Ternary copper(II) complexes with amino acid chains and heterocyclic bases: DNA binding, cytotoxic and cell apoptosis induction properties Tieliang Ma, Jun Xu, Yuan Wang, Hao Yu, Yong Yang, Yang Liu, Weiliang Ding, Wenjiao Zhu, Ruhua Chen, Zhijun Ge, Yongfei Tan, Lei Jia, Taofeng Zhu PII: DOI: Reference:

S0162-0134(14)00304-3 doi: 10.1016/j.jinorgbio.2014.12.011 JIB 9634

To appear in:

Journal of Inorganic Biochemistry

Received date: Revised date: Accepted date:

14 August 2014 9 December 2014 9 December 2014

Please cite this article as: Tieliang Ma, Jun Xu, Yuan Wang, Hao Yu, Yong Yang, Yang Liu, Weiliang Ding, Wenjiao Zhu, Ruhua Chen, Zhijun Ge, Yongfei Tan, Lei Jia, Taofeng Zhu, Ternary copper(II) complexes with amino acid chains and heterocyclic bases: DNA binding, cytotoxic and cell apoptosis induction properties, Journal of Inorganic Biochemistry (2014), doi: 10.1016/j.jinorgbio.2014.12.011

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ACCEPTED MANUSCRIPT Ternary copper(II) complexes with amino acid chains and heterocyclic bases: DNA binding, cytotoxic and cell apoptosis

b†

b†*

c

, Hao Yu , Yong Yangd, Yang Liue, Weiliang Dinga,

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a†

Tieliang Ma , Jun Xu , Yuan Wang

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induction properties

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Wenjiao Zhua, Ruhua Chenf, Zhijun Geg, Yongfei Tanh, Lei Jiab*, Taofeng Zhue*

a. Central Laboratory, the Affiliated Yixing Hospital of Jiangsu University, Yixing, Jiangsu Province, China

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b. Department of Physics and Chemistry, Henan Polytechnic University, Jiaozuo, Henan Province, China

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c. Department of Breast Surgery, the Affiliated People’s Hospital of Jiangsu University, Zhenjiang, Jiangsu Province, China

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d. Department of Brain Surgery, the Affiliated People’s Hospital of Jiangsu

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University, Zhenjiang, Jiangsu Province, China e. Department of Respiratory, the Affiliated People’s Hospital of Jiangsu University,

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Zhenjiang, Jiangsu Province, China f. Department of Respiratory, the Affiliated Yixing Hospital of Jiangsu University, Yixing, Jiangsu Province, China

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g. Department of Critical Care Medicine, the Affiliated Yixing Hospital of Jiangsu University, Yixing City, Jiangsu Province, China h. Department of Cardiac & Thoracic Surgery, the Affiliated Yixing Hospital of Jiangsu University, Yixing City, Jiangsu Province, China †: Contributed equally. *: Corresponding Author: Taofeng Zhu, E-mail: [email protected]. 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: [email protected]. Henan Polytechnic University, Jiaozuo, Henan, 454000, China. Tel.: +86-391-3987811; Fax: +86-391-3987811 Yuan Wang, Email: [email protected]. Henan Polytechnic University, Jiaozuo, Henan, 454000, China. Tel.: +86-391-3987811; Fax: +86-391-3987811

1

ACCEPTED MANUSCRIPT Abstract Nowadays, chemotherapy is a common means of oncology. However, it is difficult to find excellent chemotherapy drugs. Here we reported three new ternary copper(II)

ligand

and

heterocyclic

[Cu(dpz)(TBHP)]H2O

(2)

bases

and

(TBHP),

[Cu(phen)(TBHP)]H2O

[Cu(dppz)(TBHP)]H2O

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base

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complexes which have potential chemotherapy characteristics with reduced Schiff

(3)

(phen

(1), =

1,10-phenanthroline, dpz = dipyrido [3,2:2´,3´-f]quinoxaline, dppz = dipyrido [3,2-a:2´,3´-c]phenazine, H2TBHP = 2-(3,5-di-tert-butyl-2-hydroxybenzylamino)-2-

