DNA interactions, antitumor activities and radical scavenging properties of oxovanadium complexes with pyrazino[2,3-f][1,10]phenanthroline ligands

Article abstract of DOI:10.1007/s11243-015-9939-9

Two unsymmetrical oxovanadium complexes [VO(satsc)(dpq)] (1) (satsc = salicylaldehyde thiosemicarbazone, dpq = pyrazino[2,3-f][1,10]phenanthroline) and [VO(hntdtsc)(dpq)] (2) (hntdtsc = 2-hydroxy-1-naphthaldehyde thiosemicarbazone) have been synthesized and characterized. Both complexes show strong interactions with calf thymus DNA through an intercalative model and can efficiently cleave pBR322 DNA after irradiation with light. Both complexes also exhibit high cytotoxicities against SH-SY5Y, MCF-7 and SK-N-SH cell lines. Complex 2 was found to have higher antitumor potency, DNA-binding affinity and DNA-cleaving ability than complex 1. The abilities of these complexes to scavenge hydroxy radicals were evaluated.

Full text of DOI:10.1007/s11243-015-9939-9

Transition Met Chem
DOI 10.1007/s11243-015-9939-9
DNA interactions, antitumor activities and radical scavenging
properties of oxovanadium complexes with pyrazino[2,3-f]
[1,10]phenanthroline ligands
Pengfei Zeng1 Limin He2 Zhenfeng Ye1 Ning Yang3 Yulan Song3
Jiazheng Lu1
•
•
•
•
•
Received: 5 February 2015 / Accepted: 7 April 2015
Ó Springer International Publishing Switzerland 2015
Abstract Two unsymmetrical oxovanadium complexes
[VO(satsc)(dpq)] (1) (satsc = salicylaldehyde thiosemi-
various biological and physiological effects in the human
body [3, 5–7]. Therefore, vanadium complexes have been
investigated as drugs and found to have antitumor,
antimicrobial and insulin-enhancing properties [4–6].
Many vanadium complexes are able to suppress the growth
and spread of existing tumors by inhibiting tumor cell
proliferation, inducing apoptosis and limiting the invasion
and metastatic potential of neoplastic cells [7, 8]. For this
reason, the DNA-binding and antitumor activities of oxo-
vanadium complexes with multifunctional bridging ligands
have been investigated [9, 10]. Among these, Schiff base
complexes have exhibited significant activities with respect
to DNA binding and cleavage [9, 11–14]. Schiff base
ligands play an important role in the DNA-binding char-
acteristics of their associated complexes, because of their
electronic effects [15, 16]. In particular, a rigid aromatic
system in the Schiff base structure can give better inter-
actions with DNA, which is an important consideration in
the design of new effective DNA-binding complexes [17].
Previously, we have studied the interactions of Schiff base
oxovanadium complexes with CT-DNA and also their
cytotoxicities against myeloma cells (Ag8.653) and glio-
mas cells (U251) assessed by MTT assay. We found that
Schiff base ligands with larger aromatic or electron-with-
drawing groups generally have better DNA-binding and
antitumor activities [17–19].
carbazone,
dpq = pyrazino[2,3-f][1,10]phenanthroline)
and [VO(hntdtsc)(dpq)] (2) (hntdtsc = 2-hydroxy-1-naph-
thaldehyde thiosemicarbazone) have been synthesized and
characterized. Both complexes show strong interactions
with calf thymus DNA through an intercalative model and
can efficiently cleave pBR322 DNA after irradiation with
light. Both complexes also exhibit high cytotoxicities
against SH-SY5Y, MCF-7 and SK-N-SH cell lines. Com-
plex 2 was found to have higher antitumor potency, DNA-
binding affinity and DNA-cleaving ability than complex 1.
The abilities of these complexes to scavenge hydroxy
radicals were evaluated.
