Effects Of Osthole On Apoptosis And Autophagy Of Human Tongue Cancer Tca8113 Cells

ObjectiveTo investigate the effects of Osthole（OST）on proliferation,apoptosis and autophagy of human tongue cancer Tca8113 cells and explore its possible mechanism of action.MethodsTca8113 cells were cultured in vitro,and add the cells to the concentration of(0,20,40,80,120,160,200μmol·L^-1)OST,Detect the inhibitory effect of OST on Tca8113 cells proliferation and determine the half inhibition rate（IC50）with the tetramethylazolium salt（MTT）experiment.Clone formation test to detect the effect of OST on the clone formation ability of Tca8113 cells;Detect the effect of OST on the apoptosis of human tongue cancer Tca8113 cells by Hoechst33342staining method and Annexin V-FITC/PI method;Western blot was used to detect the expression of OST on the apoptosis-related proteins Bcl-2,Bax,Cleaved Caspase-3 and autophagy-related proteins LC3,p62,m TOR,p-m TOR,AMPK and p-AMPK in human tongue cancer cells.Results1.OST can significantly inhibit the proliferation of Tca-8113 cells,and the inhibitory rate of the drug on the cells was concentration-dependent.The proliferation inhibition rate（IC50）of OST on human tongue cancer Tca8113 cells for 24 hours was 154.1μmol·L^-1（N=5,`X±S）.2.Colony formation experiments showed that,After 1 week,Compared with the control group’s colony forming ability（99.00±1.00）%,after 40,80,120 and160μmol·L^-1OST acted on the cells,the clone forming ability decreased significantly with the increase of drug concentration,respectively（92.30±2.72）%,（67.93±3.26）%,（27.67±5.86）%and（1.33±1.53）%,and the difference was statistically significant（P<0.05）.3.The results of Hoechst33342 staining showed that the nucleus of the control group Tca8113 was complete,emitting faint blue light under a fluorescence microscope,and the fluorescence was evenly distributed;after OST treatment,the nuclear pyknosis was obvious,chromatin was shrunk,and the nucleus fragmented.Depending on the concentration of OST,the bright and dense blue fluorescence became more obvious.4.Flow cytometry results showed that after 24h,the apoptosis rate of the control group was（3.87±1.00）%.After 40,80,120 and 160μmol·L^-1OST the apoptosis rate was（11.23±5.01）%,（16.23±6.61）%,（29.47±6.70）%and（46.30±3.87）%,With the increase of drug concentration,the rate of apoptosis gradually increases and the difference was statistically significant（P<0.05）.5.Western blot results showed that osthole could up-regulate the expression of Bax and Cleaved Caspase-3 protein,and down-regulate the expression of Bcl-2 protein.At the same time increase the LC3Ⅱ,P62 and p-AMPK,reduce LC3Ⅰ,p-m TOR expression（P<0.05）.ConclusionsOsthole can inhibit the proliferation of human tongue cancer Tca8113 cells.promote cell apoptosis and may be related to the induction of autophagy through AMPK/m TOR pathway.