Efficient hydrolytic cleavage of DNA and antiproliferative effect on human cancer cells by two dinuclear Cu(II) complexes containing a carbohydrazone ligand and 1,10-phenanthroline as a coligand

Article abstract of DOI:10.1007/s00775-019-01651-8

Abstract: We report the synthesis, crystal structures and biological activities of two dinuclear Cu(II) complexes [Cu(o-phen)LCu(OAc)] (1) and [Cu(o-phen)LCu(o-phen)](OAc) (2), where o-phen = 1,10-phenanthroline, H₃L = o-HOC₆H₄C(H)=N–NH–C(OH)=N–N=C(H)–C₆H₄OH-o, and OAc=CH₃COO^?. Both compounds display strong and broad X-band EPR spectra at RT in their powder state confirming that these are paramagnetic. The intercalative DNA binding of the compounds as revealed from spectrophotometric studies was found to be consistent with the results of fluorescence spectroscopic studies for ethidium bromide displacement assay as well as enhanced viscosity of DNA in the presence of these compounds. The compounds effectively catalyze hydrolytic cleavage of supercoiled pUC19 DNA and show remarkable cytotoxicity toward human lung cancer A549 cell line (IC₅₀ values are 4.34 and 8.46?μM for 1 and 2, respectively) and breast cancer MCF7 cell line (IC₅₀ values are 6.50 and 8.68?μM for 1 and 2, respectively) and are found to be relatively less toxic toward keratinocyte HaCaT normal cell line (IC₅₀ values are 11.19 and 16.01?μM for 1 and 2, respectively). Annexin-V/PI dual staining results analyzed by flow cytometry strongly suggest the induction of apoptotic pathway for the anticancer activity of these complexes. Flow cytometry experiment for cell cycle analysis showed considerable increase in the G2/M phase in both A549 and MCF7 cell lines by these two compounds. On the other hand, compounds 1 and 2 activate reactive oxygen species (ROS) level in A549 cells, but act as scavengers or inhibitors of ROS in MCF7 cell line as analyzed by DCFDA staining using flow cytometry. Graphical abstract: [Figure not available: see fulltext.]Two dinuclear Cu(II) complexes exhibit efficient hydrolytic cleavage of DNA and display remarkable cytotoxicity against human lung cancer A549 and breast cancer MCF7cells. The ROS level in A549 cells is activated, but the ROS level in MCF7 cells is decreased in the presence of these complexes. Cell cycle analysis by flow cytometry shows G2/M phase arrest in both these cell lines.

Full text of DOI:10.1007/s00775-019-01651-8

JBIC Journal of Biological Inorganic Chemistry
https://doi.org/10.1007/s00775-019-01651-8
ORIGINAL PAPER
Efcient hydrolytic cleavage oꢀ DNA and antiproliꢀerative eꢁect
on human cancer cells by two dinuclear Cu(II) complexes containing
a carbohydrazone ligand and 1,10‑phenanthroline as a coligand
1
2
2
3
4
Sidhali U. Parsekar · Manohar Singh · Durga P. Mishra · P. K. Sudhadevi Antharjanam · Aditya P. Koley ·
Manjuri Kumar1
Received: 28 December 2018 / Accepted: 29 January 2019
©
Society for Biological Inorganic Chemistry (SBIC) 2019
Abstract
We report the synthesis, crystal structures and biological activities of two dinuclear Cu(II) complexes [Cu(o-phen)LCu(OAc)]
(
1) and [Cu(o-phen)LCu(o-phen)](OAc) (2), where o-phen = 1,10-phenanthroline, H L = o-HOC H C(H)=N–NH–
3
6
4
−
C(OH)=N–N=C(H)–C H OH-o, and OAc=CH COO . Both compounds display strong and broad X-band EPR spectra at
6
4
3
RT in their powder state conꢀrming that these are paramagnetic. The intercalative DNA binding of the compounds as revealed
from spectrophotometric studies was found to be consistent with the results of ꢁuorescence spectroscopic studies for ethidium
bromide displacement assay as well as enhanced viscosity of DNA in the presence of these compounds. The compounds
eꢂectively catalyze hydrolytic cleavage of supercoiled pUC19 DNA and show remarkable cytotoxicity toward human lung
cancer A549 cell line (IC values are 4.34 and 8.46 µM for 1 and 2, respectively) and breast cancer MCF7 cell line (IC
5
0
50
values are 6.50 and 8.68 µM for 1 and 2, respectively) and are found to be relatively less toxic toward keratinocyte HaCaT
normal cell line (IC values are 11.19 and 16.01 µM for 1 and 2, respectively). Annexin-V/PI dual staining results analyzed
5
0
by ꢁow cytometry strongly suggest the induction of apoptotic pathway for the anticancer activity of these complexes. Flow
cytometry experiment for cell cycle analysis showed considerable increase in the G2/M phase in both A549 and MCF7 cell
lines by these two compounds. On the other hand, compounds 1 and 2 activate reactive oxygen species (ROS) level in A549
cells, but act as scavengers or inhibitors of ROS in MCF7 cell line as analyzed by DCFDA staining using ꢁow cytometry.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00775-019-01651-8) contains
supplementary material, which is available to authorized users.
*
Aditya P. Koley
koleyap@yahoo.co.in
*
Manjuri Kumar
manjuri@goa.bits-pilani.ac.in
1
2
Department of Chemical Engineering, Birla Institute
of Technology and Science-Pilani, K.K. Birla Goa Campus,
Zuarinagar, Goa 403726, India
Cell Death Research Laboratory, Endocrinology Division,
CSIR-Central Drug Research Institute, Lucknow 226031,
India
3
4
Sophisticated Analytical Instrument Facility, Indian Institute
of Technology-Madras, Chennai 600036, India
Department of Chemistry, Birla Institute of Technology
and Science-Pilani, K.K. Birla Goa Campus, Zuarinagar,
Goa 403726, India
Vol.:(0
1
123456789)
3

JBIC Journal of Biological Inorganic Chemistry
Graphical abstract
Two dinuclear Cu(II) complexes exhibit eꢃcient hydrolytic cleavage of DNA and display remarkable cytotoxicity against
human lung cancer A549 and breast cancer MCF7cells. The ROS level in A549 cells is activated, but the ROS level in MCF7
cells is decreased in the presence of these complexes. Cell cycle analysis by ꢁow cytometry shows G2/M phase arrest in
both these cell lines.
Keywords Dinuclear Cu(II) complex · EPR and electronic spectra · DNA binding and cleavage · MTT assay · Cytotoxicity
and anticancer activity · Cell cycle analysis
we present the synthesis, characterization using ESI–MS,
IR, UV–visible, EPR, and X-ray crystal structures and the
biological activities of these two closely related dinuclear
Cu(II) compounds containing the enol form of the Schiꢂ
base ligand 1,5-bis(salicylidene)carbohydrazide, but dif-
fering in the types of coligand in the two Cu(II) sites. It
is to be noted here that we have reported [41] the synthe-
sis and some biological properties such as DNA binding
using UV–Vis spectroscopy, cytotoxicity using MTT assay
and apoptosis using AO/PI dual staining method for com-
pound 1, along with the corresponding heterodinuclear
Zn(II)–Cu(II) compound (3); however, we could not get
a single crystal at that time to structurally characterize it.
Herein, we conꢀrm its structure by X-ray crystallography
along with that of compound 2, and studied the hydrolytic
DNA cleavage. Also, to compare the cytotoxicity and cell
cycle analysis for 1 and 2 using human A549 and MCF7
cancer cell lines, we have carried out again the MTT assay
for 1 along with that of 2, as well as studied their apoptosis
using Annexin-V and PI double staining and cell cycle
analysis using ꢁow cytometry under identical experimental
conditions to obtain the exact diꢂerences in the biologi-
cal properties of these two closely related homodinuclear
complexes possessing only one o-phen coligand in the ꢀrst
Cu(II) site and an acetate in the second Cu(II) site of 1
versus two o-phen coligands, one each in both the Cu(II)
sites of 2.
Introduction
Studies involving DNA binding property, nucleic acid
cleavage activity and cell cytotoxicity for the metal com-
plexes, Cu(II) complexes in particular due to their bio-
compatibility and endogeneity have gained momentum in
the past two decades to ꢀnd new metallodrugs that may
serve as anticancer agents [1–22]. Many copper(II) com-
plexes cleave DNA in the presence of hydrogen peroxide
or ascorbic acid [23–28], but some copper(II) complexes
are capable of bringing about hydrolytic cleavage in the
absence of any external reagent or light [29–39] in spite
of the great stability of the phosphodiester bond in DNA
−
8
−1
(
k = 3.6 × 10
h
for unhydrolyzed ds-DNA) [33, 37].
Since hydrolytic cleavage involves the hydrolysis of the
phosphodiester bond, its great stability is a major concern
in hydrolytic cleavage. Therefore, there is a necessity of
nucleophilic activation for enormous enhancement of the
hydrolysis rate to bring about hydrolytic cleavage. Thus,
it is a diꢃcult task to ꢀnd or synthesize a suitable met-
allodrug that is capable of cleaving DNA hydrolytically
under physiological conditions [29–40]. We have syn-
thesized two dinuclear Cu(II) compounds capable of eꢃ-
cient hydrolytic cleavage of supercoiled pUC19 DNA and
also possess remarkable cytotoxicity toward human lung
cancer A549 and breast cancer MCF7 cell lines, but less
toxic toward keratinocyte HaCaT normal cell line. Here,
1
3

