PEG10基因对FAK介导的肝癌CAM-DR耐药模型中PI3K/Akt通路的影响

Effect of PEG10 gene on FAK mediated PI3K/Akt pathway in human hepatocellular carcinoma CAM-DR resistance

目的研究化疗药物顺铂(DDP)、5-氟尿嘧啶(5-Fu)、阿霉素(ADM)对PEG10基因稳定表达细胞LO2中FAK介导的肝癌CAM-DR耐药模型中PI3K/Akt通路的影响。方法选择PEG10基因稳定表达LO2细胞株,并构建细胞黏附介导的耐药LO2细胞模型(LO2/CAM-DR细胞),采用不同剂量的顺铂(DDP)、5-氟尿嘧啶(5-Fu)、阿霉素(ADM)作用LO2细胞、LO2/CAM-DR细胞,以及用FAK抑制剂预先干预的LO2/CAM-DR细胞,采用MTT法检测细胞存活率,比较各细胞组中药物的IC50及RI。Western blotting法检测药物作用后细胞中PI3K、AKT、p-AKT蛋白的表达水平;RT-PCR法检测药物处理后细胞中PI3K mRNA水平。结果 LO2细胞、LO2/CAM-DR细胞,以及用FAK抑制剂预先干预的LO2/CAM-DR细胞的存活率均与药物浓度呈负相关,LO2/CAM-DR细胞对药物的敏感性降低,各药作用后IC50均高于LO2细胞,DPP、5-FU和ADM的RI分别为1.345、2.551和2.643。采用FAK抑制剂预先干预的LO2/CAM-DR细胞对药物的敏感性高于LO2/CAM-DR细胞,差异有统计学意义(P<0.05),与LO2细胞相比,差异无统计学意义(P>0.05)。Western blotting检测结果:药物作用后LO2/CAM-DR细胞PI3K、p-AKT的表达水平均高于LO2细胞,差异有统计学意义(P<0.05),采用FAK抑制剂干预后,PI3K、p-AKT的表达水平下降,PI3K mRNA也下降。结论化疗药物与FAK抑制剂的联合使用可以提高化疗药物对肝癌耐药细胞的杀伤作用,下调FAK介导的肝癌CAM-DR耐药模型中PI3K/Akt通路。

Objective To study the effect of chemotherapeutic drug cisplatin （ DDP）, 5-fluorouracil （5-Fu） , adria- mycin （ADM） on FAK mediated PI3K/Akt pathway in hepatocellular carcinoma CAM-DR cells model LO2 cell with PEG10 gene stable expression. Methods LO2 cell lines with PEG10 gene stable expression were selected, and the LO2 cell model of adhesion mediated drug resistant （LO2/CAM-DR cells） was constructed, different dose of DDP, 5-Fu, ADM were taken in LO2 cells, LO2/CAM-DR cells, and LO2/CAM-DR cells preintervented with FAK inhibitor, cells survival rate was detected by MTT method, and the cells ICs0 and RI were calculated. The expression levels of PI3K, AKT, p-AKT protein were detected by Western blotting after treated with drugs; PI3K mRNA was detected by RT-PCR after treated with drugs. Results Survival rates of LO2 cells, LO2/CAM-DR cells, and LO2/CAM-DR cells preinter- vented with FAK inhibitor were negative correlation with the drugs concentration, the drug sensitivity of LO2/CAM-DR ceils reduced, IC50 after the drugs effect was higher than that in LO2 ceils, R[ of DPP, 5-FU and ADM were 1. 345, 2.551 and 2. 643. The drug sensitivity of LO2/CAM-DR cells preintervented with FAK inhibitor was higher than that of LO2/CAM-DR cells, and the difference was statistically significant （P 〈 0.05） , compared with LO2 cells, there was no significant difference （P 〉 0.05）. Western blotting results showed that the expression levels of PI3K, p-AKT were high- er in LO2/CAM-DR cells than that in LO2 cells, and the difference was statistically significant （P 〈 0.05）, but in LO2/ CAM-DR cells preintervented with FAK inhibitor, the expression levels of PI3K, p-AKT were decreased, expression of PI3K mRNA also was decreased. Conclusion Combination chemotherapy and FAK inhibitor can enhance the killing effect of chemotherapy drugs on liver cancer cells, through down regulation of hepatocellular carcinoma CAM-DR resist- ance in Fkt mediated PI3K/Akt pathway.