The synergistic effect of EGFR tyrosine kinase inhibitor gefitinib in combination with aromatase inhibitor anastrozole in non-small cell lung cancer cell lines

Lan Shen, Ziming Li, Shengping Shen, Xiaomin Niu, Yongfeng Yu, Zonghai Li, Meilin Liao, Zhiwei Chen, Shun Lu
Lung Cancer: Journal of the International Association for the Study of Lung Cancer 2012, 78 (3): 193-200

BACKGROUND: Several studies implicated that lung cancer progression was governed by the interaction between estrogen receptor (ER) and epidermal growth factor receptor (EGFR) signaling pathways. Combined targeting of EGFR and ER may have the synergistic effect in lung cancer treatment. The aim of this study was to explore the potential utility of inhibiting these two pathways with combination of anastrozole and gefitinib in non-small cell lung cancer (NSCLC) cell lines.

MATERIALS AND METHODS: The expression levels of ER (ER-α and ER-β) in lung cancer cell lines (A549, H460, SPC-A-1, H1299) and normal bronchus epithelial cell BEAS-2B were detected using real-time PCR and Western blot. Immunocytochemistry was used to locate ER-α and ER-β in cell line with highest ER expression levels. The cells were treated with anastrozole or gefitinib alone or in combination. The cell proliferation inhibition was detected by the CCK8 assay, cell cycle and apoptosis effects were detected by flow cytometry; the expression levels of phosphorylated-EGFR (p-EGFR), ERK, phosphorylated-ERK (p-ERK), AKT and phosphorylated-AKT (p-AKT) were detected by Western blot.

RESULTS: Among these cell lines the expression levels of ER in A549 cells were highest. In A549 cell line, ER-α was mainly localized in the cytoplasm, whereas ER-β was mainly localized in the cytoplasm and to a lesser degree in the nucleus. The combination of two drugs increased the proliferation inhibition rates for 24h, 48 h, 72 h to 37.66 ± 1.02%, 63.41 ± 2.02%, 70.50 ± 0.86%, respectively, which was closely associated with elevation of the G0/G1 phase fraction (P<0.05). Apoptosis rates of A549 cells treated with anastrozole, gefitinib alone or in combination were 10.72 ± 1.12%, 17.40±1.28%, 23.02 ± 2.32%, respectively (P<0.05). The synergistic effects of the combination therapy were accompanied by reduction of p-EGFR, p-ERK and p-AKT expression compared with individual treatment.

CONCLUSIONS: The results of this study suggest that the combination of anastrozole and gefitinib compared with either drug alone can maximally inhibit cell proliferation, induce apoptosis, and affect downstream signaling pathways. Our study supports functional interaction between the ER and the EGFR pathways in lung cancer and provides a clinically exploitable strategy for non-small cell lung cancer patients.

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