Synthesis, characterization and biological evaluation of a novel vanadium complex as a possible anticancer agent

Article abstract of DOI:10.1016/j.jorganchem.2014.08.032

Chemotherapeutics are the sole treatment modality for cancer patients at advanced condition. In the pursuit of getting an efficient, selective and non toxic anticancer agent that has not been discovered yet, we rationally synthesized a vanadium complex with a Schiff base ligand N-(-2-hydroxyacetophenone) glycinate. Characterization of the synthesized complex VO(NG)₂, was carried out with the help of various spectroscopic means. The complex proved to be a broad spectrum anticancer agent with significantly less toxicity towards normal cells as evident from cell viability assay. Furthermore, VO(NG)₂ treatment increased the life-span of ascitic and solid tumor bearing Swiss albino mice and did not show any significant symptomatic sub acute toxicity on normal Swiss albino mice.

Full text of DOI:10.1016/j.jorganchem.2014.08.032

Journal of Organometallic Chemistry 772-773 (2014) 34e41
Contents lists available at ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Synthesis, characterization and biological evaluation of a novel
vanadium complex as a possible anticancer agent
Abhinaba Sinha, Kaushik Banerjee, Arpita Banerjee, Satyajit Das,
Soumitra Kumar Choudhuri*
Department of In Vitro Carcinogenesis and Cellular Chemotherapy, Chittaranjan National Cancer Institute, Kolkata, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Chemotherapeutics are the sole treatment modality for cancer patients at advanced condition. In the
pursuit of getting an efficient, selective and non toxic anticancer agent that has not been discovered yet,
we rationally synthesized a vanadium complex with a Schiff base ligand N-(-2-hydroxyacetophenone)
glycinate. Characterization of the synthesized complex VO(NG)2, was carried out with the help of various
spectroscopic means. The complex proved to be a broad spectrum anticancer agent with significantly less
toxicity towards normal cells as evident from cell viability assay. Furthermore, VO(NG)2 treatment
increased the life-span of ascitic and solid tumor bearing Swiss albino mice and did not show any sig-
nificant symptomatic sub acute toxicity on normal Swiss albino mice.
Received 6 June 2014
Received in revised form
28 August 2014
Accepted 30 August 2014
Available online 6 September 2014
Keywords:
Anticancer agent
Ehrlich ascites carcinoma
MTT assay
© 2014 Elsevier B.V. All rights reserved.
Sarcoma 180
Vanadium complex
Introduction
inhibiting or degrading the protein tyrosine phosphatases (PTP's)
which triggers apoptosis in neoplastic cells via cell cycle arrest
Success of cancer chemotherapy relies on selective induction of
cytotoxicity to transformed self cells, but such specific drug is
elusive till date. Application of a transition metal complex, e.g., cis-
platin as anticancer drug (ACD) showed us a new direction to fight
against cancer [1,2]. Our laboratory has also participated in the
crusade against cancer by introducing various transition metal
complexes having the organic moiety N-(2-hydroxyacetophenone)
glycinate (NG) as their ligand [3]. These metal chelates viz., CuNG,
FeNG, ZnNG, MnNG imparts oxidative stress in the malignant cells
by depleting cellular glutathione and thus induce apoptosis to the
same [4e8]. Herein, we tried to explore the anticancer potential of a
vanadium complex formed with the same ligand, NG. Vanadium; a
trace element for various mammalian species has therapeutic ap-
plications to abrogate diabetes and AIDS [9,10]. Growing body of
evidences discloses that vanadium complexes are potent anticancer
agent but proves to be toxic at higher doses [11].
[13,14]. Vanadium compounds also trigger apoptosis in malignant
cells either by inducing proapototic genes or by inhibiting anti-
apoptotic gene expression [15]. Contemporary studies reveal that
transformed cells maintain an abnormal redox homeostasis that
helps them to acquire optimal genetic instability, which is instru-
mental for cancer progression [16]. Vanadium being redox-active
can generate reactive oxygen species (ROS). This heightened ROS
disrupts the redox homeostasis in neoplastic cells and imparts
redox stress leading to apoptosis either by cleaving DNA or by
facilitating mitochondrial membrane permeabilization [17].
Under this scenario, antiproliferative effect of the synthesized
complex VO(NG)2 on a number of human and murine cancer cell
lines were evaluated. The anticancer potential was further estab-
lished on ascites and solid tumor bearing mice models. The sub-
acute toxicity of the complex was also evaluated on normal mice.
Deregulated cell cycle checkpoints is one of the pivotal hall-
marks of cancer [12], and vanadium exerts its intracellular effect by
Materials and methods
Reagents
* Corresponding author. Department of In Vitro Carcinogenesis and Cellular
Chemotherapy, Chittaranjan National Cancer Institute, 37, S.P. Mukherjee Road,
Kolkata 700 026, India. Tel.: þ91 33 2476 5101/02/04x332; fax: þ91 33 2475 7606.
E-mail address: soumitra01@yahoo.com (S.K. Choudhuri).
