Restricting extracellular Ca2+ on gefitinib-resistant non-small cell lung cancer cells reverses altered epidermal growth factor-mediated Ca2+ response, which consequently enhances gefitinib sensitivity

Non-small cell lung cancer (NSCLC), one of the leading causes of cancer-related death, has a low 5-year survival rate owing to the inevitable acquired resistance toward antitumor drugs, platinum-based chemotherapy, and targeted therapy. Epidermal growth factor (EGF)-EGF receptor (EGFR) signaling activates downstream events leading to phospholipase C/inositol trisphosphate (IP 3 )/Ca 2+ release from IP 3 -sensitive Ca 2+ stores to modulate cell proliferation, motility, and invasion. However, the role of EGFR-mediated Ca 2+ signaling in acquired drug resistance is not fully understood. Here, we analyzed alterations of intracellular Ca 2+ ([Ca 2+ ]i) responses between gefitinib-sensitive NSCLC PC-9 cells and gefitinib-resistant NSCLC PC-9/GR cells, and we found that acute EGF treatment elicited intracellular Ca 2+ ([Ca 2+ ]i) oscillations in PC-9 cells but not in PC-9/GR cells. PC-9/GR cells presented a more sustained basal [Ca 2+ ]i level, lower endoplasmic reticulum Ca 2+ level, and higher spontaneous extracellular Ca 2+ ([Ca 2+ ]e) influx than PC-9 cells. Notably, restricting [Ca 2+ ]e in both cell types induced identical [Ca 2+ ]i oscillations, dependent on phospholipase C and EGFR activation. Consequently, restricting [Ca 2+ ]e in PC-9/GR cells upregulated gefitinib-mediated poly (ADP-ribose) polymerase cleavage, an increase in Bax/Bcl-2 ratio, cytotoxicity, and apoptosis. In addition, nuclear factor of activated T cell (NFAT1) induction in response to EGF was inhibited by gefitinib in PC-9 cells, whereas EGF-mediated NFAT1 induction in PC-9/GR cells was sustained regardless of gefitinib treatment. Restricting [Ca 2+ ]e in PC-9/GR cells significantly reduced EGF-mediated NFAT1 induction. These findings indicate that spontaneous [Ca 2+ ]e influx in NSCLC cells plays a pivotal role in developing acquired drug resistance and suggest that restricting [Ca 2+ ]e may be a potential strategy for modulating drug-sensitivity.