Biological evaluation of new nickel(II) metallates: Synthesis, DNA/protein binding and mitochondrial mediated apoptosis in human lung cancer cells (A549) via ROS hypergeneration and depletion of cellular antioxidant pool

Article abstract of DOI:10.1016/j.ejmech.2014.05.075

A series of novel nickel(II) thiosemicarbazone complexes(1-4) have been prepared and characterized by various spectral, analytical techniques and X-ray crystallography. Further, their efficacy to interact with CT-DNA/BSA has been explored. From the binding studies, it is inferred that complex 4 found to be more active than other complexes. The complexes bound with CT-DNA by intercalation mode. Moreover, static quenching was observed for their interaction with BSA. The new complexes were tested for their in vitro cytotoxicity against human lung adenocarcinoma (A549) cell line. The results showed that the new complexes exhibited significant degree of cytotoxicity at given experimental condition. Further, the results of LDH and NO release supported the cytotoxic nature of the complexes. The observed cytotoxicity of the complexes may be routed through ROS-hypergeneration and lipid-peroxidation with subsequent depletion of cellular antioxidant pool (GSH, SOD, CAT, GPx and GST) resulted in the reduction of mitochondrial-membrane potential, caspase-3 activation and DNA fragmentation. Thus, the data from the present study disclose that the complexes could induce apoptosis in A549 cells through mitochondrial mediated fashion and inhibited the migration of lung cancer cells and by metastasis.

Full text of DOI:10.1016/j.ejmech.2014.05.075

Accepted Manuscript
Biological evaluation of new Nickel (II) metallates: Synthesis, DNA/protein binding and
mitochondrial mediated apoptosis in human lung cancer cells (A549) via ROS hyper
generation and depletion of cellular antioxidant pool
P. Kalaivani , S. Saranya , P. Poornima , R. Prabhakaran , F. Dallemer , V. Vijaya
Padma , K. Natarajan
PII:
S0223-5234(14)00507-8
10.1016/j.ejmech.2014.05.075
EJMECH 7033
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 February 2014
Revised Date: 27 May 2014
Accepted Date: 31 May 2014
Please cite this article as: P. Kalaivani, S. Saranya, P. Poornima, R. Prabhakaran, F. Dallemer, V. Vijaya
Padma, K. Natarajan, Biological evaluation of new Nickel (II) metallates: Synthesis, DNA/protein binding
and mitochondrial mediated apoptosis in human lung cancer cells (A549) via ROS hyper generation and
depletion of cellular antioxidant pool, European Journal of Medicinal Chemistry (2014), doi: 10.1016/
j.ejmech.2014.05.075.
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Graphical Abstract
Biological evaluation of new Nickel(II) metallates: Synthesis, DNA/protein binding and
mitochondrial mediated apoptosis in human lung cancer cells (A549) via ROS hyper
generation and depletion of cellular antioxidant pool
P. Kalaivani S. Saranya, P. Poornima, R. Prabhakaran, F. Dallemer , V. Vijaya Padma
,
and K. Natarajan

ACCEPTED MANUSCRIPT
Biological evaluation of new Nickel (II) metallates: Synthesis, DNA/protein binding and
mitochondrial mediated apoptosis in human lung cancer cells (A549) via ROS hyper
generation and depletion of cellular antioxidant pool
a
b
b
a*
c
P. Kalaivani , S. Saranya , P. Poornima , R. Prabhakaran , F. Dallemer , V. Vijaya
b
a*
Padma , K. Natarajan
a
Department of Chemistry, Bharathiar University, Coimbatore- 641 046, India
Department of Biotechnology, Bharathiar University, Coimbatore- 641 046, India
Laboratoire Chimie Provence-CNRS UMR6264, Université of Aix-Marseille I, II and III –
b
c
CNRS, Campus Scientifique de Saint-Jérôme, Avenue Escadrille Normandie-Niemen, F-13397
Marseille Cedex 20, France
Abstract
A series of novel nickel(II) thiosemicarbazone complexes(1-4) have been prepared and
characterized by various spectral, analytical techniques and X- ray crystallography. Further, their
efficacy to interact with CT-DNA/BSA has been explored. From the binding studies, it is
inferred that complex 4 found to be more active other complexes. The complexes bound with
CT-DNA by intercalation mode. Moreover, static quenching was observed for their interaction
with BSA. The new complexes were tested for their in vitro cytotoxicity against human lung
adenocarcinoma (A549) cell line. The results showed that the new complexes exhibited
significant degree of cytotoxicity at given experimental condition. Further, the results of LDH
and NO release supported the cytotoxic nature of the complexes. The observed cytotoxicity of
the complexes may be routed through ROS-hypergeneration and lipid-peroxidation with
subsequent depletion of cellular antioxidant pool (GSH, SOD, CAT, GPx and GST) resulted in
the reduction of mitochondrial-membrane potential, caspase-3 activation and DNA
fragmentation. Thus, the data from the present study disclose that the complexes could induce
apoptosis in A549 cells through mitochondrial mediated fashion and inhibited the migration of
lung cancer cells and by metastasis.
Keywords
Nickel(II)thiosemicarbazone complexes, CT-DNA/BSA binding, Cytotoxicity, ROS-
hypergeneration, Mitochondrial-membrane potential, depletion of cellular antioxidant pool.
1

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Abbreviations
CT-DNA
EB
Calf thymus DNA
Ethidium bromide
BSA
Bovine serum albumin
Lung adenocarcinoma cell line
3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide
Lactate dehydrogenase
Nitric Oxide
A549
MTT
LDH
NO
ROS
DCFH-DA
GSH
OPT
SOD
CAT
GST
GPx
Reactive oxygen species
2′,7′ -dichlorofluoresceindiacetate
Reduced glutathione
Ortho-pthalaldehyde
Superoxide dismutase
Catalase
Glutathione-S-transferase
Glutathione peroxidase
Propidium iodide
PI
MPT
AIF
Mitochondrial permeability transition
Apoptosis-inducing factor
Caspase-activated deoxyribonuclease
CAD
Corresponding
authors:
Tel.:
+91-422-2428319;
Fax:
+91-422-2422387
E-mail address: rpnchemist@gmail.com (R. Prabhakaran); k_natraj6@yahoo.com (K. Natarajan)
2

