Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 128 (2014) 686–693

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DNA binding, cytotoxicity and apoptosis induction activity of a mixed-ligand copper(II) complex with taurine Schiff base and imidazole Mei Li a,b, Lin Lin kong b, Yi Gou b, Feng Yang b,⇑, Hong Liang a,b,⇑ a

College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan, China State Key Laboratory Cultivation Base for the Chemistry and Molecular Engineering of Medicinal Resources, Ministry of Science and Technology of China, Guangxi Normal University, Guilin, Guangxi, China b

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 A novel mixed-ligand copper(II)

A novel binuclear copper(II) complex (complex 1) with taurine Schiff base and imidazole has been synthesized and characterized. The interaction between 1 and DNA was investigated by UV–vis, fluorescence, circular dichroism (CD) spectra and agarose gel electrophoresis. In addition, 1 showed an antitumor effect on cell cycle and apoptosis.

complex has been synthesized and characterized.  Two copper ions are bridged by sulfonate and exhibit square pyramidal geometries.  1 could bind to CT-DNA via intercalation and show efficient cleavage activity.  1 could arrest cell cycle in the S phase and induce apoptosis of MGC-803 cells.

a r t i c l e

i n f o

Article history: Received 6 November 2013 Received in revised form 26 February 2014 Accepted 27 February 2014 Available online 14 March 2014 Keywords: Taurine Schiff base Imidazole Copper(II) complex CT-DNA Cytotoxicity Apoptosis

a b s t r a c t A novel binuclear copper(II) complex (complex 1) with taurine Schiff base and imidazole has been synthesized and structurally characterized by single crystal X-ray diffraction, elemental analysis, ESI-MS spectrometry, UV–vis and IR spectroscopy. Single-crystal analysis revealed that 1 displays the sulfonate-bridged dinuclear copper(II) centers. Both copper atoms are five-coordinated and exhibit slightly distorted square pyramidal geometries. Each of copper atom is surrounded by three oxygen atoms and one nitrogen atom from different taurine Schiff base ligands, and one nitrogen atom from one imidazole ligand. The interaction between 1 and calf thymus DNA (CT-DNA) was investigated by UV–vis, fluorescence, circular dichroism (CD) spectra and agarose gel electrophoresis. The experimental results indicated that 1 could bind to CT-DNA via an intercalative mode and show efficient cleavage activity. In addition, 1 showed an antitumor effect on cell cycle and apoptosis. Flow cytometric analysis revealed that MGC-803 cells were arrested in the S phase after treatment with 1. Fluorescence microscopic observation indicated that 1 could induce apoptosis of MGC-803 cells. Ó 2014 Elsevier B.V. All rights reserved.

Abbreviations: CT-DNA, calf thymus DNA; EB, ethidium bromide; FBS, fetal bovine serum; MTT, 3-(4,5-dimathylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide; PBS, phosphate buffered saline; PI, propidium iodide; TBE, tris-boracic-EDTA; JC-1, 5,5,6,60 -tetrachloro-1,10 ,3,30 -tetraethyl-imidacarbocyanine iodide. ⇑ Corresponding authors. Address: 15 Yucai Road, Guilin, Guangxi 541004, China. Tel./fax: +86 773 212 0958. E-mail address: [email protected] (H. Liang). http://dx.doi.org/10.1016/j.saa.2014.02.197 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.

M. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 128 (2014) 686–693

Introduction Platinum-based antitumor agents such as cisplatin, carboplatin, and oxaliplatin have achieved great successes. However, theses possess inherent limitations such as resistance, serious toxicity and other side effects [1–7]. These shortcomings make inorganic chemists attempt to replace these drugs with more effective, less toxic, target specific, and preferably noncovalently bound anticancer drugs. Copper complexes have emerged as an attractive alternative to cisplatin as anticancer substances for their essential physiological activity and oxidative nature [8–11]. As is well known, some antitumor drugs exert their activities by binding to, modifying and cleaving DNA [12]. Basically, the three non-covalent modes of the complex-DNA interaction are intercalation, groove binding and external electrostatic effects [13]. During the last few decades, the binding behavior of copper(II) complexes with DNA have been extensively studied [14–18]. Mechanism for copper(II) complexes-mediated cytotoxicity may be caused by their ability of binding to and cleaving DNA which leads to cell cycle arrest and apoptosis or generation of reactive oxygen species (ROS) then in turn to cell death [19,20]. Taurine (Fig. 1), the sulphonic acid analogue of b-alanine, has raised increasing interest to chemists as an ingredient in dietary supplements and functional foods and beverages. A few of taurine Schiff base complexes have been reported to have antiviral, anticancer and antibacterial activities [21–23]. Schiff bases derived from taurine have manifold coordination modes [24]. In a ternary complex, an aromatic-ring stacking interaction is an important characteristic, and it can stabilize the double-helical structure and the interaction between anticancer drugs and DNA [25]. Imidazole group plays an important role in numerous bioactive compounds and the pharmacological interest of the imidazole ring has already been established [26]. The study of mixed ligand complexes is becoming increasingly more important [27]. Herein, we designed and synthesized a new mixed-ligand copper(II) complex (Fig. 1) bridged by two sulfonates ligands. In order to investigate the biological activities of 1, such as DNA binding, DNA cleavage, cell cycle analysis, cell apoptosis, measurement of mitochondrial transmembrane potential, several indexes were employed. Most importantly, our current results showed the potential mechanisms of action of 1 conducting on cell proliferation and apoptosis of MGC-803 cells.

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(PI), calf thymus DNA (CT-DNA), AO/EB (Acridine orange/Ethidium bromide), RNase A and plasmid pBR322 DNA were purchased from Sigma Chemicals Co. (USA). The Tris–HCl buffer solution was prepared with triple-distilled water. Fetal bovine serum (FBS) and RPMI 1640 were obtained from Hyclone (USA). A Tris-buffer solution of CT-DNA gave a ratio of 1.8–1.9 of UV absorbance at 260 and 280 nm, indicating that the DNA was sufficiently free of protein [28]. The DNA concentration per nucleotide in base pairs was determined spectrophotometrically by employing a molar absorptivity (6600 M1 cm1) at 260 nm [29]. CT-DNA stock solution was prepared by diluting DNA to Tris–HCl/NaCl buffer (pH = 7.2, 5 mM Tris–HCl, 50 mM NaCl). Elemental analyses (C, H, N, S) were carried out on a Perkin Elmer Series II CHNS/O 2400 elemental analyzer. ESI-MS (electrospray ionization mass spectrum) spectra were recorded on a Bruker HCT Electrospray Ionization Mass Spectrometer. UV–vis absorption spectra were performed on a Varian Cary100 UV–vis spectrophotometer. Infrared spectra were obtained on a PerkinElmer FT-IR Spectrometer. Fluorescence measurements were obtained by using a Shimadzu RF-5301/PC spectrofluorophotometer. The circular dichroic spectra of CT-DNA were performed on a JASCO J-810 automatic recording spectropolarimeter operating at 25 °C. The fluorescence microscope (Nikon MF30 LED, Japan). Flow cytometry using a BectoneDickinson FACSCalibur. Synthesis of 1 Salicylaldehyde (122.12 mg, 1 mmol) was slowly added to a solution containing KOH (56.10 mg, 1 mmol) and taurine (125.15 mg, 1 mmol) in MeOH (30 mL), and the mixture was stirred for 4 h at 50 °C. Then a solution of Cu(OAc)2H2O (199.65 mg, 1 mmol) in H2O (8 mL) was added, still stirred for 2 h, and then imidazole (68.07 mg, 1 mmol) was added and refluxed for 4 h, resulting in a dark green solution. It was then filtered to discard any insoluble precipitates. The clear filtrate was left to stand at room temperature for slow evaporation, and the dark green crystals of 1 suitable for X-ray analysis were collected after several days. Yield: 419 mg, 58.4%. Selected IR (KBr cm1): 3151 (ArAH),  1628 (C@N), 1541, 1471, 1451, 1251 (SO 3 ), 1151 (SO3 ), 1033, 1 1 758; UV–vis in DMSO, k nm (e M cm ): 357 (7624), 267 (26,083); Elemental analysis (%): calc. for C24H26Cu2N6O8S2: C, 40.13; H, 3.62; N, 11.70; S, 8.92. Found: C, 40.47; H, 3.58; N, 11.60; S, 8.31. ESI-MS (in DMSO): m/z 716.9, 650.9, 588.0, 640.9, 642.9, 652.9.

