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.本研究通过单晶X射线衍射（single-crystal X-ray crystallography）对分离得到的铜(II)配合物[CuL(邻菲啰啉（o-phen）)]·H₂O（1）[其中H₂L = o-HO-C₆H₄C(H)=N-C₆H₄-SH-o，邻菲啰啉（o-phen）即1,10-菲咯啉（1,10-phenanthroline）]进行了结构表征。在液氮温度下的乙腈（CH₃CN）溶液中，配合物1呈现出典型的单体型X波段电子顺磁共振（X-band electron paramagnetic resonance）谱，其具有四方对称性，g∥=2.1479，g⊥=2.0691，A∥≈18.0 mT，A⊥≤3.9 mT。配合物1与小牛胸腺DNA（calf thymus DNA）具有较强的结合亲和力，其固有结合常数（Kb=7.88×10⁵ M⁻¹）可佐证这一点；其对溴化乙锭（ethidium bromide）的竞争性置换实验表明，该配合物以嵌入模式与DNA结合（表观结合常数Kapp=1.32×10⁶ M⁻¹）。粘度测定实验进一步证实了这一结论：随着配合物1浓度的升高，DNA溶液的粘度随之增加。配合物1可高效介导DNA的氧化与水解切割（观测到的一级反应速率常数kobs=1.987 h⁻¹）。配合物1与载体蛋白人血清白蛋白（human serum albumin, HSA）具有较强的结合亲和力（结合常数Ka=5.22×10⁵ M⁻¹）。较高的双分子猝灭常数kq=2.29×10¹³ M⁻¹·s⁻¹表明，配合物1对HSA的荧光猝灭机制为静态猝灭。荧光共振能量转移（fluorescence resonance energy transfer, FRET）理论计算显示，配合物1与HSA之间的距离（r=3.52 nm）极近。分子对接（molecular docking）研究表明，配合物1主要结合于HSA的IIA亚结构域。本研究借助蛋白-配体相互作用分析仪（protein–ligand interaction profiler）可视化展示了HSA与配合物1之间的疏水作用、氢键作用以及π-阳离子相互作用。采用HeLa细胞与MDA-MB-231细胞进行的3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）比色实验结果显示，配合物1具有显著的体外抗肿瘤活性（两种细胞的半抑制浓度IC50分别为2.63 μM与2.68 μM）。核染色实验表明，经配合物1处理的HeLa细胞发生了凋亡性细胞死亡。鬼笔环肽（phalloidin）染色可视化显示，配合物1对细胞骨架肌动蛋白丝（cytoskeletal actin filaments）造成了广泛破坏。流式细胞术（flow cytometric analysis）分析结果表明，配合物1通过将HeLa细胞周期阻滞于S期来抑制细胞增殖。蛋白质免疫印迹（Western blot analysis）结果显示，凋亡标志物蛋白半胱天冬酶3（caspase 3）、p53以及Bax的表达均出现上调。上述实验结果共同表明，配合物1通过诱导DNA损伤介导细胞凋亡，具备开发为抗肿瘤候选药物的巨大潜力。