Li Bai-He, Yuan Lei, Shi Ran-Ran, Wang Jian-Guo
Laboratory of Molecular Biology, Luohe Medical College, Luohe 462002, China.
Sheng Li Xue Bao. 2015 Dec 25;67(6):611-7.
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 shHIF-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 apoptosis. 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-1α 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.
本研究旨在探讨地高辛对人乳腺癌细胞系MCF-7/阿霉素（ADR）化疗耐药性的影响及其潜在机制。MCF-7细胞和MCF-7/ADR细胞分别作为对照组和ADR组。ADR + 地高辛组的MCF-7/ADR细胞接受48小时地高辛（10 nmol/L）处理；转染pLKO.1-shHIF-1α和pLKO.1-shcontrol质粒的MCF-7/ADR细胞分别命名为shHIF-1α组和shcontrol组。采用CCK-8法检测ADR对MCF-7/ADR细胞的细胞毒性作用，并根据CCK-8计算IC50值和耐药指数。采用RT-PCR检测缺氧诱导因子-1α（HIF-1α）和多药耐药-1（MDR1）的mRNA水平。采用蛋白质免疫印迹法分析HIF-1α和MDR1的蛋白水平。采用流式细胞术检测细胞凋亡情况。结果显示，MCF-7/ADR细胞的耐药指数为115.6，在地高辛作用下降至47.2（P < 0.05）。与对照组相比，ADR组的HIF-1α和MDR1蛋白及mRNA水平升高（P < 0.05）。地高辛降低了HIF-1α和MDR1的蛋白水平以及MDR1的mRNA水平，但不影响HIF-1α的mRNA水平。HIF-1α基因沉默后，HIF-1α和MDR1的蛋白水平下调（P < 0.05），ADR对MCF-7/ADR细胞的促凋亡作用增强。虽然也观察到地高辛在shcontrol组和shHIF-1α组中均促进细胞凋亡，但两组之间差异不显著。总之，结果表明地高辛可能通过下调HIF-1α和MDR1的表达水平，并通过不依赖HIF-1α的途径促进凋亡，从而部分逆转人乳腺癌细胞系MCF-7/ADR对ADR的耐药性。