Experimental X-ray crystallography Reagents and instrumentation All chemicals and reagents were purchased from commercial sources and were all used as received without further purification unless noted specifically. Taurine, imidazole and salicylaldehyde were purchased from Alfa Aesar Chemicals Co. (USA). Hoechst 33258, JC-1, Ethidium bromide (EB), 3-(4,5-dimathylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT), Propidium iodide

Crystal data were collected at 25 °C on a Bruker Smart Apex II CCD diffractometer equipped with graphite monochromated Mo Ka radiation (k = 0.71073 Å). The structure was solved by direct methods and refined with SHELX-97 programs [30]. The non-hydrogen atoms were located in successive difference Fourier synthesis. The final refinement was carried out by full-matrix least-squares methods with anisotropic thermal parameters for non-hydrogen atoms on F2 [30]. The hydrogen atoms were added theoretically. DNA-binding and cleavage experiments

Fig. 1. Chemical structures of taurine, taurine Schiff base and 1.

The 2.1 mM CT-DNA stock solution was stored at 4 °C for no more than 4 days before use. 1 was prepared as 2 mM DMSO stock solution and was diluted by Tris–HCl/NaCl buffer for DNA binding studies. To investigate the binding affinity between CT-DNA and 1, absorption spectra titrations were carried out in Tris–HCl/NaCl buffer at room temperature by maintaining 1 concentration as

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constant at 20 lM while adding CT-DNA solution (2.1 mM) to 1 cm path cuvette. After the mixture was incubated for 5 min, the absorption spectra were performed. EB displacement experiments were determined with an EBbound CT-DNA solution. Thus, to a solution of CT-DNA (20 lM) and EB (20 lM) in Tris–HCl/NaCl buffer (pH = 7.2, 5 mM Tris–HCl, 50 mM NaCl), aliquots of a certain amount of a solution of 1 were added. The corresponding fluorescence spectra were recorded with excitation at 350 nm and emission at 602 nm. The CD spectra of CT-DNA in the absence and presence of 1 were carried out in Tris–HCl buffer (pH = 7.2) containing 50 mM NaCl. The DNA cleavage experiments were done by agarose gel electrophoresis according to the literature method [31,32]. Briefly, supercoiled (SC) pBR322 DNA (0.1 lg lL1) in 5 mM Tris–HCl/ 50 mM NaCl buffer at pH 7.2 was incubated with 1 in absence of additives at 37 °C for 1 h, and then loading buffer was added. Then the samples were analyzed by electrophoresis for 2.5 h at 80 V on 0.8% agarose gel in TBE buffer. The pBR322 DNA was stained with 0.5 mg ml1 ethidium bromide. Bands were visualized by UV light and images were captured. Cell culture The cell lines human gastric cancer (MGC-803), human liver (HL-7702), human hepatoma (BEL-7404), human lung adenocarcinoma (A549) and human ovarian cancer (SK-OV-3) were obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were grown in a humidified atmosphere containing 5% CO2 at 37 °C in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 lg mL1 streptomycin and 100 units mL1 penicillin. Cytotoxicity evaluation Cell viability was determined by using MTT assay, which is based on the ability of the viable cells to reduce a soluble yellow tetrazolium salt to blue formazan crystals [33]. Cells were seeded in 96 well plates at a density of 1  104 cells per well and cultured for 24 h. Various concentrations of 1 between 0 and 80 lM were added to the wells and the plates were incubated in a 5% CO2 humidified atmosphere for 48 h. Then 10 lL of 5 mg mL1 MTT in phosphate buffered saline (PBS, pH 7.40) was added to each well, and cells were incubated at 37 °C for another 4 h. The formazan crystals formed were dissolved by the addition of 100 lL of DMSO and the absorbance was recorded at 570 nm using an Enzymelinked Immunosorbent Assay (ELISA) reader. The concentration required for a 50% inhibition of viability (IC50) was determined. The effect of 1 on the proliferation of all tested cells was expressed as the% cell viability, using the following formula

%cell viability ¼ A570nm of treated cells=A570nm of control cells  100%:

Apoptosis evaluation Hoechst 33258 and AO/EB double staining The nuclear morphology of cells was used to detect early apoptosis, late apoptosis and necrosis. MGC-803 cells were plated at a density of 5  105 cells in 6-well plate, 24 h later, cells were added with 1 at 0, 5, 10, 20 lM and cultured for another 24 h. Then cells were washed with PBS, and were stained with Hoechst 33258 for 5 min at room temperature. After a final wash in PBS, samples were visualized with the aid of EPI fluorescence microscopy.

