1.8–1.9:1, indicating that the DNA was
sufficiently free of protein (Reichmann et al. 1954). The
DNA concentration per nucleotide was determined by
absorption spectroscopy using the molar absorption
coefficient (6,600 M-1 cm-1) at 260 nm (Chaires et al.
1982).
¨
and Sadler 2007; Schafer et al. 2007). Ruthenium
anticancer chemistryhas alreadyyielded many promising
results. Several compounds have been described which
display an activity comparable to that of cisplatin, and in
some cases, activities are even better (Reedijk 2003;
Hotze et al. 2004; Velders et al. 2000; Habtemariam
et al. 2006; Vilaplana et al. 1994, 2006). Ruthenium
complex ImH[trans-Ru(III)Cl4(DMSO)Im] (Im = imid-
azole, NAMI-A) was the first to enter clinical trials
against metastases (Cocchietto and Sava 2000; Zorzet
et al. 2001), and KP 1019 has been introduced into phase I
clinical trials against colon carcinomas and their metas-
tases (Hartinger et al. 2006). More recent, studies
demonstrate that ruthenium(II) complexes containing
polypyridyl ligand also show high antitumor activity in
vitro (Schatzschneider et al. 2008; Liu et al. 2008, 2009;
Tan et al. 2008; Gao et al. 2007), these complexes can
effectively inhibit the cell proliferation. In this article, we
report the synthesis, characterization, DNA-binding,
cytotoxicity, apoptosis in vitro and antioxidant activity
of a new ligand DBHIP and its two ruthenium(II) com-
plexes [Ru(dmb)2(DBHIP)](ClO4)2 1 and [Ru(dmp)2
Physical measurements
Microanalysis (C, H, and N) was carried out with a
Perkin-Elmer 240Q elemental analyzer. Fast atom
bombardment (FAB) mass spectra were recorded on a
VG ZAB-HS spectrometer in a 3-nitrobenzyl alcohol
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741
Scheme 1 The synthetic route for ligand and complexes
matrix. Electrospray mass spectra (ES–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 200ꢁC, respectively, and the quoted m/z values
are for the major peaks in the isotope distribution. 1H
NMR spectra were recorded on a Varian-500 spec-
trometer. All chemical shifts were given relative to
tetramethylsilane (TMS). UV/Vis spectra were
recorded on a Shimadzu UV-3101PC spectrophotom-
eter at room temperature.
Anal. Calcd. for C19H10N4OBr2: C, 48.54; H, 2.14;
N, 11.92; Found: C, 48.51; H, 2.18; N, 11.89%.
FAB–MS: m/z = 471 [M ? 1]?. IR (KBr, cm-1):
3437, 2927, 1604, 1589, 1476, 1449, 1357, 1173,
1109, 1073, 805, 736, 670. 1H NMR (500 MHz,
DMSO-d6): 9.02 (d, 2H, J = 8.5 Hz), 8.33 (d, 2H,
J = 8.4 Hz), 8.01 (d, 2H, J = 8.6 Hz), 7.22 (d, 2H,
J = 8.2 Hz), 3.26 (s, 1H).
Synthesis of [Ru(dmb)2(DBHIP)](ClO4)2 (1)
A mixture of cis-[Ru(dmb)2Cl2]ꢀ2H2O (0.280 g,
0.5 mmol) (Sullivan et al. 1978) and DBHIP
(0.185 g, 0.5 mmol) in ethanol (30 cm3) was heated
to reflux under argon for 8 h to give a clear red solution.
Upon cooling, a red precipitate was obtained by
dropwise addition of saturated aqueous NaClO4 solu-
tion. The crude product was purified by column
chromatography on a neutral alumina with a mixture
of CH3CN–toluene (3:1, v/v) as eluant. The mainly red
band was collected. The solvent was removed under
reduced pressure and a red powder was obtained.
Yield: 71%. Anal. Calcd. for C43H34N8Cl2Br2O9Ru:
C, 45.36; H, 3.01; N, 9.84; Found: C, 45.31; H, 3.05; N,
9.87%. ES–MS [CH3CN, m/z]: 939.1 ([M–2ClO4–
H]?), 470.3 ([M–2ClO4]2?). IR (KBr, cm-1): 3434,
2925, 1619, 1445, 1366, 1121, 1090, 876, 729, 636.
