Investigation of antitumor potential of Ni(II) complexes with tridentate PNO acylhydrazones of 2-(diphenylphosphino)benzaldehyde and monodentate pseudohalides

Article abstract of DOI:10.1007/s00775-015-1315-x

Square-planar azido Ni(II) complex with condensation product of 2-(diphenylphosphino)benzaldehyde and Girard's T reagent was synthesized and its crystal structure was determined. Cytotoxic activity of the azido complex and previously synthesized isothiocyanato, cyanato and chlorido Ni(II) complexes with this ligand was examined on six tumor cell lines (HeLa, A549, K562, MDA-MB-453, MDA-MB-361 and LS-174) and two normal cell line (MRC-5 and BEAS-2B). All the investigated nickel(II) complexes were cytotoxic against all tumor cell lines. The newly synthesized azido complex showed selectivity to HeLa and A549 tumor cell lines compared to the normal cells (for A549 IC₅₀ was similar to that of cisplatin). Azido complex interferes with cell cycle phase distribution of A549 and HeLa cells and possesses nuclease activity towards supercoiled DNA. The observed selectivity of the azido complex for some tumor cell lines can be connected with its strong DNA damaging activity.

Full text of DOI:10.1007/s00775-015-1315-x

J Biol Inorg Chem (2016) 21:145–162
DOI 10.1007/s00775-015-1315-x
ORIGINAL PAPER
Investigation of antitumor potential of Ni(II) complexes
with tridentate PNO acylhydrazones of 2‑(diphenylphosphino)
benzaldehyde and monodentate pseudohalides
Božidar Čobeljic´1 · Milica Milenkovic´1 · Andrej Pevec2 · Iztok Turel2 ·
Miroslava Vujcˇic´3 · Barbara Janovic´3 · Nevenka Gligorijevic´4 · Dušan Sladic´1 ·
Siniša Radulovic´4 · Katarina Jovanovic´4 · Katarina Anđelkovic´1
Received: 15 July 2015 / Accepted: 14 November 2015 / Published online: 26 November 2015
© SBIC 2015
Abstract Square-planar azido Ni(II) complex with con-
densation product of 2-(diphenylphosphino)benzalde-
hyde and Girard’s T reagent was synthesized and its crys-
tal structure was determined. Cytotoxic activity of the
azido complex and previously synthesized isothiocyanato,
cyanato and chlorido Ni(II) complexes with this ligand
was examined on six tumor cell lines (HeLa, A549, K562,
MDA-MB-453, MDA-MB-361 and LS-174) and two nor-
mal cell line (MRC-5 and BEAS-2B). All the investigated
nickel(II) complexes were cytotoxic against all tumor cell
lines. The newly synthesized azido complex showed selec-
tivity to HeLa and A549 tumor cell lines compared to the
normal cells (for A549 IC50 was similar to that of cisplatin).
Azido complex interferes with cell cycle phase distribution
of A549 and HeLa cells and possesses nuclease activity
towards supercoiled DNA. The observed selectivity of the
azido complex for some tumor cell lines can be connected
with its strong DNA damaging activity.
Keywords Nickel(II) complexes · Girard’s T reagent ·
Phosphine ligands · Cytotoxicity · DNA interactions
Introduction
Hydrazones of 2-(diphenylphosphino)benzaldehyde repre-
sent an interesting group of phosphine ligands which pos-
sess a combination of soft and hard electron donor atoms
and exhibit versatile coordination modes depending on
reaction conditions and the nature of central metal ion
[1–13]. Of particular importance is the observed biologi-
cal activity (antimicrobial and antitumor) of these ligands
and their complexes [13–21]. Recently, we synthesized two
series of square-planar Ni(II) complexes with tridentate
PNO acylhydrazones of 2-(diphenylphosphino)benzalde-
hyde and studied their antimicrobial and antitumor activity
[20, 21]. In Ni(II) complexes with the condensation product
of 2-(diphenylphosphino)benzaldehyde and ethyl carbaz-
ate (HL1) (Scheme 1) [20] as well as in Ni(II) complexes
with 2-(diphenylphosphino)benzaldehyde 4-phenylsemi-
carbazone (HL2) (Scheme 2) [21] the monoanionic ligand
is coordinated through the phosphorus, imine nitrogen
and carbonyl oxygen atoms while the fourth coordination
place is occupied with one of three different pseudohalides:
cyanate, thiocyanate or azide. All Ni(II) complexes with
HL1 ligand showed activity to all investigated tumor cell
lines (A549—lung adenocarcinoma cells, MDA-MB-361—
breast cancer cell line, HeLa—human cervix carcinoma
cells, FemX—melanoma cell line, LS-174—colon can-
cer cells and K562—human myelogenous leukemia cells)
and were more active than the ligand. The azido complex
showed activity to K562 leukemia cells of similar intensity
as cisplatin and was less cytotoxic to the normal MRC-5
cell line (human fetal lung fibroblast cells) than cisplatin.
Electronic supplementary material The online version of this
article (doi:10.1007/s00775-015-1315-x) contains supplementary
material, which is available to authorized users.
* Katarina Anđelković
kka@chem.bg.ac.rs
1
Faculty of Chemistry, University of Belgrade, Studentski trg
12-16, 11000 Belgrade, Serbia
2
Faculty of Chemistry and Chemical Technology, University
of Ljubljana, Vecˇna pot 113, 1000 Ljubljana, Slovenia
3
Institute of Chemistry, Technology and Metallurgy,
University of Belgrade, Njegoševa 12, P.O. Box 815,
11000 Belgrade, Serbia
4
Laboratory for Experimental Pharmacology, Department
of Experimental Oncology, Institute for Oncology
and Radiology of Serbia, Pasterova 14, Belgrade, Serbia
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Scheme 1 Synthesis of con-
densation product of 2-(diphe-
nylphosphino)benzaldehyde and
ethyl carbazate (HL1) (a) and
its Ni(II) complexes (b)
All Ni(II) complexes exhibited higher cytotoxicity to K562
cells than to other cell lines. The most cytotoxic complex
to K562 and MDA-MB-361 cells was the azido complex,
to LS-174 cell line the isothiocyanato complex, and to the
normal MRC-5 cells the cyanato complex, while there was
no significant difference in cytotoxic activity between the
complexes to the other investigated cell lines. Complexes
of Ni(II) with HL1 interfered with the cell cycle of tumor
cells and induced plasmid DNA cleavage [20]. All Ni(II)
complexes with HL2 ligand showed moderate to strong
activity to all investigated tumor cell lines (HeLa, FemX,
A549, LS-174, MDA-MB-453 and K562). Azido complex
and HL2 ligand induced perturbation in cell cycle pro-
gression (arrest in the S phase, decrease of percentage of
cells in G1 phase and no significant increase of apoptotic
fraction of cells) [21]. The observed cytotoxicity of azido
complex is a consequence of DNA damaging. Previously
synthesized octahedral and square-planar Ni(II) complexes
with the condensation product of 2-(diphenylphosphino)
benzaldehyde and Girard’s T reagent (HLCl) and differ-
ent monodentates, i.e., chloride, cyanate and thiocyanate
[22] (Scheme 3), showed a moderate antibacterial activity
not only against laboratory control strains but also on clini-
cal isolates of E. coli and P. aeruginosa strains which were
resistant to most of the clinically used antibiotics [23].
In this study, the synthesis and characterization of
square-planar azido Ni(II) complex with condensa-
tion product of 2-(diphenylphosphino)benzaldehyde and
Girard’s T reagent is described. As a continuation of our
previous investigation of biological activity of Ni(II) com-
plexes with acylhydrazones of 2-(diphenylphosphino)ben-
zaldehyde [20–23] cytotoxic activity and DNA damaging
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147
Scheme 2 Synthesis of
2-(diphenylphosphino)benza-
ldehyde 4-phenylsemicarba-
zone (HL2) (a) and its Ni(II)
complexes
potential of azido, isothiocyanato, cyanato and chlorido
Ni(II) complexes with 2-(diphenylphosphino)benzaldehyde
and Girard’s T reagent has been examined.
