DNA binding, antitumor activities, and hydroxyl radical scavenging properties of novel oxovanadium(IV) complexes with substituted isoniazid

Article abstract of DOI:10.1007/s00775-013-1046-9

Four novel oxovanadium(IV) complexes-[VO(PAHN)(phen)] (1; PAHN is 4-pyridinecarboxylic acid, 2-[(2-hydroxy)-1-naphthalenylene] hydrazide, phen is 1,10-phenanthroline), [VO(PAHN)(bpy)] (2; bpy is 2,2′- bipyridine), [VO(PAH)(phen)] (3; PAH is 4-pyridinecarb

Full text of DOI:10.1007/s00775-013-1046-9

J Biol Inorg Chem (2013) 18:975–984
DOI 10.1007/s00775-013-1046-9
ORIGINAL PAPER
DNA binding, antitumor activities, and hydroxyl radical
scavenging properties of novel oxovanadium(IV) complexes
with substituted isoniazid
•
Xiangwen Liao Jiazheng Lu Peng Ying
•
•
•
•
•
Ping Zhao Yinliang Bai Wenjie Li
Mingpei Liu
Received: 1 June 2013 / Accepted: 1 September 2013 / Published online: 28 September 2013
Ó SBIC 2013
Abstract Four novel oxovanadium(IV) complexes—
[VO(PAHN)(phen)] (1; PAHN is 4-pyridinecarboxylic
acid, 2-[(2-hydroxy)-1-naphthalenylene] hydrazide, phen is
1,10-phenanthroline), [VO(PAHN)(bpy)] (2; bpy is 2,20-
bipyridine), [VO(PAH)(phen)] (3; PAH is 4-pyridinecarb-
oxylic acid, 2-[(2-hydroxy)-1-phenyl]methylene hydra-
zide), and [VO(PAH)(bpy)] (4)—have been synthesized
and characterized by elemental analysis, UV–vis spec-
troscopy, electrospray ionization mass spectrometry, IR
spectroscopy, 1H-NMR spectroscopy, and 13C-NMR
spectroscopy. Their interactions with calf thymus DNA
were investigated. The results suggest that these complexes
bind to DNA in an intercalative mode. All four complexes
exhibited highly cytotoxic activity against tumor cells (SH-
SY5Y, MCF-7, and SK-N-SH), with 50 % inhibitory
concentrations of the same order of magnitude as for cis-
platin or of lower order of magnitude. Complex 1 exhibited
the highest interaction ability and was found to be the most
potent antitumor agent among the four complexes. It can
cause G2/M phase arrest of the cell cycle, induces signifi-
cant apoptosis in SK-N-SH cells, and displays typical
morphological apoptotic characteristics. In addition, their
hydroxyl radical scavenging properties have been tested,
and complex 1 was the best inhibitor.
Electronic supplementary material The online version of this
article (doi:10.1007/s00775-013-1046-9) contains supplementary
material, which is available to authorized users.
Keywords Oxovandium complexes ꢀ DNA binding ꢀ
Cleavage ꢀ Antitumor ꢀ Scavenging hydroxyl radical
X. Liao ꢀ J. Lu (&) ꢀ P. Ying
School of Pharmacy,
Guangdong Pharmaceutical University,
Guangzhou 510006,
Guangdong, People’s Republic of China
e-mail: lujia6812@163.com
Abbreviations
CT-DNA Calf thymus DNA
DMF
N,N-Dimethylformamide
DMSO
ESI-MS
Dimethyl sulfoxide
P. Zhao (&)
School of Medicine Chemistry and Chemical Engineering,
Guangdong Pharmaceutical University,
Guangzhou 510006, Guangdong, People’s Republic of China
e-mail: zhaoping666@163.com
Electrospray ionization mass spectrometry
H4EDTA N,N’-1,2-Ethanediylbis[N-
(carboxymethyl)]glycine
IC50
50 % inhibitory concentration
3-(4,5-Dimethylthiazoyl-2-yl)-2,5-
diphenyltetrazolium bromide
Y. Bai ꢀ M. Liu
MTT
Department of Pharmacy, Lanzhou University Second Hospital,
No. 82, Cuiyingmen, Lanzhou 730030, Gansu, People’s
Republic of China
PAH
4-Pyridinecarboxylic acid, 2-[(2-hydroxy)-1-
phenyl]methylene hydrazide
W. Li
PAHN
4-Pyridinecarboxylic acid, 2-[(2-hydroxy)-1-
naphthalenylene] hydrazide
Department of Clinical Pharmacology,
School of Pharmaceutical Sciences,
Zhengzhou University, Zhengzhou 450001,
Henan, People’s Republic of China
PI
Propidium iodide
Tris
Tris(hydroxymethyl)aminomethane
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Introduction
(hntdtsc is 2-hydroxy-1-naphthaldehyde thiosemicarba-
zone, phen is 1,10-phenanthroline) was found to be a
promising compound with potential in the treatment of
cancer cells, and its 50 % inhibitory concentration (IC50) is
very close to that of cisplatin [29]. Our previous results also
indicated that with increasing the appending aromatic
moiety, the activity of vanadium complexes would much
better [29–31].
