Autophagy Inhibition Overcomes the Antagonistic Effect Between Gefitinib and Cisplatin in Epidermal Growth Factor Receptor Mutant Non--Small-Cell Lung Cancer Cells

Abstract

Background: Four large clinical trials have shown that concurrent administration of an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), such as gefitinib or erlotinib, with chemotherapy agents does not improve overall survival (OS) in unselected patients with advanced non-small-cell lung cancer (NSCLC). In the present study, the role of autophagy in the combination of gefitinib and cisplatin on EGFR-TKI-sensitive human lung cancer cell line was investigated. Moreover, whether simultaneous autophagy inhibition treatment increases the antitumor activity was explored.

Materials and methods: The PC9 cell was exposed to either gefitinib or cisplatin alone or together. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The cytotoxic interaction between gefitinib and cisplatin was determined using coefficient of drug interaction (CDI) analysis. The cell cycle distribution and apoptosis were measured using flow cytometry. Alterations in the autophagy and apoptosis signaling pathway were measured using Western blot assays.

Results: Coadministration of gefitinib and cisplatin resulted in antagonistic activity to tumor cell proliferation. However, the addition of chloroquine (CQ), an autophagy inhibitor, overcame the antagonistic effects, as demonstrated by CDI analysis and Annexin V-FITC/propidium iodide (PI) assays. In addition, gefitinib administration led to cell G1 phase arrest, which might have contributed to the antagonistic activity between gefitinib and cisplatin. However, the addition of CQ did not deregulate cell cycle arrest, indicating that other mechanisms might be involved. The Annexin V-FITC/PI assays showed that the addition of CQ significantly the increased the ratio of apoptosis cells. Also, immunoblotting assays exhibited increased Bax and decreased Bcl-2, suggesting that autophagy inhibition by CQ could increase cell apoptosis and thus overcome the antagonistic effects.

Conclusion: The combination of gefitinib with cisplatin generates antagonistic effects on EGFR-TKI-sensitive cells. However, inhibiting autophagy produces a synergistic effect, suggesting that gefitinib and cisplatin combined with an autophagy inhibitor (especially CQ) might be a beneficial strategy to overcome the antagonistic effects between EGFR-TKIs and chemotherapeutic agents.

Keywords: Apoptosis; Autophagy; Epidermal growth factor receptor; Non–small-cell lung cancer; Tyrosine kinase inhibitor.