All spectra were measured at room temperature. UV–Vis
spectra were recorded at Shimadzu 1800 UV–Vis spec-
trometer. Elemental analyses (C, H, N) were performed
by standard micro-methods using the ELEMENTARVario
ELIII C.H.N.S.O analyzer. Molar conductivity was meas-
ured at room temperature (23 °C) on a digital conductivity-
meter JENWAY-4009.
Experimental
Materials and methods
Synthesis of HLCl
2-(Diphenylphosphino)benzaldehyde (97 %) and Girard’s
T reagent (99 %) were obtained from Aldrich. IR spectra
were recorded on a Perkin–Elmer FT-IR 1725X spectrom-
Ligand HLCl was obtained by condensation reaction of
2-(diphenylphosphino)benzaldehyde and Girard’s T rea-
gent using a previously reported method [19]. Yield: 110 mg
(47 %) Mp 216 °C. IR (vs-very strong, s-strong, m-medium,
w-weak): 3524 (w), 3403 (w), 3308 (w), 3050 (m), 3018
(m), 2970 (m), 2930 (m), 2890 (m), 2817 (w), 1686 (vs),
eter using the ATR technique in the region 4000–400 cm−1
.
1H NMR (500 MHz) and 13C NMR (125 MHz) spectra
were recorded on a Bruker Avance 500 spectrometer in
1
DMSO–d6 using TMS as internal standard for H and 13C.
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Scheme 3 Synthesis of condensation product of 2-(diphenylphosphino)benzaldehyde and Girard’s T reagent (HLCl) (a) and its Ni(II) com-
plexes
1657 (m), 1602 (w), 1491 (w), 1475 (s), 1433 (s), 1410 (s),
1342 (w), 1306 (s), 1277 (w), 1212 (w), 1133 (w), 1123 (m),
1095 (w), 1044 (w), 992 (w), 948 (w), 929 (w), 876 (w), 848
(w), 765 (w), 746 (vs), 699 (s), 620 (w), 585 (w), 505 (w),
481 (w), 437 (w). Anal. Calcd. for C24H27ClN3O2P·EtOH
(%): N 8.65, C 64.26, H 6.84. Found: N 8.78, C 64.17, H
20 mL methanol and then 0.05 g (0.77 mmol) NaN3 was
added to it. The mixture was refluxed at 72 °C for 2 h. The
reaction solution was left to stand at room temperature
for 2 days while reddish crystals arose from the solution.
Yield 0.10 g (73.6 %). IR: 3309 (w), 3058 (w), 2034 (vs),
1609 (w), 1568 (m), 1482 (w), 1436 (w), 1412 (w), 1330
(w), 1293 (w), 1208 (w), 1086 (s), 1036 (s), 924 (w), 771
(w), 751 (m), 694 (m), 540 (w), 487 (w) (Fig. S1). Ele-
mental analysis calcd for C24H26BF4N6NiOP: C 48.78 %,
H 4.43 %, N 14.22 %, found: C 48.53 %, H 4.64 %, N
14.31 %. ΛM (1 mM, DMSO): 43.7 Ω−1 cm2 mol−1. λmax
(nm) (H2O): 296, 327, 344, 362, λmax (nm) (DMSO):
298, 326, 344, 362. The same pattern of electronic spec-
tra in DMSO and aqua solution is observed in the case of
[NiL(NCS)]BF4, [NiL(NCS)]SCN, [NiL(NCO)]BF4 and
[NiLCl]BF4 complexes. In electronic spectra of square-
planar nickel(II) complexes the strong band due to the
d–d electronic transition (dx2–y2 ← dxy), is observed in
the 17,000–22,000 cm−1 or 600–450 nm region, while the
band observed at higher energies (23,000–30,000 cm−1 or
1
6.92. H NMR (Chemical shift (ppm), multiplicity, number
of H-atoms, coupling constant J in Hz): 3.30 (s, 9 H), 4.76
(s, 2 H) 8.79 (d, 1 H, J = 5.0 Hz) 6.83 (m, 1H) 7.42 (m, 1 H)
7.49 (m, 1 H) 8.05 (dd, 1 H, J = 5.0 Hz) 7.19 (m, 4 H) 7.42
(m, 4 H) 7.42 (m, 2 H) 12.10 (s, 1 H). 13C NMR (chemi-
cal shift (ppm), coupling constant J in Hz): 53.4, 62.4, 143.4
(J = 25.0 Hz), 135.4 (J = 10 Hz), 133.2, 130.5, 129.4, 126.3
(J = 3.75 Hz), 136.5 (J = 18.75 Hz), 137.2 (J = 18.75 Hz),
133.5 (J = 18.75 Hz), 129.1 (J = 7.5 Hz), 129.4, 165.5.
Synthesis of [NiLN3]BF4
A mixture of 0.08 g (0.23 mmol) Ni(BF4)2·6H2O and
0.11 g (0.23 mmol) of HLCl ligand was dissolved in
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149
434–333 nm) is a charge transfer band [24]. The mentioned
bands related to the d–d transition of the Ni(II) square-pla-
nar geometry complexes are not observed in visible region,
so it is possible that change in the geometry to octahe-
dral occurs, because the characteristic bands in electronic
spectra of octahedral Ni(II) complexes are in the Vis–NIR
region (25,000–4000 cm−1 or 400–250 nm). Additional
support for change of geometry in DMSO was obtained
by NMR spectrometry, since line broadening and loss of
hyperfine splitting occurred in contrast to sharp signals in
CDCl3 (for soluble complexes). This indicates the presence
of paramagnetic Ni(II) species in DMSO solution.
cancer cell lines (MDA-MB-453 and MDA-MB-361),
human bronchial epithelial cells (BEAS-2B) and human
fetal lung fibroblast cells (MRC-5) cells were maintained
as monolayer culture in the Roswell Park Memorial Insti-
tute (RPMI) 1640 nutrient medium (Sigma Chemicals
Co, USA). Human myelogenous leukemia cells (K562)
were maintained in suspension culture. RPMI 1640 nutri-
ent medium was prepared in sterile ionized water, supple-
mented with penicillin (192 U/mL), streptomycin (200 µg/
mL), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid
(HEPES) (25 mM), l-glutamine (3 mM) and 10 % of heat-
inactivated fetal calf serum (FCS) (pH 7.2). The cells were
grown at 37 °C in 5 % CO2 and humidified air atmosphere,
by twice weekly subculture.
Synthesis of [NiHL(NCS)3]·H2O, [NiL(NCS)]BF4,
[NiL(NCS)]SCN, [NiL(NCO)]BF4 and [NiLCl]BF4
Determination of cell survival (MTT assay)
Complexes of Ni(II) with HLCl ligand, [NiHL(NCS)3],
[NiL(NCS)]BF4, [NiL(NCS)]SCN, [NiL(NCO)]BF4 and
[NiLCl]BF4 were synthesized using previously described
methods [22, 23].
Cytotoxicity of the investigated nickel(II) complexes,
the appropriate ligand, nickel(II) salt and salts of cor-
responding monodentates in comparison to cisplatin,
was determined using the 3-(4,5-dimethylthiazol-2yl)-
2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich)
assay. The MTT colorimetric assay is based on the meas-
urement of mitochondrial enzyme succinate dehydroge-
nase activity, as an indication of cell viability [27]. Cells
were seeded in 96-well cell culture plates (NUNC): HeLa
(4000 c/w), A549 (6000 c/w), LS-174 (7000 c/w), MDA-
MB-453 (5000 c/w), MDA-MB-361 (7000 c/w), BEAS-
2B (7000 c/w) and MRC-5 (5000 c/w) in culture medium
and grown for 24 h. K562 (6000 c/w) cells were seeded
2 h before treatment. Stock solutions of investigated agents
were made in DMSO at concentration of 10 mM, and after-
wards diluted with nutrient medium to desired final con-
centrations (in range up to 100 µM). Cisplatin (CDDP)
stock solution was made in 0.9 % NaCl at concentration
of 1.66 mM and afterwards diluted with nutrient medium
to desired final concentrations (in range up to 100 µM).