In the last few decades, metal complexes has received
much attention owing to their huge potential as antitumor
agents, such as ruthenium complexes, cobalt(II) com-
plexes, copper complexes, gold complexes, and platinum
complexes [1–5]. Vanadium, as a trace bioelement,
exhibits a variety of biological activities, such as antitumor,
antimicrobial, and insulin-enhancing effects, and exhibits
potent anti-HIV effects toward infected immortalized
T cells [6–12]. Many oxovanadium(IV) complexes with
various coordination modes have been prepared, viz.,
VO(O4), VO(S2N2), VO(S4), VO(N3O), and VO(N2O2),
and the relationship between their structures and insulin-
mimetic activities has been examined by evaluating both
in vivo and in vitro results [13–16]. Bis(ethylmaltola-
to)oxovanadium(IV) has completed a phase I clinical trial
in humans for the treatment of type 1 and type 2 diabetes
mellitus [17–19]. More recently, vanadium has attracted
the attention of researchers because of its wide distribution
and broad biological activity [20–22], particularly after the
discovery of its involvement in DNA-repair systems [23,
24]. Vanadium-containing compounds have also shown
great potential as inhibitors of chemically induced tumors
in test animals and cell cultures in vitro, being efficient, for
example, in the treatment of leukemia, breast adenocarci-
noma, and Ehrlich tumors in marines, and lung, breast,
ovarian, testicular, renal, gastrointestinal, and nasopha-
ryngeal carcinomas in humans [23–26]. The role of vana-
dium in the prevention of DNA–protein cross-links, DNA
chain breaks, and chromosomal aberrations has been doc-
umented. Research on the cancer therapeutic role of
vanadium has progressed to the preclinical stage in vivo
[27, 28].
To further our studies in this model system, four new
oxovanadium(IV) complexes—[VO(PAHN)(phen)] (1;
PAHN is 4-pyridinecarboxylic acid, 2-[(2-hydroxy)-1-
naphthalenylene] hydrazide), [VO(PAHN)(bpy)] (2; bpy is
2,20-bipyridine), [VO(PAH)(phen)] (3; PAH is 4-pyridine-
carboxylic
acid,
2-[(2-hydroxy)-1-phenyl]methylene
hydrazide), and [VO(PAH)(bpy)] (4)—have been synthe-
sized (Scheme 1) and characterized by elemental analysis,
UV–vis spectroscopy, electrospray ionization mass spec-
1
trometry (ESI-MS), IR spectroscopy, H-NMR spectros-
copy,and 13C-NMR spectroscopy. The interaction of these
complexes with calf-thymus DNA (CT-DNA) was inves-
tigated using UV–vis absorption spectroscopy, fluores-
cence spectroscopy, viscosity measurements, and thermal
denaturation studies. Their cytotoxicity toward
a
human breast cancer cell line (MCF-7) and human neuro-
blastoma cells (SH-SY5Y and SK-N-SH) was investigated
by 3-(4,5-dimethylthiazoyl-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay. In addition, their hydroxyl radical
scavenging properties were investigated.
Materials and methods
Materials
Keeping in view the significant bioactive nature of a
Schiff base as well as the biological functioning of vana-
dium, it was thought valuable to merge the chemistry of
Schiff bases with that of vanadium to form a novel class of
vanadium-metal-based Schiff bases that could serve as
potential antitumor agents against resistant cancer cells
[16, 28]. It is known that complexes causing photocleavage
of DNA and exhibiting photocytotoxic properties in visible
light are of importance as chemotherapeutic agents [20–
26]. Great efforts have, therefore, been made to synthesize
oxovanadium(IV) complexes of high biological activity
and low toxicity which are readily absorbed [24, 25, 27].
Previously, we reported that four oxidovanadium(IV)
complexes interact with DNA through an intercalative
mode and can efficiently cleave plasmid pBR 322 DNA.
More interestingly, these oxidovanadium(IV) complexes
exhibit highly cytotoxic activities against myeloma cell
(Ag8.653) and glioma cell (U251) lines. Especially, among
complexes with thiosemicarbazones, [VO(hntdtsc)(phen)]
VO(acac)2 (acac is acetylacetonate), 1,10-phenanthroline,
2,20-bipyridine, and isonicotinic acid hydrazide were pur-
chased from Shanghai Jingchun. CT-DNA was obtained
from Sigma. SH-SY5Y, SK-N-SH (derived from human
neuroblastoma cell lines), and MCF-7 (breast cancer cell
line isolated from human mammary glands) cell lines were
purchased from the Cell Bank of Type Culture Collection
of the Chinese Academy of Sciences (Shanghai, China).
Hoechst 33342 staining solution was purchased from
Beyotime Institute of Biotechnology (China). MTT and
rhodamine 123 were purchased from Sigma (USA). Other
materials were obtained from commercial sources and used
as received (analytical reagents). Tris(hydroxy-
methyl)aminomethane (Tris)–HCl buffer A (5 mM Tris–
HCl and 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 DNA-cleavage experiments. Buffer C
(1.5 mM NaHPO4, 0.5 mM NaH2PO4, and 0.25 mM
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977
Scheme 1 The synthetic routes
for the ligands and complexes
Na2H2EDTA pH 7.0; H4EDTA is N,N0-ethane-1, 2-diyl-
bis[N-(carboxymethyl)glycine]) was used for thermal
denaturation. All buffers were prepared using double-dis-
tilled water. A solution of CT-DNA in the buffer gave a
ratio of the UV absorbance at 260 and 280 nm of
approximately 1.8–1.9, indicating that the DNA was suf-
ficiently free of protein [32–34]. The DNA concentration
per nucleotide was determined by absorption spectroscopy
using the molar absorption coefficient (6,600 M–1 cm–1) at
260 nm [35–37]. DNA stock solutions were stored at 4 °C
and used after no more than 3 days. Solutions of com-
pounds were freshly prepared 1 h prior to biochemical
evaluation.
chemical shifts are given relative to tetramethylsilane. UV–
vis absorption spectra were recorded with a Shimadzu UV-
3101 PC spectrophotometer at room temperature. Emission
spectra were recorded with a Perkin-Elmer Lambda 55
spectrofluorophotometer. Molar conductivities in 1mM
N,N-dimethylformamide (DMF) solution at room temper-
ature were measured using a DDS-307 digital direct read-
ing conductivity meter.