Introduction
A number of transition metal complexes have been shown
to have the ability to bind and cleave DNA, especially
those with polyaromatic ligands [1–4].Vanadium exhibits
& Limin He
he791225@163.com
& Jiazheng Lu
lujia6812@163.com
In continuation of our precious work, two new oxo-
vanadium complexes [VO(satsc)(dpq)] (1) (satsc = sali-
cylaldehyde thiosemicarbazone, dpq = Pyrazino[2,3-
f][1,10]phenanthroline) and [VO(hntdtsc)(dpq)] (2) (hnt-
dtsc = 2-rhydroxy-1-naphthaldehyde thiosemicarbazone)
have been synthesized and characterized by spectroscopic
and physicochemical methods. The interactions of these
complexes with CT-DNA have been investigated. Their
photocleavage reactions with pBR 322 supercoiled plasmid
1
School of Pharmacy, Guangdong Pharmaceutical University,
Guangzhou 510006, People’s Republic of China
2
Department of Pharmaceutical Engineering, School of
Pharmacy, Guangdong Pharmaceutical University,
Guangzhou 510060, People’s Republic of China
3
Department of Clinical Pathology, First Affiliated Hospital,
Guangdong Pharmaceutical University, Guangzhou 510060,
People’s Republic of China
123

Transition Met Chem
DNA were also investigated, and the cytotoxicities toward
human breast cancer line (MCF-7) and human neuroblas-
toma cells (SH-SY5Y and SK-N-SH) were investigated by
MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-te-
trazolium bromide) assay. In addition, their hydroxyl rad-
ical scavenging properties were investigated.
recorded on a PerkinElmer Lambda 55 spectra fluoropho-
tometer. Cell assays were performed with a microplate
reader (model 680, Bio-Rad, USA).
Synthesis of satsc
satsc was synthesized through a method similar to that
described earlier. [17] A stirring solution of salicylalde-
hyde (1.2212 g, 10 mM) in 10 mL of absolute EtOH was
added dropwise to a solution of thiosemicarbazide (0.9114,
10 mM) in absolute EtOH (15 mL). The mixture was
continuously stirred at 80 °C for 3 h to give a white pre-
cipitate, which was used without further purification.
Yield: 56 %. Anal. Calcd. For C8H9N3OS: C, 49.2; H, 4.6;
N, 21.5; S, 16.4 %; Found: C, 49.1; H, 4.7; N, 21.5; S,
16.4 %. ES-MS (CH3OH, m/z): 196.0 (100 %) ([M?1]?).
IR (KBr) (mmax/cm): 3444 (vs), 3321 (vs, –NH2), 3175(vs,
N–H), 3033 (m, C–H), 1617 (vs, C=N), 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).
Experimental
Materials and methods
All reagents and solvents were of AR grade and purchased
commercially. VO(acac)2 (acac = acetylacetonate) and
1,10-phenanthroline were used as purchased (AR grade).
CT-DNA and pBR322 supercoiled plasmid DNA were
obtained from Sigma Company (Guangzhou, China).
Human breast cancer line (MCF-7) and human D cells
(SH-SY5Y and SK-N-SH) were purchased from the Cell
Bank of Type Culture Collection of the Chinese Academy
of Sciences (Shanghai, China). MTT was purchased from
Sigma. Tris buffer A (Tris = tris(hydroxylmethyl)aminom
ethane) containing 5 mM Tris–HCl and 50 mM NaCl (pH
7.2) was used for absorption titration, fluorescence emis-
sion and viscosity experiments. Tris buffer B containing
50 mM Tris–HCl and 18 mM NaCl (pH 7.2) was used for
the gel electrophoresis experiments. Buffer C (1.5 mM
NaHPO4, 0.5 mM NaH2PO4 and 0.25 mM Na2H2 EDTA
[N,N’-1,2-ethanediylbis[N-(carboxymethyl)]glycine) (pH
7.0)] was used for thermal denaturation studies. All of the
above buffer solutions were prepared using double-distilled
water. A solution of CT-DNA in buffer A gave a ratio of
UV absorbance at 260 and 280 nm of 1.8–1.9:1, indicating
that the DNA was sufficiently free of protein [20, 21]. The
DNA concentration per nucleotide was determined by
absorption spectroscopy using the molar absorption coef-
ficient (6600 M-1cm-1) at 260 nm [20, 22]. DNA stock
solutions were stored at 4 °C and employed after no more
than 3 days. Compounds were dissolved in dimethyl sul-
foxide (DMSO) and diluted with buffer solution to the
required concentrations prior to use. Solutions of com-
pounds were freshly prepared 2 h prior to biochemical
evaluation.
