Synthesis and characterization of unsymmetrical oxidovanadium complexes: DNA-binding, cleavage studies and antitumor activities

Article abstract of DOI:10.1016/j.jinorgbio.2012.02.034

Four oxidovanadium(IV) complexes, [VO(hntdtsc)(phen)] (1), [VO(hntdtsc)(bpy)] (2) (hntdtsc = 2-hydroxy-1-naphthaldehyde thiosemicarbazone, phen = 1,10-phenanthroline), [VO(satsc)(phen)] (3) and [VO(satsc)(bpy)] (4) (satsc = salicylaldehyde thiosemicarbazo

Full text of DOI:10.1016/j.jinorgbio.2012.02.034

Journal of Inorganic Biochemistry 112 (2012) 39–48
Contents lists available at SciVerse ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Synthesis and characterization of unsymmetrical oxidovanadium complexes:
DNA-binding, cleavage studies and antitumor activities
a
a
b,
d
Jiazheng Lu a, , Haiwei Guo , Xiandong Zeng , Yongli Zhang , Ping Zhao , Jing Jiang a,c, Linquan Zang a
⁎
⁎
a
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, 510006, People's Republic of China
school of basic courses, Guangdong Pharmaceutical University, Guangzhou, 510006, People's Republic of China
State Key Laboratory of Optoelectronic Material and Technologies & School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou, 510275,
b
c
People's Republic of China
d
School of Medicine Chemistry and Chemical Engineering, Guangdong Pharmaceutical University, Guangzhou, 510006, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 October 2011
Received in revised form 17 February 2012
Accepted 20 February 2012
Available online 13 March 2012
Four oxidovanadium(IV) complexes, [VO(hntdtsc)(phen)] (1), [VO(hntdtsc)(bpy)] (2) (hntdtsc=2-hy-
droxy-1-naphthaldehyde thiosemicarbazone, phen=1,10-phenanthroline), [VO(satsc)(phen)] (3) and
[VO(satsc)(bpy)] (4) (satsc=salicylaldehyde thiosemicarbazone, bpy=2,2′-bipyridine) have been synthe-
sized and characterized. The results show that complexes 1, 2, 3 and 4 interact with DNA through intercala-
tive mode and can efficiently cleave the plasmid pBR 322 DNA. It is interesting to note that these four
complexes present highly cytotoxic activities against Myeloma cell (Ag8.653) and Gliomas cell (U251)
lines. Complex 1 was found to be the most potent antitumor agent among the four complexes.
© 2012 Elsevier Inc. All rights reserved.
Keywords:
Oxidovandium complexes
DNA-binding
Cleavage
Antitumor
1. Introduction
of neoplastic cells [18,19]. Thus, the DNA-binding and antitumor ac-
tivities of oxidovandium complexes based on vanadium-metal and
In past decade, a number of transition metal complexes have
been exploited for the design of new drugs due to their diverse
biological activities. In particular for those containing polyaromatic
ligands, great interest has been aroused on them owing to their
usage as DNA-structural probes, DNA-dependent electron transfer
and sequence-specific cleaving agents and potential anti-cancer
drugs [1–4]. In fact, most of these properties have been found to
be a direct bearing on the complexes binding DNA and cleaving
DNA [5–8]. DNA-binding metal complexes, especially those with
small molecular weight, have been extensively studied as DNA
structural and conformational probes, DNA-dependent electron
transfer and sequence-specific cleaving agents, and potential anti-
cancer drugs [9–13].
As one of the trace bioelements existing in the human body,
vanadium complexes have been found to present antibacterial,
antitumor, insulin-enhancing and antiparasitic effects [14–16]. This
bioelement takes part in various DNA maintenance reactions and
thereby prevents genomic instability which otherwise leads to cancer
[15,17]. Vanadium complexes could also suppress the growth and
spread of existing tumors by inhibiting tumor cell proliferation,
inducing apoptosis and limiting the invasion and metastatic potential
multifunctional bridging ligands have been investigated [17,20,21].
On the other hand, Schiff bases have also been extensively studied
because of their potential antibacterial, antifungal, anti-malarial
and anticancer activities [16,18,22]. Moreover, the electronic effects
of Schiff base complexes are also important and significant to carry
out some discussions in this field in order to design complexes
with better DNA-binding characteristics [23–25]. The presence of a
rigid aromatic system in the Schiff base structure gives rise to partic-
ular spectroscopic properties, which make it a potential probe for
nucleic acids. Because of their wide range of pharmacological appli-
cations, thiosemicarbazones were often chosen as such ligands for
vanadium [16–19]. Additionally, thiosemicarbazones can bind to
the nitrogen bases of DNA and so hinder or block base replication
and create lesions in DNA strands by oxidative rupture [18,25–27].
So far, vanadyl complexes incorporating thiosemicarbazones have
been studied extensively for their insulin-like effects which result
in the inhibition of glycerol release and enhancement of glucose up-
take by rat adipocytes and have been used in the treatment of tuber-
culosis [16,27,28]. In this context, it occurred to us that utilization of
intercalate moiety of thiosemicarbazones in coordinate with vanadi-
um might accentuates the biochemical activity of the so-derived
hybrids molecules, which may lead to an efficient DNA binding
and DNA cleavage. These oxidovanadium complexes may be a
promising and attractive approach in the search for new potential
drugs for the therapy of cancer [16,17].
⁎
Corresponding authors. Tel.: +86 20 39352122; fax: +86 20 39352129.
E-mail address: lujia6812@163.com (J. Lu).
0162-0134/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2012.02.034

40
J. Lu et al. / Journal of Inorganic Biochemistry 112 (2012) 39–48
In the present work, four oxidovanadium complexes, [VO
determined by absorption spectroscopy using the molar absorptivity
(6600 M−1 cm−1) at 260 nm [28,29,34]. Flash chromatography was
performed using silica gel (200–300 mesh). Unless otherwise stated,
reagents were commercially available and of analytical grade.