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benzyl-acetic acid). The DNA-binding properties of the complexes were investigated by spectrometric titrations, ethidium bromide displacement experiments and viscosity

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measurements. The results indicated that the three complexes, especially the complex 13, can strongly bind to calf-thymus DNA (CT-DNA). The intrinsic binding constants

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Kb of the ternary copper(II) complexes with CT-DNA were 1.37105, 1.81105 and

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3.21105 for 1, 2 and 3 respectively. Comparative cytotoxic activities of the copper(II) complexes

were

also

determined

by

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3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results showed that the ternary copper(II) complexes had significant cytotoxic activity against the human lung cancer (A549), human esophageal cancer (Eca109) and

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human gastric cancer (SGC7901) cell lines. Cell apoptosis were detected by AnnexinV/PI flow cytometry and by Western blotting with the protein expression of p53, Bax and Bcl-2. All the three copper complexes can effectively induce apoptosis of the three human tumor cells.

Keywords: ternary copper(II) complexes; DNA-binding; cytotoxic; AnnexinV/PI; cell apoptosis

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ACCEPTED MANUSCRIPT 1. Introduction Nowadays, the major focus of research in chemotherapy for cancer includes the

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identification, characterization and development of new cancer chemopreventive

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agents [1]. Cancer chemotherapy with platinum drugs has been used since the

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discovery of cisplatin’s anti-proliferate properties by Rosenberg et al. [2]. As they possesses inherent limitations such as significant toxic side effects, general toxicity, and acquired drug resistance, attempts are being made to replace the platinum-based

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drugs with suitable alternatives, and numerous metal based complexes are synthesized and screened for their anticancer activities [3-8].

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Copper(II) ions are very important for many organisms. Like other trace metals, copper is essential to proteins that are involved in several biological processes, including respiration, metabolism, DNA synthesis, and oxidation–reduction reactions.

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In biological systems, copper exists as a variety of complexes which due to the

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coordinated forms of copper are more stable than the corresponding ionic species [9-12]. 1,10-Phenanthroline (phen) and its substituted derivatives, both in the

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metal-free state and coordinated state, disturb the functioning of a wide variety of biological systems. The copper(II) complexes containing bpy or 1,10-phenanthroline have attracted much attention because they are much more active in the presence of

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the heterocyclic ligands [13-15]. Recently, several copper complexes containing heterocyclic bases, such as 1,10-phenanthraline, were screened for their anticancer activity [10, 13-19]. Tsang and coworkers reported that incubation of a human hepatic cell line (HepG2) with [Cu(phen)2]2+ can result in the internucleosomal DNA fragmentation[20]. It is well know that apoptosis is a crucial process related to a number of diseases. The apoptosis induced by copper and its complexes have been investigated extensively on the premise that endogenous metals may be less toxic [21-25]. Copper and its complexes can catalyze the formation of reactive oxygen species (ROS) [26]. As critical triggers in cell apoptotic pathways, the formation of ROS along with other intracellular cascade reactions (such as disruption of mitochondrial transmembrane 3

ACCEPTED MANUSCRIPT potential, upregulation of Bax, down regulation of Bcl-2, and deficiency of p-53) can finally lead to cell apoptosis [27-30]. Therefore, more accurate and indepth understanding about copper-induced apoptosis will be helpful in the development of

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copper-based antitumor drugs.

All of the above facts have stimulated our interest in the present work. Herein we

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report the synthesis, the calculated energy-minimized structure, DNA-binding, cytotoxic and apoptosis induction activities of three ternary copper(II) complexes [Cu(phen)(TBHP)]H2O (1), [Cu(dpz)(TBHP)]H2O (2) and [Cu(dppz)(TBHP)]H2O (3) shown

in

scheme

1,

phen

=

1,10-phenanthroline,

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(as

dpz

=

dipyrido

[3,2-d:2´,3´-f]quinoxaline, dppz = dipyrido[3,2-a:2´,3´-c] phenazine). In order to

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investigate the biological activities of complexes 1-3, such as DNA binding, cytotoxicity and apoptosis induction activities, several methods were employed.