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1
. Introduction
Lung cancer is the leading cause of cancer-related mortality worldwide, leading to an
estimated 1.4 million death in 2010 [1]. Lung cancer is commonly classified into two major
types, small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC). NSCLC
constitutes 75% of lung cancer cases and is subdivided further into three major histological
subtypes: adenocarcinoma (AC), squamous-cell carcinoma (SCC), and large-cell carcinoma. The
AC and SCC subtypes represent 85% of NSCLC cases [2]. Emergence of resistance to anticancer
drugs poses a major clinical challenge in successful treatment of cancer [3]. There is potential to
overcome the two overriding problems in cancer chemotherapy the common occurrence of drug-
resistant tumor cells and the lack of selectivity of cancer drugs in differentiating between
cancerous cells and normal cells [4]. Following the discovery of the anti-tumor properties of
cisplatin (cis-Pt(NH )Cl ) and related complexes, attention for the discovery of new more
3
2
efficient complexes of other metals and ligands developed. Against tumors the first to be entered
for clinical trials were the analogues to cisplatin, complexes of palladium(II), cis-Pd(en)Cl and
2
cis-Pd(DACH) Cl because palladium(II) has a very similar chemistry to platinum(II). Platinum
2
2
metallo-drugs are the most effective drugs used for the treatment of tumor though its clinical
utility is restricted due to the frequent increase of drug resistance, the limited range of tumors
against which these drugs are active and severe normal tissue toxicity being the nephrotoxicity
an important side effect which interfere with their therapeutic efficiency [5-9]. These
disadvantages have motivated the improvement of platinum-based anticancer drugs different
from the traditional cisplatin structure and which could probably have different DNA-binding
modes as well as show different biological profiles [10-14]. Several biological experiments have
demonstrated that DNA is the major intracellular target of many anticancer drugs, cancerogens
and viruses [15,16]. Interactions of small molecules at specific sites along a DNA strand as
reactive models for protein–nucleic acid bindings, provide paths toward rational drug design as
well as means to develop sensitive chemical probes for DNA [17,18]. Numerous metal
complexes have been used as probes of DNA, as agents for mediation of strand scission of
duplex DNA and as chemotherapeutic agents [19-26]. Thiosemicarbazones and their metal
complexes have been expansively studied because they have a wide range of potential medical
applications [27-30]. which include notably antiparasital [31], antibacterial [32], antitumor
activities [33], antiviral [34], fungicidal [35], and antineoplastic [36]. Such potential medical
3

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activities are owing to the strong chelating ability of thiosemicarbazones with biologically
important metal ions such as Fe, Cu, Ni and their reductive capacities [37-41]. Nickel(II)
complexes containing nitrogen and sulfur donor ligands are highly important [42-46] because
several hydrogenases and carbon monoxide dehydrogenases [47] contain such nickel complexes
as their active site. In this line, analogues nickel complexes are found to be potent in various
therapeutic applications [48-50]. With the expectation, platinum mimic nickel and palladium
complexes also exhibiting significant cytotoxicity [51-54], we have already reported the
biological evolution of 3-methoxysalicylaldehyde 4(N) substituted thiosemicarbazone complexes
of palladium [53]. In this article, the new palladium complexes were exposed to study their effect
on A549 and HepG2 cell lines. The MTT, LDH, NO and cellular uptake studies were done.
However, in the present manuscript in addition to the above studies, we have done a detailed
biological investigation of new nickel(II) complexes containing 3-methoxysalicylaldehyde 4(N)
substituted thiosemicarbazone on lung carcinoma (A549) cells and mechanism of drug action
leading to apoptosis has been explored.
2
. Experimental section
2
.1. Complex synthesis and characterization:
1
-4
The ligands (H L) and the nickel precursor [NiCl (PPh ) ] were synthesized according
2
2
3 2
to the standard literature procedures [55,56]. All the reagents used were analar grade, were
purified and dried according to the standard procedure [57]. The synthesis, analytical and
1
-4
spectral characterization of the ligands (H L) were reported by our group earlier [53]. CT-
2
DNA, EB and BSA were obtained from Sigma Aldrich, DMEM, FBS and all other cell-culture
reagents were obtained from Hi-media Laboratories, India. Reagents for enzyme assays were
obtained from Merck speciality Chemicals, India. Primary antibodies used were obtained from
Cell signalling technology, USA and Upstate, USA. HRP-conjugated secondary antibodies, was
purchased from Leinco Technologies, USA. Western-blot membranes were obtained from
Whatman, USA. DCF was obtained from Calbiochem, USA. DiOC6 and CCCP were obtained
from Sigma-Aldrich. Infrared spectra were measured as KBr pellets on a Nicolet Avatar Model
-1
FT-IR spectrophotometer in the 400-4000 cm range. Elemental analysis of carbon, hydrogen,
nitrogen, and sulfur were determined using Vario EL III CHNS at the Department of Chemistry,
Bharathiar University, Coimbatore, India. The electronic spectra of the complexes have been
recorded in dichloromethane using a JASCO V-630 Spectrophotometer in the 200-800 nm range.
4

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1
Emission spectra were recorded by using JASCO FP 6600 spectrofluorometer. H NMR spectra
were recorded in DMSO at room temperature with a Bruker 400 MHz instrument, chemical shift
relative to tetramethylsilane. Melting points were measured in a Lab India apparatus. Single
crystal data collections and corrections for the new Ni(II) complexes were done at 293 K with
CCD kappa Diffractometer using graphite mono chromated MoKα (λ=0.71073 Å) radiation [58-
6
0]. The structural solutions were done by using SHELXTL-97 [61] and refined by full matrix
2
least square on F using SHELXL-97 [62].
2
.1.1. Preparation of [Ni(Msal-tsc)(PPh )].Cl (1):
3
An ethanolic (25 ml) solution of [NiCl (PPh ) ] (0.200 g; 0.3058 mmol) was slowly
2
3 2
added to 3-methoxysalicylaldehydethiosemicarbazone [H -Msal-tsc] (0.068 g, 0.3058 mmol) in
2
dichloromethane (25 ml). The mixture was allowed to stand for 4 days at room temperature.
Reddish orange crystals formed were filtered, washed with petroleum ether (60-80 °C) and n-
hexane. Yield: 58 %. M.p. 288 °C. Anal. Calcd. for C H ClN NiO PS: C, 55.85; H, 4.34; N,
2
7
25
3
2
-
1
7
1
.24; S, 5.52. Found:C, 55.02; H, 4.23; N, 7.05; S, 5.39 %. FT-IR (cm ) in KBr: 1542 (νC=N),
-1
318 (νC-O), 770 (νC=S), 1441, 1100, 694 cm (for PPh ) ; UV- vis (CH Cl ), λmax: 257 (24360)
3
2
2
3
-1
-1
3
-
nm (dm mol cm ) (intra-ligand transition); 341 (17950), 364 (8290), 372 (8102) nm (dm mol
1
-1
1
cm ) (LMCT s→d); H NMR (DMSO-d6, ppm): 8.36 (s, 1H, CH=N), δ 9.12 (s, 1H, N(2)H-
C=S-) 3.77 (s, 3H, OCH ), 6.37–7.48 (m, aromatic) 7.90 and 8.08 (2s, 1H, NH ).
3
2
The very similar method was followed to synthesize other complexes.
2
.1.2. Preparation of [Ni(Msal-mtsc)(PPh )](2):
3
The complex 2 was prepared by the procedure as used for (1) with 3-methoxy
salicylaldehyde -4(N)-methylthiosemicarbazone [H -Msal-mtsc] (0.074 g; 0.3058 mmol) and
2
[
NiCl (PPh ) ] (0.200 g; 0.3058 mmol). Yield: 63 %. M.p. 238 °C. Anal. Calcd. for
2 3 2
C H N NiO PS: C, 60.24; H, 4.69; N, 7.52; S, 5.74. Found: C, 60.18; H, 4.63; N, 7.49; S, 5.68
28
26
3
2
-1
-1
%
. FT-IR (cm ) in KBr: 1556 (νC=N), 1359 (νC-O), 738 (ν ), 1450, 1068, 610 cm (for PPh );
C-S 3
3
-1
-1
UV- vis (CH Cl ), λ : 257 (11659), 303 (8265) nm (dm mol cm )(intra-ligand transition); 359
2
2
max
3
-1
-1
3
-1
-1
(
7140) nm, 371 (7000) nm (dm mol cm ) (LMCT s→d); 410 (6900) nm (dm mol cm )
1
(forbidden (d→d) transition);
H NMR (DMSO-d6, ppm): 8.32 (s, 1H, CH=N), 8.30 (s, 1H,
NHCH ), 3.80 (s, 3H, OCH ), 6.45-6.99 (m, aromatic), 1.13 (t, 3H, CH ).
3
3
3
5

