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Polyhedron 51 (2013) 228–234

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Polyhedron
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Synthesis, structure, interaction with DNA and cytotoxicity of a luminescent


copper(II) complex with a hydrazone ligand
Soma Mukherjee a,⇑, Santanu Chowdhury a, Atanu Ghorai b, Utpal Ghosh b, Helen Stoeckli-Evans c
a
Department of Environmental Science, University of Kalyani, Kalyani, Nadia 741 235, West Bengal, India
b
Department of Biochemistry and Biophysics, University of Kalyani, Kalyani, Nadia 741 235, West Bengal, India
c
Institute of Physics, University de Neuchatel, CH 2009, Neuchatel, Switzerland

a r t i c l e i n f o a b s t r a c t

Article history: A luminescent copper(II) complex [CuII(L)H2O]2SO4,1a was synthesized with the acyclic tridentate 2-
Received 22 August 2012 hydroxy naphthaldehyde-2-pyridylhydrazone ligand, HL, 1. The molecular structure of 1a clearly shows
Accepted 2 January 2013 that it is a monomeric copper(II) complex with CuN2OOW coordination with slightly distorted square pla-
Available online 11 January 2013
nar geometry. The asymmetric unit of 1a contains two independent complex molecules and a sulfate
anion. DNA binding interactions of 1a have been investigated by absorption, emission, viscosity and ther-
Keywords: mal denaturation studies. The cytotoxic activity of 1a was measured in vitro against the HeLa cells. The
Association constant
LD50 value was calculated as 4.97 lM. There was no nuclear fragmentation observed after treatment with
Structure
Luminescence
1a, which revealed that the copper(II) complex 1a was not capable of inducing apoptosis. The cell cycle
DNA binding analysis of 1a indicated a dose dependent decrease of G0/G1 and S-phase cell population and dose-depen-
Cytotoxicity dent increase of G2/M population compared with untreated control cell.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction family of copper(II) complexes with hydrazone ligands containing


the –N@C–CH@NH–C@N– motif, which is a potential tool for
Studies on the interaction of small molecules with DNA con- development of multidentate chelators with functional diversities
tinue to attract considerable attention due to their importance in [33–37]. Moreover, modulation of ligand frame by proper choice
cancer therapy and molecular biology [1–6]. In this respect, transi- of donor atoms significantly influences the structural and
tion metal complexes that are capable of binding DNA under functional behavior of the complexes [38–40]. Herein, we have
physiological conditions are of interest in the development of synthesized the acyclic tridentate 2-hydroxy naphthaldehyde-
metal-based anticancer agents [7–11]. The use of such complexes 2-pyridylhydrazone ligand HL, 1, with conjugated aromatic core
in footprinting studies, sequence specific DNA binding agents, and its copper(II) complex, 1a in order to increase the versatility
diagnostic agents in medicinal applications and for genomic of the hydrazone system. Interestingly the molecular structure of
research has generated current interests to develop this chemistry 1a shows that each asymmetric unit contains two CuN2OOW
further [12–17]. molecules with sulfate as an anion. It shows DNA interaction and
Copper is an essential transition metal ion in the active sites of cytotoxic activity with HeLa cells as revealed from cell viability,
many enzymes present in the human body and plays an important cell morphology.
role in various physiological processes [18–22]. Due to its bioes-
sential activity and oxidative nature, suitably designed copper(II)
complexes are of great interest in the development of tumor ther- 2. Experimental
apeutics, antibacterial and antimicrobial agents [23–26].
Copper(II) complexes containing heterocyclic bases have been 2.1. Materials
extensively explored in virtue of their strong interactions with
DNA and cytotoxic activity [27–30]. Design of metal-based phar- All starting materials and solvents were purchased from Sigma
maceuticals depends on the ligand framework, the choice of metal Aldrich Chemical Company and used without further purification
ion, and its oxidation state [31,32]. Previously, we have reported a unless otherwise stated. Ethidium bromide and DNA (CT-DNA)
were purchased from Bangalore GeNei. Propidium iodide, Hoechst
dye and RNase A were purchased from Sigma Chemicals (USA).
⇑ Corresponding author. Tel.: +91 (033) 2582 8378, +91 (033) 2582 8750x291, Culture media MEM and serum was purchased from HiMedia, In-
292; fax: +91 (033) 2582 8282. dia. Other molecular biology grade fine chemicals were procured
E-mail address: sommukh445@yahoo.co.in (S. Mukherjee). locally.