The final concentration of DMSO per well did not exceed
1 %. Solutions of various concentrations of examined com-
pounds were added to the wells, except the control wells
where only nutrient medium was added. All samples were
done in triplicate. Nutrient medium with corresponding
agent concentrations but without target cells was used as a
blank, also in triplicate.
X‑ray structure determination of [NiLN3]BF4
Crystal data: C24H26BF4N6NiOP, M = 591.00, orthorhom-
bic, space group Pbca, a = 12.5970(7) Å, b = 14.9907(12)
Å, c = 27.491(2) Å, V = 5191.4(6) Å3, Z = 8, Dc = 1.512,
μ = 0.868 mm−1. A red prism of compound [NiLN3]BF4 with
dimensions of 0.20 × 0.02 × 0.02 mm was glued to a glass
thread. X-ray intensity data were collected at room temperature
with Agilent SuperNova dual source using an Atlas detector
and equipped with mirror-monochromated Mo Kα radiation
(λ = 0.71073 Å). The data were processed using CRYSA-
LIS PRO [25]. A total of 17,842 reflections were measured,
5952 were independent and 2060 [I > 2σ(I)] were considered
observed. The structures were solved by direct methods using
SHELXS-97 [26] and refined with a full-matrix least-squares
procedure based on F2 using SHELXL-97 [26]. All of the non-
hydrogen atoms were refined anisotropically. The C6 bonded
hydrogen atom was located in a difference map and refined
with the distance restraints (DFIX) with C–H = 0.98 and with
Uiso(H) = 1.2Ueq(C). All other C-H hydrogen atoms were
included in the model at geometrically calculated positions and
refined using a riding model. The final R indices R1 = 0.0682
[I > 2σ(I)] and wR2 = 0.1314 (all data) were found.
CCDC 1,409,081 contains the supplementary crystallo-
graphic data for [NiLN3]BF4. These data can be obtained
free of charge from The Cambridge Crystallographic Data
Centre via http://www.ccdc.cam.ac.uk/data_request/cif.
Cells were incubated for 48 h with the test compounds
at 37 °C, with 5 % CO2 in humidified atmosphere. After
incubation, 10 µL of MTT solution, 5 mg/mL in phosphate
buffer solution (PBS), pH 7.2, was added to each well.
Samples were incubated for 4 h at 37 °C with 5 % CO2 in
humidified atmosphere. Formazan crystals were dissolved
in 100 µL 10 % sodium dodecyl sulfate (SDS). Absorb-
ance was recorded on the ThermoLabsystems 408 Multis-
kan EX 200–240 V after 24 h at a wavelength of 570 nm.
Cell culture
Human cervix carcinoma cells (HeLa), lung adenocarci-
noma cells (A549), colon cancer cells (LS-174), breast
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Concentration IC50 (µM) was defined as the concentration
of drug producing 50 % inhibition of cell survival. It is
determined from the cell survival diagrams.
Apoptotic assay
Flow cytometric analysis of cell death induced by investi-
gated nickel(II) complexes ([NiL(NCS)]BF4, [NiLN3]BF4,
[NiL(NCO)]BF4, [NiLCl]BF4) and cisplatin as reference
compound, was performed by Annexin V-FITC apoptosis
detection kit, according to the manufacturer’s instructions
(BD Biosciences Cat. No. 65874x, Pharmingen San Diego,
CA, USA). HeLa cells (2 × 105) were seeded into 6-well
plates (Thermo Scientific Nunc™), in 2 mL of RPMI
medium. After 24 h of growth, cells were treated with com-
plexes ([NiL(NCS)]BF4, [NiLN3]BF4, [NiL(NCO)]BF4,
[NiLCl]BF4) or cisplatin, for 48 h, at 40 μM concentration
for nickel complexes and 5 μM concentration for cispl-
atin. After treatment, cells were washed with ice-cold PBS
and then resuspended in 200 μL binding buffer (10 mM
HEPES/NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl2).
100 μL of cell suspension (105 cells) was transferred
to a 5-mL culture tube and mixed with 5 μL of Annexin
V-FITC and 5 μL of propidium iodide (PI). After incuba-
tion for 15 min, at 25 °C in the dark, 400 μL of binding
buffer was added to each tube and analyzed using a FACS
Calibur Becton-Dickinson flow cytometer and Cell Quest
computer software. A minimum of 10,000 cells were ana-
lyzed per sample.
Cell cycle analysis
Flow cytometric analysis of cell cycle phase distribution of
A549 and HeLa cells, treated with nickel(II) complexes and
cisplatin as reference compound was performed after stain-
ing fixed cells with propidium iodide (PI) [28]. Cells were
seeded at density of 2.5 × 105 cells/well at 6-well plate
(NUNC) and grown in nutrition medium. After 24 h A549
cells were continually exposed to [NiLN3]BF4, [NiL(NCS)]
BF4 and [NiLCl]BF4 complexes with concentrations that
correspond to 0.5 × IC50 and IC50 (determined for 48 h
treatment), while HeLa cells were treated with [NiL(NCS)]
BF4, [NiLN3]BF4 [NiL(NCO)]BF4 and [NiLCl]BF4 com-
plexes (40 μM) and cisplatin (5 μM). After 24 and 48 h of
continual treatment, cells were collected by trypsinization,
washed twice with ice-cold PBS, and fixed for 30 min in
70 % EtOH. After fixation, cells were washed again with
PBS, and incubated with RNaseA (1 mg/mL) for 30 min at
37 °C. Cells were than stained with PI (400 µg/mL) 15 min
before flow cytometric analysis. Cell cycle phase distribu-
tion were analyzed using a fluorescence activated sorting
cells (FASC) Calibur Becton-Dickinson flow cytometer and
Cell Quest computer software.
DNA binding experiments
Fluorescence microscopy analysis of cell death (AO/EB
staining)
Calf thymus DNA (lyophilized, highly polymerized,
obtained from Serva, Heidelberg) (CT-DNA) was dissolved
in Tris buffer (10 mM Tris–HCl, pH 7.9) overnight at 4 °C.
This stock solution was stored at 4 °C and was stable for
several days. A solution of CT-DNA in water gave a ratio of
UV absorbance at 260 and 280 nm, A260/A280 of 1.89–2.01,
indicating that DNA was sufficiently free of protein. The
concentration of DNA (2.86 mg/mL) was determined from
the UV absorbance at 260 nm using the extinction coef-
ficient ε260 = 6600 M−1 cm−1 [29]. The Ni(II) complexes
([NiLN3]BF4, [NiL(NCS)]BF4 and [NiLCl]BF4) were dis-
solved in dimethyl sulfoxide in concentrations of 10 mM.
These solutions were used as stock solutions.
To examine the mode of HeLa and A549 cell death induced
by the investigated nickel(II) complexes, morphological
analysis by microscopic examination of acridine orange/
ethidium bromide-stained target cells was performed. A549
and HeLa cells were seeded overnight on cover slips in
6-well plates (2 × 105 cells per well) in 2 mL of complete
nutrient medium. After 24 h, HeLa cells were treated with
[NiL(NCS)]BF4, [NiLN3]BF4, [NiL(NCO)]BF4, [NiLCl]
BF4 complexes (in 40 μM concentrations) and CDDP
(in 5 μM concentration) for 48 h, while A549 cells were
exposed to 0.5 × IC50 concentrations of ([NiL(NCS)]BF4,
[NiLN3]BF4, [NiLCl]BF4) complexes for 48 h. After this
period, the target cells were stained with 10 µL of a mixture
of the DNA dyes acridine orange (AO) and ethidium bro-
mide (EB) (3 mg/mL AO and 10 mg/mL EB in PBS), and
visualized under a fluorescence microscope—Carl Zeiss
PALM MicroBeam with Axio Observer.Z1 using Axi-
oCamMRm (filters Alexa Fluor 489 and Alexa Fluor 546)
using the LD Plan-NeoFluar 40 ×/0.60 objective. Images
were obtained with multidimensional acquisition using dig-
ital imaging software (AxioVision Version 4.7; Carl Zeiss
Imaging Solutions).
