普纳替尼(ponatinib)抑制SNU-449人肝癌细胞增殖并阻断MAPK和PDK1/AKT/mTOR信号通路  

Ponatinib inhibits the proliferation of SNU-449 human hepatocellular cancer cells and blocks MAPK and PDKl/AKT/mTOR signaling pathways

作　　者：刘畅[1] 裴晋红[1] 穆秀丽 于保锋 弓韬 梁文婷 UU Chang;PEI Jinhong;MU XiuH;YU Baofeng;GONG Tao;UANG Wenting(Department of Biochemistry and Molecular Biology,School of Basic Medicine,Changzhi Medical College,Changzhi 046000;Department of Biochemistry and Molecular Biology,School of Basic Medicine,Shanxi Medical University,Taiyuan 030001;Institute of Environmental Science,Department of Chemistry,Shanxi University,Taiyuan 030001,China)

机构地区：[1]长治医学院基础部生物化学与分子生物学教研室,山西长治046000 [2]山西医科大学基础医学院生物化学与分子生物学教研室,山西太原030001 [3]山西大学环境科学研究所,山西太原030001

出　　处：《细胞与分子免疫学杂志》2022年第5期425-431,共7页Chinese Journal of Cellular and Molecular Immunology

基　　金：山西省自然科学基金(201901D111190,2015011113);山西省重点研发计划(国际合作)项目(201703D421023);山西省面上青年基金项目(201801D221069,201801D221059);长治医学院博士科研启动基金(BS202007);山西省高等学校科技创新计划项目(20211339);山西省高等学校大学生创新创业训练计划项目(20210548)。

摘　　要：目的研究多靶点蛋白酪氨酸激酶抑制剂普纳替尼(ponatinib)对SNU-449人肝癌细胞增殖的影响及机制。方法先使用16种酪氨酸激酶抑制剂处理SNU-449肝癌细胞72 h,采用噻唑蓝(MTT)法检测细胞增殖情况,筛选出对SNU-449肝癌细胞最敏感的ponatinib并获得半数抑制浓度(IC50)值。SNU-449细胞分为对照组、(0.06、0.3、0.6)μmol/L ponatinib处理组。采用平板克隆形成实验和倒置显微镜观察ponatinib对肝癌细胞集落形成能力和细胞形态,流式细胞术检测肝癌细胞凋亡和周期,Western blot法检测Src、磷酸化的Src(p-Src)、丝裂原激活的蛋白激酶激酶(MEK)、磷酸化的MEK(p-MEK)、胞外信号调节激酶(ERK)、磷酸化的ERK(p-ERK)、磷脂酰肌醇3激酶(PI3K)、磷酸化的PI3K(p-PI3K)、磷酸肌醇依赖性蛋白激酶1(PDK1)、磷酸化的PDK1(p-PDK1)、蛋白激酶B(AKT)、磷酸化的AKT(p-AKT)、哺乳动物雷帕霉素靶蛋白(mTOR)和磷酸化的mTOR(p-mTOR)的表达。结果在16种酪氨酸激酶抑制剂中,ponatinib能够有效抑制SNU-449肝癌细胞增殖。Ponatinib促进SNU-449细胞凋亡,呈现浓度依赖性,同时能够将细胞周期阻滞在G1期。Ponatinib抑制SNU-449细胞中Src磷酸化,抑制丝裂原活化蛋白激酶(MAPK)信号通路和磷酸肌醇依赖性蛋白激酶1/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(PDK1/AKT/mTOR)信号通路。结论Ponatinib能够抑制肝癌细胞增殖,诱导细胞凋亡和细胞周期阻滞并阻断MAPK和PDK1/AKT/mTOR信号通路,可能是一种治疗肝癌的潜在药物。Objective To investigate the effects of ponatinib(a multi-target kinase inhibitor)on the proliferation of SNU^49 human hepatocellular cancer cells and the underlying mechanism.Methods SNU^49 hepatocellular cancer cells were treated with 16 tyrosine kinase inhibitors for 72 hours.Then MTT assay was used to detect the effects of ponatinib on the survival and proliferation of the cancer cells.Ponatinib was the most sensitive drug to SNU^49 cells and the IC_(50) value was obtained.SNU-449 cells were cultured and treated with(0.06,0.3,0.6)pmol/L ponatinib,and the control group was treated with DMSO.Colony formation assay and inverted microscope were applied to observe the effects of ponatinib on the colony formation ability and morphology of SNU-449 cells.Flow cytometry was used to detect the effects of ponatinib on the apoptosis and cell cycle of SNU-449 cells.Western blotting was performed to examine the expression of Src,phosphorylated Src(p-Src),mitogen-activated protein kinase kinase(MEK),phosphorylated MEK(p-MEK),extracellular signal-regulated kinase(ERK),phosphorylated ERK(p-ERK),phosphoinositide 3-kinase(PI3K),phosphorylated PI3K(p-PI3K),phosphoinositide-dependent protein kinase 1(PDK1).phosphorylated PDKI(p-PDKI),AKT,p-AKT,mammalian target of rapamycin(mTOR)and phosphorylated mTOR(p-mTOR).Results MTT assay showed that ponatinib displayed the best inhibitory effects on SNU-449 cells in 16 tyrosine kinase inhibitors.Ponatinib promoted cell apoptosis in a concentration-dependent manner and induced cell cycle arrest at the GI phase in SNU 449 cells.A number of kinase signaling pathways were inhibited by ponatinib,including the Src signaling pathway,MAPK pathway and PDKI/AKT/mTOR pathway.Conclusion Ponatinib can inhibit the proliferation,promote the apoptosis and cell cycle arrest of hepatocellular cancer cells and block MAPK and PDKI/AKT/mTOR signaling pathways,which might be a potential agent for liver cancer treatment.

关 键 词：普纳替尼(pormtinib) 细胞增殖 细胞凋亡 细胞周期 肝癌细胞 信号通路 

分 类 号：Q279[生物学—细胞生物学] R965[医药卫生—药理学] R735.7[医药卫生—药学] R34R392-33