Effect Of Brazilin On Apoptosis And Autophagy Of Tongue Cancer Tca8113 Cells And Its Molecular Mechanism

ObjectiveTo investigate the effects of Brazilin on the proliferation,apoptosis and autophagy of human tongue squamous cell carcinoma Tca8113 cells and its molecular mechanism.Furthermore,to provide a theoretical basis for clinical drug use.MethodsTca8113 cells were treated with different concentrations of Brazilin（0,1,5,10,20,30,40,80,100,200μmol/L）.Then the effect of Brazilin on the growth of Tca8113 cells was detected by MTT.The value of half inhibition rate was calculated and the curve was made.The apoptosis of Tca8113 cells was observed by Hoechst33342 staining.The apoptosis rate of each group was detected by flow cytometry.Expression of apoptotic protein bax,bcl-2,cleaved caspase3 and autophagy-related proteins p-AMPK,p-mTOR,LC3B,p62 was detected by western blotting.The AMPK pathway inhibitor was used to interact with the Brazilin in Tca8113 cells,and the expression of the pathway-related proteins p-AMPK,p-mTOR,and LC3B was detected.Results1.Brazilin inhibits proliferation of Tca8113 cellsMTT results showed that different concentrations of Brazilin inhibited the growth of Tca8113 cells,and the inhibitory effect increased with the Brazilin concentration increasing.The calculated IC₅₀ was 31.17μmol/L at 24 hours.2.Effect of Brazilin on apoptosis of Tca8113 cellsHoechst33342 staining showed that after 24 hours of Brazilin treatment,the nuclear membrane disappeared,the nucleus was fragmented,and some nuclei were"new moon"and"petal",and the degree of morphological change was positively correlated with drug concentration.Flow cytometry was used to detect the apoptosis rate of each group.The results showed that the apoptosis rate increased with the increase of drug concentration after 24 hours of different concentrations of Brazilin,and the apoptosis rate of each group was higher than that of the control group.3.Effect of Brazilin on apoptosis-related proteinsThe results of Western blot showed that after the action of Brazilin,the expressions of proapoptotic proteins bax and cleaved-caspase3 in Tca8113cells increased significantly with the increase of Brazilin concentration（P<0.05）,and the expression of anti-apoptotic protein bcl-2 was significantly decreased.4.Effect of Brazilin on autophagy-related proteinsMeanwhile,the expression of autophagy positive correlation proteins p-AMPK and LC3B increased with increasing drug concentration.The expression of p-mTOR and p62 was gradually decreased（P<0.05）.After combing the AMPK inhibitor with Brazilin,the expression levels of p-AMPK and LC3B were still slightly higher than that of the control group,but they were significantly lower than those of the Brazilin alone,while the expression of p-mTOR was significantly higher than that of the Brazilin group.Conclusions1.Brazilin can inhibit the growth of Tca8113 cells and promote its apoptosis through the mitochondrial pathway.2.Brazilin can induce autophagy in the cells through the AMPK/mTOR pathway.