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地高辛逆转乳腺癌MCF-7/阿霉素细胞的耐药性及其机制

李伯和, 袁磊, 时冉冉, 王建国^*

漯河医学高等专科学校分子医学实验室，漯河 462002

摘要

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shRNA技术沉默HIF-1α基因的MCF-7/ADR细胞为shHIF-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shRNA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shHIF-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。

关键词： 地高辛; MCF-7/ADR细胞; 缺氧诱导因子-1α; 多药耐药基因1; 细胞凋亡

分类号：R737.9

[Reversal of adriamycin resistance by digoxin in human breast cancer cell line MCF-7/adriamycin and its mechanism.] [Article in Chinese]

LI Bai-He, YUAN Lei, SHI Ran-Ran, WANG Jian-Guo^*

Laboratory of Molecular Biology, Luohe Medical College, Luohe 462002, China

Abstract

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.

Key words: digoxin; MCF-7/ADR; hypoxia inducible factor-1α; multidrug resistance gene 1; apoptosis

收稿日期：2015-05-25　　录用日期：2015-07-30

通讯作者：王建国　　E-mail: wr0395@sina.com

引用本文：

李伯和, 袁磊, 时冉冉, 王建国. 地高辛逆转乳腺癌MCF-7/阿霉素细胞的耐药性及其机制[J]. 生理学报 2015; 67 (6): 611-617.

LI Bai-He, YUAN Lei, SHI Ran-Ran, WANG Jian-Guo. [Reversal of adriamycin resistance by digoxin in human breast cancer cell line MCF-7/adriamycin and its mechanism.] [Article in Chinese]. Acta Physiol Sin 2015; 67 (6): 611-617 (in Chinese with English abstract).