AO/EB double staining was used to detect the apoptosis of complexes against cells as described previously [34]. Briefly, 5  105 cells per well of MGC-803 cells in a 6-well plate were incubated with various concentrations (0, 5, 10, 20 lM) of 1 at 37 °C for 24 h. At the end of the culture period, cells were washed with PBS, and were stained with AO/EB solution (100 mg mL1 AO, 5 lL; 100 mg mL1 EB, 5 lL) for 10 min at 37 °C. After a final wash with PBS, samples were observed under an inverted fluorescence microscope. Measurement of apoptosis by Annexin V-FITC/PI analysis An Annexin V-FITC/PI assay was used to evaluate cellular apoptosis. Briefly, cells were harvested after the target 1 treatment with different concentrations (0, 5, 10 and 20 lM) for 24 h, and washed with PBS, then resuspended in 100 lL 1  binding buffer. Then 5 lL of annexin V-FITC and 5 lL of propidium iodide (PI) were added to the Cells (100 lL) and incubated for 1 h at room temperature in the dark. After 400 lL 1  binding buffer was added into each tube, the stained cells were analyzed by flow cytometry. The rate of cell apoptosis was analyzed. Cell cycle analysis Flow cytometry was used to evaluate DNA contents and cell cycle analysis [35]. Briefly, 5  105 MGC-803 cells were incubated with 1 at 0, 50, 10 and 20 lM at 37 °C for 48 h. Cells were then harvested and washed with cold PBS, centrifuged, resuspended in 1 mL of PBS and fixed by the dropwise addition of 9 mL precooled 75% EtOH overnight at 20 °C. For staining, cells were further washed with cold PBS, digested by 500 lL RNase A (100 lg/mL) at 37 °C for 30 min, and stained with 25 lL PI (50 mg/mL) in the dark at room temperate for 30 min. The distribution of the cell cycle was analyzed by using a BectoneDickinson FACSCalibur. Measurement of mitochondrial transmembrane potential (Dwm) The cells were stained with JC-1 to determine the mitochondrial membrane potential. First, MGC-803 cells (5  105) were seeded in 6-well plates and treated with four concentrations of 1 (0, 5, 10, and 20 lM) for 24 h. Then the medium was removed and the cells were washed with PBS twice, stained with JC-1. After 20 min of incubation at 37 °C in a 5% CO2 humidified atmosphere, the cells were washed with PBS twice and observed with an inverted fluorescence microscope. Statistical analysis All data were expressed as means ± SD (standard deviation). Statistical significance (P < 0.05) was performed by one via analysis of ANOVA followed by an assessment of differences using SPSS 16.0 software. Results and discussion Spectral analysis In the IR spectrum of 1 (Fig. S1), the stretching frequency at 1628 cm1 is due to (C@N) [36]. The band at 3151 cm1 is assigned to the aromatic (ArAH) stretching. The frequencies characteristics of the (SAO) stretching modes in 1 are observed at 1251 and 1151 cm1 [37]. In the UV–vis electronic spectrum (Fig. S2), the intense band at about 267 nm is attributed to the p–p* electron transition of the aromatic structure of taurine Schiff-base, while the less intense band at about 357 nm is typical of charge transfer between ligand and metal (ligand-to-metal charge transfer) [38]. The ESI-MS spectrum of 1 in DMSO solution (Fig. S3) shows the molecular ion peaks at m/z 716.9 which is assignable to [M-H]+. The loss