UV–visible measurements
For a UV–Vis measurement, to DNA solution (10 µL of
CT-DNA) was added a small volume of a stock solution of
Ni(II) complex (final concentration 30, 50 or 70 µM) and
the volume was adjusted up to 1 mL with 40 mM bicar-
bonate buffer, pH 8.4. Reaction mixtures were incubated
at 37 °C for 90 min with occasional vortexing. UV–vis
spectra were recorded on a UV-1800 Shimadzu UV/Vis-
ible spectrophotometer operating from 200 to 800 nm in
1.0 cm quartz cells. Spectra of Ni(II) complexes of the
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151
same concentrations were also recorded, as well as spectra
of CT-DNA.
plasmid DNA (512 ng/µL) was determined by measuring
the absorbance of the DNA-containing solution at 260 nm.
One optical unit corresponds to 50 µg/mL of double-
stranded DNA.
Fluorescence measurements
The cleavage reaction of supercoiled pUC19 DNA with
different concentration of ([NiLN3]BF4, [NiL(NCS)]BF4
and [NiLCl]BF4) was investigated by incubation of 512 ng
of plasmid in a 20 µL reaction mixture in 40 mM bicarbo-
nate buffer (pH 8.4) at 37 °C, for 90 min. The reaction mix-
tures were vortexed from time to time. The reaction was
terminated by short centrifugation at 10,000 rpm and addi-
tion of 5 µL of loading buffer (0.25 % bromophenol blue,
0.25 % xylene cyanol FF and 30 % glycerol in TAE buffer,
pH 8.24 (40 mM Tris–acetate, 1 mM EDTA)).
The competitive interactions of Ni(II) complexes and
the fluorescence probe, either ethidium bromide (EB)
or Hoechst 33258 (H), with CT-DNA have been studied
by measuring the change of fluorescence intensity of the
probe–DNA solution after addition of the complex. Reac-
tion mixtures containing 100 µM of CT-DNA (calculated
per phosphate) in 1 mL of 40 mM bicarbonate solution
(pH 8.4) were pretreated with 1.5 µL of 1 % H probe solu-
tion (28 µM final concentration) or 1 µL of 1 % EB solu-
tion (25 µM final concentration) (in separate experiments)
for 20 min and the mixture was analyzed by fluorescence
measurement. Then the increasing concentrations (0.5, 1,
1.5, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 µM) of the complex
were successively added and the change in the fluorescence
intensity was measured using a Thermo Scientific Lumina
Fluorescence spectrometer (Finland) equipped with a
150 W Xenon lamp. The slits on the excitation and emis-
sion beams were fixed at 10 nm. All measurements were
performed by excitation at 350 nm for Hoechst 33258, and
by excitation at 500 nm for EB in the range of 390–600 nm.
The control was probe–CT-DNA solution. Complexes
[NiL(NCS)]BF4, [NiLCl]BF4 and [NiLN3]BF4 did not
have fluorescence under applied conditions. The obtained
fluorescence quenching data were analyzed according to
the Stern–Volmer equation [30]:
Digestion of plasmid pUC19 DNA by restriction
enzymes
Enzyme digestions were carried out by incubating the
untreated and [NiLN3]BF4, [NiL(NCS)]BF4 and [NiLCl]
BF4 treated pUC19 plasmid samples with the restric-
tion endonucleases BamHI or HindIII (Sigma, USA).
512 ng of plasmid DNA treated or untreated with 1 µL of
10 mM Ni(II) complex were incubated with 1 µL (10 U)
of the restriction endonucleases at 37 °C for 9 h or 16 h in
20 µL of the digestion buffer (10 mM Tris–HCl, pH 8.0,
containing 100 mM NaCl, 5 mM MgCl2, 1 mM 2-mercap-
toethanol). The samples were then analyzed by agarose
gel electrophoresis. In the second set of experiments, plas-
mid samples were first treated with Ni(II) complexes, and
the obtained products were subsequently treated with the
restriction nucleases.
I0/I = 1 + Kr
(1)
where I0 and I represent the fluorescence intensi-
ties of probe–CT-DNA in absence and presence of
Agarose electrophoresis
the Ni(II)complex, respectively,
K is the quench-
ing constant. The K value is calculated from the
The samples were subjected to electrophoresis on 1 % aga-
rose gel (Amersham Pharmacia-Biotech, Inc) prepared in
TAE buffer pH 8.24. The electrophoresis was performed
at a constant voltage (80 V) until bromophenol blue had
passed through 75 % of the gel. A Submarine Mini-gel
Electrophoresis Unit (Hoeffer HE 33) with an EPS 300
power supply was used. After electrophoresis, the gel
was stained for 30 min by soaking it in an aqueous eth-
idium bromide solution (0.5 µg mL−1). The stained gel
was illuminated under a UV transilluminator Vilber-Lour-
mat (France) at 312 nm and photographed with a Nikon
Coolpix P340 Digital Camera through filter DEEP YEL-
LOW 15 (TIFFEN, USA).
ratio of the slope to the intercept from the plot of
ꢀ
ꢁ
I0/I versus r(r = Ni(II)complex /[CT − DNA].
Primary spectra of all spectrometric measurements were
imported into OriginPro 8.0 and were processed by this
software package.
DNA cleavage experiments
For DNA cleavage experiments the plasmid pUC19
(2686 bp, purchased from Sigma-Aldrich, USA) was
prepared by its transformation in chemically competent
cells Escherichia coli strain XL1 blue. Amplification of
the clone was done according to the protocol for grow-
ing E. coli culture overnight in LB medium at 37 °C [31]
and purification was performed using Qiagen Plasmid
plus Maxi kit. Finally, DNA was eluted in 10 mM Tris–
HCl buffer and stored at −20 °C. The concentration of
Phosphatase activity
Phosphatase activity of the complexes was measured using
disodium p-nitrophenyl phosphate hexahydrate (pNPP)
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Scheme 4 Synthesis of
[NiLN3]BF4 complex
according the literature [32]. The solvent chosen for
this study was 97.5 % DMSO. Solution of substrate and
[NiLN3]BF4, [NiL(NCS)]BF4 and [NiLCl]BF4 complexes
were freshly prepared and all spectra were recorded for
2 h from solution containing 1 mmol of the substrate and
0.05 mmol of the Ni(II) complexes in 1 mL of solution.
X‑ray crystallographic analysis
The nickel complex [NiLN3]BF4 crystallized in orthorhom-
bic crystal system with space group Pbca. ORTEP pres-
entation of the structure is given in Fig. 1, while bond
lengths and angles are listed in Table 1. The complex cat-
ion in [NiLN3]BF4 consists of one tridentate molecule of
deprotonated PNO ligand coordinated to the Ni(II) ion and
coordinated azide anion forming a square-planar geom-
Results and discussion
Synthesis
−
etry around Ni(II) ion. BF4 ion acts as counterion in the
crystal structure complex. Bond distances and angles in
Ligand HLCl was obtained by condensation reaction of
2-(diphenylphosphino)benzaldehyde and Girard’s T rea-
gent [19]. Complexes of Ni(II) with condensation product
of 2-(diphenylphosphino)benzaldehyde and Girard’s T rea-
gent [NiHL(NCS)3], [NiL(NCS)]BF4, [NiL(NCS)]SCN,
[NiL(NCO)]BF4 and [NiLCl]BF4 were synthesized using
previously described methods [22, 23].