3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(MTT), glycine, N-(2-Hydroxy) acetophenone, vanadyl sulphate
were purchased from Sigma Chemical Company (St. Louis, USA). All
http://dx.doi.org/10.1016/j.jorganchem.2014.08.032
0022-328X/© 2014 Elsevier B.V. All rights reserved.

A. Sinha et al. / Journal of Organometallic Chemistry 772-773 (2014) 34e41
35
other chemicals used were of highest purity and purchased from
commercial sources.
mouse normal fibroblast cell line NIH 3T3 (provided by Dr. M. M.
Gottesman, NIH, USA) were maintained in DMEM medium (GIBCO),
supplemented with 10% FBS, additional glutamine (0.15%), HEPES
(25 mM) and gentamycin (50 mg/ml). Cells were grown in plastic
tissue culture flasks (Greiner Bio-One, Frickenhausen, Germany) in
a 5% CO2 atmosphere at 37 ꢀC. Cells were passaged twice weekly.
Cells from exponentially growing cultures of 15th passage were
used for all experiments.
EAC (Ehrlich ascites carcinoma) and S180 (Sarcoma 180) cells
were cultured in vitro by isolating the ascitic fluid from EAC or S180
bearing mice within 7e10 days of inoculation with the cancer cells.
Cells were collected and washed thrice in PBS and then seeded
accordingly in each well of flat bottomed 96-well plate for MTT assay
with RPMI 1640 (for EAC) or DMEM (for S180) medium (GIBCO),
containing HEPES (Sigma), penicillinestreptomycin and 10% FBS.
Synthesis of the ligand, potassium N-(2-hydroxy acetophenone)
glycinate (NG)
The ligand, potassium N-(2-hydroxy acetophenone) glycinate
(NG) was prepared following the method of Majumder et al. as
reported earlier [18]. In brief, KOH (18.39 mM) solution was added
drop wise to glycine (18.4 mM) solution. The mixture was kept in an
ice bath and stirred continuously at 15e20 ꢀC. An ethanolic solution
(10%) of 2 hydroxyacetophenone was added slowly to the mixture.
Stirring was continued for 1 h following addition and the mixture
was kept at room temperature for 5 h. The solvent was removed
from the mixture (deep yellow) by a rotary evaporator. The yellow
mass so obtained was washed with pet-ether and precipitated with
methanol-diethyl ether mixture (1:1). The crude product was
recrystallised from methanol. The pure crystallized NG deposited,
yield 75%, m.p. 258e260 ꢀC.
Isolation of PBMC
Heparinized human blood (peripheral) was collected and
diluted with RPMI 1640 (1:1, v/v). The Lymphocyte-enriched
mononuclear cell layer [Peripheral Blood Mononuclear Cells
(PBMC)] were then isolated with the aid of Histopaque 1077
(Sigma). The procured cells were washed and finally resuspended
in cold RPMI 1640 with above-mentioned supplements.
Synthesis of the vanadium complex of NG
Vanadium N-(2-hydroxy acetophenone) glycinate [VO(NG)2] was
synthesized from the ligand, potassium N-(2-hydroxy acetophe-
none) glycinate by its reaction with vanadyl sulphate. The aqueous
solution of vanadyl sulphate (1 mM) was added to the aqueous so-
lution of NG (1.1 mM) drop-wise. The solution was stirred magneti-
cally at room temperature for 2 h and kept at 4 ꢀC for 1 week. Brown
crystalline precipitate was deposited from the solution and was
filtered off and dried. Yield 67%, m.p. 191e193 ꢀC. Anal. Calc.
Treatment
For in vitro assay a solution of VO(NG)2 (1 mg/ml) was prepared
just before experiments by dissolving the lyophilized compounds in
DMSO (0.01%) following serial dilution in FBS containing medium to
reduce the concentration of DMSO to nontoxic level i.e. less than
0.01%. For MTT assay, VO(NG)2 treatments were performed with a
C20H20O7N2V: C, 53.21, H, 4.42, N, 6.2; Found: C, 52.46; H, 4.04; N, 5.9.
Chemical characterization and instrumentation
concentration range from 2 to 28 mg/ml. For in vivo experiments a
solution of VO(NG)2 (1 g/ml) was prepared just before experiments
by dissolving the lyophilized compounds in DMSO (0.01%) following
serial dilution in PBS containing medium to reduce the concentra-
tion of DMSO to nontoxic level i.e. less than 0.01%).