JBIC Journal of Biological Inorganic Chemistry
Materials and methods
Materials
0.001 mol) and Cu(OAc)2·H2O (0.2 g, 0.001 mol) in
methanol (30 mL) was very slowly added to a solution of
1,5-bis(salicylidene)carbohydrazide (0.298 g, 0.001 mol)
in 50 mL of methanol during 30 min, while the reaction
mixture turned bright green. This was stirred at RT for
3 h, then a solution of Cu(OAc)2·H2O (0.2 g, 0.001 mol)
dissolved in methanol (20 mL) was slowly added to this
bright green reaction mixture when the solution turned
dark green and a dark green compound started separating.
This solution was stirred at RT for another 20 h and the
dark green compound was ꢀltered through a G-4 cruci-
ble, and the mother ꢀltrate was collected. The compound
in the crucible was washed well with methanol and air
dried, then washed thoroughly with water followed by
methanol and dried in vacuo. Yield: 80%. Anal. Calc. for
Carbohydrazide and salicylaldehyde were obtained from
Aldrich. Calf thymus DNA was obtained from Sigma. N,N-
dimethylformamide (DMF, GR), absolute ethanol, metha-
nol (GR), DMSO, 1,10-phenanthroline monohydrate, and
Cu(OAc) ·H O (GR) were obtained from Merck. Super-
2
2
coiled (SC) plasmid pUC19 DNA was purchased from
Genetix Biotech. All other chemicals were of reagent grades.
Physical measurements
C H N O Cu : C, 52.65; H, 3.35; N, 12.70. Found: C,
Microanalyses (C, H, N) were performed in a Perkin-Elmer
29 22
6
5
2
5
2.55; H, 3.37; N, 12.71%. ESI–MS in DMSO: m/z 683.82
2
40C elemental analyzer. Mass spectra were recorded on a
+
+
[
M+Na] and m/z 701.77 [M+K+H] .
PerkinElmer (USA) Flexer SQ 300 MS Mass Spectrometer
operating in ESI mode. Infrared spectra were recorded with
a Shimadzu IR Aꢃnity-1 FT-IR spectrometer using KBr pel-
let. Electronic spectra were recorded on a Jasco V-570 UV/
VIS/NIR spectrophotometer using a pair of matched quartz
cell of path length 1 cm. Electron paramagnetic resonance
A part of the mother ꢀltrate was collected in a long
tube and left in air for very slow evaporation. Green crys-
tals suitable for X-ray diꢂraction were obtained within
3
0 days. We have characterized this compound by X-ray
crystal structure (vide infra). These crystals lose their
solvent of crystallization (both methanol and water) and
become amorphous on long standing at RT or in vacuum.
(
EPR) spectra were recorded on a JEOL, Japan Model: JES-
FA200 ESR spectrometer with X and Q band. Fluorescence
measurements were performed using a Jasco spectroꢁuor-
ometer model FP-8500.
Preparation oꢀ [Cu(o‑phen)LCu(o‑phen)](OAc) (2)
1
,10-Phenanthroline monohydrate (0.398 g, 0.002 mol)
Syntheses
in 20 mL of methanol was slowly added to a solution of
Cu(OAc) ·H O (0.398 g, 0.002 mol) dissolved in methanol
2
2
Preparation oꢀ 1,5‑bis(salicylidene)carbohydrazide,
(
20 mL) and the solution was stirred at RT for 30 min and
(
o‑HOC H CH=NNH) C=O (H L)
6 4 2 3
stored at 4 °C. Then this cold blue solution was slowly
added to a solution of 1,5-bis(salicylidene)carbohydrazide
This compound was prepared by the reported method of
Koley et al. [41]. To a stirring solution of carbohydrazide
(
0.298 g, 0.001 mol) in 50 mL of methanol during 30 min,
while the reaction mixture turned dark green. This was
stirred for 20 h at RT and the dark green compound was
(
5 mmol) dissolved in 30 mL of absolute ethanol, salicy-
laldehyde (10 mmol) was added dropwise at RT and the
reaction mixture was stirred at RT for 2 h while a white com-
pound separated. This was ꢀltered through a G-3 crucible
and the pale white solid was washed well with ethanol and
air dried. This compound was recrystallized from metha-
nol–ethanol (1:1). Yield 70%; mp 226 °C. Anal. Calc. for
ꢀ
ltered through a G-4 crucible and the dark green solid
was washed very well with methanol and air dried. Then
the compound was washed with water and ꢀnally washed
again with methanol and dried in vacuo. Anal. Calc. for
C H N O Cu : C, 58.44; H, 3.59; N, 13.30. Found: C,
4
1
30
8
5
2
5
8.49; H, 3.59; N, 13.37%. ESI–MS in CH CN: m/z 843.7
3
C H N O : C, 60.40; H, 4.73; N, 18.78. Found: C, 60.57;
1
5
14
4
3
+
+
[
M+2H] and m/z 865.5 [M+Na+H] .
H, 4.76; N, 18.81%.
A saturated methanol solution of this green compound
was collected in a 5 mL Corning tube that was placed
inside a 15 mL Corning tube containing ~ 2 mL toluene
and was covered with a cap and left for slow evaporation
at RT when blue crystals were obtained within 30 days and
were used for X-ray crystallography. It was noted that on
long standing (or in vacuum), these crystals also lose their
solvent of crystallization and become amorphous.
Preparation oꢀ [Cu(o‑phen)LCu(OAc)] (1)
This compound was also prepared following the method
reported by Koley et al. [41]. In brief, a cold (~ 5°C)
solution of 1,10-phenanthroline monohydrate (0.2 g,
1
3

JBIC Journal of Biological Inorganic Chemistry
X‑ray crystallography
riding model with C–H or N–H 0.93 Å, (C–H=0.96 Å for
CH ) Uiso(H)=1.2Ueq(C) (Uiso(H)=1.5Ueq(C) for CH
3
3
[
Cu(o‑phen)LCu(OAc)] ·½CH OH·½H O
and N–H). The acetate anion is triply disordered with site
occupancies in the ratio of 42:24:34. Hydrogens on disor-
dered solvent molecules could not be located. Crystal data
and structure reꢀnement for 1 and 2 are presented in Table 1.
2
3
2
X-ray data collection was performed with Bruker AXS
Kappa Apex III CMOS Diꢂractometer equipped with graph-
ite monochromated Mo (Kα) (λ=0.71073 Å) radiation. The
automatic cell determination routine, with 24 frames at two
diꢂerent orientations of the detector, was employed to col-
lect reꢁections for unit cell determination. Further, intensity
data for structure determination were collected through an
optimized strategy which gave an average fourfold redun-
dancy. The program APEX23-SAINT [42] was used for
integrating the frames. Fourfold redundancy per reꢁection
was utilized for achieving good multi-scan absorption cor-
rection using the program SADABS [42]. The structure
was solved by SHELXT-2014 [43] and refined by full-
matrix least-squares techniques using SHELXL-2014, [43]
computer program. Molecular graphics were drawn using
ORTEP3 [44]. Hydrogens on all carbon atoms were ꢀxed
at calculated positions and reꢀned as riding model with
DNA binding experiments
Absorption spectral studies
Absorption titrations were performed by adding incremental
amounts of calf thymus DNA (CT-DNA) to a complex solu-
−5
tion having concentration of 5×10 M in 10 mM Tris–HCl
buꢂer at pH 7.4. The concentration of CT-DNA was calcu-
lated from the absorbance at 260 nm with the molar extinc-
−
1
−1
tion coeꢃcient for CT-DNA as 6600 M cm at 260 nm.
After every incremental addition of DNA, the reaction mix-
ture was allowed to equilibrate for 2 min at room tempera-
ture after which the absorption spectra was recorded. The
experimental data were used to calculate the intrinsic bind-
C–H=0.93 Å, (C–H=0.96 Å for CH ) Uiso(H)=1.2Ueq(C)
3
(Uiso(H)=1.5Ueq(C) for CH ), whereas hydrogens on nitro-
ing constant K .
3
b
gen were ꢀxed from the diꢂerence electron density peaks
and allowed to reꢀne freely. Since methanol and water sol-
vents are disordered, its site occupancy is constrained at 0.5.
Also, the positions of hydrogen on these solvents could not
be located and reꢀned satisfactorily and hence ignored. The
crystal data belong to a non-merohedrally twinned crystal.
The twin law [− 1 0 0, 0 − 1 0.466, 0 0 1] was identiꢀed
by TwinRotMat program and is used for the generation of
HKL5 reꢁection format which was used for reꢀnement. The
percentage of twin is given by a batch-scale factor (BASF)
of 0.14.
Viscosity measurements
Viscosity measurements were carried using CT-DNA
(100 µM) with increasing concentrations (0–100 µM) of
the complexes 1 and 2 using Ostwald viscometer. Viscosity
values were calculated from the ꢁow time of buꢂer alone
(t ) and ꢁow time of each sample (t) using the equation
0
η=(t−t )/t . Data were presented by plotting values of rela-
0
0
1
/3
tive viscosity (η/η ) against [complex]/[DNA], where η is
0
the viscosity of DNA in the presence of complex and η is
0
[Cu(o‑phen)LCu(o‑phen)](OAc)·H2O
the viscosity of DNA alone.
X-ray data collection was performed with Bruker AXS
Kappa Apex III CMOS dual source diꢂractometer with Cu
Competitive DNA binding ꢂuorescence measurements
(
Kα) (λ = 1.54178 Å) radiation. The automatic cell deter-
mination routine, with 40 frames at two diꢂerent orienta-
tions of the detector, was employed to collect reꢁections
for unit cell determination. Further, intensity data for struc-
ture determination were collected through an optimized
strategy which gave an average fourfold redundancy. The
program APEX3-SAINT [42] was used for integrating the
frames. Fourfold redundancy per reꢁection was utilized
for achieving good multi-scan absorption correction using
the program SADABS [42]. The structure was solved by
SHELXT-2014 [43] and reꢀned by full-matrix least-squares
techniques using SHELXL-2014 [43] computer program.
Molecular graphics were drawn using ORTEP3 [44]. All
hydrogens were ꢀxed at calculated positions and reꢀned as
The competitive binding studies using ethidium bromide
(EB)-bound CT-DNA was investigated using ꢁuorescent
spectrometry. The experiments were carried out by adding
incremental amounts of complexes 1 and 2 into EB–DNA
solution (2 µM EB and 50 µM CT-DNA) in 10 mM Tris
HCl buꢂer (pH 7.9). The solutions were incubated at room
temperature for 5 min to fully react, prior to recording the
spectra. The eꢂect of addition of increasing amounts of 1
and 2 to the EB–DNA complex was monitored by recording
the variation of ꢁuorescence emission spectra with excita-
tion at 510 nm and emission at 597 nm. The behavior was
analyzed through the Stern–Volmer equation and the appar-
ent binding constant (K ) was calculated.
app
1
3

