Experimental Research On The Effect Of C-Met On Radiosensitivity Of Eca109 Esophageal Squamous Cell Carcinoma Cell

Background:Esophageal cancer is the sixth most common cause of cancer deaths worldwide and is more common in the developing nations.Esophageal cancers are histologically classified as squamous cell carcinoma(SCC)or adenocarcinoma.SCC is the most common histology in Eastern Europe and Asia,and 95%of esophageal cancer is pathologically diagnosed as SCC in China.Radiotherapy is major components of therapy for patients with esophageal squamous cell carcinoma(ESCC),however,due to the tolerance of normal tissues,the radiation dose for optimal therapy is limited.Numerous studies have investigated the impact of radiotherapy dose modifications.It is a great hotspot for investigating new strategies to increase radiosensitivity in esophageal cancer.In recent years,the HGF/c-MET signaling pathway becomes the hot research area in tumor which plays an important role in the occurrence and development of cancer,and involved in tumor invasion and metastasis.MET activates HGF/c-MET signaling pathway which is associated with DNA damage repair and apoptosis,thus affecting the sensitivity of radiotherapy.The hepatocyte growth factor receptor(c-Met)signaling pathway is getting wide attention for the relationship with tumor radiosensitivity at present.The activation of c-Met signaling pathway is not only closely related to the development and metastasis of many kinds of tumors,but also participates in the mechanism of DNA repair and then imparts cellular resistance to radiation.In this study,the expression of c-Met in ESCC was detected by immunohistochemistry(IHC),then the relationship between the expression of c-Met protein and the clinicopathological features and prognosis of ESCC were discussed,as well as the effect on the efficacy of radiation therapy;Study the changes of the biological characteristics in ESCC cell line with the use of c-Met inhibitor and the effect combined irradiation in vitro to investigate the radiosensitization effect of c-MET inhibitor on ESCC cell line and its possible mechanism;And through animal experiments,to further verify the role of c-Met inhibitor in the radiosensitization which provides new ideas in the precise multidisciplinary treatment of ESCC.Methods:Expression of c-Met was detected by IHC in 180 paraffin-embedded tissue samples from postoperative patients of ESCC.The correlation between c-Met and the patient characteristics and prognosis of disease was analyzed,as well as the survival status of different c-Met expression in radiotherapy and without radiotherapy.In vitro experiments,the cells were divided into four groups:control group.BPI-9016M group,irradiation group(IR)and irradiation group plus BPI-9016M(IR+BPI-9016M).The radiosensitization of the c-Met inhibitor BPI-9016M was detected by colony formation assay.The cell survival curve was obtained and the Do value(mean lethal dose),Dq value(threshold dose)and radiosensitization ratio(SER)were calculated using the Origin 7.5 software according to the formula SF = 1-(1-e-D/DO)N based on the multi-target single-hit model.Flow cytometry was used to detect cell cycle and apoptosis.Western blot was used to detect the expression of c-Met,P53,BCl-2,cleaved-Caspase 3 and cleaved-Caspase 9 in Eca109 cell.The phosphorylation levels of DNA damage repair proteins include ATM,ATR,Chk1,Chk2 and H2AX were furtherly detected with Western blot.In vivo experiments,Eca109 cell was subcutaneously inoculated in nude mice,and after the tumorigenesis of cell,nude mice were treated with control group,BPI-9016M group,irradiation group(IR)and irradiation plus BPI-9016M group(IR+ BPI-9016M),respectively.Body weight and tumor volume were measured once per three days,and tumor volume was determined according to the formula:(length[L]×width[W]2)/2.The efficacy of each treatment was evaluated by the volume change during the treatment period.Growth delay time(GD)was calculated as the time for treated tumors to reach double in volume minus the time for control tumors to reach double in volume.The enhancement factor(EF)was then determined as follows:EF =(GDIR+BPI-9016M-GDBPI-9016M)/GDIR.The overall survival(OS)rates were calculated according to the Kaplan-Meier method,and the log-rank test was used to evaluate differences between survival curves.T-test was adopted in measurement data.All p-values reported are two tailed,and a p-value of<0.05 was considered statistically significant.All analysis was performed using SPSS 21.0 software(IBM,Armonk,NY,USA).Results:The expression of c-Met in ESCC patients was detected by IHC.There was a significant difference in OS between patients with c-Met high expression and low expression(median OS:41.9 months vs.56.7 months:p=0.001).In subgroup of patients with radiotherapy,high expression of c-Met was correlated with poor disease prognosis(p=0.002).The results showed that c-Met inhibitor BPI-9016M significantly inhibited the proliferation and growth of Eca109 cell in vitro,with a dose-response relationship between them.Flow cytometry assay showed BPI-9016M had little effect on cell cycle of the Eca109 cell.Apoptosis of Eca109 cell was induced after BPI-9016M,irradiation or irradiation plus BPI-9016M compared with the control group,the difference was statistically significant(p<0.05).The growth of Eca109 cell was inhibited by irradiation with a dose-response relationship and c-Met inhibitor enhanced the radiosensitivity of Ecal09 cell that the SER was 1.40 calculated by the multi-target