肠胃清对长春新碱诱导的人结肠癌耐药细胞株Y盒结合蛋白核移位及P-糖蛋白表达的影响

Effects of Changweiqing on Nuclear Translocation of Y-Box Binding Protein-1 and Expression of P-glycoprotein in Human Colon Cancer Cell Line with Drug-resistance Induced by Vincristine

目的观察肠胃清对长春新碱诱导的人结肠癌耐药细胞株HCT8/V的Y盒结合蛋白(YB-1)核移位、多药耐药基因MDR1及P-糖蛋白(P-gp)表达的影响,进一步探讨该方逆转肿瘤多药耐药的分子机制。方法制备大鼠灌胃后的肠胃清药物血清,以Western blot方法检测肠胃清药物血清作用下HCT8/V细胞胞浆、胞核YB-1表达情况,EMSA法分析YB-1与MDR1基因启动子结合活性,RT-PCR检测MDR1、YB-1、多药耐药相关蛋白(MRP)mRNA转录水平,流式细胞仪检测细胞膜表面P-gp表达。结果在1.25%、2.5%、5%药物血清作用下,HCT8/V细胞核内YB-1表达逐渐减弱,细胞质内YB-1表达则逐渐增强;YB-1与MDR1基因启动子结合活性逐渐下降(P<0.01);MDR1 mRNA转录水平逐渐降低(P<0.05,P<0.01),YB-1和MRPmRNA的水平无明显变化(P>0.05);细胞膜表面P-gp表达荧光强度值减弱(P<0.05,P<0.01)。结论肠胃清可通过影响耐药细胞YB-1的核移位、降低MDR1/P-gp的表达,逆转结肠癌细胞的耐药。

Objective To evaluate the effects and molecular mechanism of action of Changweiqing （CWQ） in reversing multidrug resistance by observing its impacts on nuclear translocation of Y-box binding protein-1 （YB-1）, multi-drug resistance gene （ MDR1 ） expression and P-glycoprotein （P-gp） expression in human colon cancer cell line HCT8/V with drug-resistance induced by vincristine. Methods Cultured HCT8/V cells were exposed to the CWQ-containing rat serum prepared by drug perfusion. YB-1 expressions in cell plasma and nuclei were examined by Western blot; the binding activity of YB-1 to MDR1 gene promoter sequences was detected by electrophoretic mobility shift assay （EMSA） ; the mRNA transcription levels of MDR1, YB-1 and multi-resistance related protein （MRP） were examined by RT-PCR; the expression of P-gp on cell membrane was determined by flow cytometry. Results Along with the increasing drug＇s concentration of CWQ-containing serum from 1.25% up to 2.5% and 5%, the expressions of YB-1 decreased in HCT8/V cell nuclear and increased in cytoplasm gradually; the binding activity of YB-1 to MDR1 gene promoter weakened （P 〈0.01 ）, MDR1 mRNA expression and fluorescence intensity of P-gp on cell membrane attenuated （ P 〈 0.05 or P 〈 0.01 ）, while YB-1 and MRP mRNA unchanged （ P 〉 0. 05）. Conclusion CWQ could reverse the drug-resistance of colon cancer cells by influencing nuclear translocation of YB-1 and reducing the expression of MDR1/P-gp.