JBIC Journal of Biological Inorganic Chemistry
a
Table 1 Crystal data and structure reꢀnement for 1 and 2
Identiꢀcation code
Compound 1
Compound 2
Empirical formula
Formula weight
Temperature
Wavelength
Crystal system
Space group
C117 H88 Cu N O22
C41 H32 Cu N O
6
859.82
296(2) K
1.54178 Å
Triclinic
P-1
8
24
2
8
2690.43
296(2) K
0.71073 Å
Triclinic
P-1
Unit cell dimensions
a=12.4593(2) Å
b=16.0360(2) Å
c=16.0585(3) Å
α=76.521(6)°
β=89.935(7)°
γ=67.478(6)°
a=12.0174(7) Å
b=12.1041(6) Å
c=14.5463(7) Å
α=107.890(3)°
β=97.769(4)°
γ=107.645(4)°
3
3
Volume
2868.20(16) Å
1857.06(18) Å
Z
1
2
3
3
Density (calculated)
Absorption coeꢃcient
F(000)
Crystal size
Theta range for data collection
Index ranges
Reꢁections collected
Independent reꢁections
Completeness to theta
Absorption correction
Max. and min. transmission
Reꢀnement method
Data/restraints/parameters
Goodness-of-ꢀt on F2
Final R indices [I>2sigma(I)]
R indices (all data)
Extinction coeꢃcient
Largest diꢂ. peak and hole
1.558 mg/m
1.538 mg/m
1.923 mm
880
1.535 mm−
1
−1
1366
3
3
0.100×0.100×0.050 mm
2.678°–21.910°
−12≤h≤12, −16≤k≤16, −16≤l≤16
33,612
33,612 [R(int)=?]
(=21.910°) 98.5%
Semi-empirical from equivalents
0.7457 and 0.5487
Full-matrix least-squares on F
33,612/64/789
1.047
0.100×0.100×0.050 mm
3.299°–66.992°
−13≤h≤14, −14≤k≤14, −17≤l≤17
48,548
6616 [R(int)=0.2148]
(=66.992°) 99.9%
Semi-empirical from equivalents
0.75 and 0.63
Full-matrix least-squares on F
6616/250/600
1.022
R1=0.0785, wR2=0.1802
R1=0.1550, wR2=0.2245
0.0004(2)
2
2
R1=0.0823, wR2=0.2295
R1=0.1249, wR2=0.2627
n/a
1.478 and −0.816 e Å−
3
0.623 and −0.531 e Å
−3
a
Crystallographic data for the structural analysis have been deposited with the Cambridge Crystallographic Data Center; numbers are CCDC
1
887320 for compound 1 and CCDC 1887321 for compound 2, respectively
DNA cleavage experiments
in hydrolytic DNA cleavage experiments were quantiꢀed
using image J. The ability of the complexes to convert the
supercoiled (SC, form I) to nicked circular (NC, form II)
DNA was used to measure the cleavage eꢃciency.
The interaction of the complexes 1 and 2 with supercoiled
pUC19 DNA was studied using agarose gel electrophoresis.
The cleavage of pUC19 DNA was accomplished by treating
SC plasmid pUC19 DNA (200 ng) with diꢂerent concen-
trations of complexes 1 and 2 in 50 mM Tris–HCl buꢂer
Oxidative DNA cleavage by the complexes was studied
by performing reactions in dark using hydrogen peroxide
(0.1 mM) as the oxidizing agent in the absence and presence
of the complex (0–25 µM). The solutions were incubated
at 37 °C for 1 h, subjected to electrophoresis and analyzed
for DNA cleaved products. For mechanistic investigations,
experiments were also conducted in the presence of radical
scavengers such as DMSO (2 µL), a hydroxyl radical scav-
(
pH 8.0), the samples were incubated for 3 h at 37 °C in
dark, after which a loading buꢂer containing 25% bromo-
phenol blue, 0.25% xylene cyanol and 30% glycerol was
added, electrophoresis was performed at 60 V for 1.5 h and
analyzed for cleaved products. The gel was stained with
0
.5 µg/mL ethidium bromide and viewed under UV light.
enger, and NaN (100 µM), a singlet oxygen quencher, which
3
The bands originating from SC (form I) and NC (form II)
were added to pUC19 DNA prior to the addition of complex.
1
3

