BACKGROUND: Transformed phenotypes are common to cell lines derived from various cancers. Proteome profiling is a valuable tool that may reveal uncharacteristic cell phenotypes in transformed cells. Changes in expression of glutathione S-transferases (GSTs) and other proteins interacting with glutathione (GSH) in model cell lines could be of particular interest. METHODS: We compared the phenotypes of breast cell lines EM-G3, HCC1937, MCF7 and MDA-MB-231 using 2-D electrophoresis (2-DE). We further separated GSH-binding proteins from the cell lines using affinity chromatography with GSH-Sepharose 4B, performed 2-DE analysis and identified the main protein spots. RESULTS: Correlation coefficients among 2-DE gels from the cell lines were lower than 0.65, pointing to dissimilarity among the cell lines. Differences in primary constituents of the cytoskeleton were shown by the 2-D protein maps and western blots. The spot patterns in gels of GSH-binding fractions from primary carcinoma-derived cell lines HCC1937 and EM-G3 were similar to each other, and they differed from the spot patterns of cell lines MCF7 and MDA-MB-231 that were derived from pleural effusions of metastatic mammary carcinoma patients. Major differences in the expression of GST P1-1 and carbonyl reductase [NADPH] 1 were observed among the cell lines, indicating differential abilities of the cell lines to metabolize xenobiotics. CONCLUSIONS: Our results confirmed the applicability of targeted affinity chromatography to proteome profiling and allowed us to characterize the phenotypes of four breast cancer cell lines.

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic vvi Prague Czech Republic

Zobrazit více v PubMed

Lacroix M, Leclercq G. Relevance of breast cancer cell lines as models for breast tumours: an update. Breast Cancer Res Treat. 2004;83:249–289. doi: 10.1023/B:BREA.0000014042.54925.cc. PubMed DOI

Burdall SE, Hanby AM, Lansdown MRJ, Speirs V. Breast cancer cell lines: friend or foe? Breast Cancer Res. 2003;5:89–95. doi: 10.1186/bcr577. PubMed DOI PMC

Patterson SD, Aebersold RH. Proteomics: the first decade and beyond. Nature Genet. 2003;33:311–323. doi: 10.1038/ng1106. PubMed DOI

Slany A, Haudek VJ, Zwickl H, Gundacker NC, Grusch M, Weiss TS, Seir K, Rodgarkia-Dara C, Hellerbrand C, Gerner C. Cell characterization by proteome profiling applied to primary hepatocytes and hepatocyte cell lines Hep-G2 and Hep-3B. J Proteome Res. 2010;9:6–21. doi: 10.1021/pr900057t. PubMed DOI

Hathout Y, Gehrmann ML, Chertov A, Fenselau C. Proteomic phenotyping: metastatic and invasive breast cancer. Cancer Lett. 2004;210:245–253. doi: 10.1016/j.canlet.2004.01.019. PubMed DOI

Gorg A, Drews O, Luck C, Weiland F, Weiss W. 2-DE with IPGs. Electrophoresis. 2009;30(Suppl 1):S122–S132. PubMed

Weiss W, Gorg A. High-resolution two-dimensional electrophoresis. Methods Mol Biol. 2009;564:13–32. full_text. PubMed

Zuo X, Echan L, Hembach P, Tang HY, Speicher KD, Santoli D, Speicher DW. Towards global analysis of mammalian proteomes using sample prefractionation prior to narrow pH range two-dimensional gels and using one-dimensional gels for insoluble and large proteins. Electrophoresis. 2001;22:1603–1615. doi: 10.1002/1522-2683(200105)22:9<1603::AID-ELPS1603>3.0.CO;2-I. PubMed DOI

Lee WC, Lee KH. Applications of affinity chromatography in proteomics. Anal Biochem. 2004;324:1–10. doi: 10.1016/j.ab.2003.08.031. PubMed DOI

Collinsova M, Castro C, Garrow TA, Yiotakis A, Dive V, Jiracek J. Combining combinatorial chemistry and affinity chromatography: highly selective inhibitors of human betaine: homocysteine S-methyltransferase. Chem Biol. 2003;10:113–122. doi: 10.1016/S1074-5521(03)00008-5. PubMed DOI

Di Pietro G, Magno LA, Rios-Santos F. Glutathione S-transferases: an overview in cancer research. Expert Opin Drug Metab Toxicol. 2010;6:153–170. doi: 10.1517/17425250903427980. PubMed DOI

Ballatori N, Krance SM, Notenboom S, Shi S, Tieu K, Hammond CL. Glutathione dysregulation and the etiology and progression of human diseases. Biol Chem. 2009;390:191–214. doi: 10.1515/BC.2009.033. PubMed DOI PMC

Marahatta SB, Punyarit P, Bhudisawasdi V, Paupairoj A, Wongkham S, Petmitr S. Polymorphism of glutathione S-transferase omega gene and risk of cancer. Cancer Lett. 2006;236:276–281. doi: 10.1016/j.canlet.2005.05.020. PubMed DOI

