The Impact Of KPT-330 On Radiosensitivity Of Esophageal Squamous Cancer Cells And Its Mechanism

Objective:Radiochemotherapy is the main treatment of advanced esophagus carcinoma?EC?,however,due to radiation resistance,the survival rate is still low.Nuclear export protein?exportin 1,also called chromosome region maintenance 1,CRM1?is overexpressed in EC associated with poor prognosis.It has been confirmed that CRM1inhibited increased radiation sensitivity in rectal cancer cell lines and non-small cell lung cancer?NSCLC?cell lines,while it is unclear currently whether inhibition CRM1increases radiation response to radiotherapy in EC.KPT-330 is a selective CRM1inhibitor which has been in the I/II clinical trials in non-Hodgkin lymphoma and acute myeloid leukemia and many hematological malignant tumors,and the trials confirmed that KPT-330 improved cancer therapy safely.To observe KPT-330 whether improved radiosensitivity of EC and to study its mechanism,we combined KPT-330 and radiation therapy firstly in esophageal squamous cell carcinoma?ESCC?.Methods:ECA109 esophageal cancer cells were treated with different concentrations of KPT-330?0,0.01,0.05,0.1,0.5,1,10,50umol/L?for 72 hours for cell viability with MTT assays and then its IC₅₀0 was calculated.ECA109 cells survival fraction was analyzed with colony forming assay after cells with or without 0.1umol/L KPT-330pretreatment for 12h and then accepted different doses of radiation?0,2,4,6,8Gy?.In order to study its mechanism,ECA109 cells were divided into control group,KPT-330group,irradiation group and KPT-330 combination with irradiation group:ECA109 cells were treated with KPT-330?0,0.1,0.3umol/L?for 12 hours and then accepted 0 or 4Gy for 24h,the protein levels of CRM1,p53 and nuclear-cytoplasmic p53 of the above groups were detected with Western Blot;Cells apoptosis rate of the above groups was detected by Flow cytometry after ECA109 cells were treated with KPT-330?0,0.1,0.3umol/L?for 12h and then accepted 0 or 4Gy for 48h;and cell cycle distribution of the above groups was detected by Flow cytometry after cells were treated with KPT-330?0,0.1,0.3umol/L?for 12h and then accepted 0 or 4Gy for 24h.Results:The IC₅₀0 of ECA109 cells treated with gradient concentrations of KPT-330 for72h was 0.9umol/L,and cell viability of ECA109 cells was gradually decreased with increasing concentrations of KPT-330.Colony forming assay shows that KPT-330significantly enhanced radiation therapy effects and decreased cell proliferation of ECA109 cells.The mean lethal dose?D₀?of single radiation group and KPT-330?0.1umol/L?combination with irradiation group in ECA109 cells was respectively 3.36and 1.65;the survival fraction of 2Gy?SF₂?was respectively 56.71%and 44.89%;the sensitization enhancement ratio?SER?of ECA109 cells was 2.04.Apoptosis assay reveals that 0.1umol/L KPT-330 group didn't impact ECA109 cells apoptosis rate significantly compared with control group?P>0.05?,however,KPT-330 combination with radiation group compared with irradiation group significantly increased cell apoptosis rate in ECA109 cells,especially in 0.3umo/L KPT-330 combination with radiation group?P<0.05?.Cell cycle assay shows that KPT-330 alone group increased ECA109 cells G2/M phase arrested than control group,and the difference is statistically significant?P<0.05,P<0.05?;irradiation combination with KPT-330 group increased G2/M phase arrested in ECA109 cells than irradiation group and the difference is statistically significant?P<0.01,P<0.01?.Western Blot results indicate that CRM1 levels decreased significantly in KPT-330 group compared with control group,and the difference is statistically significant?P<0.05,P<0.05?;Concurrently,KPT-330 group increased p53 protein expression levels,and the difference is statistically significant?P<0.05,P<0.01?.Compared with radiation alone group,KPT-330 combination with irradiation group increased p53 protein expression levels,the difference is statistically significant?P<0.05,P<0.05?.Nuclear-cytoplasmic protein assay shows that KPT-330group increased nuclear p53 protein levels than control group?P<0.05,P<0.01?;and nuclear p53 protein levels increased in KPT-330 combination with radiation group compared with radiation alone group,the difference is statistically significant?P<0.05,P<0.01?.Conclusion:CRM1 inhibition by KPT-330 increased radiosensitivity of ESCC.And the mechanism may be KPT-330 increased nuclear p53 protein levels;incurred G2/M phase arrested and increased apoptosis induced by radiation therapy,which may creat a novel clinical therapy strategy for esophageal cancer.