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

doi:10.1016/j.jinorgbio.2014.12.011

Journal of Inorganic Biochemistry

Volume 144, March 2015, Pages 38-46

https://doi.org/10.1016/j.jinorgbio.2014.12.011 Get rights and content

Highlights

•
The biological activity of three dinuclear copper(II) complexes was investigated.
•
The three complexes can cause DNA damage.
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Cell apoptosis was detected by AnnexinV/PI flow cytometry and Western blotting.
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All the complexes can effectively induce apoptosis of the human tumor cells.

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) complexes which have potential chemotherapy characteristics with reduced Schiff base ligand and heterocyclic bases (TBHP), [Cu(phen)(TBHP)]H₂O (1), [Cu(dpz)(TBHP)]H₂O (2) and [Cu(dppz)(TBHP)]H₂O (3) (phen = 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-benzyl-acetic acid). The DNA-binding properties of the complexes were investigated by spectrometric titrations, ethidium bromide displacement experiments and viscosity 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 K_b of the ternary copper(II) complexes with CT-DNA were 1.37 × 10⁵, 1.81 × 10⁵ and 3.21 × 10⁵ for 1, 2 and 3 respectively. Comparative cytotoxic activities of the copper(II) complexes were also determined by 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 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.

Graphical abstract

Three new ternary copper(II) complexes which have potential chemotherapy characteristics with reduced Schiff base ligand and heterocyclic bases, and the ternary copper(II) complexes had significant cytotoxic activity against the human cancer cell lines. They also can induce the cancer cell apoptosis.

Introduction

Nowadays, the major focus of research in chemotherapy for cancer includes the identification, characterization and development of new cancer chemopreventive agents [1]. Cancer chemotherapy with platinum drugs has been used since the 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 drugs with suitable alternatives, and numerous metal based complexes are synthesized and screened for their anticancer activities [3], [4], [5], [6], [7], [8].

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. In biological systems, copper exists as a variety of complexes which due to the coordinated forms of copper are more stable than the corresponding ionic species [9], [10], [11], [12]. 1,10-Phenanthroline (phen) and its substituted derivatives, both in the 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 the heterocyclic ligands [13], [14], [15]. Recently, several copper complexes containing heterocyclic bases, such as 1,10-phenanthraline, were screened for their anticancer activity [10], [13], [14], [15], [16], [17], [18], [19]. Tsang et al. 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], [22], [23], [24], [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 potential, upregulation of Bax, down regulation of Bcl-2, and deficiency of p-53) can finally lead to cell apoptosis [27], [28], [29], [30]. Therefore, more accurate and indepth understanding about copper-induced apoptosis will be helpful in the development of copper-based antitumor drugs.

All of the above facts have stimulated our interest in the present work. Herein we 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) (as shown in Scheme 1, phen = 1,10-phenanthroline, dpz = dipyrido [3,2-d:2′,3′-f]quinoxaline, dppz = dipyrido[3,2-a:2′,3′-c] phenazine). In order to investigate the biological activities of complexes 1–3, such as DNA binding, cytotoxicity and apoptosis induction activities, several methods were employed. Furthermore, the primary aim of the current study is to determine the cancer 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 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.

Section snippets

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^− 3 M. The A549

Syntheses

The mixed-ligand complexes of general formula [Cu(TBHP)(phen)]·(H₂O), [Cu(TBHP)(dpz)]·(H₂O) and [Cu(TBHP)(dppz)]·(H₂O) were synthesized by a straightforward synthesis which yielded a blue or green microcrystalline powder. The reduced molar ratio of Schiff base ligand with corresponding polypyridyl ligands and copper(II) salt is 1:1:1. All the attempts to grow single crystals of diffractometric quality for these complexes failed. The new coordination complexes were soluble in water/ethanol in

Conclusion

Finally, we found that our copper complexes possess the potential for development as an agent for human cancer therapy. Further experiments are necessary to analyze the action of copper complexes in vivo.

Abbreviations

CT-DNA

calf-thymus DNA

ROS

reactive oxygen species

ethidium bromide

MTT

3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide

PVDF

polyvinylidene fluoride

DMF

dimethylformamide

DFT

density functional theory

ECP

effective core potential

HOMO and LUMO

highest occupied and the lowest unoccupied molecular orbitals, respectively

Acknowledgments

This work was supported in part by the fund of Science and Technology of Yixing (2013-21), the fund of the Natural Science Foundation of Jiangsu (BK20141122), the National Natural Science Foundation of China (21404033, 21401046, 21001040), Doctoral Scientific Fund Project of Henan Polytechnic University (72515/086, 61307/003), and the Foundation of State Key Laboratory of Solid Lubrication (LSL-1207).

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¹: Contributed equally.

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

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Materials and instrumentation

Syntheses

Conclusion

Abbreviations

Acknowledgments

Eur. J. Med. Chem.

Coord. Chem. Rev.

J. Inorg. Biochem.

Inorg. Chim. Acta

Eur. J. Med. Chem.

J. Inorg. Biochem.

J. Inorg. Biochem.

Inorg. Chim. Acta

Polyhedron

J. Inorg. Biochem.

Polyhedron

Mutat. Res.

Bioorg. Med. Chem. Lett.

Inorg. Chem. Commun.

Toxicology

J. Inorg. Biochem.

Inorg. Chim. Acta

Inorg. Chim. Acta

J. Inorg. Biochem.

Inorg. Chim. Acta

Bioorg. Med. Chem.

J. Inorg. Biochem.

J. Inorg. Biochem.

J. Inorg. Biochem.

J. Photochem. Photobiol. B

Eur. J. Pharmacol.