| Background and ObjectiveNon-small cell lung cancer(NSCLC)accounts for 80 to 85 percent of lung cancer is one of the most mortality tumors[1,2].Radiotherapy is one of the main treatment of NSCLC,not only for the different stages of NSCLC[3,4],but also to be treatment options of local progression or central nervous system metastases after acquired resistance to EGFR-TKIs[5-7].Unfortunately,it is lack of radiotherapy curative effect predictor at present.Both clinical study and basic research have shown that NSCLC harboring EGFR mutations sensitive to radiation than EGFR wild-type[8-15].Our preliminary clinical retrospective analysis also showed that EGFR mutation type of NSCLC patients with brain metastases from radiation efficiency higher[16,17].Suggested EGFR gene mutation may be a potential predictor of high specificity radiation sensitivity.However,a recent study showed that EGFR mutation status is not correlation with NSCLC cells radiosensitivity[18].It is suggest that correlation with EGFR gene mutation and NSCLC radiosensitivity still need advance research.Local therapy includes radiotherapy as one of the main treatment strategies in local progress or central nervous system metastasis after acquired resistance to EGFR-TKIs.NSCLC patients with local advanced or central nervous system metastasis after initial TKIs treatment can get longer PFS and OS by continued EGFR-TKIs and local radiotherapy in clinical research[5,19,20].Previous studies found that EGFR TKIs-acquired drug resistance cell lines after radiation increased sensitivity to gefitinib[21-24],and displayed Mesenchymal Epithelial Transition(MET)feature after radiation[25].However,the mechanism of radiation reverse EGFR-TKIs acquired resistance still has not been ful y elucidated.Based on the previous research background and early clinical retrospective study,looking for new radiosensitivity predictors with high specificity and optimizing individualized treatment strategy of NSCLC patients have significance advantage for improve treatment o f NSCLC patients effectively and prolong survival patients contact of NSCLC.Methods1.EGFR gene mutation and the correlation with NSCLC radiosensitivity: we firstly prepared cell lines as below: EGFR wild-type squamous NSCLC cell H226,adenocarcinoma cell A549 and large cell lung cancer cell H460,EGFR exon 19 deletion mutant NSCLC cell lines PC–9 and HCC827,EGFR exon 21 L858 R mutation NSCLC cell lines H1975 and H3255,and EGFR-TKIs acquired resistance cell lines PC-9/AB and PC-9/ZD.We investigate radiosensitivity of cells by clonogenic survival assay and fitting to linear-quadratic model and “Multitarget-single hitting“ model to obtained radiation biology parameters of cell lines.2.The possible mechanisms of EGFR gene mutation sensitive to radiatio n: we draw the growth curve to test each cells proliferation after radiation,test cell cycle distribution and apoptosis of cells after radiation by flow cytometry assay,immunofluorescence assay is used to explore double stranded DNA breaks(DSBs)formation of cells after radiation and western-blot assay is used to detected apoptosis protein,repair protein and anti-apoptotic proteins expression of cells after radiation.3.Explore effect and possible mechanism of EGFR-TKIs acquired resistance NSCLC cell lines TKIs sensitivity after radiation: radiat EGFR-TKIs acquired resistant cell line PC-9/AB and PC-9/ZD 4 times with 6 Gy doses and estabolish radiation cell lines PC-9/AB IR and PC-9/ZD IR.Detect cells sensitivity to gefitinib with CCK8,NGS assay test gene mutation and amplification after radiation and western-blot assay test E – cadherin and vimentin protein expression of cells.4.Statistical analysis: Statistical analysis was done with IBM SPSS 20.0.All data are presented as mean ± SEM except other stated.The t-test was used(P < 0.05 was considered significant)to compare two groups.Each experiment was repeated three times,depending upon the particular