Chen Siying, Cai Jiangxia, Zhang Weipeng, Zheng Xiaowei, Hu Sasa, Lu Jun, Xing Jianfeng, Dong Yalin
Department of Pharmacy, The First Affiliated Hospital of Medical College, Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.
Department of Pharmacy, College of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.
Int J Oncol. 2014 Jul;45(1):448-58. doi: 10.3892/ijo.2014.2389. Epub 2014 Apr 16.
Methotrexate (MTX), as a chemotherapeutic drug, is widely used in the therapy of several cancer types. The efficiency of drug treatment is compromised by the appearance of multidrug resistance (MDR), and the underlying molecular mechanisms remain incompletely understood. We investigated the mechanism of MDR in the MTX-induced breast cancer MCF-7 cells (MCF-7/MTX) using proteomic analysis. MCF-7 drug-sensitive cells (MCF-7/S) were exposed in progressively increasing concentrations of MTX to establish the drug-resistant cell line MCF-7/MTX. The biological characteristics of the cells were analyzed by MTT, flow cytometry, quantitative PCR, western blotting and the global protein profiles of MCF-7/MTX and MCF-7/S were compared using a proteomic approach. The resistance factor of MCF-7/MTX cells was 64, and it possessed significant MDR. Seventeen differentially expressed proteins between MCF-7/MTX and MCF-7/S cells were identified, seven proteins were upregulated and 10 proteins were downregulated in MCF-7/MTX cells. We verified that the protein levels of nucleophosmin (NPM), α-enolase (ENO1) and vimentin (VIM) were upregulated, and heterogeneous nuclear ribonucleoprotein (hnRNP C1/C2), phosphoglycerate mutase 1 (PGAM1) and proteasome subunit α type-2 (PSMA2) were downregulated in MCF-7/MTX cells. The mRNA levels of NPM, VIM, hnRNP C1/C2, PGAM1 and PSMA2 were consistent with the protein expressions, but the gene expression of ENO1 was slightly downregulated. Surprisingly, knockdown of NPM by siRNA sensitized MCF-7/MTX cells to MTX and attenuated the multidrug resistance. The proteins identified, particularly NPM provides new insights into the mechanism of MDR and is expected to become a crucial molecular target for breast cancer treatment.
甲氨蝶呤（MTX）作为一种化疗药物，广泛应用于多种癌症类型的治疗。多药耐药（MDR）的出现降低了药物治疗的效果，其潜在的分子机制仍未完全明确。我们采用蛋白质组学分析方法，研究了MTX诱导的乳腺癌MCF-7细胞（MCF-7/MTX）中多药耐药的机制。将MCF-7药物敏感细胞（MCF-7/S）暴露于浓度逐渐增加的MTX中，建立耐药细胞系MCF-7/MTX。通过MTT法、流式细胞术、定量PCR、蛋白质免疫印迹法分析细胞的生物学特性，并采用蛋白质组学方法比较MCF-7/MTX和MCF-7/S的整体蛋白质谱。MCF-7/MTX细胞的耐药因子为64，具有显著的多药耐药性。鉴定出MCF-7/MTX和MCF-7/S细胞之间17种差异表达蛋白，其中7种蛋白在MCF-7/MTX细胞中上调，10种蛋白下调。我们验证了核磷蛋白（NPM）、α-烯醇化酶（ENO1）和波形蛋白（VIM）在MCF-7/MTX细胞中的蛋白水平上调，而异质性核核糖核蛋白（hnRNP C1/C2）、磷酸甘油酸变位酶1（PGAM1）和蛋白酶体亚基α-2型（PSMA2）下调。NPM、VIM、hnRNP C1/C2、PGAM1和PSMA2的mRNA水平与蛋白表达一致，但ENO1的基因表达略有下调。令人惊讶的是，通过小干扰RNA（siRNA）敲低NPM可使MCF-7/MTX细胞对MTX敏感，并减弱多药耐药性。所鉴定出的蛋白，尤其是NPM，为多药耐药机制提供了新的见解，有望成为乳腺癌治疗的关键分子靶点。