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of imidazole ion forms a base peak at m/z 650.9. Few other intense peaks are also obtained for 1 at m/z 588.0, 640.9, 642.9, and 652.9. Description of the structure The imidazole related drugs showed high therapeutic properties (such as anticancer, antimicrobial, antioxidant, antiviral, antitubercular activities) [39]. Imidazole also could serve as a bridging ligand or a terminal ligand [40]. So, to prepare a mixedligand complex, imidazole was added. After the former complex has formed by the taurine Schiff base which derived from salicylaldehyde and taurine, the imidazole was added. In the reported complexes which prepared by the same or similar taurine Schiff base [21,41], the Cu atom was surrounded by three oxygen atoms from different taurine Schiff base ligands, one nitrogen atom from taurine Schiff base ligand, and one oxygen atom from a terminal water. So in this work, after the imidazole was added, the terminal water might be replaced by one imidazole. 1 crystallizes in the orthorhombic system, space group Pca21. The details of crystallographic data and structure refinement parameters are summarized in Table 1. Selected bond angles and distances are listed in Table 2. As shown in Fig. S4 there are two taurine Schiff base ligands, two Cu atoms and two imidazole ligands in the unit. 1 displays the sulfonate-bridged dinuclear copper(II) centers with the Cu1  Cu2 distance of 5.510 Å. Both Cu atoms (Cu1 and Cu2) are five-coordinated and exhibit slightly distorted square pyramidal geometries. Interestingly, the s values [42] are 0.17 and 0.19 for Cu1 and Cu2, respectively. Cu1 atom is surrounded by three oxygen atoms from different taurine Schiff base ligands, one nitrogen atom from taurine Schiff base ligand (Cu1AN2 2.019(15) Å), and one imidazole ligand (Cu1AN5 2.041(15) Å). Cu2 atom is defined by three oxygen atoms from different taurine Schiff base ligands, one nitrogen atom from taurine Schiff base ligand (Cu2AN3 1.969(15) Å), and one imidazole ligand (Cu2AN1 1.950(15) Å). Such bond lengths are practically identical with those for the sulfonate-bridged dimeric copper(II) complexes [41]. Interestingly, the discrete dimeric molecule of 1 is further linked by four intramolecular N–HO hydrogen bonds [O1  N4i 2.994 Å, N4  O1ii 2.994 Å, O5  N6iii 2.957 Å and N6  O5iv 2.957 Å, (symmetrycodes: (i) 0.5 + x, 1  y, z; (ii) x  0.5, 1  y, z; (iii) x  0.5, y, z and (iv) 0.5 + x, y, z)] between a imidazole group and an adjacent phenoxy oxygen atom as depicted in Fig. S5a. Table 1 Crysallographic data and structure refinement parameters for 1.

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Table 2 Selected bond lengths (Å) and angles (°) for 1. Cu1AO4 Cu2AO1 S2AO2 O5ACu1AO4 O6ACu1AO4 N2ACu1AO4 N5ACu1AO4 O1ACu2AO2 O2ACu2AO7 N1ACu2AO2 N3ACu2AO2 O2AS2AO3

2.347(13) 1.936(13) 1.479(14) 88.9(5) 89.6(6) 94.5(7) 101.8(6) 173.8(5) 89.7(6) 93.1(7) 85.7(6) 108.5(8)

Cu1AN2 Cu2AN1 S2AO4 O3AS2AC9 O4AS2AO2 O6AS3AC18 O7AS3AC18 C1AO1ACu2 S2AO2ACu2 S3AO6ACu1 S3AO7ACu2 C7AN1ACu2

2.020(15) 1.950(15) 1.440(13) 105.3(8) 112.8(8) 102.0(8) 106.1(9) 128.1(9) 133.6(8) 133.3(8) 150.3(8) 128.4(15)

