Anticancer activity and DNA-binding investigations of the Cu(II) and Ni(II) complexes with coumarin derivative

Article abstract of DOI:10.1111/cbdd.12418

Two new copper(II) (2) and nickel(II) (3) complexes with a new coumarin derivative have been synthesized and structurally characterized. The DNA-binding activities of the two complexes have been investigated by spectrometric titrations, ethidium bromide d

Full text of DOI:10.1111/cbdd.12418

Received Date : 26-Apr-2014
Revised Date : 09-Jul-2014
Accepted Date : 13-Aug-2014
Article type
: Research Article
Anticancer activity and DNA-binding investigations of the Cu(II) and
Ni(II) complexes with Coumarin Derivative
Taofeng Zhu1†, Yuan Wang2†, Weiliang Ding3†, Jun Xu2, Ruhua Chen1, Jing Xie4, Wenjiao Zhu3,
Lei Jia2*, Tieliang Ma3*
1. Department of Respiratory, the Affiliated Yixing Hospital of Jiangsu University, Yixing,
Jiangsu Province, China
2. Department of Physics and Chemistry, Henan Polytechnic University, Jiaozuo, Henan
Province, China
3. Central Laboratory, the Affiliated Yixing Hospital of Jiangsu University, Yixing, Jiangsu
Province, China
4. Department of Geriatrics, the Affiliated Yixing Hospital of Jiangsu University, Yixing,
Jiangsu Province, China
†: Contributed equally.
This article has been accepted for publication and undergone full peer review but has
not been through the copyediting, typesetting, pagination and proofreading process
which may lead to differences between this version and the Version of Record. Please
cite this article as an 'Accepted Article', doi: 10.1111/cbdd.12418
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*: Corresponding Author: Tieliang Ma, E-mail: matieliang@foxmail.com. The Affiliated Yixing
Hospital of Jiangsu University, #75 Tongzhenguan Road, Yixing, Jiangsu, 214200, China.
Tel: +86-510-87921196; Fax: +86-510-87921110
Lei Jia, Email: jlxj@hpu.edu.cn. Henan Polytechnic University, Jiaozuo, Henan, 454000, China
. Tel.: +86-391-3987818.
Abstract
Two new copper(II) (2) and nickel(II) (3) complexes with a new coumarin derivative have
been synthesized and structurally characterized. The DNA-binding activities of the two
complexes have been investigated by spectrometric titrations, ethidium bromide
displacement experiments, CD (circular dichroism) spectral analysis and viscosity
measurements. The results indicate that the two complexes, especially the complex 2, can
strongly bind to calf-thymus DNA (CT–DNA). The intrinsic binding constants Kb of the
complexes with CT–DNA are 2.99×105 and 0.61×105 for 2 and 3, respectively. Comparative
cytotoxic activities of the two complexes are also determined by MTT assay. The results
show that the drugs designed here have significant cytotoxic activity against the human
hepatic (HepG2), human promyelocytic leukemia (HL60) and human prostate (PC3) cell lines.
Cell apoptosis was detected by AnnexinV/PI flow cytometry and the results show that the
two copper complexes can induce apoptosis of the three human tumor cells. In conclusions,
the two complexes show considerable cytotoxic activity against the three human cancer and
induce apoptosis of the threes.
Keywords: anticancer activity; coumarin derivative; DNA-binding properties; human tumor
cell lines; cell apoptosis
Introduction
Currently, cancer is the second cause of death, accounting for about a quarter of all deaths
[1].
During past decades, the synthesis and studies of metal complexes with active drugs as
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ligands increase the interest for inorganic, pharmaceutical and medicinal chemistry and
concentrate much attention as an approach to new drug development [2-4]. Transition metals
appear more appealing for this purpose because they can support a multitude of
coordination numbers and geometries that go far beyond sp, sp2 and sp3 hybridization of
[5]
carbon and the metal complexes have been found to be more active and desirable drugs
than the ligands themselves which may be due to the in vivo formation of metallic
complexes [6]
.