0277-5387/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.poly.2013.01.002
S. Mukherjee et al. / Polyhedron 51 (2013) 228–234 229

2.2. Physical measurements and an average flow time was calculated. The rate of flow of the
Tris buffer (pH 7.2), DNA (100 lM) and DNA with the complex,
A Perkin Elmer 2400 C Elemental Analyzer was used to collect 1a at various concentrations were measured. The relative specific
microanalytical data (C, H, N). Sartorius CP64 balance was used viscosity was calculated using the equation g = (t  t0)/t0, where
for weighing purpose. FTIR data were collected with the help of t0 is the flow time for the buffer and t is the observed flow time
FTIR Perkin Elmer L 120-000A. UV–Vis spectra were recorded on for DNA in the absence and presence of the complex 1a. Data are
Shimadzu UV-1700 spectrophotometer and corrected for back- presented as (g/g0)1/3 versus 1/R (where R = [complex]/[DNA]), g
ground due to solvent absorption. Emission spectra were carried is the viscosity of DNA in the presence of the complex and g0 is
out with the help of Perkin Elmer LS 50B Luminescence Spectrom- the viscosity of DNA alone [42,43].
eter. For binding constant measurements solutions were prepared
in methanol at a fixed concentration of HL, 1 (1.0  105 M) and at 2.5. Cell culture
a concentration of metal ions ranging from (1.0–10.0)  106 M at
room temperature. FACS Calibur™ (BD Bio-science, USA) fluores- Human cervical cancer cell line (HeLa) was obtained from Na-
cence-activated cell sorter was used for cell cycle analysis. Axio- tional Centre for Cell Sciences, Pune, India. HeLa cells were grown
skop 2 plus (Carl Zeiss) fluorescence microscope was used to study in MEM supplemented with 10% bovine serum (complete medium)
cell morphology and apoptosis. at 37 °C in humidified atmosphere containing 5% CO2.

