Analytical and Bioanalytical Chemistry

, Volume 410, Issue 30, pp 7827–7835 | Cite as

Rapid screening of drug candidates against EGFR/HER2 signaling pathway using fluorescence assay

  • Farkhondeh Khanjani
  • Reza H. SajediEmail author
  • Sadegh Hasannia
Paper in Forefront


Over the recent decade, the calcium-based assays have gained much popularity in order to discover new drugs. Since breast cancer is the second cause of death in the female population, rapid and effective methods are needed to screen drug compounds with fewer side effects. Human epidermal growth factor receptor 2 (HER2) increases intracellular free Ca2+ on its signaling pathways. In the present study, BT474 cell line, which overexpresses HER2 receptor, was selected and using fura-2-AM, intracellular Ca2+ release was investigated. The changes in the concentration of intracellular Ca2+ were evaluated by variation in the amount of fluorescence intensity. In the presence of epidermal growth factor (EGF), an increase in fluorescence intensity was observed so that after 20 min it raised to the maximum level. After treatment of BT474 cells by lapatinib, as a tyrosine kinase inhibitor (TKI), the signaling pathway of EGFR/HER2 heterodimer was significantly inhibited, which resulted in a decrease in Ca2+ entry into the cytoplasm and fluorescence emission decreased. The IC50 value for the effect of lapatinib on BT474 cells was 113.2 nmol/L. Our results suggest this method is a simple, efficient and specific approach and can potentially be useful for screening new drug candidates against EGFR/HER2 heterodimer signaling pathways.

Graphical abstract


Breast Cancer EGFR/HER2 heterodimer Fura-2-AM Intracellular Ca2+ Tyrosine kinase inhibitor (TKI) 



This work was supported by the research council of Tarbiat Modares University and Ministry of Sciences, Researches, and Technology, Iran.