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2
.1.3. Preparation of [Ni(Msal-etsc)(PPh )] (3):
3
The complex 3 was prepared by the procedure as used for (1) with 3-methoxy
salicylaldehyde-4(N)-ethylthiosemicarbazone [H -Msal-etsc] (0.078 g; 0.3058 mmol) and
2
[
NiCl (PPh ) ] (0.200 g; 0.3058 mmol). Yield: 64 %. M.p. 244 °C. Anal. Calcd for
2 3 2
C H N O SNiP: C, 60.86; H, 4.93; N, 7.34; S, 5.60. Found: C, 60.81; H, 4.87; N, 7.29; S, 5.52
29
28
3
2
-1
-1
%
. FT-IR (cm ) in KBr: 3310 (ν ), 1536(νC=N), 1276 (νC-O), 795 (ν ), 1452, 1089, 697 cm
OH
C-S
3
-1
-1
(
for PPh ) ; UV- vis (CH Cl ), λmax: 232 (21150) nm (dm mol cm ) (intra-ligand transition),
3
2
2
3
-1
-1
1
3
8
3
2
62 (18560) nm, 371 (17450) nm (dm mol cm ) (LMCT s→d); H NMR (DMSO-d6, ppm):
.54 (s, 1H, CH=N), 7.78 (br s, 1H, NHC H ), 3.75 (s, 3H, OCH ), 6.79-7.78 (m, aromatic),
2
5
3
.13-3.14 (m, 2H, CH ), 1.04 (t, 3H, CH ).
2
3
.1.4. Preparation of [Ni(Msal-ptsc)(PPh )] (4):
3
The complex 4 was prepared by the procedure as has been used for (1) with 3-methoxy
salicylaldehyde-4(N)-phenylthiosemicarbazone [H -Msal-ptsc] (0.092 g; 0.3058 mmol) and
2
[
NiCl (PPh ) ] (0.200 g; 0.3058 mmol).Yield: 74 %. M.p. 261 °C. Anal. Calcd. for
2 3 2
C H N O SNiP: C, 63.89; H, 4.55; N, 6.77; S, 5.17. Found: C, 63.82; H, 4.49; N, 6.73; S,
33
28
3
2
-1
-1
5
.11%. FT-IR (cm ) in KBr: 1554 (νC=N), 1310 (νC-O), 743 (ν ), 1435, 1096, 694 cm (for
C-S
3
-1
-1
PPh ) ;UV- vis (CH Cl ), λ : 238 (15270) nm (dm mol cm ) (intra-ligand transition); 366
3
2
2
max
3
-1
-1
3
-1
-1
(
14360), 375 (13650) nm (dm mol cm ) (LMCT s→d), 424 (12800) nm (dm mol cm )
1
(
forbidden (d→d) transition); H NMR (DMSO-d6, ppm): 8.70 (s, 1H, CH=N), 9.44 (s, 1H,
NHPh), 3.41 (s, 3H, OCH ), 6.61-7.87 (m, aromatic).
3
2
.2. DNA binding study:
Various concentrations (0.05-0.5 µM) of CT-DNA solution was dissolved in a tris HCl
buffer (pH 7) were added to the nickel complexes 1-4 (1 µM). Absorption spectra were recorded
after equilibrium at 20 °C for 10 min. Emission measurements were carried out by using a
JASCO FP- 6600 spectrofluorimeter. Tris–buffer was used as a blank to make preliminary
adjustments. The excitation wavelength was fixed and the emission range was adjusted before
measurements. All measurements were made at 20 °C. For emission spectral titrations, complex
concentration was maintained constant as 1 µM and the concentration of DNA was varied from
0
.05 to 0.5 µM. The emission enhancement factors were measured by comparing the intensities
at the emission spectral maxima under similar conditions. In order to find out the mode of
6

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attachment of CT DNA to the complexes (1-4) fluorescence quenching experiments of EB-DNA
were carried out by adding 10 µM nickel(II) complexes with 10 µl every time to the samples
containing 12 µM EB, 12 µM DNA and tris-buffer (pH=7). Before measurements, the system
was shook and incubated at room temperature for ~5 min. The emission was recorded at 530–
7
50 nm.
2
.3. Bovine serum albumin binding study:
The protein binding study was performed by tryptophan fluorescence quenching
experiments using bovine serum albumin (BSA, 1µM) as the substrate in phosphate buffer (pH
). Quenching of the emission intensity of tryptophan residues of BSA at 347 nm (excitation
7
wavelength at 278 nm) was monitored using complexes 1–4 as quenchers with increasing
complex concentration. Synchronous fluorescence spectra of BSA with various concentrations of
complexes were obtained from 300 to 400 nm when ∆λ = 60 nm and from 290 to 500 nm when
∆
λ = 15 nm. The excitation and emission slit widths were 5 and 6 nm, respectively. Fluorescence
and synchronous measurements were performed using a 1 cm quartz cell on an JASCO FP 6500
spectrofluorimeter.
2
.4. Cell-culture:
Human lung adenocarcinoma cells, A549 were obtained from NCCS, Pune, India. Cells
were grown in DMEM and 10% FBS (v/v), containing 100 units/ml penicillin, 30µg/ml
streptomycin and 20µg/ml gentamycin in a CO incubator with 5% CO .
2
2
2
.4.1. Cell proliferation (MTT) assay:
Effect of the complexes on the viability of human lung cancer cells (A549) was assayed
by the 3-(4,5-dimethylthiazol-2-yl)- 2,5- diphenyltetrazolium bromide (MTT) assay [63]. The
cells were seeded at a density of 10,000 cells per well, in 200 µl DMEM medium and were
allowed to attach overnight in a CO incubator. Then flick off the media and the complexes (1-4)
2
dissolved in DMSO and diluted in cell culture media were added to the cells at a final
concentration of 1, 10, 25 and 50 µM. After 48 hours, the wells were treated with 20 µl MTT (5
mg/ml PBS) and incubated at 37 °C for 4 hours. The purple formazon crystals formed were
dissolved in 200 µl DMSO and read at 570 nm in a micro plate reader.
7