Elemental (C, H, N) analysis was performed with the help of Per-
kineElmer 2400 Series II CHN analyzer. UVeVis spectra for the
complex and the ligand were recorded in Varian Cary 100 Scan (range
800e200 nm) in dimethyl sulfoxide (DMSO). IR spectra were recor-
ded in PerkineElmer RX 1 FT spectrophotometer in KBR discs, range
4000e400 cmꢁ1. Mass spectrum was recorded in Waters Micromass
Quattro micro API mass spectrometer, in DMSO.1H NMR and 13C NMR
spectra of the complex were recorded at room temperature in a
Bruker Avance 300 MHz spectrophotometer using 5 mm BBO probe in
DMSO-d6 solution. Thermogravimetric analysis was performed on a
TA instrument (SDT Q600) thermal analyzer in a dynamic atmosphere
of ultra high pure dinitrogen (flow rate: 100 cc minꢁꢁ11). The sample
was heated in platinum crucible at a rate of 10 ꢀC min from 25 ꢀC to
900 ꢀC. Cyclic voltametric study was done in BASi Epsilone EC in-
strument (USA). Electrodes used were Ag/AgCl (reference electrode),
platinum auxilaryelectrode and platinumworking electrode in DMSO
using tetrabutylammonium hexafluorophosphate, [N-(n-Bu)4] PF6;
0.1 M as supporting electrolyte at a scan rate of 100 mv/s (25 ꢀC),
reference against Ferrocinium/Ferrocene (Fcþ/Fc). The complex so-
lution was purged with N2 for 30 min before each experiment.
In vitro viability assay (MTT assay)
Cell viability was determined by the method as described pre-
viously [19]. In brief, cells were seeded in 96-well plates at a density
4 ꢂ 104 of cells per well. For single-agent studies, cells were seeded
and allowed to acclimatize for 24 h before treatment with
increasing concentrations of VO(NG)2 and was incubated for
further 48 h or 72 h with 5% CO2 at 37 ꢀC. Following completion of
incubation cells were further incubated with 5 mg/ml of MTT dye
for 4 h at 37 ꢀC. The cells were suspended in DMSO (0.1 ml) and the
absorbance was read at 540 nm in an ELISA reader (Tecan 200) and
the control value corresponding to drug untreated (solvent-
treated) cells was taken as 100%. The viability of treated samples
was expressed as percent of control. The IC50 values were deter-
mined as the concentration of VO(NG)2 that reduced cell viability
by 50% compared to control cells.
Cell culture
Animals
The human T-cell acute lymphoblastic leukemia cell line CCRF-
CEM (gifted by Prof T. Efferth, University of Mainz, Germany) and
human colorectal carcinoma HCT-116 (ATCC) were maintained in
RPMI 1640 medium (GIBCO Invitrogen Corp, Carlsbad, California,
USA) supplemented with 10% foetal bovine serum (FBS) (GIBCO),
glutamine (0.15%), HEPES (25 mM) and gentamycin (50 mg/ml).
Furthermore the human breast cancer cell line MCF-7 and human
astrocytoma (glioblastoma) cell line U373MG (NCCS, Pune) and
Swiss albino mice (originally obtained from National Institute of
Nutrition, Hyderabad, India and reared in the institute animal fa-
cilities) were used for all the experiments with prior approval
(Approval ID: IAEC-1.2/SKC-7/2007/4 dated 22.01.2008) of the
institutional animal ethics committee (IAEC). The experimental
protocols described herein were in accordance with the IAEC
(Registration No.: 175/99/CPCSEA, dated 28.01.2000) guidelines
laid down by the committee for the purpose of control and

36
A. Sinha et al. / Journal of Organometallic Chemistry 772-773 (2014) 34e41
supervision of experiments on animals (CPCSEA) by the Ministry of
Social Justice and Empowerment, Government of India. Adult fe-
male Swiss albino mice (weighing 18e20 g, 4e6 weeks old) were
acclimatized to the experimental room having temperature
25 2 ꢀC, controlled humidity conditions, and with photo cycle of
12 h light/12 h dark. The mice were housed in sterile polypropylene
cages containing sterile paddy husk as bedding material, fed on
autoclaved standard mice food pellets and water ad libitum.
albino mice.14 days after the treatment the experimental mice
were anaesthetized and the femur bone was cut with the help of a
vertebrate scissor. Bone marrow was flushed with KCl solution
(0.56%) and centrifuged at 3000 rpm for 15 min at 37 ꢀC. Cell
viability was tested by trypan blue solution (0.4%) and cells were
counted in a phase contrast microscope. The experiment was
repeated thrice. The average value of viable bone marrow cells was
calculated (percentage) from three independent experiments.
Cell line, tumor transplantation and experimental protocol
Isolation of spleen cell
Both EAC and S180 cells were maintained as an ascitic tumor in
female Swiss albino mice. Various groups (each group containing
six mice) of animals were taken for animal survival studies. Each
mouse was inoculated with either 106 EAC cells intraperitoneally
(i.p), or 106 S180 cells subcutaneously (on the right hind leg quar-
ters) to develop ascitic or solid tumor respectively. Following 9 days
inoculation, various VO(NG)2 doses (20, 30, 40, 50 mg/kg of body
weight) as well as vehicle control (DMSO) were administered i.p. to
EAC bearing and intratumorally (i.t.) to S180 bearing mice groups.