Synthesis of hntdtsc
This compound was prepared using a similar procedure to
that described for satsc, using 2-hydroxy-1-naphthaldehyde
(1.7218 g, 10 mmol) in place of salicylaldehyde to give a
light yellow precipitate, which was used without further
purification. Yield: 84 %. Anal. Calcd. For C12H11N3OS:
C, 58.8; H, 4.5; N, 17.1; S, 13.1 %; Found: C, 58.7; H, 4.6;
N, 17.1; S, 13.0 %. ES-MS (CH3OH, m/z): 246.0 (100 %)
([M?1]?). IR (KBr) (mmax/cm-1): 3450 (vs), 3253 (vs, –
NH2), 3167 (vs, N–H), 3053 (m, C–H), 1625 (s, C=N),
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).
Microanalyses (C, H and N) were obtained using a
PerkinElmer 240Q elemental analyzer. Electrospray mass
spectra (ES-MS) were recorded on a LCQ system (Finni-
gan MAT, USA) using methanol as mobile phase. 1H NMR
spectra were recorded on a Varian-500 spectrometer. All
chemical shifts are given relative to tetramethylsilane
(TMS). Infrared spectra were recorded on a Bomem FTIR
model MB102 spectrometer using KBr pellets. UV–Vis
spectra were recorded on a Shimadzu UV-3101 PC spec-
trophotometer at room temperature. Emission spectra were
Synthesis of dpq
dpq was synthesized through a modification of a reported
procedure [23]. To a solution of 1,10-phenanthroline-5,6-
dione in water was added 1,2-ethanediamine, and the
resulting suspension was refluxed for 12 h at 60 °C. The
product was collected and washed with water (10 mL) and
the minimum volume of diethyl ether. Yield: 68.7 %. Anal.
123

Transition Met Chem
Calcd. For C14H8N4: C, 70.4; H, 3.5; N, 24.1 %; Found: C,
70.4 H, 3.5; N, 24.1 %. ESI–MS (CH3OH, m/z):
233.0([M?1]?). IR (KBr) (mmax/cm-1):3000(m, =CH),
1581(m, C=N), 1571(m), 1519(m), 1467(s, C–H), 1390
(vs), 1078 (s), 804 (s), 740 (vs). 1H NMR (500 MHz,
DMSO-d6): 9.46 (dd, 2H, J = 10.25, 2.25), 9.24 (dd, 2H,
J = 5.375, 2.25), 9.19 (s, 2H), 7.96 (dd, 2H, J = 10.25,
5.375).
oxovanadium complex (20 lM) to which the DNA stock
solution was added. The resulting mixtures were allowed to
incubate for 5 min before the absorption spectra were
recorded. In order to further elucidate the binding strength
of the complexes, values of the intrinsic binding constant
Kb with CT-DNA were obtained by monitoring the change
in the absorbance of the ligand transfer band with
increasing amounts of DNA. The data were then fitted to
the following equation to obtain the value of Kb [17–20].