These intermolecular hydrogen-bonding interactions further connect the discrete copper(II) dimers to give a two-dimensional (2D) structure. Interestingly, using the dimmers as nodes, 1 exhibits a 4-connected sql topological net with the Schlafli symbol of (44  62) (Fig. S5b). DNA binding and cleavage activities UV absorption spectroscopy studies It is generally accepted that DNA is the primary pharmacological target of many metal based antitumor agents. The binding activities of DNA–metal complexes have been a clue of paramount importance for understanding the mechanism of effective metalbased chemotherapeutic drugs [43]. Herein, the interaction between 1 and CT-DNA was characterized by UV–vis absorption spectroscopy. The absorption spectra of 1 in the absence and presence of CT-DNA at different concentrations are given in Fig. 2. The maximal peak at 267 nm and the secondary peak at 357 nm are ascribed to intraligand p–p* charge transfer. With the concentration of CT-DNA being increased, hypochromism of 31.8% and a blueshift of 8 nm are observed at maximal peak, while hypochromism of 52.9% and a blue-shift of 3 nm are observed at secondary peak. These results suggest that hypochromism is probably owning to stacking interaction between an aromatic ring and the base pairs of DNA [44]. The extent of the hypochromism reflected the strength of intercalative interaction [45]. The intrinsic binding constant Kb was determined from a plot of [DNA]/(ea  ef) vs. [DNA] using the equation: [DNA]/(ea  ef) = [DNA]/(eb  ef) + 1/Kb(eb  ef), where [DNA] is the concentration of DNA in base pairs. The apparent absorption coefficients ea, ef and eb correspond to Aobsd./[complex], the extinction coefficient for the free compound and

1 CCDC number Empirical formula Formula weight Temperature/K Crystal system Space group a/Å, b/Å, c/Å a/°, b/°, c/° Volume/Å3 Z qcalc/mg mm3 l/mm1 F(000) Crystal size/mm3 2H range for data collection Index ranges Reflections collected Independent reflections Data/restraints/parameters Goodness-of-fit on F2 Final R indexes (I > 2r (I)) Final R indexes (all data) Largest diff. peak/hole/e Å3

955430 C24H26Cu2N6O8S2 717.71 296.15 Orthorhombic Pca21 10.082(10), 9.777(10), 28.07(3) 90.00, 90.00, 90.00 2767(5) 4 1.723 1.748 1464 0.30  0.15  0.08 2.9 to 50.22° 9 6 h 6 11, 11 6 k 6 11, 33 6 l 6 33 9311 3299[R(int) = 0.0550] 3299/1147/355 1.087 R1 = 0.0990, wR2 = 0.2532 R1 = 0.1240, wR2 = 0.2723 2.562/0.917

Fig. 2. Absorption spectra of 1 ([complex] = 2.0  105 M) in the absence (dashed line) and presence (solid line) of increasing amounts of CT-DNA in the range from 1:1 to 18:1.

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extinction coefficient for the compound in the fully bound form, respectively [46]. Kb is given by the ratio of slope to the intercept. From the absorption data, the binding constant Kb for 1 is 4.23  105 M1. The Kb value is lower than for a classical intercalator (e.g. EB-DNA, 106 M1) [47] and is close to those of DNA-intercalative dicopper(II) complexes [48–50], but higher than those of mononuclear copper(II) complexes [51–53]. The results suggest that the interaction of the 1 with DNA is a strong intercalative mode. Fluorescence spectroscopy studies Fluorescence titration were performed to investigate interaction mode of 1 with DNA. EB is one of the most sensitive fluorescence probes, its fluorescence intensity can be greatly enhanced in the presence of DNA due to its strong intercalation between the adjacent DNA base pairs [54,55]. A complex’s competitive binding to EB-bound DNA can reduce the fluorescent intensity due to displacement of bound EB from DNA [56,57]. The interaction of 1 with CT-DNA was carried out. As shown in Fig. 3, the fluorescence emission intensities at 590 nm (350 nm excitation) decreased with the increasing complex concentrations, which suggested that 1 could displace DNA-bound EB and bind to CT-DNA at the intercalation sites with almost the same affinity [58]. The relative binding intensity of 1 to CT-DNA was determined by the classical Stern– Volmer equation: I0/I = 1 + K[Q], where I0 and I are the fluorescence intensities in the absence and presence of complex, respectively, K is a linear Stern–Volmer constant, and [Q] is the concentration of complex to that of DNA. K was calculated from the slope of plot I0/I vs. [Q] [59]. The apparent binding constant K of 1 at 25 °C was calculated to be 1.08  104 M1. It may be due to 1 interacting with DNA through intercalation binding, so releasing some free EB from the EB-DNA complex, which is consistent with the above absorption spectral result. Circular dichroism absorption spectral analysis CD spectroscopic technique is useful and sensitive in detecting the conformational conformation altered by the complex in solution. In the CD spectrum of CT-DNA, a positive band at 276 nm due to base stacking and a negative band at 246 nm due to the right-handed helicity can be observed, which indicates B form of DNA in solution system [60]. So only small molecules bind to CTDNA by intercalation or covalent binding, that may result in the DNA conformational modifications or induce significant CD spectral perturbation of CT-DNA. The CD spectra of DNA modified by 1 are shown in Fig. 4. With addition of 1, the intensities of the negative and positive bands decrease by a slight redshift. These

Fig. 3. Fluorescence quenching curves of EB bound to CT-DNA by 1 ([complex] = 0  16.0  105 M). The arrow shows the intensity changes on increasing the complex concentration, Inset: plot of I0/I vs. [complex].

