目的 研究薯蓣皂苷元对盐酸米托蒽醌的抗肿瘤增敏作用及其机制,为临床联合用药提供指导。方法 以维拉帕米为阳性对照,利用四甲基偶氮唑蓝(MTT)法测定薯蓣皂苷元和抗癌药盐酸米托蒽醌联用后对耐药细胞株MES-SA/MX2增殖的影响;蛋白印迹法(WB)和实时荧光定量PCR(RT-qPCR)法研究薯蓣皂苷元对细胞P-糖蛋白(P-gp)的表达影响,流式细胞仪测定候选化合物对P-gp底物四甲基罗丹明甲酯(TMRM)在细胞中的摄取,试剂盒测定ATP酶活性。结果 薯蓣皂苷元可增加盐酸米托蒽醌对耐药细胞株MES-SA/MX2的增殖抑制,逆转指数为维拉帕米的2.4倍,同时降低P-gp蛋白达22.2% (P<0.01)的表达,但对P-gp的基因表达无影响,能提高TMRM的胞内荧光强度达14.5%,且不影响ATP酶活性。结论 薯蓣皂苷元增强米托蒽醌对耐药子宫瘤细胞的敏感性,其作用与抑制P-gp功能有关。
Abstract
OBJECTIVE To investigate the sensitizing effect and mechanism of diosgenin on anti-proliferation activity of mitoxantrone hydrochloride on MDR cells. METHODS Verapamil was used as positive control. MTT method was used to evaluate the proliferation inhibition effect of diosgenin combined with mitoxantrone hydrochloride on MES-SA/MX2 cells. Western blot method (WB) and RT-qPCR were used to evaluate the effect of diosgeninon protein and gene expression of P-gp respectively. The uptake activity of TMRM was measured by flow cytometry(FCM). ATPase activity was determined by kit. RESULTS Diosgenin can ehhance proliferation inhibition of mitoxantrone hydrochloride in MDR cell lines (MES-SA/MX2). The reversing index was 2.4 times of verapamil. Diosgenin decreased P-gp protein expression by 22.2% (P<0.01), but had marginal effect on P-gp mRNA. Diosgenin increased the fluorescence intensity of TMRM by 14.5%, and did not affect ATPase activity. CONCLUSION Diosgenin can increase the sensitivity of mitoxantrone hydrochloride by inhibiting P-gp.
关键词
薯蓣皂苷元 /
P-糖蛋白 /
蛋白印迹法 /
ATP酶
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Key words
diosgenin /
P-glycoprotein /
Western blot /
ATPase
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中图分类号：
R965
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参考文献
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脚注
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基金
国家自然科学基金资助项目(81073063,81573612);广东省自然科学基金资助项目(2015A030313357)
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