(hntdtsc)(phen)] (1), [VO(hntdtsc)(bpy)] (2) (hntdtsc=2-hydroxy-
1-naphthaldehyde thiosemicarbazone, phen=1,10-phenanthroline),
[VO(satsc)(phen)] (3) and [VO(satsc)(bpy)] (4) (satsc=salicylaldehyde
thiosemicarbazone, bpy=2,2′-bipyridine) (see Scheme 1), have been
synthesized and characterized by elemental analysis, UV–vis, ES-MS, IR,
1H NMR and magnetic moment measurement. The interactions of these
four complexes with calf-thymus DNA (CT-DNA) were investigated
using UV–vis absorption titration, fluorescence spectra, viscosity mea-
surements and thermal denaturation. Their photocleavage reactions
with pBR322 supercoiled plasmid DNA were investigated by gel electro-
phoresis. In addition, the cytotoxicity of these four complexes against the
Myeloma cell (Ag8.653) and Gliomas cell (U251) was assessed by MTT
assay.
2.2. Physical measurements
Microanalysis (C, H, and N) was carried out with a Perkin-Elmer
240Q elemental analyzer. 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
200 °C, 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 spectrometer. All chemical shifts are given relative to
2. Materials and methods
tetramethylsilane (TMS). Infrared spectra were recorded on a
Bomen FTIR model MB102 instrument using KBr pellets. UV/Vis spec-
tra were recorded on a Shimadzu UV-3101 PC spectrophotometer at
room temperature. Emission spectra were recorded on a Perkin-
Elmer Lambda 55 spectrofluorophotometer. Magnetic susceptibility
measurements were recorded on a MPMSXL-7(Quantum Design,
USA), at room temperature. Molar conductivities in DMF (1 mM/L)
solution at room temperature were measured using DDS-307 digital
direct reading conductivity meter.
2.1. Materials
VO (acac)
(acac=acetylacetonate), 1,10-phenanthroline and
2
2,2′-dipyridyl were purchased from Shang hai jingchun company.
CT-DNA and pBR 322 DNA were obtained from Sigma. Other materials
were obtained from commercial sources and used as received (ana-
lytical reagents). Tris–HCl buffer A (5 mM Tris (hydroxymetylamino-
methane)–HCl, 50 mM NaCl, pH=7.2) was used for absorption
titration, luminescence titration, and viscosity experiments. Tris–HCl
buffer B (50 mM Tris–HCl and 18 mM NaCl, pH=7.2) was used for
2.3. Synthesis of the ligands and complexes
DNA-cleavage experiments. Buffer
C
(1.5 mM NaHPO4, 0.5 mM
2.3.1. Synthesis of 2-hydroxy-1-naphthaldehyde thiosemicarbazone
(hntdtsc)
hntdtsc was synthesized through a modification of a previously
reported procedure [33–36]. A stirring solution of 2-hydroxy-1-
naphthaldehyde (0.8609 g, 5 mmol) in 10 mL of absolute alcohol
was added dropwise to thiosemicarbazide (0.4557 g, 5 mmol)
NaH2PO4 and 0.25 mM Na2H2EDTA (pH=7.0)) was used for thermal
denaturation. All buffers were prepared using double-distilled water.
A solution of CT-DNA in the buffer gave a ratio of UV absorbance at
260 and 280 nm of ca. 1.8–1.9, indicating that the DNA was sufficient-
ly free of protein [29–34]. The DNA concentration per nucleotide was
Scheme 1. Synthesis of ligands and complexes.

J. Lu et al. / Journal of Inorganic Biochemistry 112 (2012) 39–48
41
which was dissolved in 10 mL of absolute alcohol, then the mixture
was continuously stirred at 50 °C for 3 h and gave a white gossypine
precipitate, which was used without further purification. Yield: 88%.
Anal. Calcd. for C12H11N3OS: C, 58.76; H, 4.52; N, 17.13; S, 13.07%;
Found: C, 58.70; H, 4.61; N, 17.08; S, 13.01%. ES-MS (CH3OH, m/z):
246.0 (100%) ([M+1]+). IR (KBr) (νmax/cm−1): 3450 (vs), 3253
(vs, \NH2), 3167 (vs, N\H), 3053 (m, C\H), 1625 (s), 1593 (vs),
1572 (s), 1509 (vs), 1472 (s), 1452 (m), 1033 (m, C\N), 1240 (s,
C\O), 821 (s), 753 (s, C_S). 1H NMR (500 MHz, DMSO-d6): 11.42
(s, 1H, NHCS), 10.51 (s, 1H, OH), 9.06 (s, 1H, CH_N), 7.85 and 8.25
(2br s, 1H,NH2), 8.53 (d, 1H, ArH, J=8.7 Hz), 7.89 (d, 1H, ArH,
J=7.6 Hz), 7.86 (d, 1H, ArH, J=8.2 Hz), 7.57 (t, 1H, ArH, J=8.0 Hz),
7.38 (t, 1H, ArH, J=7.6 Hz), 7.21 (d, 1H, ArH, J=8.4 Hz). UV–vis
(30 mL) in place of hntdtsc in absolute methanol (100 mL). Yield:
57%. Anal. Calcd. For C20H15N5O2SV: C, 54.55; H, 3.43; N, 15.90; S,
7.28%; Found: C, 54.45; H, 3.69; N, 15.71; S, 7.21%. ES-MS (CH3OH,
m/z): 441.0 (100%) ([M+1]+). IR (KBr) (νmax/cm−1): 3448 (s),
3288 (s, \NH2), 3056 (w, C\H), 1603 (vs), 1541 (m), 1500 (s),
1433 (m), 1201 (m, C\O), 1043 (m, C\N), 945 (s, VO), 838 (s),
759 (w, C\S). 1H NMR (500 MHz, DMSO-d6): 9.11 (s, 1H, CH_N),
8.50 (m, 2H, ArH), 8.00 (br m, 3H), 7.84(br m, 5H), 6.81 (br m, 2H,
ArH), 6.73 (br m, 2H, ArH). UV–vis λmax, nm (ε, M−1 cm−1) in
DMSO: 265 (41,770), 346 (15,255), 375 (6525). Magnetic moment:
μeff: 1.68BM.