Buser K, Joncourt F, Altermatt HJ, Bacchi M, Oberli A, Cerny T. Breast cancer: pretreatment drug resistance parameters (GSH-system, ATase, P-glycoprotein) in tumor tissue and their correlation with clinical and prognostic characteristics. Ann Oncol. 1997;8:335–341. doi: 10.1023/A:1008202723066. PubMed DOI

Chekhun VF, Zhylchuk VE, Lukyanova NY, Vorontsova AL, Kudryavets YI. Expression of drug resistance proteins in triple-receptor-negative tumors as the basis of individualized therapy of the breast cancer patients. Exp Oncol. 2009;31:123–124. PubMed

Mahajan S, Atkins WM. The chemistry and biology of inhibitors and pro-drugs targeted to glutathione S-transferases. Cell Mol Life Sci. 2005;62:1221–1233. doi: 10.1007/s00018-005-4524-6. PubMed DOI PMC

Soule HD, Vazguez J, Long A, Albert S, Brennan M. A human cell line from a pleural effusion derived from a breast carcinoma. J Natl Cancer Inst. 1973;51:1409–1416. PubMed

Cailleau R, Young R, Olive M, Reeves WJ Jr. Breast tumor cell lines from pleural effusions. J Natl Cancer Inst. 1974;53:661–674. PubMed PMC

Horie K, Urano T, Ikeda K, Inoue S. Estrogen-responsive RING finger protein controls breast cancer growth. J Steroid Biochem Mol Biol. 2003;85:101–104. doi: 10.1016/S0960-0760(03)00209-7. PubMed DOI

Rochefort H, Glondu M, Sahla ME, Platet N, Garcia M. How to target estrogen receptor-negative breast cancer? Endocrine-Related Cancer. 2003;10:261–266. doi: 10.1677/erc.0.0100261. PubMed DOI

Tomlinson GE, Chen TTL, Stastny VA, Virmani AK, Spillman MA, Tonk V, Blum JL, Schneider NR, Wistuba II, Shay JW, Minna JD, Gazdar AF. Characterization of a breast cancer cell line derived from a germ-line BRCA1 mutation carrier. Cancer Res. 1998;58:3237–3242. PubMed

Brozova M, Kleibl Z, Netikova I, Sevcik J, Scholzova E, Brezinova J, Chaloupkova A, Vesely P, Dundr P, Zadinova M, Krasna L, Matouskova E. Establishment, growth and in vivo differentiation of a new clonal human cell line, EM-G3, derived from breast cancer progenitors. Breast Cancer Res Treat. 2006;103:247–257. doi: 10.1007/s10549-006-9358-x. PubMed DOI

Selicharova I, Smutna K, Sanda M, Ubik K, Matouskova E, Bursikova E, Brozova M, Vydra J, Jiracek J. 2-DE analysis of a new human cell line EM-G3 derived from breast cancer progenitor cells and comparison with normal mammary epithelial cells. Proteomics. 2007;7:1549–1559. doi: 10.1002/pmic.200600907. PubMed DOI

Matouskova E, Dudorkinova D, Krasna L, Vesely P. Temporal in vitro expansion of the luminal lineage of human mammary epithelial cells achieved with the 3T3 feeder layer technique. Breast Cancer Res Treat. 2000;60:241–249. doi: 10.1023/A:1006409605067. PubMed DOI

Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–254. doi: 10.1016/0003-2697(76)90527-3. PubMed DOI

Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem. 1974;249:7130–7139. PubMed

Rabilloud T. A Comparison Between Low Background Silver Diammine and Silver-Nitrate Protein Stains. Electrophoresis. 1992;13:429–439. doi: 10.1002/elps.1150130190. PubMed DOI

Candiano G, Bruschi M, Musante L, Santucci L, Ghiggeri GM, Carnemolla B, Orecchia P, Zardi L, Righetti PG. Blue silver: A very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis. 2004;25:1327–1333. doi: 10.1002/elps.200305844. PubMed DOI

Challapalli KK, Zabel C, Schuchhardt J, Kaindl AM, Klose J, Herzel H. High reproducibility of large-gel two-dimensional electrophoresis. Electrophoresis. 2004;25:3040–3047. doi: 10.1002/elps.200405979. PubMed DOI

Williams K, Chubb C, Huberman E, Giometti CS. Analysis of differential protein expression in normal and neoplastic human breast epithelial cell lines. Electrophoresis. 1998;19:333–343. doi: 10.1002/elps.1150190231. PubMed DOI

Vydra J, Selicharova I, Smutna K, Sanda M, Matouskova E, Bursikova E, Prchalova M, Velenska Z, Coufal D, Jiracek J. Two-dimensional electrophoretic comparison of metastatic and non-metastatic human breast tumors using in vitro cultured epithelial cells derived from the cancer tissues. BMC Cancer. 2008;8:107. doi: 10.1186/1471-2407-8-107. PubMed DOI PMC

Gilbert L, Elwood LJ, Merino M, Masood S, Barnes R, Steinberg SM, Lazarous DF, Pierce L, d'Angelo T, Moscow JA. A pilot study of pi-class glutathione S-transferase expression in breast cancer: correlation with estrogen receptor expression and prognosis in node-negative breast cancer. J Clin Oncol. 1993;11:49–58. PubMed