JBIC Journal of Biological Inorganic Chemistry
To ensure that the copper complexes 1 and 2 as a whole
are responsible for cleavage of DNA, control experi-
ments were performed with Cu(OAc) ·H O (500 µM), free
(5 µL PI and 3 µL Annexin-V FITC in the 1× binding buꢂer)
for 15 min at room temperature. The volume was made up
to 300 µL by 1× binding buꢂer and apoptosis was measured
using ꢁow cytometry FACS caliber and data were analyzed
with Modꢀt software.
2
2
ligands 1,5-bis(salicylidene)carbohydrazide (500 µM) and
1
,10-phenanthroline monohydrate (500 µM).
For kinetic measurements, the DNA cleavage rate was
measured keeping ꢀxed concentrations of DNA (200 ng)
and 1 or 2 (200 µM) at 37 °C for diꢂerent time intervals.
The decrease in form I was plotted against time and the data
Cell cycle analysis
The different phases of cell cycle were analyzed by flow
cytometry as in a previously described protocol [45, 46]. A549
and MCF7 cells were cultured in DMEM media and treated
with 1 µM, 2.5 µM, 5 µM and 10 µM concentration of com-
pounds 1 and 2 for 24 h. In brief, cells were ꢀxed in 70% ice
cold ethanol for 1 h on ice and washed with 1× PBS twice.
Fixed cells were incubated in 300 µL staining solution (50 µg/
mL propidium iodide and 50 µg/mL RNaseA in 1× PBS for
30 min at room temperature in dark). Speciꢀc cell cycle phase
arrest was assessed by FACS caliber and data were analyzed
with Modꢀt software.
ꢀ
tted into a single exponential decay curve; thereafter, the
hydrolysis rate constant K was calculated to determine
obs
the rate enhancement in comparison to that of unhydrolyzed
rate of ds-DNA.
Cell culture and treatment
Cancer cell lines A549, MCF7 and normal human keratino-
cyte HaCaT cells were obtained from American Type Cul-
ture Collection (ATCC; Manassas, VA, USA). HaCaT and
MCF7 cell lines were cultured in DMEM, while A549 cell
line was cultured in DMEM F12, supplemented with 10%
Reactive oxygen species (ROS) detection assay
FBS and antibiotic–antimycotic at 37 °C with 5% CO in a
2
CO incubator. The cells were subcultured after they had
The generation of reactive oxygen species (ROS) in A549 and
MCF7 cells was detected using ROS-sensitive ꢁuorescent
2
reached 70–80% conꢁuence by trypsinization and used for
further experimental purpose.
probe 2′,7′-dichlorodihydroꢁuorescein diacetate (H DCFDA).
2
In cells, H DCFDA is hydrolyzed by esterases into H DCF and
2
2
Cell viability assay
further oxidized by ROS to 2′,7′-dichlorodihydroꢁuorescein
(
DCF) which exhibits intense green ꢁuorescence. Brieꢁy, the
Cell viability was assessed by MTT assay according to a
previously standardized protocol [45, 46]. Brieꢁy, A549,
cultured cells were treated with diꢂerent concentrations (1,
2.5, 5 and 10 µM) of compounds 1 and 2 for 24 h, untreated
cells were maintained as control and cells treated with freshly
prepared H O were used as positive control. After comple-
3
MCF7 and HaCaT cells (5×10 cells/well) were seeded in
96-well plates and treated with 1, 2.5, 5, 10, 15 and 20 µM of
2
2
compounds 1 and 2 for 24 h. After completion of treatment,
cells were incubated with 500 µg/mL MTT (3-(4,5-dimeth-
ylthiazol-2-yl)-2-5-diphenyltetrazolium) at 37 °C for 2 h.
Culture media were removed and the formazan crystals
were dissolved in 100 µL of DMSO. The color intensity was
measured in a BMG ꢁuostar Optima plate reader at 450 nm
and percentage cell viability was calculated. The cytotox-
icity of the free Schiꢂ base ligand and the free coligand
tion of treatment, cells were trypsinized and washed with 1×
PBS. Subsequently, the samples were incubated in 10 µM
DCFDA for 30 min at room temperature in dark. Cellular
reactive oxygen species (ROS) generation was measured by
ꢁow cytometry by DCFDA staining at 488 nm excitation and
535 emission laser.
Statistical analysis
1
,10-phenanthroline were also studied using the human lung
cancer A549 cell line with diꢂerent concentrations (0.5 µM,
.0 µM, 2.5 µM, 5 µM and 10 µM) of these free ligands.
All assays with established cell lines were repeated three times.
Data are expressed as the mean± standard deviation and ana-
lyzed by one-way analysis with diꢂerences considered signiꢀ-
cant at *<0.05, **<0.01, and ***<0.001 (GraphPad Prism 5).
1
Quantifcation oꢀ apoptosis by Annexin‑V and PI
double staining
A549 and MCF7 cells were cultured in DMEM media and
treated with diꢂerent concentrations (1, 2.5, 5, 10, 15 and
Results and discussion
2
0 µM) of compounds 1 and 2 for 24 h. Apoptosis was meas-
Synthesis and characterization
ured by ꢁow cytometry using Annexin-V/PI labeling according
to the previously described protocol [45, 46]. Brieꢁy, A549
and MCF7 cells were incubated in 100 µL staining solution
The free Schiꢂ base ligand 1,5-bis(salicylidene)carbohy-
drazide (A) which is in keto form in the solid [41] can be
1
3

JBIC Journal of Biological Inorganic Chemistry
in its enol form in solution that can exist either in syn (B)
or anti (B′) as shown in Scheme 1. B′ possesses two dis-
tinctly diꢂerent tridentate coordination sites: the ꢀrst site
and 2.10 for 2, suggesting they are paramagnetic. Also, the
presence of forbidden ΔMs=± 2 line around g ~4 in both
of them (Fig. S2, inset) clearly suggests that it results from
a dimer Cu–Cu interaction. Based on their physico-chemi-
cal properties, elemental analysis, mass spectra and X-band
EPR results, the proposed structures of 1–2 are shown in
Scheme 2.
−
−
is characterized by one phenolate O , one enolate O and
one imine N, and the second site is characterized by other
−
phenolate O , other imine N and the secondary NH donor
atom. Coordination of the ligand in its B′ form in both 1
and 2 is conꢀrmed from the crystal structures of both the
compounds (vide infra).
The IR spectrum of the powder sample of the free ligand
1,5-bis(salicylidene)carbohydrazide (A) clearly shows the
−
1
−1
The reaction of Cu(OAc) ·H O with 1,5-bis(salicylidene)
ν(C=O) at 1690 cm and ν(C=N) at 1625 cm (ꢀgure not
shown), respectively, suggesting it exists in its keto form
which was conꢀrmed previously from its crystal structure
2
2
carbohydrazide (A) in methanol in the presence of
1
,10-phenanthroline (o-phen) in the ratio of 2:1:1 resulted
−
1
in the dinuclear Cu(II) complex containing one coordinated
o-phen coligand to the ꢀrst Cu(II) center and one acetate
coordinated to the second Cu(II) center in compound 1,
and the reaction in the ratio of 2:1:2 yielded compound 2,
both the Cu(II) centers containing one o-phen each and one
acetate counteranion.
[41]. The ν(C=O) band at 1690 cm is found to be absent
−
1
in the IR spectra of both 1 and 2, and the band at 1625 cm
−
1
due to ν(C=N) of the free ligand is shifted to 1605 cm in
the IR spectra of both the complexes indicating the coordina-
tion of the imine N to the metal ion. This is conꢀrmed from
the crystal structures of 1 and 2.
The solubility of the complexes is low in methanol and
acetonitrile, but they are readily soluble in DMF and DMSO.
The ESI-MS spectra for complexes 1–2 have been recorded
in the positive-ion mode and the results are presented in
Fig. S1 (Supplementary material). Complex 1 showed peaks
The electronic absorption spectra of both the compounds
in DMSO are found to be very similar (Fig. S3, Supple-
mentary material) suggesting their very similar electronic
structures. Both the compounds display ligand ꢀeld tran-
sitions around 622 nm, followed by strong LMCT in the
visible region apart from other charge transfer transitions
in the UV region.
+
+,
at m/z 683.82 [M+Na] and m/z 701.77 [M+K+H] while
+
compound 2 showed peaks at m/z 843.7 [M+2H] and m/z
+
8
65.5 [M+Na+H] , respectively. Both the compounds dis-
play strong X-band EPR at RT in their powder state (Fig.
Crystal structures oꢀ [Cu(o‑phen)
S2, Supplementary material) with g values of 2.09 for 1
LCu(OAc)] ·½CH OH·½H O and [Cu(o‑phen)
2
3
2
LCu(o‑phen)] (OAc)·H O
2
The structures of the compounds 1 and 2 have been
determined by X-ray crystallography. Figure 1 shows the
ORTEP representation of the molecule of 1 with 40%
Scheme 1 Proposed structures of the ligand in its keto and enol
forms
Scheme 2 Proposed structures of the compounds 1–2
1
3

JBIC Journal of Biological Inorganic Chemistry
probability ellipsoids [44]. The crystal for 1 is indexed in
triclinic system with space group P-1 and lattice parame-
ters a=12.4593(2) Å, b=16.0360(2) Å, c=16.0585(3) Å,
α=76.521(6)°, β=89.935(7)°, γ=67.478(6)°.
hexadentate ligand connects two copper atoms with a
Cu–Cu distance of 4.75 Å. The acetate anion is co-ordi-
nated to Cu1 and Cu3 through its bidentate binding mode
and the 1,10-phenanthroline moiety is co-ordinated to Cu2
and Cu4. The two binuclear copper complexes in the asym-
metric unit are held (Fig. 2) together by N(3)-H(3A)…
O(10) hydrogen bond interaction with a d(H…A) distance
of 1.94(5) Å. In addition to this, the crystal lattice is sta-
bilized by CH…O and CH…π interactions and also by the
The asymmetric unit of the crystal contains two crystal-
lographically independent molecules of binuclear copper
complex, half molecule of methanol and half molecule of
water. The central copper atom shows penta-coordination
forming distorted square pyramidal geometry. The unique
Fig. 1 ORTEP
diagram
of
the
compound
[Cu(o-phen)
N(6), 169.7(4); O(1)–Cu(2)–O(2), 163.1(4); N(1)–Cu(2)–O(2),
81.5(4); N(6)–Cu(2)–O(2), 88.2(4); O(1)–Cu(2)–N(5), 110.5(4);
N(1)–Cu(2)–N(5), 99.3(4); N(6)–Cu(2)–N(5), 80.0(4); O(2)–Cu(2)–
N(5), 86.3(4); O(8)–Cu(3)–N(10), 91.2(5); O(8)–Cu(3)–O(9),
93.2(4); N(10)–Cu(3)–O(9), 170.4(4); O(8)–Cu(3)–N(8), 165.5(5);
N(10)–Cu(3)–N(8), 80.1(4); O(9)–Cu(3)–N(8), 97.2(4); O(6)–Cu(4)–
N(7), 94.5(5); O(6)–Cu(4)–N(11), 95.5(4); N(7)–Cu(4)–N(11),
169.7(5); O(6)–Cu(4)–O(7), 162.7(4); N(7)–Cu(4)–O(7), 81.1(4);
N(11)–Cu(4)–O(7), 88.6(4); O(6)–Cu(4)–N(12), 111.4(4); N(7)–
Cu(4)–N(12), 99.5(4); N(11)–Cu(4)–N(12), 79.2(4); O(7)–Cu(4)–
N(12), 85.9(4)
LCu(OAc)] ·½CH OH·½H O. Selected bond lengths (Å) and angles
2
3
2
(
°):N(1)–Cu(2), 1.918(10); N(2)–Cu(1), 1.978(11); N(4)–Cu(1),
1
.964(10); N(5)–Cu(2), 2.266(12); N(6)–Cu(2), 1.985(11); N(7)–
Cu(4), 1.944(11); N(8)–Cu(3), 1.999(11); N(10)–Cu(3), 1.958(11);
N(11)–Cu(4), 1.996(11); N(12)–Cu(4), 2.264(11); O(1)–Cu(2),
1
1
1
.899(9); O(2)–Cu(2), 2.013(9); O(3)–Cu(1), 1.880(10); O(4)–Cu(1),
.956(9); O(6)–Cu(4), 1.895(10); O(7)–Cu(4), 2.018(9); O(8)–Cu(3),
.883(10); O(9)–Cu(3), 1.958(9); O(3)–Cu(1)–O(4), 93.5(4); O(3)–
Cu(1)–N(4), 90.9(4); O(4)–Cu(1)–N(4), 169.8(4); O(3)–Cu(1)–N(2),
1
66.0(5); O(4)–Cu(1)–N(2), 96.7(4); N(4)–Cu(1)–N(2), 80.7(4);
O(1)–Cu(2)–N(1), 94.5(4); O(1)–Cu(2)–N(6), 95.3(4); N(1)–Cu(2)–
1
3

