阿法替尼联合Mithramycin A对人肝癌 HepG2细胞增殖?凋亡及基因表达的影响

目的:观察阿法替尼(afatinib)联合Mithramycin A(MIT)对人肝癌 HepG2 细胞增殖?凋亡的作用以及相关因子表达的影响?方法:将afatinib与MIT单独或联合作用于肝癌HepG2细胞,采用MTT法测定药物对细胞生长的抑制率,并运用倒置显微镜观察药物作用后细胞形态学变化;以流式细胞技术测定药物对细胞周期和凋亡的影响;以实时荧光定量聚合酶链反应(qRT-PCR) 定量测定细胞内表皮生长因子受体(EGFR)?Sp1?Sp3以及增殖?凋亡相关因子 Cyclin-D1?Cyclin-E2?Bcl-2?Caspase3?Caspase9和p53的表达变化?结果:afatinib与MIT均能有效抑制肝癌HepG2 细胞的生长,并且呈现时间依赖性,两药联合作用能明显增加抑制率(P均 < 0.05);联合用药48 h后,可诱导HepG2细胞产生G0/G1期阻滞并诱发凋亡,抑制作用及凋亡率均较单药组增高(P均 < 0.05);另外,给药72 h后,单药组均出现不同程度的Cyclin-D1?Cyclin-E2?Bcl-2 mRNA 表达量下降,并伴有Caspase3基因上调?单用afatinib组同时出现Caspase9和p53的表达上调,MIT组检测到EGFR?Sp1和Sp3的同步减少,联合用药组以上改变较单药组明显(P均 < 0.05)?结论:afatinib联合MIT能有效抑制肝癌 HepG2 细胞增殖?促进凋亡,这可能与药物作用后Cyclin-D1?Cyclin-E2?Bcl-2下调以及Caspase3?Caspase9和p53的表达上调相关?此项研究可能为以EGFR为中心的肝癌联合治疗提供新方向?

The influence on cell proliferation and apoptosis of combined afatinib with Mithramycin A in human hepatocellular carcinoma cell line HepG2

Objective:To explore the effect on proliferation and apoptosis after treatment with afatinib and Mithramycin A(MIT)in hepatocellular carcinoma cell line HepG2,and detect the aberrant expression of related factors. Methods: HepG2 cells were treated by afatinib,MIT,and combination of two. MTT and flow cytometry(FCM)were used to observe cell viability and apoptosis. EGFR,Sp1,Sp3 and genes,which were related to proliferation and apoptosis like Cyclin-D1,Cyclin-E2,Bcl-2,Caspase3,Caspase9 and p53,were analyzed by qRT-PCR. Results:With the administration time increasing,the inhibition rate of HepG2 cells apparently raised,while the group treated by afatinib and MIT exhibited dramatic ascending (P < 0.05). In addition,FCM also confirmed that Combination of afatinib and MIT arreasted HepG2 cells in G0/G1 phase,and the highest apoptosis rate appeared in combined group (P < 0.05). The level of Cyclin-D1,Cyclin-E2 and Bcl-2 in HepG2 cells incubated with both afatinib and MIT were obviously down-regulated, while Caspase3 up-regulated. Besides,afatinib enhanced expression of Caspase9 and p53,while MIT down-regulated the level of EGFR (P < 0.05). Conclusion:Afatinib combined with MIT significantly suppressed proliferation and induced apoptosis in HepG2 cells by inhibiting of Cyclin-D1,Cyclin-E2 and Bcl-2 coupled with increase of Caspase3,Caspase9 and p53. Moreover,our resultes probably provide a novel EGFR-centered strategy for future combined targeting therapy in HCC.