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Furthermore, the primary aim of the current study is to determine the cancer

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chemotherapeutic potential of metal-free phen and its substituted derivatives by using three human-derived cancer model cell lines of human lung cancer (A549), human

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esophageal cancer (Eca109) and human gastric cancer (SGC7901). Most importantly, our current results suggest the complexes 1-3 own the potential activities of conducting cell proliferation and apoptosis of three human cancer cells.

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2 Materials and method 2.1 Materials and instrumentation 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. UV-visible (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 5 % CH3OH and 95 % Tris-HCl buffer (5 mM Tris-HCl, 50 mM NaCl, pH 7.1) at concentration 110-3M. The A549 human lung cancer, Eca109 human esophageal cancer and SGC7901 human gastric cancer cell lines were obtained from the Cell Culture Center of the Basic Institute of Medical Sciences, Peking Union Medical 4

ACCEPTED MANUSCRIPT College. Cell culture reagents were purchased from Gibco (CA, USA). Annexin VFITC and PI double staining was purchased from BD Biosciences (NJ, USA). 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT), DMSO were

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purchased from Sigma Aldrich (MO, USA). Polyvinylidene fluoride (PVDF) membranes and non-fat dry milk were obtained from Millipore (MA, USA). p53, Bax

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and Bcl-2 polyclonal antibody were obtained from Biovision (SF, USA). GAPDH polyclonal antibody and the secondary antibody were purchased from Beyotime (Nantong, China).

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The UV-vis absorption spectra were recorded using a Varian Cary 100 spectrophotometer and fluorescence emission spectra were recorded using a Hitachi

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F-4500 spectrofluorophotometer. The elemental analyses were performed in the microanalytical laboratory, Department of Chemistry, Lanzhou University. The 1H

(KBr

pellet)

were

recorded

using

an

FTS165

Bio-Rad

FTIR

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spectra

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NMR spectra was recorded with a Bruker ACF300 FT-NMR instrument. The infrared

spectrophotometer in the range of 4000-400 cm-1.

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Cell lines were cultured in the incubator of Thermo Fisher under the condition of 100% humidity, 5% CO2 at 37℃. RPMI-1640 culture medium was purchased from Hyclone Company. Olympus CKX51 microscope and TOUPCAM FMA050 were used to take

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photos of cell lines. MTT assay was performed using a microplate reader Infinite F50(Tecan, Männedorf, Switzerland). Flow Cytometry BD Accuri C6 was purchased from BD Company. Western blotting was executed with Biorad Mini Trans-Blot Cell. 2.2 Synthesis of the ligand (H2TBHP) A mixture of 3,5-di-tert-butyl-2-hydroxybenzaldehyde (11.7g, 0.05 mol) and phenylalanine (8.25 g, 0.05 mol) was taken in 250 ml of ethanol and NaOH (2 g, 0.05 mol) was added. The yellow solution was stirred for 6 h at room temperature prior to cooling in an ice bath. The intermediate Schiff base that formed was reduced with an excess of NaBH4 (1.95 g, 0.6 mol) in MeOH containing a few drops of NaOH solution. The yellowish color was slowly discharged, after stirring for another 3 h, the solution was acidified with acetic acid to a pH of 5.0-6.0. The resulting colorless solid was filtered off, washed with dry MeOH and Et2O, and recrystallized from H2O/EtOH 5

ACCEPTED MANUSCRIPT (1:3) after drying in air. Yield: 95%. M.P.: 235℃. H2TBHP (D2O) (ppm) 1.228 (s, 9H), 1.321 (s, 9H), 3.737 (s, 1H), 3.959 (d, 1H), 4.136 (d, 1H),4.826(d, 1H), 4.929 (d, 1H),6.865 (d, 1H), 7.027 (d, 1H), 7.130～7.265 (b, 5H). IR (KBr) cm-1: (NH) 3226,

75.16; H, 8.67; N, 3.65. Found: C, 75.31; H, 8.56; N, 3.81.

