华蟾素诱导人胃癌MKN-45细胞凋亡

Cinobufacin Induces the Apoptosis of Human Gastric Cancer MKN-45 Cells

本研究旨在探讨华蟾素诱导人胃癌MKN-45细胞凋亡及其机制。分别用终浓度0、60、70、80μg/L的华蟾素作用于人胃癌MKN-45细胞48 h,采用激光共聚焦显微镜观察细胞形态结构,流式细胞术检测细胞凋亡率、线粒体膜电位,RT-qPCR和Western blot分别检测Bax、Bcl-2、Cyt C、Caspase 9和Caspase 3基因表达水平。结果显示,与对照组相比,0、60、70、80μg/L的华蟾素作用人胃癌MKN-45细胞48 h,呈现细胞皱缩、细胞核裂解、染色质凝集等形态学变化,早期凋亡细胞和晚期凋亡细胞所占百分比均显著增加,线粒体膜电位(ΔΨm)显著降低。Bax、Cyt C、Caspase 9和Caspase 3基因的m RNA及蛋白表达水平随着药物浓度的升高均显著升高(P<0.01),Bcl-2基因mRNA及蛋白表达水平随着药物浓度的升高显著降低(P<0.01),提示华蟾素可通过上调Bax、Cyt C、Caspase 9和Caspase 3基因表达,下调Bcl-2基因表达诱导人胃癌MKN-45细胞凋亡。

This study was to investigate the effect of cinobufacin on human gastric cancer MKN-45 apoptosis.After treatment with 0, 60, 70, and 80 μg/L concentrations of cinobufacin for 48 h, the morphology of the cells was observed by laser confocal microscopy. The apoptosis, mitochondrial membrane potential( ΔΨm)were assayed by flow cytometry. The m RNA and protein levels of Bax, Bcl-2, Cyt C, Caspase 9 and Caspase 3 were measured by RT-qPCR and Western blot. Compared with the control group, the results showed that 0,60, 70, and 80 μg/L concentration of cinobufacin on human gastric cancer MKN-45 cells for 48 h, morphological changes such as cell shrinkage, nuclear lysis, chromatin condensation were observed by laser confocal microscopy, and the percentage of early apoptotic cells and late apoptotic cells increased significantly and the level of ΔΨm decreased. In addtion, the mRNA and protein expressions of Bax, Cyt C, Caspase 9 and Caspase 3 significantly increased(P<0.01), while the mRNA and protein expression of Bcl-2 significan-tly decreased(P<0.01). The results suggested that cinobufacin induced the apoptosis of human gastric cancer MKN-45 cells through up-regulation of Bax, Cyt C, Caspase 9 and Caspase 3 and down-regula-tion of Bcl-2.