摘要/Abstract

DNA是抗肿瘤药物的重要靶点，研究药物分子与DNA之间的作用有助于设计靶向DNA类抗肿瘤药物.合成和表征了新的三元铜（II）配合物[Cu（Sf）（PyTA）（H₂O）]·ClO₄·3.5H₂O[Sf=司帕沙星，5-氨基-1-环丙基-7-（顺-3，5-二甲基-1-哌嗪基）-6，8-二氟-1，4-二氢-4-氧-3-喹啉羧酸、PyTA=2，4-二氨基-6-（2'-吡啶基）-1，3，5-均三嗪].利用电子吸收光谱、KI荧光猝灭光谱、粘度测定以及分子对接技术研究了配合物与DNA之间作用，发现配合物以插入模式与DNA结合，结合常数K_b=1.23×10⁴ L/mol.此外，应用MTT[3-（4，5-二甲基噻唑-2）-2，5-二苯基四氮唑溴盐]比色法检测了配合物的细胞毒性作用，发现配合物对癌细胞A549、Bel-7402和Eca-109等表现出良好的抑制作用[IC₅₀=（57.0±1.6）~（77.6±1.4）μmol/L].尤为重要的是，通过单细胞凝胶电泳、Hoechst 33342染色、Annexin V-FITC/PI双染流式细胞术、测定线粒体膜电位、检测细胞色素C和胞内Ca²⁺水平及细胞周期分析探究了配合物抗肿瘤作用机制，结果表明，配合物通过DNA结合及线粒体功能障碍途径诱导细胞凋亡，使细胞S和G2/M周期发生阻滞并造成DNA损伤.
关键词: 铜(II)配合物, 司帕沙星, DNA, 抗肿瘤活性
DNA is an important target for antitumor drugs, hence investigation of the interaction between drug molecules and DNA can help to design targeted DNA antitumor drugs. New ternary copper(II) complex[Cu(Sf)(PyTA)(H₂O)]·ClO₄· 3.5H₂O[Sf=sparfloxacin, 5-amino-1-cyclopropyl-7-(cis-3,5-dimethyl-1-piperazinyl)-6,8-difluoro-1,4-dihydro-4-oxoquino-line-3-carboxylic acid, PyTA=2,4-diamino-6-(2'-pyridyl)-1,3,5-triazine] was synthesized and characterized by elemental analyses, molar conductivity measurement and various spectroscopic techniques such as infrared, ultraviolet-visible, and electrospray ionization mass spectra. The interaction of the complex with DNA was investigated using electronic absorption spectroscopy, KI fluorescence quench, viscosity measurement and molecular docking techniques. It was found that the complex could bind to DNA through an intercalation mode being related with the quinoline ring of ligand Sf, and the corresponding binding constant K_b is 1.23×10⁴ L/mol. Moreover, the antitumor activity of the complex was evaluated using the MTT[3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide] method, revealing that the complex displayed favorable cytotoxic effects[IC₅₀=(57.0±1.6)~(77.6±1.4) μmol/L] toward cancer cells (A549, Bel-7402 and Eca-109) and less toxic towards normal cells (3T3). Most importantly, the cytotoxic mechanism of the complex towards Eca-109 cells was explored by single cell gel electrophoresis assay, Hoechst 33342 staining, Annexin V-FITC/PI double dye flow cytometry, measurement of mitochondrial membrane potential change, detection of intracellular cytochrome C and Ca²⁺ levels, and test of cell cycle arrest. Single cell gel electrophoresis assay (comet assays) demonstrated that the complex could damage DNA and cause apoptosis. Double staining analysis showed that the complex could induce apoptosis in Eca-109 cells. Cell cycle arrest studies revealed the cell growth arrest at S and G2/M phases. The complex also could induce a reduction in the mitochondrial membrane potential and release of the cytochrome C, and increase the intracellular Ca²⁺ level. The results demonstrated that the complex could induce apoptosis in Eca-109 cells through DNA-binding mitochondrial dysfunctional pathways, which was accompanied by the cell growth arrest at S and G2/M phases and damage of DNA.
Key words: copper(II) complex, sparfloxacin, DNA, antitumor activity


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