Various VO(NG)2 doses (5, 10, 15 mg/kg body weight) and vehicle
control (DMSO) were given intramuscularly (i.m.) to S180 bearing
mice groups, while 1 group each from EAC and S180 inoculated
individuals were kept as untreated control (Each experimental EAC
bearing mice groups were kept in duplicate). Animals were
inspected daily for the assessment of ascitic load, and body weights
were measured on every 3rd day. Two weeks (at 15th day) after
drug treatment, one set from the EAC bearing experimental groups
of mice were sacrificed to measure the total ascitic fluid (TAF) and
packed cell volume (PCV) while the other set was kept to record
their mean survival time (MST). The MSTs for each group of animals
were documented by noting the time of death of each mice. The
MST of each experimental group was compared with the MST of
untreated control group to obtain the treated/control (T/C) ratio
(percent) for every treated groups separately. This T/C value for
each experimental group is instrumental for the in vivo validation
of the effect of VO(NG)2 on animal survival. National Cancer Insti-
tute (NCI, USA) considers the T/C ratios around 120% to be ‘‘mar-
ginal’’, between 120 and 150% as ‘‘clear’’ and equal or superior to
150% as ‘‘marked’’ in terms of anticancer activity [20].
Preliminary spleenic toxicity (sub-acute) of VO(NG)2 treated
[(i.p; 20, 30, 40, 50 mg/kg body weight) and (i.m; 5,10,15 mg/kg
weight)] female Swiss albino mice was further investigated keeping
appropriate vehicle and untreated control. Mice of different groups
were anaesthetized and 70% alcohol was sprayed on abdominal
region. Spleen was removed aseptically and small amount of PBS
was injected to it; Spleen was rubbed against the fine wire mesh of
the tissue grinder. The cell suspension formed is spun at
1000e1500 rpm for 5e10 min. The supernatant was discarded and
the cells were washed by spinning in PBS twice at room tempera-
ture. Cell viability was tested by trypan blue solution (0.4%) and
cells were counted in a phase contrast microscope. The experiment
was repeated thrice .The average value of viable spleen cells was
calculated from three independent experiments.
Statistical analysis
The values of the arithmetic mean from three independent ex-
periments performed in triplicate, i.e., mean S.D. are reported in
the present communication. The unpaired Student's t-test was used
to evaluate the significance of differences between treated versus
control groups, accepting P < 0.05 as a level of significance. The data
were analyzed using the Prism software (GraphPad, San Diego, CA).
Results
UVeVIS spectral study
UV bands for the ligand appeared at
423.
UV bands for the complex appeared at
and 326.
The change in the UV peak from 318 in the ligand to 231 in the
complex indicates * transition. The shift of the peak from 347
in the ligand to 264 in the complex also indicates * transition.
The bathocromatic shift of the peak from 423 in the ligand to 326 in
l max (DMSO): 318, 347 and
In vivo toxicity assay for VO(NG)2
l max (DMSO): 231, 264
To determine the sub-acute toxicity of VO(NG)2, different
experimental groups of normal female Swiss albino mice (6 mice in
each group) were treated i.p with various doses of VO(NG)2 (20, 30,
40 and 50 mg/kg body weight). Some animals were treated with
VO(NG)2 (5, 10 and 15 mg/kg body weight) through i. m. route
Appropriate vehicle control was maintained by injecting the animals
with DMSO through i.p. and i.m. routes. Some animals were also
kept untreated as control. Following 14 days treatment, blood was
collected via closed cardiac puncture with the help of a 22-guage
hypodermic needle [21]. Blood from each group was then pooled
into separate glass tubes and treated with anticoagulant (heparin) or
left untreated for serum collection. Hematological parameters were
then analyzed by automated haematology analyzer (Sysmex, KX-21)
and hematologic biochemical analyses (blood urea, creatinin, alka-
line phosphatase, ALT, AST) was performed by the help of automated
clinical chemistry analyzer (Olympus, AU400).
pep
pep
the complex also indicates pep* transition.
IR spectrum
Important infrared (IR) bands for the ligand appear at:
3410e3360, 1689, 1619, 1524, 1466, 1421, 1395, 1318, 1269, 1205,
1163, 969, 931, 752 and 730 cmꢁ1
.
Important IR bands for the complex appear at: 1645, 1609, 1443,
1379, 1324, 1252, 1131, 982, 936, 834, 745 and 436 cmꢁ1
.