Among the ligands employed for drug design, the compounds known as coumarin
(1,2-benzopyrone) serves as an important pharmacophore in drug discovery [7]. As their
ability to inhibit human immunodeficiency virus integrase, coumarin derivatives have also
been evaluated in the treatment of human immunodeficiency virus [8,9]. Further it is evident
from the literature [10-13] that, the transition and rare earth complexes of hydroxycoumarin
derivatives are also subjects of increasing interest in bioinorganic and coordination
chemistry [14].
In this paper, we focused our interests in synthesizing and evaluating the key DNA-binding
interactions of a new coumarin derivative containing a large planar aromatic ring systems
and its novel copper(II) and nickel(II) complexes. In our studies, the interaction of the two
complexes with calf-thymus (CT) DNA was investigated using different kinds of
spectrophotometric methods and viscosity measurements. The studies suggest that
intercalative binding mode appears to be acceptable. The work represents the first
assessment of the potential application of the two coumarin-metal complexes as novel
therapeutic agents for the treatment of cancer.
1. Materials and Methods
1.1 Materials
All starting materials were obtained commercially and used as received. Calf thymus DNA
(CT-DNA) and ethidium bromide (EB) were obtained from Sigma Chemical Co. All the
measurements involving the interactions of the three metal complexes with CT DNA were carried
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out in doubly distilled water buffer containing 5 mM Tris and 50 mM NaCl, and adjusted to pH 7.1
with hydrochloric acid. UV-vis spectrometer was employed to check the solution of CT-DNA purity
(A260: A280 > 1.80) and the concentration (ε = 6600 M-1 cm-1 at 260 nm) in the buffer . The ternary
copper(II) complexes were dissolved in a mixture solvent of 1 % CH3OH or 1 % DMF and 99 %
Tris-HCl buffer (5 mM Tris-HCl, 50 mM NaCl, pH 7.1) at concentration 1×10-3M. The A549
human lung cancer, SGC7901 human Gastric cancer and ECA109 human Esophageal cancer
cell lines were obtained from the Cell Culture Center of the Basic Institute of Medical
Sciences, Peking Union Medical College. Cell culture reagents were purchased from Gibco
(CA, USA). Annexin V- FITC and PI double staining was purchased from BD Biosciences (NJ,
USA). 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide
(DMSO) were purchased from Sigma Aldrich (MO, USA).
1.2 Methods
1.2.1 General Experimental
The UV-vis absorption spectra were recorded using a Varian Cary 100 spectrophotometer
and fluorescence emission spectra were recorded using
a
Hitachi F-4500
spectrofluorophotometer. The elemental analyses were performed in the microanalytical
laboratory, Department of Chemistry, LanZhou University. The 1H NMR spectra were
recorded with a Bruker ACF300 FT-NMR instrument using TMS as an internal reference in
DMSO-d6 (dimethyl sulfoxide) for the ligand. The infrared spectra (KBr pellet) were recorded
using an FTS165 Bio-Rad FTIR spectrophotometer in the range of 4000-400 cm-1.
Conductivity measurements were performed in DMF (N,N-dimethylformamide) with a
DDS-11A conductometer at 25.0 °C. Viscosity experiments were carried out on an
Ubbelodhe viscometer. The CD spectra were recorded on a Jasco J-810 spectropolarimeter.
Absorption titration experiments were performed by fixing concentrations of 2 and 3 as
constant at 10 μM while varying the concentration of ct DNA. While measuring the
absorption spectra, equal quantity of CT-DNA was added to the complexes and the
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reference solution to eliminate the absorbance of DNA itself. Fluorescence spectra of the
competitive binding experiments were carried out by maintaining the EB and ct DNA
concentration at 3 μM and 30 μM, respectively, while increasing the concentrations of the
compounds. Fitting was completed using an Origin 6.0 spreadsheet, where values of the
binding constants Kb were calculated. Viscosity experiments were carried out on an
Ubbelodhe viscometer, immersed in a thermostated water-bath maintained at 25.0±0.1 °C.
Titrations were performed for the compounds (1-6 μM), and each compound was
introduced into DNA solution (50 μM) present in the viscometer. Flow time was measured
with a digital stopwatch and each sample was measured three times and an average flow
time was calculated. Data were presented as (η/ η0)1/3 versus the ratio of the concentration
of the compound and DNA, where η is the viscosity of DNA in the presence of compound,
and η0 is the viscosity of DNA alone. Viscosity values were calculated from the observed flow
time of DNA-containing solution corrected from the flow time of buffer alone (t0), η = t-t0.
