黄芪多糖联合多柔比星抗白血病细胞HL-60/A耐药的机制研究

Mechanisms of Astragalus Polysaccharids Synergized with Doxorubicin against Drug Resistance in HL-60/A Cells

目的:探讨黄芪多糖联合多柔比星对耐药白血病细胞HL-60/A的协同抗肿瘤机制。方法:RT-PCR、Western blot及CCK-8试验鉴定急性早幼粒细胞耐药株HL-60/A的耐药表型;然后通过CCK-8试验检测黄芪多糖协同多柔比星对HL-60/A的抗肿瘤作用;Annexin-Ⅴ细胞凋亡实验检测不同药物处理后的细胞凋亡情况;Western blot检测细胞内caspase级联系统激活情况;实时定量PCR及Western blot实验分析黄芪多糖对多药耐药蛋白M RP的表达及功能影响。结果:与敏感细胞相比,HL-60/A表现出明显的耐药特征,对多柔比星的耐药倍数约为HL-60的27倍。CCK-8试验证实,黄芪多糖可有效促进多柔比星对耐药细胞HL-60/A的杀伤,且黄芪多糖与多柔比星联用可显著诱导HL-60/A细胞凋亡,同时伴随凋亡级联信号系统激活。进一步研究发现,黄芪多糖可降低HL-60/A细胞表面的MRP表达,进而抑制MRP的外排功能,增加进入耐药细胞中的药物浓度。结论:黄芪多糖联合多柔比星可有效发挥对耐药白血病细胞HL-60/A的协同抗肿瘤作用,该作用可能是通过黄芪多糖抑制HL-60/A细胞表面的MRP表达、抑制药物外排,从而增加耐药细胞内药物浓度,诱导HL-60/A细胞凋亡实现的。

Objective： To explore the synergistic anti-tumor mechanism of astragalus polysaccharids （APS） combined with doxorubicin in HL-60/A cells. Methods ： The phenotype of HL-60/A was identified by using RT-PCR, Wester blot and CCK-8, the antitumor effect of astragalus polysaccharids combined with doxorubicin on HL-60/A cells was detected by CCK-8, the apoptosis of HL-60/A after treating with different drugs was detected by Annexin-V, the caspase casade activation was detected by Wester blot, the effect of astragalus polysaccharids on expression and function of multidrug resistance protein （MRP） was detected by using real-time quantitative PCR and Western blot. Results： HL-60/A cells displayed obvious characteristics of resistance to doxorubicin, whose resistance were about 27 times that of its sensitive cell line HL-60. In addition, it was further found that astragalus polysaccharids could obviously increase the cell growth inhibition, induce cell apoptosis and caspase cascade activation, decrease the expression of MRP and increase the drug concentration in HL-60/A when combined with doxorubicin. Conclusion： Astragalus polysaccharids combined with doxorubicin can effectively overcome the drug resistance of HL-60/A. The mechanism may be associated with decreasing the expression of MRP, inhibiting drug efflux and increasing the intracellular drug concentration, then inducing HL-60/A cell apoptosis.