Cell viability assay was performed with a microplate
reader (model 680, Bio-Rad, USA). Cell cycle analysis,
annexin V–fluorescein isothiocyanate/propidium iodide
(PI) assay of apoptotic cells and detection of mitochondrial
membrane potential were performed with a FACScan flow
cytometer (BD, USA). Fluorescence microscopy of apop-
tosis assays was performed with an OX31 fluorescence
microscope (Olympus, Japan).
Physical measurements
Microanalysis (C, H, S, and N) was conducted with a
PerkinElmer 240Q elemental analyzer. Electrospray ioni-
zation mass spectra were recorded with an LCQ system
(Finnigan MAT, USA) using methanol as the mobile phase.
IR spectra were recorded with a PerkinElmer Lambda 35
instrument using KBr pellets. 1H-NMR and 13C-NMR
spectra were recorded with a Varian 500 spectrometer. All
Data were expressed as the mean the standard devia-
tion from three independent experiments. Statistical analysis
was performed using SPSS 13.0 for Windows. Comparisons
between two groups were performed by an unpaired t test.
Multiple comparisons between more than two groups were
performed by one-way analysis of variance. Significance
was accepted at a P value lower than 0.05.
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Synthesis of PAHN
phenanthroline (0.0901 g, 0.5 mmol) in absolute methanol
(100 mL) at 80 °C under argon for 2 h. Then the solid
powder was isolated from the hot solution and washed with
absolute methanol and diethyl ether, respectively, and dried
in vacuo. Yield: 0.1747 g, 65.2 %. Anal. Calcd for
C29H19N5O3V (%): C, 64.93; H, 3.57; N, 13.06; O, 8.95.
Found (%): C, 64.75; H, 3.51; N, 13.03; O, 8.90. ESI-MS
(CH3OH, m/z): 537.0([M ? H]?). IR (KBr) (mmax, cm-1):
1,589 (s) (C=N), 1,568 (m) (C=O), 955 (s) (V–O), 702
PAHN was synthesized with a method similar that
described earlier (see Scheme 1) [29–32].To a stirring
solution of 2-hydroxy-1-naphthaldehyde (0.8609 g,
5 mmol) in 10 mL of absolute alcohol was added dropwise
isonicotinic acid hydrazide (0.685 g, 5 mmol), which was
dissolved in 10 mL of absolute alcohol. Then the mixture
was stirred continuously at 50 °C for 3 h, resulting in a
yellow gossypine precipitate, which was used without
further purification. Yield: 0.7569 g, 52 %. Anal. Calcd for
C17H13N3O2 (%): C, 70.09; H, 4.50; N, 14.42; O, 10.98.
Found (%): C, 70.01; H, 4.49; N, 14.31; O, 10.87. ESI-MS
(CH3OH, m/z): 292.1([M ? H]?). IR (KBr) (mmax, cm-1):
3,450 (s) (O–H), 3,169 (s) (N–H), 1,610 (s) (C=N), 1,571
(m) (C=O). 1H-NMR (500 MHz, dimethyl-d6 sulfoxide,
DMSO-d6, ppm):12.52 (s, 1H, NHCO), 12.40 (s, 1H, OH),
9.48 (s, 1H, CH=N), 8.80 (dd, 2H, ArH, J = 8.0 Hz), 8.31
(d, 1H, ArH, J = 7.8 Hz), 7.97 (d, 1H, ArH, J = 8.2 Hz),
7.88 (br, 3H, ArH, J = 8.7 Hz), 7.62 (t, 1H, ArH,
J = 8.3 Hz), 7.41 (t, 1H, ArH, J = 8.1 Hz), 7.26 (d, 1H,
ArH, J = 7.9 Hz). 13C-NMR (500 MHz, DMSO-d6,
ppm):108.5 C (j), 118.8 C (h), 120.9 C (b), 121.4 C (n, p),
123.6 C (f), 127.8 C (g), 127.9 C(e), 128.9 C (d), 131.6 C
(c), 133.2 C (i), 139.8 C (m), 147.9 C (k), 150.4 C (o,q),
158.1 C (l), 161.0 C (a). UV–vis kmax, nm (e, M-1 cm-1) in
DMSO: 340 (25,320).
1
(s) (V–N). H-NMR (500 MHz, DMSO-d6, ppm): 9.15 (s,
1H, CH=N), 8.62 (br, 3H, ArH, J = 8.5 Hz), 8.50 (t, 6H,
ArH, J = 8.2 Hz), 8.09 (s, 2H, ArH), 7.87 (br, 3H, ArH,
J = 7.9 Hz), 7.61 (m, 3H, ArH, J = 8.2 Hz), 6.82(s, 1H,
ArH). UV–vis kmax, nm (e, M-1 cm-1) in DMSO: 264
(43,870), 341 (14,246), 385 (6,535). Magnetic moment
leff: 1.68 lB. XM (S m2 mol-1): 30.9.
Synthesis of complex 2
Complex 2 was synthesized by a procedure similar to that
used for complex 1, with 2,20-bipyridine (0.0781 g,
0.5 mmol) in place of 1,10-phenanthroline. Yield:
0.1634 g, 63.8 %. Anal. Calcd for C27H19N5O3V (%): C,
63.29; H, 3.74; N, 13.67; O, 9.37. Found (%): C, 63.01; H,
3.51; N, 13.03; O, 9.35. ESI-MS (CH3OH, m/z):
513.1([M ? H]?). IR (KBr) (mmax, cm-1): 1,618 (s) (C=N),
1
1,584 (s) (C=O), 954 (s) (V–O), 722 (s) (V–N). H-NMR
(500 MHz, DMSO-d6, ppm): 9.46 (s, 1H, CH=N), 8.63 (d,
2H, ArH, J = 8.0 Hz), 8.38 (m, 2H, ArH, J = 8.3 Hz),
7.93 (br, 6H, ArH, J = 8.7 Hz), 7.54 (br m, 6H, ArH,
Synthesis of PAH
PAH was prepared using a procedure similar to that used
for PAHN, with salicylaldehyde (0.6106 g, 5 mmol) in
place of 2-hydroxy-1-naphthaldehyde. This gave a white
precipitate, which was used without further purification.