The CD spectra of DNA were recorded on a Jasco J-810 spectropolarimeter at 25.0±0.1 ºC.
Calf thymus DNA used were 200 μM in concentration and compounds solutions was added
to a ratio of 1:1 (DNA/compound). Each sample solution was scanned in the range of
200–350 nm. CD spectrum was generated which represented the average of three scans
from which the buffer background had been subtracted.
1.2.2 Cytotoxic activity
To evaluate their cancer chemotherapeutic potential, the ability of the DMSO soluble copper
complexes 2 and 3 to kill human derived cancer cells were investigated using the human
hepatocarcinoma cancer (HepG2), human myeloid leukemia cancer (HL-60), human prostate
(PC3) cell lines and were determined by calculation of IC50.The IC50 was measured by MTT
assay. Briefly, the three kinds of tumor cells (HepG2. HL-60. PC3) were plated at a density of
1×104 cells per well in 96 -well plates overnight and then treated with different
concentrations of copper complexes 2 and 3 after 72h. Twenty microliters of MTT solution
were added to each well and the cells were cultured for another 4 h at 37℃. The medium
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was completely removed and 100 µL DMSO was added to solubilize MTT formazan crystals.
The plates were then agitated and the optical density was determined at 570 nm (A570)
using an ELISA plate reader. At least three independent experiments were performed.
1. 2.3 Quantification of apoptosis by Annexin V and PI double staining
Apoptotic rates were determined by flow cytometry using an Annexin V/PI apoptosis kit.
Briefly, the three kinds of tumor cells were seeded at a density of 1×106 cells per well in
6-well plates overnight and then treated by copper complexes 2 and 3 with each
corresponding IC50 dose for 24 h. Cells (1×106) were collected by centrifugation and washed
twice with cold PBS. Staining was performed according to the manufacturer's instructions
and the cells were analyzed using a FACS can flow cytometer and analyzed using CellQuest
software. At least three independent experiments were performed.
1.2.4 The synthesis of the compounds
Synthesis of the compound 1 and the Ligand H2L shown in scheme 1: The compound 1 was
prepared according to the literature.[15] An ethanol solution (30 ml) containing
salicylaldehyde (1.22 g, 10 mmol) was added dropwise to another ethanol solution (30 ml)
containing the compound 1 (2.54 g, 10 mmol). After refluxing for 12 h, the mixture was
cooled to room temperature, the yellow precipitate solid was collected by filtration and
washed with cool ethanol. Recrystallization from anhydrous methanol gave the ligand H2L,
1
which was dried in vacuum. Yield, 82.7%. H-NMR (DMSO-d6, ppm) d : 12.18 (1H, s, NH),
11.20 (1H, s, OH), 8.78 (1H, s, CH=N), 8.72 (1H, s, ), 6.95-7.70 (10H, ph-H). IR (KBr, cm-1):
ν(OH) 3435, ν (N-N) 2991, ν (C=O) 1632, ν(C=N) 1578, ν (C-N) 1317.
Synthesis of the complexes: The complex 2 was prepared as follows. The ligand H2L (1 mmol,
0.358 g) containing (1 mmol, 0.04g) NaOH was dissolved in 10mL anhydrous ethanol,
CuClO4·6H2O (0.5 mmol, 0.185g) in anhydrous ethanol (10 ml) was then added dropwise with
stirring. After stirring for 8 h at room temperature, a large amount of blue precipitate
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appeared. They were separated from the solution by suction filtration, purified by washing
several times with ethanol, and dried for 24 h in vacuum. Yield: 88.5%. Anal. Calcd for 2
C42H28CuN4O10: C, 62.11; H, 3.47; N, 6.90. Found: C, 61.52; H, 3.69; N, 7.11. IR (KBr, cm-1):
ν(OH) 3430, ν (N-N) 2987, ν (C=O) 1612, ν(C=N) 1536, ν (C-N) 1302. ESI-MS Calc. for
C42H24CuN4O8: 776.21. Found: 775.1 (Figure S1).