single-hit model.Western Blot showed that Eca109 cell treated with irradiation in combination with BPI-9016M could decrease the expression of Bcl-2,P53 and increase the expression of cleaved-Caspase 9,cleaved-Caspase 3.The phosphorylation level of ATM,ATR,Chkl,Chk2 and H2AX was obviously up-regulated in the irradiation group.While in the irradiation plus BPI-9016M group,the phosphorylation level of ATM,ATR,Chk1,Chk2 and H2AX was inhibited.In vivo,Eca109 cell was subcutaneously inoculated in nude mice,and after the tumorigenesis of the cell,nude mice were divided into four groups:control group,BPI-9016M group,irradiation group and irradiation plus BPI-9016M group randomly.Irradiation combined with BPI-9016M produced significant tumor volume regression,compared with BPI-9016M or irradiation alone(p<0.05).The combined treatment significantly prolonged the time required for tumor volume doubling relative to radiation alone,the enhancement factor(EF)was 1.51.Discussion:It has been reported that c-Met overexpression is associated with the malignant biological behavior of esophageal cancer.In studies of western countries,c-Met overexpression related to the prognosis of esophageal adenocarcinoma,however,the relationship in ESCC is still unclear.In our study,the high expression of c-Met in ESCC was significantly associated with poor prognosis.The result was consistent with other relevant domestic studies,suggesting that c-Met may be a potential target for ESCC..In subgroup analysis,the patient with high c-Met protein expression had poorer radiotherapy effecacy for c-Met may be involved in a variety of mechanisms of radiation resistance.In the followed experiments in vitro,we found that the growth and proliferation of Eca109 cell was inhibited by irradiation with a dose-response relationship and c-Met inhibitor BPI-9016M enhanced the radio sensitivity of Eca109 cell.The results of the western blot also confirmed that the c-Met inhibitor BPI-9016M was able to inhibit the expression of c-Met.In addition,the western blot showed that c-Met inhibitor BPI-9016M treated Eca109 cell could increase the apoptosis-related proteins and reduce the DNA damage repair proteins after irradiation.Compared with the control group.P53 in Eca109 cell was reduced slightly in BPI-9016M group and was equal in the radiation group,while in BPI-9016M plus irradiation group its level was significantly decreased.It was suggested that BPI-9016M combined with irradiation can downregulate the expression of P53 to regulate cell apoptosis.In irradiation plus BPI-9016M group,Bcl-2,which is similar in function to P53.was also significantly reduced.The activated cleaved-Caspase 9 and cleaved-caspase 3 was significantly increased in irradiation plus BPI-9016M group.The activity of Caspase 9 and Caspase 3 could promote cell apoptosis in Eca109 cell.Therefore,it is suggested that the mechanism of c-MET inhibitor to increase the radiosensitivity of ESCC cell line Ecal09 cell may be mediated by inhibition of Bcl-2.P53 and activation of Caspase 9,Caspase 3 and other proteins that regulate cell apoptosis.In the other hand,radiotherapy can lead to cell death by inducing DNA fragmentation,and the main reason for radiotherapy resistance is due to endogenous or acquired radiation resistance of tumor cells mainly in the enhancement of DNA damage repair ability.When DNA damage occurs,tumor cell will activate the corresponding repair pathway which is mainly composed of sensory factors,conduction pathways and effect factors.After DNA damage,the RAD9-HUS1-RAD1 complex is raised to the DNA damage site by a protein complex containing RAD17,to promote the phosphorylation and activation of ATR-mediated effector protein kinase Chk1,thereby modulating the progress of the S phase and G2/M phase arrest in cell cycle.The other sensory factor MRE11-RAD50-NBS1(MRN)complex which recruited ATM around the DNA double-strand breaks by detecting double-strand breaks promotes ATM-mediated histone H2AX phosphorylation,followed by a series of repair-related factors and proteins recruitment to the DNA damage area,the formation of damage points,involved in DNA damage repair process.The results in our study showed that the phosphorylation levels of DNA damage repair proteins ATM,ATR,Chkl,Chk2 and H2AX were significantly increased after irradiation in Eca109 cell.However,they were significantly inhibited if the cell was previously treated with c-Met inhibitor BPI-90161M.It is suggested that the high expression of c-Met may be an important reason for the radiotherapy resistance of ESCC,and the target inhibition of c-Met can effectively increase the sensitivity of tumor to irradiation.The radiosensitization effect is achieved by inhibiting the DNA damage repair ability partially.More importantly,we also obtained similar results in animal experiments,indicating that c-Met may an important target for clinical radiation sensitization in ESCC.and targeted inhibition of c-Met combined with radiotherapy has important research significance in treatment of esophageal cancer.Conclusion:The expression of c-Met in patients with ESCC was related to the prognosis.The results of our study indicated that the radiation sensitization of c-Met inhibitor BPI-9016M in ESCC is achieved by inhibiting the DNA damage repair partially and furthurly inducing cell apoptosis.To sum up,c-Met may an important target for clinical radiation sensitization in ESCC,and targeted inhibition of c-Met combined with radiotherapy has great research significance in treatment of esophageal cancer.