The Study Of The Mechanism Of Docetaxel's Radiosensitization Effects On Human Esophageal Caner Cell Line EC-9706

Objective1,To investigate the in vitro anti-human esophageal cancer cell line activity of docetaxel against EC-9706 and explore initially the effects and mechanism on cell cycle distribution.2,To observe the growth inhibition of human esophageal cancer cell line EC-9706 in vitro after treated by docetaxel and X-ray and determine the potential radiosensitizing effects and its possible mechanism of docetaxel.Provide new ideas and methods for the treatment of esophageal cancer.3,To observe the changes in expression of Bcl-2 after treated by different concentrations of docetaxel through RT-PCR and to explore further the mechanism in docetaxel induced growth inhibition,apoptosis and radiosensitization at gene level.Methods1,Seeded cells in culture plates and then treated them with different concentration of docetaxel after 24 hours. The cells were cultured at the incubator for 9 days. Manually counted the colonies after stained with Giemsa to determine the growth inhibition effects of docetaxel.2,During exponential growth phase, cells were treated with different doses of docetaxel(0,0.25×10-9,0.5×10-9,10-9,10-8,10-7,10-6 mol/L)for 24,48 and 72 hours and harvested with 0.25%trypsin and resuspended in media.Treated them according to the certain operation steps. The DNA content of each cell was measured using flow cytometer and analyzed with software to observe the cell cycle distribution of EC-9706 after treated by docetaxel.3,Treated the exponential growth phase cells with 10-6 mol/L docetaxel for 24 hours, and then the cells were irradiated at doses ranging from 0 to 2 Gy and harvested at 24 hours and 48 hours after irradiation.Treated them with certain operation steps.Measured the DNA content of each cell with flow cytometer and analyzed with software to observe the cell cycle distribution of EC-9706 again after the interaction of docetaxel and X-ray. 4,The cells were irradiated at doses raging from 0 to 6 Gy.The total cellular RNA were extracted at 1,6 and 24 hours after irradiation.The Bcl-2 gene expression changes were detected by RT-PCR. Result1,The colony forming ability decreased gradually with increasing dose of docetaxel treatment,when the docetaxel concentration was increased to 10-8mol/L,the colony forming inhibition rate attained 100% with IC50 5×10-10M(0.5 nM).2,Flow cytometric analysis demonstrated docetaxel induced cells G2/M phase arrest.3,G2/M phase arrest can be lifted by radiation at the dose of 2 Gy.There was time-dependent with the lifting which appeared at group 24h and became significant after 48 hours.4,The expression of Bcl-2 gene is dropped suddenly after irradiation without obvious dose-dependent and time-dependent.Conclusion1,Docetaxel shows radiosensitization in human esophageal cancer cell line EC-9706 in vitro.2,Irradation dose at 4Gy and 6Gy can cause the expression of human esophageal cancer cell line EC-9706 decreased significantly.