In the reaction of Ni(BF4)2·6H2O and NaN3 with HLCl
ligand in methanol [NiLN3]BF4 complex was obtained
(Scheme 4). Square-planar surroundings of Ni(II) ion con-
sist of the acylhydrazone ligand, in deprotonated zwitter-
ionic form, coordinated as a tridentate through phospho-
rus, imine nitrogen and carbonyl oxygen atoms and azido
ligand in the fourth coordination place.
Fig. 1 ORTEP plot of [NiLN3]BF4 from the X–ray crystal structure
with thermal ellipsoids at 30 % probability for non-H atoms and open
circles for H-atoms. BF4− is omitted for the reason of clarity
IR spectra
In the IR spectrum of [NiLN3]BF4 complex a new
band corresponding to ν(−O–C=N) of the deproto-
nated hydrazide moiety appeared at 1568 cm−1, instead
of the carbonyl band from the uncoordinated ligand at
1686 cm−1. Coordination of azomethine nitrogen atom
resulted in bathochromic shift of ν(C=N) vibration from
1657 cm−1 in the spectrum of HLCl to 1609 cm−1 in the
spectrum of [NiLN3]BF4 complex. A band at 2033 cm−1 in
the spectrum of [NiLN3]BF4 complex originates from the
coordinated azido ligand. A band originating from tetra-
fluoroborate anion in the outer sphere of the complex is
located at 1036 cm−1 [33, 34].
Table 1 Selected bond lengths (Å) and angles (°) for [NiLN3]BF4
Ni1–N1
Ni1–N4
Ni1–O1
Ni1–P1
N1–N2
N1–C6
N2–C5
O1–C5
1.859 (4)
1.867 (5)
1.888 (4)
2.1478 (16)
1.422 (5)
1.270 (7)
1.312 (7)
1.262 (6)
P1–Ni1–N1
P1–Ni1–N4
P1–Ni1–O1
N1–Ni1–N4
N1–Ni1–O1
N4–Ni1–O1
Ni1–N1–N2
N1–N2–C5
95.82 (14)
86.56 (17)
178.96 (15)
177.2 (2)
84.57 (18)
93.1 (2)
112.1 (3)
108.8 (4)
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153
compound [NiLN3]BF4 are in accordance with the reported
values for the similar square-planar complexes where lin-
ear thiocyanate [22], isocyanate [21], or azide [11, 20, 21]
anion is coordinated through nitrogen atom to the Ni metal
center. The sum of the nickel-containing angles in com-
plex [NiLN3]BF4 is 360°. The position of the Ni(II) ion is
0.0035(27) out of the best plane that contained the −coor-
dination sphere of four atoms. H-bonds between BF4 and
the complex ion C–H···F, as well as between two complex
ions C14–H14···N6 are weak and do not play an important
role in the crystal structure of [NiLN3]BF4 complex (Table
S1).
newly synthesized azido complex showed selectivity to two
tumor cell lines (HeLa and A549) compared to the nor-
mal cells (for A549 IC50 was similar to that of cisplatin).
The best cytotoxic activity was observed on K562, A549
and MDA-MB-361 cell lines for [NiL(NCS)]BF4 and for
[NiHL(NCS)3] on LS-174 cell line.
The ligand itself (HLCl) did not exhibit as strong cyto-
toxic effect against tumor cells as nickel(II) complexes,
which leads to conclusion that metal–ligand complexa-
tion is crucial for cytotoxic properties of the compounds.
The examined nickel(II) salt and salts of correspond-
ing monodentates after 48 h of incubation did not reach
IC50 values in the range of applied concentrations (up to
100 μM) in all investigated cell lines.
Determination of cell survival (MTT assay)
Comparison of cytotoxic activity of Ni(II) complexes
with HLCl ligand with the activity of the two previously
synthesized series of Ni(II) complexes with HL1 [20] and
HL2 [21] ligands indicates that the nature of the hydrazone
ligand and monodentates has important influence on cyto-
toxicity of the complexes (Table 3).
The observed trend of cytotoxicity of hydrazone ligands
on normal MRC-5 cell line was HL1 < HLCl < HL2.
Comparison of toxicity of Ni(II) complexes with 2-(diphe-
nylphosphino)benzaldehyde acylhydrazones (HLCl, HL1
and HL2) and different pseudohalogenides with cisplatin
toxicity on MRC-5 cell line is presented in Table 4 [20, 21].
Having in mind the results of cytotoxic activity of
investigated Ni(II) complexes it is clear that their activity
depends on the synergistic effect of 2-(diphenylphosphino)
benzaldehyde acylhydrazones, monodentate ligand at the
remaining coordination site and anions in the case of elec-
trolyte complexes. Different activity of [NiL(NCS)]BF4
and [NiL(NCS)]SCN is the consequence of differences in
Cytotoxic activity of the investigated nickel(II) complexes
and cisplatin (CDDP) as standard cytotoxic agent was
determined by MTT assay after 48 h treatment of six tumor
cell lines (HeLa, A549, K562, MDA-MB-453, MDA-
MB-361 and LS-174) and two normal cell line (MRC-5
and BEAS-2B). The results are shown in Table 2 in terms
of IC50 values for 48 h incubation period. IC50 values were
calculated as mean values obtained from two to three inde-
pendent experiments and presented with their standard
deviations.
Results of this assay indicate that all investigated
nickel(II) complexes showed a moderate to strong cyto-
toxic activity to all tumor cell lines in comparison with
cisplatin. Complexes [NiL(NCS)]BF4, [NiLN3]BF4 and
[NiLCl]BF4 have shown a more prominent antiprolifera-
tive activity in A549 cells, even stronger than cisplatin,
which is worth mentioning, because of lower sensitivity of
A549 cell line to treatments in general. Nevertheless, the
Table 2 Results of MTT assay presented as IC50 (μM) values obtained after 48-h treatment
HeLa
K562
A549
LS-174
MDA-MB-361 MDA-MB-453 MRC-5
BEAS-2B
HLCl
51.25 2.98 86.88 2.92 71.13 3.04
>100
>100 >100 45.40 1.16 n.t.
[NiHL(NCS)3] 34.22 3.80 52.10 16.40 72.20 0.17 35.62 1.18 40.99 0.19 48.18 0.83 18.96 0.90 n.t.
[NiL(NCS)]BF4 28.59 4.12 25.16 3.61 14.62 2.99 >100 19.62 3.31 46.34 3.58 19.61 4.26 55.12 7.68
[NiL(NCS)]SCN 25.87 4.16 76.27 6.84 50.80 12.08 97.28 1.53 36.64 3.95 44.31 2.12 25.54 0.53 n.t.
[NiLN3]BF4 22.18 2.04 63.18 6.63 22.98 2.13 70.39 4.24 39.39 4.37 49.74 0.23 32.00 0.84 49.11 0.56
[NiL(NCO)]BF4 24.29 0.97 68.99 5.97 57.62 3.85 78.94 3.69 33.54 3.88 45.60 4.84 15.58 5.64 47.63 13.44
[NiLCl]BF4
Ni(BF4)2·6H2O
NaOCN
24.25 0.44 50.17 2.59 14.94 3.78
>100
>100
>100
>100
>100
27.22 7.34 72.01 4.08 16.33 3.63 48.57 6.84
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
n.t.
n.t.
n.t.
n.t.
KSCN
NaN3
CDDP
5.17 0.29 18.56 3.28 26.21 5.36 22.41 7.18 14.7 1.2
13.51 2.56 12.13 0.92 14.19 1.43
IC50 values were calculated as mean values obtained from two to three independent experiments and presented with their standard deviations. A
sign (>) indicates that IC50 value is not reached in the examined range of concentrations (the sign is in front of the maximum value of the con-
centration in the examined range of concentrations).n.t.-not tested
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Table 3 Selectivity of Ni(II) complexes with 2-(diphenylphosphino)
benzaldehyde acylhydrazones (HLCl, HL1 and HL2) and differ-
ent pseudohalogenides towards investigated tumor cell lines (HeLa,
K562, A549, LS-174, MDA-MB-361, MDA-MB-453)
doubling the concentration of active substance does not
change the percentage of dead cells. At the same incubation
period decrease in G2 fraction of A549 cells was also noted.