2.3.6. Synthesis of [VO (satsc) (bpy)] (4)
λ
max, nm (ε, M−1 cm−1) in DMSO: 342 (25,320).
This complex was synthesized by a similar procedure as for the
complex 3, with bpy (0.0781 g, 0.5 mmol) in place of 1, 10-
phennanthroline. Yield: 49%. Anal. Calcd. For C18H15N5O2SV: C,
51.93; H, 3.63; N, 16.82; S, 7.70%; Found: C, 51.70; H, 3.89; N, 16.47;
S, 7.67%. ES-MS (CH3OH, m/z): 416.0 (95%) ([M+1]+). IR (KBr)
(νmax/cm−1): 3436 (s), 3276 (s, \NH2), 3060 (w, C\H), 1611 (s),
1538 (m), 1497 (s), 1203 (m, C\O), 1047 (w, C\N), 941 (s, VO),
823 (s), 760 (s, C\S). 1H NMR (500 MHz, DMSO-d6): 8.69 (m, 3H),
8.40 (m, 4H), 7.94 (br m, 3H), 7.45 (br m, 3H, ArH), 7.22 (br, 1H,
ArH), 6.85 (br, 1H, ArH). UV–vis λmax, nm (ε, M−1 cm−1) in DMSO:
2.3.2. Synthesis of salicylaldehyde thiosemicarbazone (satsc)
This compound was prepared by a similar procedure as for the
compound hntdtsc, with salicylaldehyde (0.6106 g, 5 mmol) in place
of 2-hydroxy-1-naphthaldehyde and gave a white precipitate, which
was used without further purification. Yield: 56%. Anal. Calcd. for
C8H9N3OS: C, 49.21; H, 4.65; N, 21.55; S, 16.42%; Found: C, 49.13; H,
4.69; N, 21.53; S, 16.39%. ES-MS (CH3OH, m/z): 196.0 (100%) ([M+
1]+). IR (KBr) (νmax/cm−1): 3444 (vs), 3321 (vs, \NH2), 3175
(vs, N\H), 3033 (m, C\H), 1627 (vs), 1537 (vs), 1491 (s), 1465 (s),
1237 (m, C\O), 1036 (m, C\N), 830 (s), 752 (s, C_S). 1H NMR
(500 MHz, DMSO-d6): 11.40 (s, 1H, NHCS), 9.89 (s, 1H, OH), 8.38 (s,
1H, CH_N), 7.92 and 8.13 (2br s, 1H each NH2), 7.92 (s, 1H, ArH),
7.21(t, 1H, ArH, J=8.4 Hz), 6.88 (d, 1H, ArH, J=7.8 Hz), 6.81 (t, 1H,
ArH, J=8.2 Hz). UV–vis λmax, nm (ε, M−1 cm−1) in DMSO: 340
(26,310).
265 (41,770), 345 (16,255), 381 (6435). Magnetic moment: μeff
:
1.67BM.
2.4. DNA binding and cleavage
Absorption titrations of the oxidovanadium complexes in buffer A
were performed using a fixed concentration of the oxidovanadium
complex (20 μM) to which the DNA stock solutions were added. The
oxidovanadium-DNA solutions were incubated at room temperature
for 5 min before the absorption spectra were recorded. In order to
further elucidate the binding strength of the complexes, the intrinsic
binding constant Kb with CT-DNA was obtained by monitoring the
change in the absorbance of the ligand transfer band with increasing
amounts of DNA. Kb was then calculated using the following
equation [29–35]:
2.3.3. Synthesis of [VO (hntdtsc) (phen)] (1)
A
mixture of hntdtsc (0.1225 g, 0.5 mmol) and 1, 10-
phennanthroline (0.0901 g, 0.5 mmol) in absolute methanol
(100 mL) was heated at 80 °C under argon for 2 h. After dissolution,
a 10 mL of methanol solution of VO (acac)2 (0.1325 g, 0.5 mmol)
was added dropwise to this mixture. The mixture was refluxed for an-
other 4 h to give a reddish-brown precipitate. The solid powder was
isolated from the hot solution and washed with absolute methanol
and diethyl ether respectively, and dried in vacuo. Yield: 85.2%.
Anal. Calcd. for C24H17N5O2SV: C, 58.78; H, 3.49; N, 14.28; S, 6.54%;
Found: C, 58.71; H, 3.67; N, 14.26; S, 6.52%. ES-MS (CH3OH, m/z):
491.0 (96%) ([M+1]+). IR (KBr) (νmax/cm−1): 3436 (s), 3265 (m,
\NH2), 3047 (m, C\H), 1606 (vs), 1529 (vs), 1459 (s), 1193 (m,
C\O), 1039 (m, C\N), 948 (s, VO), 836 (s), 757 (m, C\S). 1H NMR
(500 MHz, DMSO-d6): 9.12 (s, 1H, CH_N), 8.50 (m, 6H), 8.00 (m,
3H), 7.78 (br m, 2H, ArH), 7.5 (br m, 2H, ArH), 6.96 (br m, 3H, ArH).
UV–vis λmax, nm (ε, M−1 cm−1) in DMSO: 264 (41,770), 340
(15,255), 385 (6525). Magnetic moment: μeff: 1.68BM.
½DNAꢀ
½DNAꢀ
1
¼
þ
εa−εf εa−εf Kbðεb−εfÞ
where [DNA] is the concentration of DNA in the base pairs, and εa, εf
and εb refer to the corresponding apparent absorption coefficient
Aobsd/[Vanadium], the extinction coefficient for the free oxidovana-
dium complex and the extinction coefficient for the oxidovanadium
complex in the fully bound form, respectively. In plots of [DNA]/
(εa −εf) versus [DNA], Kb is obtained by the ratio of the slope to
the intercept.