1H NMR (500 MHz, DMSO-d6): d 9.01 (d, 2H,
J = 8.2 Hz), 8.72 (d, 2H, J = 8.8 Hz), 8.23 (s, 4H),
Synthesis of the ligand and complexes
Synthesis of 2-(3,5-dibromo-4-
hydroxyphenyl)imidazo[4,5-f][1,10]phenanthroline
(DBHIP)
A mixture of 1,10-phenanthroline-5,6-dione (0.315 g,
1.5 mmol), 3,5-dibromo-4-hydroxyphenylaldehyde
(0.419 g, 1.5 mmol), ammonium acetate (2.31 g,
30 mmol) and glacial acetic acid (30 cm3) was
refluxed with stirring for 2 h. The cooled solution
was diluted with water and neutralized with concen-
trated aqueous ammonia. The precipitate was col-
lected and purified by column chromatography on
silica gel (60–100 mesh) with ethanol as eluent to
give the compound as yellow powder. Yield: 81%.
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Biometals (2010) 23:739–752
7.99 (s, 2H), 7.85 (d, 4H, J = 8.2 Hz), 7.67(d, 2H,
J = 8.0 Hz), 7.17 (d, 4H, J = 8.0 Hz), 3.38 (s, 1H,),
2.56 (s, 6H), 2.51 (s, 6H).
relative to the control. Each experiment was repeated at
least three times to get the mean values. Four different
tumor cell lines were the subjects of this study: BEL-
7402 (hepatocellular), C-6 (Rat glioma), HepG-2
(hepatocellular) and MCF-7 (breast cancer) (purchased
from American Type Culture Collection).
Synthesis of [Ru(dmp)2(DBHIP)](ClO4)2 (2)
This complex was synthesized in an manner identical
to that described for complex 1, with cis-[Ru(dmp)2-
Cl2]ꢀ2H2O (0.312 g, 0.5 mmol) (Collin and Sauvage
1986) in place of cis-[Ru(dmb)2 Cl2]ꢀ2H2O. Yield:
70%. Anal. Calcd. for C47H34Cl2Br2N8O9Ru: C,
47.57; H, 2.89; N, 9.44; Found: C, 47.54; H, 2.94;
N, 9.48%. ES–MS [CH3CN, m/z]: 987.0 ([M–2ClO4–
H]?), 494.2 ([M–2ClO4]2?). IR (KBr, cm-1): 3436,
2926, 1628, 1586, 1444, 1366, 1121, 1084, 857, 728,
Apoptosis studies
Apoptosis studies were performed with a staining
method utilizing acridine orange AO) and ethidium
bromide (EB) (Spector et al. 1998). According to the
difference in membrane integrity between necrotic
and apoptosis, AO can pass through cell membrane,
but EB cannot. Under fluorescence microscope, live
cells appear green, necrotic cells stain red but have a
nuclear morphology resembling that of viable cells,
apoptosis cells appear green. And morphological
changes such as cell blebbing and formation of
apoptotic bodies will be observed.
1
636. H NMR (500 MHz, DMSO-d6): d 8.91 (d, 2H,
J = 8.6 Hz), 8.84 (d, 2H, J = 8.5 Hz), 8.42 (t, 4H,
J = 7.8 Hz), 8.24 (d, 2H, J = 8.2 Hz), 8.14 (s, 4H),
7.97 (d, 2H, J = 8.2 Hz), 7.38 (d, 4H, J = 8.4 Hz),
3.35 (s, 1H), 2.51 (s, 6H), 2.50 (s, 6H).
Caution Perchlorate salts of metal compounds
with organic ligands are potentially explosive, and
only small amounts of the material should be
prepared and handled with great care.
A monolayer of BEL-7402 cells was incubated in
the absence or presence of complex 2 at concentra-
tion of 25 lM at 37ꢁC and 5% CO2 for 24 h. After
24 h, each cell culture was stained with AO/EB
solution (100 lg ml-1 AO, 100 lg ml-1 EB). Sam-
ples were observed under a fluorescence microscope.