2.3. Ligand and complex 2.5.1. Cell viability and morphology


After treatment with different concentrations of the complex 1a
2.3.1. Synthesis of HL, 1 (0–10 lM) for 17 h the HeLa cells were harvested, washed with
The ligand HL, 1, was prepared by the 1:1 condensation of 2- phosphate buffered saline (PBS) twice. Then the cells were incu-
hydrazinopyridine (0.021 g, 0.20 mmol) and 2-hydroxy naphthal- bated with 0.2% trypan blue for 5 min at room temperature and
dehyde (0.036 g, 0.20 mmol) in methanol. On recrystallization counted by hemocytometer under light microscope. Each experi-
from methanol, pale yellow crystals separated out which was dried ment was repeated four times and the mean of % viable cells at
over fused CaCl2. Yield: 0.044 g (82%). C16H13N3O, 1a: C, 73.00; H, each dose was compared with the mean of untreated control using
4.94; N; 15.96. Found: C, 73.04; H, 4.96; N, 15.99%. FTIR (KBr disc): one way ANOVA with a post hoc test viz. Dunnett’s test. The p-val-
m–OH, m–NH (3106, 3009), m–CH@N (1596), 1459, 1379, 1277, 1237, ues were denoted in the figures.
1185, 1141, 1029, 988, 952, 899, 848, 814, 767, 738, 671, 648, Cell morphology study was performed according to our previ-
636, 620 cm1. ous procedure with slight modification [37]. In brief, cells grown
over cover slips for overnight, was treated with different concen-
2.3.2. Synthesis of [Cu(L)H2O]2SO4, 1a trations of 1a (0–10 lM) for 17 h. The cells over cover slips were
To a methanolic solution of copper(II) sulfate pentahydrate washed with phosphate buffered saline (PBS) twice and placed
(0.20 mmol, 0.07 g) was added a solution of HL, 1, (0.20 mmol, over clean microscopic slides. The photographs were taken using
0.04 g) in methanol (5 ml) and the resulting green solution was Axio vision 3 software package (Carl Zeiss).
stirred for 0.5 h. The solvent was evaporated by rotary evaporator
and the solid obtained was recrystallized from acetonitrile. Yield: 2.5.2. Detection of apoptosis by nuclear fragmentation
0.08 g (84%). Anal. Calc. for C32H29Cu2N6O8S, 1a: C, 48.97; H, Detection of nuclear fragmentation was done as reported earlier
3.69; N; 10.71. Found: C, 48.95; H, 3.67; N, 10.69%. Found: C, [44] with some modification. Briefly, HeLa cells were grown over
26.58; H, 2.82; N, 11.27%. FTIR (KBr disc): 3091(m–NH), 1542(m–CH- cover slips (placed inside the culture plates) overnight and then
@N), 1483, 1460, 1388, 1361, 1340, 1290, 1261, 1247, 1193, 1145, treated with different concentrations of 1a (0–10 lM) for 17 h.
1112, 1043, 967, 921, 880, 870, 855, 824, 785, 759, 703, 645 cm1. Now the cover slips were washed twice with PBS buffer and the
cells were fixed using 2 ml of fixative solution of methanol: ace-
2.4. DNA binding experiments tone (1:1) for 1 h at 4 °C. Again the cells were washed twice with
PBS buffer. Finally it was stained with Hoechst dye (1 lM), incu-
The DNA binding experiments were performed at 25.0 ± 0.2 °C. bated for 10 min in dark, and washed with PBS. Cells were exam-
The concentration of CT-DNA was determined from its absorption ined by fluorescence microscope (Carl Zeiss) using the
intensity using the known molar extinction coefficient value of appropriate filter. Apoptotic cells were distinguished by nuclear
6600 M1 cm1 at 260 nm [41]. The UV–Vis titration of 1a was per- fragmentation and chromatin condensation. The apoptotic cells
formed in buffer (50 mM NaCl–50 mM Tris–HCl, pH 7.2) medium as well as the normal cells were randomly counted and percentage
using a fixed complex concentration to which increments of the of apoptotic cells was calculated at each dose. The mean percent-
DNA stock solution (0.0  105–4.0  105 M) was added. The age of apoptotic cells with standard deviation was calculated from
resulting solution was incubated for 10 min before absorption four independent experiments.
spectra were recorded.
During the fluorescence quenching experiment, the DNA was 2.5.3. Cell cycle analysis by flow cytometer
pretreated with ethidium bromide (EB) for 0.5 h and the complex The cells were prepared for FACS analysis as our earlier studies
1a was added to this mixture. On excitation at 490 nm, emission [37,45]. In brief, after 17 h treatment, the cells were trypsinized,
peak was observed between 500 and 700 nm. washed twice with cold phosphate buffered saline (PBS), fixed with
DNA-melting experiments were carried out by monitoring the 70% chilled ethanol in PBS for 5 h at 4 °C, and then stained with a
absorbance of CT-DNA (100 lM) at 260 nm with varying tempera- solution containing 10 lg/ml propidium iodide (PI), 100 lg/ml of
ture in the absence and presence of complex 1a, in a 2:1 ratio of DNase-free RNase A (Sigma), and 0.1% (v/v) Triton X-100 in the
DNA to complex with a ramp rate of 0.5 °C min1 in Tris buffer dark for 30 min at room temperature. Cell cycle analysis was done
(pH 7.2) using a peltier system attached to the UV –Vis by a FACS Calibur (BD Bioscience, USA) [46] at 488 nm excitation.
spectrophotometer. 20,000 cells were taken for each set and fluorescence data
For viscosity measurements, the Ubbelohde viscometer was were plotted using CELLQUEST software (Becton Dickinson). Using
thermostated in water-bath maintained at 25 °C. The flow time this raw data, the percentages of cells in each phase of the cell
for each sample was measured three times using digital stopwatch cycle was obtained using ModFit LT software. Three independent
230 S. Mukherjee et al. / Polyhedron 51 (2013) 228–234

experiments were performed. The p-values were calculated using Table 1


one way ANOVA with a post hoc test viz. Dunnett’s test. Crystallographic data for [CuII(L)H2O]2SO4, 1a.