Viscosity measurements were carried out with an Ubbelohde vis-
cometer maintained at a constant temperature of (28.0 0.1) °C in
a thermostatic bath. Flow time was measured with a digital stop-
watch and each sample was measured five times to obtain the aver-
age flow time. Data are presented as (η/η0)1/3 versus binding ratio
[28], where η is the viscosity of DNA in the presence of complexes
while η0 is the viscosity of DNA alone [30,33].
2.3.4. Synthesis of [VO (hntdtsc) (bpy)] (2)
This complex was synthesized by a similar procedure as for the
complex 1, with bpy (0.0781 g, 0.5 mmol) in place of 1,10-phennan-
throline. Yield: 83.8%. Anal. Calcd. For C22H17N5O2SV: C, 56.65; H,
3.67; N, 15.02; S, 6.87%; Found: C, 56.36; H, 3.86; N, 14.88; S, 6.83%.
ES-MS (CH3OH, m/z): 467.0 (100%) ([M+1]+). IR (KBr) (νmax
/
cm−1): 3428 (m), 3266 (m, \NH2), 3064 (m, C\H), 1610 (vs),
1496 (vs), 1436 (w), 1187 (m, C\O), 1037 (m, C\N), 960 (s, VO),
879 (m), 738 (m, C\S). 1H NMR (500 MHz, DMSO-d6): 9.13 (s, 1H,
CH_N), 8.69 (m, 4H), 8.39 (m, 5H), 7.95 (br m, 4H), 7.42 (br m, 3H,
ArH). UV–vis λmax, nm (ε, M−1 cm−1) in DMSO: 264 (41,650), 341
(16,250), 376 (5322). Magnetic moment: μeff: 1.66BM.
Thermal denaturation studies were carried out with Shimadzu
UV-3101 PC spectrophotometer equipped with
a
Peltier
temperature-controlling programmer ( 0.1 °C). The melting curves
were obtained by measuring the absorbance at 260 nm for solutions
of CT-DNA (80 μM) in the absence and presence of oxidovanadium
complex [20 μM] as a function of the temperature. The temperature
was scanned from 50 to 90 °C at a speed of 5 °C min−1
.
2.3.5. Synthesis of [VO (satsc) (phen)] (3)
This complex was synthesized by a similar procedure as for the
complex 1, with satsc (0.0975 g, 0.5 mmol) in absolute methanol
The cleavage of supercoiled pBR322 DNA by the complexes was
studied by the gel electrophoresis experiment, pBR322 DNA (0.1 μg)
was treated with the oxidovanadium complexes in buffer B, and the

42
J. Lu et al. / Journal of Inorganic Biochemistry 112 (2012) 39–48
solution was incubated at 37 °C in the incubator. The samples were
analyzed by electrophoresis for 1.5 h at 85 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 μg/ml ethidium bromide and photographed on an
Alpha Innotech IS-5500 fluorescence chemiluminescence and visible
imaging system [33,34].
coordinated to vanadium [16,28]. All other frequencies founded in the
IR spectra of complexes are in accordance to the literature data for the
same type of complexes [28,36–38].
In the 1H NMR spectra of Schiff bases of thiosemicarbazone
showed peaks of (NHCS), hydroxyl (OH), amine (NH2) and imine
(CH_N) proton. However, in the 1H NMR spectra of complexes, the
peaks of (NHCS) and hydroxyl (OH) were not observed, which
affirmed that the free ligand was coordinated to vanadium.
2.5. Antitumor assay in vitro
In the electronic spectra, the complexes show an intense band at
ca. 265 nm assignable to π−π* transitions of aromatic rings of phe-
nanthroline [15,20,28,34]. A medium band is observed near 400 nm,
which is attributed to a ligand-to-metal charge-transfer transition
(LMCT) as a charge transfer from a p-orbital on the lone-pair of ligand
oxygen atoms to the empty d-orbital of the vanadium atom [33]. The
remaining bands appearing in the UV-region (320–350 nm) are as-
signable to the intraligand transitions of the Schiff base [15,16,33].
Complexes of oxidovanadium (IV) with coordination numbers 5 and
6 are usually square pyramidal/trigonal bipyramidal and distorted oc-
tahedral, respectively [14–19]. From the above obtained spectral data,
it indicates that the Schiff bases bonded through the phenolate oxy-
gen, imine nitrogen and thiolate sulfur atoms leaving the thiomethyl
as the pendant group. This implies that these complexes bear the cen-
tral V (IV) atom in a square-pyramidal geometry [16,17,33,38].
In the ES-MS spectra, for Schiff bases ligands showed peak at m/z
246.0 ([M+1]+) (hntdtsc) and 196.0 ([M+1]+) (satsc), respective-
ly. The molecular ion peaks of complexes at m/z 491.0 ([M+1]+) (1),
467.0 ([M+1]+) (2), 441.0 ([M+1]+) (3) and 417.0 ([M+1]+) (4),
respectively. Elemental analysis, ES-MS, IR and 1H NMR data of all the
compounds are in good agreement with the expected structures.
The molar conductance values of the four complexes in DMF are
given in Table 1, which indicates these four oxidovanadium com-
plexes show nonelectrolytic nature. As can be seen from Table 1, the
complexes are one-electron paramagnetic giving a magnetic moment
value of ~1.60 BM at room temperature. These values of magnetic
susceptibility also confirm that the vanadium complexes are in the
V (IV) states, with d1 configuration [33,39,40].
2.5.1. Cytotoxicity assays
The capacities of compounds to interfere with the growth of
Myeloma cells (Ag8.653) and Gliomas cells (U251) were determined
with the aid of MTT dye assay. Compounds were dissolved in DMSO
and diluted with RPMI 1640 to the required concentrations prior to
use. The control was well prepared by addition of culture medium
(100 μL). Wells containing culture medium without cells were used
as blanks. Myeloma cells (Ag8.653) and Gliomas cells (U251) with a
density 2×104 cells per well were precultured into 96-well microtiter
plates for 48 h at 37 °C, 5% CO2. Upon completion of the incubation,
then stock MTT dye solution was added to each well. After 4 h incuba-
tion, a solution containing N, N-dimethylformanmide (50%) sodium
dodecyl sulfate (20%) was added to solubilize the MTT formazan.
