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

Chen,Siying; Cai,Jiangxia; Zhang,Weipeng; Zheng,Xiaowei; Hu,Sasa; Lu,Jun; Xing,Jianfeng; Dong,Yalin

doi:10.3892/ijo.2014.2389

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

Authors:
- Siying Chen
- Jiangxia Cai
- Weipeng Zhang
- Xiaowei Zheng
- Sasa Hu
- Jun Lu
- Jianfeng Xing
- Yalin Dong
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Affiliations: 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
Published online on: April 16, 2014 https://doi.org/10.3892/ijo.2014.2389
Pages: 448-458

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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.

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1.	Jemal A, Bray F, Center MM, Ferlay J, Ward E and Forman D: Global cancer statistics. CA Cancer J Clin. 61:69–90. 2011. View Article : Google Scholar
2.	Bhoo-Pathy N, Yip CH, Hartman M, et al: Breast cancer research in Asia: adopt or adapt Western knowledge? Eur J Cancer. 49:703–709. 2013. View Article : Google Scholar : PubMed/NCBI
3.	Longley DB and Johnston PG: Molecular mechanisms of drug resistance. J Pathol. 205:275–292. 2005. View Article : Google Scholar : PubMed/NCBI
4.	Baguley BC: Multiple drug resistance mechanisms in cancer. Mol Biotechnol. 46:308–316. 2010. View Article : Google Scholar : PubMed/NCBI
5.	Olsen EA: The pharmacology of methotrexate. J Am Acad Dermatol. 25:306–318. 1991. View Article : Google Scholar
6.	Assaraf YG: Molecular basis of antifolate resistance. Cancer Metastasis Rev. 26:153–181. 2007. View Article : Google Scholar
7.	de Almagro MC, Selga E, Thibaut R, Porte C, Noe V and Ciudad CJ: UDP-glucuronosyltransferase 1A6 overexpression in breast cancer cells resistant to methotrexate. Biochem Pharmacol. 81:60–70. 2011.PubMed/NCBI
8.	Selga E, Morales C, Noe V, Peinado MA and Ciudad CJ: Role of caveolin 1, E-cadherin, Enolase 2 and PKCalpha on resistance to methotrexate in human HT29 colon cancer cells. BMC Med Genomics. 1:352008. View Article : Google Scholar : PubMed/NCBI
9.	Mencia N, Selga E, Noé V and Ciudad CJ: Underexpression of miR-224 in methotrexate resistant human colon cancer cells. Biochem Pharmacol. 82:1572–1582. 2011. View Article : Google Scholar : PubMed/NCBI
10.	Mencia N, Selga E, Rico I, et al: Overexpression of S100A4 in human cancer cell lines resistant to methotrexate. BMC Cancer. 10:2502010. View Article : Google Scholar : PubMed/NCBI
11.	Subramanian A and Miller DM: Structural analysis of alpha-enolase. Mapping the functional domains involved in down-regulation of the c-myc protooncogene. J Biol Chem. 275:5958–5965. 2000. View Article : Google Scholar : PubMed/NCBI
12.	Takashima M, Kuramitsu Y, Yokoyama Y, et al: Overexpression of alpha enolase in hepatitis C virus-related hepatocellular carcinoma: association with tumor progression as determined by proteomic analysis. Proteomics. 5:1686–1692. 2005. View Article : Google Scholar
13.	Chang GC, Liu KJ, Hsieh CL, et al: Identification of alpha-enolase as an autoantigen in lung cancer: its overexpression is associated with clinical outcomes. Clin Cancer Res. 12:5746–5754. 2006. View Article : Google Scholar : PubMed/NCBI
14.	Tsai ST, Chien IH, Shen WH, et al: ENO1, a potential prognostic head and neck cancer marker, promotes transformation partly via chemokine CCL20 induction. Eur J Cancer. 46:1712–1723. 2010. View Article : Google Scholar : PubMed/NCBI
