醉茄素A诱导人胆囊癌GBC-SD细胞凋亡及其可能的机制

Apoptosis of human gallbladder carcinoma GBC-SD cells induced by withaferin A and its possible mechanism

目的:探讨醉茄素A体外诱导人胆囊癌GBC-SD细胞凋亡及其可能的分子机制。方法:以不同浓度醉茄素A处理人胆囊癌GBC-SD细胞后,CCK-8法检测GBC-SD细胞的增殖情况,Hoechst33258荧光染色和透射电子显微镜下观察GBC-SD细胞的凋亡形态和超微结构变化,FCM法检测GBC-SD细胞的凋亡率和周期分布,蛋白质印迹法检测GBC-SD细胞中多聚ADP核糖聚合酶[poly(ADP-ribose)polymerase,PARP]、cyclin D1、cyclin B1、细胞周期蛋白依赖性激酶1(cyclin-dependent kinase1,Cdk1)和p21蛋白的表达。结果:醉茄素A对GBC-SD细胞的增殖有明显的抑制作用(P<0.05),并且这种抑制作用呈浓度和时间依赖性。醉茄素A能诱导GBC-SD细胞出现典型的凋亡形态学改变及超微结构变化。与未加药的阴性对照组比较,醉茄素A作用后GBC-SD细胞的凋亡率明显增加(P<0.01),细胞阻滞于G2/M期,细胞周期相关蛋白cyclin D1和Cdk1的表达下调(P<0.05),cyclin B1和p21蛋白的表达上调(P<0.05),PARP蛋白剪切体增多(P<0.05)。结论:醉茄素A可能通过下调细胞周期相关蛋白cyclin D1、Cdk1的表达和上调cyclin B1、p21的表达,使GBC-SD细胞阻滞于G2/M期,进而抑制GBC-SD细胞的增殖并诱导其凋亡。

Objective： To investigate WA （withaferin A）-induced apoptosis of human gallbladder carcinoma GBC-SD cells in vitro and to explore its possible mechanism. Methods： The proliferation of GBC-SD cells after treatment with different concentrations of WA was detected by CCK-8 （cell counting kit-8） assay. The apoptotic morphology and super-microstructural changes were observed by using Hoechst 33258 fluorescent staining and under a transmission electron microscope. The apoptosis rate and the cell cycle distribution of GBC-SD cells were detected by flow cytometry. The expressions of PARP [poly （ADP-ribose） polymerase], cyclin D1, cyclin B1, Cdkl （cyclin-dependent kinase 1） and p21 in GBC- SD cells were detected by Western blotting. Results： WA could significantly inhibit the proliferation of GBC-SD cells in a dose- and time-dependent manner （P 〈 0.05）. WA could also induce typical apoptotic morphological and super-microstructural changes of GBC-SD cells. As compared with the GBC-SD cells without WA treatment, WA could significantly increase the percentage of apoptotic GBC-SD cells （P 〈 0.01） and caused the cell arrest at G2/M phase. WA could also significantly down-regulate the expression levels of cyclin D1 and Cdkl proteins （P 〈 0.05）, whereas up-regulate the expression levels of cyclin B1 and p21 proteins （P 〈 0.05） and promote the cleavage of PARP （P 〈 0.05）. Conclusion： WA could induce the GBC-SD cell arrest at G2/M phase by down-regulating the expression levels of cyclin D1 and Cdkl and up-regulating the expression levels of cyclin B1 and p21. These effects may lead to the suppression of cellular proliferation and the aDoDtosis of GBC-SD cells.