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

Abstract

TOPIC: Lung Cancer TYPE: Original Investigations PURPOSE: 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 (IP3)/Ca2+ release from IP3-sensitive Ca2+ stores to modulate cell proliferation, motility, and invasion. However, the role of EGFR-mediated Ca2+ signaling in acquired drug resistance is not fully understood. METHODS: We analyzed the intracellular calcium ([Ca2+]i) signals in PC-9 cells and PC-9/GR (Gefitinib Resistant) cells under following conditions. We observed hEGF (200ng/ml)-mediated [Ca2+]i oscillations response. Calcium influx, endoplasmic reticulum (ER) calcium contents, hEGF (200 ng/ml)-mediated [Ca2+]i oscillations, and effects of inhibitors were studied in absence of extracellular calcium ([Ca2+]e). We compared gefitinib-induced cytotoxicity and cell viability according to the presence or absence of [Ca2+]e. RESULTS: NSCLC PC-9 cells and gefitinib resistant NSCLC PC-9/GR cells, and we found that acute EGF treatment elicited intracellular Ca2+ ([Ca2+]i) oscillations in PC-9 cells but not in PC-9/GR cells. PC-9/GR cells presented a more sustained basal [Ca2+]i level, lower endoplasmic reticulum Ca2+ level, and higher spontaneous extracellular Ca2+ ([Ca2+]e) influx than PC-9 cells. Notably, restricting [Ca2+]e in both cell types induced identical [Ca2+]i oscillations, dependent on phospholipase C and EGFR activation. Consequently, restricting [Ca2+]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 [Ca2+]e in PC-9/GR cells significantly reduced EGF-mediated NFAT1 induction. CONCLUSIONS: These findings indicate that spontaneous [Ca2+]e influx in NSCLC cells plays a pivotal role in developing acquired drug resistance and suggest that restricting [Ca2+]e may be a potential strategy for modulating drug-sensitivity. CLINICAL IMPLICATIONS: resistant mechanism in EGFR TKI DISCLOSURES: no disclosure on file for Misung Kim; no disclosure on file for Sohui Kim; no disclosure on file for Minsuk Kim; No relevant relationships by Seihoon Yang, source=Web Response