Fig. 4. CD spectra of CT-DNA (3 mL solution, 2.0  104 M) in the absence (dash line) and presence of 1 (solid line), with [complex]/[CT-DNA] = 0.25 in Tris–HCl buffer, pH = 7.2.

alterations indicate that the intercalative effect of 1 in base stacking of the DNA and the complex having effect on the helicity structure of the DNA [61]. DNA cleavage activities When circular plasmid DNA is subjected to electrophoresis, the information of relatively fast migration will be obtained for the covalently closed circular form (Form I). If one strand is nicked, the supercoils will relax to generate a slower-moving open circular form (Form II). If both strands are nicked, a linear form (Form III) that migrates between Form I and Form II will be generated [31,32]. Fig. S6 shows the gel electrophoresis separation of pBR 322 DNA after incubation with 1. No DNA cleavage was observed for control in the absence of 1. With increasing concentrations of 1 at 25, 50, 100, 200, and 250 lM, the Form I of pBR322 DNA was gradually converted into Form II, and at high concentration groups (lane 3 to lane 5) linear form (Form III) could be clearly observed. This result indicates that 1 can efficiently cleave plasmid DNA. Cytotoxicity evaluation The cytotoxicity of 1 in vitro against tumor cells was evaluated by MTT assay with five cell lines: MGC-803, BEL-7404, HL-7702,

Fig. 5. Effects of 1 on the survival of the five tested cells. j (MGC-803); d (SK-OV3); N (BEL-7404); . (A549);  (HL-7702).

M. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 128 (2014) 686–693 Table 3 The cytotoxic activity of 1. Compound

1 Cisplatin

IC50 values (lM) MGC-803

SK-OV-3

BEL-7404

A549

HL-7702

13.2 ± 1.2 26.4 ± 1.1

>50 16.1 ± 1.5

34.1 ± 1.7 96 ± 0.8

>50 16.9 ± 1.8

115.9 ± 1.3 86.3 ± 2.1

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SK-OV-3, A549. Cisplatin was used as the positive control. After cells were treated with different concentrations of 1 for 48 h, the viable cells were measured by MTT assay on an enzyme labeling instrument. Fig. 5 represents the comparative study of of 1 with the five different cell lines. 1 inhibited the growth of all tested cells in a dose-dependent manner in the range 0–80 lM, which is to say that, the cell viability decreased with increasing concentrations of 1. At the higher concentration of 80 lM, the cell viability for the five cell lines was 9.22% (MGC-803), 49.99% (SK-OV-3), 19.41% (BEL-7404), 43.02% (A549), and 55.57% (HL-7702), respectively. Further, as revealed by the observed IC50 values (Table 3), the IC50 values of tested SK-OV-3 and A549 cells are even at more than the concentration of 50 lM. Among the tested cells, 1 showed the highest cytotoxicities against MGC-803 cells (IC50 13.2 lM ± 1.2), and exhibits potency approximately 2 times more than cisplatin does for 48 h incubation, so further investigations were carried out only with MGC-803 cells. 1 towards the normal human liver HL-7702 cells displayed lower cytotoxicities (IC50 115.9 lM ± 1.3) than that of them to the tested tumor cell lines, and also lower than that of cisplatin. Apoptosis evaluation

Fig. 6. Annexin V/propidium iodide assay of MGC-803 cells treated with 1 measured by flow cytometry at concentrations of 1 (5, 10, and 20 lM) for 24 h.

