DSF-Cu通过影响线粒体功能和细胞骨架诱导鼻咽癌CNE-2Z细胞凋亡

DSF-Cu induces apoptosis of human nasopharyngeal carcinoma CNE-2Z cells by affecting function of mitochondria and cytoskeleton

目的探测双硫仑(disulfiram,DSF)螯合氯化铜(DSF-Cu)通过影响线粒体功能和细胞骨架诱导人低分化鼻咽癌CNE-2Z细胞凋亡。方法采用流式细胞术检测细胞周期、凋亡率、活性氧(reactive oxygen species,ROS)的生成以及线粒体膜电位(mitochondrial membrane potential,MMP)的改变。AFM探测细胞表面形貌和超微结构、细胞高度、宽度及粗糙度的变化。激光扫描共聚焦显微镜(laser scanning confocal microscope,LSCM)观察细胞骨架F-actin的分布和重组。结果细胞周期检测显示DSF-Cu将CNE-2Z细胞周期阻滞于G2/M期。Annexin-V/PI检测细胞凋亡显示随药物浓度增加,与对照组相比,实验组早凋率和晚凋率、坏死率细胞均明显增加。进一步检测凋亡相关信号发现DSF-Cu可促进CNE-2Z细胞内活性氧水平的增加及诱导MMP下降。AFM成像表明,与对照组相比,随着药物浓度增大,细胞逐渐变小,胞质浓缩,高度增加,膜表面粗糙度降低,变得平整光滑;细胞伪足由对照组的丰富密集,逐渐变短、萎缩,甚至完全破坏。LSCM进一步观察发现DSF-Cu引起F-actin荧光强度减弱,结构重排,严重影响微丝的功能。结论 DSF-Cu可诱导CNE-2Z细胞依赖线粒体的凋亡途径,利用AFM在单细胞水平上分析DSF-Cu对细胞膜的毒性作用,为鼻咽癌的治疗提供新的思路。

Aim To study the mechanism of DSF-Cu induced apoptosis of human nasopharyngeal carcinomaCNE-2Z cells by affecting the function of mitochondria and cytoskeleton. Methods The cell cycle,the rate of apotosis,the levels of intracellular ROS and MMP in CNE-2Z cells were tested by flow cytometry after treated with different concentration of DSF-Cu. The changes of the cell surface morphology,ultrastructure,cell height,width and roughness were detected by AFM.The distribution and reorganization of cytoskeleton Factin were observed by Laser scanning confocal microscope. Results Cells were incubated with different concentration of DSF-Cu（ 0 ~ 200 nmol ·L- 1） for 24 h,the apoptotic ratio increased significantly and the treatment of DSF-Cu resulted in a concentration-dependent accumulation of CNE-2Z cells in G2/ M phase.Furthermore,the treatment of DSF-Cu was able to increase the production of intracellular ROS and decrease the MMP in CNE-2Z cells. In addition,AFM imaging showed that compared to the control group,with the in-crease of DSF-Cu concentration,the CNE-2Z cells became smaller,cytoplasm condensed,the height increased,and the surface roughness reduced. Moreover,the filopodia became shorter,shrinked and even completely destroyed after treated with different concentration of DSF-Cu. At last,the LSCM image showed that the fluorescence intensity of F-actin networks was decreased,then the structure was rearranged and destroyed obviously by treated with DSF-Cu. Conclusion DSF-Cu can induce apoptosis and arrest cell cycle at G_2/ M phase in CNE-2Z cell through a mitochondriadependent pathway. Above findings highlight the applications of AFM at the single cell level for the investigation of antineoplastic drug in nasopharyngeal carcinoma therapy.