The Effect Of Apatinib On The Radiosensitivity Of Esophageal Squamous Cell Carcinoma ECA-109 Cells And Their Stem Cells And Its Mechanism

Objective To evaluate the radiosensitivity effects of apatinib on the of esophageal cancer cell line ECA-109 and its cancer stem-like cell,and preliminary investigate the underlying mechanism.Method A serum-free medium（SFM）suspension was used to culture esophageal cancer stem cell lines and enrich the esophageal stem-like spheres.The ECA-109 and its stem-like cells were divided into control group,simple drug group,simple radiation group and drug plus irradiation group.cck-8 method was used to determine the proliferation of ECA-109 and its stem-like cells.The concentration of VEGF in the cell culture medium was determined by enzyme-linked immunosorbent assay.Flow cytometry method was adopted to detect the changes of cell cycle and apoptosis.Western-bolt method was used to test the expression of CHK2,P-STAT3protein in cells.Result After apatinib acts on ECA-109 parental cells and stem-like cells for 24,48,72 h,the half of the inhibitory concentration(IC₅₀)in the ECA-109 stem-like cell(IC₅₀)was significantly higher than that in the parent cell(IC₅₀)（t=8.17、9.29、18.85,P<0.05）,difference was statistically significant,and the Inhibition effect was time-dose dependent（parental cell:r²=0.94⁰.97,P<0.05;stem-like cells:r²=0.94⁰.98,P<0.05）.when different concentrations of apatinib（parental cell:10and20μmol/L;stem-like cells,30and40μmol/L）combined with different dose of X ray（6and 8 Gy）,the ECA-109 parental cells and stem-like cell proliferation inhibition rate were significantly enhanced compared with simple radiation group,the difference is statistically significant（t=39.68、25.62、20.01、25.20、20.58、22.84、13.55、5.20、23.24、18.44、11.49,P<0.05）.VEGF secretion of unit cell of ECA-109parental cells and stemlike cells were significant different（t=7.454,P<0.05）.Compared with control group（0μmol/L）,the expression of VEGF of simple drug group and drug plus irradiation group for each kind of cell was decreased,and the differences of parental cells were statistically significant（parental cells:t=14.51,26.40,P<0.05）.Compared with control group,the cell apoptosis rate and the percentages of cells in G2/M phase were significantly increased in drug plus irradiation group,the difference was statistically significant（t=8.83,11.59,P<0.05）.Compared with control group,the expression of CHK2 and P-STAT3 was decreased in simple drug group,the difference was statistically significant（t=3.355、4.104,P<0.05）;Compared with simple radiation group,the expression of CHK2 and P-STAT3 was decreased in drug plus irradiation group,the difference was statistically significant（t=9.053、2.357,P<0.05）.Conclusion Apatinib can enhance the radiosensitivity of the eca-109 parental cells and its stem-like cells,and it has a more significant effect on parental cells.The mechanism may be related to the inhibition of VEGF signal of esophageal carcinoma cells can further inhibit cell proliferation,promote cell apoptosis and induce cell cycle redistribution.The reasons for the radioresistance of ECA-109 stem-like cells may be related to the higher expression of VEGF protein in stem-like cells.