Compliance with ethical standards

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. 1.
    Howell A, Anderson AS, Clarke RB, Duffy SW, Evans DG, Garcia-Closas M, et al. Risk determination and prevention of breast cancer. Breast Cancer Res. 2014;16(5):446.CrossRefGoogle Scholar
  2. 2.
    Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin. 2016;66(1):7–30.CrossRefGoogle Scholar
  3. 3.
    Holliday DL, Speirs V. Choosing the right cell line for breast cancer research. Breast Cancer Res. 2011;13(4):215.CrossRefGoogle Scholar
  4. 4.
    Perou CM, Jeffrey SS, Van De Rijn M, Rees CA, Eisen MB, Ross DT, et al. Distinctive gene expression patterns in human mammary epithelial cells and breast cancers. Proc Natl Acad Sci. 1999;96(16):9212–7.CrossRefGoogle Scholar
  5. 5.
    Pegram BMD, Lipton A, Hayes DF, Weber BL, Baselga JM, Tripathy D, et al. Phase II study of receptor-enhanced chemosensitivity using recombinant humanized anti-p185HER2/neu monoclonal antibody plus cisplatin in patients with HER2/neu-overexpressing metastatic breast cancer refractory to chemotherapy treatment. J Clin Oncol. 1998;16(8):2659–71.CrossRefGoogle Scholar
  6. 6.
    Yarden Y, Sliwkowski MX. Untangling the ErbB signalling network. Nat Rev Mol Cell Biol. 2001;2(2).CrossRefGoogle Scholar
  7. 7.
    Baselga J, Swain SM. Novel anticancer targets: revisiting ErbB2 and discovering ErbB3. Nat Rev Cancer. 2009;9(7):463.CrossRefGoogle Scholar
  8. 8.
    Cerra M, Cecco L, Montella M, Tuccillo F, Bonelli P, Botti G. Epidermal growth factor receptor in human breast cancer comparison with steroid receptors and other prognostic factors. Int J Biol Markers. 1994;10(3):136–42.CrossRefGoogle Scholar
  9. 9.
    Lenferink AEG, Pinkas-Kramarski R, van de Poll MLM, van Vugt MJH, Klapper LN, Tzahar E, et al. Differential endocytic routing of homo- and hetero-dimeric ErbB tyrosine kinases confers signaling superiority to receptor heterodimers. EMBO J. 1998;17(12):3385–97.CrossRefGoogle Scholar
  10. 10.
    Ullrich A, Schlessinger J. Signal transduction by receptors with tyrosine kinase activity. Cell. 1990;61(2):203–12.CrossRefGoogle Scholar
  11. 11.
    Hynes NE, Lane HA. ErbB receptors and cancer: the complexity of targeted inhibitors. Nat Rev Cancer. 2005;5(5):341–54.CrossRefGoogle Scholar
  12. 12.
    Noh D-Y, Shin SH, Rhee SG. Phosphoinositide-specific phospholipase C and mitogenic signaling. Biochim Biophys Acta Rev Cancer. 1995;1242(2):99–113.CrossRefGoogle Scholar
  13. 13.
    Thomas D, Hanley MR. Pharmacological tools for perturbing intracellular calcium storage. Methods Cell Biol. 1994;65.Google Scholar
  14. 14.
    Roderick HL, Cook SJ. Ca2+ signalling checkpoints in cancer: remodelling Ca2+ for cancer cell proliferation and survival. Nat Rev Cancer. 2008;8(5):361–75.CrossRefGoogle Scholar
  15. 15.
    Y Maximov P, M Lee T, Craig Jordan V. The discovery and development of selective estrogen receptor modulators (SERMs) for clinical practice. Curr Clin Pharmacol. 2013;8(2):135–55.CrossRefGoogle Scholar
  16. 16.
    Van Cutsem E, Kang Y, Chung H, Shen L, Sawaki A, Lordick F. Efficacy results from the ToGA trial: a phase III study of trastuzumab added to standard chemotherapy in first-line HER2-positive advanced gastric cancer. J Clin Oncol. 2009;15s:27.Google Scholar
  17. 17.
    Schroeder RL, Stevens CL, Sridhar J. Small molecule tyrosine kinase inhibitors of ErbB2/HER2/neu in the treatment of aggressive breast cancer. Molecules. 2014;19(9):15196–212.CrossRefGoogle Scholar
  18. 18.
    Geyer CE, Forster J, Lindquist D, Chan S, Romieu CG, Pienkowski T, et al. Lapatinib plus capecitabine for HER2-positive advanced breast cancer. N Engl J Med. 2006;355(26):2733–43.CrossRefGoogle Scholar
  19. 19.
    Konecny GE, Pegram MD, Venkatesan N, Finn R, Yang G, Rahmeh M, et al. Activity of the dual kinase inhibitor lapatinib (GW572016) against HER2-overexpressing and trastuzumab-treated breast cancer cells. Cancer Res. 2006;66(3):1630–9.CrossRefGoogle Scholar
  20. 20.