Synthesis of complex 1
½DNAŠ ½DNAŠ
1
¼
þ
ea À ef eb À ef Kbðeb À efÞ
A mixture of satsc (0.0975 g, 0.5 mmol) and dpq (0.116 g,
0.5 mmol) in absolute MeOH (15 mL) was refluxed under
where [DNA] is the concentration of DNA in the base pairs
and ea, ef and eb refer to the corresponding apparent
absorption coefficient Aobsd/[vanadium], the extinction
coefficient for the free oxovanadium complex and the
extinction coefficient for the oxovanadium complex in the
fully bound form, respectively. Plots of [DNA]/(ea - ef)
versus [DNA] yielded values of Kb by the ratio of the slope
to the intercept.
argon. After dissolution,
a solution of VO(acac)2
(0.1325 g, 0.5 mmol) in MeOH (10 mL) was added drop-
wise to the refluxing solution. A brown precipitate was
formed after refluxing for another 4 h. The precipitate was
collected by filtration, washed with absolute methanol and
then dried in vacuo. Yield: 82.6 %. The crude product was
purified by recrystallization such that the material was
dissolved in hot MeOH and cooled to room temperature;
whereupon the pure product precipitated out. Anal. Calcd.
For C22H16N7O2SV: C, 53.6; H, 3.3; N, 19.9; O, 6.5; S, 6.5;
V, 10.3 %; Found: C, 53.6 H, 3.3; N, 19.9; O, 6.5; S, 6.5;
V, 10.4 %. ES-MS (CH3OH, m/z): 493.05 (100 %)
([M?1]?). IR (KBr) (mmax/cm-1): 3436 (s), 3290 (s, –
NH2), 3113 (m, C-H), 1601 (vs, C=N), 1540 (s), 1507 (s),
1469 (s), 1207 (m, C–O), 1151 (m, C–N), 940 (s, VO), 815
Viscosity and thermal denaturation experiments
The viscosity experiments were conducted using an
Ubbelohde viscometer, immersed in a thermostatic water
bath maintained at a constant temperature (28.0 0.1) °C.
A digital stopwatch was used for measurement of flow
times, and each sample was measured five times to obtain
the average flow time. Data are presented as (g/g0)1/3
versus binding ratio, where g is the viscosity of DNA in the
presence of the complex, while g0 is the viscosity of DNA
alone [17, 25, 26].
1
(m), 734 (m, C–S), 599 (m). H NMR (500 MHz, DMSO-
d6): 9.03 (s, 1H, CH=N), 7.82 (br, m, 3H), 7.53–7.35 (br m,
6H), 7.20 (2H, NH2).
DNA-melting experiments were done by monitoring the
absorption intensity of CT-DNA (80 lM) at 260 nm at
various temperatures, both in the absence and in the pres-
ence of the complexes (10 lM). Measurements were taken
using a Shimadzu UV-3101 PC spectrophotometer attached
to a Peltier temperature controller by increasing the tem-
perature of the solution by 5 °C per min from 50 to 90 °C.
Synthesis of complex 2
Complex 2 was synthesized by a similar procedure as for
the complex 1, using hntdtsc (0.123 g, 0.5 mmol) in place
of satsc. The precipitate was reddish brown. Yield: 81.2 %.
The recrystallization procedure was same as for complex 1.
Anal. Calcd. For C26H18N7O2SV: C, 57.46; H, 3.34; N,
18.04; O, 5.89; S, 5.90; V, 9.37; Found: C, 57.48 H, 3.36;
N, 18.07; O, 5.89; S, 5.88; V, 9.39 %. ES-MS (CH3OH, m/
z): 543.07 (100 %) ([M?1]?). IR (KBr) (mmax/cm-1): 3332
(s, NH2), 3193 (m), 2963 (m, C–H), 1614 (s, C=N), 1597
(s), 1537 (vs), 1499 (s), 1193 (m, C–O), 1085 (m, C–N),
DNA photocleavage studies
The DNA cleavage activities of the complexes were
assayed by agarose gel electrophoresis. Supercoiled plas-
mid pBR322 DNA (0.1 lg) was treated with the required
complex in buffer B, and the solution was incubated at
37 °C. The samples were analyzed by electrophoresis for
1.5 h at 81 V on 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 ethidium bromide and photographed on an
Alpha Innotech IS-5500 fluorescence chemiluminescence
and visible imaging system. The efficiency of DNA
cleavage was measured by determining the ability of the
1
956 (s, VO), 811 (s), 732 (m, C–S). H NMR (500 MHz,
DMSO-d6): 9.33 (s, 1H, CH=N), 7.87 (br, m, 3H),
7.57–7.34 (m, 6H), 7.20 (2H, NH2).