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2
.4.2. Release of lactate dehydrogenase (LDH):
LDH activity was determined by the linear region of a pyruvate standard graph using
regression analysis and expressed as percentage (%) leakage as described previously [64]. Briefly, to a
set of tubes, 1ml of buffered substrate (lithium lactate) and 0.1ml of the media or cell extract
were added and tubes were incubated at 37 °C for 30 min. After adding 0.2 ml of NAD solution,
the incubation was continued for another 30 min. The reaction was then arrested by adding 0.1ml
of DNPH reagent and the tubes were incubated for further period of 15 min at 37 °C. After this,
0
.1ml of media or cell extract was added to blank tubes after arresting the reaction with DNPH.
.5 ml of 0.4 N sodium hydroxide was added to all the tubes. The colour developed was
3
measured at 420 nm in a Shimadzu UV/visible spectrophotometer. The amount of LDH released
was expressed as percentage.
2
.4.3. Nitric oxide (NO) assay:
The amount of nitrite was determined by the method of Stuehr and Marletta [65]. Nitrite
reacts with Griess Reagent to give a coloured complex measured at 540 nm. To 100 µl of the
medium, 50 µl of Griess reagent I was added, mixed and allowed to react for 10 min. This was
followed by 50 µl addition of Griess reagent II, the reaction mixture was mixed well and incubated for
another 10 min at room temperature. The pink colour developed was measured at 540 nm in a
microquant plate reader (Biotek Instruments).
2
.4.4. Wound healing assay:
For cell motility determination, A549 cells (1 x10 cells/ml) were plated in six well tissue
5
culture plates and grown to 80–90% confluence. After aspirating the medium, the centers of the
cell mono layers were scraped with a sterile micropipette tip to create a denuded zone (gap) of
constant width. Subsequently, cellular debris was washed with PBS, and A549 cells were
exposed to complexes 1- 4. The wound closure was monitored and photographed at 48 h with an
Olympus inverted microscope and camera.
2
.4.5. Reactive Oxygen Species (ROS) generation:
Relative changes in intracellular reactive oxygen species in A549 cells were monitored
using a fluorescent probe, 2′,7′ -dichlorofluoresceindiacetate (DCFH-DA) [66].The treatment
8

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groups were maintained as described for LDH leakage and a time course for ROS generation by
complexes was done. Cells were incubated with 10µM of DCFH-DA and incubated for 30
min, followed by incubation with complexes (1-4) for different time periods(15min, 30min and
1
hour). One treatment group with 50 µM H O was included to serve as a positive
2 2
control. Cells were then harvested, centrifuged, washed and re suspended in PBS and read
in a Hitachi spectrofluorimeter using excitation 480 nm, emission 520 nm. The estimations
were carried out thrice in triplicate, ensuring each time that the number of cells per treatment
group were the same to ensure reproducibility. The values were expressed as % relative
fluorescence as compared to the control.
2
.4.6. Cellular antioxidant status:
After completing the treatment schedule, cell extracts were prepared by sonication in
Tris-EDTA-PMSF buffer and the protein content of the supernatant was determined by Lowry
method [67] and used for antioxidant assays. Both reagent blanks and enzyme blanks were
measured for all assays. All assays were carried out in the linear range and expressed as specific
activities of enzymes. The intracellular concentration of reduced glutathione was estimated by
the fluorometric assay as described by Pereira-Caro et al. [68] using ortho-pthalaldehyde (OPT).
Superoxide dismutase (SOD) [69], Catalase (CAT) [70], Glutathione-S-transferase (GST) [71],
and Glutathione peroxidase (GPx) [72] activities were determined as described previously.
2
.4.7. Measurement of mitochondrial transmembrane potential:
Mitochondrial energization was determined as the retention of the dye 3, 3′
dihexyloxacarbocyanine iodide [DiOC (3)] in normal cells and exclusion by the cells with
6
mitochondrial membrane damage. Cells were treated with complexes 1-4 as described in LDH
leakage experiment. After the treatment period the medium was flicked off and washed twice with
o
PBS. Then the cells were treated with 50 nM [DiOC (3)] at 37 C for 30 min and the cells were washed
6
twice with PBS. The cells were collected, re-suspended in 2 ml PBS and fluorescence intensity was
measured in a fluorescent spectrophotometer using Excitation/Emission as 488nm/500nm.
2
.4.8. Apoptotic Cell Detection by Propidium iodide:
After treatment with complexes for 48 hours, the cells were collected, washed with PBS,
o
fixed overnight with 70% ethanol at 4 C, and incubated with propidium iodide at room
temperature for 30 minutes. Nuclear morphology was examined using fluorescence microscope.
9

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2
.4.9. Western blot analysis:
Cells were collected and washed twice in PBS, then lysed in ice-cold lysis buffer (50 mM
Tris–HCl, pH-7.4, 150 mM NaCl, 5 mM EDTA, 50 mM NaF, 1% Triton X-100, 1mM sodium-
orthovanadate, 1mM phenylmethanesulfonylfluoride, 1mg/ml aprotinin, 2 µg/ml pepstatin-A,
and 2 µg/ml leupeptin) on ice for 1h. Cell lysates were then centrifuged for 15 min at 13,000 rpm
at 4°C. Proteins were separated using SDS-PAGE and transferred to PVDF membrane. The blots
were blocked with 5% non-fat milk in TBST at RT for 1h and incubated overnight with the
o
appropriate primary antibody at 4 C. After wash, the blots were incubated with peroxidase-
conjugated secondary antibody for 1h. Bands were monitored using DAB system. GAPDH were
used as an internal control.
2
.5. DNA fragmentation:
DNA fragmentation analysis was carried by the method of Gong et al [73]. The cells are
prefixed in 70% ethanol, DNA is extracted with 0.2 M phosphate-citrate buffer at pH-7.8, and
the extract is sequentially treated with RNase A and proteinase K and then subjected to
electrophoresis.
3
. Results and discussion
The stoichiometric reactions of [NiCl (PPh ) ] with a series of 3-methoxy-4(N)-
2
3 2
1
–4
substituted thiosemicarbazones (H2L)
in 1 : 1 ethanol/dichloromethane resulted in the
formation of new complexes 1-4 (Scheme 1), where the substituted thiosemicarbazones acted as
1
2-4
a tridentate ONS ligand. Among them, H2L acted as a monobasic tridentate and (H2L) acted
as dibasic tridentate ligands. The analytical data of which confirmed the stoichiometry of the
complexes (1–4). The structures of the complexes (1, 2 and 4) were confirmed by the X-ray
crystallographic study. The new complexes (1–4) are soluble in common organic solvents such
as dichloromethane, chloroform, benzene, acetonitrile, ethanol, methanol, dimethylformamide
and dimethylsulfoxide.
3
.1. Spectroscopic studies:
The infra red spectra of the ligands (H2L) exhibited ν(OH) vibration in the region
1
–4
-1
3
339–3458 cm , which disappeared completely after complexation with the nickel(II) ion
showing deprotonation prior to coordination through oxygen atom in all the four complexes (1–
-1
4
). It is further confirmed with the downfield shift of 28–43 cm for ν
[74]. An azomethine
(C–O)
10

