Induction of cell death by ternary copper(II) complexes of L-tyrosine and diimines: role of coligands on DNA binding and cleavage and anticancer activity

Abstract

The mononuclear mixed ligand copper(II) complexes of the type [Cu(l-tyr)(diimine)](ClO₄), where tyr is l-tyrosine and diimine is 2,2'-bipyridine (bpy) (1), 1,10-phenanthroline (phen) (2), 5,6-dimethyl-1,10-phenanthroline (5,6-dmp) (3), and dipyrido[3,2-d:2',3'-f]quinoxaline (dpq) (4), have been isolated and characterized by analytical and spectral methods. In the X-ray crystal structure 3 Cu(II) possesses a distorted square pyramidal coordination geometry with the two nitrogen atoms of 5,6-dmp ligand and the amine nitrogen and carboxylate oxygen atoms of l-tyrosine located at the equatorial sites and the coordinated water molecule present in the apical position. The electronic absorption and electron paramagnetic resonance (EPR) spectral parameters reveal that the complexes retain their square-based geometries even in solution. All of the complexes display a ligand field band in the visible region (600-700 nm) in Tris-HCl/NaCl buffer (5:50 mM) at pH 7.2 and also axial EPR spectra in acetonitrile at 77 K with g > g⊥ indicating a d_x²-y² ground state. The g and A values of 2.230 and (170-180) × 10^-4 cm^-1, respectively, conform to a square-based CuN₃O coordination chromophore, which is consistent with the X-ray crystal structure of 3. The interaction of the complexes with calf thymus DNA (CT DNA) has been explored by using physical methods to propose modes of DNA binding of the complexes. Absorption (K_b) and emission spectral studies and viscosity measurements indicate that 4 interacts with DNA more strongly than all of the other complexes through partial intercalation of the extended planar ring of dpq with DNA base stack. Interestingly, complex 3 exhibits a DNA binding affinity that is higher than that of 2, which suggests the involvement of 5,6-dimethyl groups on the phen ring in hydrophobic interaction with DNA surface. In contrast with the increase in relative viscosities of DNA bound to 2-4, the viscosity of DNA bound to 1 decreases, indicating the shortening of the DNA chain length by means of the formation of kinks or bends. All complexes exhibit effective DNA (pUC19 DNA) cleavage at 100 μ M complex concentrations, and the order of DNA cleavage ability varies as 3 > 2 > 4 > 1. Interestingly, 3 exhibits a DNA cleavage rate constant that is higher than that of the other complexes only at 100 μ M concentration, whereas 4 exhibits the highest cleavage rate constant at 80 µM complex concentration. The oxidative DNA cleavage follows the order 4 > 3 > 2 > 1. Mechanistic studies reveal that the DNA cleavage pathway involves hydroxyl radicals. Interestingly, only 4 displays efficient photonuclease activity upon irradiation with 365 nm light, which occurs through double-strand DNA breaks involving hydroxyl radicals. Furthermore, cytotoxicity studies on the nonsmall lung cancer (H-460) cell line show that the IC₅₀ values of 2-4 are more or less equal to cisplatin for the same cell line, indicating that they have the potential to act as very effective anticancer drugs in a time-dependent manner. The study of cytological changes reveals the higher induction of apoptosis and mitotic catastrophe for 4 and 3, respectively. The alkaline single-cell gel electrophoresis (comet assay), DNA laddering, and AO/EB and Hoechst 33258 staining assays have also been employed in finding the extent of DNA damage. Flow cytometry analysis shows an increase in the percentage of cells with apoptotic morphological features in the sub-G₀/G₁ phase for 4, whereas it shows mitotic catastrophe for 3.