DNA-binding studies
DNA-binding experiments were performed at room tem-
perature. Absorption titrations of the complexes in buffer A
were performed by using a fixed concentration of
123

Transition Met Chem
complex to form open circular or nicked circular DNA
from its supercoiled form [17, 20–24].
(ANOVA). Significance was accepted at a P value lower
than 0.05.
Cytotoxicity assays
Results and discussion
The abilities of the complexes to interfere with the growth
of SH-SY5Y, MCF-7 and SK-N-SH human cancer cell
lines were determined by the 3-(4,5-dimethylthiazol-2-yl)-
2,5 diphenyltetrazolium bromide (MTT) dye assay. Com-
pounds were dissolved in 0.1 % DMSO and diluted with
RPMI 1640 to the required concentrations prior to use [25–
30]. A control well was prepared by the 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 with a density 2 9 104 cells per well were
precultured into 96-well microtiter plates for 48 h at 37 °C
in a 5 % CO2 incubator. On completion of the incubation,
stock MTT dye solution was added to each well. After 4-h
incubation, a solution containing N,N-dimethylformamide
(50 %) and sodium dodecyl sulfate (20 %) in water 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 ASCENT microplate reader.
IC50 values were determined by plotting the percentage
viability versus concentration on a logarithmic graph and
reading off the concentration at which 50 % of cells
remained viable relative to the control [25, 29–32].
Synthesis and characterization
The free ligands satsc and hntdtsc were prepared by the
reaction of thiosemicarbazide with 2-hydroxy-1-naph-
thaldehyde and salicylaldehyde, respectively. Complexes 1
and 2 were prepared by refluxing a mixture of the required
ligand, VO(acac)2 and dpq in absolute methanol, and then
purified by recrystallization. The structures of the com-
plexes were deduced by elemental analysis, ES-MS, UV–
1
Vis, IR and H NMR spectroscopy. The syntheses of the
free ligands and their complexes are summarized in
Scheme 1.
The IR spectra of the free Schiff bases were compared
with the spectra of their oxovanadium complexes. The
frequency of absorption of the azomethine (–CH=N) group
was reduced by 16 and 11 cm-1 for complexes 1 and 2,
respectively. A signal for the V–S bond was observed for
complexes 1 and 2 at 733 and 732 cm-1, respectively, and
V–O stretching for the complexes occurred at 940 and
956 cm-1, respectively, as reported for other oxovanadium
1
complexes. The H NMR spectra of the free ligands were
recorded in DMSO-d6, and their tentative assignments are
given in the experimental section. All the proton and car-
bon signals were observed in the expected regions.
The electronic spectra of the oxovanadium complexes
and the free ligands are reported in the experimental sec-
tion. The ESI–MS spectra of the free Schiff bases (satsc
and hntdtsc) and dpq showed peaks at m/z 196 [M?H]?,
216.0 [M?H]? and 233.0 [M?H]?, respectively. The
oxovanadium complexes ([VO(satsc)(dpq)] and [VO(hnt-
dtsc)(dpq)]) gave peaks at m/z 493.0 [M?H]? and 543.0
[M?H]?, respectively.
Radical scavenger measurements
Hydroxyl radicals were prepared in aqueous media using
the Fenton system [33]. A solution of each complex was
prepared with DMF (N,N-dimethylformamide). Each 4 mL
aliquot of assay mixture contained the following reagents:
the test compound (2.0–5.0 lM), safranin (28.5 lM),
EDTA-Fe(II) (100 lM), H2O2 (44.0 lM) and 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 tests were run in triplicate
and are expressed as the mean. The absorbance in the
presence of the test complex Ai, the absorbance in the
absence of the complex A0 and the absorbance in the
absence of the test complex and EDTA-Fe(II) AC were
measured. The scavenging ratio (%) was calculated using
the following equation [34].