15.	Tu SH, Chang CC, Chen CS, et al: Increased expression of enolase alpha in human breast cancer confers tamoxifen resistance in human breast cancer cells. Breast Cancer Res Treat. 121:539–553. 2010. View Article : Google Scholar : PubMed/NCBI
16.	Chuthapisith S, Layfield R, Kerr ID, Hughes C and Eremin O: Proteomic profiling of MCF-7 breast cancer cells with chemoresistance to different types of anti-cancer drugs. Int J Oncol. 30:1545–1551. 2007.PubMed/NCBI
17.	Mizukami Y, Iwamatsu A, Aki T, et al: ERK1/2 regulates intracellular ATP levels through alpha-enolase expression in cardiomyocytes exposed to ischemic hypoxia and reoxygenation. J Biol Chem. 279:50120–50131. 2004. View Article : Google Scholar
18.	de Sousa Abreu R, Penalva LO, Marcotte EM and Vogel C: Global signatures of protein and mRNA expression levels. Mol Biosyst. 5:1512–1526. 2009.PubMed/NCBI
19.	Ivaska J, Pallari HM, Nevo J and Eriksson JE: Novel functions of vimentin in cell adhesion, migration, and signaling. Exp Cell Res. 313:2050–2062. 2007. View Article : Google Scholar : PubMed/NCBI
20.	Singh S, Sadacharan S, Su S, Belldegrun A, Persad S and Singh G: Overexpression of vimentin: role in the invasive phenotype in an androgen-independent model of prostate cancer. Cancer Res. 63:2306–2311. 2003.PubMed/NCBI
21.	Karihtala P, Auvinen P, Kauppila S, Haapasaari KM, Jukkola-Vuorinen A and Soini Y: Vimentin, zeb1 and Sip1 are up-regulated in triple-negative and basal-like breast cancers: association with an aggressive tumour phenotype. Breast Cancer Res Treat. 138:81–90. 2013. View Article : Google Scholar : PubMed/NCBI
22.	Tseng YH, Chang KW, Yang CC, et al: Association between areca-stimulated vimentin expression and the progression of head and neck cancers. Head Neck. 34:245–253. 2012. View Article : Google Scholar : PubMed/NCBI
23.	Kim JJ, Yin B, Christudass CS, et al: Acquisition of paclitaxel resistance is associated with a more aggressive and invasive phenotype in prostate cancer. J Cell Biochem. 114:1286–1293. 2013. View Article : Google Scholar : PubMed/NCBI
24.	Sun S, Wong TS, Zhang XQ, et al: Protein alterations associated with temozolomide resistance in subclones of human glioblastoma cell lines. J Neurooncol. 107:89–100. 2012. View Article : Google Scholar : PubMed/NCBI
25.	Liu H, Zhang HW, Sun XF, et al: Tamoxifen-resistant breast cancer cells possess cancer stem-like cell properties. Chin Med J (Engl). 126:3030–3034. 2013.PubMed/NCBI
26.	Krecic AM and Swanson MS: hnRNP complexes: composition, structure, and function. Curr Opin Cell Biol. 11:363–371. 1999. View Article : Google Scholar : PubMed/NCBI
27.	Carpenter B, MacKay C, Alnabulsi A, et al: The roles of heterogeneous nuclear ribonucleoproteins in tumour development and progression. Biochim Biophys Acta. 1765:85–100. 2006.PubMed/NCBI
28.	Brockstedt E, Rickers A, Kostka S, et al: Identification of apoptosis-associated proteins in a human Burkitt lymphoma cell line. Cleavage of heterogeneous nuclear ribonucleoprotein A1 by caspase 3. J Biol Chem. 273:28057–28064. 1998. View Article : Google Scholar
29.	Rahman-Roblick R, Johannes Roblick U, Hellman U, et al: p53 targets identified by protein expression profiling. Proc Natl Acad Sci USA. 104:5401–5406. 2007. View Article : Google Scholar : PubMed/NCBI