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2.3 Synthesis of the complexes

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(CH2) 2968, (C=O) 1712, 1492, (phen-OH) 1230. Anal. Calcd for C24H33NO3: C,

To the green solution formed from Cu(ClO4)26H2O (0.371 g, 1 mmol) in MeOH (8 mL) and heterocyclic bases (1 mmol) in MeOH (8 mL) was added a filtered solution

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of H2TBHP (0.293 g, 1 mmol) in H2O (20 mL) containing LiOH. 1,10-phenanthroline (phen, for complex 1), dipyrido[3,2-d:2´,3´-f]quinoxaline (dpz, for complex 2)

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dipyrido [3,2-a:2´,3´-c] phenazine (dppz, for complex 3). The resulting blue solution was stirred for 1 hour and then filtered and left for a week, after which time the blue

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micro-crystals or powders were isolated by filtration.

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Complex 1: Yield: 86.5 %. Anal. Calcd for C36H41CuN3O4: C, 67.22; H, 6.42; N, 6.53. Found: C, 67.15; H, 6.61; N, 6.43. IR (KBr, cm-1): (NH) 3122, (CH2) 2959, ( C=O)

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1625, 1410.

Complex 2: Yield: 88.2 %. Anal. Calcd for C38H41CuN5O4: C, 65.64; H, 5.94; N, 10.07. Found: C, 65.58; H, 6.05; N, 10.28. IR (KBr, cm-1): (NH) 3139, (CH2) 2952,

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( C=O) 1630, 1383.

Complex 3: Yield: 89.8 %. Anal. Calcd for C42H43CuN5O4: C, 67.68; H, 5.81; N, 9.40. Found: C, 67.85; H, 5.68; N, 9.54. IR (KBr, cm-1): (NH) 3098, (CH2) 2956, ( C=O) 1582, 1358. Caution. Perchlorate salts of metal complexes containing organic ligands are potentially explosive. Only small quantity of material should be prepared and handled with suitable safety measures. 2.4 Spectroscopic studies on DNA interaction 2.4.1. Electronic absorption spectra Electronic absorption titration of the ternary copper(II) complex samples in the aqueous buffer solution (50 mM NaCl-5 mM Tris-HCl, pH 7.1) were performed at a fixed complex concentration (10 M) while gradually increasing the concentration of 6

ACCEPTED MANUSCRIPT CT-DNA. The absorption data were analyzed to evaluate the intrinsic binding constant Kb, which can be determined from Eq. (1) [31], [DNA]/(εa-εf)=[DNA]/(εb-εf)+1/Kb(εb-εf) (1)

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

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coefficient εa, εf and εb correspond to Aobsd/[M], absorption coefficient of the free

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compound and the extinction coefficient of the compound when fully bound to DNA, respectively. In plots of [DNA]/(εa-εf) versus [DNA], Kb is given by the ratio of slope to the intercept.

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2.4.2. Fluorescence spectra

Further support for the ternary copper(II) complexes binding to DNA is given through

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the emission quenching experiment. EB is a common fluorescent probe for DNA structure and has been employed in examinations of the mode and process of metal

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complex binding to DNA. The mixed solution of DNA and EB was titrated by

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ternary copper(II) complexes (ex = 500 nm, em = 520.0-650.0 nm). According to the classical Stern-Volmer equation (2) [32]: (2)

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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

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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. 2.4.3. Viscosity measurements Viscosity experiments were carried on an Ubbelodhe viscometer, immersed in a thermostated water-bath maintained at 28.0±0.5 ºC. Flow time was measured with a digital stopwatch for different concentrations of the complexes, maintaining the initial DNA concentration. Each sample was measured three times and an average flow time was calculated. Data were presented as (η/η0)1/3 versus binding ratio r (r = [complex]/DNA]) [26], where η is the viscosity of DNA in the presence of complex, and η0 is the viscosity of DNA alone. The relative viscosity values for DNA in the presence (η) and absence (η0) of the complexes were calculated using the relation η = 7

ACCEPTED MANUSCRIPT (t - t0)/t0, where t is the observed flow time in seconds [33, 34].

