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

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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 effect of GNA combined with Adriamycin (ADR) on the proliferation of human breast cancer cell line MCF-7 and to explore the related mechanisms. Methods: The human breast cancer cell line MCF-7 was cultured in vitro. The survival rate of cells treated with GNA and ADR alone or in combination was detected by MTT assay. The drug combination of GNA and ADR was evaluated by Chou-Talalay index method. Annexin V-FITC / PI double staining flow cytometry was used to detect the apoptosis rate of MCF-7 cells; the change of mitochondrial membrane potential of JC-1 staining cells was detected by flow cytometry; Western blot was used to detect MCF-7 The changes of the protein expression of ASK-1, p-ASK-1, JNK, p-JNK, Cyt-c, Caspase-9 and Caspase-3 were detected by Western blotting and the changes of related proteins after treatment with JNK inhibitor SP600125 . Results: The cell viability of human breast cancer cell line MCF-7 decreased with the dose of GNA (0.0625-4.0000μmol / L) and ADR (0.5-16.0μmol / L) at a certain concentration range (P <0.01 ), And the cell viability of the combination group was lower than that of the drug alone group (P <0.01). Combined index method analysis and calculation of combined index CI <1, suggesting that synergies between the two drugs combined. Apoptosis of MCF-7 cells was induced by GNA and ADR alone and in combination, and the apoptosis rate in combination group was significantly higher than that in the drug alone group (P <0.01). The mitochondrial membrane potential of MCF-7 cells was decreased (P <0.01), but the membrane potential of MCF-7 cells in combination group was significantly lower than that in MCF-7 alone group (P <0.01). Compared with the normal control group, ASK-1, p-ASK-1, JNK, p-JNK, Cyt-c, Caspase-9 and Caspase-3 proteins were all activated and the expression of related proteins increased Combination therapy group protein expression was significantly increased (P <0.01). The protein expression of ASK-1 and p-ASK-1 had no significant difference with that of untreated group after treatment with JNK pathway inhibitor SP600125. The expression of Cyt-c and Caspase-9 protein decreased slightly and the expression of Caspase-3 protein did not change significantly. However, SP600125 inhibited the JNK and p-JNK protein expression, and the combined treatment group protein expression was reduced (P <0.01). CONCLUSION: Both GNA and ADR can induce the apoptosis of human breast cancer cell line MCF-7 both alone and in combination. The combination of GNA and ADR can produce synergistic effect, which may be related to the regulation of JNK signaling pathway.
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