Proteomic identification of differentially expressed proteins associated with the multiple drug resistance in methotrexate-resistant human breast cancer cells

Abstract

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.

Salvianolic acid A reverses the paclitaxel resistance and inhibits the migration and invasion abilities of human breast cancer cells by inactivating transgelin 2

“…Paclitaxel and salvianolic acid A (12 μM) were purchased from Sike Cell viability was measured on the basis of MTT assay as described previously 35 .…”

Histone deacetylase inhibitor reverses multidrug resistance by attenuating the nucleophosmin level through PI3K/Akt pathway in breast cancer

“…The MCF-7/S sensitive human breast cancer cell line was obtained from Shanghai Institute of Cell Biology in the Chinese Academy of Sciences (Shanghai, China). The methotrexate-resistant breast cancer cell line (MCF-7/MTX) was successfully established as previously described (5) with the concentration of 220 nM MTX. Both cell types were cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (PAA, Austria) and 1% penicillin/streptomycin in a humidified atmosphere of 5% CO 2 at 37˚C.…”

“…For in vitro knockdown experiments, the double-stranded small interfering RNA (siRNA) against human NPM1 and non-specific siRNA (control siRNA) were purchased from Shanghai GenePharma Company Ltd. (China). They were used for transient transfection as previously described (5). MCF-7/MTX cells were seeded at a density of 5x10 5 per well into a 6-well plate and cultured in the medium until cell confluence reached ~40%.…”

“…They were used for transient transfection as previously described (5). MCF-7/MTX cells were seeded at a density of 5x10 5 per well into a 6-well plate and cultured in the medium until cell confluence reached ~40%. The following day, cells were transfected with 50 nM NPM1 siRNA and Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions.…”

“…In our previous study, we established the methotrexateresistant breast cancer cells (MCF-7/MTX) and identified nucleophosmin that is a new drug-resistant target related to the MDR of breast cancer (5). Nucleophosmin (NPM, also known as B23, numatrin, or N038) was originally discovered as a non-ribosomal nucleolar phosphoprotein that was located primarily in the nucleoli and shuttled between the nucleoli and cytoplasm during the cell cycle (6).…”