雷公藤甲素促进三阴性乳腺癌细胞凋亡的机制研究

Effect of triptolide on apoptosis of triple-negative breast cancer cells and its mechanism

目的 研究雷公藤甲素(Tpl)对人三阴性乳腺癌(TNBC)细胞凋亡的促进作用及可能的分子机制。方法 体外培养TNBC细胞株MDA-MB-453细胞并分为4组,对照组正常培养,Tpl低、中、高剂量组分别添加10、20和40 nmol/L的Tpl;MDA-MB-453细胞分别转染胎盘特异性8基因(PLAC8)过表达质粒和空载体质粒,添加或不添加20 nmol/L处理,分别记作过表达组、过表达+Tpl组、空载组、空载+Tpl组。采用细胞增殖和毒性检验(CCK-8)法检测细胞增殖能力,采用克隆形成实验分析克隆形成能力,采用Transwell实验分析细胞迁移能力;应用流式细胞术检测细胞凋亡情况;利用Western blot法分析切割型半胱氨酸蛋白酶(Cle-Caspase)-3、Cle-Caspase-9、B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、PLAC8、Wnt3a及β-连环蛋白(β-catenin)相对表达量。结果 与对照组比较,低、中、高剂量组细胞克隆形成率及迁移率均降低,凋亡率均升高,Cle-Caspase-3、Cle-Caspase-9、Bax蛋白相对表达量均升高,Bcl-2、Wnt3a、β-catenin、PLAC8蛋白相对表达量均下降(均P<0.05)。与空载组比较,空载+Tpl组的上述指标表达趋势一致,过表达组上述指标表达均相反(均P<0.05)。与过表达组比较,过表达+Tpl组各指标差异均无统计学意义(均P>0.05)。结论 Tpl能促进MDA-MB-453细胞株凋亡,该机制可能与PLAC8介导的Wnt/β-catenin信号通路有关。

Objective To investigate the effect of triptolide(Tpl)on the proliferation of human triple-negative breast cancer(TNBC)cells and its mechanism.Methods TNBC MDA-MB 453 cells were cultured in vitro and treated with 0(control group),10(low-dose group),20(medium-dose group)and 40 nmol/L(high-dose group)Tpl,respectively.MDA MB-453 cells were transfected with placenta-specific protein 8(PLAC8)over-expression plasmid or empty vector plasmid to establish PLAC8-overexpression cell line(OE-PLAC8)and control cell line(Vector),then treated with 20 nmol/L Tpl,respectively.Cell count kit(CCK-8)assay was used to detect cell proliferation capacity.Clone formation assay was used to analyze clone formation capacity and Transwell assay was used to analyze cell migration capacity.Flow cytometry was used to detect the apoptosis.Western blot analysis was performed to detect the expression level of cleaved Caspase 3,cleaved Caspase 9,Bcl 2,Bax,PLAC8,Wnt 3a andβ-catenin proteins.Results Compared with the control group,the clone formation rate and mobility of MDA-MB 453 cells in the low-,medium-and high-dose Tpl groups were decreased,the apoptosis rate was increased,and the protein expressions of cleaved Caspase 3,cleaved Caspase 9 and Bax were increased,the expression of Bcl 2,Wnt3a,β-catenin and PLAC8 protein decreased(all P<0.05).Compared with Vector group,the expression trend of the above indicators in the Vector+Tpl group was consistent,while the above indicators in OE-PLAC8 group were opposite(P<0.05).Compared with OE-PLAC8 group,there was no significant difference in all indexes in OE-PLAC8+Tpl group(P>0.05).Conclusion Tpl can promote apoptosis in MDA-MB 453 cells,which may be related to PLAC8-mediated Wnt/β-catenin signaling pathway.