2. 5 Cytotoxic activity

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To evaluate their potential cancer chemotherapeutic ability, the DMSO soluble copper complexes 1–3 were used to kill the human lung cancer (A549), human esophageal

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cancer (Eca109) and human gastric cancer (SGC7901), and the calculation of IC50 was employed to determine the ability by MTT assay. Briefly, the three kinds of tumor cells (A549, Eca109 and SGC7901) were plated at a density of 1104 cells per

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well in 96 -well plates overnight and then treated with different concentrations of copper drug after 72h. Twenty microliters of MTT solution were added to each well

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and the cells were cultured for another 4 h at 37℃. The medium was completely removed and 100 µL DMSO was added to solubilize MTT formazan crystals. The

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plates were then agitated and the optical density was determined at 570 nm (A570)

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using an Infinite F50 Microplate Reader (TECAN). At least three independent experiments were performed.

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2.6 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

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well in 6-well plates overnight and then treated by copper complexes 1–3 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 flow cytometer and analyzed using Cell Quest software. At least three independent experiments were performed. 2.7 Western blotting analysis. The three kinds of tumor cells (1×106) were seeded in 6 -well plates overnight. Cells were treated by copper complexes 3 with each corresponding IC50 dose for 24 h. Cells were then washed twice with PBS (phosphate buffered saline). The total proteins were solubilized and extracted with lysis buffer (20mM HEPES, pH 7.9, 20% glycerol, 200mM KCl, 0.5mM EDTA, 0.5%NP40, 0.5mM DTT (Dithiothreitol), 1% protease inhibitor cocktail). Protein concentration was determined by bicinchoninic acid 8

ACCEPTED MANUSCRIPT protein assay. The samples were separated by SDS - PAGE, transferred to PVDF membranes by electroblotting and probed with antibodies against p53, Bax, Bcl-2 and GAPDH. The membranes were incubated in the second antibody of IgG (Merck, USA)

scanned for the relative value of protein expression.

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2.8 Statistical analysis

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marked by alkaline phosphatase (AP) and colored by ECL at room temperature. It was

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

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analyzed using a one-way analysis of variance followed by post hoc comparisons.

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Statistical significance was defined as p-values < 0.05.

3 Results and discussion

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3.1 Syntheses

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The mixed-ligand complexes of general formula [Cu(TBHP)(phen)](H2O), [Cu(TBHP)(dpz)](H2O) and [Cu(TBHP)(dppz)](H2O) were synthesized by a straight

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forward synthesis in which the simultaneous reaction of reduced Schiff base ligand with corresponding polypyridyl ligands and copper(II) salt in a 1:1:1 molar ratio, which yielded a blue or green microcrystalline powder. All the attempts to grow single

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crystals of diffractometric quality for these complexes failed. The new coordination complexes were soluble in water/ethanol in 1:1 as well as in DMSO and dimethylformamide (DMF) but insoluble in nonpolar solvents. The analytical data C, H, N confirmed the composition of the complexes and the stoichiometry proposed. The stability of the copper(II) complexes in the buffer systems had been studied by observing the UV-vis spectrums and estimating the molar conductivities at different time intervals for any possible change. The tested copper(II) complexes were prepared in DMF solution and for experiments freshly diluted in phosphate buffer system. Then, the UV-vis spectrums and molar conductivities were registered at different time intervals. The investigations revealed that the UV-vis spectra remained unaltered for the solutions and no obvious change were found of the molar conductance values for very freshly prepared and for over the whole experiment (72 h). It indicated that the 9

ACCEPTED MANUSCRIPT copper(II) complexes were quite stable in solution. Evidence of the mode of bonding of the ligand was gathered from the FTIR spectra. The characteristic bands of the carboxylate groups were observed in the range 1580-1630 cm-1 for asymmetric

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stretching and 1358-1410 cm-1 for symmetric stretching, respectively [35]. The

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difference between as(COO-) and the sym(COO-) stretching frequencies in complexes

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is  200 cm-1, thus suggesting a terminal coordination mode for the carboxylate group [36]. Weak absorptions observed in the range of 2930-2990 cm-1 can be attributed to the CH2 of the reduced Schiff base ligand and the vibration observed in the range of

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2970-3120 cm-1 can be attributed to the NH of the ligand. The broad bands at 3430 cm-1 were ascribed to the vibration of the O-H stretching of the water ligands. Bands

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corresponding to the vibrational modes of heterocyclic bases were also detected. Strong bands in the 900–700 cm-1 region in spectra of aromatic hydrocarbons had

