Liu X-Y, Liu S-P, Jiang J, Zhang X, Zhang T
Department of Oncology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China.
Eur Rev Med Pharmacol Sci. 2016;20(6):1098-108.
The resistance of hepatocellular carcinoma (HCC) to chemotherapy may be mediated by the c-Jun N-terminal kinase (JNK) pathway. We wished to verify the involvement of this pathway in resistance of HCC cells to cisplatin.
We used HepG2 cell line and cisplatin-resistant clone (HepG2/DDP). Expressions of drug resistance and apoptosis-related genes were analyzed by qPCR. Protein expressions were assessed by Western blot. The JNK pathway was assessed as total JNK1/2 and JNK1/2 phosphorylation. Cell growth kinetics was quantified by the CCK-8 assay, and cell apoptosis (Annexin V / propidium iodide) by flow cytometry.
HepG2/DDP cells were more resistant and less apoptotic on cisplatin. Expression of drug-resistance genes MDR1, MRP1 and MPR2 was significantly up-regulated in HepG2/DDP cells (p < 0.05), with up-regulation of MDR1 being the highest. This was confirmed by Western blot analysis of P-glycoprotein (P-gp), MRP1 and MRP2 proteins, the proteins encoded by the above genes. Expression of anti-apoptotic genes Bcl-2 and Bcl-XL was significantly up-regulated, and expression of pro-apoptotic genes Bak and Bad was significantly reduced, in HepG2/DDP cells (p < 0.05). Cisplatin treatment of HepG2 led to increased phosphorylation of JNK1/2; the trend reversed by the inhibitor SP600125. Furthermore, cisplatin increased expression of P-gp, which was also attenuated by SP600125. Cell growth was inhibited more substantially, and cell apoptosis promoted, when HepG2 cells were exposed to both cisplatin and SP600125.
Inhibition of the JNK signaling pathway enhances the sensitivity of HCC cells to cisplatin by down-regulating the expression of P-gp.
肝细胞癌（HCC）对化疗的耐药性可能由c-Jun氨基末端激酶（JNK）通路介导。我们希望验证该通路在HCC细胞对顺铂耐药中的作用。
我们使用了HepG2细胞系和顺铂耐药克隆（HepG2/DDP）。通过qPCR分析耐药性和凋亡相关基因的表达。通过蛋白质印迹法评估蛋白质表达。以总JNK1/2和JNK1/2磷酸化来评估JNK通路。通过CCK-8测定法定量细胞生长动力学，并通过流式细胞术检测细胞凋亡（膜联蛋白V/碘化丙啶）。
HepG2/DDP细胞对顺铂更具耐药性且凋亡较少。耐药基因MDR1、MRP1和MPR2在HepG2/DDP细胞中的表达显著上调（p<0.05），其中MDR1的上调最为明显。这通过对上述基因编码的蛋白质P-糖蛋白（P-gp）、MRP1和MRP2进行蛋白质印迹分析得到证实。抗凋亡基因Bcl-2和Bcl-XL在HepG2/DDP细胞中的表达显著上调，而促凋亡基因Bak和Bad的表达显著降低（p<0.05）。顺铂处理HepG2导致JNK1/2磷酸化增加；抑制剂SP600125可逆转这一趋势。此外，顺铂增加了P-gp的表达，SP600125也可使其减弱。当HepG2细胞同时暴露于顺铂和SP600125时，细胞生长受到更显著的抑制，细胞凋亡增加。
抑制JNK信号通路通过下调P-gp的表达增强了HCC细胞对顺铂的敏感性。