Compound 1a
2.6. X-ray diffraction studies Empirical formula 2(C16H14CuN3O2)SO41.5(H2O)
Formula mass 810.77
A rod shaped single crystal of 1a (color green, 0.20  0.25  T (K) 293
k (Å) 0.71073
0.40 mm) was mounted on a glass fiber. Intensity data were
Crystal system triclinic
collected at 296 K on a Bruker CCD Diffraction System by the Space group  (No. 2)
P 1Þ
x-scan method over the 2h range, 2.4–25.2° on a Bruker APEXII a (Å) 10.1966(18)
CCD diffractometer using Mo Ka radiation [47] equipped with a b (Å) 11.872(2)
four-circle goniometer and using Mo-Ka graphite monochromated c (Å) 13.858(3)
radiation (k = 0.71073 Å). Structure solution and refinement were a (°) 82.936(2)
b (°) 76.815(2)
done by direct methods using SHELXS-97 and SHELXL-97 programs c (°) 84.639(2)
[48]. The structure was successfully solved in space group P 1.  Of V (Å3) 1617.2(5)
the 4844 independent reflections, 3742 satisfying I > 2r(I) were Z 2
used for structure solution. The water H-atoms were located from Dcalc (g/cm3) 1.655
l (mm1) 1.448
Fourier difference maps and refined with distance restraints. The
F(0 0 0) 822
remainders of the H-atoms were included in calculated positions Crystal dimension (mm) 0.20  0.23  0.50
and treated as riding atoms using SHELXL default parameters. All h Range (°) 1.5, 23.6
non-H atoms were refined anisotropically using weighted full- Limiting indices 11 < h < 11, 13 < k < 13, 15 < l < 15
Reflections measured 13 707
matrix least-squares on F2. A semi-empirical absorption correction
Reflections unique, Rint 4844, 0.0444
(multi-scan) was applied using SADABS. A region of disordered Reflections observed [I > 2r(I)] 3742
electron density was equated to 1.5H2O per molecule of the Refinement method Full-matrix least-squares on F2
complex 1a, using the SQUEEZE routine in PLATON [49]. Total potential Data/parameters 4844/454
solvent accessible void volume = 129.4 Å3; Electron count/cell = Goodness-of-fit (GOF) on F2 1.033
R1a, wR2b [I > 2r(I)] 0.0449, 0.1096
26; equated to 1.5H2O per molecule of 1a. Fig. 1 illustrates the
R1a, wR2b [all data] 0.0606, 0.1158
molecular structure and crystallographic numbering scheme, Residual electron density (e Å3) 0.47, 0.80
drawn using the program PLATON [49]. Details concerning crystal a
R1 = [R||Fo|  |Fc||]/R|Fo| (based on F).
data and refinement are given in Table 1. b
wR2 = [[Rw (|Fo2  Fc2|)2]/[Rw(Fo2)2]]1/2 (based on F2).

3. Results and discussion

The ligand HL, 1, is tridentate in nature with NNO as potential elemental analysis data are consistent with the proposed empirical
donor sites. The 1:1 reaction of HL, 1 with copper(II) sulfate penta- formulae. The room temperature (298 K) magnetic moment of the
hydrate in methanol at room temperature afforded green colored complex 1a is found to be 1.84 lb (S = 1/2, t26e3) which is expected
complex [CuII(L)H2O]2SO4, 1a in excellent yield. The ligand and for d9 system. The complex 1a is non-conducting in acetonitrile.
the complex are soluble in methanol and were characterized by There is no change in absorption and emission pattern of 1a in so-
elemental analyses, FTIR, absorption, emission spectroscopy. The lid and solution phases, indicating the stability of the structure in a

Fig. 1. Perspective view and atom labeling scheme for [CuII(L)H2O]2SO4, 1a. The thermal displacement ellipsoids are drawn at the 50% probability level.
S. Mukherjee et al. / Polyhedron 51 (2013) 228–234 231

solution phase. The single crystal of the complex 1a has been iso- Table 3
lated and its crystal structure was solved successfully. Hydrogen bonding (Å) and angles (°) for [CuII(L)H2O]2SO4, 1a.

Complex, 1a
D–H  A D–H H  A D  A D–H  A
O1W–H1WA  O12 0.89(3) 1.71(3) 2.587(4) 168(5) 1_455
O1W–H1WB  O2 0.86(4) 1.92(4) 2.767(5) 171(5) 2_656
N2–H2 N  O14 0.8600 1.9000 2.729(5) 161.00 2_665
O2W–H2WA. O1 0.86(3) 1.87(3) 2.714(4) 170(6) 2_656
O2W–H2WB  O11 0.90(2) 1.69(3) 2.571(4) 167(4) 2_656
N5–H5 N  O13 0.8600 1.8800 2.716(5) 164.00
C8–H8  O11 0.9300 2.3600 3.292(6) 177.00 2_665
C11–H11  O11 0.9300 2.3700 3.271(5) 163.00 2_665
C16–H16  O12 0.9300 2.5800 3.421(6) 150.00 1_455
C24–H24  O12 0.9300 2.4200 3.308(5) 160.00
C27–H27  O12 0.9300 2.3200 3.238(5) 171.00

