Different Expression of Extracellular Signal-Regulated Kinases (ERK) 1/2 and Phospho-Erk Proteins in MBA-MB-231 and MCF-7 Cells after Chemotherapy with Doxorubicin or Docetaxel

Document Type : Original Article


Kashan Anatomical Research Centre, Kashan University of Medical Science, Kashan, Iran


Curative treatment of breast cancer patients using chemotherapy often fails as a result of intrinsic or acquired resistance of the tumor to the drug. ERK is one of the main components of the Ras/Raf/MEK/ERK cascade, which mediates signal from cell surface receptors to transcription factors to regulate different gene expression. In this study, cytotoxicity and the expression of Erk1/2 and phospho-ERK was compared in MDA-MB-231 (ER-) and MCF-7 (ER+) cell lines after treatment with doxorubicin (DOX) or docetaxel (DOCT).
Materials and Methods
Cell cytotoxicity of DOX or DOCT was calculated using MTT assay. Immonofluorescent technique was used to show MDR-1 protein in MDA-MB-231 and MCF-7 cells after treatment with DOX or DOCT. The expression of ERK1/2 and phpspho-ERK was assayed with immunoblotting.
Comparing IC50 values showed that MDA-MB-231 cells are more sensitive than MCF-7 cells to DOX or DOCT. Immonofluorescent results confirmed the expression of MDR-1 in these two cell lines after DOX or DOCT treatment. In MDA-MB-231 cells the expression of ERK1/2 and phospho-ERK was decreased after DOX treatment in a dose-dependent manner. In contrast in MCF-7 cells the expression of ERK1/2 and phospho-ERK was increased after DOX treatment. DOCT treatment demonstrated the same result with less significant differences than DOX.
The heterogeneity seen in cell lines actually reflects the heterogeneity of breast cancers. That is why, patients categorized in one group respond differently to a single treatment. These results emphasize the importance of a more accurate classification and a more specific treatment of breast cancer subtypes.


1. Falandry C, Canney PA, Freyer G, Dirix LY. Role of combination therapy with aromatase and cyclooxygenase-2 inhibitors in patients with metastatic breast cancer. Ann Oncol 2009; 20:615-620.
2. Rouzier R, Rajan R, Wagner P, Hess KR, Gold DL, Stec J, et al. Microtubule-associated protein tau: a marker of paclitaxel sensitivity in breast cancer. Proc Natl Acad Sci USA 2005; 102:8315-8320.
3. Fitzgibbons PL, Page DL, Weaver D, Thor AD, Allred DC, Clark GM, et al. Prognostic factors in breast cancer. College of American Pathologists Consensus Statement 1999. Arch Pathol Lab Med 2000; 124:966-978.
4. Elston CW, Ellis IO. Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up. Histopathology 1991; 19:403-410.
5. Veronesi U, Zurrida S, Viale G, Galimberti V, Arnone P, Nole F. Rethinking TNM: a breast cancer classification to guide to treatment and facilitate research. Breast J 2009; 15:291-295.
6. Humber CE, Tierney JF, Symonds RP, Collingwood M, Kirwan J, Williams C, et al. Chemotherapy for advanced, recurrent or metastatic endometrial cancer: a systematic review of Cochrane collaboration. Ann Oncol 2007; 18:409-420.
7. Biganzoli L, Minisini A, Aapro M, Di Leo A. Chemotherapy for metastatic breast cancer. Curr Opin Obstet Gynecol 2004; 16:37-41.
8. Fornari FA, Randolph JK, Yalowich JC, Ritke MK, Gewirtz DA. Interference by doxorubicin with DNA unwinding in MCF-7 breast tumor cells. Mol Pharmacol 1994; 45:649-656.
9. Lomovskaya N, Otten SL, Doi-Katayama Y, Fonstein L, Liu XC, Takatsu T, et al. Doxorubicin overproduction in Streptomyces peucetius: cloning and characterization of the dnrU ketoreductase and dnrV genes and the doxA cytochrome P-450 hydroxylase gene. J Bacteriol 1999; 181:305-318.
10.  Lyseng-Williamson KA, Fenton C. Docetaxel: a review of its use in metastatic breast cancer. Drugs 2005; 65:2513-2531.
11.  Yvon AM, Wadsworth P, Jordan MA. Taxol suppresses dynamics of individual microtubules in living human tumor cells. Mol Biol Cell 1999; 10:947-959.
