Asiatic Acid Induces Endoplasmic Reticulum Stress In Apoptosis Of Tongue Cancer Squamous Tca8113 Cells

ObjectiveTo investigate the effects of Asiatic acid（AA）on proliferation and apoptosis of tongue squamous cancer Tca8113 cells,and to explore the effect of endoplasmic reticulum stress and the changes of related pathway proteins.Methods1.MTT assay was used to detect the inhibitory effect of AA on proliferation of Tca8113 cells.2.Colony formation test was used to detect the effect of AA on the clonal formation of Tca8113 cells.3.Hoechst33342 staining was used to detect the effect of AA on apoptosis of Tca8113 cells.4.The apoptosis rate of Tca8113 cells was detected by flow cytometry.5、The expression of Bcl-2,Bax and cleaved caspase-3 protein in Tca8113cells before and after AA treatment was detected by Western blot.The expression levels of Calpain,Grp78,IRE1α,P-IRE1α,JNK and P-JNK in endoplasmic reticulum stress were detected.6.Fluo-4/AM was used as Ca²⁺fluorescence probe to observe the change of intracellular calcium concentration in Tca8113 cells.7.Immunofluorescence was used to detect the changes of Grp78 protein expression in tongue cancer cells induced by AA.8.Establish xenograft tumor model and analyze the effect of AA on apoptosis in tumor tissue by TUNEL method.Results1.MTT assay showed that AA inhibited the proliferation of Tca8113 cells,and the cell viability of the drug-added group was lower than that of the control group（P<0.01）.2.The ability of cell colony formation was lower than that of control group.3.The number of apoptotic cells with blue and white fluorescence stained by Hoechst33342 increased,and the number of cells with typical apoptotic morphological characteristics increased.4.Annexin V/PI staining showed that compared with the control group,the apoptosis rate of tongue cancer cells in the drug-added group was significantly higher（P<0.01）.5.Compared with the control group,Western blot showed that the expression of Bax and cleaved-caspase3 protein increased and the expression of Bcl-2 protein decreased（P<0.05）.The expression of endoplasmic reticulum stress-related proteins Calpain,Grp78,P-IRE1α,P-JNK was up-regulated,while IRE1α,JNK had no obvious change（P<0.05）.6.Immunofluorescence assay showed that the content of Grp78 increased after AA treatment.7.Fluo-4AM staining showed that intracellular calcium fluorescence increased after AA treatment.8.The xenograft tumor model showed that AA could obviously inhibit tumor growth and promote apoptosis of tongue cancer cells（P<0.01）.ConclusionAsiatic acid inhibits proliferation and induces apoptosis of human tongue squamous cancer Tca8113 cells,and its mechanism may be related to activation of IRE1α/JNK pathway of endoplasmic reticulum stress.