In vitro cytotoxicity assay
Standard 3-(4,5-dimethylthiazole)-2,5-diphenyltetrazo-
lium bromide (MTT) assay procedures were used
(Mosmann 1983). Cells were placed in 96-well micro-
assay culture plates (1 9 104 cells per well) and grown
overnight at 37ꢁC in a 5% CO2 incubator. Compounds
tested were dissolved in DMSO and diluted with RPMI
1640 to the required concentrations prior to use. Control
wells were prepared by addition of culture medium
(100 ll). Wellscontainingculturemediumwithoutcells
were used as blanks. The plates were incubated at 37ꢁC
in a 5% CO2 incubator for 48 h. Upon completion of the
DNA binding and photoactivated cleavage
The DNA-binding and photoactivated cleavage
experiments were performed at room temperature.
Buffer A [5 mM tris(hydroxymethyl)aminomethane
(Tris) hydrochloride, 50 mM NaCl, pH 7.0] was used
for absorption titration, luminescence titration and
viscosity measurements. Buffer B (50 mM Tris–HCl,
18 mM NaCl, pH 7.2) was used for DNA photoclea-
vage experiments. Buffer C (1.5 mM Na2HPO4,
0.5 mM NaH2PO4, 0.25 mM Na2EDTA, pH 7.0)
was used for thermal DNA denaturation experiments.
Buffer D (0.9% of physiological saline) was used for
retardation assay of pGL 3 plasmid DNA.
incubation, stock MTT dye solution (20 ll, 5 mg ml-1
)
was added to each well. After 4 h incubation, buffer
(100 ll)containingN,N-dimethylformamide(50%)and
sodium dodecyl sulfate (20%) was added to solubilize
the MTT formazan. The optical density ofeachwellwas
then measured on a microplate spectrophotometer at a
wavelength of 490 nm. The IC50 values were deter-
mined by plotting the percentage viability versus
concentration on a logarithmic graph and reading off
the concentration at which 50% of cells remain viable
The absorption titrations of the complexes in buffer
were performed using a fixed concentration (20 lM)
for complexes to which increments of the DNA stock
solution were added. Ru–DNA solutions were allowed
to incubate for 5 min before the absorption spectra
were recorded. The intrinsic binding constants K,
based on the absorption titration, were measured by
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Biometals (2010) 23:739–752
743
monitoring the changes of absorption in the MLCT
band with increasing concentration of DNA using the
following equation (Carter et al. 1989).
ethidium bromide and photographed on an Alpha
Innotech IS-5500 fluorescence chemiluminescence
and visible imaging system. The integrated density
values (IDV) were given by FluorChem 5500 soft-
ware. The percentage of cleavage (C) was calculated
according to Equation, where DI, DII and DIII are the
IDVs of Form I (supercoil form), Form II (nicking
form) and Form III (linear form), respectively.
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ea ꢁ ef
eb ꢁ ef
b ꢁ ðb2 ꢁ 2K2Ct½DNAꢂ=sÞ
2KCt
¼
ð1aÞ
ð1bÞ
b ¼ 1 þ KCt þ K½DNAꢂ=2s
where [DNA] is the concentration of CT-DNA in
base pairs, the apparent absorption coefficients ea, ef
and eb correspond to Aobsed/[Ru], the absorbance for
the free ruthenium complex, and the absorbance for
the ruthenium complex in fully bound form, respec-
tively. K is the equilibrium binding constant, Ct is the
total metal complex concentration in nucleotides and
s is the binding site size.
DII þ 2DIII
C ¼
ð2Þ
DI þ DII þ DIII
Scavenger measurements of hydroxyl radical
(OH•)
The hydroxyl radical (OH•) in aqueous media was
generated by the Fenton system (Li et al. 2008). The
solution of the tested complexes was prepared with
DMF. The 4 ml of assay mixture contained following
reagents: safranin (28.5 lM), EDTA–Fe(II) (100 lM),
H2O2 (44.0 lM), the tested compounds (0.5–3.5 lM)
and a phosphate buffer (67 mM, pH = 7.4). The assay
mixtures were incubated at 37ꢁC for 30 min in a water
bath. After which, the absorbance was measured at
520 nm. All the tests were run in triplicate and
expressed as the mean. Ai was the absorbance in the
presence of the tested compound; A0 was the absor-
bance in the absence of tested compounds; Ac was the
absorbance in the absence of tested compound, EDTA–
Fe(II), H2O2. The suppression ratio (ga) was calculated
on the basis of (Ai - A0)/(Ac - A0) 9 100%.