JBIC Journal of Biological Inorganic Chemistry
solvent-mediated hydrogen bond interactions. The overall
structure of this compound is found to be similar to that of
the compound Zn(o-phen)LCu(OAc)·0.5H O·0.5CH OH
group P-1. The lattice parameters are: a = 12.0174(7) Å,
b = 12.1041(6) Å, c = 14.5463(7) Å, α = 107.890(3)°,
β = 97.769(4)°, γ = 107.645(4)°. The asymmetric unit of
the crystal contains one binuclear copper complex, one
molecule of acetate anion and one molecule of water.
The copper atoms show penta-coordination forming dis-
torted square pyramidal geometry. The unique hexadentate
ligand connects two copper atoms with a Cu–Cu distance
of 4.73 Å. The disordered acetate anion does not show
any coordination to metal atoms; instead, it participates in
H-bond interaction with the N–H moiety of the hexaden-
tate ligand. Solvent-mediated H-bond interactions hold the
binuclear copper complexes together in the crystal lattice
2
3
we have reported earlier [41].
Figure 3 shows the ORTEP representation of com-
pound 2 with 40% probability ellipsoids [42]. The mol-
ecule of 2 crystallizes in triclinic crystal system with space
(
Fig. 4).
DNA binding studies
Absorption spectroscopic studies
The binding aꢃnity of complex 2 toward calf thymus DNA
(
CT-DNA) has been studied by electronic absorption spec-
troscopy. In general, the absorption spectra of metal com-
plexes binding to CT-DNA through intercalative mode show
decrease in molar absorptivity due to strong stacking inter-
action between the aromatic chromophore of the ligand and
Fig. 2 Packing of the molecules of 1 in the unit cell
Fig. 3 ORTEP diagram of
the compound [Cu(o-phen)
LCu(o-phen)] (OAc)·H O.
2
Selected bond lengths (Å)
and angles (°):N(1)–Cu(1),
1
.948(6); N(3)–Cu(2), 1.936(6);
N(4)–Cu(2), 2.024(6); N(5)–
Cu(2), 2.306(6); N(6)–Cu(1),
2
.021(6); N(7)–Cu(1), 2.326(7);
N(8)–Cu(1), 2.005(6); O(1)–
Cu(1), 1.901(5); O(2)–Cu(2),
1
.959(5); O(3)–Cu(2), 1.902(5);
O(1)–Cu(1)–N(8), 162.4(3);
N(1)–Cu(1)–N(8), 79.8(2);
O(1)–Cu(1)–N(6), 90.0(2);
N(1)–Cu(1)–N(6), 174.2(3);
N(8)–Cu(1)–N(6), 99.8(2);
O(1)–Cu(1)–N(7), 103.1(2);
N(1)–Cu(1)–N(7), 97.2(2);
N(8)–Cu(1)–N(7), 93.4(2);
N(6)–Cu(1)–N(7), 77.0(2);
O(3)–Cu(2)–N(3), 93.7(2);
O(3)–Cu(2)–O(2), 163.3(2);
N(3)–Cu(2)–O(2), 81.4(2);
O(3)–Cu(2)–N(4), 92.0(2);
N(3)–Cu(2)–N(4), 174.3(2);
O(2)–Cu(2)–N(4), 93.0(2);
O(3)–Cu(2)–N(5), 95.3(2);
N(3)–Cu(2)–N(5), 103.1(2);
O(2)–Cu(2)–N(5), 101.3(2);
N(4)–Cu(2)–N(5), 76.8(2)
1
3

JBIC Journal of Biological Inorganic Chemistry
of [DNA]/(ε −ε ) versus [DNA]. The observed K value was
a
f
b
5
−1
found to be 2.86×10 M for 2 which is slightly higher than
5
−1
that reported (2.63 × 10 M ) for 1 [41], suggesting that
complex 2 has a higher DNA binding aꢃnity than 1.
Viscosity measurements
As a means to further support the intercalative mode of
DNA binding results obtained from spectroscopic anal-
ysis, viscosity measurements were carried out to study
the eꢂect of increasing concentrations of both 1 and 2
on CT-DNA. A classical intercalative mode of binding
causes signiꢀcant increase in viscosity, because insertion
of intercalator causes the base pairs of DNA to separate
and unwind resulting in an increase in the overall length of
DNA, which increases the viscosity. Based on our obser-
Fig. 4 Packing of the molecules of 2 in the unit cell
the DNA base pairs. The UV–Vis absorption spectrum of
compound 2 is signiꢀcantly perturbed on addition of incre-
mental amounts of CT-DNA (Fig. 5). The absorption band at
1
/3
vations, the data plotted for the relative viscosity (η/η )
0
3
96 nm shows hypochromism of 67.3% for 2. The observed
against [complex]/[DNA] (Fig. 6) reveal that there is
steady increase in viscosity in case of both copper com-
plexes 1 and 2 which conꢀrms the intercalative mode of
binding of the copper(II) complexes to CT-DNA. It is to
be noted that increase in viscosity is more prominent in 2
than 1 which is found to be in line with spectral studies.
spectroscopic changes thus imply intercalation of 2 into the
DNA base stack. A similar result was observed for 1 as we
have reported earlier [41]. The intrinsic binding constant
(
K ) for the association of 2 with CT-DNA was determined
b
quantitatively using the Eq. (1):
DNA] [DNA]
[
1
=
+
ꢀ − ꢀ ) (ꢀ − ꢀ ) K (ꢀ − ꢀ )
a f b f b b f
,
Competitive DNA binding: ꢂuorescence studies
(1)
(
Ethidium bromide (EB) is a planar intercalating dye which
emits ꢁuorescence upon binding to calf thymus DNA. We
performed ethidium bromide displacement assay to examine
the ability of the complexes 1 and 2 to replace EB from the
EB–DNA complex. The interaction of complexes 1 and 2
with ethidium bromide-bound CT-DNA in 10 mM Tris–HCl
buꢂer (pH 7.9) was carried out and it was found that the
where [DNA] denotes the concentration of CT-DNA, and
the apparent extinction coeꢃcient (ε ) was obtained by
a
calculating Abs/[complex]. The terms ε and ε denote the
f
b
extinction coeꢃcients of free (unbound) and the fully bound
complexes, respectively. The binding constant was calcu-
lated from the ratio of the slope to the intercept of the plot
3
2
2
1
1
0
.0
.5
.0
.5
.0
.5
1
2
+ CT DNA
+ CT DNA
0
.0
0.2
0.4
0.6
0.8
1.0
1.2
[
Complex]/[DNA]
Fig. 5 Change in electronic absorption spectra of 2 [5×10− M]
upon titration with CT-DNA (0–30 µM) dissolved in 10 mM
Tris–HCl buꢂer (pH 7.4). The ꢀgure in inset shows the linear
5
Fig. 6 Eꢂect of increasing amount of 1 and 2 on the relative viscosity
of CT-DNA (100 µM) in 10 mM Tris–HCl buꢂer (pH 7.4) at room
temperature
ꢀ
t of [DNA]/(ε −ε ) versus [DNA]. Intrinsic binding constant
a f
5 −1 2
K =2.86 ×10 M (R =0.99874 for 11 points)
b
1
3