3.1. Crystal structure of [CuII(L)H2O]2SO4, 1a

The molecular structure of 1a clearly shows that it is a mono-


meric copper(II) complex with CuN2OOW coordination (Fig. 1).
The ligand binds in a tridentate manner utilizing the pyridyl-N,
imine-N and naphthol-O atoms as potential donor sites. The asym-
metric unit contains two independent complex molecules and a
sulfate anion. Copper(II) atoms in each molecule of 1a have slightly
distorted square planar geometry. The sum of the angles in the Cu-
N2OOw plane is ca. 360° for each molecule. Atom Cu1 is displaced
from the mean plane through atoms O1/N1/N3/O1w [maximum
deviation 0.006(3) Å] by 0.0249(6) Å. Atom Cu2 is displaced from
the mean plane through atoms O2/N4/N6/O2w [maximum devia-
tion 0.090(3) Å] by 0.0227(6) Å. Both the molecules are strongly
hydrogen bonded with the sulfate oxygen present in the crystal
lattice. The bond distances of the coordinating atoms with the cen-
Fig. 2. Absorbance spectra of HL, 1, (1.0  105 M) upon addition of Cu2+ ion
tral metal atoms are Cu1–O1 1.878(3) Å, Cu1–O1W 1.925(3) Å, (1.0  106–1.0  105 M) in methanol (pH 7.0) at 298 K.
Cu1–N1 1.915(3) Å, Cu1–N3 1.968(3) Å, Cu2–O2 1.881(3) Å,
Cu2–N4 1.912(4) Å, Cu2–N6 1.973(4) Å, Cu2–O2W 1.921(3) Å. The
angles at Cu1 between the cis-positioned donor pairs span the
range 82.98(15)–97.37(15)° and those between the trans- the visible region (300–470 nm) (Fig. 2). On excitation at 290 nm,
positioned pairs are 174.29(15)–178.80(14)°. The angles at Cu2 ligand strongly emits at 450 nm due to an intraligand 1(p–p⁄) tran-
between the cis- positioned donor pairs span the range sition. Upon complexation with copper(II) the emission peak is
82.79(15)–96.94(14)° and those between the trans-positioned slightly blue shifted with a quenching in the emission intensity
pairs are 171.36(14)–176.03(15)°. Selected bond distances and as expected for bivalent copper [35,50].
bond angles are listed in Table 2. Details of the hydrogen bonding
are given in Table 3.
3.3. Thermodynamics of binding
3.2. Spectroscopic studies
The association constant (Kass) of the complex 1a can be esti-
mated via UV–Vis titrations according to the Eq. (1) [51,52], where
UV–Vis titration was carried out in methanol at room tempera-
X represents the absorption intensity,
ture. The multiple bands appearing in the UV region (200–380 nm)
are characteristics of the ligand, HL, 1. In the complex 1a the ligand
centered bands are accompanied by multiple bands extending in X ¼ X o þ ðX lim  X o Þ=2C o fC H þ C G þ 1=K ass  ½ðC H þ C G
þ 1=K ass Þ2  4C H C G 1=2 g ð1Þ
Table 2 Xlim represents the absorption intensity at full complexation,
Selected bond lengths (Å) and bond angles (°) for [CuII(L)H2O]2SO4, 1a.
Co is the initial concentration of the ligand, CH and CG are
Complex, 1a the corresponding concentrations of the ligand and metal ion
Cu1–O1 1.879(3) Cu2–O2 1.881(3) during titration. The association constant was found to be
Cu1–N1 1.915(3) Cu2–N4 1.912(4) (5.06 ± 0.004)  104 at 298 K. The temperature dependence of
Cu1–N3 1.968(3) Cu2–N6 1.973(4) binding constants was studied between 293 and 308 K and the
Cu1–O1W 1.925(3) Cu2–O2W 1.921(3) values of the thermodynamic parameters for the binding were
N1–N2 1.391(5) N4–N5 1.384(5)
O1–Cu1–N1 91.58(14) O2–Cu2–N4 91.92(14)
obtained by variable temperature UV–Vis titration in methanol–
O1–Cu1–N3 174.29(15) O2–Cu2–N6 171.36(14) water mixture. The standard Gibb’s free energy change, DG0,
N1–Cu1–N3 82.98(15) N4–Cu2–N6 82.79(15) the standard enthalpy change, DH0 and the standard entropy
O1–Cu1–O1W 88.03(14) O2–Cu2–O2W 88.81(13) change, DS0 were calculated using Van’t Hoff’s Equation and
N1–Cu1–O1W 178.80(14) N4–Cu2–O2W 176.03(15)
found to be 6012.11 ± 0.56 cal M1, 434.55 ± 0.30 cal M1 and
N3–Cu1–O1W 97.37(15) N6–Cu2–O2W 96.94(14)
23.12 ± 0.004 cal M1, respectively.
232 S. Mukherjee et al. / Polyhedron 51 (2013) 228–234