The preparation of the complex 3 is similar to that of complex 2, by using NiClO4·6H2O in
place of CuClO4·6H2O. Anal. Calcd for 3 C42H28CuN4O10: C, 62.48; H, 3.50; N, 6.94. Found: C,
62.86; H, 3.83; N, 7.25. IR (KBr, cm-1): ν(OH) 3428, ν (N-N) 2992, ν (C=O) 1617, ν(C=N) 1538,
ν (C-N) 1306. ESI-MS Calc. for C42H24NiN4O8: 771.36. Found: 770.0 (Figure S2).
1. 2.5 Statistical analysis
All data were analyzed by SAS 6.12 software and the results were expressed by mean–SD. To
compare the differences between the groups, statistical significance was analyzed using a
one-way analysis of variance followed by post hoc comparisons. Statistical significance was
defined as p-values < 0.05.
2. Results
2.1 Chemical Characterization
The synthetic route of the ligands is show in scheme 1. The ligand is soluble in methanol and
ethanol, while the complexes are slightly soluble in methanol, insoluble in ethanol. The two
complexes are air stable for extended periods. Since the crystal structure of the complexes
have not been obtained yet, we characterized the complex and determined its possible
structure by elemental analyses, molar conductivities, and IR data. The elemental analyses
show that the formulas of the complexes conform to M(HL)2·2H2O (M= Cu, Ni). The likely
structure of the metal-complex is shown in Figure. 1. The tested metal-complexes were
prepared in DMF and freshly diluted in tris buffer system (at pH 7.4, 7.8). These complexes
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are quite stable, showing ignorable dissociation in the tris buffer, as the UV-visible
absorption spectra have no obvious change for the solutions very freshly prepared and the
stored minutes or days.
2.2 biological activities
To evaluate their cancer chemotherapeutic potential, the ability of the DMSO soluble copper
complexes 2 and 3 to kill human derived cancer cells was investigated using the human
hepatic (HepG2), human promyelocytic leukemia (HL60) and human prostate (PC3) cell lines
and was determined by calculation of IC50 (the drug concentration causing a 50% reduction
in cellular viability). Cells were continuously exposed to test agent for 72 h, and their effects
on cellular viability was evaluated. It was intended that the results from these studies would
allow the identification of those copper and nickel derivatives with cancer chemotherapeutic
potential. Comparison of IC50 values, allowed the relative potency of each of the test
compounds to be determined and ranked. The IC50 values obtained for all tested compunds
are presented in Table 1. The morphology examination also show that the proliferation of
the cells are significant inhibited and the cells exhibit morphological change such as cell
shrinkage and cell detachment (Figure 5).
Apoptotic rates were determined by flow cytometry using an Annexin V/PI apoptosis kit, as
shown in Figure 6. The number of apoptotic cells induced by complexs 2 and 3 were more
than control cell lines（p<0.05, especially by complex 2.
2.3 DNA-Binding
The absorption spectra of 2 and 3 in the absence and presence of ct DNA are given in Figure
2. In the absence of ct DNA. Upon increasing DNA concentrations, the hypochromisms
increased up to 29.3 % at 299 nm and 34.5 % at 423 nm for complex 2; 21.4 % at 299 nm and
23.3 % at 423 nm for complex 3.
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In order to further investigate the interaction mode between the two complexes and ct
DNA, the fluorescence titration experiments were performed. The intrinsic fluorescence
intensities of DNA and that of EB in Tris-HCl buffer are low, while the fluorescence intensity
of EB will be enhanced on addition of DNA owing to its intercalation into the DNA. If the
complexes can intercalate into DNA, the binding sites of DNA available for EB will be
decreased, and hence the fluorescence intensity of EB will be quenched [16]. In our
experiments, as depicted in Figure 3a for complex 2 and Figure 3b for complex 3, the
fluorescence intensity of EB at 584 nm shows a remarkable decreasing trend with the
increasing concentration of the complex 2 or 3.
