Mixed Ligand Mononuclear Copper(II) Complex as a Promising Anticancer Agent: Interaction Studies with DNA/HSA, Molecular Docking, and In Vitro Cytotoxicity Studies

The isolated copper­(II)
complex [CuL­(o-phen)]·H2O (1) [H2L = o-HO-C6H4C­(H)N-C6H4-SH-o, o-phen = 1,10-phenanthroline] was structurally
characterized using single-crystal X-ray crystallography. 1 in CH3CN at liquid nitrogen temperature displayed a characteristic
monomeric X-band electron paramagnetic resonance spectrum having a
tetragonal character with g∥ =
2.1479 and g⊥ = 2.0691 and A∥ ≈ 18.0 mT and A⊥ ≤ 3.9 mT, respectively. 1 showed a strong binding affinity toward calf thymus DNA as reflected
from its intrinsic binding constant (Kb = 7.88 × 105 M–1), and its competitive
displacement of ethidium bromide suggested an intercalative DNA-binding
mode (Kapp = 1.32 × 106 M–1). This was confirmed from the viscosity study
that showed an increase in the viscosity of DNA with an increasing
concentration of 1. Complex 1 is highly
efficient in promoting oxidative and hydrolytic DNA cleavage (kobs = 1.987 h–1). 1 showed a strong binding affinity with the carrier protein human
serum albumin (HSA) (Ka = 5.22 ×
105 M–1). A high bimolecular quenching
constant kq = 2.29 × 1013 M–1s–1 indicated a static quenching
mechanism involved in the fluorescence quenching of HSA by 1. Fluorescence resonance energy transfer theory suggested that the
distance (r = 3.52 nm) between 1 and
HSA is very close. Molecular docking studies suggested that 1 primarily binds to HSA in subdomain IIA. A protein–ligand
interaction profiler was used to visualize hydrophobic, hydrogen bonds,
and π–cation interactions between HSA and 1. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
assay using HeLa and MDA-MB-231 cells showed a significant in vitro
anticancer activity of 1 (IC50 2.63 and 2.68
μM, respectively). Nuclear staining assays suggested apoptotic
cell death in HeLa cells treated with 1. The effect of 1 on the cytoskeletal actin filaments visualized using phalloidin
staining showed extensive destruction of actin filaments. Flow cytometric
analysis indicated that 1 inhibits the growth of HeLa
cells through cell cycle arrest in the S phase. Western blot analysis
showed upregulation in the expression of apoptotic marker proteins
caspase 3, p53, and Bax. These results collectively indicate that 1 induces apoptosis by promoting DNA damage and has a high
potential to act as an anticancer agent.