Fig. 3. Variation of UV–Vis absorption spectra of 1a (1.0  105 M) in presence of


CT-DNA (4.0  105 M) in buffer (Tris–HCl–NaCl, pH 7.2) medium at room
temperature. Inset: plot of [DNA]/(ea  ef) vs. [DNA]. Fig. 5. Thermal melting curves of CT-DNA (red) and CT-DNA + 1a (blue). (For
interpretation of the references to color in this figure legend, the reader is referred
to the web version of this article.)
3.4. DNA binding studies

3.4.1. Absorption spectral studies of CT-DNA with 1a 3.4.2. Ethidium bromide induced emission spectral studies
UV–Vis spectroscopy is an effective tool to determine the bind- Competitive ethidium bromide (EB) binding study was carried
ing modes of metal complexes with DNA. The spectral changes out to understand the mode of DNA interaction of 1a. The molecu-
(hypochromism or hyperchromism) reflect the corresponding lar fluorophore EB emits intense fluorescence at 600 nm in the
alteration of DNA in its conformation and structure after the metal presence of CT-DNA due to its strong intercalation between the
complex bound to DNA [53]. We have investigated the DNA bind- adjacent DNA base pairs. Addition of a second molecule, which
ing activity of the ligand HL, 1 and the complex 1a. In the case of 1, binds to DNA more strongly than EB, would quench the DNA-in-
no significant interaction was observed. In copper(II) complex, 1a, duced EB emission [57,58]. The extent of quenching of the fluores-
the decrease in absorption intensity (hypochromism) with increas- cence of EB bound to DNA would reflect the extent of DNA binding
ing concentration of CT-DNA (0.0–4.0  105 M) was observed of the second molecule. Here, upon addition of the complex, 1a, in
(Fig. 3) [54]. Tris HCl–NaCl buffer (pH 7.2) to CT-DNA (4.0  105 M) pretreated
In order to further investigate the intensity of interactions be- with EB the emission intensity decreases almost up to 50% (Fig. 4).
tween 1a and CT-DNA, we have calculated the intrinsic binding The quenching of the EB bound to DNA by the complex 1a is in
constant, Kb, from the plot of [DNA]/(ea  ef) versus [DNA] using agreement with the linear Stern–Volmer equation, Eq. (3) [58],
the following Eq. (2) [55,56] where, I0 and I represent the fluorescence.

½DNA=ðea  ef Þ ¼ ½DNA=ðeb  e  fÞ þ 1=K b ðeb  ef Þ ð2Þ I0 =I ¼ 1 þ K sv r ð3Þ

where [DNA] is the concentration of DNA in base pairs, the molar


absorption coefficients ea represent the apparent absorption coeffi- intensities in the absence and presence of the complex 1a, r is the
cient for the complex and the extinction coefficient for the complex concentration ratio of 1a to DNA. Ksv is the linear Stern Volmer
in the fully bound form and the extinction coefficient for the free quenching constant. The Ksv value is obtained from the slope of I0/
complex, respectively. The Kb is obtained by the ratio of the slope I versus r linear plot and is found to be 2.90, which indicates a
to the intercept and is found to be 2.05  104 M1 for 1a (Fig. 3). strong interaction of 1a with CT-DNA [59].

