新藤黄酸联合阿霉素通过JNK信号通路诱导人乳腺癌细胞凋亡的相关机制研究

Mechanism study on human breast cancer cells apoptosis induced by neogambogic acid coupled with Adriamycin through JNK pathway

目的:研究新藤黄酸(GNA)与阿霉素(ADR)联合应用对人乳腺癌细胞MCF-7增殖的影响,并进一步探讨两药联用的相关机制。方法:体外培养人乳腺癌细胞MCF-7细胞株,采用MTT法检测GNA与ADR单独及联合处理后细胞的存活率;Chou-Talalay联合指数法评价GNA与ADR的药物联合作用;Annexin V-FITC/PI双染流式细胞仪检测给药后MCF-7细胞凋亡率;流式细胞仪检测JC-1染色细胞线粒体膜电位变化;Western blot法分别检测GNA与ADR单独及联合处理MCF-7细胞后ASK-1、p-ASK-1、JNK、p-JNK、Cyt-c、Caspase-9和Caspase-3通路蛋白表达的变化,同时检测经JNK通路抑制剂SP600125处理后相关蛋白指标的变化。结果:在一定浓度范围内,人乳腺癌细胞MCF-7的细胞存活率随着GNA(0.0625-4.0000μmol/L)与ADR(0.5-16.0μmol/L)给药剂量增加而降低(P<0.01),且联合用药组细胞存活率低于单独用药组(P<0.01)。联合指数法分析计算后得出合用指数CI<1,提示两药联合具有协同作用。GNA与ADR单独和联合作用均能诱导MCF-7细胞凋亡,且联合用药组细胞凋亡率与单独用药组比较,显著增加(P<0.01)。单独给药组MCF-7细胞线粒体膜电位有所降低(P<0.01),而联合用药组与单独给药组相比则膜电位明显降低(P<0.01)。与正常对照组比较,单独给药组中ASK-1、p-ASK-1、JNK、p-JNK、Cyt-c、Caspase-9和Caspase-3蛋白均被激活,相关蛋白表达量增加,而联合用药组蛋白表达显著上调(P<0.01)。使用JNK通路抑制剂SP600125处理后,ASK-1和p-ASK-1蛋白表达与未处理组无明显差异,Cyt-c和Caspase-9蛋白表达量略微下降,Caspase-3蛋白表达无显著变化,而SP600125抑制了JNK,p-JNK蛋白表达上调,且联合用药组蛋白表达与单独给药组相比有所降低(P<0.01)。结论:GNA与ADR单独和联合应用均能诱导人乳腺癌细胞MCF-7细胞凋亡,且两者联合应用可产生协同效应,其作用机制可能与调控JNK信号通路相关。

Objective: To study the combination effects of gambogenic acid(GNA) and Adriamycin(ADR) on the proliferation of human breast cancer MCF-7 cells, and its mechanism. Methods: Human breast cancer MCF-7 cells were cultured in vitro and cell viability after being treated with GNA and ADR singly and jointly was measured by MTT assay. The Chou-Talalay combination index was used to analyze the drug combination effect. The Flow cytometry Annexin V-FITC/PI double staining was applied to observe the apoptosis rate of MCF-7 cells. The Flow cytometry JC-1 staining was used to detect the change of mitochondrial membrane potential and Western blot was used to measure the protein expressions of ASK-1, p-ASK-1, JNK, p-JNK, Cyt-c, Caspase-9 and Caspase-3. Meanwhile, the changes of these protein after the treatment of JNK inhibitor SP600125 were measured. Results: The proliferative activity of human breast cancer MCF-7 cells was decreased with the increasing dose of GNA(0.0625-4.0000μmol/L) and ADR(0.5-16.0μmol/L) in a certain range, and the cell survive rate was lower in the combined group(P<0.01). The results of Chou-Talalay analysis showed that combination index was lower than 1, indicating that the two drugs had a synergistic inhibitory effect. GNA and ADR could induce apoptosis in either single or combined group, and the apoptosis rate of combination group was apparently increased than that the treatment of GNA or ADR singly(P<0.01). The mitochondrial transmembrane potential of single groups was decreased(P<0.01) and the combined group showed a better result(P<0.01). Compared with control group, the related proteins such as ASK-1, p-ASK-1, JNK, p-JNK, Cyt-c, Caspase-9 and Caspase-3 in the single groups were activated, and these proteins were significantly increased by the administration of drug combination(P<0.01). The treatment of JNK inhibitor SP600125 slightly decreased the protein expressions of Cyt-c and Caspase-9, and inhibited the upregulation of protein expressions of JNK and p-JNK, but the protein expressions of ASK-1, p-ASK-1 and Caspase-3 were no obviously changed by the treatment of JNK inhibitor SP600125. The treatment of JNK inhibtor SP600125 inhibited the upregulation of JNK, P-JNK. The protein expressions in the combination groups were lower than those in the single groups(P<0.01). Conclusion: The single and combination use of GNA and ADR can induce the apoptosis of human breast MCF-7 cells, and the combination of two drugs have a better synergistic effect, the mechanism of which may be related to the JNK pathway.