experiment.Results 1.EGFR gene mutation NSCLC cell lines are sensitive to radiation.EGFR mutation cells PC-9,HCC827,H1975,H3255 and EGFR-TKIs acquired resistance cells PC-9/AB,PC-9/ZD were sensitive to radiation compared with EGFR wild-type NSCLC cells A549 and H226.The radiosensitivity is no difference between EGFR mutation cells and EGFR-TKIs acquired resistance cells.EGFR wild-type cell H460 compared with EGFR wild-type NSCLC cells A549 and H226 sensitive to radiation,and displayed similar radiosensitivity to EGFR mutation cells and EGFR-TKIs acquired resistance cells.Radiation biology parameters showed that H226 and A549 cell are significantly higher than other cells with the D0 which represented 37% damage of radiation and Dq which represented quasithreshould damage dose.A549,H226 and HCC827 cell significantly lower than the other cells with the?/? which represented repair capacity,suggested that A549,H226 and HCC827 cell repair capacity after radiation stronger than other cel s.2.The preliminary mechanism of EGFR mutation NSCLC cell with high radiosensitivityThe growth curve showed that EGFR mutation cell lines group proliferation after radiation lower than H226 and A549 cell.Flow cytometry assay detect cell cycle distribution and apoptosis of cells show that EGFR mutation cells,EGFR-TKIs acquired resistance cells and EGFR wild-type cells H460 appeared to significant apoptosis after radiation 24 hours and EGFR wild-type cells A549 and H226 don't show apoptosis after radiation.EGFR wild-type cells A549 and H226 appeared to cell cycle G0/G1 and G2/M phase arrest after radiation and cell cycle fraction keep growing until 48 hours after radiation.EGFR mutation cells,EGFR-TKIs acquired resistance cells and EGFR wild-type cell H460 merely after appear cell cycle G2/M phase arrest without G0/G1 phase arrest after radiation and the cell cycle fraction of G2/M phase increase the peak after radiation 24 hours and decline on 48 hours.Immunofluorescence assay showed that EGFR wild-type cells A549 and H226 appear obvious?-H2 AX focus formation after radiation 30 minutes and disappeared on 6 hours after radiation.EGFR mutation cells,EGFR-TKIs acquired resistance cells and EGFR wild-type cell H460 showed significant ?-H2 AX focus formation after radiation 15 minutes and continued until 24 hours after radiation.Western-blot assay showed that EGFR mutation cells express pro-apoptosis proteins Bax,apoptosis protein expression Caspase-3 and without express anti-apoptotic proteins Bcl-2,repair protein DNA-PKcs after radiation.A549 and H226 expression Bcl-2,Caspase-3 without Bax after radiation and expression DNA-PKcs until at least 6 hours after radiation.H460 Bax expressed Bax,Bcl-2,Caspase-3 and DNA-PKcs after radiation.3.Effect of reverse gefitinib resistant of PC-9/AB and PC-9/ZD after radiation and preliminary mechanisms.PC-9/AB IR and PC-9/ZD IR cell form had change by characterized of circular and smaller.CCK8 method showed that PC-9/AB IR and PC-9/ZD IR returned sensitivity to gefitinib to PC-9/AB and PC-9/ZD.NGS assay found that without any gene mutation,amplification or fusion of gene,only PC-9/ZD disappeared AKT1 gene exon 11 F349 L mutation after radiation.Western-blot assay showed that PC-9/AB IR and PC-9/ZD IR upregulate expression of E-cadherin,downregulate expression vimenten with MET compared to PC-9/AB and PC-9/ZD.ConclusionEGFR mutations correlation with NSCLC radiosensitivity and possible mechanism include: repair capacity declined,inactivation or lack of cell cycle checkpoint,cell proliferation capacity declined and susceptible to damage caused by radiation.EGFR-TKIs acquired resistance cells did not displayed significant different radiosensitivity,but NSCLC cells harboring EMT expressed Bcl-2 after radiation.Radiation can reverse resistance to gefitinib of EGFR-TKIs acquired resistance cells,the mechanism may be MET after radiation. |
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