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-1
nitrogen ν(C=N) band appeared at the region 1536–1593 cm in the ligands has been shifted to
-1
lower frequency in all the complexes (1536–1556 cm ) indicating the coordination of
azomethine nitrogen atoms [75]. As the ligand contains a thioamide (-NH-C=S) functional group
it may exist in thione- thiol tautomerization. The absence of the ν(S-H) stretch in the region 2500-
-1
-1
2
600 cm and presence of ν
stretch in the region 3150-3350 cm in the IR spectrum of the
N-H)
(
ligands indicate thione form in the solid state. This is further confirmed from the presence of a
-1
strong band in the region 771-795 cm corresponding to the ν(C=S) stretching which was
completely disappeared in the spectra of the new complexes 2-4 and the appearance of a new
-1
band at 738-743 cm due to ν
vibration, indicated the coordination of the sulfur atom after
(C-S)
enolisation followed by deprotonation [76,77]. However, the absence of the ν(S-H) stretch and
-1
presence of ν(N-H) stretch at 3055 cm for complex 1 indicate the involvement of the thione
sulphur in the coordination rather than thiolate [51]. Moreover, the characteristic absorption
bands corresponding to the presence of triphenylphosphine were also present in the expected
region [75]. The electronic spectra of nickel(II) complexes displayed four to five bands in the
region around 232-424 nm. The band appeared at 232-303 nm has been assigned to intra ligand
transition and the band at 341-375 nm to LMCT (s→d) and the shoulder at 410-424 nm to
1
1–4
forbidden (d→d) transition [78,79]. The H-NMR spectra of the ligands (H2L)
and the
corresponding complexes (1-4) recorded in DMSO showed all the expected signals. In the
1
–4
spectra of (H2L) , a sharp singlet corresponding to the phenolic –OH group has appeared at
1.34-11.76 ppm (Fig. S1-S8). However, this singlet completely disappeared in all the four
complexes confirmed the involvement of phenolic oxygen in coordination [54]. The spectra of
1
1
–4
(
H2L) showed a singlet at δ 9.13-10.0 ppm corresponding to (N(2)H-C=S) group [80], but in
complexes 2 -4 there was no resonance attributable to N(2)H, indicating coordination of the
thiolate sulphur atom of the ligand in the anionic form after deprotonation at N(2). However, in
the spectrum of complex 1, a singlet occurred at δ 9.12 ppm corresponding to (N(2)H-C=S-)
1
–4
group indicating the thionic form of ligand even after complexation [51]. In the spectra of H2L
and the complexes, a complex multiplet appeared at 6.37–7.87 ppm was assigned to aromatic
protons of the ligands and triphenylphosphine [75] and a singlet corresponding to the –OCH3
group occurred at the 3.41–3.80 ppm range [53]. Two singlets observed at 8.37–8.40 ppm and
8
.37–9.40 ppm have been assigned to azomethine and terminal -NH protons of the ligands 2–4.
Whereas, in the complexes a singlet at 8.30-8.70 ppm was observed for the presence of
11

ACCEPTED MANUSCRIPT
azomethine protons and another singlet observed around 8.12-9.20 ppm in the complexes 2, 3
and 4 was assigned to the terminal NH protons of the ligands. Moreover, in complex 1, there
were two broad singlets appeared at 7.90 and 8.08 ppm corresponding to NH protons of the
2
2
3
coordinated ligand. A triplet observed around 1.03–2.90 ppm in the spectra of H2L , H2L and
the complexes (2 and 3), was assigned to the presence of methyl group of protons. Further, a
3
multiplet at 3.12–3.58 ppm was also observed corresponding to the methylene protons of H2L
and complex 3 [53].
3
.2. X-ray crystallography:
The ORTEP diagram of the complexes 1, 2 and 4 with the numbering scheme is shown in
Fig. 1–3 and S9. The most significant parameters for these compounds are shown in Tables 1 and
2
. Crystallographic analysis revealed that complexes 1, 2 and 4 crystallized in the monoclinic
1
, 2 , 4
crystal system with space group, C2/c, Cc and P21 respectively. Among the ligands (H2L)
,
1
2
4
H2L acted as a monobasic tridentate whereas, (H2L) and (H2L) acted as a dibasic tridentate. In
1
the complex 1, the ligand (H2L) coordinated to nickel by utilizing its phenolic oxygen, N1
nitrogen and thione sulphur atoms by forming one six member and another five member ring
with a bite angle of [88.0(1) (S-Ni-N(1)]. The presence of a chloride ion outside the coordination
2
+
sphere compensates the charge of nickel as Ni , giving ionic nature to the complex (1). The Ni–
S 2.144(1) Å, Ni–P 2.2130 Å, Ni-N 1.892(3) Å and Ni–O 1.850(3) Å bond lengths are
1
comparable with the values of already reported ionic complex [51]. From the H-NMR spectral
studies, there was a detectable proton signal at δ 9.12 ppm corresponding to the (N(2)H-C=S)
group. However, the position of this proton (hydrogen) could not be located in the X-ray
crystallographic analysis. While dealing with the hydrogen bonding, we could be able to find the
(D…A) distance corresponding to the N(2)…Cl(1) bond between the imine nitrogen and the
chloride ion which was present outside the coordination sphere. The chloride ion involved in one
intra molecular hydrogen bonding with imine nitrogen and intermolecular hydrogen bonding
with (H3A) and (H3B) protons of N(3) amine of second and third molecules respectively (Table
S1). Interestingly, this intra and inter molecular hydrogen bonding resulting a 2D network which
contains parallelogram like structure generated by two chloride ions and two amine nitrogen
atoms (Fig. S9). The variation in the Ni-N, Ni-O, Ni-S and Ni-P bond distances and significant
deviation from ideal angle S(1)-Ni-O(1) [177.52(9) °] and P(1)-Ni-N(1) [176.8(1) °] brought a
distorted square planar geometry to the molecule.
12

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In the complexes 2 and 4, the Ni(II) ion is coordinated to the dibasic tridentate ligands
through thiolate sulfur (Ni–S bond distances of 2.131(2) A ˚ and 2.158(1) A ˚respectively),
phenolic oxygen (Ni–O bond distances of 1.855(5) A ˚ and 1.845(3) A ˚ respectively) and the
nitrogen atom (Ni–N bond distances of 1.911(7) A ˚ and 1.889(4) A ˚ respectively). The
remaining binding site is occupied by the triphenylphosphine unit (Ni–P(1) bond distances of
2
.224(2) A ˚ and 2.226(1) A ˚ respectively) with a bite angle [S(1)–Ni(1)–N(1)] of 86.4(2) ° for
2
and 85.8(1) ° for 4. The Ni–S, Ni–P, Ni–O and Ni–N bond lengths are comparable with those
of the reported complexes [51,52,74,81]. The [S(1)–Ni(1)–O(1)] bond angles found are 178.5(2)
°
for 2 and 178.6(1) ° for 4 and [P(1)–Ni(1)–N(1)] bond angles found are 173.3(2) ° for 2 and
71.6(1) ° for 4 which deviate considerably from the ideal angle of 180° causing significant
1
distortion in the square planar geometry of the complexes. It is observed from the trans angle
P(1)–Ni(1)–N(1)] of the above said complexes, the deviation from the ideal geometry is a bit
more in 4 as compared with 1 and 2.
[
3
.3. DNA binding studies:
For evaluating the anticancer property of any newly synthesized complex, DNA binding
is the predominant property looked for in pharmacology and hence, the interaction between DNA
and metal complexes is of paramount importance in understanding the mechanism. Thus, the
mode and propensity for binding of the new nickel(II) complexes 1- 4 to CT DNA were studied
with the help of electronic absorption and fluorescence quenching techniques.
The absorption spectra of complexes (1–4) at constant concentration (1 µM) and with
different concentrations of CT-DNA (0.05–0.50 µM) are given in Fig. 4. The absorption spectra
of complex 1 mainly consist of four resolved bands [intra ligand (IL) and charge transfer (CT)
transitions] centered at 257 nm (IL), 341nm, 364 nm and 372 nm (CT). As the DNA
concentration is increased, the hypochromism of 85.45 % with a blue shift of 3 nm was observed
in the intra ligand band. The CT bands at 341, 364 and 372 nm showed modest hyperchromism
with negligible absorption shifts. In addition, the binding of complex 1 to CT DNA led to
isosbestic spectral change with the isosbestic point at 302 nm. For complex 2, upon addition of
DNA, the intra ligand band at 257 nm exhibited hypochromism of 92.79 % with a 9 nm blue
shift. The CT band at 359 nm showed 22.35 % of hyperchromism without the wavelength shift in
the absorption maxima. The binding behaviour of complexes 3 and 4 is quite similar. As the
DNA concentration is increased, the intra ligand band at 258 nm (for 3) and 257 nm (for 4)
13