The IR spectrum of the ligand showed broad band at
3360e3410 cmꢁ1 indicating the presence of phenolic nOH group. The
free nOH is generally observed between 3400 and 3500 cmꢁ1. The
observed low value is due to intramolecular H-bond formation be-
tween H and nitrogen [18]. In vanadium complex the band dis-
appeared and thus indicating complexation involving phenolic eOH
group of the ligand. The alkyl groups CH2, CH3 show characteristic
deforming bands at 1466e1395 cmꢁ1 and the rocking modes at
730 cmꢁ1 in the ligand. In complex the deforming bands and rocking
Isolation of bone marrow cell
Preliminary bone marrow toxicity was assayed following
administration of various doses of VO(NG)2 [(i.p; 20, 30, 40, 50 mg/
kg body weight) and (i.m; 5,10,15 mg/kg weight)] on female Swiss

A. Sinha et al. / Journal of Organometallic Chemistry 772-773 (2014) 34e41
37
modes are shifted to 1443 cmꢁ1 and 745 cmꢁ1 respectively. The
13C NMR analysis
characteristic imine band at 1619 cmꢁ1 (ligand) is shifted towards
1609 cmꢁ1 further conforming complex formation. The sharp band at
1395 cmꢁ1 in the ligand may be assigned due to nOCO symmetric
stretching that has been shifted to 1379 cmꢁ1 in the complex and
thus indicating coordination with the V atom through the eCOOꢁ
group of the ligand. The band at 1269 cmꢁ1 in the ligand may be
assigned due to nPh(CO) which has been shifted to 1252 cmꢁ1 in the
complex indicating coordination of the phenolic oxygen to the VO
moiety. The band at 1689 cmꢁ1 in the ligand may be assigned due to a
n(OCO) asymmetric vibration and in complex this vibration appears as
broad band at 1646 cmꢁ1 [22]. Two new bands only in the complex at
982 cmꢁ1 and 436 cmꢁ1 confirm the presence of vanadyl and VeN
functionality respectively [23,24] The sharp peak in the spectra of the
complex at 1609 cmꢁ1 is characteristic of the imine group and the
band at 1443 cmꢁ1 is due to skeletal vibration of the aromatic moiety.
These bands were observed at a relatively lower frequency than in
ligand NG which is indicative of complex formation. Thus, the IR
spectra results provide strong evidence for the complex formation.
13C NMR of VO(NG)2 (DMSO-d6, 300 MHz, 300 K):
116 (C-2),133 (C-3),123 (C-4),129 (C-5),119 (C-6) 164 (C-7),19.2 (C-
8), 57 (C-9), 171 (C-10).
The peaks appeared at 116-133 had been assigned for the aro-
matic carbons. The peaks for eCOOH carbon appeared at 171. The
peak for eCH]N carbon, ]CH2 carbon and eCH3 carbon were
noted 164, 57 and 19 respectively in the spectrum (Supplementary
material). The 13C NMR peaks were in agreement with the skeletal
structure of VO(NG)2.
d
157.8 (C-1),
Thermogravimetric analysis of VO(NG)2
Thermogravimetric analysis of complex, VO(NG)2, have been
performed to elucidate the solid state thermal behavior, to inden-
tify the end products and thereby the composition of the original
species. The analysis revealed that upon gradual heating the com-
plex decomposes in two steps. The first step occurs at ~220 ꢀC is
probably due to a partial decomposition of the ligand (observed
33%) (calculated weight loss for 1 molecule of ligand NG: 42.57%).
The second step of the thermal decomposition, which occurs at
~410 ꢀC, was assigned to the loss of the second ligand molecule
(observed 82%) (calculated.85.14%) with the formation of VO2
(observed 19%) (calculated.18.40%).
Mass spectral study
Mass spectral data is presented in Fig. 1. The formation of molec-
ular ion peaks indicates that the structure of the complex is A in Fig. 1.
1H NMR analysis
Cyclic voltammetry analysis
1H NMR of VO(NG)2 (DMSO-d6, 300 MHz, 300 K):
d 7.8 (d, 1H,
J ¼ 0.028), 7.4 (t, 1H, J ¼ 0.025), 6.8 (dd, 2H, J ¼ 0.018, 0.026), 3.527
Cyclic voltammetric assay of VO(NG)2 was performed to find out
whether it has the ability to alter cellular redox status. The
(s, 2H), 2.51 (s, 3H).
Fig. 1. Mass spectral analysis. Mass fragmentation pattern of VO(NG) 2, depicting the chemical structure of the complex (A).

38
A. Sinha et al. / Journal of Organometallic Chemistry 772-773 (2014) 34e41
Table 1
compound exhibits irreversible voltammogram with an oxidation
peak at 0.150 V and reduction peak at ꢁ0.348 V. The result shows
that the compound possibly generates ROS at very low volt
(ꢁ0.348 V); thus the compound has very low reduction potential
and is capable of generating ROS in physiological condition.
Comparative account of antiproliferative effect of VO(NG)2 on various cells.
Cell line
IC50 dose (
m
g/ml)
Growth inhibition (%) at
highest conc. of VO(NG)2
HCT 116
MCF-7
U373MG
CCRF-CEM
S180
EAC/S
Normal human PBMC
NIH3T3
e
9.9 2.7
57.57 7.9
79.79 3.4
82.82 4.1
74.98 7.0
47.86 5.4
11.94 0.68
e
1.51 0.11
28 1.9
17.10 0.93
12.21 0.84
e
e
e
VO(NG)2 exhibits antiproliferative effect selectively on malignant
cell lines in time and dose dependent manner
MTT
(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide) assay was conducted on a number of VO(NG)2 treated
malignant cells such as, MCF 7, HCT 116, U373 MG, CCRF-CEM, EAC,
Sarcoma 180, human normal PBMC and NIH 3T3 (Fig. 2).
normal human PBMC (Fig. 2) and no significant cytotoxicity on NIH
3T3 cells (Fig. 2). Furthermore, administration of the ligand (NG) to
all the malignant cells herein at identical doses imposed negligible
cytotoxicity.
