摘要
目的探讨双花母草素(PRO)调节蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路对非小细胞肺癌(NSCLC)细胞恶性生物学行为的影响。方法体外培养人非小细胞肺癌(NSCLC)细胞系A549,用CCK8检测PRO对A549细胞活性的影响;将A549细胞分为NC组、PRO低浓度处理组(PRO-L组,2.5μmol/L)、PRO中浓度处理组(PRO-M组,5μmol/L)、PRO高浓度处理组(PRO-H组,10μmol/L)、PRO-H+Akt/mTOR通路激活剂(740Y-P)组(10μmol/L PRO+30μmol/L 740Y-P);克隆形成实验检测各组A549细胞增殖情况,TUNEL染色法检测各组A549细胞凋亡情况,划痕实验和Matrigel侵袭实验分别检测各组A549细胞迁移和侵袭能力;qRT-PCR和Westren Blot实验检测上皮间质转化(EMT)和Akt/mTOR通路相关基因的表达情况。构建NSCLC裸鼠移植瘤模型,将造模成功的裸鼠分为:模型(Model)组、PRO组、PRO+740Y-P组,观察各组裸鼠肿瘤组织生长情况,并检测各组肿瘤组织中Akt、p-Akt、mTOR、p-mTOR蛋白表达情况。结果与NC组相比,PRO-L组、PRO-M组和PRO-H组的A549细胞克隆形成数目、划痕愈合率和侵袭细胞数减少,Akt、mTOR、Vimentin mRNA及p-Akt、p-mTOR、Vimentin蛋白表达量明显下降,细胞凋亡率、E-cadherin mRNA和其蛋白表达量显著上升(P<0.05);与PRO-H组相比,PRO-H+740Y-P组A549细胞克隆形成数目、划痕愈合率和侵袭细胞数明显增多,Akt、mTOR、Vimentin mRNA和p-Akt、p-mTOR、Vimentin蛋白表达量明显上升,细胞凋亡率、E-cadherin mRNA和蛋白表达量明显下降(P<0.05)。与模型组相比,PRO组裸鼠肿瘤组织质量、体积及p-Akt、p-mTOR蛋白表达量显著降低(P<0.05);与PRO组相比,PRO+740Y-P组裸鼠肿瘤组织质量、体积及p-Akt、p-mTOR蛋白表达量显著升高(P<0.05)。结论PRO可通过抑制Akt/mTOR信号通路的激活从而实现对NSCLC细胞恶性生物学行为的抑制作用。
Objective To investigate the effect of prunetin(PRO)on the malignant biological behaviors of non-small cell lung cancer(NSCLC)cells by regulating the protein kinase B(Akt)/mammalian target of rapamycin(mTOR)signaling pathway.Methods Human NSCLC cell line A549 was cultured in vitro.The Cell Counting Kit-8(CCK-8)assay was used to detect the effect of PRO on A549 cell viability.A549 cells were divided into five groups:negative control(NC)group,low-concentration PRO treatment group(PRO-L group,2.5μmol/L),medium-concentration PRO treatment group(PRO-M group,5μmol/L),high-concentration PRO treatment group(PRO-H group,10μmol/L),and PRO-H+Akt/mTOR pathway activator(740Y-P)group(10μmol/L PRO+30μmol/L 740Y-P).Colony formation assay was performed to evaluate cell proliferation;TUNEL staining was used to detect cell apoptosis;wound-healing assay and Matrigel invasion assay were conducted to measure cell migration and invasion abilities,respectively.Quantitative real-time polymerase chain reaction(qRT-PCR)and Western blot were used to detect the expression levels of genes related to epithelial-mesenchymal transition(EMT)and the Akt/mTOR pathway.A nude mouse xenograft model of NSCLC was established,and successfully modeled nude mice were divided into three groups:Model group,PRO group,and PRO+740Y-P group.The growth of tumor tissues in nude mice was observed,and the protein expression levels of Akt,phosphorylated Akt(p-Akt),mTOR,and phosphorylated mTOR(p-mTOR)in tumor tissues were detected.Results Compared with the NC group,the PRO-L,PRO-M,and PRO-H groups showed decreased colony formation number,wound healing rate,and number of invasive cells in A549 cells;significantly reduced mRNA expression levels of Akt,mTOR,and Vimentin,as well as protein expression levels of p-Akt,p-mTOR,and Vimentin;and significantly increased cell apoptosis rate,mRNA expression level of E-cadherin,and its protein expression level(all P<0.05).Compared with the PRO-H group,the PRO-H+740Y-P group exhibited significantly increased colony formation number,wound healing rate,and number of invasive cells;remarkably elevated mRNA expression levels of Akt,mTOR,and Vimentin,as well as protein expression levels of p-Akt,p-mTOR,and Vimentin;and significantly decreased cell apoptosis rate,mRNA expression level of E-cadherin,and its protein expression level(all P<0.05).Compared with the Model group,the PRO group showed significantly reduced tumor weight,volume,and protein expression levels of p-Akt and p-mTOR in nude mice(all P<0.05).Compared with the PRO group,the PRO+740Y-P group had significantly increased tumor weight,volume,and protein expression levels of p-Akt and p-mTOR(all P<0.05).Conclusion PRO can inhibit the malignant biological behaviors of NSCLC cells by suppressing the activation of the Akt/mTOR signaling pathway.
作者
赵晓静
常丽薇
崔永丽
李燕涛
薛艳超
ZHAO Xiaojing;CHANG Liwei;CUI Yongli;LI Yantao;XUE Yanchao(Respiratory and Critical Care Medicine Department Ⅱ Ward,Handan Central Hospital,Handan,Hebei 056000,China)
出处
《临床肺科杂志》
2026年第1期82-89,共8页
Journal of Clinical Pulmonary Medicine
基金
邯郸市科学技术研究与发展计划项目(21422083138)。
