鸦胆子素A诱导非小细胞肺癌细胞株H460凋亡及机制的初步研究  

Bruceine A induces apoptosis in non-small-cell lung cancer cell line H460 by regulating mitochondria-related apoptosis signaling

作　　者：李聪 王燕[1] 赵晓丽[2] 王真[2] 蒋建东[1] LI Cong;WANG Yan;ZHAO Xiao-li;WANG Zhen;JIANG Jian-dong

机构地区：[1]中国医学科学院北京协和医学院药物研究所,北京100050 [2]中国医学科学院医药生物技术研究所,北京100050

出　　处：《中国医药生物技术》2019年第4期294-301,共8页Chinese Medicinal Biotechnology

基　　金：国家自然科学基金(500101206)

摘　　要：目的考察鸦胆子素A(BA)对人非小细胞肺癌细胞系H460细胞凋亡的影响和作用机制。方法用BA对H460细胞进行处理,采用MTT法检测细胞增殖情况,并利用Annexin V-FITC/PI双染法检测BA对H460细胞凋亡的影响。Hoechst 33342染色和免疫荧光染色观察细胞凋亡时染色质的凝集以及DNA损伤标志蛋白γ-H2AX表达情况。Western blot和RT-PCR检测H460细胞内凋亡相关蛋白表达水平。结果MTT结果表明,与对照组相比,BA能显著抑制H460细胞增殖。对A549、H1299、H1355三株非小细胞肺癌细胞系进行抑制增殖作用检测,结果显示BA对三株细胞系均有不同程度抑制作用。BA作用48h后,各浓度BA处理H460细胞凋亡率均高于对照组,具有时间依赖性,差异具有统计学意义。BA处理组可见明显的染色质凝集,细胞核碎片化,DNA损伤标志蛋白γ-H2AX表达明显增加。Western blot结果显示,BA作用48h后,促凋亡蛋白Bax表达升高,抗凋亡蛋白Bcl-2表达降低,与对照组相比,Bax/Bcl-2比值显著升高。同时与对照组相比,凋亡通路中的cleaved caspase-9、cleaved caspase-3表达明显增加,并引起凋亡标志蛋白PARP切割。RT-PCR结果显示,BA处理H460细胞后,与对照组相比,Bcl-2基因表达显著降低。结论BA能抑制人非小细胞肺癌细胞株H460细胞增殖,显著诱导其凋亡,该作用与BA引起DNA损伤并激活线粒体凋亡通路有关。Objective We aim to investigate whether bruceine A (BA) induces apoptosis in human non-small-cell lung cancer cell (NSCLC) H460 cells and to learn the mechanism.Methods The proliferation of NSCLC cell lines,including H460,A549,H1299 and H1355,was detected by the MTT assay after treatment with BA.Effect of BA on apoptosis in the H460 cells was measured by FACS analysis with Annexin V-FITC/PI staining.Hoechst 33342 and immunofluorescence (IF) staining were used to observe the chromatin condensation and the expression of the DNA damage marker protein γ-H2AX,respectively.Western blot and real-time PCR were used to detect the expression of apoptosis-related protein and mRNA in the H460 cells,respectively.Results Based on MTT result,the proliferation was significantly inhibited in H460 cells after treatment with BA for 48 h.The proliferation inhibition of other NSCLC cell lines A549,H1299 and H1355 upon BA treatment was detected,and BA inhibited the proliferation of the three cell lines in a dose-dependent manner.Significant induction of apoptosis was observed in a time-dependent fashion upon BA treatment in the H460 cells.Also,remarkable nuclear condensation was observed following BA treatment in H460 cells using Hoechst 33342 staining.DNA damage marker γ-H2AX was increased by BA as evidenced by IF staining.Furthermore,BA increased and decreased the expression of Bax and Bcl-2,respectively,and cleaved caspase-9,caspase-3 and PARP protein,as demonstrated by Western blot.Also,Bcl-2 gene expression in the H460 cells was significantly decreased after BA treatment.Conclusion BA inhibits the H460 cells proliferation by inducing apoptosis,which may be mediated through the mitochondria-dependent apoptotic signaling pathways.

关 键 词：鸦胆子素 细胞凋亡 癌 非小细胞肺 H460细胞 

分 类 号：R285[医药卫生—中药学]