The Effect Of Gefitinib In Radiosensitization Through Autophagy Regulation In Human Non-small-cell Lung Cancer A549 Cell Line

Objective: Observing the effect of autophagy regulation by Gefitinib or 3-Methyladenine(3-MA) in radiosensitization of human non-small-cell lung cancer A549 cell line so as to investigate:①The influence on modulation of autophagy level induced by Gefitinib, 3-MA andγ-ray on A549 cell.②The relationship among autophagy, apoptosis and cell cycle after different treatment.③The influence on radiosensitization caused by autophagy level and cell cycle blocking.Methods: Human non-small-cell lung cancer A549 cell line was chosen as target cells. The subjects were divided into six groups: control group(C), radiotherapy group(R), Gefitinib-treated group(G), 3-MA-treated group(M), Gefitinib plus radiotherapy group(G+R), 3-MA plus radiotherapy group(M+R). MTT assay method was used to determine the proliferation inhibition of A549 cell which was respectively treated with radiotherapy, Gefitinib or 3-MA alone. The half inhibitory concentration(IC50) and the ten percents inhibitory concentration(IC10) were determined. First, group R, G+R and M+R were treated withγ-ray ranging from 0Gy to 12Gy, cell plating efficiency(PE) and survival fraction(SF) were determined using clonogenic assay, and radiosensitivity parameters were calculated. Second, group R, G and M were treated withγ-ray(IC50), Gefitinib(IC50) and 3-MA(IC50) respectively, group G+R and M+R were pretreated with IC10 Gefinitib or 3-MA beforeγ-ray(IC50), PE and SF were determined. Flow cytometry was used to detect cell cycle and apoptotic rate of A549 cell after different treatment. MAPLC3-Ⅱexpression in A549 cell in each group was analyzed by Western blot. Autophagy body was observed by transmission electron microscope. Results:①Proliferation of A549 cell was inhibited in group G. The inhibition rate was dose-dependent when Gefitinib concentration was lower than 80μg/ml(P<0.05). Both group R and M were assayed for inhibitory activity against A549 cell growth and the cell viability were dose-dependent(P<0.05).②G roup R, G, M had lower SF than group C; group G had markedly lower SF than group M, group R had the lowest SF among above groups. The SF of group G+R was lower than that of group C, R, G. The SF of group M+R was lower than of group C, R, M. Inhibition of clone formation in group M+R was more obvious than in group G+R. When treated with 2Gy or 4Gyγ-ray radiotherapy, the SF of group G+R could decrease obviously. Treatment withγ-ray ranging from 2Gy to 12Gy, the SF of group M+R could greatly decreased. Pretreatment with IC10 Gefinitib or IC10 3-MA before the radiatiotherapy had obvious radiotherapy sensitization effect, the D0 of sensitizing enhancement ratios(SER) were 1.236 and 1.170 while the Dq of SER were 1.482 and 2.487 respectively.③Cell cycle of group R was arrested at G2～M phase while proportions of G0～G1 phase and S phase cells were declined comparing with group C(P<0.05). Group G blocked more cells at G0～G1 phase and has lower proportion of G2～M phase cells than group C(P<0.05), but there is no significant difference in the decline of proportion of S phase cells(P>0.05). The difference between group M and group C was not significant(P>0.05). Group G+R arrested cell cycle at G2～M phase, remarkably decreased of G0～G1 phase cells was also shown(P<0.05), but the decrease of S phase cells was not significant comparing with group C(P>0.05). Group M+R has no significant decrease of G0～G1 phase cells(P>0.05), increasing the percentage of G2～M phase cells and reducing the percentage of cells at S phase compared with group C (P<0.05). Groups R, M, G+R and M+R induced obvious apoptosis in A549 cell compared with group C(P<0.05), but the difference between group G and group C was not significant(P>0.05).④MAPLC3-Ⅱhas small expression in group C. Expression of MAPLC3-Ⅱin group R and G was increased(P<0.05) and decreased in group M. MAPLC3-Ⅱexpressed more in group G+R than in group G and R while expressed less in group M+R than in group R on the contrary. Linear correlation analysis showed that there was no obvious correlation between expression of MAPLC3-Ⅱand apoptotic rate(P>0.05).Conclusion:①Gefitinib,γ-ray and 3-MA could inhibit proliferation of A549 cell, in a dose-dependent.②Both IC10 Gefinitib and IC10 3-MA have obvious radiotherapy sensitization effect.③Gefitinib blocked cell cycle at G0～G1 phase,γ-ray blocked cell cycle at G2～M phase.④Gefitinib may induce A549 cell to produce autophagic cell deathinstead of apoptosis.⑤Both Gefitinib andγ-ray could up-regulate the expression of autophagy in A549 cell. Gefitinib could enhance the effect of autophagy causes byγ-ray, and 3-MA could suppression the effect.