索拉菲尼诱导的肝癌耐药细胞株的建立及其生物学特性

[目的]建立索拉菲尼诱导的PLC/PRF/5和Huh7耐药细胞株,并研究其耐药细胞的生物学特性.[方法]通过药物敏感性实验CCK8法测定索拉菲尼的半数抑制浓度IC50值;采用体外间歇诱导法,使用PLC/PRF/5和Huh7各自IC50值作为筛选浓度,建立索拉菲尼诱导的耐药细胞株模型;Real time-PCR检测5种耐药基因(GST-π、LRP、MDR1、MRP、TopoⅡ)在耐药组和对照组中的表达差异;Western blot检测MAPK信号通路中关键信号因子细胞外调节蛋白激酶(extracellular regulated protein kinases,ERK)ERK1/2在不同组别细胞中的磷酸化水平差异.[结果]所筛选的PLC/PRF/5和Huh7耐药组细胞与对照组细胞相比较,其细胞生存率有显著性提高(P＜0.05);5种耐药基因中MDR1的表达有显著性上调(P＜0.05);索拉菲尼作用靶点的下游信号因子ERK1/2的磷酸化水平上调.[结论]成功地在体外建立索拉菲尼诱导的PLC/PRF/5和Huh7耐药细胞株,其耐药机制可能与ERK1/2的磷酸化水平上调导致耐药基因MDR1高表达有关.

Establishment and biological characteristics of sorafenib-resistant hepatocellular carcinoma cell lines

Objective To establish sorafenib-indeuced drug resistant hepatoma cell lines and investigate their biological characteristics.Methods CCK8 assay was used for drug sensitivity of hepatocellular carcinoma cell lines PLC/PRF/5 and Huh7 to sorafenib.By using intermittent induction in vitro and the obtained IC₅0 value as a screening concentration,we screened and established drug resistant cell lines.Expression of drug resistant genes,GST-π,LRP,MDR1,MRP and Topo Ⅱ,were examined by real time-PCR.Western blot analysis was applied to examine the phosphorylation level of extracellular regulated protein kinases 1/2（ERK1/2）.Results Sorafenib-resistant PLC/PRF/5 and Huh7 cells had a longer survival rate compared with corresponding control cells（P<0.05）.MDR1 was the only one from the 5 resistance genes who had significantly increased expression（P<0.05）.Furthermore,sorafenib was found to induce the phosphorylation of ERK1/2.Conclusion Sorafenib resistant hepatoma cell lines PLC/PRF/5 and Huh7 are successfully established.The underlying mechanism may be associated with sorafenib-induced ERK1/2 activation that leads to high expression of drug resistant gene MDR1.