Fig. 4. Variations of emission spectra of EB with CT- DNA (4.0  105 M) on addition of the complex, 1a. Inset: plot of I0/I vs r.
S. Mukherjee et al. / Polyhedron 51 (2013) 228–234 233

3.5. Cytotoxicity studies

3.5.1. Cell viability and morphology


The percentage of viable HeLa cells was decreased dose-depen-
dently with increase of concentration of 1a. The LD50 value was
found to be 4.97 lM. The decrease of % cell viability at 1 lM was
not statistically significant (p = 1.000) but significant at 3 lM
(p = 0.000) and at the higher doses (3–10 lM). Morphology of HeLa
cells was observed to be changed with increase in concentration of
1a.

3.5.2. Detection of apoptosis by nuclear fragmentation


Fig. 6. Effect of the increasing amount of 1a on the relative viscosity of CT-DNA at
25 °C, [DNA] = 100 lM. Nuclear fragmentation is one of the characteristic features of
apoptotic cell death, a special type of cell death [44,63]. We mon-
itored nuclear fragmentation of HeLa cells after treatment with
3.4.3. Thermal denaturation studies various doses (0–10 lM) of 1a. There was no nuclear fragmenta-
The denaturation of DNA from double-strand to single strand tion observed after treatment with 1a. Therefore, this data revealed
results in absorption hyperchromism at 260 nm. The binding of that although 1a exhibited a cell-killing effect, but they were not
metal complexes to the double-stranded DNA usually stabilizes capable of inducing apoptosis. Our result implied that the complex
the duplex structure to some extent, depending on the strength 1a under investigation triggered some unfavorable environment to
of the interaction with the nucleic acid. The binding should lead the cells so that as the dosage increased the cells became more
to an increase in the melting temperature (DTm) of DNA as com- damaged resulting in more small and deformed shaped nucleus
pared to DNA itself [60]. Herein we have performed the thermal (Fig. 7B) than untreated one (Fig. 7A), but it could not induce
denaturation studies of CT-DNA with the complex 1a. The melting apoptosis.
curves of CT-DNA in the absence and presence of 1a is presented in
Fig. 5. A moderate increase of melting temperature (3 °C) in pres- 3.5.3. Cell cycle analysis
ence of 1a possibly due to a non-intercalative binding behavior as The cell cycle distribution in HeLa cells treated with 1a was ana-
reported earlier [61]. lyzed using ModFit LT software and pictures for a typical experi-
ment. The percentage of cell population in different phases were
obtained using ModFit LT software and the mean of % cell popula-
3.4.4. Viscosity measurements tion with standard deviation at each phase obtained from three
The DNA binding mode of 1a was further investigated by viscos- independent experiments were given in Table 4. The p-values were
ity measurements. Herein the relative specific viscosity of DNA was calculated at each dose with respect to untreated control and
examined by varying the concentration of 1a. It was found that the shown within bracket as shown in Table 4.
viscosity of DNA remain almost unchanged upon addition of 1a There was a dose dependent decrease of G0/G1 and also S-phase
(Fig. 6), suggesting a non-intercalative, and probably groove bind- cell population and dose-dependent increase of G2/M population
ing mode of the copper(II) complex [62]. compared with untreated control. Such decrease and increase in

Fig. 7. Nuclear fragmentation detection under fluorescence microscope – (A) untreated control HeLa cells and (B) represents HeLa cells treated with 10 lM of 1a for 17 h.

Table 4
Cell cycle distribution in HeLa cells by FACS. Cells were treated with different concentrations of 1a (0–10 lM). The percentages of cell cycle distribution were evaluated by ModFit
LT software using PI staining. Values represent the means ± SD of three independent experiments. p-Values were calculated with respect to untreated control for each phase and
shown within the brackets.

Dose (lM) G0/G1 S G2/M


0 74.42 ± 0.42 19.15 ± 0.62 6.43 ± 0.20
1 70.43 ± 0.50 (p = 0.000) 13.90 ± 0.57 (p = 0.010) 15.65 ± 0.36 (p = 0.000)
3 27.48 ± 0.42 (p = 0.000) 34.70 ± 0.92 (p = 0.000) 37.80 ± 0.81 (p = 0.000)
6 29.43 ± 0.13 (p = 0.000) 43.04 ± 3.45 (p = 0.000) 27.53 ± 3.53 (p = 0.000)
10 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
234 S. Mukherjee et al. / Polyhedron 51 (2013) 228–234

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