Ternary dinuclear copper(II) complexes of a hydroxybenzamide ligand with diimine coligands: The 5,6-dmp ligand enhances DNA binding and cleavage and induces apoptosis

Article abstract of DOI:10.1021/ic1024185

The dinuclear copper(II) complexes [Cu₂(LH)₂(diimine) ₂(ClO₄)₂](ClO₄)₂ (1-4), where LH = 2-hydroxy-N-[2-(methylamino)ethyl]benzamide and diimine = 2,2′-bipyridine (bpy; 1), 1,10-phenanthroline (phen; 2), 5,6-dimethyl-1,10-phenanthroline (5,6-dmp; 3), and dipyrido[3,2-d:2′, 3′-f]quinoxaline (dpq; 4), have been isolated and characterized. The X-ray crystal structure of complex 1 contains two copper(II) centers bridged by the phenolate moiety of the amide ligand. All of the complexes display a ligand-field band (630-655 nm) and the PhO^--to-Cu^II ligand-to-metal charge-transfer band (405-420 nm) in solution. Absorption and emission spectral studies and viscosity measurements indicate that complex 4 interacts with calf thymus DNA more strongly than all of the other complexes through strong partial intercalation of the extended planar ring (dpq) with a DNA base stack. Interestingly, 3 exhibits a DNA binding affinity higher than 2, suggesting the involvement in hydrophobic interaction of coordinated 5,6-dmp with the DNA surface. In contrast to the increase in relative viscosities of DNA bound to 2-4, a decrease in viscosity of DNA bound to 1 is observed, indicating a shortening of the DNA chain length through formation of kinks or bends. All of the complexes exhibit an ability to cleave DNA (pUC19 DNA) in a 5% DMF/5 mM Tris-HCl/50 mM NaCl buffer at pH 7.1 in the absence of an oxidant at 100 μM complex concentration, which varies as 4 > 2 > 1 > 3. The order of DNA the cleavage ability at 30 μM concentration in the presence ascorbic acid is 4 > 2 > 1 > 3, and, interestingly, 4 alone shows an ability to convert supercoiled DNA into nicked-coiled DNA even at 6 μM concentration, beyond which complete degradation is observed and the pathway of oxidative DNA cleavage involves hydroxyl radicals. In the presence of distamycin, all of the complexes, except 3, show decreased DNA cleavage activity, suggesting that the complexes prefer to bind in the DNA minor groove. All of the complexes exhibit prominent DNA cleavage even at very low concentrations (nM) in the presence of H₂O₂ as an activator, with the order of cleavage efficiency being 3 > 2 > 4 > 1. Studies on the anticancer activity toward HEp-2 human larynx cell lines reveal that the ability of the complexes to kill the cancer cell lines varies as 3 > 4 > 2 > 1. Also, interestingly, the IC₅₀ value of 3 is lower than that of cisplatin, suggesting that the hydrophobicity of methyl groups on the 5 and 6 positions of the complex enhances the anticancer activity. The mode of cell death effected by the complex has been explored by using various biochemical techniques like comet assay, mitochondrial membrane potency, and Western blotting. The complex has been found to induce nuclear condensation and fragmentation in cell lines. Also, it triggers activation of caspases by releasing cytochrome c from mitochondria to cytosol, suggesting that it induces apoptosis in cells via the mitochondrial pathway.

Full text of DOI:10.1021/ic1024185

ARTICLE
pubs.acs.org/IC
Ternary Dinuclear Copper(II) Complexes of a Hydroxybenzamide
Ligand with Diimine Coligands: the 5,6-dmp Ligand Enhances
DNA Binding and Cleavage and Induces Apoptosis
Sethu Ramakrishnan,† Dhanasekaran Shakthipriya,† Eringathodi Suresh,‡ Vaiyapuri Subbarayan Periasamy,§
Mohammad Abdulkader Akbarsha,§ and Mallayan Palaniandavar*,†
†Centre for Bioinorganic Chemistry, School of Chemistry, and §Department of Animal Science, Bharathidasan University,
Tiruchirapalli 620 024, India
‡Analytical Science Discipline, Central Salt and Marine Chemical Research Institute, Bhavnagar 364 002, India
S Supporting Information
b
ABSTRACT: The dinuclear copper(II) complexes [Cu2(LH)2-
(diimine)2(ClO4)2](ClO4)2 (1ꢀ4), where LH = 2-hydroxy-
N-[2-(methylamino)ethyl]benzamide and diimine = 2,20-bipyr-
idine (bpy; 1), 1,10-phenanthroline (phen; 2), 5,6-dimethyl-1,
10-phenanthroline (5,6-dmp; 3), and dipyrido[3,2-d:20,30-
f]quinoxaline (dpq; 4), have been isolated and characterized.
The X-ray crystal structure of complex 1 contains two copper-
(II) centers bridged by the phenolate moiety of the amide
ligand. All of the complexes display a ligand-field band
(630ꢀ655 nm) and the PhOꢀ-to-CuII ligand-to-metal charge-
transfer band (405ꢀ420 nm) in solution. Absorption and emission spectral studies and viscosity measurements indicate that
complex 4 interacts with calf thymus DNA more strongly than all of the other complexes through strong partial intercalation of the
extended planar ring (dpq) with a DNA base stack. Interestingly, 3 exhibits a DNA binding affinity higher than 2, suggesting the
involvement in hydrophobic interaction of coordinated 5,6-dmp with the DNA surface. In contrast to the increase in relative
viscosities of DNA bound to 2ꢀ4, a decrease in viscosity of DNA bound to 1 is observed, indicating a shortening of the DNA chain
length through formation of kinks or bends. All of the complexes exhibit an ability to cleave DNA (pUC19 DNA) in a 5% DMF/
5 mM Tris-HCl/50 mM NaCl buffer at pH 7.1 in the absence of an oxidant at 100 μM complex concentration, which varies as 4 > 2 >
1 > 3. The order of DNA the cleavage ability at 30 μM concentration in the presence ascorbic acid is 4 > 2 > 1 > 3, and, interestingly,
4 alone shows an ability to convert supercoiled DNA into nicked-coiled DNA even at 6 μM concentration, beyond which complete
degradation is observed and the pathway of oxidative DNA cleavage involves hydroxyl radicals. In the presence of distamycin, all of
the complexes, except 3, show decreased DNA cleavage activity, suggesting that the complexes prefer to bind in the DNA minor
groove. All of the complexes exhibit prominent DNA cleavage even at very low concentrations (nM) in the presence of H2O2 as an
activator, with the order of cleavage efficiency being 3 > 2 > 4 > 1. Studies on the anticancer activity toward HEp-2 human larynx cell
lines reveal that the ability of the complexes to kill the cancer cell lines varies as 3 > 4 > 2 > 1. Also, interestingly, the IC50 value of 3 is
lower than that of cisplatin, suggesting that the hydrophobicity of methyl groups on the 5 and 6 positions of the complex enhances
the anticancer activity. The mode of cell death effected by the complex has been explored by using various biochemical techniques
like comet assay, mitochondrial membrane potency, and Western blotting. The complex has been found to induce nuclear
condensation and fragmentation in cell lines. Also, it triggers activation of caspases by releasing cytochrome c from mitochondria to
cytosol, suggesting that it induces apoptosis in cells via the mitochondrial pathway.
’ INTRODUCTION
successful in treating cases lacking cross resistance.4ꢀ6 A few
dinuclear platinum complexes have been found to act as anti-
cancer drugs better than cisplatin, particularly for treating metas-
tasis. Very recently, the antitumor activity of several cationic
trinuclear platinum complexes have been investigated by Farrell
et al., and one (BBR-3464) of the complexes has undergone both
phase I7 and phase II8,9 clinical trials. The trinuclear complex has
Since the discovery of the antitumor activity of cisplatin,1
numerous mononuclear platinum(II) complexes have been
synthesized and their anticancer activities carefully evaluated
on appropriate biological models.2 Several polynuclear platinum
complexes of multidentate ligands have been isolated now and
are found to form adducts with DNA, which are substantially
different from those of cisplatin.3 Certain classes of platinum(II)
complexes with two or three platinumꢀamine units linked by a
diamine chain of variable length have already been shown to be
Received: December 3, 2010
Published: June 14, 2011
r
2011 American Chemical Society
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been shown to be cytotoxic at concentrations that are 20-fold
lower than that of cisplatin, and the increased potency is attrib-
uted to its unique DNA binding profile in comparison to mono-
nuclear complexes.10 All of these polynuclear complexes are
involved in novel types of both intra- and interstrand cross-
links11 with DNA, which indeed may have different biological
effects. Unfortunately, in contrast to general expectations, most
of the platinum(II) complexes investigated so far exhibit bio-
logical activities very similar to those of cisplatin with almost
superimposable properties and, thus, with no significant thera-
peutic advantage. Also, the types of cancer that can be treated
with platinum drugs are very few, and the drugs suffer from side
effects and resistance phenomena.12,13 So, many other novel
transition-metal complexes as well as small-molecule-based anti-
tumor agents have been developed, and some of them are under
clinical trial.14aꢀe
Scheme 1. Dinuclear Copper(II) Complexes 1ꢀ4 of Diimine
Ligands
Copper(II) complexes are considered the best alternatives to
cisplatin because copper is biocompatible and exhibits many
significant roles in biological systems. Also, synthetic copper(II)
complexes have been reported to act as pharmacological agents
and as potential anticancer and cancer-inhibiting agents.15ꢀ20
Also, very recently, certain mixed-ligand copper(II) complexes,
which strongly bind and cleave DNA, exhibit prominent anti-
cancer activities and regulate apoptosis.21ꢀ25 Very recent studies
have demonstrated that certain multinuclear copper(II) com-
plexes can efficiently promote DNA cleavage by selectively
oxidizing deoxyribose or nucleobase moieties.26 The copper
centers in some multinuclear complexes act synergistically to
cleave DNA with higher efficiency or selectivity.26ꢀ30 Karlin and
co-workers31,32 have shown that nuclearity is a crucial parameter
in oxidative DNA cleavage, with the synergy between the metal
ions contributing to the high nucleolytic efficiency of polynuclear
copper(II) compounds. Therefore, designing suitable multinuc-
lear copper complexes for DNA binding and cleavage under
both oxidative and hydrolytic conditions, depending upon the
recognition elements in the ligand, is of remarkable importance
in considering the advantages of processes that produce frag-
ments similar to those formed by restriction enzymes.26,27
So, in the present investigation, we have isolated redox-active
mixed-ligand dinuclear copper(II) complexes of the general
formula [Cu2(LH)2(diimine)2(ClO4)2](ClO4)2 (1ꢀ4), where
LH is 2-hydroxy-N-[2-(methylamino)ethyl]benzamide and dii-
mine is 2,20-bipyridine (bpy; 1), 1,10-phenanthroline (phen; 2),
5,6-dimethyl-1,10-phenanthroline (5,6-dmp; 3), or dipyrido-
[3,2-d:20,30-f]quinoxaline (dpq; 4) (Scheme 1), and investigated
their DNA binding and cleavage properties and anticancer
activities. The bidentate diimine coligand is expected to function
as a DNA recognition element, while the tridentate benzamide
functions as the primary ligand. There has been considerable
attention focused on the use of small metal complexes containing
diimines as metal-based synthetic nucleases.26ꢀ29 Very recently,
we have found that the diimine coligands in mixed-ligand
copper(II)24,25 and ruthenium(II)33 complexes play a pivotal
role in the mechanism underlying induction of cell death.