    Fan F, Wood KV. Bioluminescent assays for high-throughput screening. Assay Drug Dev Technol. 2007;5(1):127–36.CrossRefGoogle Scholar
  21. 21.
    Kuo MS, Auriau J, Pierre-Eugene C, Issad T. Development of a human breast-cancer derived cell line stably expressing a bioluminescence resonance energy transfer (BRET)-based phosphatidyl inositol-3 phosphate (PIP3) biosensor. PLoS One. 2014;9:e92737.CrossRefGoogle Scholar
  22. 22.
    Zamani P, Sajedi RH, Hosseinkhani S, Zeinoddini M, Bakhshi B. A luminescent hybridoma-based biosensor for rapid detection of V. cholerae upon induction of calcium signaling pathway. Biosens Bioelectron. 2016;79:213–9.CrossRefGoogle Scholar
  23. 23.
    Le Poul E, Hisada S, Mizuguchi Y, Dupriez VJ, Burgeon E, Detheux M. Adaptation of aequorin functional assay to high throughput screening. J Biomol Screen. 2002;7(1):57–65.CrossRefGoogle Scholar
  24. 24.
    Regehr WG, Tank DW. Selective fura-2 loading of presynaptic terminals and nerve cell processes by local perfusion in mammalian brain slice. J Neurosci Methods. 1991;37(2):111–9.CrossRefGoogle Scholar
  25. 25.
    Hartford Svoboda KK, Reenstra WR. Approaches to studying cellular signaling: a primer for morphologists. Anat Rec. 2002;269(2):123–39.CrossRefGoogle Scholar
  26. 26.
    Zamani P, Sajedi RH, Hosseinkhani S, Zeinoddini M. Hybridoma as a specific, sensitive, and ready to use sensing element: a rapid fluorescence assay for detection of Vibrio cholerae O1. Anal Bioanal Chem. 2016;408(23):6443–51.CrossRefGoogle Scholar
  27. 27.
    Nuccitelli R. A practical guide to the study of calcium in living cells. In: Academic Press. 1st ed; 1994.Google Scholar
  28. 28.
    Blagodatski A, Cherepanov V, Koval A, Kharlamenko VI, Khotimchenko YS, Katanaev VL. High-throughput targeted screening in triple-negative breast cancer cells identifies Wnt-inhibiting activities in Pacific brittle stars. Sci Rep. 2017;7(1):11964.CrossRefGoogle Scholar
  29. 29.
    Kumar S, Bajaj S, Bodla RB. Preclinical screening methods in cancer. Indian J Pharmacol. 2016;48(5):481.CrossRefGoogle Scholar
  30. 30.
    Banappagari S, Corti M, Pincus S, Satyanarayanajois S. Inhibition of protein–protein interaction of HER2–EGFR and HER2–HER3 by a rationally designed peptidomimetic. J Biomol Struct Dyn. 2012;30(5):594–606.CrossRefGoogle Scholar
  31. 31.
    Liu W, Xu J, Wu S, Liu Y, Yu X, Chen J, et al. Selective anti-proliferation of HER2-positive breast cancer cells by anthocyanins identified by high-throughput screening. PLoS One. 2013;8(12):e81586.CrossRefGoogle Scholar
  32. 32.
    Pottle J, Sun C, Gray L, Li M. Exploiting MCF-7 cells’ calcium dependence with interlaced therapy. J Cancer Ther. 2013;4(7):32.CrossRefGoogle Scholar
  33. 33.
    Hotta Y, Ando H, Fujita M, Nakagawa J, Takeya K, Sakakibara J. Different effects of isoproterenol and dihydroouabain on cardiac Ca2+ transients. Eur J Pharmacol. 1995;282(1–3):121–30.CrossRefGoogle Scholar
  34. 34.
    Korutla L, Cheung JY, Medelsohn J, Kumar R. Inhibition of ligand-induced activation of epidermal growth factor receptor tyrosine phosphorylation by curcumin. Carcinogenesis. 1995;16(8):1741–5.CrossRefGoogle Scholar
  35. 35.
    Rusnak DW, Lackey K, Affleck K, Wood ER, Alligood KJ, Rhodes N, et al. The effects of the novel, reversible epidermal growth factor receptor/ErbB2 tyrosine kinase inhibitor, GW2016, on the growth of human normal and tumor-derived cell lines in vitro and in vivo. Mol Cancer Ther. 2001;1(2):85–94.Google Scholar
  36. 36.
    Li X, Yang C, Wan H, Zhang G, Feng J, Zhang L, et al. Discovery and development of pyrotinib: A novel irreversible EGFR/HER2 dual tyrosine kinase inhibitor with favorable safety profiles for the treatment of breast cancer. Eur J Pharm Sci. 2017;110:51–61.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Farkhondeh Khanjani
    • 1
  • Reza H. Sajedi
    • 1
    Email author
  • Sadegh Hasannia
    • 1
  1. 1.Department of Biochemistry, Faculty of Biological SciencesTarbiat Modares UniversityTehranIran

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