TOPK抑制剂HI-TOPK-032对胰腺神经内分泌肿瘤BON-1细胞体外恶性表型的影响  

Effect of ToPK inhibitor Hi-ToPK-032 on malignant phenotype of pancreatic neuroendocrine tumor BoN-1 cells in vitro

作　　者：郭鑫 李刚[1] 叶辰[1] 阿卜杜·海拜尔·萨杜拉 任思谦 袁蒙 孟猛 钱海利 原春辉[1] GUO Xin;LI Gang;YE Chen;ABUDU Haibaier Sadula;REN Si-qian;YUAN Meng;MENG Meng;QIAN Hai-li;YUAN Chun-hui(Department of General Surgery,Peking University Third Hospital,Beijing 100191,China;National Cancer Center/National Clinical Research Center for Cancer/State Key Laboratory of Molecular Oncology,Cancer Hospital,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100021,China)

机构地区：[1]北京大学第三医院普通外科,北京100191 [2]国家癌症中心/国家肿瘤临床医学研究中心/中国医学科学院北京协和医学院肿瘤医院分子肿瘤学国家重点实验室,北京100027

出　　处：《肝胆胰外科杂志》2021年第5期285-291,297,共8页Journal of Hepatopancreatobiliary Surgery

基　　金：国家自然科学基金项目(81672862)。

摘　　要：目的杀伤性T细胞来源的蛋白激酶(T-lymphokine activated killer cell-originated protein kinase,TOPK)高表达与肿瘤增殖、凋亡、侵袭和转移密切相关。本研究旨在探讨使用TOPK抑制剂HI-TOPK-032对胰腺神经内分泌肿瘤细胞BON-1体外恶性表型的抑制作用。方法通过蛋白免疫印迹实验检测人正常胰腺导管上皮细胞和不同胰腺肿瘤细胞系中TOPK的蛋白表达水平;CCK-8实验检测HI-TOPK-032对BON-1细胞增殖的影响;克隆形成实验检测HI-TOPK-032对BON-1细胞体外克隆形成的影响;Transwell实验检测HI-TOPK-032对BON-1细胞迁移和侵袭能力的影响;流式细胞仪检测HI-TOPK-032对BON-1细胞周期的影响;Annexin V检测HI-TOPK-032对BON-1细胞凋亡和坏死的影响。结果与正常胰腺导管上皮细胞相比,胰腺神经内分泌肿瘤细胞BON-1中TOPK蛋白表达显著上调;在体外实验中,与对照组相比,在含1、2.5、5μmol/L浓度的HI-TOPK-032培养基中,BON-1细胞增殖能力依次减弱(22.2±8.2)%、(90.4±1.0)%、(89.7±0.9)%(P<0.001),克隆形成依次减少(19.1±2.1)%、(42.5±5.7)%、(87.0±5.6)%(P<0.001),迁移能力依次减弱(9.3±5.6)%、(70.5±4.0)%、(87.5±3.5)%(P<0.01),侵袭能力依次减弱(23.0±4.2)%、(60.7±5.4)%、(93.6±3.0)%(P<0.01);在含2.5、5μmol/L浓度HI-TOPK-032培养基中,G0/G1期的BON-1细胞比例分别增加(12.2±2.0)%、(18.3±1.4)%(P<0.001),在5μmol/L浓度时,S期的细胞比例减少(18.4±6.1)%(P<0.01),在2.5、5μmol/L浓度时,G2/M期的细胞比例分别减少(17.6±8.6)%、(16.4±4.5)%(P<0.001);HI-TOPK-032促进BON-1细胞凋亡和坏死,凋亡依次增加(60.6±30.9)%、(79.5±27.5)%、(165.8±34.9)%(P<0.05),5μmol/L浓度时,坏死显著增加,增加(385.8±67.3)%(P<0.001)。结论TOPK靶向抑制剂HI-TOPK-032显著抑制BON-1细胞的体外恶性表型,抑制效果呈剂量依赖式。HI-TOPK-032能调控BON-1细胞周期,促进凋亡和坏死。TOPK抑制剂HI-TOPK-032可能在胰腺神经内分泌肿瘤的靶向治疗中发挥重要�Objective T-lymphokine activated killer cell-originated protein kinase(TOPK)high expression is closely related to tumor proliferatio,apoptosis,invasion and metastasis.The purpose of this study is to investigate the inhibitory effect of TOPK inhibitor HI-TOPK-032 on malignant phenotype of pancreatic neuroendocrine tumor BON-1 cells in vitro.Methods Western blotting was used to detect the expression of TOPK protein in human normal pancreatic ductal epithelial cells and different pancreatic tumor cell lines.CCK-8 experiment was employed to detect the effect of HI-TOPK-032 on the proliferation of BON-1 cells.Clone formation assay was applied to detect the effect of HI-TOPK-032 on the clone formation of BON-1 cells in vitro.Transwell assay was used to detect the effect of HI-TOPK-032 on migration and invasion of BON-1 cells.Flow cytometric was employed to detect the effect of HI-TOPK-032 on cell cycle of BON-1 cells.Annexin V was used to detect the effect of HI-TOPK-032 on apoptosis and necrosis of BON-1 cells.Results Compared with that in pancreatic ductal epithelial cells,the expression of TOPK protein in pancreatic neuroendocrine tumor BON-1 cells was significantly upregulated.Compared with the control group,in a medium containing HI-TOPK-032 at a concentration of 1,2.5,and 5μmol/L,the proliferation of BON-1 cells was reduced by(22.2±8.2)%,(90.4±1.0)%,(89.7±0.9)%(P<0.001),clone formation decreased by(19.1±2.1)%,(42.5±5.7)%,(87.0±5.6)%(P<0.001),the migration ability was decreased by(9.3±5.6)%,(70.5±4.0)%,(87.5±3.5)%(P<0.01),and the invasion ability was reduced by(23.0±4.2)%,(60.7±5.4)%,(93.6±3.0)%(P<0.01).After using HI-TOPK-032 at the concentration of 2.5 and 5μmol/L,the proportion of BON-1 cells in G0/G1 phase increased by(12.2±2.0)%and(18.3±1.4)%(P<0.001).At the concentration of 5μmol/L,the proportion of BON-1 cells decreased by(18.4±6.1)%in phase S(P<0.01).At the concentration of 2.5 and 5μmol/L,the proportion of cells in the G2/M phase decreased by(17.6±8.6)%and(16.4±4.5)%(P<0.001).HI-TOPK-

关 键 词：杀伤性T细胞来源的蛋白激酶(TOPK) TOPK抑制剂(HI-TOPK-032) 胰腺神经内分泌肿瘤 BON-1细胞 

分 类 号：R735.9[医药卫生—肿瘤]