12.  Coley HM, Labeed FH, Thomas H, Hughes MP. Biophysical characterization of MDR breast cancer cell lines reveals the cytoplasm is critical in determining drug sensitivity. Biochim Biophys Acta  2007; 1770:601-608.
13.  Akan I, Akan S, Akca H, Savas B, Ozben T. Multidrug resistance-associated protein 1 (MRP1) mediated vincristine resistance: effects of N-acetylcysteine and Buthionine sulfoximine. Cancer Cell Int 2005; 5:22.
14.  Choi CH. ABC transporters as multidrug resistance mechanisms and the development of chemosensitizers for their reversal. Cancer Cell Int 2005; 5:30.
15.  Vasilevskaya I, O'Dwyer PJ. Role of Jun and Jun kinase in resistance of cancer cells to therapy. Drug Resist Updat 2003; 6:147-56.
16.  Pommier Y, Redon C, Rao VA, Seiler JA, Sordet O, Takemura H, et al. Repair of and checkpoint response to topoisomerase I-mediated DNA damage. Mutat Res 2003; 532:173-203.
17.  McCubrey JA, Steelman LS, Abrams SL, Lee JT, Chang F, Bertrand FE, et al. Roles of the RAF/MEK/ERK and PI3K/PTEN/AKT pathways in malignant transformation and drug resistance. Adv Enzyme Regul 2006; 46:249-279.
18. Brantley-Finley C, Lyle CS, Du L, Goodwin ME, Hall T, Szwedo D, et al. The JNK, ERK and p53 pathways play distinct roles in apoptosis mediated by the antitumor agents vinblastine, doxorubicin, and etoposide. Biochem Pharmacol 2003; 66:459-469.
19.  Sebolt-Leopold JS. Development of anticancer drugs targeting the MAP kinase pathway. Oncogene 2000; 19:6594-6599.
20.  Hall RE, Lee CS, Alexander IE, Shine J, Clarke CL, Sutherland RL. Steroid hormone receptor gene expression in human breast cancer cells: inverse relationship between oestrogen and glucocorticoid receptor messenger RNA levels. Int J Cancer 1990; 46:1081-1087.
21.  Thompson EW, Paik S, Brunner N, Sommers CL, Zugmaier G, Clarke R, et al. Association of increased basement membrane invasiveness with absence of estrogen receptor and expression of vimentin in human breast cancer cell lines. J Cell Physiol 1992; 150:534-544.
22. Zhang RD, Fidler IJ, Price JE. Relative malignant potential of human breast carcinoma cell lines established from pleural effusions and a brain metastasis. Invasion Metastasis 1991; 11:204-215.
23.  Sommers CL, Byers SW, Thompson EW, Torri JA, Gelmann EP. Differentiation state and invasiveness of human breast cancer cell lines. Breast Cancer Res Treat 1994; 31:325-35.
24.  Tong D, Czerwenka K, Sedlak J, Schneeberger C, Schiebel I, Concin N, et al. Association of in vitro invasiveness and gene expression of estrogen receptor, progesterone receptor, pS2 and plasminogen activator inhibitor-1 in human breast cancer cell lines. Breast Cancer Res Treat 1999; 56:91-97.
25.  Fabbro D, Kung W, Roos W, Regazzi R, Eppenberger U. Epidermal growth factor binding and protein kinase C activities in human breast cancer cell lines: possible quantitative relationship. Cancer Res 1986; 46:2720-2725.
26.  Broxterman HJ, Gotink KJ, Verheul HM. Understanding the causes of multidrug resistance in cancer: a comparison of doxorubicin and sunitinib. Drug Resist Updat 2009; 12:114-126.
27. Germann UA, Pastan I, Gottesman MM. P-glycoproteins: mediators of multidrug resistance. Semin Cell Biol 1993; 4:63-76.
28.  Baselga J, Mendelsohn J. The epidermal growth factor receptor as a target for therapy in breast carcinoma. Breast Cancer Res Treat  1994; 29:127-138.
29.  Llovet JM. Updated treatment approach to hepatocellular carcinoma. J Gastroenterol  2005; 40:225-235.
30. Gariboldi MB, Ravizza R, Molteni R, Osella D, Gabano E, Monti E. Inhibition of Stat3 increases doxorubicin sensitivity in a human metastatic breast cancer cell line. Cancer Lett 2007; 258:181-188.
