索拉菲尼诱导的肝癌耐药细胞株的建立及其生物学特性

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

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(extra-cellular 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。

Objective To establish sorafenib-indeuced drug resistant hepatoma cell lines and investi- gate their biological characteristics. Methods CCK8 assay was used for drug sensitivity of hepatocellular car- cinoma 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-IT, LRP, MDR1, MRP and Topo I] , 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 sig- nificantly 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 estab- lished. The underlying mechanism may be associated with sorafenib-induced ERK1/2 activation that leads to high expression of drug resistant gene MDRI.