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exhibited hypochromism of 93.01% and 73.84 % respectively with 2-5 nm blue shift. The CT
bands showed slight hyperchromism at 362, 371 nm (for 3) and 375, 424 nm (for 4) without any
shift upon addition of CT-DNA. A distinct isosbestic point appeared at 302 nm and 300 nm for
complexes 3 and 4. The observed hypochromic effect with blue shift suggested that complexes
(
1–4) bind to CT-DNA by intercalation mode [82]. The intrinsic binding constant K was
b
determined by using Stern volmer equation (1) [83,84].
DNA] ⁄ [ε -ε ]) = [DNA] ⁄ [ε -ε ]+ 1 ⁄ Kb[ε -ε ]
[
(1)
a
f
b
f
b
f
The absorption coefficients ε , ε , and ε correspond to Aobsd/[complex], the extinction
a
f
b
coefficient for the free complex and the extinction coefficient for the complex in the fully bound
form, respectively. The slope and the intercept of the linear fit of the plot of [DNA]/[ε - ε ]
a
f
versus [DNA] give 1/[ε - ε ] and 1/K [ε - ε ], respectively. The magnitude of the binding
a
f
b
b
f
strength of compounds with CT DNA was calculated by the intrinsic binding constant Kb (Table
), which can be determined by monitoring the changes in the absorbance in the IL band at the
3
corresponding λmax with increasing concentration of DNA and is given by the ratio of the slope to
the Y intercept in plots of [DNA]/(ε - ε ) versus [DNA] (Fig. 5). From the binding constant
a
f
values, it is inferred that complex 3 exhibited better binding than other complexes.
The emission spectra of new nickel (II) complexes 1-4 showed fluorescence emission at
4
12, 715, 509 and 409 nm respectively (Fig. S10). Addition of CT DNA to the complex solution
resulted in hypochromism of 37.46 %, 63.97 %, 81.26 % and 16.69 % respectively for
complexes 1-4 by decreasing intensity with 1-2 nm blue shifts in the absorption maxima. The
marked decreases in the fluorescence intensity of complexes indicate the intercalative binding
mode of DNA. The binding of the complexes 1-4 to CT- DNA through intercalation mode has
further been confirmed by competitive fluorescence quenching experiments. EB (3,8-diamino-5-
ethyl-6-phenylphenanthrium bromide) is an intercalator that gives a significant increase in
fluorescence emission when bound to DNA and it can be quenched by the addition of second
DNA binding molecule by either replacing the EB and/or by accepting the excited state electron
of the EB through a photoelectron transfer mechanism. Upon addition of complexes 1-4 to CT-
DNA which was pretreated with EB caused significant reduction in the emission intensity at 605
nm (Fig. S11). Which indicates that the replacement of the EB fluorophore by respective
complexes. The quenching extents of 1-4 were evaluated qualitatively by employing Stern–
Volmer equation (2).
14

ACCEPTED MANUSCRIPT
I /I = K [Q] + 1
(2)
0
sv
Where I is the emission intensity in the absence of compound, I is the emission intensity in the
o
presence of compound, K is the quenching constant, and [Q] is the concentration of the
sv
compound. The K values have been obtained as a slope from the plot of I /I vs. [Q] (Fig. S12).
sv
0
3
3
3
-1
3
-1
It was found to be 4.33 x10 , 5.19 x10 , 5.31 x 10 M and 3.87 x 10 M respectively for
complexes 1-4, Further, the apparent DNA binding constants (Kapp) were calculated using eqn
(3):
K [EB] = K [complex]
(3)
EB
app
where [complex] is the value at 50 % reduction in the fluorescence intensity of EB, KEB (1.0
7
-1
x10 M ) is the DNA binding constant of EB, [EB] is the concentration of EB = 12 µM). K
app
5
5
5
5
-1
values were 5.19 x 10 , 6.22 x 10 , 6.37 x 10 and 4.64 x 10 M for complexes 1–4 respectively.
From these experimental data, it is seen that the complex 3 replaces the EB more effectively than
the other complexes, which is in agreement with the results observed from DNA binding study.
Furthermore, the observed quenching constants and binding constants of the new complexes
suggest that the interaction of all the complexes with DNA may be intercalative.
3
.4. Quenching mechanism of BSA by complexes:
Bovine serum albumin (BSA) is the most extensively studied serum albumin, due to its
structural homology with human serum albumin (HSA). Binding of Schiff base metal complexes
with the most abundant carrier proteins (serum albumins) have also been an area of interest as
such drug–protein binding greatly influences absorption, drug transport, storage, metabolism and
excretion properties of typical drugs in vertebrates [85]. In addition, it has been shown that
binding of Schiff-base metal complexes to bovine serum albumin (BSA) have enhanced the
antioxidant capacity of BSA in ROSs scavenging for about more than 10-times [86]. Since serum
albumins are well known to bind with small aromatics, the possible binding interactions of the
nickel(II) thiosemicarbzone complexes (1-4) with BSA have been investigated by absorption
/
emission-titration experiments at room temperature. In the absorption spectra of BSA, the
addition of 10 µM concentrations of the four complexes (1-4) (Fig. S13) showed enhanced
absorption intensity of BSA with a blue shift of complex–BSA spectrum (from 273 to 269 nm).
The formation of non-fluorescence ground-state complex induced the change in absorption
spectrum of fluorophore and possible quenching mechanism of BSA by complexes (1-4) was
found as static quenching [87].
15

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3
.4.1. Fluorescence quenching studies of BSA:
A solution of BSA (1 µM) was titrated with various concentrations of the complexes (0–
2
5 µM). Fluorescence spectra were recorded in the range of 290–500 nm upon excitation at 280
nm. The changes observed on the emission spectra of BSA by the addition of increasing amounts
of the complexes (1-4) which are shown in Fig. 6. Upon the addition of complexes 1–4 to BSA, a
significant decrease in the fluorescence intensity was observed at 347 nm with hypochromism of
8
7.74 %, 93.36 %, 93.67 % and 86.12 % respectively accompanied by 1-6 nm blue shifts. The
observed quenching may be attributed to the possible changes in secondary structure of protein
and this indicating the binding of complex to BSA [88]. According to Stern−Volmer quenching
5
-1
5
-1
6
-1
equation, eq 2. The K value was found to be 6.37 x 10 M , 6.49 x 10 M , 1.33 x 10 M and
SV
5
-1
2
.94 x 10 M corresponding to complexes 1, 2, 3 and 4 respectively. The observed linearity in
the plots(Fig. S14; Table 4) indicates the ability of the complexes to quench the emission
intensity of BSA. From K values, the complex 3 exhibited better protein-binding ability with
SV
enhanced hydrophobicity. The results obtained from protein binding are reliable with its strong
DNA binding affinity. For the static quenching interaction, if it is assumed that there are similar
and independent binding sites in the biomolecule, the binding constant (K ) and the number of
b
binding sites (n) can be determined according to the method [89] using the Scatchard equation
(4).
log [(I −I)/I]= log K + n log [Q]
(4)
0
b
where, in the present case, K is the binding constant for the complex–protein interaction and ‘n’
b
is the number of binding sites per albumin molecule, which can be determined by the slope and
the intercept of the double logarithm regression curve of log [(I −I)/I] versus log[Q] (Fig. S15;
0
Table 4).
3
.4.2. Synchronous fluorescence spectroscopic studies of BSA:
Synchronous fluorescence spectral study involves simultaneous scanning of the
excitation and emission monochromators while maintaining a constant wavelength interval
between them. It was used to obtain information about the molecular environment in the vicinity
of the fluorophore moieties of BSA [90]. Synchronous fluorescence spectra show tyrosine
residues of BSA only at the wavelength interval ∆λ of 15 nm whereas tryptophan residues of
BSA at ∆λ of 60 nm. The amount of complexes (1-4) added to BSA (1µM) is increased; there is
16