Our data clearly indicated that VO(NG)2 exhibits time and dose
dependent antiproliferative activity on the above mentioned cancer
cells (Table 1). But under the influence of identical VO(NG)2 con-
centrations we noticed miniscule inhibition of proliferation of
Fig. 2. Comparison of the cytotoxic effect of the vanadium complex and its ligand on different cells.

A. Sinha et al. / Journal of Organometallic Chemistry 772-773 (2014) 34e41
39
Table 3
Dose response curves prepared for vanadium complex
(VO(NG)2) and the ligand (NG) on (A) HCT116, (B) MCF7, (C)
U373MG, (D) CCRF-CEM, (E) S180, (F) EAC cell lines and VO(NG)2
alone on (G) Human PBMC and (H) NIH3T3 cells according to MTT
assay. All the cancer cells herein were treated with similar doses of
NG and was incubated further for 72 h. Results are expressed as
percentage viability of treated cells compared to control (solvent-
treated) cells. Value represents the mean SD of three independent
experiments with four replicates in each (Fig. 2).
Survivability assay of VO(NG)2 treated cell lines showing their
respective IC50 values and or survivability of cells receiving 28 mg/
ml VO(NG)2 (72 h incubation). Results are expressed as percentage
viability of treated cells compared to control (solvent-treated) cells.
Effect of VO(NG)2 on toxicological parameters for hepatic and renal function in fe-
male Swiss albino mice blood.
Groups
Urea
(mg/dL)
Creatinine
(mg/dL)
Alkaline
phosphatase (U/L)
(U/L)
SGOTa
SGPTb
(U/L)
Untreated
I.P (VC)
54 1.9
57 1.9
55 1.4
47 1.0
49 2.1
53 1.4
51 1.0
0.4 0.01
0.3 0.02
200 6.9
181 6.1
182 5.9 53 1.6
183 5.6 41 0.9
170 4.8 66 1.9
188 5.8 62 2.1
196 6.2 71 2.4
189 5.9 68 2.1
188 5.7 56 1.5
168 6.0 47 1.3
178 5.3 56 1.6
181 5.5 61 1.9
I.P (20 mg/kg)
I.P (30 mg/kg)
I.P (40 mg/kg)
I.P (50 mg/kg)
I.M (VC)
I.M (5 mg/kg)
I.M (10 mg/kg) 52 1.6
I.M (15 mg/kg) 59 1.5
0.3 0.019 259 7.6
0.4 0.023 239 7.1
0.3 0.026 242 6.7
0.4 0.011 271 7.7
0.4 0.021 193 5.9
43 01.7 0.2 0.013 176 5.5
0.4 0.03
0.3 0.017 204 6.7
187 6.3
Values represent the mean
SD of 3 independent experiments
with 4 replicates in each (Table 1).
a
Serum glutamate ortho-transferase (SGOT).
Serum glutamine pyruvate transaminase (SGPT).
b
VO(NG)2 imparts no significant toxicity on female Swiss albino mice
Table 4
Effect of VO(NG)2 on spleen and bone marrow of female Swiss albino mice.
A preliminary 14 days (sub-acute) toxicity study was carried out
on normal female Swiss albino mice model receiving VO(NG)2 doses
either i.p. (20, 30, 40 or 50 mg/kg body weight) or i.m. (5, 10, 15 mg/
kg body weight). Herein we observed that no significant toxic death
occurred following vehicle (DMSO) and VO(NG)2 treatment. Hema-
tological (Table 2) and other biochemical parameters that are in-
dicators for hepatic and renal toxicity (Table 3) remained unchanged
following VO(NG)2 application (up to highest dose of 50 mg/kg body
weight) compared to untreated control. Furthermore no spleen and
bone marrow toxicity in vehicle (DMSO) as well as VO(NG)2 treated
groups compared to control was noticed (Table 4).
Groups
Bone marrow cell survival (%)
Spleen cell survival (%)
Untreated
I.P (VC)
95.3 3.2
96.2 3.2
91.1 2.7
92.7 2.9
84.1 2.8
83.5 1.7
93.2 3.1
92.7 2.8
91.3 2.3
90.5 2.5
97.2 3.8
90.8 2.5
93.4 3.2
91.7 2.6
90.3 3.0
87.5 2.5
94.3 3.7
93.2 2.8
94.5 3.6
89.7 2.8
I.P (20 mg/kg)
I.P (30 mg/kg)
I.P (40 mg/kg)
I.P (50 mg/kg)
I.M (VC)
I.M (5 mg/kg)
I.M (10 mg/kg)
I.M (15 mg/kg)
VO(NG)2 (all the doses) through both i.p and i.m routes showed
no hematological toxicity to normal female Swiss albino mice in
comparison to untreated control mice (P < 0.05). Herein vehicle
control (VC) is solvent (DMSO)-treated normal female Swiss albino
VO(NG)2 enhanced the life-span of various malignant cell bearing
mice
mice. Values represent mean
standard deviation (S.D.) from 3
independent experiments for 6 mice in each group (Table 2).
