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

发布时间：2018-05-04 07:05

  本文选题：新藤黄酸 + 阿霉素　； 参考：《中华中医药杂志》2017年06期

【摘要】：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(P0.01),且联合用药组细胞存活率低于单独用药组(P0.01)。联合指数法分析计算后得出合用指数CI1,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(P0.01)。单独给药组MCF-7细胞线粒体膜电位有所降低(P0.01),而联合用药组与单独给药组相比则膜电位明显降低(P0.01)。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(P0.01)。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(P0.01)。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。
[Abstract]:Aim: to study the effect of the combination of GNAA and adriamycin on the proliferation of human breast cancer cell line MCF-7, and to explore the mechanism of the combination of the two drugs. Methods: human breast cancer cell line MCF-7 was cultured in vitro. MTT assay was used to detect the survival rate of the cells treated with GNA and ADR alone and in combination with Chou-Talalay index method. The combined effect of GNA and ADR was evaluated by Annexin V-FITC/PI double staining flow cytometry to detect the apoptosis rate of MCF-7 cells. The changes of mitochondrial membrane potential in JC-1 stained cells were detected by flow cytometry and the expression of Caspase-9 and Caspase-3 pathway proteins in MCF-7 cells treated with GNA and ADR alone and in combination with ADR were detected by flow cytometry. At the same time, the changes of related protein indexes were detected after treatment with JNK pathway inhibitor SP600125. Results: within a certain concentration range, the cell survival rate of human breast cancer cell line MCF-7 decreased with the increase of GNA(0.0625-4.0000 渭 mol / L and ADR(0.5-16.0 渭 mol / L administration, and the cell survival rate of the combined treatment group was lower than that of the single drug group. The combined index CI1 was calculated by the combined index method, which indicated that the combination of GNA and ADR could induce apoptosis of MCF-7 cells both alone and in combination, and the apoptosis rate of the combined treatment group was significantly higher than that of the control group. The mitochondrial membrane potential of MCF-7 cells in the single administration group was lower than that in the control group (P 0.01), while the membrane potential in the combination group was significantly lower than that in the control group. Compared with the control group, the expression of Caspase-9 and Caspase-3 in ASK-1 + p-ASK-1 group was activated, and the expression of related proteins was increased, while the expression of histone in combination group was significantly up-regulated (P 0.01). After treatment with JNK pathway inhibitor SP600125, the expression of ASK-1 and p-ASK-1 protein was not significantly different from that of untreated group. The expression of Caspase-3 protein decreased slightly, while SP600125 inhibited the up-regulation of JNK-p-JNK protein expression. The expression of histone in combined drug group was lower than that in control group (P 0.01). Conclusion ADR alone or in combination can induce apoptosis of human breast cancer MCF-7 cells, and the synergistic effect can be produced by the combination of them. The mechanism may be related to the regulation of JNK signaling pathway.
【作者单位】： 安徽中医药大学科研实验中心省部共建教育部新安医学重点实验中心;
【基金】：国家自然科学基金项目(No.81173600,No.81673650)~~
【分类号】：R737.9
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本文编号：1842071