JBIC Journal of Biological Inorganic Chemistry
emission band at 597 nm of the EB–DNA system decreased
in intensity with increase in complex concentration (Fig. 7).
This observation implies that both the complexes have been
able to displace EB from EB–DNA complex and such char-
acteristic decrease is observed in DNA interaction by the
intercalative mode. The quenching of EB bound to DNA by
the copper complexes 1 and 2 is in agreement with the linear
Stern–Volmer equation (2) (Fig. 7 inset):
complex concentration at 597 nm was used to calculate
the apparent binding constant from 50% quenching using
Eq. (3):
KEB[EB] = Kapp[complex]50,
(3)
where [complex] is the complex concentration at
50
5
0% reduction of the fluorescence intensity of EB-
bound CT-DNA, K is the binding constant of EB
EB
7
−1
(
K
=1.0×10 M ) and [EB] is the concentration of eth-
EB
Fo
idium bromide used (2 µM). The calculated apparent bind-
=
1 + KSV[Q],
(2)
6
−1
F
ing constant (K ) for the complexes are 1.02×10 M for
app
6
−1
complex 1 and 1.43×10 M for 2, thus it is observed that
where F and F are the emission intensities of EB-bound
0
K
of 2 > 1, which shows a similar trend to the K val-
CT-DNA in the absence and presence of the quencher
app
b
ues obtained from UV–Vis absorption spectral studies. It is
(
complex) concentration [Q], respectively, and is the K
SV
also to be noted that the K of the complexes 1 and 2 are
Stern–Volmer quenching constant. The plot of F/F vs.
app
0
observed to be tenfold lower than the binding constant of
7
−1
classical intercalator EB (1×10 M ).
Cleavage activity with pUC19 DNA
The ability of the complexes 1 and 2 to cleave DNA was
assessed by performing gel electrophoresis on supercoiled
plasmid DNA pUC19 as the substrate in a medium of
5
0 mM Tris–HCl buꢂer (pH 8.0) in the absence of any exter-
nal additives or light. Both complexes were found to cleave
DNA hydrolytically. The distribution of the supercoiled (SC)
and nicked forms (NC) in the agarose gel electrophoresis
was used to measure the extent of hydrolysis of the phospho-
diester bond. At a complex concentration of 30 µM and an
incubation time of 3 h, 1 cleaves 19% of SC DNA (Fig. 8a,
lane 5), while 90% cleavage is observed for 2 (Fig. 8c, lane
5
). Again at 50 µM concentration, 1 cleaves 87% of SC DNA
(
Fig. 8b, lane 2), while complete cleavage is observed for
2
(Fig. 8d, lane 2). This greater nuclease activity of 2 as
compared to 1 can be possibly rationalized on the basis of
its higher binding aꢃnity toward CT-DNA as reꢁected from
DNA binding studies.
The nuclease activity of the complexes has also been
studied in the presence of H O . A ꢀgure representing oxi-
2
2
dative DNA cleavage by complex 2 has been incorporated
(
Fig. 8e, lanes 1–4), which shows that with increasing
concentration (0–25 μM) of complex 2, supercoiled DNA
is gradually converted completely to the nicked form. To
elucidate the mechanistic aspects of the chemical-induced
DNA cleavage, experiments were conducted in the pres-
ence of inhibitors. Oxidative DNA cleavage performed in
the presence of DMSO, a known scavenger of hydroxyl
ion, showed considerable inhibition of cleavage (Fig. 8e,
Fig. 7 Eꢂect of the addition of complexes 1 (a) and 2 (b) on the
lane 5); on the other hand, the presence of NaN , a singlet
3
emission intensity of ethidium bromide-bound CT-DNA at diꢂerent
oxygen quencher, did not have any considerable eꢂect on
the DNA cleaving ability of the complex (Fig. 8e, lane 6).
Thus, it may be concluded that hydroxyl ion is associated
with DNA cleavage exhibited by the complexes.
concentrations in 10 mM Tris–HCl buꢂer (pH 7.9). λ =510 nm.
ex
The arrow shows the change in ꢁuorescence intensity with increasing
complex concentration. The inset shows the linear ꢀt of F /F versus
0
[
complex]
1
3

JBIC Journal of Biological Inorganic Chemistry
Fig. 8 Hydrolytic and oxidative cleavage of supercoiled pUC19
DNA (200 ng) at 37 °C in 50 mM Tris–HCl buꢂer (pH 8.0). a
Lane 1, DNA control; lane 2, DNA+1 (5 µM); lane 3, DNA+1
control; lane 2, DNA+2 (50 µM); lane 3, DNA+2 (100 µM);
lane 4, DNA+2 (150 µM); lane 5, DNA+2 (200 µM). e Lane
1, DNA+H O control; lane 2, DNA+2 (5 µM)+H O ; lane
2
2
2
2
(
10 µM); lane 4, DNA+1 (20 µM); lane 5, DNA+1 (30 µM); lane
3, DNA+2 (15 µM)+H O ; lane 4, DNA+2 (25 µM)+H O ;
2 2 2 2
6
, DNA+1 (40 µM); lane 7, DNA+1 (50 µM). b Lane 1, DNA
control; lane 2, DNA+1 (50 µM); lane 3, DNA+1 (100 µM); lane
, DNA+1 (150 µM); lane 5, DNA+1 (200 µM). c Lane 1, DNA
lane 5, DNA+2 (25 µM)+H O +DMSO (2 µL); lane 6,
2 2
DNA+2 (25 µM)+H O +NaN (100 µM).
f Lane 1, con-
2
2
3
4
trol DNA; lane 2, DNA+Cu(OAc) ·2H O (500 µM); lane 3,
2 2
control; lane 2, DNA+2 (5 µM); lane 3, DNA+2 (10 µM); lane
DNA+1,5-bis(salicylidene)carbohydrazide (500 µM); lane 4,
DNA+1,10-phenanthroline monohydrate (500 µM)
4
, DNA+2 (20 µM); lane 5, DNA+2 (30 µM). d Lane 1, DNA
To ensure that the copper complexes 1 and 2 as a whole
This amounts to an enormous enhancement of cleavage rate
8
are responsible for cleavage of DNA, control experiments
were performed with Cu(OAc) ·H O (500 µM), free
of 3.21–3.23×10 in comparison to the unhydrolyzed rate of
−8
−1
ds-DNA (k=3.6×10 h ). This rate enhancement is much
higher than the rate enhancements reported for most of the
transition metal-based synthetic hydrolases [29, 30, 36, 45].
2
2
ligands 1,5-bis(salicylidene)carbohydrazide, (500 µM)
and 1,10-phenanthroline monohydrate (500 µM). No
cleavage was observed with either Cu(OAc) ·H O,
2
2
ligand 1,5-bis(salicylidene)carbohydrazide or coligand
Cytotoxicity
1
,10-phenanthroline monohydrate (Fig. 8f, lanes 2–4),
even at a concentration of 500 µM. This conꢀrms that nei-
ther Cu(OAc) ·H O nor the free ligands alone are capable
Cell proliꢀeration
2
2
of bringing about DNA strand cleavage.
The cytotoxicity of the complexes was tested against cells
from A549 lung cancer cell line, MCF7 breast cancer cell
line and HaCaT human keratinocyte normal cell line using
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay. From the percentage cell viability
versus complex concentration plot (Fig. 10), a dose-depend-
ent cell death-inducing ability was observed for both the
complexes 1 and 2. It is observed that complex 1 exhib-
its more cytotoxicity than 2 in both A549 and MCF7cell
Further, the kinetics of DNA cleavage were studied by
monitoring the % DNA cleavage by the complexes 1 and 2
(
200 µM) with time (Fig. 9). The time-dependent decrease
of form I and increase of form II were found to ꢀt well to a
single-exponential decay and increase, respectively. A plot of
log (% SC DNA) with time gave a linear ꢀt from which the
hydrolytic rate constant (k ) was obtained. The k value
obs
obs
obtained were 11.55 h− for 1 and 11.63 h for 2, respectively.
1
−1
1
3

