Radiosensitizing effects of gefitinib at different administration times in vitro

Abstract

The optimal administration time for applying epidermal growth factor receptor inhibitors combined with radiotherapy has been unclear. We investigated the efficacy of combining gefitinib with radiation in different treatment schedules. We demonstrated that gefitinib was administered to A549 lung cancer cells in three ways (administration before irradiation, administration upon irradiation, administration after irradiation) to establish the radiosensitizing effect. Cell-survival rates were evaluated by colony-forming assays. Cell apoptosis and cell-cycle distribution were investigated using flow cytometry; meanwhile, the expression of P21, Cdc25c, Bcl-2, Bax, Rad51 and phosphorylated DNA-PKcs (phospho-DNA-PK) after 6 Gy irradiation and/or gefitinib were determined by Western blot analysis. The sensitizer enhancement ratios of the gefitinib administration before irradiation, administration upon irradiation, and administration after irradiation groups were 2.23, 1.51 and 1.30, respectively. A higher apoptosis rate and G(2)/M phase arrest were observed in cells at 48 h after exposure to 6 Gy irradiation when gefitinib was administrated before irradiation. Increased cell apoptosis and cell cycle arrest were further supported by the expression changes of Bcl-2, Bax, P21, Cdc25c, Rad51 and phospho-DNA-PK at the same time. The best radiosensitizing effect was obtained when gefitinib was delivered before irradiation. Apoptosis might be an important way of cell killing and G(2)/M phase arrest might be an important mechanism of apoptosis. The expression proportion changes of P21/Cdc25c proteins may play an important role in G(2)/M cell cycle arrest. Moreover, the pro-apoptotic/antiapoptotic and DNA repair factors may be important modulators taking part in the molecular events of the radiosensitizing effect of gefitinib combined with irradiation.

Figure 1

EGFR and phospho‐EGFR expressions in A549 cells. Western blots of EGFR, phospho‐EGFR and β‐actin as a control. A549 cells expressed high levels of EGFR and phospho‐EGFR (lanes A). The A431 skin cancer cell was as a positive control (lanes B) and the striated muscle cell of nude mouse was as the negative control (lanes C).

Figure 2

Dose–survival curves of A549 cells after irradiation with and without gefitinib at different administration times. A, Radiation; B, gefitinib administration after irradiation; C, gefitinib administration upon irradiation; D, gefitinib administration before irradiation. Compared with the other groups, the cells exposed to gefitinib before irradiation revealed more sensitivity to X‐rays.

Figure 3

A549 cell apoptosis rates of combining gefitinib with radiation in different treatment schedules. The apoptotic rates were detected by flow cytometry. There were few apoptotic cells in (a) control group, (d) administration before irradiation group, (e) administration upon irradiation group and (f) administration after irradiation group. A549 cells had higher apoptosis rates when compared with (c) irradiation or (b) gefitinib‐alone treated group, and the highest was in gefitinib preradiation treatment group.

Figure 4

Effects of irradiation and/or gefitinib‐treatment on cell cycle distribution in A549 cells. Flow cytometric analysis revealed that the fraction of cells in G₀/G₁ increased after treatment with gefitinib (b) while those in G₂/M increased after treatment with irradiation (c) compared with untreated cells (a). In administration before 6‐Gy irradiation group (d), a sharp increase in the fraction of cells in the G₂/M phase was observed. The G₂/M phase arrests in administration upon irradiation group (e) and administration after irradiation group (f) were lower than that in group D, but sustained at a relatively high level.

Figure 5

Expressions of Bcl‐2, Bax, P21, Cdc25c, Rad51 and phospho‐DNA‐PK in A549 cells subjected to 6 Gy irradiation and/or gefitinib‐treatment. Control groups (lanes A), gefitinib‐treated groups (lanes B), irradiation‐treated groups (lanes C), gefitinib preradiation treatment groups (lanes D), concurrent treatment groups (lanes E), gefitinib postradiation treatment groups (lanes F). The results suggested that the radiosensitizing effect of gefitinib combined with irradiation might be influenced by the protein related to cell cycle redistribution, the pro‐/antiapoptotic factors regulation, and DNA repair factors changes.