It is proposed to employ electronic absorption and emission
spectral and viscosity studies to diagnose the mode and extent of
interaction of the dinuclear copper(II) complexes with DNA and
then to understand the DNA cleavage abilities and anticancer
activities of the complexes in terms of their DNA binding affinity.
Also, we have chosen to study the in vitro cytotoxicity of the
DNA-cleaving dinuclear copper(II) complexes toward HEp-2
human larynx cancer cell lines. It is remarkable that the 5,6-dmp
complex, which binds to DNA through hydrophobic forces of
interaction, exhibits a strong potential to act as a novel antitumor
agent in micromolar concentrations.
’ RESULTS AND DISCUSSION
Syntheses and Structures of Complexes. Mixed-ligand
dinuclear copper(II) complexes of the type [Cu2(LH)2-
(diimine)2(ClO4)2](ClO4)2 (1ꢀ4), where LH = 2-hydroxy-
N-[2-(methylamino)ethyl]benzamide and diimine = 2,20-bipyr-
idine (bpy; 1), 1,10-phenanthroline (phen; 2), 5,6-dimethyl-
1,10-phenanthroline (5,6-dmp; 3), and dipyridoquinoxaline
(dpq; 4),35 have been isolated by adding a methanolic solution
of hydrated copper(II) perchlorate to a mixture of the ligand LH
and diimine in a methanol solution. All of the complexes have
been obtained in good yields and characterized by elemental
analysis, electrospray ionization mass spectrometry (ESI-MS),
and UVꢀvisible spectral technique. The X-ray crystal structure
of 1 reveals that it contains a dimeric copper unit, and related
complexes 2ꢀ4 are suggested to contain the same structure,
which is supported by ESI-MS data. Complexes 1 and 2 are
soluble in a 5 mM Tris-HCl/50 mM NaCl buffer at pH 7.1, while
3 and 4 are soluble in a 5% DMF/5 mM Tris-HCl/50 mM NaCl
buffer at pH 7.1. For the present study, solutions of the com-
plexes were prepared in a 5% DMF/5 mM Tris-HCl/50 mM
NaCl buffer at pH 7.1. In a 5% DMF/5 mM Tris-HCl/50 mM
NaCl buffer at pH 7.1, all of the complexes exhibit only one broad
band in the visible region (630ꢀ655 nm) with very low
absorptivity (Table S1 in the Supporting Information), which
is consistent with the square-based geometry of copper(II)
observed in the solid-state structure of 1. The intense absorption
bands observed in the regions of 239ꢀ282 and 405ꢀ420 nm are
attributed to the intraligand π f π* transitions within the
coordinated phenolate moiety and diimines and the PhOꢀ
f
copper(II) ligand-to-metal charge-transfer (LMCT) transitions,
respectively. ESI-MS spectral data (Figure S1 in the Supporting
Information) suggest that the perchlorate anions are strongly
bound to copper in the dimeric complex. This is supported
by conductivity measurements in aqueous solution and in an
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Table 1. Crystal Data and Structure Refinement Details for
Complex 1
1
empirical formula
Cu2C40H44 Cl4N8O20
1225.73
fw
cryst syst
triclinic
space group
P1
a, Å
9.6719(12)
11.4218(14)
11.9689(15)
109. 261(2)
106. 116(2)
94. 474(2)
1178.0(3)
1
b, Å
c, Å
R, deg
β, deg
γ, deg
V, Å
Z
λ, Å
Mo KR, 0.710 73
1.728
Dcalc, g cmꢀ3
GOF on F2
1.093
θ for data collection
final R indices [I > 2σ(I)]
R1a
1.9ꢀ25.0
0.0656
0.1317
Figure 1. ORTEP representation of complex cation 1 showing the atom
numbering scheme and displacement ellipsoids (50% probability level).
wR2a
a R1 = ∑||Fo| ꢀ |Fc||/∑|Fo|; wR2 = {∑w[(Fo ꢀ Fc2)2/∑w[(Fo ) ]}1/2
.
2
2 2
aqueous N,N-dimethylformamide (DMF; 2%) solution (ΛM,
210ꢀ325 Ωꢀ1 cm2 molꢀ1; Table S2 in the Supporting Infor-
mation), which fall in the range34 for 1:2 electrolytes. The
electron paramagnetic resonance spectra of the complexes are
silent, suggesting that their two copper(II) centers are strongly
antiferromagnetically coupled.
Table 2. Selected Interatomic Distances and Angles for
Complex 1
bond distances (Å)
bond angles (deg)
Cu1ꢀN1
1.979(5)
1.993(4)
1.902(3)
2.421(4)
1.938(4)
2.582(4)
O1ꢀCu1ꢀN1
93.75(17)
172.74(17)
84.47(14)
91.83(15)
103.22(15)
84.80(15)
170.19(17)
93.63(16)
91.82(15)
86.08(16)
82.06(16)
171.66(13)
81.55(18)
96.74(16)
90.57(15)
Description of the Crystal Structure of [Cu2(LH)2-
(bpy)2(ClO4)2](ClO4)2 (1). An ORTEP view of the complex
cation [Cu2(LH)2(bpy)2(ClO4)2]2þ with the atom numbering
scheme is shown in Figure 1. Selected bond lengths and angles
for 1 are listed respectively in Tables 1 and 2. The distorted
octahedral environment around each copper(II) in the dimeric
complex is constituted of the two nitrogen atoms (N1 and N2) of
2,20-bipyridine, the carbonyl (O1) and phenolate (O2) oxygen
atoms of the N-protonated bidentate amide ligand, the oxygen
atom (O6) of the perchlorate anion, and the coordinated
phenolate oxygen atom (O1_a) of the second amide ligand
coordinated to the other copper(II). The CuꢀNbpy distances
observed [Cu1ꢀN1, 1.978(5) Å; Cu1ꢀN2, 1.992(4) Å] fall
within the range for CuꢀNimine distances observed for other
diimine complexes already reported.32 The amide O1 [Cu1ꢀO1,
1.902(3) Å] and phenolate (O2) oxygen atoms [Cu1ꢀO2,
1.939(4) Å] are bound to copper(II) in the equatorial plane at
distances shorter than those of the phenolate [Cu1ꢀO1_a,
2.420(4) Å] and perchlorate oxygen atoms [Cu1ꢀO6,
2.583(4) Å] occupying the axial positions, which is expected
from the presence of two electrons in the dz2 orbital of copper-
(II). The tetragonally distorted (T = 0.781)36 octahedral com-
plex shows static JahnꢀTeller distortion as a consequence of
crystal packing. The dimerization in the complex is because of the
absence of a strong donor atom in the equatorial position37 of the
neutral complex, which leads to the coordinated phenolate group
in the neighboring molecule to default to the equatorial position
in the first molecule. Interestingly, the two phenolates are
coordinated in a cis-axial/equatorial fashion, with the equatorial
Cu1ꢀN2
Cu1ꢀO1
Cu1ꢀO1_a
Cu1ꢀO2
Cu1ꢀO6
O1ꢀCu1ꢀN2
O1ꢀCu1ꢀO1_a
O1ꢀCu1ꢀO2
O1ꢀCu1ꢀO6
O2ꢀCu1ꢀO6
O2ꢀCu1ꢀN1
O2ꢀCu1ꢀN2
O1_aꢀCu1ꢀO2
O6ꢀCu1ꢀN1
O6ꢀCu1ꢀN2
O1_aꢀCu1ꢀO6
N1ꢀCu1ꢀN2
O1_aꢀCu1ꢀN1
O1_aꢀCu1ꢀN2
CuꢀO1 bond [1.902(3) Å] being shorter than the axial Cuꢀ
O1_a bond [2.420(4) Å].
DNA Binding Studies: Absorption Spectral Studies. Upon
the addition of a solution of calf thymus (CT) DNA to the
copper(II) complexes, a decrease in the absorption intensities
(hypochromism, Δε, 66ꢀ96%; Figure 2) of the π f π*
absorption bands of 2ꢀ4 with red shifts (2ꢀ8 nm) in the band
positions is observed, indicating strong DNA binding of the
complexes. In contrast, an increase in the intensity (hyper-
chromism, Δε, 57%; Figure S2 in the Supporting Information)
of the band with no shift in the band position is observed for 1,
suggesting a mode of DNA binding for the complex different
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from that of others. Because the extent of hypochromism is
commonly associated with the strength of intercalative inter-
action,38ꢀ40 the observed trend in hypochromism, 4 > 3 > 2,
reflects a decrease in the DNA binding affinities of the complexes
in this order (Figure S2 in the Supporting Information). From
the observed spectral changes, the value of the intrinsic equilib-
rium DNA binding constant (Kb) was determined for 2ꢀ4 by
using the equation41
expected to hinder the partial intercalation of the phen ring and,
hence, lead to a DNA binding affinity lower than that of the phen
complex 2. However, a higher DNA binding affinity is observed
for 3, suggesting that the complex is involved in DNA groove
binding rather than intercalative DNA binding. This is supported by
the lower red shift observed for 3 (2 nm). Similar observations were
made by us earlier for the simple mononuclear complexes [Cu(5,6-
dmp)3]2þ, [Zn(5,6-dmp)3]2þ 36 and [Ru(5,6-dmp)3]2þ 44
,
,
the mixed-ligand mononuclear complexes [Ru(5,6-dmp)-
(NH3)4]2þ 45 [Cu(imda)(5,6-dmp)],46 [Cu(dipica)(5,6-
dmp)]2þ 47 and [Cu(L-tyr)(5,6-dmp)]þ (L-tyr = L-tyrosine),25
½DNAꢁ=ðεa ꢀ εf Þ ¼ ½DNAꢁ=ðεb ꢀ εf Þ þ 1=Kbðεb ꢀ εf Þ
,
,
and the dinuclear complex [{(5,6-dmp)2Ru}2(bpm)]4þ48 bound
to CT DNA. Complex 1 with two noncoplanar pyridine rings is
obviously not involved in partial intercalative interaction but
in the electrostatic binding to DNA via the exterior of phos-
phate esters. Thus, the number of aromatic rings in the diimine
coligand and the substituent on the phen ring dictate the DNA
binding affinity and binding structure of the present mixed-ligand
complexes. Also, the diimine coligands rather than the primary
ligand play a vital role in determining the mode as well as the
extent of DNA binding of the complexes.