Yield: 0.7351 g, 61 %. Anal. Calcd for C13H11N3O2 (%):
C, 64.72; H, 4.60; N, 17.42; O, 13.26. Found (%): C, 64.13;
H, 4.51; N, 17.33; O, 13.03. ESI-MS (CH3OH, m/z):
242.0([M ? H]?). IR (KBr) (mmax, cm-1): 3,443 (s) (O–H),
3,208 (s) (N–H),1,603 (s) (C=N), 1,538 (s) (C=O). 1H-
NMR (500 MHz, DMSO-d6, ppm): 12.29 (s, 1H, NHCO),
11.07(s, 1H, OH), 8.80 (dd, 2H, ArH, J = 8.1 Hz), 8.68 (s,
1H, CH=N), 7.78(d, 1H, ArH, J = 8.3 Hz), 7.61(m, 1H,
ArH, J = 7.9 Hz), 7.32 (dd, 2H, ArH, J = 8.4 Hz), 6.92
(m, 2H, ArH, J = 8.0 Hz). 13C-NMR (500 MHz, DMSO-
d6, ppm): 116.4 C(b), 118.7 C(f), 119.4 C(d), 121.5 C(j, l),
129.2 C(e), 131.7 C(c), 139.9 C(i), 148.9 C(g), 150.4
C(k,m), 157.5 C(a), 161.3 C(h). UV–vis kmax, nm (e,
M-1 cm-1) in DMSO: 341 (25,320).
J = 8.4 Hz), 7.25 (m, 2H, ArH, J = 8.0 Hz). UV–vis kmax
,
nm (e, M-1 cm-1) in DMSO: 264 (43,650), 341 (14,250),
376 (6,532). Magnetic moment leff: 1.72 lB. XM
(S m2 mol–1): 28.5.
Synthesis of complex 3
Complex 3 was synthesized by a procedure similar to that
used for complex 1, with PAH (0.120 g, 0.5 mmol) in
absolute methanol (30 mL) in place of PAHN in absolute
methanol (100 mL). Yield: 0.1385 g, 57 %. Anal. Calcd
for C25H17N5O3V (%): C, 61.74; H, 3.52; N, 14.40; O,
9.87. Found (%): C, 61.13; H, 3.51; N, 14.23; O, 9.83. ESI-
MS (CH3OH, m/z): 487.0 ([M ? H]?). IR (KBr) (mmax
,
cm-1): 1,610 (s) (C=N), 1,589 (s) (C=O), 955 (s) (V–O),
727 (s) (V–N). 1H-NMR (500 MHz, DMSO-d6, ppm): 9.87
(s, 1H, CH=N), 8.75 (br, 1H, ArH, J = 8.7 Hz), 8.42 (m,
3H, ArH, J = 7.8 Hz), 7.95 (br, 3H, ArH, J = 8.4 Hz),
7.86 (d, 2H, ArH, J = 8.2 Hz), 7.57 (br m, 3H, ArH,
J = 8.0 Hz), 7.31 (m, 2H, ArH, J = 8.0 Hz), 7.08 (m, 2H,
ArH, J = 8.2 Hz). UV–vis kmax, nm (e, M-1 cm-1) in
DMSO: 265 (41,760), 345 (15,245), 378 (6,475). Magnetic
moment leff: 1.69 lB. XM (S m2 mol-1): 40.1.
Synthesis of complex 1
Complex 1 was obtained as reddish-brown powder by re-
fluxing a mixture of PAHN (0.145 g, 0.5 mmol) and 1,10-
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979
Synthesis of complex 4
(20 lM) as a function of temperature. The temperature was
scanned from 50 to 90 °C at a speed of 5 °C min-1
.
Complex 4 was synthesized by a procedure similar to that
used for complex 3, with 2,20-bipyridine (0.0781 g,
0.5 mmol) in place of 1,10-phenanthroline. Yield:
0.1053 g, 45.6 %. Anal. Calcd for C23H17N5O3V (%): C,
59.75; H, 3.71; N, 15.15; O, 10.38. Found (%): C, 59.73; H,
3.41; N, 15.13; O, 10.23. ESI-MS (CH3OH, m/z): 463.0
([M ? H]?). IR (KBr) (mmax, cm-1): 1,610 (s) (C=N),
Cytotoxicity assay
The cytotoxicity of the oxovanadium(IV) complexes was
tested in SH-SY5Y, MCF-7, and SK-N-SH human cancer
cell lines using MTT assay. The compounds were first
dissolved in DMSO (less than 0.1 %) and were then diluted
with RPMI 1640 to the required concentrations prior to use.
The control was prepared by addition of culture medium
(100 lL). Wells containing culture medium without cells
were used as blanks. SH-SY5Y, MCF-7, and SK-N-SH
cells at a density 2 9 104 cells per well were precultured in
96-well microtiter plates for 48 h at 37°C, 5 % CO2. On
completion of the incubation, stock MTT dye solution was
added to each well. After 4 h incubation, a solution con-
taining DMF (50 %) and sodium dodecyl sulfate (20 %)
was added to solubilize the MTT formazan. The cell via-
bility 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 per-
centage viability versus concentration on a logarithmic
graph and reading off the concentration at which 50 % of
cells remain viable relative to the control cells (0.5 %
DMSO) [28, 29, 41–43]. Cultures were duplicated for each
experimental point in at least three independent experi-
ments. The same batches of culture medium, sera, and
reagents were used throughout the study.
