异紫堇碱衍生物COM33对Huh7细胞增殖和凋亡的影响

Effect of isocorydine derivative, COM33, on proliferation and apoptosis of Huh7 cells

目的观察异紫堇碱衍生物COM33对肝癌Huh7细胞增殖和凋亡的影响。方法以0.625、1.25、2.5、5、10、20、40、80μmol/L COM33分别处理正常肝细胞LO2和肝癌细胞Huh724 h后,采用细胞计数法(CCK-8)检测细胞吸光度(A)值,并在Graphpad prism软件中通过非线性回归分析获得半数抑制浓度(IC50)。随后采用COM33处理Huh7细胞,以无COM33组设为对照组;细胞克隆实验用于评估COM33对Huh7细胞增殖的影响;10μmol/L COM33处理Huh7细胞24 h后,在倒置显微镜下观察细胞形态变化,用膜联蛋白V-异硫氰酸荧光素(Annexin V-FITC)/碘化丙锭(PI)双染试剂盒检测细胞凋亡情况,免疫印迹实验检测给药前后细胞中Gasdermin D(GSDMD)蛋白、B细胞淋巴瘤/白血病-2(bcl-2)蛋白、bcl-2相关X蛋白(bax)、半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-9剪切体(cleaved Caspase-9)的表达水平。组间比较采用Student’st检验。结果COM33剂量依赖性地抑制Huh7细胞增殖,且在Huh7细胞中的IC50值明显低于LO2细胞[(11.99±0.53)μmol/L比(33.33±3.10)μmol/L,t=11.810,P<0.01];给药组细胞形成的克隆集落少于对照组[(681±13)个比(387±29)个,t=15.950,P<0.01];Huh7细胞经COM33处理后出现皱缩变圆、贴壁不良;给药组Annexin V阳性细胞比例高于对照组[(7.28±0.86)%比(1.37±0.09)%,t=11.820,P<0.01];两组细胞GSDMD蛋白表达并无差异,且未检测到GSDMD剪切体;bax蛋白相对表达量给药组高于对照组(1.02±0.04比0.50±0.06,t=12.250,P<0.01);cleaved Caspase-9蛋白相对表达量给药组高于对照组(0.39±0.11比0.07±0.03,t=4.726,P<0.01;0.39±0.11比0.16±0.07,t=2.942,P<0.05);bcl-2蛋白相对表达量给药组低于对照组(0.54±0.06比0.82±0.12,t=3.551,P<0.05)。结论COM33对LO2细胞毒性不明显,可有效抑制Huh7细胞增殖,并诱导其凋亡。

Objective To study the effect of the isocorydine derivative,COM33,on the proliferation and apoptosis of human hepatocellular carcinoma Huh7 cells.Methods Cell counting kit 8(CCK-8)assays were performed to measure the absorbance of hepatocellular carcinoma cell line Huh7 and normal liver cell line LO2 treated with 0.625,1.25,2.5,5,10,20,40,80μmol/L COM33 for 24 h.The half maximal inhibitory concentration(IC50)was obtained using nonlinear regression in Graphpad prism.Huh7 cells were treated with COM33,and COM33-free group was considered as control group.Colony formation assays were performed to evaluate the proliferation of Huh7 cells treated with COM33.After treatment of Huh7 cells with 10μmol/L COM33 for 24 h,the morphological changes were observed under inverted microscope,apoptosis was assessed using an annexin V-fluoresceine isothiocyanate(Annexin V-FITC)/Propidium Iodide(PI)double-staining kit,and the expression of Gasdermin D(GSDMD),B cell lymphoma/leukemia-2(bcl-2),bcl-2 associated X(bax)and cleaved cysteinyl aspartate-specific protease(Caspase)-9 was detected by Western blotting.Statistical analysis was performed using Student’s t-test.Results COM33 inhibited Huh7 cell proliferation in a dose-dependent manner with an IC50 of(11.99±0.53)μmol/L,lower than that in LO2 cells[(11.99±0.53)μmol/L vs.(33.33±3.1)μmol/L,t=11.810,P<0.01].The colonies of COM33 group were smaller and less than in control group(681±13 vs.387±29,t=15.950,P<0.01).Morphological changes after treatment with COM33 included cell shrinkage,rounding,and poor adherence.The percentage of Annexin V positive Huh7 cells in COM33 group significantly increased from 1.37%±0.09%(control group)to 7.28%±0.86%(t=11.820,P<0.01).Western blotting assays showed that the expression of GSDMD was not statistically significant and its N-terminal fragment(GSDMD-N)was not detected.However,treatment with COM33 increased bax expression(1.02±0.04 vs.0.50±0.06,t=12.250,P<0.01)and cleaved Caspase-9 expression(0.39±0.11 vs.0.07±0.03,t=4.726,P<0.01;0.39±0.11 vs.0.16±0.07,t=2.942,P<0.05),and decreased bcl-2 expression(0.54±0.06 vs.0.82±0.12,t=3.551,P<0.05)in Huh7 cells.Conclusion COM33 inhibits Huh7 cell proliferation by inducing apoptosis with low cytotoxicity in LO2 cells.