EthBr Displacement Assay. Upon the addition of complexes
1ꢀ4 (0ꢀ60 μM) to CT DNA pretreated with EthBr ([EthBr]/
[DNA] = 0.1) in a 5% DMF/5 mM Tris-HCl/50 mM NaCl
buffer at pH 7.1, the emission intensity of DNA-bound EthBr
decreases. From a plot of the observed intensities against the
complex concentration, the values of the apparent DNA binding
constant (Kapp) were calculated using the equation49
where [DNA] is the concentration of CT DNA in the base pairs,
εa is the apparent extinction coefficient obtained by calculating
Aobs/[complex], εf corresponds to the extinction coefficient of
the complex in its free form, and εb refers to the extinction
coefficient of the complex in the bound form. Each set of data,
when fitted to the above equation, gives a straight line with a
slope of 1/(εb ꢀ εf) and a y intercept of 1/Kb(εb ꢀ εf), and the
ratio of the slope to intercept gives the value of the intrinsic
binding constants (Figure 2). The Kb values of the complexes
[(0.5ꢀ7.6) ꢂ 104] follow the order 4 > 3 > 2 (Table 3), which is
consistent with the above order of hypochromism. They are
much lower than those observed42 for the typical classical
intercalator ethidium bromide (EthBr; Kb, 4.94 ꢂ 105 Mꢀ1 in
a 25 mM Tris-HCl/40 mM NaCl buffer, pH 7.9) and the partially
intercalating complex cation43 [Ru(bpy)2(dpq)]2þ (Kb, 1.4 ꢂ
106 Mꢀ1 in a 25 mM Tris-HCl/40 mM NaCl buffer, pH 7.9),
both bound to CT DNA. So, it is obvious that the highest DNA
binding affinity as well as the largest red shift (2, 6 nm; 3, 2 nm; 4,
8 nm) observed for 4 are due to the larger surface area of the
aromatic ring of the dpq ligand, which enhances the extent of its
stacking with DNA base pairs. The incorporation of the methyl
groups on the 5 and 6 positions of the phen ring as in 3 would be
KEthBr½EthBrꢁ ¼ Kapp½complexꢁ
)
where KEthBr (4.94 ꢂ 105 Mꢀ1 42 is the DNA binding constant
of EthBr, [EthBr] is the concentration of EthBr (12.5 μM),
and [complex] is the concentration of the complex used to
obtain 50% reduction in the fluorescence intensity of EthBr.
The observed decrease in the emission intensity of EthBr
(Figure 3) and, hence, the DNA binding affinities of the
complexes follow the order 4 > 3 > 2 > 1 (Table 3), which is
in conformity with the order of DNA binding affinities obtained
from the absorption spectral (cf. above) and viscosity studies
(cf. below). Both the electron transfer from excited EthBr to
copper(II) and the EthBr displacement mechanisms would
account for the highest value of Kapp determined for the partially
intercalating complex 4. At higher concentrations, the latter
would compete efficiently with the intercalatively bound EthBr
for DNA binding and quench the EthBr emission more strongly
than 1ꢀ3. We propose that hydrogen-bonding interactions,
Figure 2. Absorption spectra of 3 (2 ꢂ 10ꢀ5 M) in a 5% DMF/5 mM
Tris-HCl/50 mM NaCl buffer at pH 7.1 in the absence (R = 0) and
presence of (R = 25) of increasing amounts of CT DNA. Inset: Plot of
[DNA] vs [DNA]/(εa ꢀ εf) at R = 25 of 3.
þ
which would occur between the ligand ꢀN(Me)H2 moieties
in the dinuclear copper(II) complex and DNA, would also
Table 3. Ligand-Based Absorption Spectral Properties and Fluorescence Spectral Properties of Copper(II) Complexes Bounda to
CT DNA
complex
λmax (nm)
R
change in the absorbance
Δε (%)
red shift (nm)
Kb ꢂ 104 (Mꢀ1
)
Kapp ꢂ 105 (Mꢀ1 b
)
[Cu2(LH)2(bpy)2(ClO4)2]2þ
[Cu2(LH)2(phen)2(ClO4)2]2þ
[Cu2(LH)2(5,6-dmp)2(ClO4)2]2þ
[Cu2(LH)2(dpq)2(ClO4)2]2þ
239
273
282
257
25
25
25
25
hyperchromism
hypochromism
hypochromism
hypochromism
57
66
81
96
0
6
2
8
0.5
0.7
1.3
3.4
0.5 ( 0.1
2.3 ( 0.5
7.6 ( 0.8
a Measurement made at R = 25, where R = [DNA]/[Cu complex] and the concentrations of copper(II) complex solutions are 3.0 ꢂ 10ꢀ5 M (1), 2.0 ꢂ
10ꢀ5 M (2), 1.0 ꢂ 10ꢀ5 M (3), and (4). b Measurement at 1/R = 25, where 1/R = [Cu complex]/[DNA] and the concentration of the DNA solution is
125 μM.
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Figure 5. DNA cleavage of (40 μM) complexes 1ꢀ4 with an incubation
time of 1 h in a 5% DMF/5 mM Tris-HCl/50 mM NaCl buffer at pH 7.1:
lane 1, DNA control; lane 2, DNA þ 1; lane 3, DNA þ 2; lane 4, DNA þ
3; lane 5, DNA þ 4; lane 6, DNA þ [Cu(dpq)2]2þ. The complex
concentration is 100 μM for lanes 2ꢀ6. SC and NC are supercoiled and
nicked-circular forms of DNA, respectively.
Table 4. Self-Activated Cleavage Data of SC pUC19 DNA
(40 μM in Base Pairs) by Complexes 1ꢀ4 (0.1 mM) in the
Absence of Any Reducing Agent for an Incubation Time of 1 h
Figure 3. Effect of the addition of complexes 1ꢀ4 on the emission
intensity of the CT DNA-bound EthBr at different complex concen-
trations in a 5% DMF/5 mM Tris-HCl/50 mM NaCl buffer at pH 7.1
at 25 °C.
form %
serial no.
reaction conditions
DNA control
SC
NC
1
2
3
4
5
6
92.1
71.5
63.2
74.2
11.5
10.0
7.9
28.5
36.8
25.8
88.6
90.0
DNA þ 1
DNA þ 2
DNA þ 3
DNA þ 4
DNA þ [Cu(dpq)2(H2O)](ClO4)2
2ꢀ4, complex 1 shortens the DNA chain length by forming
bends or kinks on the DNA double helix upon binding to the
DNA surface, which is in conformity with its lowest DNA
binding affinity (cf. above). Thus, viscosity studies are in con-
formity with the hypochromism and Kb and Kapp values observed
for the complexes (cf. above) and confirm that the central rings
of dpq (4) and phen (2) are involved in the partial intercala-
tive mode of DNA binding while the 5,6-dmp (3) and bpy (1)
complexes are involved in DNA groove and electrostatic binding,
respectively.
Figure 4. Effect of the addition of complexes 1ꢀ4 to CT DNA: relative
viscosities vs 1/R; [CT DNA] = 500 μM.
contribute significantly to the higher DNA binding affinity of 4.
The complex 3 interacts with DNA through hydrophobic methyl
groups on the 5 and 6 positions of the phen ring, which is more
important than the partial intercalation of the phen ring in 2 in
determining the extent of EthBr quenching.
DNA Cleavage Studies: DNA Cleavage without Added
Reductant. To explore the DNA cleavage abilities of 1ꢀ4,
supercoiled (SC) pUC19 DNA (40 μM in base pairs) was
incubated at 37 °C with the complexes in a 5% DMF/5 mM
Tris-HCl/50 mM NaCl buffer at pH 7.1 for 1 h in the absence of
an activator. All of the complexes cleave SC DNA (form I) into
NC DNA (form II) (Figure 5), and the DNA cleavage efficien-
cies follow the order 4 (88.6) > 2 (36.8) > 1 (28.5) > 3 (25.8%)
(Table 4). This reveals that 4 cleaves DNA more efficiently
than the other complexes, obviously because of involvement
in the strong partial intercalation of the extended aromatic ring
of the dpq ligand. Also, the DNA cleavage efficiencies of 1ꢀ4
are lower than those of their corresponding bis(diimine)copper-
(II) complexes like [Cu(dpq)2]2þ, which exhibits ∼100%
Viscosity Measurements. In order to understand the nature
and mode of DNA binding of the dinuclear complexes, viscosity
measurements were carried out. The values of the relative specific
viscosity (η/η0), where η and η0 are the specific viscosities of
DNA in the presence and absence of the complexes, are plotted
against 1/R (=[complex]/[DNA] = 0.05ꢀ0.5; Figure 4). The
ability of the complexes to increase the viscosity of DNA depends
upon the diimine ligand: dpq (4) > 5,6-dmp (3) > phen (2) >
bpy (1). The increase in the viscosity observed for the dpq
complex is significantly higher than that for the phen complex,
but it is less than that for the potential intercalator EthBr.50 The
partial insertion51 of the coordinated dpq ligand of 4 in between
the DNA base pairs (cf. above) is deeper than that of the
coordinated phen ligand of 2. The DNA groove binding of 3
via the hydrophobic interaction of the coordinated 5,6-dmp
ligand is more effective than the partial intercalation of the phen
ring in 2 in lengthening the DNA duplex. A similar enhanced
viscosity has been observed for simple (5,6-dmp)3FeII,52 -CoII,53
-NiII,53 -CuII,36 and -ZnII 36 complexes and mixed-ligand copper-
(II) complexes with 5,6-dmp as the coligand.45ꢀ48 In contrast to
DNA cleavage.54 Control experiments with ligand, Cu(ClO4)2
3
6H2O, or DNA alone did not reveal any cleavage. Complex 4,
which exhibits the strongest DNA binding affinity and effective
DNA cleavage efficiency, was subjected to further cleavage
studies. Upon variation of the concentration of 4 with a
constant concentration of SC pUC19 DNA (40 μM) under
“pseudo-MichaelisꢀMenton”, concentration-dependent DNA
cleavage is observed in a 5% DMF/5 mM Tris-HCl/50 mM
NaCl buffer at pH 7.1 and 37 °C (Table 5). The kinetic
parameters kcat (1.61 ( 0.13 hꢀ1) and KM (1.35 μM) at an
incubation time of 60 min were calculated based on the plots
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obtained (Figure 6A,B). The rate of cleavage of ds-DNA [(7.7 (
0.6) ꢂ 106 hꢀ1] for 4 is enormously higher than the uncatalyzed
rate (3.6 ꢂ 10ꢀ8 hꢀ1).55 The cleavage reaction was performed
also by variation of the DNA concentration and keeping the
complex concentration constant under a “true Michaelisꢀ
Menton” kinetic condition with an incubation period of 60 min.
