Combination effects of gefitinib (‘Iressa’, ZD1839) with other anticancer agents on non-small-cell lung cancer cells

4538 Gefitinib (‘Iressa’, ZD1839) is an orally active EGFR-TKI (epidermal growth factor receptor tyrosine kinase inhibitor), which inhibits EGFR downstream signaling, including mitogen-activated protein kinase (MAPK), signal transducer and activator of transcription (STAT), and Akt pathways. Since the Akt signaling pathway mediates anti-apoptotic signaling, it is expected that gefitinib should enhance the cytotoxic effects of other anticancer drugs. In an attempt to establish new combination chemotherapy regimens using gefitinib, we evaluated combination effects between gefitinib and standard anticancer drugs on human non-small-cell lung cancer (NSCLC) cell lines in vitro. We used two NSCLC cell lines, PC-9, highly gefitinib sensitive (IC50: 17 nM), and PC-14, intrinsic gefitinib resistant (IC50: 20 μM). We examined the combination effects of gefitinib with vinorelbine (VNR), vincristine (VCR), paclitaxel (TAX), mitomycin C (MMC), irinotecan (CPT-11), 5-fluorouracil (5-FU), etoposide (VP-16), or cisplatin (CDDP). Cytotoxicity of the two drug combinations was determined simultaneously by MTT assay, using continuous drug exposure for 4 to 5 days. The combination effects were analyzed by an improved isobologram analysis. Drug-mediated apoptotic cell death was measured by fluorescent activated cell sorting (FACS) analysis, using propidium iodide staining and TdT-mediated dUTP-biotin nick end labeling (TUNEL) methods. Caspase-3 activity was measured by cleavage of the colorimetric substrate DEVD-pNA. In PC-9 cells, the combined effect of gefitinib with VCR was evaluated as supra-additive, and the effects with VNR, CPT-11, TAX, MMC, and 5-FU were evaluated as additive in isobologram analysis. Combinations of gefitinib with VP-16/CDDP were evaluated as a weak additive effect. All of these validated combinations in isobologram analysis induced apoptotic cell death synergistically in FACS analysis. When 10 nM gefitinib was combined with IC50 concentrations of VNR, VCR, CPT-11, TAX, and 5-FU drug-mediated apoptosis was 3 to 12-fold higher than that caused by monotherapy in PC-9 cells. The drug-mediated caspase-3 activation was measured at 24 hours after drug exposure. When 10 nM gefitinib was combined with VNR, VCR, CPT-11, and 5-FU (this concentration of gefitinib did not activate caspase-3 within this time course in PC-9 cells), drug-mediated caspase-3 activity was significantly enhanced by gefitinib (1.5 to 2-fold higher than that caused by monotherapy). Gefitinib did not enhance CDDP-mediated caspase-3 activation. In PC-14 cells, gefitinib did not enhance any drug-mediated apoptosis or caspase-3 activation. Combination therapy of gefitinib with VNR, VCR, CPT-11, TAX, MMC, and 5-FU may be effective in patients with NSCLC, especially in tumors that have gefitinib sensitivity. ‘Iressa’ is a trademark of the AstraZeneca group of companies.