Thermal denaturation studies were carried out with
a
Perkin-Elmer Lambda 35 spectrophotometer
equipped with a Peltier temperature-controlling pro-
grammer ( 0.1ꢁC). The melting temperature Tm,
which is defined as the temperature where half of the
total base pairs is unbonded, is usually introduced.
The melting curves were obtained by measuring the
absorbance at 260 nm for solutions of CT-DNA
(80 lM) in the absence and presence of the Ru(II)
complex (32 lM) as a function of the temperature.
The temperature was scanned from 50 to 90ꢁC at a
speed of 1ꢁC min-1
.
Viscosity measurements were carried out using an
Ubbelodhe viscometer maintained at a constant
temperature at 25.0 ( 0.1)ꢁC in a thermostatic bath.
DNA samples approximately 200 base pairs in
average length were prepared by sonicating in order
to minimize complexities arising from DNA flexibil-
ity (Chaires et al. 1982). Flow time was measured
with a digital stopwatch, and each sample was
measured three times, and an average flow time was
calculated. Data were presented as (g/g0)1/3 versus
binding ratio (Cohen and Eisenberg 1969), where g is
the viscosity of DNA in the presence of complexes
and g0 is the viscosity of DNA alone.
For the gel electrophoresis experiment, super-
coiled pBR 322 DNA (0.1 lg) was treated with the
Ru(II) complexes in buffer B, and the solution was
then irradiated at room temperature with a UV lamp
(365 nm, 10 W). The samples were analyzed by
electrophoresis for 1.5 h at 80 V on a 0.8% agarose
gel in TBE (89 mM Tris–borate acid, 2 mM EDTA,
pH = 8.3). The gel was stained with 1 lg/ml
Results and discussion
Synthesis and characterization
The ligand DBHIP was prepared through condensation
of 1,10-phenanthroline-5,6-dione with 3,5-dibromo-4-
hydroxyphenylaldehyde using a similar method to that
described by Steck and Day (1943). The corresponding
ruthenium(II) complexes were synthesized by direct
reaction of DBHIP with the appropriate precursor com-
plexes in ethanol and purified by column chromatog-
raphy.
1
For each of the two complexes, two sets of H
NMR signals were observed. One set corresponds to
the ancillary ligands, dmb or dmp, and the other set
corresponds to the intercalative ligand, DBHIP. In
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Biometals (2010) 23:739–752
each of the two complexes, the chemical shifts of
protons on the nitrogen atom of imidazole ring were
not observed, probably because the protons are very
active and easy to be exchanged quickly between the
two nitrogens of the imidazole ring in solution.
The complexes were also characterized by elec-
trospray mass spectrometry (ES–MS). In the ES–MS
spectra for the complexes 1 and 2, as expected, the
intense signals for [M–2ClO4–H]? and [M–2ClO4]2?
were observed, the obtained molecular weights are
consistent with the expected values.