The cell viability was determined by measuring the absorbance of
each well at 490 nm using a Multiskan SSCENT microplate reader.
IC50 values were determined by plotting the percentage viability ver-
sus concentration on a logarithmic graph and reading off the concen-
tration at which 50% of cells remain viable relative to the control
[49–51].
2.5.2. Cell cycle analysis
Analysis of the cell cycle of control and treated cancer cells was
determined. Using standard methods, the DNA of cells were stained
with PI, and the proportion of non-apoptotic cells in different phases
of the cell cycle was recorded [50,51]. The cancer cells were treated
with the complex, harvested by centrifugation at 1000×g for 5 min,
and then washed with ice-cold PBS. The collected cells were fixed
overnight with cold 70% ethanol, and then stained with PI solution
consisting of 50 μg/mL PI, 10 μg/mL RNase. After 10 minute incuba-
tion at room temperature in the dark, fluorescence-activated cells
were sorted in a FACScan flow cytometer using Cell Quest 3.0.1 soft-
ware. The percentage of cells in each phase of the cell cycle was deter-
mined as least in triplicate and expressed as mean SD.
3.2. DNA-binding studies
3.2.1. Electronic absorption titration
Electronic absorption spectroscopy is one of the most common
ways to investigate the interaction of complexes with DNA. The inter-
calation of complexes into the base pairs of DNA usually results in
hypochromism and bathochromism (red shift), which is correlated
with the stacking interaction between the planar aromatic chromo-
phore of complexes and the base pairs of DNA. The extent of the
hypochromism is commonly consistent with strength of intercalative
binding interaction [34,41,42].
3. Results and discussion
3.1. Synthesis and characterization
The ligands, hntdtsc and satsc were prepared by the reaction of
thiosemicarbazide with 2-hydroxy-1-naphthaldehyde and salicylal-
dehyde, respectively, in the appropriate mole ratios, using absolute
methanol as the solvent [33–35]. The complexes [VO(hntdtsc)
(phen)] (1), [VO(hntdtsc)(bpy)] (2), [VO(satsc)(phen)] (3) and
[VO(satsc)(bpy)] (4) were prepared by refluxing of ligand, VO(acac)2
with 1,10-phennanthroline and 2,2′-bipyridine in absolute methanol,
respectively. The desired complexes were purified by recrystalliza-
tion. The structures of these compounds were confirmed by elemen-
tal analysis, ES-MS, UV–vis, IR, and 1H NMR spectroscopy. Synthetic
routes of ligands and their complexes are shown in Scheme 1.
In the IR spectra of Schiff bases of thiosemicarbazone showed ab-
sorption bands at 3450–3167 cm−1 for (\NH2) and (\NH\), at
2989–3053 cm−1 for aromatic (C\H), at 1593–1627 cm−1 for azo-
methine group (\CH_N) and (C_C), at 750–760 cm−1 confirms
the group (\C_S) and at 1237–1240 cm−1 possible for group
(C\O), But in the IR spectra of complexes, the peak of the azomethine
group (\CH_N) has reduced by 14–19 cm−1 as compared with their
ligands, respectively, which is assigned to the azomethine (\CH_N)
For metallo-intercalators, DNA-binding is associated with hypo-
chromism and a red shift in the MLCT and ligand bands [16,17].
Fig. 1 shows the absorption spectra of complexes (1), (2), (3) and
(4) in the presence of increasing concentration of DNA. As can be
seen in Fig. 1, upon increasing the CT-DNA concentration, appreciable
hypochromism and bathochromism for the complexes 1 at 263.5 nm,
2 at 273 nm, 3 at 264 nm and 4 at 280.5 nm exhibit hypochromism of
29.7%, 25.7%, 24.3% and 19.3%, and bathochromism of 3 nm, 2.5 nm,
2 nm and 2 nm, respectively. According to previous reported results
Table 1
Conductivity and magnetic data of oxidovanadium complexes.
Compounds
ΩM (Ω−1 cm2 mol−1
)
B.M (μeff)
1
2
3
4
9.43
13.9
8.36
13.1
1.65
1.66
1.64
1.67

J. Lu et al. / Journal of Inorganic Biochemistry 112 (2012) 39–48
43
(a)
(b)
2.0
1.8
1.6
1.4
1.2
1.0
2.0
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.5
1.6
1.2
0.8
0.4
0.0
1.2
0.9
0
2
4
6
8
10
0.0
0.5
1.0
1.5
2.0
2.5
5
[DNA]x105M
0.6
0.3
0.0
[DNA]x105M
250
300
350
400 450 500 550 600
250
300
350
400
450 500 550 600
Wavelength/nm
Wavelength/nm
(c)
(d)
0.9
0.6
0.3
0.0
7.5
6.0
4.5
3.0
1.5
0.0
9.0
7.5
6.0
4.5
3.0
1.5
0.0
1.2
0.9
0.6
0.3
0.0
0
4
8
12
16
0
1
2
3
4
5
6
7
[DNA]x105M
[DNA]x105M
250
300
350
400
450 500 550 600
250
300
350
400 450 500 550 600
Wavelength/nm
Wavelength/nm
Fig. 1. Absorption spectral of the complexes 1 (a), 2 (b), 3 (c) and 4 (d) in Tris–HCl buffer A upon increasing amounts of CT-DNA. [V]=20 μM, [DNA]=(0–100) μM. Arrow shows
the decreasing absorbance upon increasing CT-DNA concentrations. Insert: Plots of [DNA]/(εa-εf) versus [DNA] for the titration of [V] with CT-DNA.
