摘要
Nutlin-3是代表性小分子MDM2-p53拮抗剂,能通过破坏p53和MDM2的相互作用,稳定p53状态,从而诱导p53信号通路发挥抗肿瘤作用。本研究以HCT-116、H460、HepG2、MCF-7、A549、SJSA-1六种野生型p53肿瘤细胞系为研究对象,采用噻唑蓝(methyl thiazolyl tetrazolium,MTT)法、平板克隆实验检测nutlin-3对6种不同野生型p53癌细胞增殖的影响;通过流式细胞术检测nutlin-3对H460细胞周期和细胞凋亡的影响;通过蛋白印迹实验检测泛素特异蛋白酶7(ubiquitin-specific protease 7,USP7)、细胞死亡结构域相关蛋白(death domain-associated protein,DAXX)、鼠双微体2(murine double minute 2,MDM2)、鼠双微体4(murine double minute 4,MDMX/MDM4)、p53的表达,探讨nutlin-3的抗肿瘤作用机制;通过免疫共沉淀(co-immunoprecipitation,Co-IP)实验检测nutlin-3对MDM2与MDMX、p53相互作用的影响。结果显示,nutlin-3呈时间和浓度依赖性地抑制H460的增殖;细胞周期和凋亡结果显示,nutlin-3能阻滞H460细胞周期于G0/G1期,通过激活cleaved-PARP并诱导细胞凋亡;蛋白印迹结果显示,nutlin-3能够上调H460细胞中USP7、DAXX、MDM2、MDMX、p53蛋白的表达;Co-IP结果表明,nutlin-3抑制MDM2与p53、MDM2与MDMX的蛋白相互作用。综上所述,nutlin-3能够显著抑制野生型p53癌细胞的增殖,并诱导细胞周期阻滞和细胞凋亡,其机制可能与破坏MDM2/MDMX与p53相互作用,从而激活MDM2-p53信号通路有关。
Nutlin-3 is a representative small molecule MDM2-p53 antagonist,which can stabilize the p53 state by disrupting the interaction between p53 and MDM2,thereby inducing the p53 signaling pathway to exert antitumor effects.In this study,six wild-type p53 tumor cell lines,HCT-116,H460,HepG2,MCF-7,A549 and SJSA-1,were used as research objects,and the effects of nutlin-3 on the proliferation of six wild-type p53 cancer cells were detected by methyl thiazolyl tetrazolium(MTT)method and plate cloning assay.The effects of nutlin-3 on H460 cell cycle and apoptosis were detected by flow cytometry.Western blot assay was used to detect ubiquitinspecific protease 7(USP7),death domain-associated protein(DAXX),murine double minute 2(MDM2),murine double minute 4(MDMX/MDM4),p53,to explore the anti-tumor mechanism of nutlin-3;co-immunoprecipitation(Co-IP)assay was used to detect the effect of nutlin-3 on the interaction between MDM2,MDMX and p53.The results showed that nutlin-3 inhibited the proliferation of H460 in a time-and concentration-dependent manner.The results of cell cycle and apoptosis showed that nutlin-3 could block the H460 cell cycle in the G0/G1 phase,and induce apoptosis by activating cleaved-PARP.Western blot results showed that nutlin-3 could up-regulate the expression of USP7,DAXX,MDM2,MDMX and p53 in H460 cells.Co-IP results showed that nutlin-3 inhibited the protein interactions between MDM2 and p53 and MDM2 and MDMX.In conclusion,nutlin-3 can significantly inhibit the proliferation of wild-type p53 cancer cells and induce cell cycle arrest and apoptosis,which may be related to the disruption of MDM2/MDMX's interaction with p53 to activate the MDM2-p53 signaling pathway.
作者
马明远
刘莹莹
钱宇卿
周思雨
李铭东
MA Ming-yuan;LIU Ying-ying;QIAN Yu-qing;ZHOU Si-yu;LI Ming-dong(School of Pharmacy,Jiangxi University of Chinese Medicine,Nanchang 330006,China)
出处
《药学学报》
北大核心
2025年第5期1407-1413,共7页
Acta Pharmaceutica Sinica
基金
江西省教育厅科学技术研究项目(GJJ2400827,GJJ2400835)。
