Gefitinib radiosensitizes non-small cell lung cancer cells by suppressing cellular DNA repair capacity

Abstract

Purpose: Overexpression of the epidermal growth factor receptor (EGFR) promotes unregulated growth, inhibits apoptosis, and likely contributes to clinical radiation resistance of non-small cell lung cancer (NSCLC). Molecular blockade of EGFR signaling is an attractive therapeutic strategy for enhancing the cytotoxic effects of radiotherapy that is currently under investigation in preclinical and clinical studies. In the present study, we have investigated the mechanism by which gefitinib, a selective EGFR tyrosine kinase inhibitor, restores the radiosensitivity of NSCLC cells.

Experimental design: Two NSCLC cell lines, A549 and H1299, were treated with 1 micromol/L gefitinib for 24 h before irradiation and then tested for clonogenic survival and capacity for repairing DNA double strand breaks (DSB). Four different repair assays were used: host cell reactivation, detection of gamma-H2AX and pNBS1 repair foci using immunofluorescence microscopy, the neutral comet assay, and pulsed-field gel electrophoresis.

Results: In clonogenic survival experiments, gefitinib had significant radiosensitizing effects on both cell lines. Results from all four DNA damage repair analyses in cultured A549 and H1299 cells showed that gefitinib had a strong inhibitory effect on the repair of DSBs after ionizing radiation. The presence of DSBs was especially prolonged during the first 2 h of repair compared with controls. Immunoblot analysis of selected repair proteins indicated that pNBS1 activation was prolonged by gefitinib correlating with its effect on pNBS1-labeled repair foci.

Conclusions: Overall, we conclude that gefitinib enhances the radioresponse of NSCLC cells by suppressing cellular DNA repair capacity, thereby prolonging the presence of radiation-induced DSBs.

Figures

Fig. 1

Gefitinib radiosensitizes NSCLC cells and suppresses their signaling pathways downstream of EGFR. A549 (A and C) and H1299 (B and D) cells were treated with gefitinib (1 or 2 μmol/L for 24 h) and assessed for radiosensitization by clonogenic cell survival (A and B) immediately after irradiation or harvested for immunoblot analysis (C and D) 2 h after irradiation. For the survival curves, each data point represents the average of three independent experiments each plated in triplicate: solid line, control; dotted line, 1 μmol/L; dashed line, 2 μmol/L. Bar, SE. The effects of gefitinib treatment on the downstream signaling of the EGFR pathway proteins EGFR, pEGFR, AKT, pAKT, ERK, and pERK were analyzed by immunoblot analysis. Actin was used as a loading control.

Fig. 2

Gefitinib suppresses HCR in A549 (A) and H1299 (B) cells. Cells (2 × 104) were seeded in each well of six-well plates and were either left untreated or were treated with 1 μmol/L gefitinib for 24 h. They were then infected with unirradiated or irradiated (4,000 Gy) Ad-βgal for another 24 h. The cells were then stained for β-gal, and the β-gal – positive cells were counted and recorded. The percentage of positive cells were normalized to controls for comparison. Columns, average of at least three independent experiments; bars, SE.

Fig. 3

Gefitinib suppresses the repair of radiation-induced DSBs detected on the basis of γ-H2AX and pNBS1 foci. A549 (A and B) and H1299 (C and D) cells growing on coverslips in 35-mm dishes were exposed to gefitinib (1 μmol/L) for 24 h, irradiated (2 Gy), and fixed at the specified times for analysis of nuclear γ-H2AX and pNBS1 foci using immunofluorescence microscopy. Columns, average of three independent experiments; bars, SE.

Fig. 4

Gefitinib suppresses the repair of radiation-induced DSBs detected on the basis of the comet assay and PFGE. A549 (A and C) and H1299 cells (B and D) were exposed to gefitinib (1 μmol/L) for 24 h, irradiated with either 20 Gy for the comet assay (A and B) or 40 Gy for PFGE (C and D) and harvested at the indicated times. Columns, average of three independent experiments; bars, SE.

Fig. 5

Gefitinib does not affect the nuclear levels of EGFR, DNA-PK, ATM, or their activated forms after irradiation but does enhance the activation of NBS1. A549 and H1299 cells were exposed to gefitinib (1 μmol/L) for 24 h, irradiated or not with 5 Gy, and harvested 30 min after irradiation for the results in A or at the indicated times for B. Actin served as a loading control.