Circular dichroic spectral technique is useful in diagnosing changes in DNA morphology
during drug-DNA interactions, as the band due to base stacking (275 nm) and that due to
right-handed helicity (248 nm) are quite sensitive to the mode of DNA interactions with
small molecules. The circular dichroic spectrum of CT DNA (Figure 4) exhibits a positive band
at 275 nm due to base stacking and a negative band at 246 nm due to helicity of B DNA.
3. Discussion
3.1 Chemical Characterization
The elemental analyses show that the formulas of the complexes conform to M(HL)2·2H2O
(M= Cu, Ni). The likely structure of the metal-complex is shown in Figure 1. The tested
metal-complexes were prepared in DMF and freshly diluted in tris buffer system (at pH 7.4,
7.8). These complexes are quite stable, showing ignorable dissociation in the tris buffer, as
the UV-visible absorption spectra have no obvious change for the solutions very freshly
prepared and the stored minutes or days. The IR spectra of the two complexes are very
similar. The νC=O of the free ligand are at 1632 cm-1, for complex 2 and 3 this peak shifted to
1612 and 1617 cm-1, ν(ligand–complexes) is equal to 20 and 15 cm-1, respectively. The band at 617
and 599 cm-1 for 2 and 3 is assigned to νM-O. This data strongly indicates that the oxygen of
the carbonyl has formed a coordinative bond with the metal ions. The band at 1578 cm-1 for
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the free ligand is assigned to the νC=N stretch [17], which shifts to 1536 and 1538 cm-1 for the
complexes 2 and 3. These further confirm that the nitrogen of the imino group bonds to the
metal ions.
3.2 DNA-Binding Mode and Affinity
The observed hyperchromism in Figure 2 for complexes 2 and 3 unambiguously revealed the
active participation of coumarine moieties in associating with the DNA[18]. The
[19]
hypochromicity, characteristic of intercalation
has been usually attributed to the
interaction between the electronic states of the compound chromophores and those of the
DNA bases [20], while the red shift has been associated with the decrease in the energy gap
between the highest and the lowest molecular orbitals (HUMO and LUMO) after binding of
the complex to DNA [13]. However, the lack of red shift suggests that the binding mode of
both 2 and 3 was not classical intercalation. The binding constants [21], Kb for the complexes 2
and 3 have been determined from the plot of [DNA]/ (εa - εf) vs. [DNA] and found to be 2.99
×105 M-1 and 0.61 ×105 M-1 respectively. The results indicate that the binding strength of
complex 2 is stronger than that of 3.
The fluorescence titration experiments indicated that some EB molecules are released from
EB-DNA after an exchange with the complex 2 or 3 which result in the fluorescence
quenching of EB. This observation is often the characteristic of intercalation. And he
quenching plots (inset) followed the classical Stern-Volmer equation, F0/F=Kq[Q]+1, where F0
is the emission intensity in the absence of quencher, F is the emission intensity in the
presence of quencher, Kq is the quenching constant, and [Q] is the quencher concentration.
The shape of Stern-Volmer plots can be used to characterize the quenching as being
predominantly dynamic or static. Plots of F0/F versus [Q] appear to be linear and Kq depends
on temperature. The quenching plots illustrate that the quenching of EB bound to DNA by
the compounds is in good agreement with the linear Stern-Volmer equation (Fig. 3(a) and
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3(b), inset). In the plots of F0/F versus [Q], Kq is given by the ratio of the slope to the
intercept. The Kq value for 2 is 1.9 × 104 M-1 while the compound 3 is 0.85 × 104 M-1, which
shows that 2 is more able than 3 in replacing the strong DNA intercalators EB, in consistent
with the higher value of Kb spectrophotometrically determined.
Incubation of the DNA with the present compounds induced changes in the CD spectrum. On
addition of all the complexes to CT-DNA, faint red shift with intensity increase in the positive
or negative bands are observed. When addition of 2 to CT-DNA, it is observed that the
negative-band position was shifted to 246 nm with more evident increase than 2 in molar
ellipticity, while the intensity of the positive band in the CD spectrum of DNA is perturbed
remarkably with no shift. This phenomenon may be due to the intercalation of the complex
through π-stacking which stabilizes the right-handed B form DNA [22,23]
.