DNA-binding studies
Electronic absorption spectroscopy is one of the most
common ways to investigate the interaction of complexes
with DNA. The intercalation of complexes into the base
pairs of DNA usually results in hypochromism and bath-
ochromism (red shift) in the MCCT and ligand bonds,
which is correlated with the stacking interaction between
the planar aromatic chromophore of the complexes and the
base pairs of DNA. The extent of the hypochromism is
commonly consistent with the strength of the intercalative
binding [17–20]. Figure 1 shows the absorption spectra of
complexes 1 and 2 in the presence of increasing concen-
trations of DNA. Thus, upon increasing the CT-DNA
ðAi À A0Þ=ðAc À A0Þ Â 100 %
The data are expressed as the mean SD from these
independent experiments. Statistical analysis was per-
formed using SPSS 13.0 for Windows. Comparisons
between the two groups were performed using an unpaired
t test. Multiple comparisons between more than two groups
were performed by one-way analysis of variance
123

Transition Met Chem
These observations suggest that both oxovanadium com-
plexes interact with DNA, most likely through a stacking
mode between the aromatic chromophore and the base
pairs of DNA.
NH2
HN
OH
S
MeOH,50°C,3h
+
N
HN
OH
S
NH2
The intrinsic binding constants Kb were calculated by
monitoring the changes in absorbance of the ligand transfer
bands with increasing amounts of CT-DNA. The values of
Kb obtained for complexes 1 and 2 were 9.87 9 103 and
11.0 9 104 M-1, respectively. The differences in their
binding strengths may be due to the presence of an extra
aromatic moiety in hntdtsc, resulting in larger binding
affinities of the corresponding complexes compared to
those of satsc.
CHO
NH2
sctsc
N
N
N
N
N
N
O
O
N
V
N
VO(acac)2,MeOH,reflex
N
SH
HN
The interaction of the complexes with CT-DNA was
also studied using fluorescence emission titration experi-
ments in Tris buffer A at room temperature. The emission
spectra of the complexes in the absence and presence of
CT-DNA are shown in Fig. 2. The maximum emission
intensities were observed at 704 and 705 nm for complexes
1 and 2, respectively. With increasing concentrations of
CT-DNA, the emission intensities of complexes 1 and 2
increase by factors of about 1.36 and 1.24, respectively.
The emission intensities of complexes 1 and 2 grow to
about 13.3 and 12.2 % larger than those in the absence of
DNA. The results indicate that both complexes can inter-
calate into the base pairs of the DNA and so be protected
from the surrounding water molecules, thus lengthening
their luminescence lifetimes. Our experiments also showed
that there is a competition between ethidium bromide and
the complexes for DNA binding.
NH2
(1)
NH2
HN
OH
+
S
MeOH,50°C,3h
OH
N
HN
NH2
CHO
S
NH2
(hntdtsc)
N
N
N
N
N
N
O
O
N
V
N
VO(acac)2,MeOH,reflex
N
SH
HN
NH2
Next, the interactions between the complexes and DNA
were investigated by viscosity measurements. A classical
intercalative mode is expected to cause a significant
increase in the viscosity of the DNA solution due to an
increase in the separation of base pairs at the intercalation
sites and hence an increase in the overall DNA length. In
contrast, a partial, non-classical intercalation of the ligand
could bend (or kink) the DNA helix and reduce its effective
length and, concomitantly, its viscosity [26].
(2)
Scheme 1 Synthesis of ligands and complexes
concentration, the absorption band of complex 1 at 275 nm
exhibited hypochromism of 15.9 % and bathochromism of
about 3 nm. Complex 2 also exhibited hypochromism of
19.2 % at 263 nm and bathochromism of about 5 nm.