Forrester LM, Hayes JD, Millis R, Barnes D, Harris AL, Schlager JJ, Powis G, Wolf CR. Expression of glutathione S-transferases and cytochrome P450 in normal and tumor breast tissue. Carcinogenesis. 1990;11:2163–2170. doi: 10.1093/carcin/11.12.2163. PubMed DOI

Bateman RL, Rauh D, Tavshanjian B, Shokat KM. Human carbonyl reductase 1 is an S-nitrosoglutathione reductase. J Biol Chem. 2008;283:35756–35762. doi: 10.1074/jbc.M807125200. PubMed DOI PMC

Johansson M, Lundberg M. Glutathionylation of beta-actin via a cysteinyl sulfenic acid intermediary. BMC Biochem. 2007;8:26. doi: 10.1186/1471-2091-8-26. PubMed DOI PMC

Ejiri S. Moonlighting functions of polypeptide elongation factor 1: from actin bundling to zinc finger protein R1-associated nuclear localization. Biosci Biotechnol Biochem. 2002;66:1–21. doi: 10.1271/bbb.66.1. PubMed DOI

Koonin EV, Mushegian AR, Tatusov RL, Altschul SF, Bryant SH, Bork P, Valencia A. Eukaryotic translation elongation factor 1 gamma contains a glutathione transferase domain--study of a diverse, ancient protein superfamily using motif search and structural modeling. Protein Sci. 1994;3:2045–2054. doi: 10.1002/pro.5560031117. PubMed DOI PMC

Zhang W, Wang L, Liu Y, Xu J, Zhu G, Cang H, Li X, Bartlam M, Hensley K, Li G, Rao Z, Zhang XC. Structure of human lanthionine synthetase C-like protein 1 and its interaction with Eps8 and glutathione. Genes Dev. 2009;23:1387–1392. doi: 10.1101/gad.1789209. PubMed DOI PMC

Chung CH, Kurien BT, Mehta P, Mhatre M, Mou S, Pye QN, Stewart C, West M, Williamson KS, Post J, Liu L, Wang R, Hensley K. Identification of lanthionine synthase C-like protein-1 as a prominent glutathione binding protein expressed in the mammalian central nervous system. Biochemistry. 2007;46:3262–3269. doi: 10.1021/bi061888s. PubMed DOI

Warleta F, Campos M, Allouche Y, Sanchez-Quesada C, Ruiz-Mora J, Beltran G, Gaforio JJ. Squalene protects against oxidative DNA damage in MCF10A human mammary epithelial cells but not in MCF7 and MDA-MB-231 human breast cancer cells. Food Chem Toxicol. 2010;48:1092–1100. doi: 10.1016/j.fct.2010.01.031. PubMed DOI

Pichot CS, Hartig SM, Xia L, Arvanitis C, Monisvais D, Lee FY, Frost JA, Corey SJ. Dasatinib synergizes with doxorubicin to block growth, migration, and invasion of breast cancer cells. Br J Cancer. 2009;101:38–47. doi: 10.1038/sj.bjc.6605101. PubMed DOI PMC

Aroui S, Brahim S, Waard MD, Kenani A. Cytotoxicity, intracellular distribution and uptake of doxorubicin and doxorubicin coupled to cell-penetrating peptides in different cell lines: a comparative study. Biochem Biophys Res Commun. 2010;391:419–425. doi: 10.1016/j.bbrc.2009.11.073. PubMed DOI

Huang Y, Hager ER, Phillips DL, Dunn VR, Hacker A, Frydman B, Kink JA, Valasinas AL, Reddy VK, Marton LJ, Casero RA Jr, Davidson NE. A novel polyamine analog inhibits growth and induces apoptosis in human breast cancer cells. Clin Cancer Res. 2003;9:2769–2777. PubMed PMC

Susila A, Chan H, Loh AX, Phang HQ, Wong ET, Tergaonkar V, Koh CG. The POPX2 phosphatase regulates cancer cell motility and invasiveness. Cell Cycle. 2010;9:179–187. PubMed

Mineva ND, Wang X, Yang S, Ying H, Xiao ZX, Holick MF, Sonenshein GE. Inhibition of RelB by 1,25-dihydroxyvitamin D3 promotes sensitivity of breast cancer cells to radiation. J Cell Physiol. 2009;220:593–599. doi: 10.1002/jcp.21765. PubMed DOI PMC

Wilchek M, Miron T, Kohn J. Affinity chromatography. Methods Enzymol. 1984;104:3–55. full_text. PubMed

Unravelling heterogeneous effects of cancer‑associated fibroblasts on poor prognosis markers in breast cancer EM‑G3 cell line: In vitro‑targeted treatment (anti‑IL-6, anti‑VEGF-A, anti‑MFGE8) based on transcriptomic profiling

Fibroblasts prepared from different types of malignant tumors stimulate expression of luminal marker keratin 8 in the EM-G3 breast cancer cell line