氯氰碘柳胺钠通过抑制蛋白酪氨酸磷酸酶SHP2诱导RAS突变型肺癌细胞衰老和死亡  

Closantel sodium induces RAS mutation lung cancer cell senescence and death by inhibiting protein tyrosine phosphatase SHP2

作　　者：席芮颖 付乃洁 李润择 张国林[1] 王飞[1] XI Ruiying;FU Naijie;LI Runze;ZHANG Guolin;WANG Fei(Chengdu Institute of Biology,Chinese Academy of Sciences,Chengdu 610041,China;University of Chinese Academy of Sciences,Beijing 100049,China)

机构地区：[1]中国科学院成都生物研究所,成都610041 [2]中国科学院大学,北京100049

出　　处：《应用与环境生物学报》2022年第2期365-371,共7页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金项目(21977092,21861142007)资助。

摘　　要：含Src同源2结构域蛋白酪氨酸磷酸酶2(Src homology region 2-containing protein tyrosine phosphatase 2,SHP2)具有调控细胞增殖分化、维持周期、调节免疫等功能,与众多肿瘤的发生发展密切相关.SHP2是RAS蛋白(ratsarcoma protein,RAS)信号通路的重要调控蛋白,研究表明抑制SHP2活性能减缓RAS突变肺癌的进展.因此筛选特异性的SHP2抑制剂,有助于为肺癌的治疗提供新的手段.通过建立不同蛋白酪氨酸磷酸酶(protein tyrosine phosphatase,PTP)体外筛选体系,从化合物文库中筛得SHP2特异性抑制剂氯氰碘柳胺钠(closantel sodium,CS),其IC_(50)值为84.83μmol/L.采用Western Blot检测CS对SHP2磷酸化的抑制作用;采用细胞热转移试验(cellular thermal shift assay,CETSA)检测在不同温度及浓度下CS对SHP2的热稳定性的影响;采用CCK8检测CS对人肺癌细胞A549、NCI-H358、NCI-H1944的细胞毒作用;采用β-半乳糖苷酶染色检测CS在无血清(0%)、低血清(3%)及常规血清(10%)3种条件下诱导肺癌细胞衰老表型.结果显示,CS可特异性抑制SHP2的活性,并显著抑制SHP2的磷酸化;在52-60℃的温度范围内,CS可稳定胞内SHP2构象,并具有浓度依赖性;CS可抑制肺癌细胞增殖,并具有浓度依赖性;10%血清条件下,CS作用使得细胞衰老表型显著增加,可诱导肺癌细胞衰老.本研究表明CS可通过抑制SHP2活性及磷酸化诱导肺癌细胞衰老、促进肺癌细胞死亡,结果可为开发新的治疗肺癌等疾病的特异性SHP2抑制剂提供新的化合物骨架.(图5表2参36)Src homology region 2-containing protein tyrosine phosphatase 2(SHP2),which regulates cell proliferation and differentiation,maintains the cell cycle,and regulates immunity.SHP2 is closely related to tumorigenesis and development.SHP2 is also an important regulatory protein in the RAS signaling pathway.Many studies have shown that inhibiting SHP2 activity can slow down the progression of RAS-mutant lung cancer.Therefore,screening for effective SHP2 inhibitors can provide a new approach for the treatment of lung cancer.We established different protein tyrosine phosphatases using in vitro screening systems,with closantel sodium(CS)as an SHP2 specific inhibitor,in the screening of our compound library,with an IC_(50) of 84.83μmol/L.The inhibitory effect of CS on SHP2 phosphorylation was tested by western blotting.The thermal stability of CS to SHP2 was analyzed using a cellular thermal shift assay at different temperatures and concentrations.The cytotoxicity of CS on A549,NCI-H358,and NCI-H1944 human lung cancer cells was tested by the CCK8 assay.The senescence phenotype of lung cancer cells induced by CS was measured byβ-galactosidase staining under serum-free(0%),low serum(3%),and conventional serum(10%)conditions.CS specifically inhibited the activity and significantly suppressed the phosphorylation of SHP2.Simultaneously,stabilized intracellular SHP2 conformation at temperatures ranging from 52℃to 60℃,and inhibited lung cancer cell proliferation in a concentration-dependent manner,inducing lung cancer cell senescence.The findings demonstrate that CS can induce lung cancer cell senescence and death by inhibiting the activity and phosphorylation of SHP2.CS is implicated as a new compound skeleton for the development of new specific SHP2 inhibitors for the treatment of lung cancer and other diseases.

关 键 词：氯氰碘柳胺钠 SHP2 肺癌 细胞衰老 

分 类 号：R734.2[医药卫生—肿瘤]