DNA-binding of ruthenium(II) complexes
Fig. 1 Effect of increasing amounts of complexes EB (filled
triangle), 1 (filled circle) and 2 (filled square) on the relative
viscosity of calf thymus DNA at 25 ( 0.1)ꢁC. [DNA] =
0.30 mM
Viscosity measurements
The viscosity of a DNA solution was sensitive to the
addition of organic drugs and metal complexes bound
by intercalation. And relative viscosity measurements
have proved to be a reliable method for the assign-
ment of the mode of binding compounds to DNA. In
general, intercalation of a ligand into DNA is known
to cause a significant increase in the viscosity of a
DNA solution due to an increase in the separation of
the base pairs at the intercalation site and, hence, an
increase in the overall DNA molecular length; A
partial and/or nonclassical intercalation of ligand
could bend (or kink) the DNA helix, reduces its
effective length and, concomitantly, its viscosity
(Satyanarayana et al. 1993; Waring 1965). The
relative change in viscosity was measured using
CT-DNA with increasing concentrations of the
complexes 1 and 2. The effects of complexes 1 and
2, together with ethidium bromide (EB) on the
relative viscosity of rod-like DNA are shown in
Fig. 1. It is well-known that the DNA intercalator EB
increases the viscosity of DNA with increments of the
concentration. On increasing the concentration of
complexes 1 and 2, the relative viscosity of the DNA
increased steadily. These behaviors are similar to that
of EB. The results suggest that complexes 1 and 2
intercalate between the base pairs of CT-DNA.
intercalation. The absorption spectra of complexes 1
and 2 mainly consist of three resolved bands in the
range 200–600 nm. The bands below 300 nm are
attributed intraligand (IL) p–p* transitions, the bands
at the range of 300–400 nm are attributed to p–p*
transitions, and the lowest energy bands at 464 nm for
1 and 463 nm for 2 are assigned to the metal-to-ligand
charge transfer (MLCT) transition.
Figure 2 shows the absorption spectra of complexes
1 and 2 in the presence of increasing concentration of
DNA. As increasing the concentration of CT-DNA, the
MLCT transition band of complexes 1 at 464 nm and 2
at 463 nm exhibit hypochromism of 17.5 and 16.8%,
and bathochromism of 2 and 3 nm, respectively. The
intrinsic constants Kb were determined by monitoring
the changes of absorbance in the MLCT band with
increasing concentration of CT-DNA. The values of Kb
are determined to be 8.64 0.16 9 104 (s = 1.34)
and 2.79 0.21 9 104 (s = 2.17) M-1 for 1 and 2,
respectively. These values are comparable to that of
complexes [Ru(phen)2(PBIP)]2? (5.91 9 104 M-1
)
(Xu et al. 2004) and [Ru(bpy)2PIP]2? (4.7 0.2
9 105 M-1) (Wu et al. 1997), but smaller than that
of complexes [Ru(bpy)2(dppz)]2? (4.90 9 106 M-1
)
(Friedman et al. 1990).
Electronic absorption spectra studies
Thermal denaturation studies
Due to the intercalative mode involving a stacking
interaction between the aromatic chromophore and the
DNA base pairs, the large change in absorbance can be
observed while a complex binds to DNA through
When the temperature in solution increases, the
double-stranded DNA gradually dissociates to single
strands, and generates a hyperchromic effect on the
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745
Fig. 2 Absorption spectra of complexes 1 (a) and 2 (b) in
Tris–HCl buffer upon addition of CT-DNA. [Ru] = 20 lM.
Arrow shows the absorbance change upon the increase of DNA
concentration. Plots of (ea - ef)/(eb - ef) versus [DNA] for the
titration of DNA with Ru(II) complexes
absorption spectra of DNA base pairs (k = 260 nm).
A large change in the Tm of DNA is an indication of
strong interaction between the complexes and DNA.
The interactions of all ruthenium(II) complexes with
CT-DNA are characterized by measuring the effects
on melting temperature of DNA (Fig. 3). In the
absence of any added complexes, the thermal dena-
turation determined for DNA gave a Tm of 61.30
0.54ꢁC under our experimental conditions. The
observed melting temperatures in the presence of
complexes 1 and 2 were 67.30 0.50 and 66.99
0.53ꢁC, respectively. The increases in Tm of the two
Ru(II) complexes (the DTm is 6.00 and 5.69ꢁC for 1
and 2) are comparable to that observed for Ru(II)
complexes (Chen et al. 2008; Tan et al. 2009; Liu et al.
2006) and lend strong support for intercalation into the
helix of DNA. The experimental results also indicate
that complex 1 exhibits larger DNA-binding affinity
than complex 2 does.
