Role Of EMT In Radiosensitivity Of NSCLC With Acquired Resistance Of EGFR-TKI

Background Epidermal growth factor receptor-tyrosine kinase inhibitors(EGFR-TKI)is one of the most important achievement for non-small cell lung cancer's treatment in recent years.EGFR-TKI has been the first line treatment to those non-small cell lung cancer(NSCLC)patients with EGFR-mutant type.However,the therapeutic effect is greatly interfered by the acquired resistance of EGFR-TKI.Radiation therapy is one of the most important treatment strategy for EGFR-TKI resistant patients as local therapy.Clinic study indicated that EGFR-TKI resistant patients treat with RT would have a long PFS and OS.However,radiation resistance is a significant barrier affecting the therapeutic effect of this procedure.What is the radiosensitivity of NSCLC with the acquired resistance of EGFR-TKI? And it is still an open question that the predict markers are absent to prognosis of radiation therapy for those NSCLC with the acquired resistance of EGFR-TKI.Epithelial-mesenchymal transformation(EMT)played an important role in the process of the invasion and metastasis of tumor.Previous studies showed that the resistant mechanisms including EMT were closely related to the acquired resistance of EGFR-TKI.In recent years,increasing evidence suggests that EMT is closely linked with NSCLC radiosensitivity.The study of the relation between EMT and NSCLC radiosensitivity is expected to contribute to the prevention of radioresistance and thus improve efficacy of radiation therapy to provide benefit to NSCLC patients.To explore the influence of epithelial-mesenchymal transition(EMT)in radiosensitivity of non-small cell lung cancer NSCLC cell with acquired resistance to EGFR-TKI.We presumed,accordingly,that EMT would impact the radiosensitivity of NSCLC with the acquired resistance of EGFR-TKI,that could be used as one predictive indicator for RT.Materials and methods In this study,Human lung adenocarcinoma cell line PC-9 and Gefitinib acquired resistance cell line PC-9/AB were used.TGF-?1 was used to induce cell line PC-9/TGF.And generated cell line PC-9/AB-CDH1 by stably transfected with CDH1.EMT-related proteins were assessed by western blotting assay.Cell migration was tested with wound healing assay.The sensitivity to irradiation was tested with both CCK8 and colony formation assay.Flow cytometry for apoptosis analysis was used for survival fraction within the cell lines.Cell lines The human EGFR-mutant lung adenocarcinoma cell lines PC-9 and PC-9/AB were from Sun-Yat-Sen University Cancer Center(SYSUCC,Guangzhou,China).PC-9/AB-CDH1,a cell line with stable E-cadherin over-expression,was established by transforming gene-CDH1 with lentivirus in PC-9/AB.PC-9/TGF cell line was from PC-9 treated with TGF-?1 for about 14 days.Cell lines PC-9 and PC-9/AB-CDH1 were maintained in RPMI-1640 medium(GIBCO),containing 10% fetal bovine serum.All cells were cultured in a humidified atmosphere at 37 °C containing 5% CO2.Medium replacement was done every third day with fresh medium.Western blotting Cold RIPA lysis buffer was used to lyse cells,which containing protease inhibitors and phosphatase inhibitors.Lysates(100?l per lane)were homogenized in the loading buffer.Protein was boiled about 5 min,and then separated by SDS-PAGE.Using a PVDF membrane(Millipore,U.S.A)to transfer protein from gel.The membrane,being blocked with 5% fat-free dry milk,was treated with antibodies: E-cadherin(Cell Signaling),Vimentin(Cell Signaling),?-actin(Cell Signaling),Snail(Cell Signaling),and kept overnight at a tempreture of 4 °C.After being washed twice with TBST,the membranes were incubated with rabbit secondary antibodies for 1h,and finally visualized with ECL plus detection reagents(Beyotime,China).?