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been identified with motions of the ring hydrogen atoms. In heterocyclic bases, and

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also in its complex, two strong bands appeared approximately near 850 and 720 cm-1 [37, 38]. The observed FTIR changes suggested the presence of carboxylate and

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nitrogen donor atoms around the metal center. In order to conform the selectivity and the configuration of H2TBHP, we carried out density functional theory (DFT) calculations with B3LYP exchange functionals using

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the Gaussian 09 C.01 package. The 6-31G(d,p) Cu basis sets were used except for Cu2+, where LANL2DZ effective core potential (ECP) was employed. The optimized configuration was shown in Fig. 1, which showed that the complex 1 consists of mononuclear units with the Cu(II) ion assuming a distorted square pyramid geometry with N3O2 donor set. The four basal positions were occupied by two N atoms of the phen ligand, the phenolic O and amine N atoms of the H2TBHP ligand. The Cu–N(phen) bond length are 2.096 and 2.505 Å and the Cu–N(amino

acid)

is 1.917 Å. The

Cu–O(carbonyl group) bond length is 1.892 Å, and the Cu–O(phenolic group) bond length is 1.909 Å.

3.2 DNA-binding activities Electronic absorption spectroscopy is an effective technique for DNA binding studies 10

ACCEPTED MANUSCRIPT of metal complexes [39]. Transition metal centers are particularly attractive moieties for such research since they exhibit well-defined coordination geometries and also possess distinctive electrochemical or photophysical properties, thus enhancing the

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functionality of the binding agents. Hypochromicity is the characteristic of intercalation [40], which is usually attributed to the interaction between the electronic

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states of the complexes and those of the DNA bases [41]. While the red shift has been associated with the decrease in the energy gap between the highest occupied and the lowest unoccupied molecular orbitals (HUMO and LUMO) after binding of the

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complexes to DNA [42]. The binding of ternary copper(II) complexes to DNA helix were characterized through absorption spectral titrations, by following changes in

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absorbance and shift in wavelength, unambiguously revealed the active participation in associating with the DNA. Fig. 2 showed the absorption spectra of ternary

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copper(II) complexes in the absence and presence of CT-DNA. It is simply noticed

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that with the increasing DNA concentration, the absorption bands of the three complexes displayed decrease in molar absorptivity (hypochromism) as well as slight

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bathochromism. The observed red-shift is an evidence of the stabilization of the CT DNA duplex and the hypochromism might be attributed to interaction between the

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aromatic rings of the complexes and DNA base pairs.

The Kb values of ternary copper(II) complexes were 1.37105, 1.81105, and 3.21105 for 1, 2 and 3, respectively. The significant difference in DNA binding affinity of the three ternary copper(II) complexes could be understood that the extended planar structure of the dpz and dppz ligands greatly facilitating non-covalent interactions with the DNA molecule. The results suggested that all the metal complexes reported here interacted with CT DNA quite probably by intercalating the complexes into DNA base pairs. Interestingly, the Kb values obtained for the above ternary copper(II) complexes were higher than those for the other known mononuclear and binuclear copper(II) complexes containing 1,10-phenanthroline ([Cu(dipsH2)(phen)]·H2O (Kb, 6.35103 M-1, dipsH2 = 3,5-diisopropylsalicylic acid) [43], [Cu2(phen)2Cl4] (Kb, 4.75104 M-1) 11

ACCEPTED MANUSCRIPT [44]. This showed that comparatively the ternary copper(II) complex samples can bind to CT-DNA very strongly. In order to further investigate the interaction mode between the complexes and

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CT-DNA, the fluorescence titration experiments were performed. Fluorescence quenching refers to the process in which the fluorescence intensity of EB-DNA

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decreases upon adding the three copper complexes as quenchers. Stern-Volmer constant (Kq) was used to evaluate the fluorescence quenching efficiency. The intrinsic fluorescence intensities of DNA and that of EB in Tris-HCl buffer were

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low, while the fluorescence intensity of EB enhanced on addition of DNA as its intercalation into the DNA. Therefore, EB can be used to probe the interaction of

