Effect Of Anlotinib Combined With Radiotherapy On The Proliferation、Apoptosis And Radiotherapy Sensitivity Of Esophageal Squamous Cell Carcinoma KYSE-150 Cells

BackgroundThe morbidity of esophageal cancer is higher than other countries.Esophageal squamous carcinoma,accounts for 88%of China’s EC.As one of the important treatment methods for esophageal cancer,radiotherapy is often combined with surgery,chemotherapy and other treatment methods in clinical treatment.Anlotinib is a new type of oral Tyrosine Kinase Inhibitor（TKI）,which plays an anti-tumor effect by inhibiting tumor vascular growth.The purpose of this research was to make clear the growth inhibition,apoptosis promotion and radiotherapy sensitization of ESCC cells in vitro.ObjectiveTo observe the effect of anlotinib unite with radiotherapy on the multiplication,apoptosis and radiotherapy sensitivity of ESCC.Methods1.The esophageal squamous cell carcinoma KYSE-150 cells in the logarithmic growth phase were cultured respectively with culture medium containing final concentrations of 0,2,4,8,16,32,64,128μmol·L^-1anlotinib.Use the Cell Counting Kit-8（CCK-8）method to test the multiplication ability of KYSE-150 cells in the eight groups of different consistences of anlotinib,and the cell multiplication inhibition rate was calculated.The cell multiplication inhibition curve was draw by Graphpad Prism8and obtain the half inhibitory concentration（IC50）.The consistence and treatment time of anlotinib at the minimum IC50 value were selected as the drug treatment conditions for the subsequent experiments.2.The esophageal squamous cell carcinoma KYSE-150 cells in the logarithmic growth phase were collected and randomly divided into control group,2,4,6 Gy single irradiation group,simple anlotinib group,2,4,6Gy combined irradiation group.The cells in the control group,2,4,6 Gy single irradiation group were cultured with anlotinib-free medium for 48 hours,and then replaced with anlotinib-free medium,and X-ray irradiation of 2,4,6 Gy was given respectively;the cells in the simple anlotinib group,2,4,6 Gy combined irradiation group were cultured for 48 hours with culture medium containing anlotinib with a final concentration of 4μmol·L-1,and then replaced with culture medium without anlotinib,and X-ray irradiation of 2,4,6 Gy was given respectively.The clonogenic ability of cells in the single irradiation group and the combined irradiation group with different X-ray irradiation doses were detected by the plate Clone Formation Experiment,and the survival score（SF）of every group was counted.The sensitivity of anlotinib combined with radiotherapy to KYSE-150 cell radiotherapy was detected by the single-click multi-target model.3.Take control group,4Gy single irradiation group,simple anlotinib group and4Gy combined irradiation group.After the intervention,use Flow Cytometry to detect,and use flowjo software to calculate the apoptosis rate.Results1.when anlotinib intervened for 24 hours,the inhibition rate of cell proliferation increased with the increase of anlotinib concentration in the 4,8,16,32,64 and 128μmol·L^-1anlotinib group（P<0.05）:when anlotinib interfered for 48 and 72 hours,the inhibition rate of cell proliferation increased with the increase of anlotinib concentration in the control group,2,4,8,16,32,64 and 128μmol·L^-1anlotinib group（P<0.05）;The IC50 values of anlotinib treatment of esophageal squamous cell carcinoma KYSE-150 cells for 24,48,72h were 12.7±0.5μmol·L^-1,3.7±1.0μmol·L^-1,6.8±0.7μmol·L^-1respectively.2.The SF of cells in the 2,4,6Gy combined irradiationgroup was significantly lower thanthat in the single irradiation group with the same dose（P<0.05）;The D₀values of the single irradiation group and the combined group were 5.84 and 4.11,respectively,and the sensitizer enhancement ratio was 1.42.3.The apoptosis rate of cells in the 4 Gy irradiation group,simple anlotinib group and 4 Gy combined group was significantly higher than that in the control group（P<0.05）;the apoptosis rate in the 4 Gy combined group was significantly higher than that in the 4 Gy irradiation group and the simple anlotinib group（P<0.05）.ConclusionAnlotinib combined with radiotherapy can restrain the multiplication of ESCC KYSE-150 cells and enhance the sensitiveness of ESCC to radiotherapy,and its machine-processed probable by promoting cell apoptosis.