NVP-TNKS656通过调节Hippo信号通路抑制肝细胞肝癌的生长

NVP-TNKS656 inhibits the growth of hepatocellular carcinoma cell lines by regulating Hippo signaling pathway

目的:研究NVP-TNKS656对肝细胞癌(HCC)细胞系生长的影响以及相应分子机制。方法:采用2.5、5、10.0μmol/L剂量NVP-TNKS656处理5种HCC细胞系,选取HLE及HLF细胞系,分为四组:二甲基亚砜(DMSO)组、NVP-TNKS6562.5.0μmol/L组、NVP-TNKS6565.0μmol/L组和NVP-TNKS65610μmol/L组,细胞培养48 h进行后续实验。采用结晶紫染色法计数细胞克隆形成数目;蛋白质印迹法检测是相关蛋白(YAP)、血管动蛋白样1(AMOTL1)、血管动蛋白样2(AMOTL2)蛋白表达水平;实时荧光定量聚合酶链式反应(qRT-PCR)法检测YAP及其下游结缔组织生长因子(CTGF)和富含半胱氨酸的血管生成诱导剂61(Cyr61)的mRNA表达;采用双荧光素酶报告基因检测转录增强因子域家族(TEAD)荧光素酶活性。结果:在HLE、HLF、Huh7、MHCC97-H、MHCC97-L细胞系中,NVP-TNKS656以剂量依赖的方式抑制了5种HCC细胞系的生长,DMSO对照组与不同剂量给药组间细胞克隆形成计数均差异有统计学意义(F=90.46、68.58、191.8、114.6、201.4,均P<0.05)。在HLE和HLF细胞系中,NVP-TNKS656可呈剂量依赖性显著降低YAP蛋白水平,降低YAP下游靶基因CTGF(HLE细胞分别为1.02±0.02、0.90±0.03、0.57±0.02、0.38±0.03,HLF细胞分别为0.98±0.03、0.86±0.02、0.66±0.02、0.43±0.01)及Cyr61(HLE细胞分别为1.00±0.01、0.86±0.02、0.74±0.03、0.44±0.03,HLF细胞分别为0.99±0.02、0.87±0.01、0.72±0.02、0.54±0.01)的表达(均P<0.05),并抑制YAP/TEAD荧光素酶分子的活性。同时NVP-TNKS656以剂量依赖的方式上调了YAP的两个主要负调控因子,即AMOTL1/2蛋白,促进HLE和HLF细胞的凋亡。结论:NVP-TNKS656可以通过稳定AMOTL1/2下调YAP下游靶基因从而抑制HCC的增殖,可能成为治疗HCC的潜在药物。

Objective To investigate the effect of a tankyrase inhibitor NVP-TNKS656 on the growth of hepatocellular carcinoma(HCC)cell lines and the involved molecular mechanisms.Methods Five HCC cell lines were treated with 0,2.5,5.0,10.0μmol/L of NVP-TNKS656.The cell lines of HLE and HLF were selected and divided into four groups:0.0μmol/L(control or DMSO),2.5μmol/L,5.0μmol/L,10.0μmol/L of NVP-TNKS656 groups.Cells were cultured for 48 h for subsequent experiments.Crystal violet staining was used to count the number of the newly formed cell clones.Western blotting was used to detect the protein expression levels of Yes-associated protein(YAP),angiomotin-like 1(AMOTL1)and AMOTL2.The real-time qRT-PCR was used to detect the mRNA expression of YAP and its downstream connective tissue growth factor(CTGF)and cysteine-rich 61(Cyr61).Dual luciferase reporter gene was used to detect the luciferase activity of transcriptional enhancer activator domain(TEAD)family.Results NVP-TNKS656 inhibited the growth of 5 HCC cell lines in a dose-dependent manner in HLE,HLF,Huh7,MHCC97-H,and MHCC97-L cell lines.There were significant differences in the newly formed cell clone numbers between control(0μM of NVP-TNKS656)and each of 2.5μmol/L,5.0μmol/L,10.0μmol/L of NVP-TNKS656 groups in a dose-dependent manner(F=90.46,68.58,191.8,114.6 and 201.4,all P<0.05).In HLE and HLF cell lines,NVP-TNKS656 significantly reduced the protein level of YAP in a dose-dependent manner and decreased the YAP target gene CTGF(HLE cells:1.02±0.02,0.90±0.03,0.57±0.02,0.38±0.03,HLF cells:0.98±0.03,0.86±0.02,0.66±0.02,0.43±0.01)and Cyr61(HLE cells:1.00±0.01,0.86±0.02,0.74±0.03,0.44±0.03 and HLF cells:0.99±0.02,0.87±0.01,0.72±0.02,0.54±0.01)(P<0.05),and inhibited the activity of YAP/TEAD luciferase.At the same time,NVP-TNKS656 up-regulated two major negative regulators of YAP,namely AMOTL1 and AMOTL2 proteins,and promoted the apoptosis of HLE and HLF cells in a dose-dependent manner.Conclusion NVP-TNKS656 can inhibit the proliferation of HCC by stabilizing AMOTL1/AMOTL2 and down-regulating the YAP target gene.This study indicates that NVP-TNKS656 can be used as a potential drug for treating HCC.