华蟾素注射液联合放疗对食管癌细胞增殖与周期的影响

Effect of Cinobufacini Injection Combined with Radiotherapy on Proliferation and Cell Cycle of Human Esophageal Carcinoma

目的:观察华蟾素注射液对食管癌细胞ECA109增殖的抑制作用,分析华蟾素联合X射线照射对细胞周期的影响,探讨华蟾素注射液与放疗联合效应的机制。方法:实验分空白组、华蟾素组、放疗组、华蟾素+放疗组,作用食管癌ECA109细胞48 h后,应用噻唑蓝(MTT)比色法检测华蟾素注射液对ECA109细胞增殖的影响;流式细胞术(FCM)检测细胞周期的变化,反转录-PCR(RT-PCR)和免疫印迹法(Western blot)检测各组泛素连接酶(RNF2),p16和周期蛋白依赖性激酶(CDK4)mRNA和蛋白表达。结果:华蟾素可显著抑制食管癌ECA109细胞的增殖,且呈浓度和时间依赖性。与空白组比较,各药物组中DNA合成前期(G0/G1期)细胞比例明显升高(P<0.05),S期细胞比例明显下降(P<0.05)。与空白组比较,华蟾素+放疗组中RNF2 mRNA和蛋白明显降低(P<0.05),且各药物组均能促进p16 mRNA和蛋白表达(P<0.05),抑制CDK4 mRNA和蛋白水平(P<0.05)。结论:华蟾素注射液能有效抑制人食管癌ECA109细胞增殖,并将细胞阻滞于G0/G1期,机制可能与降低RNF2,CDK4水平,上调p16基因水平相关。

Objective： To investigate the effects of Cinobufacini （Cino） injection on the proliferation of human esophageal carcinoma cells ECA109, analyze the effect of Cin combined with irradiation on cell cycle, and explore the potential mechanisms of Cino injection combined with radiotherapy. Method： ECA109 cells were divided into blank group, Cino group, radiotherapy group, and combined group （Cino＋radiotherapy）. After the esophageal carcinoma cells ECA109 were treated for 48 h, methylthiazolyldiphenyl-tetrazolium bromide （MTT） assay was used to detect the effects of Cino on proliferation of ECA109 cells, flow cytometry （FCM） was used to detect the changes in cell cycles, reverse transcription-polymerase chain reaction（RT-PCR） and Western blot were used to respectively detect the mRNA expressions and protein expressions of ring finger protein 2 （RNF2）, p16 and cyclin-dependent kinase 4 （CDK4） in ECA109 cells. Result： Cinobufacini injection significantly inhibited ECA109 cells proliferation in doses and time dependent ways. As compared with the blank group, the proportion of cells in G0/G1 phase were significant increased （P〈0.05） and the proportion of cells in S phase were significant decreased （P〈0.05） in various treatment groups. As compared with the blank group, the mRNA and protein expressions of RNF2 in the combined group were significantly decreased （P〈0.05）, mRNA and protein expressions of p16 were higher in various treatment groups （P〈0.05）, while mRNA and protein expressions of CDK were lower in the various treatment groups （P〈0.05）. Conclusion： Cino injection can effectively inhibit the proliferation of human esophageal carcinoma ECA109 cells and arrest cell cycle at G0/G1 phase. The mechanism might be partly related to the down-regulation of RNF2, CDK4 levels, and the up-regulation of p16 mRNA expressions.