The rate constant observed for 4 is (6.7 ( 0.3) ꢂ 106 hꢀ1
(Figure S3A,B in the Supporting Information). However, the rate
of DNA cleavage is slightly lower than those of [Cu(dpq)2]2þ 54
and mononuclear mixed-ligand copper(II) complexes.24,25 The
extent of DNA cleavage varies exponentially with the incubation
time. The plots of disappearance of form I (SC DNA) and the
appearance of form II (NC) versus time follow pseudo-first-
order kinetic profiles and fit well into a single-exponential curve
at constant complex and DNA concentrations (Figure S4 and
Table S3 in the Supporting Information).
participates in the cleavage process. However, the addition of
the minor groove binder distamycin decreases the ability of the
complex to cleave DNA, suggesting that the complex prefers to
bind in the DNA minor groove. Further, the addition of super-
oxide dismutase (superoxide scavenger) to the reaction mixture
does not quench the cleavage reaction significantly, revealing that
superoxide anion is also not the active species.56
DNA Cleavage with Added Reductant. The DNA strand
scission by 1ꢀ4 was studied in a 5% DMF/5 mM Tris-HCl/
50 mM NaCl buffer at pH 7.1 using plasmid SC pUC19 DNA as a
substrate in the presence of activators57ꢀ59 (ascorbic acid or
H2O2), and the reaction mixture was incubated at 37 °C for 1 h.
The cleavage abilities of the complexes in the presence of
ascorbic acid under identical conditions were assessed from
conversion of DNA from the supercoiled form (SC DNA, form
I) to the nicked-circular form (NC DNA, form II). At 30 μM
complex concentration, 4 completely degrades SC DNA into
undetectable minor fragments (Figure 7 and Table 6) while 1
(NC form, 97%) and 2 (NC form, 98%) cause DNA cleavage
more prominent than 3 (NC form, 92%). In the control
experiments with DNA alone or ascorbic acid alone, no DNA
cleavage is observed. The concentration-dependent DNA clea-
vage experiments were carried out for 1, 2, and 4 with a constant
concentration of DNA (40 μM in base pairs) and ascorbic acid
(30 μM). It is clearly seen that 1 and 2 effect efficient DNA
cleavage at 30 μM (NC DNA, 96%) and 20 μM (NC DNA, 90%)
concentrations, respectively (Figure S5A in the Supporting
Information), while 4 cleaves DNA completely (100%) even
at 6 μM concentration (Figure S5B in the Supporting Infor-
mation). As the concentration of 4 is increased, the amount of
NC DNA (form II) increases, reaches a maximum (complete
conversion) at nearly 6 μM, and remains constant until 8 μM
(Figure S5B in the Supporting Information), where the linear
The preliminary mechanism of DNA strand scission by 4 has
been investigated in the presence of several addends under an
inert atmosphere. When the hydroxyl radical scavenger dimethyl
sulfoxide (DMSO) is added to the reaction mixture, inhibition of
DNA cleavage is observed, revealing that the cleavage reaction
•
involves OH radicals. The addition of NaN3 scarcely protects
DNA against strand breakage induced by 4, which suggests that
neither 1O2 nor any other singlet oxygen-like entity participates
in the oxidative DNA cleavage. This attenuation of the cleavage
activity can be explained by taking into consideration the
reduction of CuII ion in the reactive oxygen species (ROS)
production process. Also, the addition of catalase enzyme blocks
the breakdown of DNA, suggesting that hydrogen peroxide
Table 5. Concentration-Dependent DNA Cleavage Data of
SC pUC19 DNA (40 μM in Base Pairs) by Complex 4 in the
Absence of External Agent
form (%)
serial no.
reaction conditions
SC
NC
1
2
3
4
5
6
7
8
9
DNA control
89.1
75.1
86.8
50.8
32.4
4.4
10.9
24.9
13.2
49.2
67.6
95.6
91.7
88.8
92.0
DNA þ 4 (10 μM)
DNA þ 4 (20 μM)
DNA þ 4 (30 μM)
DNA þ 4 (40 μM)
DNA þ 4 (80 μM)
DNA þ 4 (100 μM)
DNA þ 4 (120 μM)
DNA þ 4 (160 μM)
Figure 7. DNA cleavage of SC pUC19 DNA (40 μM) by copper(II)
complexes in a buffer containing a 5% DMF buffer in the presence of
ascorbic acid (H2A) at 37 °C: lane 1, DNA control; lane 2, DNA þ H2A;
lane3, DNAþ H2A þ 1; lane 4, DNAþ H2A þ 2; lane 5, DNA þ H2A þ
3; lane 6, DNA þ H2A þ 4; lane 7, DNA þ H2A þ [Cu(dpq)2]þ. The
complex concentration is 30 μM for lanes 3ꢀ7. SC and NC are
supercoiled and nicked-circular forms of DNA, respectively.
8.3
11.2
8.0
Figure 6. (A) Concentration-dependent cleavage of SC pUC19 (40 μM in base pairs) with different concentrations of complexes: lane 1, DNA; lane 2,
DNA þ 4 (0.01 mM); lane 3, DNA þ 4 (0.02 mM); lane 4, DNA þ 4 (0. 03 mM); lane 5, DNA þ 4 (0.04 mM); lane 6, DNA þ 4 (0.08 mM); lane 7,
DNA þ 4 (0.1 mM); lane 8, DNA þ 4 (0.12 mM); lane 9, DNA þ 4 (0.16 mM). SC and NC are supercoiled and nicked-circular forms of DNA,
respectively. (B) Plot of complex concentrations against the rate of DNA cleavage.
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Table 6. Oxidative Cleavage Data of SC pUC19 DNA (40 μM
in Base Pairs) by Complexes 1ꢀ4 (30 μM) in the Presence of
Ascorbic Acid (10 μM) for an Incubation Time of 1 h
Table 7. Oxidative Cleavage Data of SC pUC19 DNA (40 μM
in Base Pairs) by Complexes 1ꢀ4 (10 μM) in the Presence of
H2O2 (200 μM) for an Incubation Time of 1 h
form %
form %
serial no.
reaction conditions
DNA control
SC
NC
serial no.
reaction conditions
DNA control
SC
NC
1
2
3
4
5
6
7
90.6
89.8
3.0
9.4
1
2
3
4
5
6
7
92.6
86.0
45.0
7.4
DNA þ H2A
10.2
97.0
98.0
DNA þ H2O2
14.0
55.0
DNA þ H2A þ 1
DNA þ H2O2 þ 1
DNA þ H2A þ 2
2.0
DNA þ H2O2 þ 2
2.0
a
98.0
a
DNA þ H2A þ 3
8.0
a
92.0
a
DNA þ H2O2 þ 3
DNA þ H2A þ 4
DNA þ H2O2 þ 4
4.0
a
96.0
a
DNA þ H2A þ [Cu(dpq)2(H2O)](ClO4)2
2.0
98.0
DNA þ H2O2 þ [Cu(dpq)2(H2O)](ClO4)2
a Completely degraded.
a Completely degraded.
form of DNA (form III) is not observed at all under the present
conditions. This suggests that DNA cleavage occurs randomly
because a significant portion of plasmid DNA is already con-
verted to the NC form without concurrent formation of the SC
form. Above a 8 μM concentration, DNA is degraded completely.
The (100%) DNA cleavage efficiency shown by complex 4
(6 μM) is higher than its corresponding Cu(dpq)2þ 60
,
Cu-
Figure 8. DNA cleavage of SC pUC19 DNA (40 μM) by copper(II)
complexes in a buffer containing 5% DMF/5 mM Tris HCl and 50 mM
NaCl in the presence of H2O2 at 37 °C: lane 1, DNA control; lane 2,
DNA þ H2O2; lane 3, DNA þ H2O2 þ 1; lane 4, DNA þ H2O2 þ 2;
lane 5, DNA þ H2O2 þ 3; lane 6, DNA þ H2O2 þ 4; lane 7, DNA þ
H2O2 þ [Cu(dpq)2]þ. The complex concentration is 10 μM for lanes
3ꢀ7. SC and NC are supercoiled and nicked-circular forms of DNA,
respectively.
