JIB-04对肝癌细胞株Huh7周期及凋亡的影响及其机制

Effect and mechanism of JIB-04 on hepatoma cell line Huh7 cycle and apoptosis

目的观察JIB-04对肝癌细胞Huh7周期、凋亡的影响。方法用浓度为(0、4、8、16μmol/L)的JIB-04分别处理正常肝细胞L02及肝癌细胞Huh7均由广东医科大学附属医院临床技能中心肝胆实验室提供,用细胞计数试剂盒(CCK-8)检测细胞增殖变化;用流式细胞仪检测Huh7细胞周期分布及凋亡;蛋白质印迹法(Western blot)检测肝癌细胞Huh7凋亡蛋白表达。采用单因素方差分析或两样本均数比较。结果JIB-04对正常肝细胞L02抑制作用轻微,经JIB-04培养48 h(8μmol/L)后,抑制率仅为5.6%,而不同浓度JIB-04对肝癌细胞Huh7有不同程度的抑制作用,且其抑制作用呈现时间及浓度依赖性,半数抑制浓度(IC50)为3.5μmol/L;在周期检测结果中,肝癌细胞Huh7分期中S、M期比例明显减少,相反G1比例明显升高且呈药物浓度依赖性;Western blot中随着JIB-04浓度的增加,肝癌细胞Huh7凋亡率明显升高,在最高浓度8μmol/L培养48 h后其凋亡率达(28.38±0.73)%,且不同浓度组与对照组两两比较其凋亡率差异有统计学意义(t=2.767,P<0.05);在肝癌细胞凋Huh7亡蛋白检测中,凋亡蛋白p53、B细胞淋巴瘤/白血病-2相关X蛋白(bax)明显上调,相反B细胞淋巴瘤/白血病-2(bcl-2)下调。结论JIB-04对正常肝细胞无明显抑制作用,可抑制肝癌细胞Huh7增殖,有干扰其周期分布及促进凋亡作用。

Objective To study the effects of JIB-04 on the proliferation and apoptosis of hepatocellular carcinoma(HCC)Huh7 cells.Methods JIB-04,histone demethylase inhibitor,with different concentrations(0,4,8,16μmol/L)were used to treat Huh7 cells and normal hepatocyte L02 cells.Cell counting kit-8(CCK-8)kit was used to detect cell proliferation of HCC cells.The flow cytometry was used to detect cell cycle and apoptosis of Huh7 cells.The expression of apoptotic proteins was detected by Western blotting in Huh7 cells.Results JIB-04 had no obvious inhibitory effect on L02 cells.After treatment with 16μmol/L JIB-04 for 48 h,the inhibition rate was only 5.6%and different concentrations of JIB-04 had different inhibitory effects on Huh7 cells.The inhibitory effects of JIB-04 on Huh7 cells were time-and concentration-dependent,and IC50 was 3.5μmol/L.The flow cytometry detection of hepatocellular carcinoma cells Huh7,cell cycle detection showed that the proportion of S phase and M phase of hepatocellular carcinoma cells were significantly reduced,while the proportion of G1 phase was significantly increased.At the same time,with the increase of JIB-04 concentration,the apoptosis rate of Huh7 was significantly increased.Which were respectively(28.38±0.73)%after 48 h treatmenting with the highest JIB-04 concentration of 8μmol/L.And there was statistically significant between control group and different concentration groups(t=2.767,P<0.05);In the detection of hepatocarcinoma cell Huh7 apoptosis-related protein,the results suggest that the expression of protein B cell lymphoma/leukemia-2 associated X protein(bax)and p53 increased,and the expression of B cell lymphoma/leukemia-2(bcl-2)was down-regulated.Conclusion The inhibitory effect of JIB-04 on normal hepatocytes was not obvious,but it had obvious inhibition on Huh7 hepatoma cells.JIB-04 has no obvious inhibitory effect on normal hepatocytes,which can inhibit the proliferation Huh7 hepatoma cells and interfere with their cycle distribution and promote apoptosis.