ACCEPTED MANUSCRIPT
a decrease in the intensity of the fluorescence spectral band corresponding to tyrosine residue
with a red shift (1-5 nm) (Fig. S16). In addition, significant decrease of fluorescence intensity of
tryptophan residues together with a small red shift (1-2 nm) in the emission wavelength was also
observed for the complexes except 4 which displayed hypochromism (Fig. S17). These
experimental results indicate that the metal complexes do affect the microenvironment of both
tyrosine and tryptophan residues during the binding process and synchronous measurements
confirmed the effective binding of all the complexes with BSA.
3
.5. Effect of Ni(II) thiosemicarbazones complexes on cell proliferation:
The effect of new nickel (II) thiosemicarbazone complexes (1-4) on A549 cell line was
determined by MTT. Cells were treated with different concentrations (1-50 µM) of
thiosemicarbazones for different time periods. No cytotoxicity was observed up to 10µM
concentration. The cell proliferation declined in a dose dependent manner when the cells were
exposed to more than 10µM for an incubation period of 12 and 24 hours and there were no
significant reduction in cell proliferation by the complexes when compared with control cells. In
turn, the newly synthesized complexes significantly decreased the A549 cell proliferation with
an exposure time of 48 h. Hence, we selected 48 hour time period as optimum for the action of
the nickel complexes used in the present study (Fig. 7). When the A549 cells were exposed to
complexes (1-4) for 48 h the IC values were nearly 30 µM as shown in table 5. Hence 15 and
5
0
3
0 µM concentrations were used for further studies. Among the four complexes, ethyl substituted
1
6c
thiosemicarbazone effectively declined the proliferation of lung adenocarcinoma cell line.
Light microscopic analyses of the cells treated with nickel complexes showed significant
morphological changes as shown in Fig. 8.
3
.5.1. Cytotoxic effect of Ni(II) thiosemicarbazone on A549 cells:
The cytotoxic effect of complexes 1-4 on A549 cell line was determined by Lactate
dehydrogenase (LDH) leakage and nitric oxide release into the extracellular medium. Even
though there are several cytotoxic enzyme markers are reported, their use has been limited by the
presence of low amount in many cells and hence an elaborate kinetic assay is required to
measure most enzyme activities. LDH is a stable cytoplasmic enzyme which is released into the
culture medium following loss of membrane integrity resulting from apoptosis. LDH activity,
therefore, can be used as an indicator of cell membrane integrity and serves as a general means to
assess cytotoxicity resulting from chemical compounds or environmental toxic factors [91,92]. In
17

ACCEPTED MANUSCRIPT
the present study, significant level of LDH leakage was observed in the cell culture medium of
A549 cell line, when the complexes were treated with two different concentrations for a period
of 48 hours. Complexes had the tendency to increase LDH leakage even at low dose such as
1
5µM whereas LDH liberation after treatment with 30µM concentration was highly significant
in comparison with control indicating that the effect was dose dependent. Complex 3 showed
significant level of LDH leakage in A549 cells compared to cisplatin. This authenticate that
complex 3 was more effective than complex 1, 2 and 4.
NO plays an important role in many physiological processes including vascular
regulation, immune responses and neural communication. Nitric Oxide (NO), produced
endogenously from L-Arginine by the action of NO synthase (NOS), an enzyme existing in three
isoforms. Among them, two isoforms exist constitutively and the third isoform is an inducible
form (iNOS) which is expressed in response to stress. During oxidative stress, it has been shown
that the expression of iNOS was increased which in turn leads to the nitric oxide accumulation.
When excess, nitric oxide damages most of the biomolecules, including DNA and protein [93].
NO is extremely unstable and undergoes rapid oxidative degradation to the stable product
-
nitrite (NO ) which can be spectrophotometrically determined. The level nitric oxide release
2
was significantly increased by the complexes 1-4 treated on lung adenocarcinoma cell line
(A549) compared with control cells. Among them complex 3 is more effective in the induction
of cytotoxicity in terms of both LDH and NO release in A549 cells (Fig. S18).
3
.5.2. A549 cell migration inhibition studies by Ni(II) thiosemicarbazones:
Metastatic cancers have several important characteristics, including the migratory and
invasive activities of tumour cells. In order to examine the inhibitory effect of complexes 1-4 on
cancer cell migration, A549 cells were incubated in the absence or presence of these complexes
for wound migration assay. As shown in Fig. 9, complex treatment strongly suppressed A549
cell migration to the wounded area in a concentration-dependent manner. This indicates that the
complexes could suppress the A549 cell migration, thereby metastasis.
3
.5.3. Ni(II) thiosemicarbazones induced ROS hypergeneration and lipid peroxidation:
Reactive oxygen species (ROS) is a term which encompasses all highly reactive, oxygen-
containing molecules, including free radicals. The role of oxidative stress in vascular diseases,
diabetes, renal ischemia, atherosclerosis, pulmonary pathological states, inflammatory diseases,
and cancer has been well established. ROS are highly reactive metabolites generated during
18

ACCEPTED MANUSCRIPT
normal cell metabolism; however, elevated intracellular ROS could be sufficient to trigger
apoptosis [94,95]. Moreover, apoptotic cell death is reported to be preceded by the following
sequential facts: ROS production, loss of mitochondrial trans-membrane potential, release of
cytochrome C, and activation of caspase-3 [96]. To determine whether ROS are involved in
complex induced apoptotic machinery, the level of ROS in the cells treated with the complexes
1
-4 was measured. The ROS generation was determined for short time periods (15 min, 30 min
and 1 hour) since ROS production is an early event in apoptosis. The results demonstrate that the
ROS generation was significantly increased within 15min and peaked at 30 min (Fig. 10). With
incubation period of 60 min there was decline in the ROS generation. Our results correlate with
the finding of Pelosi et al. [97] in which thiosemicarbazone increased ROS generation in 41M
and SK-BR-3 cell lines. These results revealed the fact that nickel (II) complexes (1-4) mediate
apoptosis by ROS hypergeneration. Ethyl substituted complex induced ROS hypergeneration
more efficiently than the other substitutions.
It has been shown that lipid hydroperoxides and oxygenated products of lipid
peroxidation can participate in the signal transduction cascade, control of cell proliferation and
apoptosis [98]. Hence we further examined the effect of the complexes on lipid peroxidation.
Lipid peroxidation was significantly increased in lung cancer cell lines treated with complex 3
than other complexes (Fig. S19). The complex induced ROS hypergeneration might be the
reason for the production of peroxide free radicals in the drug treated cancer cells compared with
control cells. Level of lipid peroxidation by cisplatin was lower in A549 cells than the
complexes.
3
.5.4. Depletion of intracellular antioxidant pool by nickel(II) thiosemicarbazones:
The presence of antioxidants in cells as a protective mechanism during oxidative stress
and apoptotic cell death has been reported [99]. Oxidative stress indicates an imbalance state
between production of ROS and antioxidant defenses. These defenses include superoxide
dismutase, which converts O to H O , glutathione peroxidase, and/or catalase, which in turn
2
2
2
convert H O to water. Antioxidants also play an important role against oxidant injury induced
2
2
by an excess of ROS generation, by scavenging these species before they affect any
biomolecules. An imbalance in these oxidant defenses leads to depletion of reduced glutathione
(GSH), dissipation of mitochondrial transmembrane potential, lipid peroxidation, altered enzyme
activities, DNA damage and eventual cell death by apoptosis [100]. Several earlier studies have
19