-actin was used as a control in the experiments.Irradiation Cells were seeded and incubated for 24 h at 37°C in 5%CO2.The following day cells were irradiated with doses ranging as indicted,using X-ray Irradiation System(CLINAC 2300C/D,Varian,U.S.A),6MV,angle=180,field=10cm×10cm,distance=100cm,dose rate=200c Gy/min,at room temperature,Cell density in different irradiate doses were done as described.Cell proliferation assay We used CCK8 assay to investigate the sensitivity of Gefitinib and evaluate relative viable cells.Being seeded into 96-well plates at a density of 2000,cells were cultured for 24 h before being treated with Gefitinib or exposed to different doses of irradiation.24 h after treatment,10?l CCK8 solution(Dojindo,Kumamoto,Japan)and 100?l RPMI-1640 were added to each well and incubated for 3h at 37 °C.The absorbance at ?=450 nm was measured.Wound healing assay Cells were seeded on six-well plates at a high density,and were cultured for about 3 days to produce a monolayer,respectively.Confluent cell monolayers were then scratched using a plastic 200 ?l-pipette tip,as wound areas of cells.Wounded monolayers were then washed three times with RPMI-1640 medium(Gibco)to remove cell debris and incubated in culture medium.Images were captured at the indicated times(0,24,48 h),and the wound healing rate was calculated.Healing rate =(width of wound at 0 h – width of wound at x h)/width of wound at 0 h.Vitro Clonogenic assays Exponentially growing cells were harvested by exposure to trypsin and were plated in 60 mm plates.Cell density in different irradiate doses(0,2,4,6,8 and 10Gy)were 200,500,1000,1000,5000,and 5000 cells per plate.The following day cells were irradiated with doses ranging from 0 to 10 Gy,using X-ray Irradiator(CLINAC 2300C/D,Varian,U.S.A)and incubated at 37°C in 5%CO2.The culture media with 10%FBS was changed every 3 days.10 to 14 days later,colonies were washed with PBS,fixed.Cells were stained with crystal violet.Colonies?50 cells were counted as surviving colonies.The plating efficiency(PE)and surviving fraction(SF)were calculated.Flow cytometry assay The cells treated with 4 Gy irradiation dose were cultured for 24 h,and harvested,washed with PBS and Cell resuspended in binding buffer for 15 min,followed by the addition of Annexin V-FITC(Dojindo,Kumamoto,Japan).Apoptosis analysis was carried out using a flow cytometer(BD FACSCalibur,Oxford,UK).Statistical analysis Data were analysed using SPSS 20 software.Experiments were performed at least three times.All values were expressed in terms of the mean±SD.For statistical analysis,the independent Student's t test was used to compare the parametric variables between groups.For the comparison of multiple groups,a one-way ANOVA test was applied.All tests were twosided and the level of significance was set at a probability of 0.05.Results 1.We detected EGFR gene statement in PC-9 and PC-9/AB,both cell line were EGFR gene exon 19 mutant-type,without T790 m mutant,without c-Met gene amplification.The sensitivities to Gifitinib of different cell lines were as follow: the sensitivity of PC-9/AB cells to the treatment of significantly lower than PC-9 cell line,IC50:9.68?mol/L VS 0.06?mol/L(P < 0.01);2.Compared with PC-9,as EMT phenotype presented,PC-9/AB displayed radiation resistance.With the increase of irradiation dose,relative survival cells of PC-9 and PC-9/AB are gradually reduced.The relative survival rate of PC-9/AB was higher than that of PC-9(P<0.05);clone forming rate of PC-9/AB cell was higher than that of PC-9(P<0.05);Under 4Gy dose irradiation,the apoptosis of PC-9/AB cells was lower than