1
1,589 (s) (C=O), 955 (s) (V–O), 699 (s) (V–N). H-NMR
(500 MHz, DMSO-d6, ppm): 9.25 (s, 1H, CH=N), 8.69 (s,
3H, ArH), 8.40 (br, 4H, ArH, J = 8.4 Hz), 7.96 (m, 3H,
ArH, J = 8.2 Hz), 7.46 (s, 2H, ArH, J = 8.0 Hz), 6.94 (br,
2H, ArH, J = 7.8 Hz), 6.79 (m, 2H, ArH, J = 8.2 Hz).
UV–vis kmax, nm (e, M-1 cm-1) in DMSO: 265 (41,770),
345 (15,258), 380 (6,455). Magnetic moment leff: 1.73 lB.
XM (S m2 mol-1): 37.9.
DNA binding
Absorption titration experiments were performed with
fixed concentrations of the complexes (20 lM), while
gradually increasing the concentration of CT-DNA.
Vanadium–DNA solutions were incubated for 5 min before
the absorption spectra were recorded. To compare the
binding strength of the complexes, their intrinsic binding
constants (Kb) were determined by monitoring the changes
in absorbance in the ligand transfer band with increasing
concentration of CT-DNA. Kb was then calculated using
the following equation [35–40]:
Cell cycle analysis
½DNAꢁ ½DNAꢁ
1
¼
þ
ð1Þ
The cell cycle of control and treated cancer cells was
analyzed. With use of standard methods, the DNA of cells
was stained with PI, and the proportions of nonapoptotic
cells in different phases of the cell cycle were recorded [28,
44–46]. The cancer cells were treated with the complexes,
harvested by centrifugation at 1,000g for 5 min, and then
washed with ice-cold phosphate buffer solution. The cells
collected were fixed overnight with cold 70 % ethanol, and
were then stained with PI solution consisting of
50 lg mL-1 PI and 10 lg mL-1 RNase. After 10 min
incubation at room temperature in the dark, fluorescence-
activated cells were sorted in a FACScan flow cytometer
using the Cell Quest 3.0.1 software program. The per-
centage of cells in each phase of the cell cycle was
determined at least in triplicate and was expressed as the
mean the standard deviation.
ea ꢂ ef eb ꢂ ef Kbðeb ꢂ efÞ
where [DNA] is the concentration of DNA in base pairs, ea
is the extinction coefficient observed for Aobs/[V], eb is the
extinction coefficient of the complex when fully bound to
DNA, and ef is the extinction coefficient of the complex
free in solution.
Viscosity measurements were performed with a Ub-
belohbe viscometer maintained at a constant temperature of
(28 0.1) °C in a thermostatic bath. A digital stopwatch
was used to measure the flow time, and each sample was
measured five times to obtain the average flow time. Data
are presented as (g/g0)1/3 versus the binding ratio, where g
is the viscosity of DNA in the presence of the complexes
and g0 is the viscosity of DNA alone [29, 30].
Thermal denaturation studies were performed with a
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 lM) in
the absence and presence of the oxovanadium complex
Fluorescence microscopy of apoptosis assays
The apoptosis assay method was modified from that in a
previous report [28, 47–50]. Briefly, after exposure to the
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oxovanadium complexes for 48 h, the cancer cells were
washed twice with phosphate buffer solution, and were
then stained with 10 lg mL-1 Hoechst 33342 staining
solution at 37 °C for 30 min according to the manufac-
turer’s instructions. Finally, the cells were observed under
a fluorescence microscope.
The electronic spectra of the oxovanadium(IV) com-
plexes and the ligands were reported in ‘‘Materials and
methods.’’ As for the ESI–MS spectra, the Schiff base
ligands, PAHN and PAH, showed peaks at m/z 292.0
([M ? H]?) and 242.0 ([M ? H]?), respectively. The
oxovanadium(IV) complexes were found at m/z 536.0
([M ? H]?) (1), 512.0 ([M ? H]?) (2), 486.0 ([M ? H]?)
(3), and 462.0 ([M ? H]?) (4), respectively.
Scavenging of the hydroxyl radical
The molar conductivities of complexes 1–4 were
recorded in DMF and fall in the range 28–37 S m2 mol-1
(Table S2), which is low for them to be regarded as non-
electrolytic [28, 29, 32]. The magnetic moments
(1.68–1.75 lB) were obtained at room temperature
(Table S2). These values are close to the reported values
for a square-pyramidal geometry [28–34]. The elemental
analysis, UV–vis spectroscopy, ESI–MS, IR spectroscopy,
1H-NMR spectroscopy, and 13C-NMR spectroscopy results
for all the compounds are in agreement with the proposed
structures shown in Scheme 1.
The hydroxyl radical in aqueous media was prepared by the
Fenton reaction [51–54]. Solutions of the test complexes
were prepared with DMF. The 4 mL of the assay mixture
contained the following reagents: the test compounds
(2.0–5.0 lM), safranin (28.5 lM), EDTA-Fe(II) (100 lM),
H2O2 (44.0 lM), and a phosphate buffer (67 mM, pH 7.4).