DNA photocleavage
The cleavage reaction on plasmid DNA induced by
ruthenium(II) complexes can be monitored by aga-
rose gel electrophoresis. When circular plasmid DNA
is subject to electrophoresis, relatively fast migration
will be observed for the intact supercoiled form
(Form I); If scission occurs on one strand (nicked),
the supercoiled will relax to generate a slower-
moving open circular form (Form II). If two strands
are cleaved, a linear form (Form III) that migrates
between Form I and Form II will be generated
(Barton and Raphael 1984). Figure 4a shows gel
electrophoresis separation of pBR 322 DNA after
incubation with different concentrations of Ru(II)
complexes and irradiation at 365 nm for 30 min. No
obvious DNA cleavage was observed for controls in
which complex was absent, or incubation of the
plasmid with the Ru(II) complex in dark (data not
presented). With increasing concentration of the
Ru(II) complexes, the amount of Form I of pBR
322 DNA diminishes gradually, whereas that of Form
II increase. Under the same experimental conditions,
complex 1 exhibits more effective DNA cleavage
activity than complex 2. The different cleaving
Fig. 3 Thermal denaturation of CT-DNA in the absence (filled
square) and presence of complexes 1 (filled circle) and 2 (filled
triangle). [Ru] = 32 lM, [DNA] = 80 lM
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Biometals (2010) 23:739–752
Fig. 4 a Photoactivated cleavage of pBR 322 DNA in the
presence of different concentrations of Ru(II) complexes after
irradiation at 365 nm for 30 min. b Photoactivated cleavage of
supercoiled pBR 322 DNA by complexes 1 and 2 (20 lM) in
the absence and presence of different inhibitors [100 mM
mannitol, 200 mM dimethyl sulfoxide (DMSO), 1,000 U ml-1
superoxide dismutase (SOD), 1.2 mM distidine] after irradia-
tion at 365 nm for 30 min. c Bar diagram representation of the
effect of inhibitors on the photoactivated cleavage activity of
complexes 1 and 2
efficiency may be ascribed to the different
DNA-binding affinity of two Ru(II) complexes.
To explore the DNA photocleavage mechanism,
control experiments were carried out for complex 1
and 2 in the presence of the D-mannitol, dimethyl-
sulfoxide (DMSO), superoxide dismutase (SOD)
enzyme and histidine (Fig. 4b). The cleavage of the
plasmid was not inhibited in the presence of hydroxyl
radical (OH•) scavengers such as mannitol (Cheng
et al. 1993) and dimethylsulfoxide (Lesko et al.
1980), which indicated that hydroxyl radicals were
not likely to be the cleaving agent. In the presence of
superoxide dismutase (SOD), a facile superoxide
anion radical (O2•–) quencher, the cleavage was
obviously improved. In the presence of the singlet
oxygen (1O2) scavenger histidine (Nilsson et al.
1972), the DNA cleavage of the plasmid by com-
1
plexes 1 and 2 was inhibited, suggesting that O2 is
likely to be the reactive species responsible for the
cleavage reaction. Figure 4c shows the bar diagram
representation of the percentage of cleavage (C) for
complexes 1 and 2.
Retardation of pGL 3 plasmid DNA
DNA condensation into contact structures has been
received considerable attention to understand the
Fig. 5 Agarose gel electrophoresis retardation of pGL 3
plasmid DNA in the presence different concentrations of
complexes 1 and 2. 1: Lane (1) DNA alone, (2) 0.33 mM; (3)
1 mM; (4) 1.67 mM; (5) 2.33 mM. 2: (6) DNA alone, (7)
0.33 mM; (8) 1.0 mM; (9) 1.67 mM; (10) 2.33 mM.
[DNA] = 0.5 lg
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Biometals (2010) 23:739–752
747
Table 1 The IC50 values for DBHIP, 1 and 2 against selected cell lines
Compounds
IC50 (lM)
BEL-7402 (24 h)
BEL-7402 (48 h)
C-6
HepG-2
MCF-7
DBHIP
15.26 4.11
73.51 3.54
21.72 3.12
30.25 3.64
11.18 3.87
62.19 5.57
17.11 3.18
20.12 2.35
15.05 4.45
52.68 3.43
19.63 3.54
10.26 2.78
10.65 3.62
172.38 3.52
14.09 4.81
26.25 3.12
18.45 2.89
170.20 4.37
22.58 3.69
11.34 2.38
1
2
Cisplatin
mechanism of uptake of gene vectors in living cells.