JBIC Journal of Biological Inorganic Chemistry
(
36.65 µM for MCF7) [34]. Another point to be noted that
though the results of DNA binding and cleavage studies
exhibited by compounds 1 and 2 are very similar, their
cytotoxic behavior toward A549, MCF7 and HaCaT cell
lines are considerably diꢂerent. This diꢂerence may origi-
nate from the diꢂerence in their abilities to diꢂuse across
the cell membrane. Compound 1 being uncharged polar
molecule will be able to passively diꢂuse across the cell
membrane. On the other hand, compound 2 being ionic
will ꢀnd it diꢃcult to diꢂuse across the cell membrane as
compared to compound 1 [47]. Another point to be men-
tioned here is that the reported [41] neutral heterodinuclear
Zn(II)/Cu(II) compound [Zn(o-phen)LCu(OAc)] (3) con-
taining the enol form of the same carbohydrazone Schiꢂ
base (H L=o-HOC H C(H)=N–NH–C(OH)=N–N=C(H)-
3
6
4
C H OH-o), which is structurally very similar to com-
6
4
pound 1 under study, also exhibited [41] very high cyto-
toxicity against the human lung cancer A549 cell line
(
IC =4.80 μM) and for the breast cancer MCF7 cell line
50
(
IC =3.6 μM). At the same time, both these compounds
50
1
and 3 were found to be much less toxic at their 10 μM
concentrations toward HaCaT normal cell line. On the other
hand, compound 2, which also contains Cu(II) in both the
sites like compound 1, but containing o-phen coligands in
both the Cu(II) centers, exhibits relatively lesser cytotoxic-
ity, in spite of containing two o-phen coligands in the mol-
ecule, not only toward both the cancer cells but also toward
HaCaT normal cells as compared to compound 1 containing
only one o-phen coligand in the ꢀrst Cu(II) site and acetate
in the second Cu(II) site.
We have also studied the cytotoxicity of the free ligands
to compare that with their metal bound states in compounds
Fig. 9 Hydrolytic cleavage of SC pUC19 DNA (200 ng) showing the
decrease in form I (SC DNA) and the formation of form II (NC DNA)
with the incubation time (in dark at 37 °C): a using 200 µM concen-
tration of 1; inset shows the plot of log(% SC DNA) vs. time for com-
plex 1 (200 µM). b Using 200 µM concentration of 2; inset shows the
plot of log (% SC DNA) vs. time for complex 2 (200 µM)
1
and 2. The cytotoxicity study of the free Schiꢂ base ligand
and the free coligand 1,10-phenanthroline were carried out
using the human lung cancer A549 cell line as a model case
with diꢂerent concentrations (0.5 µM, 1.0 µM, 2.5 µM,
5
µM, and 10 µM) of these free ligands for 24 h. The cell
viabilities were found to be nearly 87% and 72% with 10 µM
of the free Schiꢂ base ligand and the free 1,10-phenanth-
roline ligand, respectively (Fig. S4, Supplementary mate-
rial), suggesting that the free ligands are capable of causing
individually about 13% and 28% cell death at 10 µM con-
centrations. On the other hand, the cell viabilities for the
compounds 1 and 2 were found to be nearly 21% and 48%
with 10 µM concentrations of 1 and 2, respectively. Thus,
copper chelation with these ligands certainly increased their
toxicity, as observed for many other cases [1].
lines. Most importantly, the complexes 1 and 2 appear to be
comparatively less toxic to normal HaCaT cells (Fig. 10e,
f). Especially in case of complex 2, not much toxicity is
observed till 5 µM dose in HaCaT cell line. At 10 µM con-
centration, 2 exhibited signiꢀcant cytotoxicity as seen from
cell viability of ~ 48% in both cancerous cell lines A549
and MCF7, whereas the cell viability was ~90% in normal
HaCaT cell line; this marked diꢂerence between the cyto-
toxic eꢂect in cancerous and noncancerous cell lines is of
considerable importance. The IC values are presented in
Very recently, we have reported [48] the cytotoxic-
ity of a five-coordinate Cu(II) compound [Cu(pabt)
5
0
Table 2. The IC values for 1 and 2 for both A549 and
5
0
MCF7 cell lines are found to be higher than that reported
for cisplatin (4.13 µM for A549, 3.92 µM for MCF7). How-
ever, the IC values for 1 and 2 for MCF7 cell line are
(o-phen)](ClO ) (4), (Hpabt = N-(2-mercaptophenyl)-2′-
4
pyridylmethylenimine, o-phen=1,10-phenanthroline) with
A549 cell line and the IC value was found to be 5.26 μM,
5
0
50
found to be much lower than that reported for carboplatin
suggesting it is highly potent for the A549 cells. This
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JBIC Journal of Biological Inorganic Chemistry
Fig. 10 Eꢂect of 1 and 2 on cell
viability in A549, MCF7 and
HaCaT cell lines. Cells were
treated with compounds 1 and
2
for 24 h. Eꢂect of 1 and 2 on
cell viability in a, b A549, c,
d MCF7 and e, f HaCaT was
analyzed by MTT assay. Data of
three independent experiments
are presented as mean± SD.
Diꢂerence in signiꢀcance
was determined by one-way
ANOVA. *P<0.05, **P<0.01,
*
**P<0.001, ns not signiꢀcant
Table 2 IC50 values for the
compounds 1 and 2 calculated
from the plot of cell viability
versus concentration in the
MTT assay
Anjomshoa et al. [50] have studied the anticancer activity
of a mononuclear Cu(II) complex with 5,6-diphenyl-3-(2-
pyridyl)-1,2,4-triazine ligand and the reported IC values
Cell line IC50 (µM)
Compd 1 Compd 2
5
0
A549
MCF 7
HaCaT
4.34
6.50
11.19
8.46
8.68
16.01
were 7.80 and 9.80 μM for A549 and MCF7 cells, respec-
tively. Nair and coworkers [13] have studied the cytotoxic
eꢂects of two copper(II) complexes of terpyridine deriva-
tives with human breast cancer MCF7 cell line and the IC
5
0
values were found to be 6.25 and 3.125 μM for those two
compound was found to be much less toxic toward the human
keratinocyte HaCaT normal cells. Also, we have recently
reported [49] the cytotoxicity of another ꢀve-coordinate
complexes. Abdi et al. [51] have reported the IC value
5
0
for MCF7 cells (4.57 μM) of a mononuclear copper(II)
complex with terpyridine and an extended phenanthroline
base. Meenongwa et al. [52] have studied the eꢂects of N,N-
heterocyclic ligands on the in vitro cytotoxicity of some
copper(II) chloride complexes with MCF7 cells, and a few
of these compounds were found to be cytotoxic toward this
cell line. Inspired by the ꢀrst synthetic chemical nuclease,
[
(
CuL(o-phen)] (5) and a four-coordinate [CuL(Imz)] (6),
H L=o-HOC H C(H)=NC H OH-o, o-phen=1,10-phen-
2
6
4
6
4
anthroline, and Imz=imidazole) compounds with A549 and
MCF7 cancer cell lines. Both these complexes were found
to be very toxic for A549 cells (IC values were 0.67 and
5
0
2
+
0
.59 μM, respectively) as well as quite toxic for MCF7 cells
[Cu(phen) ] (phen=1,10-phenanthroline), and based on
2
(
IC values were 6.30 and 8.88 μM, respectively), but these
50
two compounds were found to be equally toxic for HaCaT
normal cells. Lu et al. [34] have studied the cytotoxicity of
Cu(II) complexes, which also exhibited lower IC values in
Fig. 11 Apoptosis analysis by Annexin-V and propidium iodide stain-
ing. a A549 cells and b MCF7 cells treated with 1, 2.5, 5, 10, 15 and
◂
5
0
2
0 µM of compounds 1 and 2 for 24 h. Eꢂect on apoptosis was ana-
A549 as well as MCF7 cell lines, but the cytotoxic eꢂect of
lyzed by Annexin-V/PI staining using ꢁow cytometry. Representative
data of three independent experiments are presented
these compounds on any normal cell line was not reported.
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JBIC Journal of Biological Inorganic Chemistry
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3

