葫芦素B通过AKT/mTOR和MAPK信号通路诱导非小细胞肺癌细胞自噬  

The AKT/mTOR and MAPK pathway are involved in the induction of autophagy by Cucurbitacin B in non-small cell lung cancer cells

作　　者：崔启迪 吕光耀 张金杰[2] 孙杉杉 陆梅 吕文文[2,3] CUI Qidi;LYU Guangyao;ZHANG Jinjie;SUN Shanshan;LU Mei;LYU Wenwen(Department of Oncology,Binzhou Medical University Hospital,Shandong Binzhou 256600,China;Department of Pharmacy,Binzhou Medical University Hospital,Shandong Binzhou 256600,China;School of Pharmacy,Binzhou Medical University,Shandong Yantai 264600,China)

机构地区：[1]滨州医学院附属医院肿瘤科,山东滨州256600 [2]滨州医学院附属医院药学部,山东滨州256600 [3]滨州医学院药学院,山东烟台264600

出　　处：《现代肿瘤医学》2024年第16期2929-2936,共8页Journal of Modern Oncology

基　　金：国家自然科学基金(编号:81502937);吴阶平医学基金(编号:320.6750.2021-10-18);齐鲁卫生与健康杰出青年人才项目(编号:鲁卫人才字[2020]3号);山东省中医药科技发展计划项目(编号:Q-2023105);滨州医学院科研基金(编号:BY2021KJ44)。

摘　　要：目的:观察葫芦素B(Cucurbitacin B,CuB)对非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞自噬的影响,探讨其可能的机制。方法:应用CCK-8法测定不同浓度CuB对A549细胞及H1299细胞增殖能力的影响。应用丹酰戊二胺(monodansylcadaverine,MDC)染色,荧光显微镜下观察细胞自噬,应用透射电镜观察自噬小体和自噬溶酶体。另外,应用自噬双标腺病毒(mRFP-GFP-LC3)进行转染,使用共聚焦显微镜观察药物处理后的自噬流变化。Western blot方法检测自噬相关标志物LC3II/I、p62及Beclin-1的表达水平变化。应用Western blot考察AKT、mTOR、ERK、p38、JNK蛋白及其磷酸化蛋白表达水平变化。结果:CuB呈剂量依赖抑制A549及H1299细胞增殖。MDC染色荧光显微镜下观察,药物处理组可见明显绿色致密斑点;药物处理48 h后透射电镜观察可见自噬溶酶体;mRFP-GFP-LC3转染A549及H1299细胞后显示CuB组红色荧光增加,提示自噬流增加;0.04μmol/L的CuB处理A549及1299细胞48 h后,LC3II/I及Beclin-1的蛋白表达增加,p62表达水平降低,AKT及mTOR蛋白磷酸化水平表达降低,ERK、JNK、p38 MAPK磷酸化蛋白水平升高。结论:本研究首次阐明了CuB在非小细胞肺癌中具有诱导自噬的能力,这种作用可能与抑制AKT/mTOR、激活MAPK信号通路有关。Objective:To examine the impact of Cucurbitacin B(CuB)on autophagy in non-small cell lung cancer cells and explore its underlying mechanism.Methods:CCK-8 method was used to determine the effect of CuB on the proliferation of A549 cells and H1299 cells.Autophagy was observed using fluorescent staining with monodansylcadaverine(MDC)under a fluorescence microscope,and autophagosomes were observed using transmission electron microscopy.In addition,the changes of autophagic flux after transfection of autophagy double-labeled adenovirus(mRFP-GFP-LC3)were detected by confocal microscopy.The levels of autophagy-related markers including LC3II/I,p62 and Beclin-1 were measured using Western blot.Additionally,the levels of AKT,mTOR,ERK,p38,JNK,p-AKT,p-mTOR,p-ERK,p-p38 and p-JNK were also examined.Results:CuB dose-dependently suppressed the growth of A549 and H1299 cells.In the drug treatment group,clear and dense green spots were observed after MDC staining.Transmission electron microscopy indicated the presence of autophagic lysosomes as well.After transfection of A549 and H1299 cells with mRFP-GFP-LC3,the red fluorescence was increased in CuB group.The expression levels of LC3II/I and Beclin-1 in A549 and 1299 cells were increased after CuB treatment at 0.04μmol/L for 48 h,while that p62 was decreased.Furthermore,the expression of p-AKT and p-mTOR were decreased,but p-ERK,p-JNK and p-p38 MAPK were elevated.Conclusion:This study firstly elucidated the potential of CuB to induce autophagy in NSCLC,the mechanism might be related to inhibition of AKT/mTOR and activation of MAPK signaling pathway.

关 键 词：葫芦素B 非小细胞肺癌 自噬 AKT MTOR MAPK 

分 类 号：R734.2[医药卫生—肿瘤]