The absorbance of the assay mixture was measured at
520 nm after incubation at 37 °C for 15 min in a water
bath. All the tests were performed in triplicate and the
results were expressed as the mean. The scavenging ratio
was calculated on the basis of (Ai - A0)/(Ac -
A0) 9 100 %, where Ai is the absorbance in the presence of
the test complex, A0 is the absorbance in the absence of the
test complex, and Ac is the absorbance in the absence of the
test complex and EDTA-Fe(II).
In addition, no change was observed in the UV–vis
absorption spectra of all the compounds after the solutions
had been diluted with water, indicating that the compounds
are stable in water.
DNA-binding studies
Results and discussion
Absorption titrations and fluorescence spectroscopy studies
Synthesis and characterization
Absorption titration in the UV–vis range was used to
investigate the interaction of the four oxovanadium(IV)
complexes with CT-DNA. The interaction of the com-
plexes with the base pairs of DNA is accompanied by
hypochromism and bathochromism [29, 35], which is
mainly attribute to the stacking interaction between the
planar aromatic chromophore of the complexes and the
base pairs of DNA. For metal interaction, DNA binding is
associated with hypochromism and a redshift in the metal-
to-ligand charge transfer and ligand bands [35–37].
The ligands PAHN and PAH were easily synthesized in
high yield by refluxing a mixture of isonicotinic acid
hydrazide with 2-hydroxy-1-naphthaldehyde and salicyl-
aldehyde, respectively, in absolute methanol. Complexes
1–4 were prepared by refluxing VO(acac)2 with 1,10-
phenanthroline and 2,20-bipyridine in absolute methanol.
The synthesis routes of the complexes and ligands are
shown in Scheme 1. All complexes and ligands were
characterized by elemental analysis, ESI–MS, IR, UV–vis
spectroscopy, 1H-NMR spectroscopy, and 13C-NMR
spectroscopy.
In the electronic spectra, appreciable hypochromism and
bathochromism can be observed for the four complexes
with increasing amounts of DNA. The specific hypochro-
mism and bathochromism of the four complexes were
24 % and 2 nm (1), 12 % and 2 nm (2), 10.5 % and 2 nm
(3), and 8.7 % and 1 nm (4), respectively (Fig. S1).
According to previously reported results [34–39], the UV–
vis spectral characteristics suggest that complexes 1–4
interact with DNA most likely through a mode that
involves a stacking interaction between the aromatic
chromophore and the base pairs of DNA. The intrinsic
binding constant, Kb, decreases in the order 1 [ 2 [ 3 [ 4.
The differences in their binding strength may be due to the
presence of an appending aromatic moiety in PAHN, and
this explains the larger binding affinities of the
The IR spectra of the free forms of the ligands were
compared with the spectra of the vanadium complexes
(Table S1). A signal for V–N stretch was observed for
complexes 1–4 at 702, 722, 727, and 699 cm-1, respec-
tively, and m(V–O) stretching for the complexes occurred at
approximately 955 cm-1 as reported for other oxovana-
dium derivatives [14, 17, 23–25].
1
The H-NMR and 13C-NMR spectral data of the free
forms of the ligands recorded in DMSO-d6 and the possible
assignments were reported in ‘‘Materials and methods.’’
All the protons due to heteroaromatic groups and the car-
bons were found in the expected regions [24, 28].
123

J Biol Inorg Chem (2013) 18:975–984
981
corresponding complexes in comparison with those of
complexes incorporating PAH [33, 38, 44]. The results also
imply that the interactions of these complexes with DNA
may be mainly through the PAHN and PAH plane and base
pairs, and show a classic intercalative mode. Furthermore,
the electronic effect of 1,10-phenanthroline or 2,20-bipyri-
dine is one of the factors determining the binding affinities.
Probably, the absence of substituents on the phenol ring
may improve the intercalation ability of the vanadyl
complex. The DNA-binding affinity (Kb value) was slightly
smaller than for VO(SAA)(phen),VO(MOSAA)(phen), and
other previously reported complexes [29–32]. The different
results might be due to the different electrochemical per-
formances and the influence of different ligand systems
[29, 33, 38, 44].
effective length [25, 28, 29]. Therefore, viscosity mea-
surements were performed to further clarify the intercala-
tion mode with CT-DNA. When the amounts of all four
complexes were increased, the viscosity of CT-DNA
increased steadily (Fig. S3), and this is similar to the
behavior of ethidium bromide, a well-known DNA inter-
calator [29, 30, 34–36]. Here, the viscosity measurements
suggest that these complexes may bind to CT-DNA
through intercalation, in agreement with the UV–vis and
fluorescence spectroscopy studies. The experimental vis-
cosity results thus provide strong evidence for the inter-
action of complexes 1–4 with DNA by intercalation modes
and the electronic effect of 1,10-phenanthroline or 2,20-
bipyridine may be an important factor in determining the
intrinsic binding constant Kb [33, 38, 44–46].
To compare quantitatively the DNA-binding strengths
of these complexes, the intrinsic binding constant Kb was
calculated from the changes in absorbance in the ligand
charge transfer bands with increasing amounts of CT-
DNA. The values of Kb were calculated using Eq. 1 to be
(2.70 0.10) 9 104, (2.10 0.10) 9 104, (1.60 0.10)
9 103, and (1.18 0.10) 9 104 M-1 for complexes 1, 2,
3, and 4, respectively. These Kb values are in the range
observed for previously reported complexes [23].
Thermal denaturation studies provide an additional way
to detect intercalation modes [31, 37–39]. The melting
temperature (Tm) is the temperature at which the double-
stranded DNA dissociates into single strands, and is
determined by the hyperchromic effect on the absorption of
DNA base pairs (k = 260 nm). Tm of CT-DNA in the
absence of the complexes is 60.7 0.2 °C. The observed
Tm values in the presence of complexes 1–4 were
65.5 0.2, 64.2 0.1, 62.6 0.1, and 62.0 0.1 °C,
and DTm was 4.8, 3.5, 1.9, and 1.3 °C, respectively. The
Tm values of these complexes are obviously increased
compared with Tm of CT-DNA in the absence of the
complexes (Fig. S4). Furthermore, complex 1 has a larger
DNA-binding affinity than complexes 2–4.
