Sequence-dependent antiproliferative effects of gefitinib and docetaxel on non-small cell lung cancer (NSCLC) cells and the possible mechanism

Abstract

Purpose: Recent clinical trials showed that the sequential combination of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) and chemotherapy could prolong the PFS and/or OS of advanced non-small cell lung cancer (NSCLC) patients with EGFR mutation. The aim of present study was to assess the optimal combination sequence and to explore its possible mechanism.

Methods: PC-9 cells and A549 cells, the lung adenocarcinoma cells with mutant and wide-type EGFR respectively, were treated with docetaxel/gefitinib alone or in different combination schedules. The EGFR and K-ras gene status was determined by qPCR-HRM technique. Cell proliferation was detected by MTT assay. The expression and phosphorylation of EGFR, ERK, Akt and IGF-1R were detected by western blot. Cell cycle distribution was observed by flow cytometry.

Results: Only sequential administration of docetaxel followed by gefitinib (D → G) induced significant synergistic effect in both cell lines (Combination Index<0.9). The reverse sequence (G → D) resulted in an antagonistic interaction in both cell lines (CI>1.1), whereas the concurrent administration (D+G) showed additive (0.9<CI<1.1)-synergistic effect in PC-9 cells and antagonistic-additive effect in A549 cells. Mechanism studies showed that docetaxel-induced phosphorylation of EGFR and ERK was repressed by subsequently used gefitinib, but not by concurrent exposure of gefitinib. The gefitinib-repressed phosphorylation of EGFR and ERK was reversed neither by concurrent nor by subsequent administration of docetaxel. D+G reinforced their inhibition on the phosphorylation of IGF-1R in PC-9 cells.

Conclusions: The cytotoxic drugs followed by EGFR-TKIs may be the optimal combination for antiproliferative effects in EGFR-mutant NSCLC cells, and the phosphorylation of EGFR and ERK might contribute to this effect.

Figure 1. Docetaxel or gefitinib alone inhibited the proliferation of lung adenocarcinoma cells in a dose-dependent manner.

Cells were treated with gradient concentrations (10⁻⁴ M∼10⁻¹⁰ M) of gefitinib or docetaxel for 72 h. Cell proliferation was determined by MTT assay. (A) A549 cells; (B) PC-9 cells. *P<0.05 compared with control group. Bars: ± SD, n = 3.

Figure 2. Effects of different exposure schedules of gefitinib and docetaxel on cell proliferation.

Cells were treated with three different sequences of gefitinib and docetaxel (D→G; G→D; D+G). The drug doses were combined using constant ratios of IC50 values (0.125, 0.25, 0.5, 1, 2 and 4 times of IC50). (A, B) The inhibition rate was determined by MTT assay. *P<0.05, D→G versus G→D; #P<0.05 D→G versus D+G. The D→G sequence produced the most potent inhibitory effect. (C, D) The combination index (CI) was calculated using CompuSyn software. Only the D→G sequence showed synergistic effect. (D–G) docetaxel followed by gefitinib; (G–D) gefitinib followed by docetaxel; (D+G) docetaxel and gefitinib administered concurrently. Bars: ± SD, n = 3.

Figure 3. Effects of different exposure schedules of gefitinib and docetaxel on cell signaling pathways.

The expression and phosphorylation of some representative molecules in correlated cell signaling pathways including EGFR, ERK, Akt and IGF-1R were detected by western blot. (A) A549 cells; (B) PC-9 cells. (C) control group; (D) docetaxel alone; (G) gefitinib alone; (D–G) docetaxel followed by gefitinib; (G–D) gefitinib followed by docetaxel; (D+G) docetaxel and gefitinib administered concurrently.

Figure 4. Effects of different exposure schedules of gefitinib and docetaxel on cell cycle distribution.

The effects of different administration schedules of gefitinib and docetaxel on cell cycle distribution were detected by flow cytometry. (C) control group; (D) docetaxel alone; (G) gefitinib alone; (D–G) docetaxel followed by gefitinib; (G–D) gefitinib followed by docetaxel; (D+G) docetaxel and gefitinib administered concurrently. *P<0.05 compared with control group. n = 3.