It is well known that polyamine can effectively
condense DNA (Huang et al. 2006; Vijayanathan
et al. 2001; Sarkar et al. 2009). However, the studies
of small molecules to condense DNA have been less
paid attention. Ji and co-workers reported the Ru(II)
complexes [Ru(bpy)2(PIPSH)]2? and [Ru(phen)2
(PIPSH)]2? (Sun et al. 2009) can effectively con-
dense DNA at a concentration of 80 lM. The abilities
of complexes 1 and 2 to condense pGL 3 DNA were
Fig. 6 a Cell viability of DBHIP, 1 and 2 on tumor BEL-7402
(a), C-6 (b), HepG-2 (c) and MCF-7 (d) cell proliferation in
vitro. Each data point is the mean standard error obtained
from at least three independent experiments. b Light micros-
copy of C-6 cell line after treated for 48 h in the absence I
(control) and presence of different concentrations complex 1:
II: [Ru] = 6.25 lM; III: [Ru] = 12.5 lM; IV: [Ru] = 25 lM;
V: [Ru] = 50 lM; VI: [Ru] = 100 lM; VII: [Ru] = 200 lM.
Cell was observed using an inverted microscope and photo-
graphed by a digital camera
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Biometals (2010) 23:739–752
Fig. 6 continued
In vitro cytotoxicity
assessed by gel retardation assay. Figure 5 shows that
the concentrations of complexes 1 and 2 are 0.33 and
1.0 mM, complexes 1 and 2 can not condense the
DNA. However, at the high concentrations of
2.33 mM, the effects of condensation of DNA by
complexes 1 and 2 were obviously observed.
The cytotoxicity of DBHIP and complexes 1 and 2
was assayed on BEL-7402 (hepatocellular), C-6 (Rat
glioma), HepG-2 (hepatocellular) and MCF-7 (breast
cancer) by cell survival after 48 h of exposure to the
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Biometals (2010) 23:739–752
749
desired concentration (6.25, 12.5, 25, 50, 100, 200
and 400 lM) using the MTT assay. The IC50 values
were calculated after 48 h of incubation with ligand
DBHIP, complexes 1, 2 together with cisplatin in
different concentrations and are listed in Table 1.
Table 1 show that the IC50 values are time-depen-
dent. Though none of the cytotoxicity of ligand and
complexes is as high as that of cisplatin, it is clear
that the complex 2 is more sensitive against selected
four cell lines than complex 1. The IC50 values for
complex 2 (17.11 3.18, 19.63 3.54, 14.09
4.81 and 22.58 3.69 on BEL-7402, C-6, HepG-2
and MCF-7, respectively) are far lower than that of
complex 1 (62.19 5.57, 52.68 3.43, 172.38
3.52 and 170.20 4.37), under the same condition,
complex 2 show higher activity on four tumor cell
lines than complex 1. Comparing the cytotoxicity of
ligand and complex 2, we can conclude that the
antitumor activities against selected four tumor cell
lines have been slightly weakened when ligand bonds
with the Ru(II) metal center to form complexes.
Figure 6a show the results of cell viability assay for
different compounds in BEL-7402, C-6, HepG-2 and
MCF-7 cell lines. The cell viability was found to be
concentration dependent, and increasing the concen-
trations of DBHIP, complexes 1 and 2, an obvious
decrease was observed for the cell viability.
Figure 6b (I–VII) was the antiproliferative activity
for complex 1 against tumor cells of C-6, with
increasing the concentration of complex 1 (from II to
VII), the proliferation of tumor cells of C-6 was
effectively inhibited.