Fluorescence spectroscopy was used to study interac-
tions of the complexes with CT-DNA in Tris buffer A at
room temperature [29–31]. With increasing concentrations
of CT-DNA, the emission intensities of complexes 1–4
increase by about 1.40 0.01, 1.38 0.01, 1.25 0.01,
and 1.13 0.01 times at about 700 nm, respectively, in
comparison with those of the complexes alone (Fig. S2).
On the other hand, the enhancement of emission intensity
is indicative of binding of the complexes to the hydro-
phobic pocket of DNA, since the hydrophobic environment
inside the DNA helix reduces the accessibility of water
molecules to the complex and the mobility of the complex
is restricted at the binding site, leading to a decrease of the
vibrational modes of relaxation, thus lengthening the
luminescence lifetimes and increasing emission intensity
[33, 38, 44–46]. This observation is consistent with the
results of the UV–vis experiments.
Cytotoxicity assays
The antitumor activity of all the complexes and ligands
against three human-derived cell lines (SH-SY5Y, MCF-7,
and SK-N-SH) was evaluated by MTT assays. The IC50
values were calculated after 48 h incubation with isoniazid
ligands, the four complexes, and cisplatin in different
concentrations, respectively. As shown in Table 1, these
oxovanadium complexes exhibit broad inhibition of the
three human cancer cell lines tested, with IC50 values
ranging from 1.21 0.13 to 42.2 4.14 lM, and the
antitumor activities are concentration-dependent. Complex
1 possessed the most potent inhibitory effect on the two
cell lines. This is consistent with its ability to bind with
CT-DNA, indicating that the antitumor properties of the
oxovanadium complexes may be closely related to their
DNA-binding mode. Previously, we reported that the
antiproliferative activity of other oxovanadium(IV) com-
plexes was most likely associated with their DNA-binding
abilities [29]. The present results confirmed our previous
observations that the antitumor activity of oxovana-
dium(IV) complexes is associated with their binding abil-
ities and mode of intercalation with DNA.
Viscosity measurements and thermal denaturation studies
Hydrodynamics (viscosity) experiments are useful to detect
intercalation between small molecules and DNA in the
absence of crystallographic structural data. When a com-
plex binds to DNA by intercalation, the base pairs are
separated to accommodate the binding ligand, resulting in
lengthening of the DNA helix, and so increasing the vis-
cosity of the DNA solution [35–37], in contrast, partial
and/or nonclassic intercalation of a ligand may cause a
bend or twist in the DNA helix, leading to a decrease of its
123

982
J Biol Inorg Chem (2013) 18:975–984
exposed to 0.4 lM complex 1 for 48 h (Table S3). Treat-
ment with 0.1 or 0.2 lM complex 1 resulted in modest
phase arrest of SK-N-SH cells at the 48-h time point. There
was a significantly increased G0/G1 phase distribution and
a significantly decreased G2/M phase distribution in a dose-
dependent manner, indicating the induction of G2/M phase
arrest by complex 1. Moreover, the number of apoptotic
cells and the amount of cell debris significantly increased
after SK-N-SH cells had been exposed to complex 1. The
results of the cell cycle analysis in this work suggested that
the oxovanadium complexes induced proliferative sup-
pression of SK-N-SH cells via the induction of apoptosis
[47, 49–51].
Table 1 Antiproliferative effects of complexes 1–4 (see Scheme 1
for the structures), cisplatin, and ligands on different cells lines after
48 h treatment
Compounds
IC50 (lM)
SH-SY5Y
MCF-7
SK-N-SH
Cisplatin
7.72 1.34
75.2 1.24
82.3 1.28
43.61 1.54
3.95 0.14
6.90 0.38
8.86 0.31
42.2 1.04
13.3 1.32
54.0 1.12
32.4 1.04
[100
2.95 0.14
47.3 1.19
79.5 1.27
20.78 1.54
1.21 0.13
4.10 0.18
2.40 0.34
24.1 0.25
PAHN
PAH
VO(acac2)
1
2
3
4
1.97 0.21
11.5 0.94
26.5 0.62
17.4 0.45
Induction of apoptosis as evidenced by Hoechst 33342
staining
Data are expressed as the 50 % inhibitory concentration (IC50). Cells
were treated with various concentrations of the test compounds for
48 h. Cell viability was determined by 3-(4,5-dimethylthiazoyl-2-yl)-
2,5-diphenyltetrazolium bromide assay and IC50 values were calcu-
lated as described in ‘‘Materials and methods.’’ Each value represents
the mean the standard deviation of three independent experiments
Apoptosis is an important continuous process of destruc-
tion of undesirable cells during the development or
homeostasis in multicellular organisms. This process is
characterized by distinct morphological change,s including
membrane blebbing, cell shrinkage, dissipation of the
mitochondrial membrane potential, chromatin condensa-
tion, and DNA fragmentation [47, 48].
PAHN 4-pyridinecarboxylic acid, 2-[(2-hydroxy)-1-naphthalenylene]
hydrazide, PAH 4-pyridinecarboxylic acid, 2-[(2-hydroxy)-1-
phenyl]methylene hydrazide, acac acetylacetonate
Interestingly, the IC50 values of complex 1 were com-
parably lower than those of cisplatin under the same con-
ditions. From a comparison of the antitumor activities of
their ligands and VO(acac)2, the four complexes appeared
to be more cytotoxic to the SH-SY5Y, MCF-7, and SK-N-
SH cell lines. Usually, the pharmacological activity of these
types of molecules is often enhanced by complexation with
metal ions [33, 45–49]. Among the cell lines under evalu-
ation, the SK-N-SH cells proved to be the most sensitive to
treatment with the oxovanadium(IV) complexes.