[9,34,43], these spectral characteristics obviously suggest that com-
plexes 1, 2, 3 and 4 interact with DNA most likely through a mode
that involves a stacking interaction between the aromatic chromo-
phore and the base pairs of DNA.
3 and 5.84:1for 4 reached a saturating value, the emission intensities
of complexes 1 (at 458 nm), 2 (at 458 nm), 3 (at 705 nm) and 4 (at
706 nm) grow to about 53%, 48%, 38% and 29 % larger than those in
the absence of DNA, respectively. The enhancement of emission in-
tensity is indicative of binding of the complexes to the hydrophobic
pocket of DNA, since the hydrophobic environment inside the DNA
helix reduces the accessibility of water molecules to the complex
and the complex mobility is restricted at the binding site, leading to
decrease of the vibrational modes of relaxation. These results are in
agreement with the change in UV–vis spectra data.
In order to compare quantitatively the binding strength of these
complexes with DNA, the intrinsic binding constant Kb was calculated
by monitoring the changes of absorbance in the ligand transfer bands,
with the increasing amounts of CT-DNA. The intrinsic binding con-
stant Kb obtained for complexes 1, 2, 3 and 4 were 8.2×104 M−1
,
7.8×104 M−1, 1.37×104 M−1 and 9.62×103 M−1, respectively. The
Kb decreases in the order: 1>2>3>4. The differences of their bind-
ing strength may be due to the presence of an appending aromatic
moiety in hntdtsc and thus the larger binding affinities of the corre-
sponding complexes in comparison with those complexes incorporat-
ing satsc [9,33,38,44]. The results also imply that the interactions of
these complexes with DNA may be mainly through π−π stacking of
hntdtsc and satsc plane and base-pairs, and show a classic intercala-
tive mode. Meanwhile, the electronic effect of phenanthroline is one
of the factors in determining the binding affinities.
3.2.3. Viscosity measurements
To further clarify the nature of the binding interaction between
both complexes and DNA, viscosity measurements were carried out
on CT DNA by varying the concentration of the added complexes.
Spectroscopic data are necessary, but not sufficient to support a bind-
ing mode. Hydrodynamic measurements which are sensitive to
length increase (for example, viscosity, sedimentation) are regarded
as the least ambiguous and the most critical tests of binding in solu-
tion in the absence of crystallographic structure data [36–40]. A clas-
sical intercalative mode causes a significant increase in viscosity of
DNA solution due to increase in separation of base pairs at intercala-
tion sites and hence an increase in overall DNA length. In contrast, a
partial, non-classical intercalation of compounds could bend (or
kink) the DNA helix and reduce its effective length and, concomitant-
ly, its viscosity [36,41–43].
3.2.2. Fluorescence spectroscopic studies
The interaction of the complexes with CT-DNA was studied using
fluorescence emission titration experiment in the Tris buffer A at
room temperature. The emission spectra of these four complexes in
the absence and presence of CT DNA are shown in Fig. 2. As shown
in Fig. 2, an obvious increase in emission intensity was observed for
the four complexes. As increasing the concentration of CT-DNA,
when the ratio of [DNA]/[V] of 11.67:1 for 1, 9.73:1 for 2, 6.81:1 for
The effects of complexes 1, 2, 3, 4 and EB on the viscosity of CT-
DNA are shown in Fig. 3. As can be seen in Fig. 3, upon increasing

44
J. Lu et al. / Journal of Inorganic Biochemistry 112 (2012) 39–48
(a)
(b)
800
800
600
400
200
0
600
400
200
0
400
450
500
550
600
650
400
450
500
550
600
650
Wavelength/nm
Wavelength/nm
(c)
(d)
400
350
300
250
200
150
100
50
1000
800
600
400
200
0
0
690
700
710
720
730
680
690
700
710
720
730
740
Wavelength/nm
Wavelength/nm
Fig. 2. Emission spectra of complexes 1 (a), 2 (b), 3 (c) and 4 (d) in Tris–HCl buffer A in the absence and presence of CT-DNA. [V]=20 μM. Arrow shows the increasing intensity
upon increasing DNA concentrations.
the amounts of complexes 1, 2 and 3, the relative viscosity of DNA in-
creases steadily. The increasing degree of viscosity is 1>2>3>4. The
viscosity experimental results thus provide strong evidence for the
interaction of complexes 1, 2, 3 and 4 with DNA by intercalation
modes and the electronic effect of phenanthroline is an important
factor in determining the intrinsic binding constant Kb.
ratio [V]/[DNA] of 1:4, the observed Tm in the presence of complexes
1, 2, 3 and 4 are 65.9 0.2 °C, 64.5 0.2 °C, 63.9 0.2 °C and 63.7
0.2 °C, respectively. The moderate increase in Tm (ΔTm are 5.2 °C,
3.8 °C, 3.2 °C and 3.0 °C for 1, 2, 3 and 4, respectively) was comparable
1.04
1.03
1.02
1.01
1.00
3.2.4. Thermal denaturation studies
Thermal behaviors of DNA in the presence of compounds can give
insight into their conformational changes when temperature is
raised, and offer information about the interaction strength of com-
plexes with DNA. Normally, when the temperature in the solution in-
creases, the double-stranded DNA will gradually dissociate to single
strands and generate a hyperchromic effect on the absorption spectra
of DNA bases (λMax =260 nm). The melting temperature Tm, which is
defined as the temperature where half of the total base pairs are un-
bounded, is usually introduced. Generally, Tm will increase consider-
ably when intercalative binding occurs, since intercalation of the
complexes into DNA base pairs causes stabilization of base stacking
and hence raises the melting temperature of the double-stranded
DNA [36,39–43].
0.00
0.02
0.04
0.06
0.08
0.10
The changes of melting temperature of CT-DNA in the absence and
presence of the complexes 1, 2, 3 and 4 are showed in Fig. 4. The Tm of
CT-DNA in the absence of the complexes is 60.7 0.2 °C. As can be
seen from Fig. 4, when mixed with the compounds at a concentration
[V]/[DNA]
Fig. 3. Effects of increasing amounts of complexes 1 (■), 2 (●), 3 (▲) and 4 (▼) on the
relative viscosity of CT-DNA at 28 ( 0.1) °C. [DNA]=0.40 mM.