31.  Sakin V, Eskiocak U, Kars MD, Iseri OD, Gunduz U. hTERT gene expression levels and telomerase activity in drug resistant MCF-7 cells. Exp Oncol  2008; 30:202-205.
32.  Ahn SH, Son BH, Kim SW, Kim SI, Jeong J, Ko SS, et al. Poor outcome of hormone receptor-positive breast cancer at very young age is due to tamoxifen resistance: nationwide survival data in Korea--a report from the Korean Breast Cancer Society. J Clin Oncol 2007; 25:2360-2368.
33.  McDonald SL, Stevenson DA, Moir SE, Hutcheon AW, Haites NE, Heys SD, et al. Genomic changes identified by comparative genomic hybridisation in docetaxel-resistant breast cancer cell lines. Eur J Cancer  2005; 41:1086-1094.
34.  Fite A, Goua M, Wahle KW, Schofield AC, Hutcheon AW, Heys SD. Potentiation of the anti-tumour effect of docetaxel by conjugated linoleic acids (CLAs) in breast cancer cells in vitro. Prostaglandins Leukot Essent Fatty Acids 2007; 77:87-96.
35.  Corkery B, Crown J, Clynes M, O'Donovan N. Epidermal growth factor receptor as a potential therapeutic target in triple-negative breast cancer. Ann Oncol 2009; 20:862-867.
36.  Kim JH, Kim TH, Kang HS, Ro J, Kim HS, Yoon S. SP600125, an inhibitor of Jnk pathway, reduces viability of relatively resistant cancer cells to doxorubicin. Biochem Biophys Res Commun 2009; 387:450-455.
37.  Pichot CS, Hartig SM, Xia L, Arvanitis C, Monisvais D, Lee FY, et al. Dasatinib synergizes with doxorubicin to block growth, migration, and invasion of breast cancer cells. Br J Cancer 2009; 101:38-47.
38.  Garnett MJ, Marais R. Guilty as charged: B-RAF is a human oncogene. Cancer Cell 2004; 6:313-319.
39.  Platanias LC. Map kinase signaling pathways and hematologic malignancies. Blood 2003; 101:4667-4679.
40.  Sebolt-Leopold JS, Herrera R. Targeting the mitogen-activated protein kinase cascade to treat cancer. Nat Rev Cancer  2004; 4:937-947.
41.  Anonymous OT. Approved Claims for microtubule inhibitors. US Food and Drug Administration? 2006.
42.  Barrios CH, Sampaio C, Vinholes J, Caponero R. What is the role of chemotherapy in estrogen receptor-positive, advanced breast cancer? Ann Oncol 2009; 20:1157-1162.
43.  Kim SK, Yang JW, Kim MR, Roh SH, Kim HG, Lee KY, et al. Increased expression of Nrf2/ARE-dependent anti-oxidant proteins in tamoxifen-resistant breast cancer cells. Free Radic Biol Med  2008; 45:537-546.
44.  Bergqvist J, Elmberger G, Ohd J, Linderholm B, Bjohle J, Hellborg H, et al. Activated ERK1/2 and phosphorylated oestrogen receptor alpha are associated with improved breast cancer survival in women treated with tamoxifen. Eur J Cancer 2006; 42:1104-11012.
45.  Blanquart C, Karouri SE, Issad T. Implication of protein tyrosine phosphatase 1B in MCF-7 cell proliferation and resistance to 4-OH tamoxifen. Biochem Biophys Res Commun 2009; 387:748-753.
46.  Nicholson RI, Hutcheson IR, Britton D, Knowlden JM, Jones HE, Harper ME, et al. Growth factor signalling networks in breast cancer and resistance to endocrine agents: new therapeutic strategies. J Steroid Biochem Mol Biol 2005; 93:257-262.
47.  Navolanic PM, Steelman LS, McCubrey JA. EGFR family signaling and its association with breast cancer development and resistance to chemotherapy (Review). Int J Oncol 2003; 22:237-252.
48.  Nicholson RI, Hutcheson IR, Knowlden JM, Jones HE, Harper ME, Jordan N, et al. Nonendocrine pathways and endocrine resistance: observations with antiestrogens and signal transduction inhibitors in combination. Clin Cancer Res 2004; 10:346S-354S.
49. Ross JS, Linette GP, Stec J, Clark E, Ayers M, Leschly N, et al. Breast cancer biomarkers and molecular medicine. Expert Rev Mol Diagn 2003; 3:573-585.