In an attempt to elucidate whether the G2/M phase arrest
in the SK-N-SH cells induced by complex 1 was associated
with apoptosis, we identified the occurrence apoptosis by
Hoechst 33342 staining [50–52]. Figure 1 shows repre-
sentative Hoechst 33258 fluorescence photomicrographs of
cultured SK-N-SH cells treated with or without the oxo-
vanadium(IV) complexes. In control cultures (Fig. 1a),
nuclei of SK-N-SH cells appeared with regular contours
and were round and large. By contrast, the condensation of
nuclei characteristic of apoptotic cells was evident in SK-
N-SH cells treated with 60 or 120 lM complex 1 for 24 h
(Fig. 1c, d). Most nuclei of Jurkat cells treated with com-
plex 1 appeared hypercondensed (brightly stained), and the
typical apoptotic bodies were observed, which was differ-
ent from what was observed in the control cells (Fig. 1c, d).
The results indicated that the G2/M phase arrest in the SK-
N-SH cells induced by complex 1 was associated with
apoptosis and that the activities are concentration-depen-
dent (Fig. 1b). This may imply that these oxovanadium
complexes can cause proliferative suppression of cancer
cells via the induction of apoptosis [47–50]. To fully
understand the mechanism involved in the induction of
apoptosis by the oxovanadium complexes, further investi-
gations will be needed to be conducted.
The micrographs of the SH-SY5Y cell line after treat-
ment for 48 h in the absence of the complexes and in the
presence of the different complexes show that the prolif-
eration of SH-SY5Y tumor cells was effectively inhibited
(Fig. S5).
Cell cycle analysis
To further define the mechanism of the antiproliferative
effect of the oxovanadium(IV) complexes on tumor cells,
the cell cycle phase distribution was analyzed by flow
cytometry with PI staining [48–51]. According to the
results in Table 1, SK-N-SH cells exhibited the highest
sensitivity to complex 1. Thus, this cell line was used for
further investigation of the underlying mechanisms
accounting for the antiproliferative action of complex 1.
SK-N-SH cells were treated with 0.1, 0.2, and 0.4 lM
complex 1, respectively, for 48 h. The results of the cell
cycle analysis in this work showed that the G2/M phase was
arrested significantly after SK-N-SH cells had been
Scavenging of the hydroxyl radical
It is well known that as one of the most reactive products of
reactive oxygen species, the hydroxyl radical could cause
123

J Biol Inorg Chem (2013) 18:975–984
983
Fig. 1 Apoptosis of SK-N-SH
cells induced by complex 1 (see
Scheme 1 for the structure). SK-
N-SH cells were incubated with
complex 1 for 24 h and stained
by Hoechst 33342. Almost all
cells in the control group were
normal. However, apoptotic
cells appeared after 24 h
treatment with complex 1. The
arrows indicate apoptotic cells
identified by the Hoechst 33342
staining. a The blank control
group. b Cells with 30 lM
complex 1. c Cells treated with
60 lM complex 1. d Cells
treated with 120 lM complex 1
complexes reached its peak at 4.0 lM, and then decreased
gradually. To the best of our knowledge, there is no
information available on the scavenging of the hydroxyl
radical by oxovanadium(IV) complexes and/or their scav-
enging capacity [53–55]. It is well known that antioxidants
repress cancer metastasis by scavenging reactive oxygen
species [53, 56, 57], and so the above results may help
understand the mechanism involved in scavenging by
oxovanadium complexes in antitumor cells. The mecha-
nism involved in the scavenging of the hydroxyl radical by
oxovanadium complexes is under further investigation in
our laboratory.
Conclusion
Fig. 2 Scavenging effect of the complexes on hydroxyl radicals (see
Scheme 1 for the structures of the complexes 1–4)
The results reported in this work provide more data related
to the interaction of oxovanadium(IV) complexes with
DNA. The in vitro results provide experimental evidence
that oxovanadium(IV) complexes have the potential to be
anticancer complexes against human cancer cells. Under
certain conditions, these oxovanadium(IV) complexes can
scavenge the free radical hydroxyl, a topic that has been
only scarcely investigated. Thus, the results obtained from
the present oxovanadium(IV) complexes are of importance
for the development of metal-based agents for anticancer
applications. Further work is in progress to better identify
the mechanism of action and to prepare more potent and
stabler related oxovanadium compounds for the treatment
of cancer.
cell membrane disintegration, membrane protein damage,
and DNA mutation and further initiate or propagate the
development of many diseases [53–55]. Since complexes
1–4 all showed good ability to cleave DNA, it was con-
sidered necessary to investigate their scavenging effect on
the hydroxy radical. Rutin, which is known to be an
effective antioxidant agent [56, 57], was used as a positive
control, and the results are shown in Fig. 2.
As shown in Fig. 2, the scavenging effect of complexes
1–4 was concentration-dependent and the scavenging ratio
increased on increasing of the concentration of the com-
plexes. The scavenging ratio of the oxovanadium(IV)
123

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J Biol Inorg Chem (2013) 18:975–984
Acknowledgments We gratefully acknowledge financial support
for this work by the Science and Technology Research Project of
Guangdong province (no. 2012B031800431), People‘s Republic of
China, and the National Natural Science Foundation of the People‘s
Republic of China (no. 81102753 and no. 21101033).
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Conflict of interest The authors declare that there is no conflict of
interest.
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