Only in the case of [NiLN3]BF4 complex decrease in the
percentage of cells in S phase of cell cycle was observed.
After 48 h of incubation with complexes [NiL(NCS)]BF4
and [NiLCl]BF4 no changes in cell cycle phase distribu-
tion occurred compared to control population of cells. After
48 h of treatment with [NiLN3]BF4 complex the increase of
G1 and decrease of S phase fraction were observed, while
the percentage of cells in sub G1 phase was the same as in
the control.
X =
[NiLX]
[NiL1X]
[NiL2X]
pseudohalogenide
OCN−
LS-174
K562
A549
MDA-MB-361
HeLa
MDA-MB-453
SCN−
K562
LS-174
HeLa
A549
MDA-MB-453
MDA-MB-361
−
N3
A549
K562
HeLa
Analysis of perturbations of HeLa cells treated with
selected complexes [NiL(NCS)]BF4, [NiL(N3)]BF4,
[NiL(NCO)]BF4 and [NiLCl]BF4 (40 μM) and cispl-
atin (5 μM) after 48 h was also performed. Nickel com-
plexes [NiL(NCS)]BF4, [NiLN3]BF4, [NiL(NCO)]BF4 and
[NiLCl]BF4 show slight decrease of percent of cells in G1
phase and increase of percent of cells in S phase (Fig. 3).
While cisplatin, as expected according to literature data
induced more aggressive perturbations of cell cycle with
block of cell cycle in S phase in HeLa cells [21, 39].
MDA-MB-361 LS-174
MDA-MB-453
Table 4 Toxicity of Ni(II) complexes with 2-(diphenylphosphino)
benzaldehyde acylhydrazones (HLCl, HL1 and HL2) and different
pseudohalogenides on MRC-5 cell line
X = pseudohalogenide
OCN−
SCN−
[NiLX]
[NiL1X]
[NiL2X]
++
++
+
++
+
+
+++
+++
+++
−
N3
Fluorescence microscopy analysis of cell death (AO/EB
staining)
(+++ stronger, ++ similar, + lower toxicity of Ni(II) complex in
comparison with cisplatin)
In order to assess the apoptotic potential of the investi-
gated nickel(II) complexes ([NiL(NCS)]BF4, [NiLN3]
BF4, [NiL(NCO)]BF4 and [NiLCl]BF4), morphological
analysis by fluorescence microscopy of acridine orange/
ethidium bromide-stained HeLa and A549 cells was per-
formed. Typical morphological features of the apoptotic
cells are shrinkage, retraction of pseudopods, reduction
of cellular volume (pyknosis), chromatin condensation,
nuclear fragmentation, minor modification of cytoplasmic
organelles, plasma membrane blebbing [40]. In live cells
and in early apoptotic cells, considering that plasma mem-
brane integrity is preserved, only acridine orange enters the
cell while ethidium bromide is excluded and the nucleus is
stained green. In late apoptosis or necrosis along with the
loss of membrane integrity, both dyes enter the cell and the
nucleus becomes orange-red.
coordination properties of corresponding anions as well as
the possibility of thiocyanate oxidation in biological sys-
−
tems. Biological activity of OCN−, SCN− and N3 was
described in the literature [35–37]. So the possible role of
investigated Ni(II) complexes is the transport of biologi-
cally active pseudohalogenides into the cell. Also, phos-
phine acylhydrazone ligands are biologically active com-
pounds and coordination of metal ion to the phosphorus
atom can prevent oxidation of phosphine to phosphine
oxide and as a consequence of this enhance biological
activity [38].
Cell cycle analysis
Cell cycle analysis of A549 cells treated with nickel(II)
complexes ([NiLN3]BF4, [NiL(NCS)]BF4 and [NiLCl]
BF4) and cisplatin was performed by flow cytometry after
staining with propidium iodide [28]. Cells were continually
exposed to nickel(II) complexes ([NiLN3]BF4, [NiL(NCS)]
BF4 and [NiLCl]BF4) and cisplatin for 24 and 48 h with
increasing concentrations of agents (0.5 × IC50 and IC50).
After 24 h of continual treatment with complexes [NiLN3]
BF4, [NiL(NCS)]BF4 and [NiLCl]BF4, increase of apop-
totic fraction of cells (Sub-G1 fraction) was noted (Fig. 2).
This increase was not concentration-dependent because
After 48 h of treatment of HeLa cells with investigated
complexes [NiL(NCS)]BF4, [NiLN3]BF4, [NiL(NCO)]
BF4, [NiLCl]BF4 (in 40 μM concentrations) and CDDP (in
5 μM concentration) there is a prominent reduction in the
number of cells compared to control population, together
with initial retracting of pseudopods and rounding off
(Fig. 4). However, not all cells become uniformly round,
cells with spindle-shaped morphology are still present and
majority of cells has intact membrane since they colored
green. Different types of alterations were noticed after
treatment with complexes [NiL(NCS)]BF4, [NiLN3]BF4
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155
Fig. 2 Effect of the complexes [NiL(NCS)]BF4, [NiLCl]BF4 and
cisplatin (CDDP) on cell cycle progression of A549 cells following
24 and 48 h incubation with concentrations of investigated complexes
corresponding to 0.5 × IC50 and IC50. Controls were untreated cells
(incubated with nutrient medium only). The results are expressed as
mean standard deviations of three independent experiments
and [NiLCl]BF4 like chromatin condensation, membrane
blebbing and apoptotic bodies characteristic to the vari-
ous stages of apoptosis. Necrotic cells with red enlarged
nucleus without or with low chromatin condensation are
also spotted (Fig. 4 [NiL(NCS)]BF4 and [NiLCl]BF4).
Complex [NiL(NCO)]BF4 except the beginning of round-
ing off have not induced other morphological changes.
Control A549 cells presented on photomicrographs in
Fig. S2 are of light green color, short, spindle-shaped and
triangle-shaped, with significant growth density. Only after
48 h of continual action with 0.5 × IC50 concentrations
of investigated nickel(II) complexes ([NiL(NCS)]BF4,
[NiLN3]BF4 and [NiLCl]BF4) the beginning of nuclear
condensation is observed, which is considered to be an
early apoptotic marker. However, other characteristic hall-
marks of apoptosis, such as membrane blebbing and apop-
totic bodies, have not been noticed.
Apoptotic assay
Fluorescent microscopy after AO/EB staining show that
complexes [NiL(NCS)]BF4, [NiLN3]BF4 and [NiLCl]BF4
after 48 h treatment of HeLa cells have a potential of induc-
ing some apoptotic characteristics of cell death. Further
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Fig. 3 Effect of the complexes [NiL(NCS)]BF4, [NiLN3]BF4,
[NiL(NCO)]BF4, [NiLCl]BF4 and cisplatin (CDDP) on cell cycle
progression of HeLa cells after 48 h incubation with 40 μM concen-
tration for nickel(II) complexes and 5 μM concentration for cisplatin.
Controls were untreated cells (incubated with nutrient medium only).
The results are expressed as mean
independent experiments
standard deviations of three
analysis of their apoptotic potential was investigated using
flow cytometry dual staining with Annexin V-FITC and
PI, method that enables detection of translocation of phos-
phatidylserine from the inner to the outer side of plasma
membrane during apoptosis. In Fig. S3 representative dot
plot diagrams are shown, with percentages of viable cells
(Annexin V-FITC and PI negative), early apoptotic cells
(Annexin V-FITC positive and PI negative), late apoptotic
cells and necrotic cells (Annexin V-FITC negative and PI
positive) and dead cells (Annexin V-FITC negative and PI
positive). Results shown as bar graph in Fig. 5 indicate that
after 48 h treatment and 40 μM concentration of investi-
gated nickel(II) complexes ([NiL(NCS)]BF4, [NiLN3]BF4,
[NiL(NCO)]BF4 and [NiLCl]BF4) no increase of percent-
age of cells labeled with Annexin V-FITC (early apoptotic),
nor cells labeled with both dyes Annexin V-FITC and PI
(late apoptotic and necrotic cells) compared to control was
found. However, number of dead cells increased to 14.83 %
for [NiL(NCS)]BF4, 33.9 % for [NiLN3]BF4, 14.285 % for
[NiL(NCO)]BF4 and 20.765 % for [NiLCl]BF4).