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complexes with DNA [45]. In our experiments, as depicted in Fig. 3, for complex 1, 2 and 3, the fluorescence intensity of EB at 584 nm showed a remarkable decreasing

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trend with the increasing concentration of the complex 1, 2 or 3, indicating that some

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EB molecules were released from EB-DNA after an exchange with the complex 1, 2 or 3 which resulted in the fluorescence quenching of EB. We assumed the reduction

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of the emission intensity of EB on increasing the complex concertration could be caused due to the displacement of the DNA bound EB by the ternary copper(II) complexes. The Kq values of ternary copper(II) complexes were 1.67 104 M-1, 2.95 

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104 M-1, 5.04 104 M-1 for 1, 2 and 3, respectively. The magnitude order of Kq for the three complexes was very similar to the absorption titration methods and a similar explanation could be cited as stated earlier. Although spectral methods were necessary, it was not sufficient to support a binding mode. In the absence of crystallographic structural data, hydrodynamic measurement was considered as least ambiguous and the most critical test to find the binding mode of DNA with metal complexes [46]. When small planar molecules intercalated into the neighbouring base pairs of DNA, the DNA double helix loosened to accommodate the intercalation, resulting in a lengthening of the DNA helix. Since the viscosity of the DNA solution was very sensitive to the changes in DNA length, the viscosity of the DNA solution was usually increased in the intercalation binding mode as the result of DNA length increase [47]. 12

ACCEPTED MANUSCRIPT Fig. 4 depicted the effect of complexes 1-3 on the DNA viscosity. The change in relative viscosity for the dppz complexes were more than that for the phen or dpz analogues suggesting greatest DNA binding propensity of the dppz complexes.

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Furthermore, it is noteworthy that the Kb values were higher than the binding affinity

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of EB for DNA (Kb = 1.23 (0.07)105 M-1), suggesting the greater DNA binding

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propensity of the three ternary copper complexes than the EB [48]. This was consistent with the observed trend shown by other optical methods and suggested

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primarily an intercalation binding nature of the complexes.

3.3 Effect of copper complexes 1-3 on the growth of the cancer cell lines.

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Cytotoxicity is a common limitation in terms of the introduction of new compounds into the pharmaceutical industry. Schiff base complexes of copper had recently been

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investigated for their therapeutic potential [49]. The effect of the reported complexes

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on the growth of tumor cells was studied in human lung cancer (A549), human esophageal cancer (Eca109) and human gastric cancer (SGC7901) cell line. It was

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intended that the results from these studies would allow the identification of those ternary derivatives with cancer chemotherapeutic potential. Comparison of IC50 values, allowed the relative potency of each of the test complexes to be determined

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and ranked. All complexes were able to inhibit cell growth in a concentration dependent manner, with the IC50 values shown in Table 1. The three metal complexes displayed a concentration-dependent cytotoxic profile in all cell lines. Since the IC50 values for complexes 1-3 were statistically lower than that for metal-free heterocyclic bases and H2TBHP in all of the tested cells, it suggested that coordinated copper(II) ion played a major role in mediating potency of the complexes. It was evident that complex 3 exhibited higher cytotoxicity than the other two complexes against each of the selected cell lines. The better cytotoxic activity of complex 3 was in accordance with its stronger DNA binding ability, which further suggested the binding of the complexes to DNA and thus consequently leads to cell death. The morphology examinations also showed that the proliferation of the cells 13

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shrinkage and cell detachment, as shown in Fig. 5.

3.4 Effect of copper complexes 1-3 on the inducing apoptosis of the cancer cell

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lines.

To determine whether the decrease in human tumor cell growth was attributed to the induction of apoptosis in cancer cells, Annexin V/PI assay were used to detect

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apoptotic cells. As shown in Fig. 6, Annexin V/PI assay revealed that the number of

MA

apoptotic cells increased by treatment of copper complexes 1-3 (P100 15.8 50.6 35.2 4.8 2.1 1.2

AC

CE P

TE

D

MA

NU

SC R

IP

Tested complexes

31

T

human gastric cancer (SGC7901) cell line after incubation for 72 h. SGC7901 >100 8.6 29.5 26.1 5.8 2.4 0.8