VO(NG)2 (all the doses) through both i.p and i.m routes showed
no hepatic and renal toxicity over normal female Swiss albino mice
as compared with untreated control mice (P < 0.05). Herein vehicle
control (VC) is solvent (DMSO) treated normal female Swiss albino
VO(NG)2 found to be cytotoxic against MCF 7 (in vitro) and EAC
cells (ex vivo). In vivo experimentation of this complex on EAC
(originated as murine breast cancer) bearing mice model also
exhibited anticancer potential. Out of all the doses, 30 mg/kg body
weight of VO(NG)2 when administered (i.p) to EAC bearing mice
proved to be most effective showing a T/C value of 150%. The
intratumoral (i.t.) efficacy of the complex was evaluated in S180
tumor bearing mice. However, VO(NG)2 administration through i.t.
route proved to be less effective in S180.
Application of VO(NG)2 through i.m. route in S180 bearing mice
caused an abrupt increase in lymphocyte population compared to
untreated control. (VONG)2 failed to modulate the lymphocyte
population through i.m. route in EAC bearing mice (data not
mice. Values represent mean
standard deviation (S.D.) from 3
independent experiments for 6 mice in each group (Table 3).
VO(NG)2 (all the doses) through both i.p. and i.m routes were
non-toxic to spleen and bone marrow cells of treated normal fe-
male Swiss albino mice when compared with untreated control
mice (P < 0.05). Herein vehicle control (VC) is solvent (DMSO)
treated normal female Swiss albino mice. Values represent
mean standard deviation (S.D.) from 3 independent experiments
for 6 mice in each group (Table 4).
Table 2
Effect of VO(NG)2 on hematological parameters of female Swiss albino mice.
Parameters
(UT)
I.P VC
I.P
I.P
I.P
I.P
I.M VC
I.M
I.M
I.M
(20 mg/kg)
(30 mg/kg)
(40 mg/kg)
(50 mg/kg)
(5 mg/kg)
(10 mg/kg)
(15 mg/kg)
WBC (ꢂ103/
m
L)
19.1 1.3
9.65 0.9
15.0 1.1
49.3 3.2
51.1 3.8
15.5 0.8
30.4 2.3
921 38.8
88.5 7.3
19.0 1.6
10.0 0.9
15.7 1.1
51.9 3.9
51.9 3.7
15.7 0.7
30.3 2.1
1060 31.4
86.0 7.1
13.2 0.9
9.17 0.8
14.0 1.0
46.6 3.1
50.8 3.6
15.3 0.8
30.0 2.2
1303 41.1
88.4 7.4
12.4 0.8
9.58 0.9
15.2 1.1
50.8 3.8
53.0 3.2
15.9 0.9
29.9 2.0
1205 36.0
87.2 7.2
10.3 0.8
6.20 0.4
10.0 0.7
34.6 2.3
55.6 3.1
16.1 1.2
28.7 1.9
1300 37.4
74.1 6.7
18.2 1.6
7.08 0.8
10.8 0.8
35.9 1.9
50.7 3.4
15.3 0.7
30.1 2.3
1447 44.3
80.3 6.9
19.9 1.2
9.06 0.8
14.2 0.9
46.6 3.2
51.7 3.9
15.6 0.8
30.9 2.1
1049 29.7
87.4 7.3
22.9 2.4
8.89 0.7
13.4 1.1
44.2 2.8
49.7 3.8
15.0 0.9
30.3 2.1
1063 40.2
93.7 8.1
20.5 1.9
9.02 0.9
13.5 0.8
46.3 2.9
51.7 3.5
14.9 0.5
28.9 2.6
1209 35.2
88.3 7.9
17.1 1.1
7.71 0.5
10.5 0.9
36.0 2.1
52.3 2.9
13.6 0.5
29.1 2.4
922 36.4
81.5 2.1
RBC (ꢂ106/
HGB (g/dL)
HCTa (%)
m
L)
MCVb (fL)
MCHc (pg)
MCHCd (g/dL)
PLT (ꢂ103/
mL)
LYM (%)
a
Hematocrit value (HCT).
Mean corpuscular volume (MCV).
Mean corpuscular hemoglobin (MCH).
Mean corpuscular hemoglobin concentration (MCHC).
b
c
d

40
A. Sinha et al. / Journal of Organometallic Chemistry 772-773 (2014) 34e41
shown). Thus VO(NG)2 may have the host protective immune
modulatory potential on S180 bearing mice model. The result
inspired us to apply the complex through i.m route into the S180
cancer model. Our data found accordance with the above
mentioned hypothesis as VO(NG)2 treatment (i.m.; 5 mg/kg and
10 mg/kg body weight) increased the survival of S180 bearing mice
with T/C values of 150% and 162% respectively (Table 5).