2þ 61
(dpq)2
,
and [Cu(dpq)3]2þ36 complexes. The high affinity
of 4 for intercalative DNA binding could be related to the
presence of an extended aromatic ring of the dpq ligand, making
noncovalent interactions between the π system of the ligands
and DNA base pairs more favorable in association with the close
proximity of the phenolato bridge, which enhances the nuclease
ability of dinuclear complexes.26ꢀ32 Also, it has been reported
that the Cu(bpy) , ,
2þ 60 Cu(bpy)22þ 61 and mixed-ligand copper-
(II)25 complexes containing bpy as the coligand are nuclease-
inactive. However, it is interesting that the bpy complex 1 shows
complete conversion of SC DNA into NC DNA at a 30 μM
complex concentration. The prominent DNA cleavage exhibited
by 4 suggests that DNA binding in the form of partial intercala-
tion could be an important factor for the cleavage activity. It is
likely that generation of a hydroxyl radical and/or activated
oxygen mediated57 by the copper(II) complexes results in DNA
cleavage.
The ability of 1ꢀ4 to cause DNA cleavage (10 μM) in a 5%
DMF/5 mM Tris-HCl/50 mM NaCl buffer at pH 7.1 has also
been studied in the presence of H2O2 (200 μM) as an activator
by gel electrophoresis using SC pUC19 DNA (40 μM in base
pairs) after incubation of the reaction mixture at 37 °C for 1 h.
At 10 μM complex concentration, conversion of DNA from the
SC form to the NC form is observed. While 2 and 4 cause
respectively 98% and 96% DNA cleavage (Table 7), 1 exhibits
only 55% DNA cleavage. For 3, interestingly, complete degrada-
tion of SC DNA to undetectable smaller fragments is observed
(Figure 8). In the control experiment with DNA alone and DNA
with H2O2, no cleavage is observed. Because 2ꢀ4 exhibit efficient
cleavage activity in the presence of H2O2 under identical condi-
tions, they were subjected to concentration-dependent cleavage
studies. As the complex concentration is increased, the disap-
pearance of form I and appearance of form II are observed. At
concentrations of 2 and 5 μM, respectively, complexes 2 and 4
exhibit more than 90% DNA cleavage (Figure S7A,B and Tables
S4 and S5 in the Supporting Information); however, interestingly,
3 displays the same percentage of DNA cleavage even at 0.2 μM
(200 nM) concentration (Figure 9 and Table 8), and the smaller
fragments of undetectable DNA are observed at 10 μM complex
concentration. It is known that copper(II) complexes interact
with DNA in the presence of O2 or a redox agent, the ROS
generated, causing major DNA damage.62ꢀ65 When the standard
radical scavenger DMSO is added to the reaction mixture, a
dramatic reduction in DNA cleavage is observed. Also, upon the
addition of NaN3 (singlet oxygen quencher) to the reaction
mixture, the cleavage reaction is inhibited significantly. Further,
the 5,6-dmp complex 3 shows DNA cleavage more efficient
The mechanism of pUC19 DNA cleavage by all of the four
complexes was studied by the addition of the hydroxyl radical
inhibitor DMSO (Figure S6 in the Supporting Information). It
was found that the addition of DMSO significantly diminishes
the nuclease activity, indicating the involvement of the hydroxyl
radical in the cleavage process. The complexes would first
interact with DNA by intercalation to form a CuIIDNA adduct
species followed by its reduction by the added reductant to a
CuIDNA adduct, which then generates hydroxyl radicals by
3
reaction with O2. These hydroxyl radicals would then attack
DNA, causing strand scission. Also, it is observed that among all
of the complexes only the 5,6-dmp complex exhibits DNA
cleavage in the presence of a minor groove binder like distamycin,
which reveals that the 5,6-dmp complex alone binds to the DNA
major groove to cleave DNA and all of the remaining complexes
cleave DNA after binding to the DNA minor groove. On the basis
of all of these results, we propose that the complex interacts with
DNA through the minor groove, after which the copper(II)
centers are reduced in the presence of an activating agent to
copper(I) species. The latter subsequently reacts with O2 to give
rise to the hydroxyl radicals needed for DNA breakdown.
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Table 9. In Vitro Cytotoxicity Assays for Complexes 1ꢀ4 and
Ligands against HEp-2 Human Larynx Cancer Cell Line (IC50
Values in Millimolar Concentration)
aIC50 (μM)
complex/ligand
24 h
48 h
Figure 9. Concentration-dependent DNA (20 μM) cleavage by com-
plex 3 (0.05ꢀ10 μM) in 5% DMF/5 mM Tris-HCl/50 mM NaCl at
pH 7.1 and 37 °C in the presence of H2O2 (200 μM) at 37 °C: lane 1,
DNA þ H2O2; lane 2, DNA þ H2O2 þ 3 (0.05 μM); lane 3, DNA þ
H2O2 þ 3 (0.1 μM); lane 4, DNA þ H2O2 þ 3 (0.2 μM); lane 5, DNA þ
H2O2 þ 3 (0.5 μM); lane 6, DNA þ H2O2 þ 3 (1 μM); lane 7, DNA þ
H2O2 þ 3 (5 μM); lane 8, DNA þ H2O2 þ 3 (10 μM). Forms I and IIare
supercoiled and nicked-circular forms of DNA, respectively.
[Cu2(LH)2(bpy)2(ClO4)2]2þ (1)
[Cu2(LH)2(phen)2(ClO4)2]2þ (2)
[Cu2(LH)2(5,6-dmp)2(ClO4)2]2þ (3)
[Cu2(LH)2(dpq)2(ClO4)2]2þ (4)
29.4 ( 4.3
4.3 ( 0.5
1.3 ( 0.1
4.5 ( 0.3
<250
22.4 ( 3.7
9.6 ( 0.4
0.8 ( 0.1
3.1 ( 0.2
<200
bpy
phen
5,6-dmp
dpq
<200
185.5 ( 10.0
32.4 ( 3.0
94.3 ( 12.2
43.0 ( 5.0
123.5 ( 12.4
Table 8. Concentration-Dependent Oxidative Cleavage Data
of SC pUC19 DNA (40 μM in Base Pairs) by Complex 3 in the
Presence of H2O2 (200 μM)
a IC50 = concentration of the drug required to inhibit growth of 50% of
the cancer cells (in μM).
of cisplatin (IC50, 7.1 ( 2.7 μM).67 It is evident that the
hydrophobic forces of interaction of the 5,6-dmp complex 3
lead to its highest cytotoxicity. We have obtained similar results
for the mixed-ligand complexes [Cu(L-tyr)(5,6-dmp)]þ25 and
[Cu(tdp)(tmp)]þ [tmp = 3,4,7,8-tetramethyl-1,10-phenanthro-
line; H(tdp) = 2-[(2-(2-hydroxyethylamino)(ethylimino)-
methyl]phenol],21 which exhibit stronger DNA binding through
hydrophobic forces of interaction involving 5,6-dmp and tmp
coligands and show percentages of apoptosis cell death higher
than those of their bpy, phen, and dpq analogues. Because the cell
killing activity of the copper(II) complexes is similar to that of
cisplatin,68 the present complexes are suitable candidates for
potential applications as anticancer drugs.
form (%)
serial no.
reaction conditions
DNA þ H2O2
SC
NC
1
2
3
4
5
6
7
8
86.3
14.0
12.0
10.0
6.0
13.7
86.0
88.0
90.0
94.0
96.0
DNA þ H2O2 þ 3 (0.05 μM)
DNA þ H2O2 þ 3 (0.10 μM)
DNA þ H2O2 þ 3 (0.20 μM)
DNA þ H2O2 þ 3 (0.50 μM)
DNA þ H2O2 þ 3 (1.00 μM)
DNA þ H2O2 þ 3 (5.00 μM)
DNA þ H2O2 þ 3 (10.0 μM)
4.0
2.0
98.0
a
a
a Completely degraded.
Apoptosis (programmed cell death) is a normal component of
the development and maintenance of health of multicellular
organisms. Cells die in response to a variety of external and phy-
siological stimuli, and during apoptosis, they do so in a controlled
and regulated fashion. This makes apoptosis distinct from the
other form of cell death, called necrosis, in which uncontrolled
(accidental) cell death leads to lysis of cells, to inflammatory
responses, and, potentially, to serious health problems. Also,
apoptosis, by contrast, is a process in which cells play an active
role in their own death (cell suicide). Most tumor cells retain
their sensitivity to some apoptotic stimuli from chemotherapeu-
tic agents, and in this context, the apoptosis-inducing ability of
drugs seems to be a primary factor in determining their efficacy.69
To understand the mode of cell death caused by 3, we have
employed different biochemical techniques like fluorescent stain-
ing for morphological assessment, single-cell gel electrophoresis
(comet assay) for detecting DNA fragmentation, mitochondrial
membrane potential (ΔΨm) to assess changes in the membrane
potential, and a Western blotting technique to evaluate the
expression level of the pro- and antiapoptotic proteins.
Fluorescent Staining Method. The characteristic morpho-
logical changes induced by 3 have been evaluated by adopting
fluorescent microscopic analysis of Hoechst 33258- and acridine
orange/EthBr (AO/EB)-stained cells (Figure 10). The results
obtained reveal that the complexes induce cell death through
different modes like apoptosis and necrosis. Upon treatment of
the cells with IC50 concentration of 3 at different incubation
times (24 and 48 h), morphological changes such as chromatin
fragmentation, bi- and/or multinucleation, cytoplasmic vacuola-
tion, nuclear swelling, cytoplasmic blebbing, and late apoptosis
than the phen (2) and dpq (4) complexes, both bound to DNA
through partial intercalation, as is bound to the surface of DNA in
the major groove. It is also possible that the copper(I) species
produced would interact with H2O2 to generate hydroxyl radicals
(Fenton mechanism).66 The above mechanism of DNA cleavage
mediated by the complexes is similar to that proposed forother di-
and multinuclear copper(II) complexes.31,32 Thus, all of the
complexes exhibit DNA cleavage activity with an activator
like ascorbic acid or H2O2, which plays a vital role in determining
the type of DNA cleavage observed without an activator and also
•
supports the proposed mechanisms of cleavage involving OH
radicals.