JBIC Journal of Biological Inorganic Chemistry
1
3

JBIC Journal of Biological Inorganic Chemistry
◂
Fig. 12 Eꢂects of 1 and 2 on cell cycle progression. a A549 and b
using ꢁow cytometry. Our results showed drastic decrease
in viable cells (lower left quadrant Annexin-V−, PI−) in
a dose-dependent manner upon treatment with 1 and 2 as
compared to that of control cells in both A549 (Fig. 11a)
and MCF7 (Fig. 11b) cell lines studied.
MCF 7 cells treated with 1, 2.5, 5, 10, 15 and 20 µM of compounds
1
and 2 for 24 h. Eꢂect on cell cycle arrest was analyzed by PI stain-
ing using ꢁow cytometry. Representative data of three independent
experiments are presented
It is observed that A549 cells upon incubation for 24 h
with each of the complexes 1 and 2 show a general trend of
increase in early apoptotic cells with increasing complex
concentration up to 5 µM (lower right quadrant: Annexin-
V+, PI−). Then a large population of late apoptotic cells
27.75% and 21.46% (upper right quadrant) corresponding to
treatment of 10 µM concentration of 1 and 2 was observed.
Also, it is noted that from 10 µM dose onward, there is a
steady decrease in cell population in the lower right quad-
rant as most of the cells have passed onto the late apoptotic/
necrotic stage from early apoptotic stage (Fig. 11b).
In case of MCF7 cell line upon incubation with each of
the complexes 1 and 2 for 24 h, a general trend of increase
in early apoptotic cells with increasing complex concentra-
tion up to 10 µM (lower right quadrant: Annexin-V+, PI−)
is seen. Also a higher population of late apoptotic cells
(35.05%) is observed in case of 10 µM dose of 1 (upper right
quadrant) as compared to 15.19% cell population upon treat-
ment with the same dose of 2; this observation is in good
agreement with that observed in the MTT assay (Fig. 10c,
d). On further increasing the complex concentration to 15
and 20 µM, there is steady decrease in the cell population
in the lower right quadrant which represents early apoptotic
cells, as they may have passed onto next stage (late apopto-
sis/necrosis) (Fig. 11b).
the results that mononuclear [Cu(phen) (phthalate)] com-
2
plexes exhibited very good chemotherapeutic potential
against colon (HT29), breast (MCF7) and prostate (DU145)
cancer cell lines, Kellett et al. [53] have synthesized and
studied the water-soluble bis(1,10-phenanthroline) octane-
2
+
2+
dioate Cu (and Mn ) complexes that displayed extremely
high cytotoxicity toward some human cancer cell lines
(
HT29, SW480 and SW620), but these were found to be
less toxic toward normal human keratinocyte cells (HaCaT).
Copper(II) complexes with terpyridine ligands studied by
Djuran and coworkers [54] exhibited very low IC values
5
0
for A549 cells (1.2–1.9 μM). Apart from these there are
studies [55–57] by Nair and coworkers involving A549 cell
lines with Cu(II) complexes with imidazole terpyridine, with
substituted terpyridine and terpyridine ligands and some of
these complexes exhibited quite low IC values in A549
5
0
cells. Nagababu et al. [5] have studied the cytotoxicity of
Cu complexes, two of which also exhibited low IC values
5
0
in A549 cell line; however, these complexes were found to
be cytotoxic also toward normal cell lines. Another study
reported by Feng and Liu [58] for A549 cells with Cu(II)-
complexes based on tricationic metalloporphyrin salicyloyl-
hydrazone ligands, however, exhibited high IC values.
5
0
Annexin‑V FITC/PI double staining study using ꢂow
cytometry
Cell cycle analysis
During the initial stages of apoptosis, the cells lose their
membrane phospholipid asymmetry and the membrane
phospholipid phosphatidylserine (PS) is translocated from
the inner face of the plasma membrane to the cell surface.
PS can be easily detected by staining with ꢁuorescent con-
jugated Annexin-V that speciꢀcally binds to PS exposed
to the cell surface, thereby allowing the detection of early
apoptotic cells. Cells are stained in parallel with propidium
iodide (PI) which can enter the cells only when the plasma
membrane is damaged. Therefore, viable cells are Annexin-
V and PI negative (Annexin-V−, PI−). Cells undergoing
early apoptosis but having intact membranes exclude propid-
ium iodide (PI); this allows us to distinguish between early
apoptotic cells (Annexin-V+, PI−) from late apoptotic cells
The progression of cells through the diꢂerent phases of cell
cycle is controlled by check points at diꢂerent stages. Most
of the anti-proliferative drugs target distinct points in the
cell cycle and control cell cycle phase progression [46, 59,
60]. Experiments were carried out to determine the phase of
the cell cycle in which the compounds 1 and 2 arrested the
cell growth. A549 and MCF7 cells were synchronized by
serum starvation for 12 h, and after serum starvation A549
and MCF7 cells were grown in DMEM media and treated
with 1, 2.5, 5 and 10 µM concentrations of 1 and 2 for 24 h.
The DNA content of cells was determined by ꢁow cytom-
etry after the cells were stained with propidium iodide (PI).
In case of both compounds, the results showed consider-
able increase in the G2/M phase in both cell lines in a dose-
dependent manner (Fig. 12). As seen for the A549 cell line
(Fig. 12a), the cell population increased up to 27.97% and
23.10% upon treatment with 10 µM dose of 1 and 2, respec-
tively, as compared to 11.63% cell population in G2/M of
control. Similarly, for MCF7 cell line, the cell population
increased up to 21.05% and 23.23% upon treatment with
(
Annexin-V+, PI+). Necrotic cells are Annexin negative
and PI positive (Annexin-V−, PI+). To see the eꢂect of the
copper complexes under study, A549 and MCF7 cells were
treated with the indicated doses of 1 and 2 (1 µM, 2.5 µM,
5
µM, 10 µM, 15 µM and 20 µM) for 24 h and the percent-
age cell death was analyzed by Annexin-V FITC/PI staining
1
3

JBIC Journal of Biological Inorganic Chemistry
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3

JBIC Journal of Biological Inorganic Chemistry
◂
Fig. 13 Eꢂects of 1 and 2 on cellular ROS generation. a A549 and b
in the present study is not surprising and may be associated
with observed apoptosis. However, it is important to note
that both the compounds 1 and 2 show G2/M phase cell
cycle arrest in both A549 and MCF7 cell lines and activate
ROS level in A549 cell line, but they act as scavengers or
inhibitors of ROS in MCF7 cell line as analyzed by DCFDA
staining using ꢁow cytometry.
MCF 7 cells treated with 1, 2.5, 5, 10, 15 and 20 µM of compounds
1
and 2 for 24 h. Eꢂect on cellular ROS generation was analyzed by
DCFDA staining using ꢁow cytometry. Representative data of three
independent experiments are presented
10 µM dose of 1 and 2, respectively, as compared, to 15.79%
cell population in the G2/M of control (Fig. 12b). DNA dam-
age is known to be a key factor that triggers cell cycle arrest.
Nuclease activity studied by gel electrophoresis experiments
indicate that both 1 and 2 are highly capable of inducing
hydrolytic as well as oxidative DNA cleavage (Fig. 8). Thus,
extensive DNA damage caused by the treatment of 1 and 2
may have resulted in the G2/M phase arrest [45, 61, 62] in
A549 and MCF7 cells.
Conclusions
Two homodinuclear mixed ligand copper(II) complexes
were structurally characterized by X-ray crystallography
and their biological activities studied under identical experi-
mental conditions. The compounds eꢃciently bind to CT-
DNA with intercalative binding mode that is supported by
viscosity measurements as well as from the ꢁuorescence
spectroscopic studies of ethidium bromide displacement
assay. Both the compounds exhibit eꢃcient hydrolytic DNA
Eꢁect oꢀ compounds 1 and 2 on cellular ROS
generation
To assess the capability of the complexes 1 and 2 to gener-
ate intracellular reactive oxygen species (ROS), A549 and
MCF7 cells were treated with 1, 2.5, 5 and 10 µM concen-
trations of 1 and 2 for 24 h and the intracellular ROS levels
were determined by ꢁow cytometry using the redox-sensitive
cleavage of SC pUC19 DNA and the k values were found
obs
−1
−1
to be 11.55 h for 1 and 11.63 h for 2, respectively. This
amounts to an enormous enhancement of cleavage rate of
8
3
.21–3.23×10 in comparison to the unhydrolyzed rate of
−8 −1
ds-DNA (k=3.6×10 h ). Though both these compounds
exhibit strong in vitro cytotoxicity against human lung can-
cer A549 cell line and human breast cancer MCF7 cell line
as evident from signiꢀcant decrease in cell viability and are
found to be potent for both these cell lines as revealed from
their IC values, compound 2, in spite of containing o-phen
ꢁ
uorescent probe 2′,7′ dichlorodihydroꢁuorescein diacetate
2
(
H DCFDA). It was observed that in A549 cell line, there
was considerable ROS generation upon treatment with 1 and
2
. As indicated by the results at 10 µM concentration of 1
and 2, ROS detected (694.36, 705.44) was considerably high
compared to untreated A549 cells (519.27) (Fig. 13a), but
not so signiꢀcantly high to account for the total cytotoxicity
observed from their MTT assay. These observations suggest
that increased ROS level may be one of the factors respon-
sible for the observed apoptosis in A549 cells. However,
in MCF7 cells we observed that 1 and 2 decreased ROS
level in a dose-dependent manner and that the ROS level was
drastically decreased from 1332.79 (control) to 214.29 and
5
0
coligands in both the Cu(II) sites, exhibits relatively lesser
cytotoxicity not only toward both the cancer cells, but also
toward HaCaT normal cells as compared to compound 1
containing only one o-phen coligand in the ꢀrst Cu(II) site
and acetate in the second Cu(II) site. This diꢂerence may be
attributed to the diꢂerence in their abilities to diꢂuse across
the cell membrane. Annexin-V/PI dual staining results ana-
lyzed by ꢁow cytometry strongly suggest the induction of
the apoptotic pathway for the anticancer activity of these
complexes. Cell cycle analysis with compounds 1 and 2
showed cell cycle arrest in the G2/M phase in both A549
and MCF7 cell lines. However, the most important observa-
tion made in this study is that compounds 1 and 2 showed
diꢂerential behavior toward ROS in these two cancer cell
lines. While both of them were found to be activators of
ROS in A549 cells, they acted as inhibitors or scavengers of
ROS in MCF7 cells.
2
23.16 upon treatment with 10 μM dose of 1 and 2, respec-
tively (Fig. 13b), which is intriguing. There are reports that
not only the increased ROS level, but also the depletion of
ROS below a certain threshold level may lead to cancer cell
killing [63, 64]. Liou and Storz [64] have discussed elabo-
rately the role of reactive oxygen species (ROS) in cancer.
They have pointed out that though the role for ROS-activated
Erk1/2 (extracellular regulated kinase 1/2) signaling in cell
proliferation is well known [65–67], its ability to regulate
cancer cell survival appears to be very speciꢀc for cell type
[
68–70]. As an example, they have mentioned that when
Acknowledgements We thank the SAIF, IIT-Madras, for XRD and
SAIF, IIT-Bombay, for EPR facilities. A.P.K. thanks Mr. Atanu
Banerjee and Prof. Rupam Dinda, Department of Chemistry, National
Institute of Technology, Rourkela, India, for help with the ESI-MS
results. MK thanks SERB, DST, Government of India, for the Project
EMR/2017/001562.
MCF-7 and MDA-MB-435 breast cancer cells were treated
with ROS scavengers or inhibitors that target Erk1/2 or its
upstream kinase MEK (mitogen-activated protein kinase
kinase), it resulted in apoptosis and cell adhesion [71, 72].
Thus, the decrease in ROS levels by 1 and 2 in MCF7 cells
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3
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