VO(NG)2 (various doses) through intraperitonel (i.p.), intra-
tumoral (i.t.) and intramuscular (i. m.) routes increases the mean
survival time (MST) and therefore T/C value of EAC and S180 cells
bearing mice groups compared to untreated (control) groups. The
packed cell volume (PCV) and the total ascitic fluid (TAF) were also
decreased in treated groups compared to untreated EAC cell
bearing mice. Herein vehicle control (VC) is solvent (DMSO)-treated
(i.p.) (30 mg/kg body weight) on EAC bearing mice exhibited T/C
value of 150% whereas VO(NG)2 administration (i.m) at lower doses
(5 and 10 mg/kg body weight) proved to be most potent on S180
bearing mice (T/C values of 149% and 162.2%). All these doses
showed “marked activity” according to standard National Cancer
Institute protocols for screening new anticancer drugs [20]. More-
over, VO(NG)2 treatment up to 50 mg/kg of body weight exhibited
no significant toxicity on normal female Swiss albino mice.
Our group previously reported numerous transition metal
complexes viz, CuNG, FeNG, ZnNG, MnNG and all of these com-
plexes alters redox homeostatic machinery of cancer cells [3e8].
VO(NG)2 being a transition metal complex exhibits irreversible
reduction at physiological electro chemical potential range as
evident from cyclicvoltammetric analysis. Therefore, VO(NG)2
probably imparts redox stress to the cancer cells by deregulating
their redox homeostatic biochemistry in in vitro as well as in vivo
cancer models. We also cannot rule out the possibility of ROS-
mediated immune-modulation in the S180 tumor microenviron-
ment following VO(NG)2 (i.m) administration [26,27]. Thus in
agreement with our proposition, the in vitro and in vivo studies
indicate the promise of a broad spectrum anticancer agent in the
form of present vanadium complex, VO(NG)2.
mice. Values represent mean
standard deviation (S.D.) from 3
independent experiments for 6 mice in each group (Table 5).
Discussion
The present work is based on our proposition that a coordina-
tion complex of vanadium may be less toxic and potent anticancer
agent if a suitable organic moiety can be attached to vanadium
atom [25]. Under this backdrop we selected the non-toxic ligand,
NG which showed anticancer activity when bound to divalent
copper, iron, manganese and zinc atoms [3]. We synthesized and
characterized VO(NG)2 and studied it's in vitro and in vivo anti-
cancer potential. For in vitro studies we selected six transformed
cell lines viz., human colorectal carcinoma (HCT 116), human breast
carcinoma (MCF 7), human astrocytoma (U 373 MG), human T
lymphoblastic leukemia (CCRF-CEM), murine Ehrlich ascites carci-
noma (EAC) and murine sarcoma (S 180) models to determine
whether VO(NG)2 has differential antiproliferative effects on them
or not. Our studies reveled that VO(NG)2 inhibits the proliferation
of all the above mentioned cell lines differentially, in time and dose
dependent manner (Table 1). Furthermore, application of identical
VO(NG)2 dosage to normal human PBMC and normal murine NIH
3T3 cells did not hamper their survivability (Table 1). VO(NG)2
proved to be most effective on MCF7 cell line with IC50 value as low
Conclusions
This novel complex of vanadium, VO(NG)2 proved to be a potent
cytotoxic agent specifically for the malignant cells in vitro as
revealed by cell survival assay. VO(NG)2 also proved to be an
effective antineoplastic agent in vivo on both ascitic (EAC) as well as
solid tumor (S180) bearing female Swiss albino mice. However,
VO(NG)2 (5 mg/kg) when administered through i.m. route proved
to be most potent on S180 bearing mice with a ‘marked’ T/C value of
‘162.2%’. Although vanadium is toxic at higher doses, surprisingly
VO(NG)2 treatment up to 50 mg/kg body weight exhibited no sig-
nificant manifestation of toxic symptoms on female Swiss albino
mice. As our preclinical data sufficiently justified our hypothesis,
further efforts are devoted to understand the molecular mechanism
of VO(NG)2 induced cancer cell death.
as 1.60 0.53 mg/ml (at 72 h of incubation) and also significantly
cytotoxic to EAC cells (originated as murine breast cancer) (ex vivo).
Thus, we selected EAC bearing Swiss albino mice as our in vivo
model of interest. The S180 bearing Swiss albino mice were
included for the present work to serve as a solid tumor bearing
sarcoma model. Our study disclosed that VO(NG)2 administration
Acknowledgements.
Chittaranjan National Cancer Institute, Kolkata 700026 has
provided financial support for the present work.
Appendix A. Supplementary data
Table 5
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.jorganchem.2014.08.032.
Effect of VO(NG)2 on cancer bearing female Swiss albino mice.
Group
Route Total ascitic Packed cell
Mean
T/C
fluid (ml)
volume (ml) survival
value
time (days) (%)
References
EAC (UT)
EAC (VC)
e
13.5 1.7
13.9 1.8
11.5 1.4
7.9 0.9
12.7 1.6
13.3 1.5
e
e
e
e
e
e
e
e
e
e
8.0 1.2
8.3 1.3
6.2 0.6
4.7 0.5
7.6 0.7
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e
e
e
e
e
e
e
e
e
e
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23 1.8
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33 2.7
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68 5.2
59 4.0
47 3.5
54 4.7
86 7.1
79 6.7
64 5.1
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