Studies on the Anticancer Activity of Complexes. The
cytotoxicity of the strongly DNA binding and cleaving complexes
1ꢀ4 on HEp-2 human larynx cancer cell line has been evaluated
in comparison with the widely used drug cisplatin under identical
conditions by using MTT assay. The IC50 values determined
(1.3 ( 0.1ꢀ29.4 ( 4.3 μM at 24 h and 0.79 ( 0.10ꢀ22.4 (
3.7 μM at 48 h; Table 9) are found to be significant, and they
follow the trend 1 > 2 > 4 > 3. This clearly indicates that the
anticancer activity of the complexes is time-dependent and varies
with the mode and extent of their interaction with DNA. The
IC50 values determined for the free coligands (Table 9) are 9ꢀ40
times higher than those for the respective complexes, revealing
that the complex species rather than the dissociated coligands are
actually responsible for the observed antitumor activity of the
complexes. While 2 and 4 exhibit appreciable cytotoxic effects,
3 is remarkable in displaying a cytotoxic effect (IC50, 0.79 (
0.10 μM) approximately 10 times more prominent than that
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Figure 11. Comet assay of EB-stained (a) control (untreated) 3 and
(b and c) treated cells.
Figure 10. AO/EB- and Hoechst 33258-stained HEp-2 human larynx
cancer cells: (a and b) untreated cells; (c and d) at 24 h and (e and f) at
48 h incubation by the treatment of 3.
indication of dotlike chromatin condensation70 have been ob-
served by adopting Hoechst 33258 staining. The cytological
changes observed are classified into four types according to the
fluorescence emission and morphological features of chromatin
condensation in the AO/EB-stained nuclei: (i) viable cells having
uniformly green fluorescing nuclei with highly organized struc-
ture; (ii) early apoptotic cells (which still have intact membranes
but have started undergoing DNA fragmentation) having green
fluorescing nuclei but with perinuclear chromatin condensation
visible as bright-green patches or fragments; (iii) late apoptotic
cells having orange-to-red fluorescing nuclei with condensed or
fragmented chromatin; (iv) necrotic cells, swollen to large sizes,
having uniformly orange-to-red fluorescing nuclei with no in-
dication of chromatin fragmentation. All of these morphological
changes indicate that the cells are committed to death in such a
way that both apoptotic and necrotic cells increase in number in a
time-dependent manner. The apoptotic morphologies induced
by 3 are confirmed by AO/EB staining adopting fluorescence
microscopy, which suggests that the complex causes apoptosis
more efficiently than all other complexes during 24 and 48 h
treatment, and the percentage of the apoptotic mode of cell death
caused at 48 h is higher than that at 24 h. Similar observations
have been made by us for the mixed-ligand complexes [Cu-
(tdp)(tmp)]þ21 and [Cu(L-tyr)(5,6-dmp)]þ,25 which show a
percentage of apoptosis cell death higher than that of their other
phen analogues.
Single-Cell Gel Electrophoresis. The single-cell gel electro-
phoresis (comet) assay has also been used to evaluate single- and
double-stranded DNA breaks at the single-cell level because the
assay detects DNA breaks, alkali-labile lesions, and genomic
lesions that are subject to DNA repair.71 When the cancer cells
are treated with IC50 concentration of 3, statistically significant
and well-formed comets are observed, while the control (un-
treated) cells fail to show a cometlike appearance (Figure 11).
This clearly reveals that the complex indeed induces DNA
fragmentation, which is a hallmark of apoptosis.71 The high
levels of DNA damage induced by the complex reinforces the
Figure 12. (A) Mitochondrial trans membrane potency of complex 3:
control (a) (untreated) and treated with 3 (b and c) at different times of
incubation. (B) Schematic representation of major stimuli for the
induction of apoptosis.72.
above results obtained by using MTT assay and fluorescent
staining assay.
Apoptosis Detection by Mitochondrial Membrane Poten-
tial (ΔΨm) Analysis. In this analysis, the mitochondrial mem-
brane potential is monitored using JC1, a cell-permeable
fluorescent dye, which preferentially enters into mitochondria
because of the highly negative mitochondrial membrane poten-
tial and confers a green fluorescence. It is well-known that, upon a
loss of the membrane integrity, the mitochondrial potential
changes, cytochrome c located normally inside the mitochondria
is translocated into the cytosol, and the JC1 dye is released from
mitochondria, which leads to a decrease in the intracellular
fluorescence (Figure 12A). The cancer cells were treated with
3 at different incubation time intervals to understand the effect of
the complex on the mitochondrial membrane potential and,
hence, the translocation of cytochrome c. With an increase in
time, the intracellular fluorescence is found to decrease, revealing
an increase in the loss of membrane potential and leading to the
release of cytochrome c. The cytochrome c released binds with
apaf1 protein to form an apotosome complex and activates the
cascades of caspase proteins, which triggers the enzymes and
proteins to cause DNA fragmentation, leading to apoptosis.
Thus, the observed loss of fluorescence by the cancer cells upon
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treatment with 3 clearly reveals that the complex induces
apoptosis in cancer cells via the mitochondrial pathway, that is,
release of cytochrome c from mitochondria to cytosol, leading
to caspase activation during the apoptotic process. Some of
the major stimuli that can induce apoptosis are illustrated in
Figure 12B.72
involved in the cancer profile. The above MTT assay and mor-
phological changes observed reveal that the 5,6-dmp complex
possesses a very prominent cytotoxicity.
’ CONCLUSIONS
The mixed-ligand dinuclear copper(II) complexes of bpy,
phen, 5,6-dmp, and dpq exhibit prominent DNA cleavage in
the presence of activators, which is higher than those of their
corresponding mono- and bis(diimine) complexes. The minor
groove-bound dpq complex completely degrades SC DNA into
undetectable smaller fragments even at 8 μM concentration and
shows an oxidative DNA cleavage in the absence and presence of
the activator ascorbic acid with efficiency more profound and
prominent than that of the other diimine complexes. So, it is
evident that the partially intercalating coligand dpq is an excellent
DNA recognition element, which could be used to design robust
and efficient copper(II)-based chemical nucleases. On the other
hand, the 5,6-dmp complex preferentially docked in the DNA
major groove behaves as a chemical nuclease even at a lower
complex concentration of 0.2 μM in the presence of H2O2 as an
activator with efficiency higher than that of the other complexes.
Thus, the DNA binding behavior and, hence, the DNA cleavage
efficiency of the mixed-ligand dinuclear copper(II) complexes
are dictated by the size, shape, and electronic structures of the
dinuclear copper(II) complexes, as determined by the diimine
coligands. Also, while the diimine ligand could function as a DNA
recognition element, the amide group of the primary ligand and/
or a water molecule present in the axial copper site could
promote DNA binding and DNA cleavage.
The dinuclear copper(II) complexes with phen, 5,6-dmp, and
dpq coligands exhibit anticancer activities toward human larynx
cancer cell lines (HEp-2) more potent than the widely used drug
cisplatin. This reveals that a synergic combination of the ligand
and metal ion is important in the design of a potential anticancer
drug, the activity of which correlates well with the ability of the
complexes to strongly bind and cleave DNA. Thus, the hydro-
phobic forces of interaction exhibited by the 5,6-dmp ligand,
which is responsible for the stronger DNA groove binding, more
effective DNA cleavage of the complex in the presence of H2O2
as an activator, and more facile transport across the cell mem-
brane of the hydrophobic 5,6-dmp complex, account for the
enhanced cytotoxicity of the complex. Also, biochemical studies
like fluorescence staining, comet assay, mitochondrial transmem-
brane potential, and Western blotting technique reveal that the
5,6-dmp complex acts as a potent anticancer agent by inducing
phenotypical changes, DNA fragmentation, loss of mitochon-
drial membrane potential, and upregulation of proapoptotic
proteins or downregulation of antiapoptotic proteins, which is
consistent with the induction of mainly apoptotic cell death.
Thus, it emerges from the present study that the water-soluble
5,6-dmp complex can act as an efficacious antiproliferative drug
by inducing morphological changes consistent with the induction
of apoptotic cell death. Further mechanistic and cellular uptake
studies are essential to probe the higher potency of the complex
to kill cancer cells.
Western Blotting Analysis. The tumor suppressor protein
p53 is a redox-active transcription factor that coordinates and
prepares cellular mechanisms in response to a variety of stresses
that lead to genomic instability. It occupies a pivotal role in
maintaining the genomic integrity72 because it plays a vital role in
cell arrest, DNA repair, cellular senescence, differentiation, and
apoptosis. It also facilitates the repair and survival of scarcely
damaged cells and eliminates severely damaged cells from the
replicative pool to protect the organism. Also, both proteins Bcl-
2 and Bax are transcriptional targets for p53, which induces
apoptosis in response to DNA damage.73 It is widely accepted
that these three proteins are very important in deciding the issues
of cell suicide.74 Now, the expression of the proapoptotic p53 and
Bax and antiapoptotic Bcl-2 proteins in HEp-2 larynx cancer cell
lines treated for 24 and 48 h with 3 has been determined, and the
results obtained are referenced to a control experiment (without
drug treatment). It is found that the expression level of the Bcl-2
protein decreases and the Bax protein level is constitutively
expressed, suggesting that apoptosis by 3 could be mediated
through the downregulation of the antiapoptotic protein Bcl-2
(Figure S8 in the Supporting Information). However, as shown
in Figure S8 in the Supporting Information, the p53 protein level
in HEp-2 larynx cancer cell lines is markedly increased upon
treatment with 3 in a time-dependent manner (increase in the
level of the expression of the p53 status in the presence of 3),
revealing that the complex induces apoptosis by activating the
p53 apoptosis pathway. The p53 protein is activated in response
to DNA damage, which subsequently unbalances the Bax/Bcl-2
ratio. The Bcl-2 family of proteins regulates apoptosis by con-
trolling the mitochondrial permeability and the release of cyto-
chrome c. The expression level of antiapoptotic protein Bcl-2 is a
critical intracellular checkpoint of apoptosis within a distal
common cell death pathway. It is stated that the main mechanism
for apoptosis appears to be a mitochondria-mediated pathway
accompanied by loss of the mitochondrial transmembrane
potential (cf. above) followed by cytochrome c release from
mitochondria into cytosol, resulting in activation of caspase
cascades and leading to apoptosis.75 Also, the upregulation of
proapoptotic protein p53 and the downregulation of antiapop-
totic protein Bcl-2 caused by 3 could possibly activate mitochon-
dria-mediated apoptosis.