J. Lu et al. / Journal of Inorganic Biochemistry 112 (2012) 39–48
45
to those observed for classical intercalate and lends strong support for
3.3. Antitumor assay in vitro
3.3.1. Cytotoxicity assays
their binding with DNA in intercalative modes [33,42–44]. The exper-
iment results also show that complex 1 exhibits a larger DNA-binding
affinity than that of complexes 2, 3 and/or 4 does. It is consistent with
their binding abilities with CT-DNA.
The antitumor activity of all these complexes and the correspond-
ing ligands against Ag8.653 and U251 cell lines was evaluated by MTT
assay [45–48]. The IC50 values obtained of ligands and their com-
plexes against selected two tumor cell lines are shown in Table 2.
The histogram of cell viability assay for different compounds against
selected tumor cells obtained with continuous exposure for 48 h is
shown in Fig. 8. As shown in Table 2 and Fig. 8, the synthetic oxidova-
nadium complexes exhibit broad inhibition on the two tested human
cancer cell lines with the IC50 values ranging from 0.004 to 0.950 μM,
respectively. The results also indicate that all of these oxidovanadium
complexes and their corresponding ligands exhibit antitumor activi-
ties against the selected cell lines in different concentrations and
the antitumor activities are concentration-dependent [16,18]. With
comparison of the antitumor activities of the ligands and correspond-
ing complexes, oxidovanadium complexes appeared to be much cyto-
toxic against the cell lines of Ag8.653 and U251. Fig. 7 shows the
antiproliferative activity in the absence (I) and presence of complexes
1 (II), 2 (III), 3 (IV) and 4 (V), at the concentration of 200 μM. As
shown in Fig. 7, the proliferation of tumor cells of Ag8.653 was effec-
tively inhibited.
3.2.5. DNA cleavage
The cleavage reactions of complexes with plasmid DNA were
monitored by agarose gel electrophoresis. The four complexes
showed considerable DNA cleavage ability at low concentrations
(15 μM) in the presence of H2O2. When circular plasmid DNA is sub-
ject to electrophoresis, relatively fast migration will be observed for
the intact supercoiled form (Form I). If scission occurs on one strand
(nicking), the supercoil form will relax to generate a slower moving
open circular form (Form II). If both strands are cleaved, a linear
form (Form III) that migrates between Forms I and II will be generat-
ed [29–32].
Figs. 5 and 6 show the cleavage of plasmid pBR322 DNA after incu-
bation with different concentrations of oxidovanadium complexes at
37 °C for 1 h in the dark. Under the same conditions no cleavage of
pBR 322 DNA occurred for H2O2 alone (lane 2 in Figs. 5 and 6, respec-
tively). In the presence of the complexes 1, 2, 3, and 4 with 30 mM
H2O2 (lanes 3, 4 and 5 in Figs. 5 and 6, respectively) or 30 mM H2O2
and 0.02 M L-histamine being a singlet oxygen quencher (lanes 6
and 11 in Figs. 5 and 6, respectively), plasmid DNA was nicked,
being evidently observed from the formation of From II, and for that
L-Histamine slightly inhibits the cleavage of DNA. On the contrary,
complete inhibition of DNA cleavage occurred in the presence of hy-
droxyl radical scavenger DMSO (lanes 7, 12 in Figs. 5 and 6, respec-
tively), which suggests that •OH radical is likely to be the reactive
species for the cleavage reaction. This can be explained by the gener-
ation of •OH radicals obtained from the oxidation of VO2+ in the pres-
It is notable that complex 1, as compared with other tested com-
plexes, possessed the most potent inhibitory effect against the two
cell lines. And its IC50 value which is very close to that of cisplatin in-
dicated its high cytotoxic effects against human cancer cells. This is
consistent with its binding abilities with CT DNA, indicating that the
antitumor abilities of the oxidovanadium complexes may be closely
related to their DNA binding mode.
3.3.2. Cell cycle analysis
In order to further define the mechanism of anti-proliferative ef-
fect of oxidovanadium complexes on tumor cells, the cell cycle
phase distribution was analyzed by flow cytometry with PI staining
[16,49–51]. According to the results of Table 3, Ag8.653 cells exhib-
ited the higher sensitivity to complex 1. Thus this cell line was used
for further investigation on the underlying mechanisms accounting
for the antiproliferative action of complex 1. The Ag8.653 cells were
treated with 0.001 μM, 0.002 μM and 0.004 μM of complex 1 for
48 h, respectively. As shown in Table 3, the G2/M phase was arrested
significantly after Ag8.653 cells were exposed to 0.004 μM (IC50
values) complex 1 for 48 h. Treatment with 0.001 or 0.002 μM com-
plex 1 resulted in modest G2/M phase arrest of Ag8.653 cells at 48 h
time point. The results in this work showed that there were signifi-
cantly decreased G0/G1 phase distribution and increased G2/M
phase distribution in a dose-dependent manner, indicating the induc-
tion of G0/G1-phase arrest by complex 1. Moreover, apoptotic cells
and cell debris significantly increased after Ag8.653 cells were ex-
posed to the complex 1. In conclusion, the results suggested that oxi-
dovanadium complexes induced proliferative suppression of Ag8.653
cells were via the induction of apoptosis [47,49,51]. To fully under-
stand the mechanism involved in the induction of apoptosis by
ence of H2O2 through the reaction as follows: (VO2+ +H2O2 →VO2+
+
•OH+H+) [9,16–19]. These results indicate that the process of DNA
cleavage may be closely related to the oxidation of vanadyl ions
of oxidovanadium complexes and these four oxidovanadium com-
plexes can degrade pBR322 DNA through oxidative cleavage in the
presence of H2O2 [9,41,42]. Under comparative experimental condi-
tions, the cleavage ability follows the order of 1>2>3>4. This is
consistent with the magnitude of their intrinsic binding constants
(Kb), indicating that the photocleavage abilities might be closely
related to the electronic effect of the ligands, which affected the
binding ability as well.