DNA binding activity and mode of DNA interaction
The hyperchromism and hypochromism are regarded as
spectral evidence for DNA double-helix structural change
when DNA reacts with other molecules. The hyper-
chromism originates from the disruption of the DNA
duplex secondary structure and the hypochromism origi-
nates from the stabilization of the DNA duplex by either
the intercalation binding mode or the electrostatic effect
of small molecules [41, 42]. Electronic absorption spectra
of the nickel complexes [NiL(NCS)]BF4, [NiLCl]BF4 and
[NiLN3]BF4 recorded at different concentrations without or
with fixed concentration of CT-DNA are shown in Fig. 6a–
c. UV–Vis spectra of all three Ni(II) complexes displayed
similar absorption bands. It was found that the maximum
absorption of the compounds was centered at 294 nm with
shoulders at 339–343 and 358–368 nm. Upon interaction
of Ni(II) complexes with CT-DNA, the peaks at 294 nm
disappeared, while DNA absorption maximum was shifted
from 258 to 251 nm, indicating the formation of a [NiLX]
BF4─CT-DNA complex. Detailed absorption changes
induced by binding of the Ni(II) complexes to CT-DNA
were further calculated, Fig. 6d–f. The absorption value of
the sum of absorbances at 258 nm of a free Ni(II) complex
Lack of compatibility of results of AO/EB staining and
fluorescent microscopy analysis and Annexin V-FITC/propid-
ium iodide staining and analysis on flow cytometer lead to the
necessity of further research of precise mechanism of action.
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157
Fig. 4 Photomicrographs
of acridine orange/ethidium
bromide-stained HeLa cells in
control and HeLa cells exposed
for 48 h to nickel(II) complexes
[NiL(NCS)]BF4 [NiLN3]BF4
[NiL(NCO)]BF4, [NiLCl]BF4
(40 μM concentration) and
CDDP (5 μM concentration)
and free CT-DNA was a little different from the absorp-
tion value of a [NiLX]BF4─CT-DNA. The hypochromism
of about 7 % has been observed with lower concentra-
tions of [NiL(NCS)]BF4, Fig. 6d. The similar decrease
in absorption intensity at 258 nm has also been observed
after interaction of 30 µM [NiLCl]BF4 with CT-DNA (the
hypochromism was calculated as 9 %), Fig. 6e. The DNA
showed the least changes after interaction with Ni(II)
azido complex, Fig. 6f (this weak hypochromism was
calculated as −3.5 %). The percentages were determined
from (εDNA + εCOM − εDNA − COM)/(εDNA + εCOM) × 100,
where εDNA is the extinction coefficient of CT-DNA, εCOM
is the extinction coefficient of a free Ni(II) complex, and
εDNA − COM is the extinction coefficient of a DNA-bound
Ni(II) complex.
red shift. In this case, considering the blue shift and the
positive charge on the side chain, electrostatic interaction is
the probable mode of binding.
In order to further investigate the binding mode, fluores-
cent displacement experiments were carried out with two
different dyes: Hoechst 33258, a minor groove binder, and
ethidium bromide, a typical DNA intercalator. Hoechst
33258 (H) binds strongly and selectively with high affin-
ity to double-stranded B-DNA structure and like other
minor groove binders, it recognizes at least four AT base
pairs. It binds by combination of hydrogen bonding, van
der Waals contacts with the walls of the minor groove,
and electrostatic interactions between its cationic struc-
ture and the DNA [43]. Binding of Hoechst 33258 to CT-
DNA was followed by excitation at 350 nm with maxi-
mum in fluorescence at 444 nm. The fluorescence intensity
of the band at 444 nm of the Hoechst–CT-DNA system
decreased remarkably with the increasing concentrations
The weak hypochromic effect was observed for interac-
tions between [NiL(NCS)]BF4 or [NiLCl]BF4 and DNA.
Evidence for intercalation is normally hypochromism and
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Fig. 5 Bar graphs of percent of HeLa cells labeled with Annexin
V-FITC (A) and PI (P) quantified by FACS, after 48 h treatment with
nickel(II) complex ([NiL(NCS)]BF4, [NiLN3]BF4, [NiL(NCO)]BF4
and [NiLCl]BF4) and CDDP (with 40 μM concentration for nickel(II)
complexes and 5 μM concentration for cisplatin); bar graphs repre-
sent mean SD of triplicates of one experiment
Fig. 6 Changes in UV–Vis absorption spectra of CT-DNA (88 µM)
after interaction with different concentrations of Ni(II) complexes.
a–c: UV–Vis absorption spectra of [NiL(NCS)]BF4 (30, 50 and
70 µM), [NiLCl]BF4 (30, 50 and 70 µM) and [NiLN3]BF4 (30, 50
and 70 µM), respectively, before and after interaction with CT-DNA;
c–e comparison of absorption at 258 nm between the CT-DNA–Ni(II)
complexes and the sum values of CT-DNA and Ni(II) complexes
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159
Fig. 7 Displacement of DNA-
bound Hoechst 33258 (H) by
Ni(II) complexes: a changes
of fluorescence intensities at
λmax = 444 nm with concentra-
tion of the complexes and b
fluorescence quenching curves
of H bound to CT-DNA at
λmax = 440 nm by [NiL(NCS)]
BF4, [NiLCl]BF4 and [NiLN3]
BF4; r = [Ni(II) complex]/[CT-
DNA]
Fig. 8 Displacement of DNA-
bound ethidium bromide (EB)
by [NiLN3]BF4 complex: emis-
sion spectra (λex = 500 nm) of
EB alone (25 µM, bottom line),
EB bound to CT-DNA (100 µM,
top line) and quenching of
EB–CT-DNA system by the
complex at increasing concen-
trations (0.5, 1, 1.5, 2, 4, 6, 8,
10, 12, 14, 16, 18 and 20 µM,
curves from top to bottom);
inset fluorescence quenching
curves of EB bound to CT-DNA
at λmax = 600 nm by [NiLN3]
BF4; r = [NiLN3]BF4]/[CT-
DNA]
of the Ni(II) complexes (Fig. 7a). The maximal decrease of
fluorescence intensity of Hoechst—CT-DNA by [NiLCl]
BF4 was 75 %, by [NiL(NCS)]BF4, 65 % and by [NiLN3]
BF4 35 %. Results showed that the displacement of Hoe-
chst by [NiLN3]BF4 was less efficient than by other two
nickel(II) complexes. These results were consistent with
quenching curves shown in Fig. 7b that were applied to
determine K by linear regression of a plot of I0/I against r.
The quenching constants calculated from the Stern–Volmer
equation [30] were K = 10.31, K = 15.35 and K = 2.88
for [NiL(NCS)]BF4, [NiLCl]BF4 and [NiLN3]BF4, respec-
tively. These data suggest minor groove binding of chlo-
rido and isothiocyanato complexes, but for azido complex,
a possible destabilization of DNA structure, especially if
hyperchromism (about −3.5 %) occurring in UV spectra at
higher concentrations of the complex is taken into account.