Fig. 1 Absorption spectra of
the complexes 1(a) and 2(b) in
Tris–HCl buffer I upon
increasing amounts of CT-
DNA. [V] = 20 lL,
[DNA] = (0–100) lL. Arrow
shows the decrease upon
increasing CT-DNA
concentrations. Inset plots of
[DNA]/(ea - ef) versus [DNA]
for the titration of [V] with CT-
DNA
123

Transition Met Chem
Fig. 2 Emission spectra of
complexes 1(a) and 2(b) in
Tris–HCl buffer 1 in the
absence and presence of CT-
DNA. [V] = 20 lL. Arrows
shows increasing intensity upon
increasing DNA concentration
DNA cleavage experiments
The effects of addition of EB and complexes 1 and 2 on
the CT-DNA viscosity are shown in Fig. 3. Upon
increasing the amounts of complexes 1 and 2, the relative
viscosity of DNA increases steadily. EB, as a typical
intercalator, also increases the relative DNA viscosity [24,
25, 27]. These results are in agreement with the above UV
spectroscopic data and provide strong evidence for the
interaction of complexes 1 and 2 with DNA by intercala-
tive binding. The electronic effect of phenanthroline
appears to be an important factor in determining the
intrinsic binding constant Kb.
The cleavage reactions of the complexes with plasmid
DNA were monitored by agarose gel electrophoresis. Both
complexes showed considerable DNA cleavage ability at
low concentrations (15 lM) in the presence of H2O2. When
circular plasmid DNA is subject to electrophoresis, rela-
tively 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 generated [26, 27].
Thermal denaturation experiments have been carried out
in order to explore the binding of the complexes with CT-
DNA. As Fig. 4 shows, a small positive shift of DNA-
melting temperature (DTm) is observed on addition of the
complexes to CT-DNA. In contrast to the above results, the
low DTm values for 1 and 2 suggest primarily groove
binding of the complexes to CT-DNA stabilizing the DNA
double-helix structure, in preference to an intercalative
mode of binding to DNA that normally gives a large pos-
itive DTm value [17–20, 24].
Figure 5 shows the cleavage of plasmid pBR332 DNA
after incubation with complexes 1 and 2 at 37 °C for 1 h in
the dark. There was no cleavage of pBR 322 DNA for
H2O2 alone under the same conditions, as shown in lane 2.
In the presence of complexes 1 and 2 with 30 mM L-1
H2O2 (lanes 3–5 and 8–10, respectively) or 30 mM H2O2
and 0.02 M L-histidine as a singlet oxygen quencher (lanes
6 and 11), plasmid DNA was nicked as is evident from the
1.20
complex1
complex2
1.15
1.10
1.05
1.00
0.00
0.05
0.10
[V]/[DNA]
Fig. 4 Thermal denaturation of CT-DNA in the absence (filled
square) and presence of complexes 1 (filled circle) and 2 (filled
triangle). [V] = 20 lM, [DNA] = 80 lM
Fig. 3 Effects of increasing the amounts of complexes 1 and 2 on the
relative viscosity of CT-DNA at 28 ( 0.1) °C. [DNA] = 0.40 Mm
123

Transition Met Chem
formation of form II, while L-histidine slightly inhibits the
cleavage of DNA. Conversely, in the presence of the
hydroxyl radical scavenger DMSO (lanes 7, 12), complete
inhibition of DNA cleavage occurred, which suggests that
ÁOH radical is likely to be the reactive species for cleavage.
This can be explained by the generation of ÁOH radicals
obtained from the oxidation of VO2? in the presence of
H2O2 through the following reaction:
Table 1 The IC50 values for complexes and ligands against SH-
SY5Y, SK-N-SH and MCF-7 cell lines
Compounds
IC50* (lM)
SH-SY5Y
MCF-7
SK-N-SH
Cisplatin
dpq
7.72 0.24 13.36 1.25
14.04 0.65 29.34 4.23
23.79 2.15 [100
2.95 0.14
8.67 0.63
4.67 0.63
33.98 1.23
20.78 1.54
hntdtsc
satsc
49.53 4.21 [100
VO2þ þ H2O2 ! VOþ þ ÁOH þ Hþ
According to previous reports, the process of DNA
cleavage may be closely related to oxidation of vanadyl
ions in the presence of H2O2.