DNA
1.0
complex 1
complex 2
0.8
0.6
0.4
0.2
0.0
complex 3
complex 4
1
2
3
4
5
6
7
8
9
10 11 12
Form 2
Form 1
Fig. 5. Cleavage of pBR322 DNA by oxidovanadium complexes 1 and 3 (15–60 μM) in
the absence and presence of H2O2 (30 mM) in buffer B (pH 7.2). Lane 1, DNA control;
lane 2, DNA+H2O2; lane 3, DNA+1 (15 μM)+H2O2; lane 4, DNA+1 (30 μM)+
H2O2; lane 5, DNA+1 (60 μM)+H2O2; lane 6, DNA+1 (30 μM)+H2O2 +L-Histidine
(0.02 M); lane 7, DNA+1 (30 μM)+H2O2 +DMSO (2 μL); lane 8, DNA+3 (15 μM)+
H2O2; lane 9, DNA+3 (30 μM)+H2O2; lane 10, DNA+3 (60 μM)+H2O2; lane 11,
DNA+3 (30 μM)+H2O2 +L-Histidine (0.02 M); lane 12, DNA+3 (30 μM)+H2O2 +
DMSO (2 μL).
40
50
60
70
80
90
Temperature /
Fig. 4. Thermal denaturation of CT-DNA in the absence and presence of complexes 1, 2,
3 and 4. [V]=20 μM, [DNA]=80 μM.

46
J. Lu et al. / Journal of Inorganic Biochemistry 112 (2012) 39–48
1
2
3
4
5
6
7
8
9
10 11 12
Table 2
Form 2
Form 1
The IC50 values for hntdtsc, satsc, 1, 2, 3 and 4 against Ag8.653 and U251 cell lines.
Compounds
IC50
(μM)
Ag8.653
U251
Fig. 6. Cleavage of pBR322 DNA by oxidovanadium complexes 2 and 4 (15–60 μM) in
the absence and presence of H2O2 (30 mM) in buffer B (pH 7.2). Lane 1, DNA control;
lane 2, DNA+H2O2; lane 3, DNA+2 (15 μ M)+H2O2; lane 4, DNA+2 (30 μM)+
H2O2; lane 5, DNA+2 (60 μM)+H2O2; lane 6, DNA+2 (30 μM)+H2O2 +L-Histidine
(0.02 M); lane 7, DNA+2 (30 μM)+H2O2 +DMSO (2 μL); lane 8, DNA+4 (15 μM)+
H2O2; lane 9, DNA+4 (30 μM)+H2O2; lane 10, DNA+4 (60 μM)+H2O2; lane 11,
DNA+4 (30 μM)+H2O2 +L-Histidine (0.02 M); lane 12, DNA+4 (30 μM)+H2O2 +
DMSO (2 μL).
Cisplatin
hntdtsc
satsc
1
2
3
4
0.002 0.001
2.034 0.332
22.384 4.733
0.004 0.001
0.296 0.081
0.369 0.097
0.950 0.081
0.005 0.001
5.210 0.217
25.543 2.195
0.007 0.001
0.134 0.079
0.203 0.068
0.524 0.029
Cells were treated with various concentrations of tested compounds for 48 h. Cell
viability was determined by MTT assay and IC50 values were calculated as described
in Materials and Methods. Each value represents the mean SD of three independent
experiments.
oxidovanadium complexes, further investigation will be needed to be
carried out.
4. Conclusion
compound with a potential in treatment of cancers. Further work is
in progress to better identify the active species in solution and to pre-
pare more potent and stable related oxidovanadium compounds.
Four oxidovanadium complexes, [VO(hntdtsc)(phen)] (1),
[VO(hntdtsc)(bpy)] (2), [VO(satsc)(phen)] (3) and [VO(satsc)(bpy)]
(4) have been synthesized and characterized by elemental analysis,
ES-MS, IR, 1H NMR, UV–vis and magnetic moment measurement.
Their DNA-binding activities were investigated using spectroscopic
methods, viscosity measurements and thermal denaturation. The ex-
perimental results show that these four oxidovanadium complexes
bind to CT-DNA by intercalation modes and the DNA-binding affinity
follows the order 1>2>3>4. These oxidovanadium complexes show
efficiently oxidative cleavage of supercoiled plasmid DNA in the pres-
ence of H2O2. In addition, these complexes present cytotoxic activities
against Ag8.653 and U251 cell lines. Complex 1 exhibits a promising
Abbreviations
MTT
3-(4,5-dimethylthiazoyl-2-yl) 2,
5-diphenyltetrazoliumbromide;
50% inhibition concentrations;
the DNA-melting temperature where total base pairs are
unbound;
IC50
Tm
ES-MS
electrospray mass spectra;
Hntdtsc 2-hydroxy-1-naphthaldehyde thiosemicarbazone;
Satsc salicylaldehyde thiosemicarbazone;
Fig. 7. Cell viability of hntdtsc, satsc, 1, 2, 3 and 4 on tumor Ag8.653 (a, b) and U251 (c, d) cells proliferation in vitro. Each data point is the mean standard error obtained from
three independent experiments.

J. Lu et al. / Journal of Inorganic Biochemistry 112 (2012) 39–48
47
Fig. 8. Micrograph of the myeloma tumor (Ag8.653) cell line after treated for 48 h in the absence (I) (control) and presence of complexes 1 (II), 2 (III), 3 (IV) and 4 (V), respectively,
[V]=200 μM. Cell was observed using an inverted microscope and photographed by a digital camera.
Phen
Bpy
1,10-phenanthroline;
2,2′-bipyridine;
National Natural Science Foundation of P. R. China (Nos. 81102753
and 21101033).
Acac
acetylacetonate;
CT-DNA calf thymus DNA;
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