黄卡瓦胡椒素B与放线菌素D协同诱导核仁应激抑制三阴性乳腺癌细胞增殖  

Inhibition of triple-negative breast cancer cell proliferation by synergistic induction of nucleolar stress by Flavokawain B and Actinomycin D

作　　者：高自立 潘灵慧 高绮雪 贺业淞 李雪森 GAO Zili;PAN Linghui;GAO Qixue;HE Yesong;LI Xuesen(Institute for Cancer Medicine,School of Basic Medical Sciences,Southwest Medical University,Sichuan Luzhou 646000,China)

机构地区：[1]西南医科大学基础医学院肿瘤医学研究所,四川泸州646000

出　　处：《现代肿瘤医学》2024年第23期4432-4442,共11页Journal of Modern Oncology

基　　金：四川省科技计划联合创新专项(编号:2022YFS0623)。

摘　　要：目的:探究黄卡瓦胡椒素B(Flavokawain B,FKB)与放线菌素D(Actinomycin D,ActD)协同抑制三阴性乳腺癌细胞增殖的效果及其协同抗癌机制。方法:培养SUM159PT、HS578T两种三阴性乳腺癌细胞,使用MTT法评估不同浓度的FKB和ActD两种单药以及适当浓度的两药联合对这两种癌细胞增殖的抑制效果。流式细胞术检测两种三阴性乳腺癌细胞经过FKB和ActD单药及联合药物处理后对细胞周期和凋亡的影响。RT-qPCR实验检测两种三阴性乳腺癌细胞经过FKB和ActD单药及联合药物处理后未组装为核糖体的游离rRNA变化情况。细胞免疫荧光实验观察两种三阴性乳腺癌细胞经过FKB和ActD单药及联合药物处理后NPM1蛋白的核内定位,以标识细胞核仁应激触发情况。Western blot实验检测两种三阴性乳腺癌细胞经过FKB和ActD单药及联合药物处理后细胞周期和凋亡相关蛋白的变化。结果:FKB与ActD协同抑制了两种三阴性乳腺癌细胞增殖,流式细胞术结果显示联合用药显著诱导更多的三阴性乳腺癌细胞凋亡(P<0.01),并影响了细胞周期进程,且在使用凋亡抑制剂抑制凋亡信号通路和血清剥夺法控制细胞周期后可以减弱联合用药对细胞增殖的抑制(P<0.01)。RT-qPCR结果显示联合用药处理造成三阴性乳腺癌细胞游离的成熟和未成熟18S、28S rRNA显著蓄积(P<0.01),提示细胞核糖体RNA剪切加工或组装受到严重干扰,细胞免疫荧光结果显示联合用药后三阴性乳腺癌细胞NPM1蛋白出核仁弥散分布于细胞核,表明联合用药对三阴性乳腺癌细胞核糖体RNA剪切加工或组装的干扰触发了细胞核仁应激反应。Western blot结果显示联合用药显著诱导了p21蛋白上调、Bcl2蛋白下调以及Caspase3蛋白和PARP蛋白的切割。结论:FKB与ActD可能通过协同干扰核糖体RNA的剪切加工或组装破坏核仁稳态触发核仁应激反应,并诱导下游p21蛋白的升高进而导致三阴性乳腺癌细Objective:To investigate the synergistic inhibition of triple-negative breast cancer cell proliferation by Flavokawain B(FKB)and Actinomycin D(ActD),as well as their synergistic anticancer mechanism.Methods:Cultivate SUM159PT and HS578T triple-negative breast cancer cells,and employ the MTT assay to assess the inhibitory effects of different concentrations of FKB and ActD monotherapies,as well as appropriate concentrations of combination therapy,on the proliferation of these two types of cancer cells.To utilize flow cytometry to examine the effects of FKB and ActD monotherapies,as well as combination therapy,on cell cycle progression and apoptosis in the two types of triple-negative breast cancer cells.Conduct RT-qPCR experiments to analyze the changes in free rRNA that were not assembed into ribosomes following treatment with FKB and ActD monotherapies,as well as combination therapy,in the two types of triple-negative breast cancer cells.Perform cell immunofluorescence experiments to observe the nuclear localization of NPM1 protein in the two types of triple-negative breast cancer cells after treatment with FKB and ActD monotherapies,as well as combination therapy,in order to identify cellular nucleolar stress induction.Finally,to utilize Western blot experiments to assess changes in cell cycle and apoptosis-related proteins in the two types of triple-negative breast cancer cells following treatment with FKB and ActD monotherapies,as well as combination therapy.Results:FKB and ActD synergistically inhibited cell proliferation in two types of triple-negative breast cancer cells,and flow cytometry results showed that the combination therapy significantly induced more apoptosis in triple-negative breast cancer cells(P<0.01)and affected cell cycle progression,and that the combination's inhibition of cell proliferation was attenuated by inhibition of apoptotic signaling pathways using apoptosis inhibitors and by controlling the cell cycle using serum deprivation(P<0.01).RT-qPCR results indicated that combination ther

关 键 词：三阴性乳腺癌 黄卡瓦胡椒素B 放线菌素D 核仁应激 细胞凋亡 

分 类 号：R737.9[医药卫生—肿瘤]