Tuberc Respir Dis.  2011 Oct;71(4):259-265.

Enhanced Sensitivity to Gefitinib after Radiation in Non-Small Cell Lung Cancer Cells

• Choi YJ
• Rho JK
• Back DH
• Kim HR
• Lee JC
• Kim CH

Abstract

BACKGROUND
Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, gefitinib and erlotinib, are effective therapies for non-small cell lung cancer (NSCLC) patients whose tumors harbor somatic mutations in EGFR. The mutations are, however, only found in about 30% of Asian NSCLC patients and all patients ultimately develop resistance to these agents. Ionizing radiation has been shown to induce autophosphorylation of EGFR and activate its downstream signaling pathways. In the present study, we have tested whether the effect of gefitinib treatment can be enhanced after ionizing radiation.
METHODS
We compared the PC-9 and A549 cell line with its radiation-resistant derivatives after gefitinib treatment with cell proliferation and apoptosis assay. We also analyzed the effect of gefitinib after ionizing radiation in PC-9, A549, and NCI-H460 cells. Cell proliferation was determined by MTT assay and induction of apoptosis was evaluated by flow cytometry. Caspase 3 activation and PARP cleavage were evaluated by western blot analysis.
RESULTS
PC-9 cells having mutated EGFR and their radiation-resistant cells showed no significant difference in cell viability. However, radiation-resistant A549 cells were more sensitive to gefitinib than were their parental cells. This was attributable to an increased induction of apoptosis. Gefitinib-induced apoptosis increased significantly after radiation in cells with wild type EGFR including A549 and NCI-H460, but not in PC-9 cells with mutated EGFR. Caspase 3 activation and PARP cleavage accompanied these findings.
CONCLUSION
The data suggest that gefitinib-induced apoptosis could increase after radiation in cells with wild type EGFR, but not in cells with mutated EGFR.

Keyword

gefitinib; Radiation; Carcinoma, Non-Small-Cell Lung

MeSH Terms

Apoptosis
Asian Continental Ancestry Group
Blotting, Western
Carcinoma, Non-Small-Cell Lung
Caspase 3
Cell Line
Cell Proliferation
Cell Survival
Flow Cytometry
Humans
Parents
Protein-Tyrosine Kinases
Quinazolines
Radiation, Ionizing
Receptor, Epidermal Growth Factor
Erlotinib Hydrochloride
Caspase 3
Protein-Tyrosine Kinases
Quinazolines
Receptor, Epidermal Growth Factor

Figure

• 

  Figure 1 (A) Effect of gefitinib on proliferation determined by using the MTT assay. Cells were treated with the indicated concentrations of gefitinib and proliferation was determined 72 hours later. PC-9 cells having mutated EGFR and their radiation-resistant cells (PC-9/RR) showed no significant difference in cell viability. However, radiation-resistant A549 cells (A549/RR) were more sensitive to gefitinib than were their parental cells. (B) Induction of apoptosis after gefitinib treatment for 48 hours. After staining with annexin V and PI, the cells were analyzed by flow cytometry to analyze apoptosis. PC-9 cells and their radiation-resistant cells (PC-9/RR) showed a similar apoptosis fraction at each concentration. However, radiation-resistant A549 cells (A549/RR) showed a significantly higher rate of apoptosis than did their parental cells. *p<0.05, †p<0.05.

• 

  Figure 2 (A) Induction of apoptosis by gefitinib treatment in PC-9, A549, NCI-H460 cells. The cells were irradiated with 2 Gy and treated with each concentration of gefitinib (1 µM for PC-9, 20 µM for A549, 30 µM for NCI-H460) after 48 hours. A further 48 hours after the gefitinib treatment, apoptosis was analyzed by FACS. The results were compared to those of cells treated with only gefitinib without previous irradiation. G, gefitinib; RT, radiation. *p<0.05, †p<0.05. (B) The difference of apoptosis induction by gefitinib with or without previous irradiation was plotted. Gefitinib-induced apoptosis increased after radiation in cells with wild type EGFR, including A549 and NCI-H460, but not in PC-9 cells with mutated EGFR. *p<0.05, †p<0.05.

• 

  Figure 3 Western blot analysis of procaspase 3, caspase 3, and PARP. (A) PC-9 radiation-resistant cells (PC-9/RR), A549 radiation-resistant cells (A549/RR) and their parental cells were exposed to gefitinib for 24 hours in each concentration and were harvested for the results. Actin served as a loading control. Gefitinib-induced apoptosis increased in A549/RR cells compared to their parental cells. (B) PC-9 and A549 cells were either irradiated or not irradiated with 2 Gy and treated with gefitinib (1 µM for PC-9, 20 µM for A549) after 48 hours. Twenty four hours after gefitinib treatment, the cells were harvested. Gefitinib-induced apoptosis increased after irradiation in A549 but not in the PC-9 cells. C: control; G: gefitinib; RT: radiation.

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