吡唑啉酮铜配合物通过内外信号途径诱导人肝癌细胞BEL-7404凋亡

Pyrazolone copper complex induces apoptosis in BEL-7404 hepatocellular carcinoma cells through extrinsic and intrinsic signaling pathways

目的探讨新型吡唑啉酮铜配合物(P-FAH-Cu-bpy)对人肝癌细胞BEL-7404的诱导凋亡机制。方法通过溶液法制备得到吡唑啉酮铜配合物P-FAH-Cu-bpy。X-射线单晶衍射实验测定晶体结构。MTT法检测P-FAH-Cu-bpy体外对BEL-7404细胞活性的影响。倒置显微镜和Hoechst 33258染色法观察细胞及核形态变化。流式细胞术检测P-FAHCu-bpy对BEL-7404细胞凋亡、周期、ROS、线粒体膜电位的影响。划痕实验检测细胞迁移。Western blot检测P-FAHCu-bpy对BEL-7404细胞凋亡、迁移及侵袭相关蛋白表达的影响。结果 P-FAH-Cu-bpy是一个具有扭曲的四方锥构型的五配位铜(Ⅱ)配合物;呈浓度和时间依赖性地抑制了BEL-7404细胞增殖,诱导了细胞凋亡,将细胞周期阻滞在G2/M期,降低了细胞线粒体膜电位,增加了胞内ROS水平;上调了细胞Bax、细胞色素C、Cleaved-caspase-3/9/8和Cleaved-PARP表达量,而下调了caspase-7、Bcl-2表达水平;抑制了细胞的迁移和侵袭。结论 P-FAH-Cu-bpy通过内外凋亡信号途径抑制了人肝癌细胞BEL-7404生长。

Aim To investigate the mechanism of apoptosis induced by novel pyrazolone copper complex(P-FAH-Cu-bpy)on human hepatoma cell line BEL-7404.Methods P-FAH-Cu-bpy was prepared by solution method.The crystal structure of P-FAH-Cu-bpy was determined by X-ray single crystal diffraction.The effect of P-FAH-Cu-bpy on the viability of BEL-7404 cells in vitro was detected by MTT assay.The morphology and apoptotic karyotype changes of BEL-7404 cells were observed by inverted microscope and Hoechst 33258 staining,respectively.The effects of P-FAH-Cu-bpy on apoptosis,cell cycle,ROS,mitochondrial membrane potential were detected by flow cytometry.Cell migration was detected by scratch test.The effects of P-FAH-Cu-bpy on apoptosis,migration and invasion-related protein expression in BEL-7404 cells were assessed by Western blot.Results P-FAH-Cu-bpy as a pentacoordinate copper(Ⅱ)complex with a twisted tetragonal pyramidal configuration suppressed cell proliferation in a dose-and time-dependent manner,induced apoptosis,arrested the cell cycle in the G2/M phase,decreased the mitochondrial membrane potential,and increased the levels of intracellular ROS.The expression levels of Bax,cytochrome C,Cleaved-caspase-3/9/8 and Cleaved-PARP were up-regulated,while the expression levels of caspase-7 and Bcl-2 were down-regulated;cell migration and invasion were inhibited.Conclusion P-FAH-Cu-bpy suppresses BEL-7404 cell growth through both extrinsic and intrinsic apoptosis pathways.