Radiosensitization Effects And Mechanisms Of AZD9291 Alone And Combined With LC On PC-9-IR Cells

Objective:Radiosensitization effects and mechanisms of AZD9291 alone and combined with LC on PC-9-IR cells.Methods:MTS was used to detect the inhibitory effect of AZD9291 alone and combined with lysimachia capilliposide hemsl capilliposide(LC)on non-small cell lung cancer cell lines(H460,PC-9,PC-9-IR and H1975)in vitro.PC-9-IR cells treated with AZD9291 individually and combined with LC followed by ionizing radiation.Clonogenic survival assay was performed and sensitizer enhancement ratio(SER)was calculated.The levels of total and its phosphorylation protein of EGFR,ERK,AKT were detected by Westernblot.Flow cytometry and immunofluorescent technique were used to detect the levels of yH2AX and radiation-induced apoptosis of PC-9-IR cells treated by AZD9291 and ionizing radiation.Sensitizer enhancement ratio of AZD9291 on PC-9-IR cells with DNA-PKcs gene knocked out by interfering RNA technology was calculated.Nude mouse model is used to verify the AZD9291 radiation-sensitizing effect in vivo.EGFR phosphorylation protein and Ki67,DNA-PKcs,?H2AX and CC3 protein of tumor tissue expression were detected by immunohistochemistry.Results:IC50 of AZD9291 on human lung cancer cell lines(H460,PC-9,PC-9-IR and H1975)is 1.27 ?m,8.36nM,7.04nM and 10.62nM,respectively.IC50 of LC on human lung cancer cell lines(H460,PC-9,PC-9-IR and H1975)is 4.190?g/ml,1.658?g/ml,1.017?g/ml and 5.577?g/ml,respectively.0.5096?g/ml LC compared with AZD9291(1.76nM)have the best combined inhibitory effect on PC-9-IR and the combine index(CI)is 0.16.CI ranged from 0.16 to 0.65 with different concentrations combination of LC and AZD9291 and located below CI=1.0.SER was 0.87(P>0.05)and 1.03(P>0.05)with AZD9291(1.271 ?M and 6.355 ?M)on H460 cells and SER was 1.07(P>0.05)and 1.12(P>0.05)with AZD9291(8.38nM and 41.81nM)on cell line PC-9,respectively.SER was 1.16(P<0.005)and 1.29(P<0.0001)with AZD9291(7.04nM and 35.19nM)on PC-9-IR cells and SER was 1.03(P>0.05)and 1.18(P<0.005)with AZD9291(10.62nM and 53.10nM)on cell line H1975,respectively.The phosphorylation levels of EGFR,AKT and ERK decreased significantly when PC-9-IR cells pretreated with AZD9291 followed by IR.However,The phosphorylation levels of EGFR and AKT decreased significantly when H1975 cells pretreated with AZD9291 followed by IR but the phosphorylation level of ERK did not change significantly.Cell apoptosis of PC-9-IR was 50.4 ± 5.8%treated with AZD9291 combined with IR,which was higher than the control group(12.4 ± 1.2%,P<0.01),and the group treated with AZD9291(34.6±3.9%,P<0.05)or IR(25.2 ± 3.9%,P<0.05)alone.yH2AX-positive cells of PC-9-IR reach the highest(85.3 ± 4.8%)after treated with AZD9291 followed by IR(6Gy)8 hours later,and then gradually decreased.SERicso of AZD9291 was 1.09(P>0.05)when PC-9-IR cells DNA-PKcs gene knocked down by siRNA-DNA-PKcs.SER was 0.98 and 1.02 respectively when PC-9-IR cells treated with AZD9291(1.76nM)or LC(0.51 ?g/ml)alone,which was far blow than treated with AZD9291(1.76nM)and LC(0.51 ?g/ml)(P<0.05).The relative volume reached 1483.7 ± 96.8,1117.5 ± 68.0 and 645.7 ± 77.9 in IR group,AZD9291 group and combined group,respectively,which was less than 2063.5 ± 149.3 in control group.Treated with AZD9291 can significantly reduce the phosphorylation level of EGFR of PC-9-IR,despite IR has little effect on EGFR phosphoiylation level of PC-9-IR.Compared with the control group,AZD9291 treatment can reduce the expression of Ki67and increase the expression of CC3.Expression of ?H2AX protein had no significant difference in control group and AZD9291 group,but expression of yH2AX protein increased significantly in IR group and decreased in combined group compared with control group.Similarly,compared with radiotherapy alone,AZD9291 can significantly reduce the expression of DNA-PKcs protein.Conclusion:AZD9291 and LC have proliferation inhibition effects on human lung cancer cells(H460,PC-9,PC-9-IR and H1975)and AZD9291 combined with LC had a better value-added inhibition synergistic effect on PC-9-IR cells in vitro.Through inhibiting proliferation,promoting apoptosis of PC-9-IR cells and slowing repair process of cellular DNA damage caused by radiotherapy,AZD9291 can play a radiosensitization role on PC-9-IR cell.