It is expected that the DNA groove-binding complex 3 with
hydrophobic 5,6-dmp coligand, which exists as the dicationic
[Cu2(LH)2(5,6-dmp)2(ClO4)2]2þ species (cf. above), would
adhere to the plasma membrane by electrostatic attraction, get
transported across the cell membrane by a concentration gra-
dient, and then interfere with the cellular function of DNA by
binding to it. Thus, the prominent cytotoxicity of the 5,6-dmp
complex is consistent with its strong DNA binding affinity and
efficient DNA cleavage in the presence of H2O2. Also, a complex
like 4 with a higher DNA binding affinity and DNA cleavage
ability fails to show cytotoxicity higher than its DNA groove-
binding analogue 3. The complexes might exhibit cytotoxicity by
inducing apoptosis, which plays a key role in regulating the cell
cycle. So, if the drug is to exhibit cytotoxicity by inducing
apoptosis, it should target and inhibit some of the proteins
’ EXPERIMENTAL SECTION
Reagents and Materials. Copper(II) perchlorate hexahydrate
(Aldrich), 1,10-phenanthroline (Merck, India), calf thymus (CT) DNA
(highly polymerized stored at 4 °C), superoxide dismutase (SOD),
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catalase (Sigma, stored at ꢀ20 °C), and plasmid SC pUC19 DNA and
agarose (Genei, Bangalore, India) were used as received. Ultrapure Milli-
Q water (18.2 MΩ) was used in all experiments. The ligand dipyrido-
[3,2-d:20,30-f]quinoxaline (dpq)35 and ligand 2-hydroxy-N-[2-(methyl-
amino)ethyl]benzamide (LH)76 were prepared by their reported
procedure. The commercial solvents were distilled and then used for
the preparation of complexes.
HEp-2 human larynx cancer cell line was obtained from National
Center for Cell Science, Pune, India. The cells were cultured in a RPMI
1640 medium (Biochrom AG, Berlin, Germany), supplemented with
10% fetal bovine serum (Sigma, Madison, WI), in 96-well culture plates,
at 37 °C in a humidified atmosphere of 5% CO2 in a CO2 incubator
(Heraeus, Hanau, Germany). All of the experiments were performed
using cells from passage 15 or less.
Synthesis of LH. To a methanolic solution of N-methylethylene-
diamine (0.9 mL, 10 mmol) and 1-methylsalicylate (1.3 mL, 10 mmol)
was added 20 mL of acetonitrile. The resulting solution was stirred for
12 h. The colorless crystalline product obtained was filtered off and
washed with water and then ether.76 The crude product was recrystal-
lized from ethanol to give a crystalline product. MP: 138 °C. 1H NMR
(200 MHz, CDCl3): δ 7.6ꢀ6.9 (m, 4H), 6.8 (s, 1H), 3.4 (t, 2H), 2.9
(t, 2H), 2.3 (s, 1H), 2.1 (s, 3H).
Isolation of 1. A methanolic solution (5 mL) of copper(II)
perchlorate hexahydrate (0.37 g, 1 mmol) was added to a solution of
LH (0.19 g, 1 mmol) in methanol (5 mL) with constant stirring. Then a
solution of 2,20-bipyridine (0.15 g, 1 mmol) in methanol (5 mL) was
added to the reaction mixture with constant stirring. The dark-green
complex was filtered off and dried under vacuum. The concentrated
methanolic solution of complex 1 kept aside for 1 week. By the slow
evaporation of solution, green crystals were obtained. Anal. Calcd for
C40H44Cl4Cu2N8O20: C, 39.2; H, 3.62; N, 9.14. Found: C, 39.18; H, 3.6;
N, 9.12. ESI-MS: {[Cu2(LH)2(bpy)2(ClO4)2](ClO4)}þ displays a peak
at m/z 1124.53 (calcd m/z 1123.07).
Isolation of [Cu2(LH)2(phen)2(ClO4)2](ClO4)2 (2). A metha-
nolic solution (5 mL) of copper(II) perchlorate hexahydrate (0.37 g,
1 mmol) was added to a solution of LH (0.19 g, 1 mmol) in methanol
(5 mL) with constant stirring. During stirring, a solution of 1,10-
phenanthroline (0.19 g, 1 mmol) in methanol (5 mL) was added. The
dark-green precipitate obtained was filtered off and dried under vacuum.
Anal. Calcd for C44H44Cl4Cu2N8O20: C, 41.49; H, 3.48; N, 8.8. Found:
C, 41.5; H, 3.49; N, 8.82. ESI-MS: {[Cu2(LH)2(phen)2(ClO4)2]-
(ClO4)}þ displays a peak at m/z 1171.07 (calcd m/z 1171.60).
Isolation of [Cu2(LH)2(5,6-dmp)2(ClO4)2](ClO4)2 (3). A meth-
anolic solution (5 mL) of copper(II) perchlorate hexahydrate (0.37 g, 1
mmol) was added to a solution of LH (0.19 g, 1 mmol) in methanol
(5 mL) with constant stirring. To the reaction mixture was added a
methanolic solution (5 mL) of 5,6-dimethyl-1,10-phenanthroline (0.21
g, 1 mmol). The green complex obtained was filtered off and washed
with diethyl ether. Anal. Calcd for C48H52Cl4Cu2N8O20: C, 43.35; H,
3.94; N, 8.43. Found: C, 43.37; H, 3.95; N, 8.45. ESI-MS: {[Cu2-
(LH)2(5,6-dmp)2(ClO4)2](ClO4)}þ displays a peak at m/z 1227.14
(calcd m/z 1130.53).
Isolation of [Cu2(LH)2(dpq)2(ClO4)2]2(ClO4)2 (4). A methano-
lic solution (5 mL) of copper(II) perchlorate hexahydrate (0.37 g,
1 mmol) was added to a solution of LH (0.19 g, 1 mmol) in methanol
(5 mL) with constant stirring. The color of the solution changed to
green. Then a solution of dpq32 (0.23 g, 1 mmol) in methanol (5 mL)
was added with constant stirring. The green complex was filtered off and
dried under vacuum. Anal. Calcd for C48H44Cl4Cu2N12O20: C, 41.84;
H, 3.22; N, 12.2. Found: C, 41.85, H, 3.25, N, 12.2.
meter using cuvettes of 1 cm path length. Emission intensity measure-
ments were carried out using a Jasco F 6500 spectrofluorimeter.
Viscosity measurements were carried out using a Schott Gerate AVS
310 automated viscometer.
Solutions of DNA in the buffer 5 mM Tris HCl/50 mM NaCl in water
gave a ratio of UV absorbance at 260 and 280 nm, A260/A280, of 1.9,77
indicating that the DNA was sufficiently free of protein. Concentrated
stock solutions of DNA (16.5 mM) were prepared in a buffer and
sonicated for 25 cycles, where each cycle consisted of 30 s with 1 min
intervals. The concentration of DNA in nucleotide phosphate (NP)
was determined by UV absorbance at 260 nm after 1:100 dilutions.
The extinction coefficient, ε260, was taken as 6600 Mꢀ1 cmꢀ1. Stock
solutions were stored at 4 °C and used after no more than 4 days.
Plasmid SC pUC19 DNA was stored at ꢀ20 °C, and the concentration
of DNA in base pairs was determined by UV absorbance at 260 nm after
appropriate dilutions taking ε260 as 13 100 Mꢀ1 cmꢀ1. Concentrated
stock solutions of metal complexes were prepared by dissolving calcu-
lated amounts of metal complexes in the respective amount of solvent
and diluted suitably with a corresponding 5% DMF/5 mM Tris-HCl/
50 mM NaCl buffer at pH 7.1 to the required concentrations for all of the
experiments.
X-ray Crystallography. A crystal of suitable size was selected from
the mother liquor, immersed in paraffin oil, then mounted on the tip of a
glass fiber, and cemented using epoxy resin. Intensity data for the crystal
were collected using Mo KR (λ = 0.710 73 Å) radiation on a Bruker
SMART APEX diffractometer equipped with a CCD area detector at
293 K. The crystallographic data are collected in Table 1. The SMART
program78 was used for collecting frames of data, indexing reflections,
and determining lattice parameters; the SAINT79 program for integra-
tion of the intensity of reflections and scaling, the SADABS80 pro-
gram for absorption correction, and the SHELXTL81 program for space
group and structure determination and least-squares refinements on F2.
The structure was solved by a heavy-atom method. The non-hydrogen
atoms were located in successive difference Fourier syntheses. The final
refinement was performed by full-matrix least-squares analysis. Hydro-
gen atoms attached to the ligand moiety were located from the difference
Fourier map and refined isotropically.
DNA Binding and Cleavage Experiments. Concentrated stock
solutions of metal complexes were prepared by dissolving them in a 5%
DMF/5 mM Tris-HCl/50 mM NaCl buffer at pH 7.1 of metal
complexes and diluting suitably with the corresponding buffer to the
required concentrations for all of the experiments. For absorption and
emission spectral experiments, the DNA solutions were pretreated with
solutions of metal complexes to ensure no change in the concentration
of the metal complexes.
Absorption spectral titration experiments were performed on a Varian
Cary 300 Bio UVꢀvisible spectrophotometer by maintaining a constant
concentration ofthecomplex whilevarying thenucleic acid concentration.
This was achieved by dissolving an appropriate amount of the complex
and DNA stock solutions while maintaining the total volume constant
(1 mL). This results in a series of solutions with varying concentrations of
DNA but with a constant concentration of the complex. The absorbance
(A) of the most red-shifted band of each investigated complex was
recorded after successive additions of CT DNA.
Emission intensity measurements were carried out using a 5% DMF/
5 mM Tris-HCl/50 mM NaCl buffer solution as a blank to make
preliminary adjustments. The excitation wavelength was fixed and the
emission range was adjusted before measurements. DNA was pretreated
with EthBr in the ratio [DNA/EthBr] = 10 for 30 min at 27 °C. The
metal complexes were then added to this mixture, and their effect on the
emission intensity was measured.
Methods and Instruments. Microanalyses (C, H, and N) were
carried out with a Vario EL elemental analyzer. UVꢀvisible spectros-
copy was recorded on a Varian Cary 300 Bio UVꢀvisible spectrophoto-
For viscosity measurements, the Schott Gerate AVS 310 automated
viscometer was thermostatted at 25 °C in a constant-temperature
bath. The concentration of DNA was 500 μM in NP, and the flow
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ARTICLE
times were noted from the digital timer attached with the viscometer
(1/R = [Cu]/[DNA] = 0.5).
microscope as that used above. Tests were done in triplicate, counting a
minimum of 100 total cells each.