PC-9,apoptosis rate:(14.80 ± 1.06)% VS(30.52 ± 2.76)%(P<0.05);3.EMT phenotype was presented in PC-9/AB,compared with cell line PC-9,performanced an obvious change in both morphology and molecular biology: connection between cells became loose,appearanced spindle cells changed,the type of epithelial marker protein E-cadherin decreased,and mesenchymal marker protein Vimentin increased as transcription factors associated with EMT expression Snail did.The migration of PC-9/AB cells increased significantly compareing that of PC-9,Scratches healing rate: 24h(53.23±2.18)% VS(33.47±1.38)%,48h(83.88±3.35)% VS(49.84 ± 0.78)%;4.Inducing PC-9 by treating with TGF-?1,The PC-9/TGF cell line displayed EMT phenotype both in morphology and molecular biology,as presented in PC-9/AB;The migration of PC-9/TGF cells increased significantly compareing with PC-9,Scratches healing rate:24h(51.05±1.34)%VS(33.47±1.38)%,48h(80.50±3.23)% VS(49.84±0.78)%.PC-9/TGF was EGFR gene exon 19 mutant-type,without T790 m mutant,without c-Met gene amplification.The sensitivity of TGF-induced cell line PC-9/TGF cells significantly decreased,comparing with PC-9,IC50:6.21?mol/L VS 0.06?mol/L(P <0.01);5.As well as EMT phenotype presented,PC-9/TGF also displayed radiation resistance.With the increase of irradiation dose,relative survival cells of PC-9 and PC-9/TGF are gradually reduced.The relative survival rate of PC-9/TGF was higher than that of PC-9(P<0.05);clone forming rate of PC-9/TGF cell was higher than that of PC-9(P<0.05);Under 4Gy dose irradiation,the apoptosis of PC-9/TGF and PC-9/AB cells was lower than PC-9,apoptosis rate(15.24 ± 2.03)% VS(14.80 ± 1.06)% VS(30.52 ± 2.76)%(P<0.05).The difference between PC-9/TGF and PC-9/AB had no statistical significance(P>0.05);6.CDH1 gene was transfected into target cell line PC-9/AB,PC-9/AB-CDH1 cells reversed EMT,connection between cells become close,appearanced pebble shape and less pseudopods,decreased expression of E-Cadherin,increased expression of Vimentin and Snail.The migration of PC-9/AB-CDH1 cells decreased significantly compareing that of PC-9/AB,Scratches healing rate: 24h(37.16±4.15)%VS(53.23±2.18)%,48h(49.32 ± 3.12)%VS(83.88% ± 3.35)%.EMT reversed cell line PC-9/AB-CDH1 was EGFR gene exon 19 mutant-type,without T790 m mutant,without c-Met gene amplification.When PC-9/AB was tansfected with CDH1 gene,The sensitivity to Gifitinib of PC-9/AB-CDH1 cells was obviously higher than that of PC-9/AB cells,IC50:1.25?mol/L VS 9.68?mol/L(P < 0.01);7.When it came to EMT reversed cell line PC-9/AB-CDH1,the radiosensitivity was significantly higher than PC-9/AB.With the increase of irradiation dose,relative survival cells of PC-9/AB-CDH1 and PC-9/AB are gradually reduced.The relative survival rate of PC-9/AB-CDH1 was lower than that of PC-9/AB(P<0.05);clone forming rate of PC-9/AB-CDH1 was lower than that of PC-9/AB(P<0.05);Under 4Gy dose irradiation,the apoptosis of PC-9/AB-CDH1 cells was higher than PC-9/AB,apoptosis rate:(33.85±2.10)%VS(14.80±1.06)%(P<0.05).Conclusions In this study,we observed that: PC-9/AB cell line,NSCLC with acquired resistance of EGFR-TKI,appearing with EMT phenotype,displayed radioresistance.Compared with parent cell line PC-9,it showed lower radiosensitivity.Inducing PC-9 with TGF-?1,the radiosensitivity of PC-9/TGF with EMT was much lower than that of PC-9.As EMT was reversed,its radiosensitivity was recovered.we reveal in first time that: EMT plays an important role in the radiosensitivity of NSCLC cell line with acquired resistance of EGFR-TKI,and related with the resistance to irradiation in NSCLC cell line with acquired resistance of EGFR-TKI,might be one of effective predictors to the sensitivity of IR.