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demonstrated that the onset of apoptosis is associated with a fall of intracellular GSH in different
cellular systems [101]. Hence we analyzed the intracellular GSH content. The total reduced
glutathione was significantly decreased in the cells exposed to complexes (1-4) with control cells
(Fig. S20). Complex 3 effectively reduced the level of reduced glutathione in lung
adenocarcinoma cells than the other complexes. In this study the enzymatic antioxidants such as
SOD, CAT, GPx and GST were significantly decreased by thiosemicarbazone treated A549
cancel cell line compared with control cells. Complex 3 is more effective than other complexes
(Table 6). Even though the complexes could not deplete the antioxidant pool efficiently than
cisplatin, however, exert their cytotoxic effect through the depletion of cellular antioxidant pool.
3
.5.5. Mitochondrial membrane potential (ꢀψM) studies:
The collapse of the mitochondrial transmembrane potential has been shown to promote
(mitochondrial permeability transition) MPT. The MPT is a critical event in cell death following
inhibition of the mitochondrial electron transport chain and is involved in the mechanism of
mitochondrial dysfunction in apoptosis [102]. The MPT involves the formation of a non specific
pore across the inner mitochondrial membrane, permitting the free distribution of ions, solutes,
and small molecular weight molecules (1500 Da) across the membrane [103]. The opening of
MPT induces the release of proapoptotic molecules, including AIF (apoptosis-inducing factor)
and cytochrome C, from the intermembrane space in the cytoplasm [102]. Subsequently,
cytochrome C interacts with Apaf-1, dATP/ATP, and procaspase-9 to form the apoptosome
leading to activation of caspase-9 and downstream effector caspases [104]. The activated caspase
9
in turn activates the effector caspase 3 which on the other hand induces the apoptotic signaling
cascade by activating ICAD.
To check whether the apoptosis induced by nickel complexes is through mitochondrial
mediated pathway, ꢀψM of the cells treated with nickel(II) complexes 1-4 was analyzed. The
results showed that the complexes are highly catastrophic to mitochondrial membrane and
reduces the mitochondrial membrane potential as indicated by the decreased DiOC6 fluorescence
in the cells treated with these complexes in comparison with control cells (Fig. 11). It is obvious
from the results that these complexes 1-4 induces mitochondrial mediated apoptosis in human
lung adenocarcinoma cell lines.
3
.5.6. Nickel complexes induce apoptosis in lung adenocarcinoma cells:
20

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Cells die in response to a variety of stimuli and during apoptosis they do so in a
controlled, regulated fashion. Cell death can be mediated through apoptosis or necrosis.
Apoptosis is a type of programmed cell death and is now recognized as an important mode of
cell death in response to cytotoxic treatments [105]. To check whether the complexes can induce
apoptosis in lung adenocarcinoma cells, the propidium iodide staining was performed. Apoptotic
cells can be distinguished from the normal cells by cell membrane blebbing which is the
morphologic characteristic of apoptosis. Since the complexes showed a dose dependent activity
in all the assays, the further studies were carried out using only the higher concentration of the
complexes (30ꢁM). As shown in Fig. 12 apoptotic bodies were visible in the cells treated with
nickel(II) complexes 1-4. These results confirm the apoptotic inducing nature of the
thiosemicarbazone complexes.
Cleavage of nuclear DNA is regarded as a biochemical hallmark of apoptosis. DNA
fragmentation is a key feature of programmed cell death, and the process is characterized by the
activation of endogenous endonucleases with subsequent cleavage of chromatin DNA into
internucleosomal fragments [106]. In normal cells, Caspase-activated deoxyribonuclease (CAD)
binds with its specific inhibitor and form a complex called ICAD. During apoptosis, caspase-9
damages the nuclear pores in an unknown fashion so that activated caspase-3 can enter the
nucleus to cleave ICAD. This releases the CAD from the complex, which can result in DNA
degradation [107]. To further confirm the apoptotic induction by the complexes, procaspase 3
expression levels and DNA fragmentation analysis were also performed. From the results
obtained it was obvious that the nickel(II) complexes containing-3-methoxy salicylaldehyde
4
(N) substituted thiosemicarbazones induce apoptosis by reducing the levels of procaspase 3
which is an indication of activation of caspase 3 and fragmenting DNA molecule in the cell (Fig.
3 and S21). The extent of apoptosis induction was more with the higher concentration of the
complexes.
. Conclusion
New nickel(II) thiosemicarbazone complexes have been synthesized and characterized by
1
4
various spectro and analytical techniques. The true nature of coordination of the ligand with the
central metal atom was established by X-ray crystallographic studies. From the study, it is
inferred that in complex 1, the ligand coordinated as tridentate monobasic nature and the
remaining complexes the ligands coordinated as tridentate bibasic nature. In order to know about
21

ACCEPTED MANUSCRIPT
their potential binding ability of the new complexes, CT-DNA and BSA protein were taken as
models. The complexes bind to CT-DNA through intercalation mode and with BSA, the
quenching was found as static. Taken together our data entertain the notion that the newly
synthesized nickel complexes induce cytotoxicity in human lung cancer cells (A549) via LDH
and NO release into the culture medium. The complexes could encourage ROS hyper-generation
with subsequent depletion of intracellular antioxidant pool and mitochondrial membrane
depolarization leading to caspase activation and DNA fragmentation which were the hallmarks
of apoptosis. Thus the complexes could induce mitochondrial mediated apoptosis. From all the
above studies, it is clearly evident that complex 3 exhibited higher activity than all other
complexes. In addition, the present investigation lights up the antimetastatic effect of nickel
complexes by inhibiting A549 cell migration. Hence, further studies on animal models to
elucidate the clear mechanism of action of the complexes are highly warranted to unveil the
nickel complexes as anticancer drugs.
Supplementary material
Crystallographic data for [Ni(Msal-tsc)(PPh )].Cl (1) [Ni(MSal-mtsc)(PPh )] (2) and
3
3
[
Ni(Msal-ptsc)(PPh )] (4) have been deposited at the Cambridge Crystallographic Data Centre as
3
supplementary publication (CCDC No. 958106, 958104 and 958105). The data can be obtained
free of charge at www.ccdc.cam.ac.uk/conts/retrieving.html or from the Cambridge
Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK [fax: + 44-1223/336-
0
33; e-mail: deposit@ccdc.cam.ac.uk].
ACKNOWLEDGMENT
The authors gratefully acknowledge Council of Science and Industrial research
CSIR), New Delhi, India and Department of Science and Technology (DST), New Delhi,
India for the financial support.
(
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