For the further study of the binding mode between a
Ni(II) complex and DNA, fluorescence quenching of DNA-
bound ethidium bromide (EB) system by [NiL(NCS)]BF4,
[NiLCl]BF4 and [NiLN3]BF4 was investigated. Isothiocy-
anato and chlorido Ni(II) complexes did not quench the
fluorescence of EB under the applied experimental condi-
tions. The result of the displacement experiment using the
intercalator ethidium bromide by [NiLN3]BF4 complex is
shown in Fig. 8. It showed that there was significant reduc-
tion (up to 38 %) in the fluorescence intensity of DNA–
EB by increasing concentrations of [NiLN3]BF4 complex,
accompanied by blue shift of emission maximum from
600 to 596 nm, so that an isosbestic point was formed at
550 nm. These changes are typical of metal complex bound
to double-stranded DNA through non-covalent interac-
tions [44]. Fluorescence quenching data were analyzed by
according to the Stern–Volmer Eq. (1) The value K = 0.965
was calculated from the ratio of the slope to the inter-
cept from the plot of I0/I versus r (r = [NiLN3]BF4]/[CT-
DNA]), demonstrated in inset in Fig. 8.
DNA cleavage studies
The ability of Ni(II) complexes to cleave double-stranded
plasmid DNA were investigated using an agarose electro-
phoretic assay. The assay allows assessment of DNA strand
cleavage by monitoring the conversion of untreated super-
coiled form (FI) plasmid DNA into the nicked form (FII)
and linear form (FIII). As shown in Fig. 9 (lane P), plas-
mid pUC19 consisted mainly of FI and FII. An addition of
a complex to the plasmid resulted in formation of a linear
form FIII and gradual disappearance of supercoiled form
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Fig. 9 Agarose gel electrophoretic analysis of plasmid pUC19
(512 ng) without (lanes P) and pUC19 (512 ng) with 0.1, 0.2, 0.3.
0.4, 0.5, 0.6 and 0.8 mM complex [NiL(NCS)]BF4, lanes 1–7,
respectively—(a); pUC19 (512 ng) with 0.1, 0.2, 0.3, 0.4, 0.5, 0.6
and 0.8 mM complex [NiLCl]BF4, lanes 8–14, respectively—(b);
pUC19 (512 ng) with 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 and 0.8 mM complex
[NiLN3]BF4, lanes 15–21, respectively—(c)
Fig. 10 Agarose gel electrophoretic analysis of restriction endonu-
clease fragmentation of pUC19 (512 ng) by BamHI (10 U) and Hin-
dIII (10 U) after and before interactions with the Ni(II) complexes: a
the control samples pUC19—lane P, pUC19 digested with BamHI—
lane B, and pUC19 digested HindIII—lane Hi; b pUC19 after inter-
action with 0.1 mM [NiL(NCS)]BF4 digested with BamHI and Hin-
dIII—lane 1 and lane 2, respectively; pUC19 digested with BamHI
and HindIII before interaction with 0.1 mM [NiL(NCS)]BF4—lane
3 and 4, respectively; c pUC19 after interaction with of 0.1 mM
[NiLCl]BF4 digested with BamHI and HindIII—lane 5 and lane 6,
respectively, and pUC19 digested with BamHI and HindIII before
interaction with 0.1 mM [NiLCl]BF4—lane 7 and 8, respectively;
d pUC19 after interaction with of 0.1 mM [NiLN3]BF4, digested
with BamHI and HindIII—lane 9 and lane 10, respectively; pUC19
digested with BamHI and HindIII before interaction with 0.1 mM
[NiLN3]BF4—lane 11 and 12, respectively; e pUC19 after interaction
with increasing concentrations of [NiLN3]BF4, complex (0.1, 0.2,
0.3, 0.4, 0.5, and 0.6 mM, lanes 13–18, respectively), digested with
BamHI; lane P—control pUC19; lane M—molecular weight markers
(1 kb DNA ladder)
in concentration-dependent way (Fig. 9a–c). The genera-
tion of the linear form increased gradually up to 0.6 mM of
[NiL(NCS)]BF4 (Fig. 9a, lane 6), up to 0.4 mM of [NiLCl]
BF4 (Fig. 9b, lane 11) and up to 0.3 mM of [NiLN3]BF4,
(Fig. 9c, lane 17), when the bands of these plasmid forms
smeared, indicating that the complexes possess a nuclease
activity, converting DNA to shorter fragments. As can be
seen in Fig. 9, the electrophoretic patterns indicating nucle-
ase activity were very similar with small differences for
all three Ni(II) complexes, the azido complex showing the
highest activity.
To check the cleavage activity of Ni(II) complexes,
two restriction enzymes were used in reaction with
pUC19. Restriction endonucleases typically recognize
short (4–8 bp) target sequences, usually palindromic, and
cleave double-stranded DNA at specific sites within a
Restriction enzyme activity is strongly dependent on the
local conformation of DNA at the restriction site. Gel elec-
trophoresis revealing the products formed after digestion of
plasmid pUC19 by BamHI and HindIII, in the absence and
in the presence of Ni(II) complexes, is shown in Fig. 10.
Two bands corresponding to the supercoiled FI and nicked
form FII were observed for the undigested plasmid pUC19
(Fig. 10, lane P). After BamHI and HindIII digestion, the
linear forms FIII corresponding to DNA fragment of 3.0 kb
were observed (Fig. 10a, lanes B and Hi, respectively). Par-
tial enzyme inhibition of BamHI digestion was observed
after the interactions of [NiL(NCS)]BF4, [NiLCl]BF4 and
[NiLN3]BF4 with pUC19, as shown in Fig. 10b–d, lanes
1, 5 and 9 respectively. When BamHI digestion of plasmid
is done first, and then interaction with the complexes fol-
lowed, there was no further DNA damage by [NiL(NCS)]
BF4 and [NiLCl]BF4 (Fig. 10b, c, lanes 2 and 6, respec-
tively). On the contrary, the interaction of [NiLN3]BF4
with previously digested DNA produced additional dam-
age at concentration of 0.1 mM, which did not induce dam-
age in pUC19 which had not been exposed to the restric-
tion enzyme, as shown by the smear of the corresponding
band (Fig. 10d, lane 10). With increasing concentration
and
recognition site [29].
The restriction enzymes BamHI and HindIII recognize the
sequences: and, respectively, and cut it at position as shown
by the arrows.
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J Biol Inorg Chem (2016) 21:145–162
161
of [NiLN3]BF4, more extensive damage was observed
(Fig. 10e, lanes 13–18).
DNA molecule. Metabolic stability of hydrazone ligand
(the presence of amide instead of ester group) or similar-
ity with biological molecules (as in the case of Girard’s T
reagent) might be important properties for enhancement
of biological activity. Comparison of biological activity of
pseudohalide complexes with the activity of chlorido com-
plex indicates that both monodentate ligand toxicity and its
influence on stability of complex compound are responsi-
ble for biological activity.
HindIII activity was not influenced by the presence of
[NiL(NCS)]BF4, and [NiLCl]BF4, either before or after
interaction with the DNA (Fig. 10b, lanes 3, 4 and Fig. 10c,
lanes 7, 8, respectively). The complex [NiLN3]BF4 did not
inhibit the HindIII digestion, as band smear can be seen
(Fig. 10d, lane 10), but after the enzyme digestion, the
complex did not induce any further damage (Fig. 10d lane
12).
Acknowledgments This work was supported by the Ministry of
Education, Science and Technological development of the Republic
of Serbia (Grant OI 172055 and Grant III 41026). We thank the Slove-
nian Research Agency (ARRS) through program P-0175 for financial
support and EN-FIST Centre of Excellence, Dunajska 156, 1000 Lju-
bljana, Slovenia, for using SuperNova diffractometer.
The obtained results indicated that all investigated Ni(II)
complexes possess nuclease activity towards supercoiled
DNA. They preferentially bind to sequences containing
less than four A-T base pairs in double-stranded DNA.
Complex [NiLN3]BF4 showed a stronger activity than the
other two. The observed selectivity of the azido complex
for some tumor cell lines can be ascribed to the stronger
DNA damaging activity. In comparison to the similar com-
plexes without a positive charge in the side chain [20], the
damaging effect was higher, probably due to stronger elec-
trostatic binding. The generally higher cytotoxic activity of
the complexes described in this paper compared to those
without a positive charge in the side chain [20] could be
ascribed to DNA binding and damage.
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