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

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 ᅟ.