重组人内皮抑素对人肝癌细胞SMMC-7721增殖及凋亡的影响

Effect of rh-endostatin on proliferation and apoptosis in hepatocellular carcinoma cell SMMC-7721

目的初步研究Wnt通路在重组人血管内皮抑素(rh-Endostatin,ES)诱导人肝癌细胞SMMC-7721增殖、凋亡过程中的作用及可能机制。方法分别以50、100、200、400μg/ml ES体外处理人肝癌细胞株SMMC-7721,利用MTT比色法检测不同浓度、时间ES作用下的细胞增殖状态;利用流式细胞仪检测细胞周期及凋亡;Western blot检测β连环蛋白(β-catenin)、糖原合酶激酶3β(GSK-3β)表达;应用RT-PCR检测Wnt信号通路下游基因survivin及cyclin D1 mRNA表达情况。结果不同浓度的ES对人肝癌细胞SMMC-7721具有不同程度的生长抑制作用,且存在时间剂量效应,ES作用48 h和72 h后,SMCC-7721细胞抑制率分别为(10.45±0.73)%和(21.81±0.55)%(50μg/ml);(15.42±1.91)%和(27.81±0.92)%(100μg/ml);(23.64±0.43)%和(31.07±0.13)%(200μg/ml);(28.88±0.63)%和(43.76±0.55)%(400μg/ml)。与24 h各浓度组相比差异有统计学意义(P=0.000)。各浓度ES处理后,SMMC-7721细胞G1期细胞比例与对照组比较差异有统计学意义(P=0.00);SMMC-7721细胞经ES作用72 h后,随ES浓度升高,细胞凋亡率增加,与对照组相比,差异有统计学意义(P<0.05)。ES能显著降低细胞β-catenin蛋白表达(P=0.000),但对GSK-3β蛋白表达无明显影响。ES能下调Wnt信号通路下游转录因子survivin及cyclin D1 mRNA水平。结论 ES通过促进β-catenin蛋白降解,降低Wnt信号通路下游癌基因survivin及cyclin D1 mRNA的转录,从而抑制SMMC-7721细胞增殖,并诱导细胞凋亡。ES抑制增殖、诱导凋亡的作用与GSK-3β蛋白表达无关。

Objective To investigate the effect of Wnt signal pathway on proliferation and apoptosis of rh-endostatin（ES） induced hepatocellular carcinoma cell SMMC-7721 and the possible mechanism.Methods Hepatocellular carcinoma cell SMMC-7721 was treated with different concentrations of rh-endostatin（50,100,200 and 400 μg/ml,respectively） in vitro.MTT assay was used to detect cell proliferation in ES treatment at different concentrations and different time intervals.Flow cytometry was used to detect effect of ES on cell cycle and apoptosis.Protein contents of β-catenin and glycogen synthase kinase-3β（GSK-3β） were determined by Western blotting.mRNA expressions of Wnt signaling pathway downstream genes survivin and cyclin D1 were determined by reverse transcription-polymerase chain reaction（RT-PCR）.Results ES significantly inhibited the growth of SMMC-7721 cells in a time and dose dependent manner.After ES treatment for 48 and 72 h,the inhibition rates of SMCC-7721 cell were（10.45±0.73）% and（21.81±0.55）%（50 μg/ml）,（15.42±1.91）% and（27.81±0.92）%（100 μg/ml）,（23.64±0.43）% and（31.07±0.13）%（200 μg/ml） and（28.88±0.63）% and（43.76±0.55）%（400 μg/ml）,respectively,which were significantly different from that of 24 h treatment（P=0.000）.The proportion of SMMC-7721 cells arrested in G1 phase after ES treatment was significantly different from that of the control（P=0.000）.After ES treatment for 72 h,SMMC-7721 cell apoptosis rate increased in a dose dependent manner and was significantly different from that of the control（P〈0.05）.Western blotting showed that protein expression of β-catenin in SMMC-7721 cells was significantly inhibited by ES,but GSK-3β protein expression was not affected.mRNA expressions of Wnt signal pathway downstream genes survivin and cyclin D1 decreased after ES treatment.Conclusion ES can inhibit proliferation and induce apoptosis in hepatocellular carcinoma cell SMMC-7721 by inhibiting protein expression of β-catenin and mRNA expressions of survivin and cyclin D1.The inhibitory effect of ES on cell proliferation and apoptosis is not related to protein expression of GSK-3β.