Effect Of Coixenolide On Biological Behavior And Radiosensitization Of Homologous Nasopharyngeal Carcinoma Cells With Different Radioresistance

PART 1 EFFECT OF COIXENOLIDE ON BIOLOGICAL BEHAVIO OF HOMOLOGOUS NASOPHARYNGEAL CARCINOMA CELLS WITH DIFFERENT RADIORESISTANCEObjective:To investigate the effects of coixenolide(CXL)on proliferation?apoptosis and migration in homologous nasopharyngeal arcinoma cells with different radioresistance.All the above to provide a theoretical and experimental basis for the further study of the mechanism by which CXL targets asopharyngeal carcinoma cell.Methods:CCK-8 assay was applied to detect viability of CNE-2R and CNE2 cells after treated with different concentrations of CXL(0?0.5?1?5?10?20 g/L)for 24 h,48 h and 72 h respectively;DAPI staining was applied to detect morphological changesof CNE-2R and CNE2 cells after treated with different concentrations of CXL(0?1?5?10g/L)for 72h.Flow cytometry with Annexin V/PI stainingwas applied to detect the level of apoptosis of CNE-2R and CNE2 cells after treated with different concentrations of CXL(0?1?5?10g/L)for 72h.Transwell assay was applied to detect the migration ability of CNE-2R and CNE2 cells after treated with different concentrations of CXL(0?1.5?10g/L).Results:CCK-8 assay showed that CXL produced an obvious dose-time dependent inhibitory effect on the viability of CNE-2R cells with the IC50 of 273.20 g/L?10.88 g/L?4.55 g/L and CNE-2 cells with the IC50 of 256.60 g/L?5.44 g/L?2.43 g/L for 24h?48h?72h respectively.DAPI staining showed that chromatin condensation,margination and splitting into a massive structure were observed in CXL cells array.Flow cytometry with Annexin V/PI staining showed that the apoptotic rate of CNE-2R cells was apparently increased(p<0.05)from 4.51±0.03%to 11.68±3.54%compared to CNE-2 cells which was significantly increased(p<0.05)from 5.27±0.04%to 15.68±2.76%.Transwell assay showed that CXL produced an obvious dose dependent inhibitory effect on the migration ability of CNE-2R and CNE-2 cells;Conclusion:The results indicated that CXL could inhibit the proliferation?migration ability and induce the apoptosis of the homologous nasopharyngeal arcinoma cells CNE-2R and CNE-2 with different radioresistance.All the effects above obeyed the changes of a dosage dependent manner.PART TWO:THE EFFECT OF CXL ON THE EXPRESSION OF PROTEINS RELATED TO MITOCHONDRIAL PATHWAY IN HOMOLOGOUS NASOPHARYNGEAL CARCINOMA CELLS WITH DIFFERENT RADIORESISTANCEObjective:To investigate the relationships between the mechanism by which CXL induce the apoptosis of the homologous nasopharyngeal arcinoma cells with different radioresistance and Proteins expressions of apoptosis relat to mitochondria pathway.In order to provide theoretical and experimental basis for the antitumor effect of CXL on nasopharyngeal carcinomaMethods:To detect the protein expression level of several genes related to apoptosis included Bax?Bcl-2?Caspase-9?cleaved-caspase-3 and cleaved-PARP and the ratio of bcl-2 to bax by Western blot after treating CNE-2R and CNE-2 cells with CXL for 0,24 h,48 h,72 h respectively.Results:Western blot showed that after treated with CXL,the expression of Bax?Caspase-9?cleaved?caspase-3?cleaved-PARP significantly increased(p<0.05)compared to the control groups.The expression of Bcl-2 significantly decreased(p<0.05).The ratio of bcl-2 to bax was decreased(p<0.001).Conclusion:The apoptosis of the homologous nasopharyngeal arcinoma cells with different radioresistance can be mediated by the mitochondria pathway through CXL.PART THREE:EFFECT OF CXL ON RADIOSENSITIZATION OF HOMOLOGOUS NASOPHARYNGEAL CARCINOMA CELLS WITH DIFFERENT RADIORESISTANCEObjective:To investigate the effects of CXL on radiosensitization in homologous nasopharyngeal arcinoma cells with different radioresistance.In order to provide theoretical and experimental basis for the reversal of radiation resistance in nasopharyngeal carcinoma and searching for potential radiosensitizers.Methods:After treated CNE-2R and CNE-2 cells with 0.1 g/L CXL for 24 h and accepted the X ray with the doses of 0?2?4?8Gy respectively.To calculate SF by colony formation assay.To establish the single-hit multi-target model and drew the cell survival curves through Graphpad Prim.To calculated the characteristic parameters N?Do and Dq and further work out the ratio of radiosensitization.SER after treated the CNE-2R and CNE-2 cells withCXL in the end.To analyze the effects of CXL on the radiosensitization of the homologous nasopharyngeal arcinoma cells with different radioresistance.Results:Colony formation assay showed that after treated CNE-2R and CNE-2 cells with 0.1 g/L CXL for 24 h and accepted the X ray with the doses of 0?2?4?8Gy respectively,SF in experimental group was lower than SF in blank group both in CNE-2R and CNE-2 cells.The single-hit multi-target model and the cell survival curves showed that after accepting the X ray with the doses of 0?2?4?8Gy,the cell survival curve of CXL group was lower than that in blank group.The SERs of CNE-2R and CNE-2 cells were worked out as 1.55 and 1.37 respectively through the calculated characteristic parameters N?Do and Dq,which suggested that CXL had the effects on radiosensitization in CNE-2R and CNE-2 cells.Conclusion:The CXL has the effects on radiosensitization in homologous nasopharyngeal arcinoma cells with different radioresistance.