3.3 Cytotoxic activity and cell apoptosis
The two metal complexes displayed a concentration-dependent cytotoxic profile in all cell
lines. Since the IC50 values for complexes 2 and 3 are statistically lower than that for
metal-free ligand in all of the tested cells, it suggests that coordinated metal ions play a
major role in mediating potency of the complexes.
The IC50 values for complex 3 on all the cells are statistically different, it displayed a greater
effect against HL60 and PC3 cell lines, which means 3 is sensitive for the HepG2 cell line.
While the complex 2 show good cytotoxic activities for all of the tested cell lines and the
results also suggest that 2 had more significant cytotoxic activities than 3 against the three
human cancer cell lines, which is consistent with the result of DNA binding studies above.
This may be due to the complexes inducing DNA damage in cancer cells and the nature of
the compound itself.
And, in this detection, cells stained single-positive Annexin V- FITC were considered mostly
early apoptotic cells and cells stained single positive for PI were considered mostly necrotic
cells, while cells that were stained double-positive could be either necrotic or apoptotic
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cells. As shown in Fig. 6, The number of apoptotic cells induced by complex 2 was more than
the other tested cell lines, which is consistent with the result of DNA binding studies and
cytotoxic activity.
4. Conclusions
In conclusions, a new coumarin derivative and two traditional metal complexes are prepared
and characterized; the DNA-binding properties of the complexes are investigated by
absorption, fluorescence, circular dichroic spectral and viscosity measurements. The results
clearly indicate that the two complexes can bind to DNA through intercalation mode. The
reported two complexes also show considerable cytotoxic activity against three cell lines
(human hepatocarcinoma cancer HepG2, human myeloid leukemia cancer HL-60, human
prostate PC3), and the IC50 values of all the metal complexes are lower than that of the
ligand, which also show many potential practical applications, such as the development of
nucleic acid molecular probes and new therapeutic reagents for cancers. Further
experiments are necessary to analyze the action of copper complexes in vivo.
Acknowledgements
This work was supported in part by the National Natural Science Foundation of China
(21001040), Fund of Science and Technology of Yixing (2013-21), Fund of Clinical Science and
Technology of Wuxi (MD201202), Fund of the Natural Science Foundation of Jiangsu
(BK20141122), the Doctoral Scientific Fund Project of Henan Polytechnic University
(72515/086, 61307/003), the Foundation of State Key Laboratory of Solid Lubrication
(LSL-1207).
The authors declare that they have no conflict of interest.
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Figure legand:
Scheme 1. The synthetic route of the ligands.
Figure 1. The estimated structure of the metal complexes.
Figure 2. Electronic spectra of the complex 2 (a), complex 3 (b) in Tris-HCl buffer upon
addition of calf-thymus DNA. [Compound] = 10 M, [DNA] = 0-20 M. Arrow shows the
absorbance changes upon increasing DNA concentration. Inset: plots of [DNA]/ (εa-εf) vs.
[DNA] for the titration of complex 2 and 3 with CT-DNA.
Figure 3. The emission spectra of DNA-EB system (15 M and 1.5 M EB), ex =500nm, em =
520-720 nm, in the presence of the complex 2 (a), complex 3 (b). [DNA]=10 M, [Compound]
= 0-35 M. Arrow shows the emission intensity changes upon increasing complex
concentration. Inset: Stern-Volmer plot of the fluorescence titration data of the complexes.
Figure 4. CD spectra of CT-DNA (120 M) in the absence and presence of the complexes 2 and
3 (60 M).
Figure 5. Phase-contrast micrographs of cells treated with complexes 2 and 3. Inhibition rate
under different concentration of complexes 2 and 3.
Figure 6. Annexin V-FITC/PI staining detected apoptosis in three cancer cells after treatment
of the tested compounds
Figure. S1 The mass spectroscopy of the complex 2.
Figure. S2 The mass spectroscopy of the complex 3.
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Table 1. IC50 values of the tested compounds
IC50 (μM)
cell line
H2L
2
3
Cu(ClO4)2⋅6H2O
>300
Ni(ClO4)2⋅6H2O
>300
PC3
>160
>160
>160
43
38
15
>100
69
HepG2
HL60
>300
>300
>300
>300
>100
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This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.