CA-4类衍生物LGD5诱导人宫颈癌HeLa细胞发生G2/M周期阻滞和凋亡的机制研究

Study of the mechanism of combretastatin a-4 derivative LGD5 induced G2/M cycle arrest and apoptosis in human cervical cancer HeLa cells

目的:探究微管抑制剂CA-4类衍生物LGD5对人宫颈癌HeLa细胞的作用机制。方法:选用HeLa细胞,分为空白组、CA-4阳性对照组、不同浓度的LGD5组成实验组。采用MTT法考察LGD5对HeLa细胞的生长抑制情况并确定实验浓度,采用倒置显微镜和吖啶橙染色观察用药前后HeLa细胞形态学变化及细胞凋亡情况,采用DAPI免疫荧光染色考察LGD5对微管的作用,采用PI流式细胞术考察LGD5对细胞周期的影响,采用蛋白免疫印迹法考察LGD5对细胞周期蛋白和凋亡相关蛋白的影响。结果:MTT实验结果显示LGD5抑制HeLa细胞的生长抑制具有时间依赖性和剂量依赖性。定时拍照和吖啶橙染色观察到LGD5诱导HeLa细胞发生凋亡,并产生明显的染色质凝集和凋亡小体。DAPI染色观察到LGD5抑制HeLa细胞的微管聚合。PI流式细胞术结果显示LGD5诱导HeLa细胞发生G2/M周期阻滞,在12 h内具有时间依赖性和剂量依赖性,且差异有统计学意义(P<0.01),24 h以后引起细胞凋亡且具有时间依赖性;Western blot结果显示,LGD5下调Cdc2和Cdc25C,上调p-Cdc2、Cyclin B1和p-histone H3,进一步验证LGD5诱导HeLa细胞发生G2/M周期阻滞,除此之外,LGD5使HeLa细胞Caspase 3表达增加,上调Caspase 9和Bax,下调Pro-caspase 9和Bcl-2,说明LGD5诱导HeLa细胞凋亡与线粒体途径有关。结论:CA-4类衍生物LGD5可抑制HeLa细胞的微管聚合,诱导其发生G2/M周期阻滞,进而通过线粒体途径诱导细胞发生凋亡。

AIM:To explore the mechanism of action of the microtubular inhibitor of CA-4 derivative LGD5 on human cervical cancer HeLa cells.METHODS:HeLa cells were selected and divided into blank group,CA-4 positive control group,and different concentrations of LGD5 were formed into the experimental group.MTT was used to investigate the growth inhibition of LGD5 on HeLa cells and to determine the assay concentration.Cell morphology and apoptosis were observed before and after drug administration by inverted microscope and acridine orange staining.Immunofluorescence staining was used to examine the effect of LGD5 on microtubules using DAPI.The effect of LGD5 on cell cycle by PI flow cytometry.Protein immunoblotting was used to examine the effect of LGD5 on cyclins and apoptosis-related proteins.RESULTS:MTT experiments showed that LGD5 inhibited HeLa cells in a time-and dose-dependent manner.Timed photography and acridine orange staining observed that LGD5 induced apoptosis in HeLa cells and produced significant chromatin agglutination and apoptotic bodies.Inhibition of microtubule polymerization in HeLa cells by LGD5 was observed by DAPI staining.The PI flow cytometry results showed that LGD5 induced G2/M cycle arrest in HeLa cells,was time-dependent and dose-dependent within 12 h,and had a significant difference(P<0.01),apoptosis was induced after 24 h and it was time-dependent.The results of Western blot show that,LGD5 downregulates Cdc 2 and Cdc25C,upregulation of p-Cdc 2,and Cyclin B1 and p-histone H3,further verified that LGD5 induced G2/M cycle arrest in HeLa cells,besides,LGD5 caused increased Caspase 3 expression in HeLa cells,upregulated Caspase 9 and Bax,down-regulation of Procaspase 9 and Bcl-2,this result indicates that HeLa cell apoptosis induced by LGD5 is related to the mitochondrial pathway.CONCLUSION:The CA-4 derivative LGD5 inhibited microtubule polymerization in HeLa cells,induced their G2/M cycle arrest,and subsequently induced cell apoptosis through the mitochondrial pathway.