水仙环素通过调控PLK1/AKT信号通路诱导三阴性乳腺癌细胞周期阻滞和凋亡

Narciclasine induces cell cycle arrest and apoptosis in triple-negative breast cancer cells by regulating PLK1/AKT signaling pathway

目的研究水仙环素(Nas)对人三阴性乳腺癌(TNBC)细胞周期和凋亡的抑制作用及分子机制。方法采用细胞增殖-毒性检测试剂盒(CCK-8)、克隆形成、Transwell、流式细胞术等实验分析Nas对TNBC细胞株MDA-MB-231细胞增殖和侵袭能力的抑制作用,及对细胞周期和凋亡的诱导作用;利用免疫印迹(WB)实验分析Nas对MDA-MB-231细胞株cle-Caspase-3、PARP、cle-PARP、PLK1、PI3K、p-PI3K、AKT、p-AKT、CDK1、Cyclin B、Cyclin A、p-21、p-27、p-62、LC3、γ-H2AX等蛋白质的影响。结果不同浓度(0.5、1.0、2.0μmol/L)Nas能抑制MDA-MB-231细胞株的克隆形成(48 h抑制率分别为15.99%、24.76%和54.17%)、侵袭能力(48 h抑制率分别为0.68%、23.20%和45.27%),可将细胞周期阻滞于G2期(48 h阻滞率分别为75.50%、140.56%和316.06%),可对细胞凋亡起到诱导作用(48 h凋亡诱导率分别为105.39%、199.73%和375.74%;72 h凋亡诱导率分别为104.45%、244.96%和709.60%)。分子机制实验显示,Nas可以显著促进cle-Caspase-3、cle-PARP、p-21、p-27及γ-H2AX等蛋白质的表达,显著抑制PLK1、p-PI3K、p-AKT、CDK1、Cyclin B等蛋白质的表达水平,而对p-62、LC3-Ⅱ/Ⅰ等蛋白质的表达水平影响不明显。结论Nas能抑制MDA-MB-231细胞株的克隆形成、侵袭能力,对凋亡起到诱导作用,并将细胞周期阻滞于G2期,该机制可能通过调节PLK1/AKT信号通路来实现。

Objective To investigate the inhibitory effect of narciclasine(Nas)on cell cycle and apoptosis in human triple negative breast cancer(TNBC)cells and the underlying molecular mechanism.Methods Cell proliferation assay(CCK-8),colony formation,Transwell,and flow cytometry were performed to analyze the inhibitory effect of Nas on the proliferation and invasion ability,as well as its induction effects on cell cycle arrest and apoptosis of TNBC cell line MDA-MB-231.Western blot analysis was performed to investigate the effects of Nas on the protein expression levels of cle-caspase-3,PARP,cle-PARP,PLK1,PI3K,p-PI3K,AKT,p-AKT,CDK1,cyclin B,cyclin A,p-21,p-27,p-62,LC3andγ-H2AX in MDA-MB-231 cells.Results Nas at different concentrations(0.5,1.0,and 2.0μmol/L)inhibited colony formation(inhibition rates at 48 h:15.99%,24.76%,and 54.17%,respectively),and the invasion(inhibition rates at 48 h:0.68%,23.20%,and 45.27%,respectively)of the MDA-MB-231 cells.Nas arrested cell cycle at the G2 phase(arrest rates at 48 h:75.50%,140.56%,and 316.06%,respectively)and induced apoptosis(the apoptosis rates at 48 h:105.39%,199.73%,and 375.74%,respectively;at 72 h:104.45%,244.96%,and 709.60%,respectively)Molecular mechanism experiments showed that Nas significantly promoted the protein expression levels of cle-Caspase-3,cle-PARP,p-21,p-27,andγ-H2AX,but significantly inhibited the expression levels of PLK1,p-PI3K,p-AKT,CDK1,and Cyclin B.However,it had no significant effect on the expression of p-62 or LC3-Ⅱ/Ⅰratio.Conclusion Nas can inhibit the colony formation and invasion ability of MDA-MB-231 cells,induce apoptosis,and arrest the cell cycle at the G2 phase,which may be achieved by regulating the PLK1/AKT signaling pathway.