VO(acac)2
VO(satsc)(dpq)
VO(hntdtsc)(dpq)
43.61 1.54 [100
10.64 0.21 26.34 0.39 5.551 0.025
8.30 0.09 23.46 1.27 3.48 0.017
* Cells were treated with various concentrations of tested compounds
for 48 h. Cell viability was determined by MTT, and IC50 values were
calculated as described in ‘‘Materials and methods’’. Each value
represents the mean SD of three independent experiments
Cytotoxicity assays in vitro
The antitumor activities of the two complexes and the
corresponding free ligands against SH-SY5Y, MCF-7 and
SK-N-SH cell lines were evaluated by MTT assay [28–30].
The IC50 values of the free ligands and their complexes
against these tumor cells after continuous exposure for
48 h are shown in Table 1.
the antiproliferative effect of these complexes on cancer
cells will require further study.
Hydroxyl radical scavenging experiments
As shown in Table 1, the complexes exhibit broad
inhibition of all three human cancer cell lines with IC50
values ranging from 3.48 to 26.34 lM. The SK-N-SH
cells proved to be the most sensitive to treatment with
these complexes. Micrographs of the SH-SY5Y cell
lines after treatment for 48 h in the absence (I) and
presence of complexes 1 (II) and 2 (III) are shown in
Fig. 6.
As one of the most important reactive oxygen species
(ROS), hydroxyl radical can cause cell membrane disin-
tegration, membrane protein damage and DNA mutations.
The scavenging effect on hydroxy radical of complexes 1
and 2 was observed [33, 34]. Since complexes 1 and 2
showed good ability to cleave DNA, it was considered
necessary to investigate their scavenging effects on the
hydroxy radical. Rutin, which is known to be an effective
antioxidant agent [35], was used as a positive control. As
shown in Fig. 7, the scavenging effect of complexes 1 and
2 was concentration dependent. However, the scavenging
ratio decreased when the concentration reached 4.0 lM.
This may be attributed by the generation of ÁOH radicals
obtained from the oxidation of VO2? in the presence of
H2O2 through the following reaction [18, 25–28]:
In Table 1 and Fig. 6, it can be seen that the complexes
and free ligands all showed cytotoxicity against the three
tumor cell lines [30–32]. The two oxovanadium complexes
were more cytotoxic than the free ligands. Complex 2
shows the most potent inhibitory effect against these cell
lines. This is consistent with its binding abilities with CT-
DNA, indicating that the antitumor abilities of the oxo-
vanadium complexes may be related to their interactions
with DNA. These results are in line with our previous
studies [17–20, 25]. However, the detailed mechanism of
VO2þ þ H2O2 ! VOþ þ ÁOH þ Hþ
Fig. 5 Cleavage of pBR322 DNA by oxovanadium complexes 1 and
3 (15–60 lM) 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 lM) ?H2O2; lane 4 DNA ? 1(30 lM) ? H2O2; lane 5
DNA ? 1(60 lM) ? H2O2; lane 6 DNA ? 1(30 lM) ? H2O2 ? L-
histidine (0.02 M); lane
7
DNA ? 1(30 lM) ? H2O2 ? DMSO
lane
(2lL); lane DNA ? 3(15 lM) ? H2O2;
8
9
DNA ? 3(30 lM) ? H2O2; lane 10 DNA ? 3(60 lM) ? H2O2;
lane 11 DNA ? 3(30 lM) ? H2O2 ? L-histidine (0.02 M); lane 12
DNA ? 3(30 lM) ? H2O2 ? DMSO (2lL)
123

Transition Met Chem
Fig. 6 Micrograph of the SH-SY5Y cell line after treated for 48 h in the absence (I) (control) and presence of same concentration (200 lM) of
complexes 1 (II) and 2 (III), respectively
and the Scientific and Technical Department of Guangdong Phar-
maceutical University (No. 2014-36).
Conflict of interest The authors have declared that there is no
conflict of interest.
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