30.	Koryllou A, Patrinou-Georgoula M, Troungos C and Pletsa V: Cell death induced by N-methyl-N-nitrosourea, a model SN1 methylating agent, in two lung cancer cell lines of human origin. Apoptosis. 14:1121–1133. 2009. View Article : Google Scholar : PubMed/NCBI
31.	Grover R, Sharathchandra A, Ponnuswamy A, Khan D and Das S: Effect of mutations on the p53 IRES RNA structure: Implications for de-regulation of the synthesis of p53 isoforms. RNA Biol. 8:132–142. 2011. View Article : Google Scholar : PubMed/NCBI
32.	Hossain MN, Fuji M, Miki K, Endoh M and Ayusawa D: Downregulation of hnRNP C1/C2 by siRNA sensitizes HeLa cells to various stresses. Mol Cell Biochem. 296:151–157. 2007. View Article : Google Scholar : PubMed/NCBI
33.	Ren F, Wu H, Lei Y, et al: Quantitative proteomics identification of phosphoglycerate mutase 1 as a novel therapeutic target in hepatocellular carcinoma. Mol Cancer. 9:812010. View Article : Google Scholar : PubMed/NCBI
34.	Evans MJ, Saghatelian A, Sorensen EJ and Cravatt BF: Target discovery in small-molecule cell-based screens by in situ proteome reactivity profiling. Nat Biotechnol. 23:1303–1307. 2005. View Article : Google Scholar : PubMed/NCBI
35.	Gao H, Yu B, Yan Y, et al: Correlation of expression levels of ANXA2, PGAM1, and CALR with glioma grade and prognosis. J Neurosurg. 118:846–853. 2013. View Article : Google Scholar : PubMed/NCBI
36.	Jiang X, Sun Q, Li H, Li K and Ren X: The role of phosphoglycerate mutase 1 in tumor aerobic glycolysis and its potential therapeutic implications. Int J Cancer. Nov 28–2013.(Epub ahead of print). View Article : Google Scholar
37.	Chaneton B and Gottlieb E: PGAMgnam style: a glycolytic switch controls biosynthesis. Cancer Cell. 22:565–566. 2012. View Article : Google Scholar : PubMed/NCBI
38.	Tan JY, Huang X and Luo YL: PSMA7 inhibits the tumorigenicity of A549 human lung adenocarcinoma cells. Mol Cell Biochem. 366:131–137. 2012. View Article : Google Scholar : PubMed/NCBI
39.	Du H, Huang X, Wang S, Wu Y, Xu W and Li M: PSMA7, a potential biomarker of diseases. Protein Pept Lett. 16:486–489. 2009. View Article : Google Scholar : PubMed/NCBI
40.	Sakai A, Otani M, Miyamoto A, Yoshida H, Furuya E and Tanigawa N: Identification of phosphorylated serine-15 and -82 residues of HSPB1 in 5-fluorouracil-resistant colorectal cancer cells by proteomics. J Proteomics. 75:806–818. 2012. View Article : Google Scholar : PubMed/NCBI
41.	Yung BY: Oncogenic role of nucleophosmin/B23. Chang Gung Med J. 30:285–293. 2007.
42.	Wong JC, Hasan MR, Rahman M, et al: Nucleophosmin 1, upregulated in adenomas and cancers of the colon, inhibits p53-mediated cellular senescence. Int J Cancer. 133:1567–1577. 2013. View Article : Google Scholar : PubMed/NCBI
43.	Lin M, Hu J, Liu T, Li J, Chen B and Chen X: Knockdown of nucleophosmin by RNA interference reverses multidrug resistance in resistant leukemic HL-60 cells. Immunobiology. 218:1147–1154. 2013. View Article : Google Scholar : PubMed/NCBI
44.	Grisendi S, Mecucci C, Falini B and Pandolfi PP: Nucleophosmin and cancer. Nat Rev Cancer. 6:493–505. 2006. View Article : Google Scholar
45.	Karhemo PR, Rivinoja A, Lundin J, et al: An extensive tumor array analysis supports tumor suppressive role for nucleophosmin in breast cancer. Am J Pathol. 179:1004–1014. 2011. View Article : Google Scholar : PubMed/NCBI