Hoechst 33258 and AO/EB double staining Characteristic of apoptosis can be identified by visualizing nuclear changes and apoptotic body formation, MGC-803 cells were treated with 1 at different concentration for 24 h, stained with Hoechst 33258 [62] and AO/EB respectively. As shown in Fig. S7A, Hoechst 33258 staining showed the blue apoptotic cells with apoptotic features that exhibit nuclear pyknosis, showing round shapes and an enhanced fluorescent signal. The results showed that 1 can induce apoptosis of MGC-803 cells. AO/EB

Fig. 7. Effects of 1 on the MGC-803 cell cycle progression. MGC-803 cells were treated with PBS at various concentrations of 1 (5, 10, and 20 lM) for 48 h.

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double staining (Fig. S7B) also demonstrated that treatment with different of 1 can induced apoptosis of MGC-803 cells characterized by the distinctive red orange fluorescence changed to the green fluorescence gradually.

our present study suggest that 1 may be a potent antitumor drug against MGC-803 cells.

Measurement of apoptosis by Annexin V-FITC/PI analysis Annexin V- FITC/PI staining was also used to detect Apoptotic cells. The staining single-positive for Annexin V- FITC mostly reflected early apoptotic cells and the staining single positive for PI mostly reflected necrotic cells, so cells that were stained doublepositive could be either necrotic or apoptotic cells [63]. As shown in Fig. 6, Annexin V-FITC/PI staining data indicated that percentage of apoptosis was 2.5%, 6.6%, 12.3%, and 21.3%, respectively, for 24 h. The results showed that 1 could effectively induce apoptosis in MGC-803 cells in a dose-dependent manner.

This work was supported by the Natural Science Foundation of China (31060121 and 21171043) and Natural Science Foundation of Guangxi (2012GXNSFCB053001 and 2013GXNSFGA019010).

Cell cycle analysis The effect of 1 on cell cycle distribution of MGC-803 cells carried out by flow-cytometric analysis further confirmed tumor cell apoptosis. The cell cycle is the series of events that take place in a cell leading to its division and duplication (replication). The cell cycle consists of four distinct phases: G1 phase, S phase (synthesis), G2 phase (collectively known as interphase) and M phase (mitosis). The G1 stage is the stage when accumulations of energy and material for DNA replication occur. The S stage is the stage of DNA replication. The G2 stage is the stage of preparation for the M stage. The M stage is ‘‘mitosis’’, and is when nuclear and cytoplasmic division occurs [64]. As shown in Fig. 7, cytometrie profiles of the PIstained DNA show an accumulation of cells in the S phase of the MGC-803 cell cycle treated with 1 for 48 h at 0, 5, 10, 20 lM. With the increase concentrations of 1, S phase populations increased from 48.08% to 63.19%, while G1 and G2 phase decreased profoundly, which indicated that 1 could delay or inhibit cell cycle progression through S phase and the percentage of apoptotic cells presented a dose-dependent increase from 5 to 20 lM. Measurement of mitochondrial transmembrane potential (Dwm) The characteristic of a molecule event for early apoptosis could be reflected by reduction of mitochondrial membrane potential. Mitochondria form red fluorescent aggregates at high membrane potential, whereas exits mainly in cytosol forming green fluorescent at membrane potential decreased, presenting a collapse of membrane. The reduction of mitochondrial membrane potential was detected by fluorescence microscopy analysis of JC-1 stained MGC-803 cells. As shown in Fig. S8, after treatment with increasing concentrations of 1, a change from red fluorescent to green fluorescent gradually occured, indicating that 1 reduce mitochondrial membrane potential disruption of MGC-803 cells in a dose-dependent manner [65]. From these results, we can speculate that the intrinsic mitochondrial pathway may be involved in apoptosis induced by 1. Conclusions In this paper, 1 with nucleic acids and its biochemical effect on human cancer cells were studied. The crystal structure of the 1 exhibits bicopper(II) located in a 4 + 1 pyramidal coordination environment with the mixed ligand having a N, N, O, O, O chelating motif. 1 binds to CT-DNA via an intercalative mode and can efficiently cleave plasmid DNA. Cytotoxicity research unequivocally demonstrated that 1 has stronger cytotoxic properties against human tumor cells in vitro. 1 could induce S-phase cell cycle arrest and apoptosis of the MGC-803 cells. The intrinsic mitochondrial pathway may be involved in apoptosis induced by 1. The data from

Acknowledgments

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