The cleavage of DNA in the absence and presence of activating agents
like ascorbic acid (10 μM) or H2O2 (200 μM) was monitored using
agarose gel electrophoresis. Reactions using plasmid SC pUC19 DNA
(form I, 40 μM) in a 5% DMF/5 mM Tris-HCl/50 mM NaCl buffer at
pH 7.1 were treated with copper complexes. The cleavage of DNA in the
absence of activating agents was monitored using agarose gel electro-
phoresis. The samples were incubated for 1 h at 37 °C. A loading buffer
containing 25% bromophenol blue, 0.25% xylene cyanol, and 30%
glycerol (3 μL) was added and electrophoresis performed at 60 V for
5 h in a tris-acetate-EDTA (TAE) buffer (40 mM tris-base, 20 mM acetic
acid, and 1 mM EDTA) using 1% agarose gel containing 1.0 μg/mL
EthBr. The gels were viewed in a Alpha Innotech Corp. gel doc system
and photographed using a CCD camera. Densitometric calculations
were made using the AlphaEaseFC Stand Alone software. The intensities
of SC DNA were corrected by a factor of 1.47 as a result of its lower
staining capacity by EthBr.82 The cleavage efficiency was measured by
determining the ability of the complex to convert DNA from the SC
form to the NC form.
The decrease in the intensities of form I (SC), or the increase in the
intensities of form II (NC), was then plotted against catalyst concentra-
tions, and these were fitted well with a single-exponential decay curve
(pseudo-first-order kinetics) by the use of known equations.55 For
anaerobic experiments, deoxygenated water and anaerobic stock solu-
tions were prepared. In order to identify the ROS involved in the
cleavage reaction, the radical scavengers such as the hydroxyl radical
(DMSO, 10%), singlet oxygen (NaN3, 100 μM), SOD (10 unit), and
H2O2 (catalase, 0.1 unit) were introduced. The reaction products were
resolved on 1% agarose gel in a TAE buffer.
Cell Viability Assay. MTT assay was carried out as described
previously.83 Complexes 1ꢀ4, in the concentration range of 0.05ꢀ
50 μM, dissolved in H2O were added to the wells 24 h after seeding of
5 ꢂ 103 cells wellꢀ1 in 200 μL of a fresh culture medium. After 24 and
48 h, 20 μL of a MTT solution [5 mg/mL in phosphate-buffered saline
(PBS)] was added to each well, and the plates were wrapped with
aluminum foil and incubated for 4 h at 37 °C. The purple formazan
product was dissolved by the addition of 100 μL of DMSO to each well.
The absorbance was monitored at 570 nm (measurement) and 630 nm
(reference) using a 96-well plate reader (Bio-Rad, Hercules, CA). Data
were collected for three replicates each and used to calculate the
mean. The percentage inhibition was calculated, from this data, using
the formula
Comet Assay. DNA damage was quantified by means of the comet
assay as described.85,86 Assays were performed under red light at 4 °C.
Cells used for the comet assay were sampled from a monolayer during
the growing phase, 24 h after seeding. Cells were treated with copper(II)
complexes at IC50 dose, and cells were harvested by a trypsinization
process at 12 and 24 h. A total of 200 μL of 1% normal agarose in PBS
at 65 °C was dropped gently onto a fully frosted microslide, covered
immediately with a coverslip, and placed over a frozen ice pack for
about 5 min. The coverslip was removed after the gel had set. The cell
suspension from one fraction was mixed with 1% low-melting agarose at
37 °C in a 1:3 ratio. A total of 100 μL of this mixture was applied quickly
on top of the gel, coated over the microslide, and allowed to set as before.
A third coating of 100 μL of 1% low-melting agarose was placed on the
gel containing the cell suspension and allowed to set. Similarly, slides
were prepared (in duplicate) for each cell fraction. After solidification of
the agarose, the coverslips were removed, and the slides were immersed
in an ice-cold lysis solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM
Tris, NaOH; pH 10, 0.1% Triton X-100) and placed in a refrigerator at
4 °C for 16 h. All of the above operations were performed in low-lighting
conditions in order to avoid additional DNA damage. Slides, after
removal from the lysis solution, were placed horizontally in an electro-
phoresis tank. The reservoirs were filled with an electrophoresis buffer
(300 mM NaOH and 1 mM Na2EDTA, pH > 13) until the slides were
just immersed in it. The slides were allowed to stand in the buffer for
about 20 min (to allow DNA unwinding), after which electrophoresis
was carried out at 0.8 v/cm for 15 min. After electrophoresis, the slides
were removed, washed thrice in a neutralization buffer (0.4 M Tris,
pH 7.5), and gently dabbed to dry. Nuclear DNA was stained with 20 μL
of EthBr (50 μg/mL). Photographs were taken using an epifluorescence
microscope (Carl Zeiss). A total of 200 cells from each treatment were
digitalized and analyzed with image analysis (CASP Software). The
images were used to estimate the DNA content of individual nuclei and
to evaluate the degree of DNA damage representing a fraction of the
total DNA in the tail.
Mitochondrial Transmembrane Potential. The mitochon-
drial transmembrane potential was measured using the fluorescent
probe JC-1, which produces green fluorescence in the cytoplasm and
red-orange fluorescence when concentrated in respiring mitochondria
having a negative internal potential. Cells treated overnight with
complex 3 in 12-well plates were incubated for 30 min with 2 μg/mL
of JC-1 in the culture medium. The adherent cell layer was then washed
three times with PBS and dislodged with 250 μL of trypsinꢀEDTA.
Cells were collected in PBS/2% bovine serum albumin (BSA), washed
twice by centrifugation, resuspended in 0.3 mL of PBS/2% BSA, mixed
gently, and examined in the fluorescent microscope using a UV filter
(450ꢀ490 nm). A total of 300 cells per sample were counted in duplicate
for each dose point. The specific fluorescent patterns were indicative of
live and dead cells.
mean OD of untreated cells ðcontrolÞ ꢀ mean OD of treated cells
¼
mean OD of untreated cells ðcontrolÞ
ꢂ100
The IC50 values were calculated using Table Curve 2D, version 5.01.
Hoechst 33258 Staining. The cell pathology was detected by
staining the nuclear chromatin of trypsinized cells (4.0 ꢂ 104/mL) with
1 μL of Hoechst 33258 (1 mg/mL) for 10 min at 37 °C. Staining
of suspension cells with Hoechst 33258 was used to detect apoptosis.84
A drop of cell suspension was placed on a glass slide, and a coverslip was
overlaid to reduce light diffraction. At random, 300 cells were observed
in a fluorescent microscope (Carl Zeiss, Jena, Germany) fitted with a
377ꢀ355 nm filter and observed at 400ꢂ magnification, and the
percentage of cells reflecting pathological changes was calculated. Data
were collected for four replicates and used to calculate the mean and
standard deviation.
Western Blot Analysis. Total proteins from untreated and treated
cells for 12 and 24 h were determined by a modified Lowery method
using specific apoptotic markers such as p53, Bcl-2, Bax, and β-actin
(loading control) antibodies (Sigma, Madison, WI). In T75 plates, 5 ꢂ
106 cells were seeded and treated with the complexes at IC50 concen-
tration of 3. After the indicated periods, cells were washed in cold
PBS and lysed in 1% Triton X-100, 50 mmol/L Tris, pH 7.6, and
150 mmol/L NaCl containing 2 mmol of L-phenylmethylsulfonyl
fluoride. Subsequently, proteins were separated by sodium dodecyl
sulfate/polyacrylamide gel electrophoresis and transferred to a nitro-
cellulose membrane. The loading and transfer of equal amounts of
protein was confirmed by staining the membrane with Ponceau S.
Membranes were blocked with 5% nonfat dry milk powder in a Tris-
buffered saline (TBS; 10 mmol/L Tris-HCl, pH 7.4, 100 mmol/L NaCl)
AO/EB Staining. For both suspension and adherent cells, 96-well
plates were centrifuged at 1000 rpm (129 g) for 5 min using a Beckman
model TJ-6 centrifuge with inserts for 96-well plates. An EB/AO dye mix
(8 μL) was added to each well, and cells were viewed under the same
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ARTICLE
for 1 h and then incubated with the primary antibody at appropriate
dilutions overnight at 4 °C. Membranes were washed four times with
TBS/0.05% Tween-20 and incubated with secondary antibodies con-
jugated with peroxidase (Bangalore Genei, Bangalore, India) or alkaline
phosphatase for 1 h. After extensive washing, the reaction was developed
with diaminobenzidine/1ꢀ4CN substrate.
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’ ASSOCIATED CONTENT
S
Supporting Information. X-ray crystallographic data in
b
CIF format, electronic absorption spectral behavior, molar con-
ductivity, time-dependent DNA cleavage data, concentration-
dependent oxidative cleavage data, ESI-MS spectra, plot of A/A0
vs R, saturation kinetics, time course of pUC19 cleavage, and
Western blotting analyses. This material is available free of charge
via the Internet at http://pubs.acs.org.
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1999, 38, 11560.
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’ AUTHOR INFORMATION
Corresponding Author
*E-mail: palanim51@yahoo.com or palaniandavarm@gmail.com.
(22) Ng, C. H.; Kong, K. C.; Von, S. T.; Balraj, P.; Jensen, P.;
Thirthagiri, E.; Hamada, H.; Chikira, M. Dalton Trans. 2008, 447.
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Zhang, J.; Guo, Z. J. Inorg. Biochem. 2004, 98, 2099.
(24) Rajendiran, V.; Karthik, R.; Palaniandavar, M.; Evans, H. S.;
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Inorg. Chem. 2007, 46, 8208.
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’ ACKNOWLEDGMENT
The authors thank the Council of Scientific and Industrial
Research [Grants 01(2101)/07/EMR-II and SRF to S.R.] for
funding this research program. M.P. is a recipient of the Ramanna
Fellowship (Grant No. SR/S1/RFIC/01/2007-2010 & SR/S1/
RFIC/01/2010-2013) of the Department of Science and Tech-
nology, New Delhi, India. The X-ray and fluorescence spectral
facilities in the department were created by funding respectively
from the Department of Science and Technology (DST-FIST),
New Delhi, India, and the University Grants Commission (SAP),
New Delhi, India.
(27) Li, L.; Karlin, K. D.; Rokita, S. E. J. Am. Chem. Soc. 2005,
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