Apoptosis assay
Apoptosis induced by compounds is one of the
considerations in drug development. Apoptosis assay
were carried out with a staining method utilizing
acridine orange AO) and ethidium bromide (EB). The
AO/EB staining is sensitive to DNA and was used to
assess changes in nuclear morphology. Apoptotic and
necrotic cells can be distinguished from one another
Fig. 8 Scavenging effect of the ligand DBHIP and complexes
1 and 2 on hydroxyl radicals. Experiments were performed in
triplicate
Fig. 7 BEL-7402 cell were stained by AO/EB and observed
under fluorescence microscopy. BEL-7402 cell without treat-
ment (a) and in the presence of complex 2 (b) incubated at
37ꢁC and 5% CO2 for 24 h. Cells in a, b and C are apoptotic,
living and necrotic cells, respectively
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Biometals (2010) 23:739–752
Table 2 The influence of investigated compounds for OH•
Compounds
Average inhibition (%) for OH (lM)
Equation
IC50 (lM)
R2
0.5
1.0
1.5
2.0
2.5
3.0
3.5
DBHIP
1.05
6.13
3.44
28.85
34.23
26.31
37.52
49.63
41.56
43.05
53.81
47.98
46.64
56.05
53.21
50.37
59.34
56.20
52.92
60.24
57.85
Y = 47.23 ? 58.97x
Y = 56.43 ? 63.49x
Y = 52.11 ? 65.74x
1.11
0.79
0.93
0.9801
0.9678
0.9900
1
2
IC50 values were calculated from regression lines where: x was log of the tested compound concentration and Y was percent
inhibition of the tested compounds. When the percent inhibition of the tested compounds was 50%, the tested compound
concentration was IC50. R2 is the correlation coefficient
using fluorescence microscope after being stained
with AO/EB solution, apoptotic cells showed apop-
totic features such as nuclear shrinkage, chromatin
condensation. In the absence of complexes 1, the
living cells were stained bright green in spots
(Fig. 7a). However, after treatment with complex 1,
green apoptotic cells containing apoptotic bodies, as
well as red necrotic cells, were also observed
(Fig. 7b). Similar result for complex 2 was also
observed.
to form complexes. The information obtained from
the present work would be helpful to develop new
potential antioxidants and new therapeutic reagents
for some diseases.
Conclusion
A new ligand DBHIP and its two ruthenium(II)
complexes [Ru(dmb)2(DBHIP)]2? and [Ru(dmb)2(DB-
HIP)]2? were successfully prepared and characterized
1
by elemental analysis, ES–MS, IR, H NMR. These
Antioxidant activity studies
complexes have high DNA-binding affinity and excel-
lent antioxidative abilities. The results show they
interact with DNA by intercalative mode. Complex 2
shows high cytotoxicity against the selected four tumor
cell lines and the two complexes can effectively induce
the apoptosis of BET-7402 cells. Upon irradiation at
365 nm, complexes 1 and 2 can also cleave plasmid
DNA. At high concentration, complexes 1 and 2 can
effectively condense the pGL 3 DNA.
Since complexes 1 and 2 showed good DNA-binding
affinity, it was considered worthwhile to study other
potential aspects of these complexes such as antiox-
idant activity. The hydroxyl radical (OH•) was one of
the most reactive products of reactive oxygen species
(ROS), which could result in cell membrane disinte-
gration, membrane protein damage, DNA mutation
and further initiate or propagate the development of
many diseases. The antioxidant activities on hydroxyl
radical of complexes 1 and 2 together with ligand
DBHIP were investigated. As shown in Fig. 8 and
Table 2. The inhibitory effect of the ligands DBHIP
and their Ru(II) complexes 1 and 2 on OH• was
concentration-dependent and the suppression ratio
increased with increasing of sample concentration in
the range of 0.5–3.5 lM. The suppression ratio
against OH• valued from 1.05 to 52.92% for DBHIP,
6.13 to 60.24% for complex 1 and 3.44 to 57.85% for
complex 2. The antioxidant activity against hydroxyl
radical of complex 1 (IC50 = 0.79 lM) is higher than
that of complex 2 (IC50 = 0.93 lM) under the same
experimental condition. It is clear that the hydroxyl
radical scavenging activity can be enhanced when
ligand (IC50 = 1.11 lM) bonds Ru(II) metal center
Acknowledgements This work is supported by the National
Nature Science Foundation of China (30800227), the Science
and Technology Foundation of Guangdong Province (2009B030
803057) and Guangdong Pharmaceutical University for financial
support.
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