地高辛逆转乳腺癌MCF-7/阿霉素细胞的耐药性及其机制

Reversal of adriamycin resistance by digoxin in human breast cancer cell line MCF-7/adriamycin and its mechanism

本研究旨在探究地高辛对乳腺癌MCF-7/阿霉素(ADR)细胞耐药性的影响,并探讨其分子机制。将正常培养的MCF-7和MCF-7/ADR细胞分别设为对照组和ADR组;经地高辛(100 nmol/L)处理48 h后的MCF-7/ADR细胞为ADR+digoxin组;用sh RNA技术沉默HIF-1α基因的MCF-7/ADR细胞为sh HIF-1α组,并设置对照组为shcontrol组。采用CCK-8法检测ADR的细胞毒作用,以测定IC50与耐药指数;用RT-PCR法检测缺氧诱导因子-1α(HIF-1α)和多药耐药基因1(multidrug resistance-1,MDR1)的mRNA水平;用Western blot方法检测HIF-1α和MDR1蛋白的表达水平;用流式细胞术检测细胞凋亡。结果显示,ADR组细胞对ADR的耐药指数高达115.6,地高辛使其耐药指数下降至47.2(P<0.05);ADR组细胞中HIF-1α与MDR1的mRNA和蛋白水平均高于对照组细胞(均P<0.05),而地高辛可显著降低ADR组细胞HIF-1α与MDR1的蛋白水平,以及MDR1的mRNA水平(P<0.05),但对HIF-1α的mRNA水平无明显影响;经sh RNA干扰HIF-1α基因表达后,HIF-1α和MDR1的蛋白水平均显著降低(P<0.05);沉默HIF-1α基因能显著增强ADR对ADR组细胞的促凋亡作用(P<0.05)。地高辛可有效地诱导shcontrol组和sh HIF-1α组细胞凋亡(P<0.05),但二组之间无显著差异。以上结果提示,地高辛可通过下调HIF-1α蛋白表达从而在转录水平抑制MDR1的表达,也可通过不依赖HIF-1α的通路诱导MCF-7/ADR细胞凋亡,进而在一定程度上逆转MCF-7/ADR细胞的耐药性。

The aim of this study was to investigate the effects of digoxin on the chemoresistance of human breast cancer cell line MCF-7/adriamycin （ADR） and its underlying mechanism. MCF-7 and MCF-7/ADR cells were designated as control and ADR groups, respectively. MCF-7/ADR cells in ADR ＋ digoxin group received 48 h of digoxin （10 nmol/L） treatment; MCF-7/ADR cells transfected with pLKO. 1-shHIF-1α and pLKO. 1-shcontrol plasmids were named shHlF-1α and shcontrol groups, respectively. CCK-8 assay was employed to detect the cytotoxic effect of ADR on MCF-7/ADR cells, and IC50 value and resistance index were calculated according to CCK-8. RT-PCR was used to measure the mRNA levels of hypoxia inducible factor-1α （HIF-1α） and multidrug resistance-1 （MDR1）. Western blot was used to analyze the protein levels of HIF-1α and MDR1. Flow cytometry was used to determine the apop- tosis. The result showed that the resistance index of MCF-7/ADR cells was 115.6, and it was reduced to 47.2 under the action of digoxin （P 〈 0.05）. In comparison with control group, ADR groups showed increased protein and mRNA levels of HIF-1α and MDR1 （P 〈 0.05）. Digoxin reduced the protein levels of HIF-1α and MDR1, as well as the mRNA level of MDR1, but did not affect the mRNA level of HIF-1α. After HIF-la gene was silenced, the protein levels of HIF-1α and MDR1 were down-regulated （P 〈 0.05）, and the pro-apoptotic effect of ADR on MCF-7/ADR cells was enhanced. Although it was also observed that digoxin promoted cell apoptosis in both shcontrol and shHIF-1α groups, the difference between the two groups was not significant. In conclusion, the results suggest that digoxin may partially reverse the ADR resistance in human breast cancer cell line MCF-7/ADR by means of down-regulating the expression levels of HIF-1α and MDR1 and promoting apoptosis via HIF-1α-independent pathway.