Biomolecular Interaction, Anti-Cancer and Anti-Angiogenic Properties of Cobalt(III) Schiff Base Complexes

Abstract

Two cobalt(III) Schiff base complexes, trans-[Co(salen)(DA)₂](ClO₄) (1) and trans-[Co(salophen)(DA)₂](ClO₄) (2) (where salen: N,N'-bis(salicylidene)ethylenediamine, salopen: N,N'-bis(salicylidene)-1,2-phenylenediamine, DA: dodecylamine) were synthesised and characterised using various spectroscopic and analytical techniques. The binding affinity of both the complexes with CT-DNA was explored adopting UV-visible, fluorescence, circular dichroism spectroscopy and cyclic voltammetry techniques. The results revealed that both the complexes interacted with DNA via intercalation as well as notable groove binding. Protein (BSA) binding ability of these complexes was investigated by absorption and emission spectroscopy which indicate that these complexes engage in strong hydrophobic interaction with BSA. The mode of interaction between these complexes and CT-DNA/BSA was studied by molecular docking analysis. The in vitro cytotoxic property of the complexes was evaluated in A549 (human small cell lung carcinoma) and VERO (African green monkey kidney cells). The results revealed that the complexes affect viability of the cells. AO and EB staining and cell cycle analysis revealed that the mode of cell death is apoptosis. Both the complexes showed profound inhibition of angiogenesis as revealed in in-vivo chicken chorioallantoic membrane (CAM) assay. Of the two complexes, the complex 2 proved to be much more efficient in affecting the viability of lung cancer cells than complex 1. These results indicate that the cobalt(III) Schiff base complexes in this study can be potentially used for cancer chemotherapy and as inhibitor of angiogenesis, in general, and lung cancer in particular, for which there is need for substantiation at the level of signalling mechanisms and gene expressions.

Figure 1

Scheme of synthesis of complexes 1 and 2.

Figure 2

Electronic absorption spectra of cobalt(III) complexes 1 (a) and 2 (b) in the absence (dashed line) and presence (solid line) of increasing amounts of DNA. Insert: Plot of [DNA]/(ε_a − ε_f) vs [DNA]. [Complex] = 2 × 10⁻⁵ M⁻¹, [DNA] = 0–2.1 × 10⁻⁴ M⁻¹. Emission spectra (λ_ex = 450 nm) of EB - DNA: in the absence (dotted line) and in the presence of (solid line) of the complexes 1 (c) and 2 (d). Stern-Volmer (CV) plot (e) of fluorescence quenching of EB - DNA by complexes 1 and 2. Cyclic Voltammetry of complexes 1 (f) and 2 (g) in the absence ( black line) and in the presence (red line) of DNA. Circular dichroism spectra of CT-DNA (4 × 10⁻⁵ M) in the presence of complex 1 (h) and 2 (i), r = [complex]/[DNA] = 0.0, 0.1 and 0.5).

Figure 3

The fluorescence emission spectra of BSA in the presence of complexes 1 (a) and 2 (b). The dashed line shows the intensity of BSA in the absence of complexes. After addition of the complexes 1 and 2 ([complex] = 0–4 × 10⁻⁶ M; [BSA] = 1.35 × 10⁻⁶ M) to BSA, the emission intensity of BSA was decreased which is indicated by “arrow”. Stern-Volmer plot for quenching of BSA by cobalt(III) complexes 1 (c) and 2 (d). The UV-Vis absorption spectra of BSA and complexes 1 (e) and 2 (f). Plot of log[(F₀ − F)/F] vs log [Q] for BSA- cobalt(III) complexes 1 (g) and 2 (h). van’t Hoff plot for the interaction of BSA with complexes 1 and 2 (i).

Figure 4

The binding poses of the metal complexes 1 (A) and 2 (B) into the DNA duplex obtained from molecular docking analysis.The binding poses of the metal complexes 1 (C) and 2 (D) into the BSA obtained from molecular docking analysis. (For clarity the hydrogen atoms of the metal complexes are omitted).

Figure 5

(A1). Morphological assessment of control (A1 c; the cells are viable as inferred from the green fluorescence) and treated (A1, 1 complex 1; & A1, 2 complex 2) A549 lung cancer cells. (A2) The graph shows data on percentage of cells that are normal and those afflicted with apoptosis and necrosis in the control and 24 h treated groups. (A3) The effects of complexes 1 and 2 on the cell cycle progression in lung cancer A549 cells. (A4) The bar diagram displays higher percentage of cells in sub G₀ − G₁ and G₂-M phases after treatment of A549 cancer cells with complexes 1 and 2. The cell cycle distribution was analyzed using Dean-Jett-Fox software and depicted as the histogram. (A5) The vascular sprouting has been damaged (marked by arrows) on exposure to complexes 1 and 2 (10 μM) compared to control at 0 h (C, 1 and 2) after 6 h (C′, 1′ and 2′) of treatment. (A6) The different angiogenic parameters such as vessel length, vessel size and number of junctions decreased on exposure to complexes 1 and 2 compared to control. (Data are expressed in Mean ± SD).