维纳托克在肝癌细胞中的抗肿瘤活性及其机制

目的探讨维纳托克(Venetoclax)在HepG2、Hep3B 2个肝癌细胞株的抗癌活性。方法对人肝癌细胞株HepG2、Hep3B细胞株进行体外培养,采用不同浓度的Venetoclax(0、3、10、30μmol/L)处理肝癌细胞,以细胞计数试剂盒(CCK-8)检测细胞活性,0,3,10和30μmol/L Venetoclax作用细胞48 h,锥虫蓝拒染法检测细胞的死亡,0,5μmol/L Venetoclax,作用24 h,流式细胞学检测细胞凋亡率,蛋白质印迹法(Western blot)检测Venetoclax诱导半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-3活化,聚腺苷二磷酸核糖聚合酶(PARP)裂解。组间比较采用t检验。结果Venetoclax对HepG2、Hep3B细胞株均有较强活性抑制作用。不同浓度的Venetoclax处理肝癌细胞48、72 h,Venetoclax在HepG2细胞株中取得的半数细胞活性抑制率(IC50)值分别为46.5μmol/L和14.2μmol/L;在Hep3B细胞的IC50值分别为24.6μmol/L和11.2μmol/L。处理24 h,30.0μmol/L的Venetoclax在HepG2和Hep3B细胞株中诱导57%对照组、实验组凋亡率分别为(4.00±1.00)%、(56.67±8.51)%,t=-10.650,P<0.01]和54%对照组、实验组凋亡率分别为(5.67±1.53)%、(54.33±9.87)%,t=-8.440,P<0.01]的肝癌细胞发生凋亡,并能诱导肝癌细胞的Caspase-3活化,PARP裂解,差异均有统计学意义。用0、3、10和30μmol/L的Venetoclax处理48 h,在HepG2细胞株中可诱导3%(3.26±1.71)%,12%(12.36±1.99)%,(t=-12.36,P<0.05)],32%(32.38±4.57)%,(t=-10.350,P<0.01)]和67%(66.71±6.29)%,(t=-16.86,P<0.01)]的细胞死亡,差异均有统计学意义;在Hep3B细胞株诱导2%(1.91±0.68)%,16%(15.72±3.40)%,(t=-6.890,P<0.05)],42%(42.39±6.86)%,(t=-10.180,P<0.01)]和73%(73.32±2.69)%,t=-44.490,P<0.01]的细胞死亡,差异均有统计学意义。应用半胱氨酸蛋白酶(Caspase)抑制剂预处理,Venetoclax对HepG2和Hep3B细胞的死亡率分别从56%(56.71±6.29)%降低至11%(10.70±1.43)%,(t=12.350,P<0.01)和68%(68.05±10.16)%至12%(12.7±6.12)%,(t=8.080,P<0.01),差异有统计学意义。结论Venetoclax能够通过诱导Caspase的活化,诱导肝癌细胞发生凋亡,并对肝癌细胞进行杀伤。

Anticancer activity and underlying mechanism of venetoclax in hepatocellular carcinoma cells

Objective To investigate biological activity of Venetoclax in two hepatocellular carcinoma(HCC)cell lines of HepG2 and Hep3B and the potential usefulness of this new drug in HCC.Methods Liver cancer cell lines of HepG2 and Hep3B were treated with Venetoclax.The cell counting kit-8(CCK-8)assay was used to examine the cell viability inhibition.The Trypan blue exclusion assay was used to examine the cell death induction.Annexin V staining was used to examine apoptosis,and Western blotting was used to examine cysteinyl aspartate-specific protease(Caspase)activation and poly ADP-ribose polymerase(PARP)cleavage.A Caspase inhibitor was used to investigate the Caspase-dependency of Venetoclax-induced apoptotic cell death.Results Venetoclax potently inhibited cell viability in HCC HepG2 and Hep3B cell lines in a dose-dependent manner.Venetoclax achieved half maximal inhibitory concentration(IC50)values of 46.5μmol/L and 14.2μmol/L,respectively,in HepG2 cell line for 48-and 72-h treatment,and achieved IC50 values of 24.6μmol/L and 11.2μmol/L,respectively,in Hep3B cell line.Venetoclax at 30μmol/L induces 57%(4.00±1.00)%vs.(56.67±8.51)%,t=-10.650,P<0.01]and 54%(5.67±1.53)%vs.(54.33±9.87)%,t=-8.440,P<0.01]cells positively stained with Annexin-V-Fluorescein Isothiocyanate(FTIC)in HepG2 and Hep3B cell lines,respectively after 24 h-treatment.Venetoclax also triggers activation of caspase-3 and PARP cleavage in both cell lines.Venetoclax at 0,3,10 and 30μmol/L triggers 3%(3.26±1.71)%,12%(12.36±1.99)%,t=-12.360,P<0.05],32%(32.38±4.57)%,t=-10.350,P<0.01]and 67%(66.71±6.29)%,t=-16.860,P<0.01]cell death,respectively,in HepG2 cell line,and 2%(1.91±0.68)%,16%(15.72±3.40)%,t=-6.890,P<0.05],42%(42.39±6.86)%,t=-10.180,P<0.01]and 73%(73.32±2.69)%,t=-44.490,P<0.01]in Hep3B cell lines.Pretreatment with a caspase inhibitor largely attenuated Venetoclax-induced cell death in both cell lines,the dead cell rate on HepG2 and Hep3B cells decreased from 56%(56.71±6.29)%to 11%(10.70±1.43)%,(t=12.350,P<0.01)and 68%(68.05±10.16)%to 12%(12.7±6.12)%,(t=8.080,P<0.01)respectively.Conclusion Venetoclax displays potent anticancer activity by inducing apoptosis in HCC cells.