摘要
目的:探讨薯蓣皂苷(dioscin,DIO)对人乳腺癌阿霉素(doxorubicin,DOX)耐药的影响及其可能的作用机制.方法:采用MTT法检测DIO不同浓度对MCF-7/DOX细胞活力的影响,并运用MTT、Westem blot和qRT-PCR等方法考察DIO对MCF-7/DOX细胞DOX耐药的逆转作用.采用网络药理学预测DIO干预乳腺癌DOX耐药的潜在靶点,利用STRING数据库和Cytoscape 3.9.1软件构建PPI网络,筛选核心靶点,通过DAVID数据库进行GO功能富集分析和KEGG通路富集分析.最后,采用qRT-PCR实验对核心靶点进行验证.结果:DIO质量浓度在0.375～1.56 μg·mL 1时对MCF-7/DOX细胞活力无明显影响.0.75μg·mL-1 DIO与不同浓度DOX联用后,DOX对MCF-7/DOX细胞的IC50下降,DIO的逆转耐药倍数为2.02,且MCF-7/DOX细胞中MDR1、MRP1蛋白和mRNA表达下调(P＜0.05),表明DIO增强了 MCF-7/DOX细胞对DOX的敏感性,逆转MCF-7/DOX细胞耐药.网络药理学分析显示DIO逆转乳腺癌阿霉素耐药的潜在靶点78个,其中核心靶点包括AKT1、TP53、BCL2、IL-6、CASP3、JUN等,主要涉及脂质和动脉粥样硬化、癌症等信号通路.验证实验结果显示0.75 μg·mL-1 DIO与DOX联用后,上调TP53和CASP3 mRNA表达(与对照组比较,P＜0.05),下调AKT1和IL-6 mRNA表达(与DOX比较,P＜0.05),有下调BCL2mRNA表达的趋势,对JUN mRNA无明显影响.结论:DIO可通过作用于AKT1、TP53、BCL2、IL-6、CASP3等核心靶点而逆转乳腺癌DOX耐药.
Abstract
OBJECTIVE To explore the effect of dioscin(DIO)on doxorubicin(DOX)resistance in human breast cancer MCF-7/DOX cell and elucidate its possible mechanism.METHODS MTT assay was utilized for detecting the viability of MCF-7/DOX cell after treating with different concentrations of DIO.MCF-7/DOX cell was exposed to non-toxic concentrations of DIO plus DOX for observing the reversal effect of DIO on DOX resistance by methylthiazolyldiphenyl tetrazolium(MTT),West-em blot and quantitative real-time polymerase chain reaction(qRT-PCR).Network pharmacology was utilized for predicting the potential targets of DIO against DOX resistance in breast cancer.Protein-protein interaction(PPI)network was constructed through a STRING database and Cytoscape 3.9.1 software for selecting core targets.Gene Ontology(GO)and Kyoto Encyclo-pedia of Genes and Genomes(KEGG)pathway enrichment analyses were further performed through the David database.Finally qRT-PCR was performed to verify the mRNA expression of core targets.RESULTS At a concentration of 0.375-1.56 μg·mL-1,DIO displayed little cellular cytotoxicity.After exposure to DIO 0.75 μg·mL-1 plus different concentrations of DOX,half maxi-mal inhibitory concentration(IC50)of DOX declined with a reversing multitude of 2.02 and protein/mRNA expressions of MDR1/MRP1 became down-regulated(P＜0.05).It implied that DIO reversed DOX resistance.Network pharmacology prediction yielded 78 potential targets for DIO reversing DOX resistance in breast cancer.Some core targets included protein kinase B(AKT1),tumor protein P53(TP53),apoptosis regulator BCL2(BCL2),interleukin-6(IL-6),caspase 3(CASP3)and jun proto-oncogene(JUN)involved in lipid and atherosclerosis,neoplastic pathways and other signal pathways.0.75 μg·mL-1 DIO plus DOX up-regulated mRNA expressions of TP53 and CASP3(vs.control group,P＜0.05)and down-regulated mRNA expressions of AKT1 and IL-6(vs.DOX group,P＜0.05).With a tendency of down-regulating BCL2 mRNA,it had no obvi-ous effect on JUN mRNA level.CONCLUSION DIO may reverse DOX resistance in breast cancer through modulating the core targets of AKT1,TP53,BCL2,IL-6 and CASP3.
基金项目
国家自然科学基金项目(82060733)
国家自然科学基金项目(81960732)
江西省自然科学基金项目(20224BAB206111)
NETL
NSTL
万方数据