Effects Of Asiatic Acid On Proliferation And Apoptosis Of Human Hepatoma SMMC-7721 Cells In Vivo And In Vitro And Its Mechanism

Objective:To investigate the influences and mechanism related to apoptosis of asiatic acid（AA）on the proliferation and apoptosis of human hepatoma SMMC-7721cells in vitro.To investigate the inhibitory effect of AA on human hepatoma xenograft tumor in nude mice by using bioluminescence imaging technique and mechanism related to apoptosis in vivo.Methods:Human hepatoma SMMC-7721 cells were exposed to different concentrations of AA（20-60μM）for 24 h,after that,the morphological changes of cells were observed under microscope,then the cell viability,apoptosis rate,and the mitochondrial membrane potential changes were examined by CCK-8method;Annexin V-APC/7-AAD double staining flow cytometric assay and JC-1 staining respectively;in addition,the expression of the mitochondrial apoptosis related proteins including P53,Bax,Bcl-2,Cyt-C,Caspase-3 and Cleaved Caspase-3 were detected by Western blot.The SMMC-7721 cell lines with stable expression of luciferase（SMMC-7721-Luc）were established by transfection via lentiviral vector.After that,fifteen 4-to 5-week-old male BALB/c（nu-nu）nude mice were used to establish the subcutaneously xenograft tumor model bearing SMMC-7721-Luc cell lines and then be divided into 3 groups:model group,AA50 mg/Kg group and AA 100 mg/Kg group（n=5 per group）.The solvent or AA was given orally for 21 days.At 7 d,14 d and 21 d,the dynamic tumor growth of xenografted tumor was observed by using small animal in vivo imaging system.At the end of test,the weight and volume of tumors in nude mice were measured;the pathological changes of human hepatoma cells were observed under light microscope after HE staining;TUNEL assay was performed to analyze the apotosis of tumor;in addition,the expression of P53,Bax,Bcl-2,Cyt-C,Caspase-3andCleavedCaspase-3wereexaminedby immunohistochemical staining and Western blot.Results:The results of CCK-8 showed that AA inhibited the proliferation of human hepatoma SMMC-7721 cells in a dose-dependent manner with IC₅₀ of 38.31μM and induced the apoptosis-like morphological change.AnnexinV-APC/7-AAD double staining streaming results showed that,compared with the control group,AA dose-dependently increased the apoptosis rate of human hepatoma SMMC-7721 cells（P<0.01）.JC-1 results showed that,compared with the control group,AA significantly decreased the red/green fluorescence ratio under fluorescence microscope in a dose-dependent manner（P<0.01）,showing that AA could dose-dependently reduced mitochondrial membrane potential.In addition,Western blot results showed that at the concentration of 40μM,AA significantly up-regulated the protein expression levels of Bax,Cyt-C,and Cleaved Caspase-3（P<0.05 or P<0.01）,down-regulated the expression level of Bcl-2 and Caspase-3（P<0.05 or P<0.01）,but had no influence on the expression of P53 compared with those of the control group.The nude mice xenografted tumor model bearing SMMC-7721-Luc cell lines were successfully established in vivo.In vivo imaging detection showed that the fluorescent area and intensity of AA groups were all apparently lower than those of the model group（P<0.05）.Compared with the model group,the tumor weight and volume of AA 100mg/kg group were significantly reduced（P<0.01）.Moreover,HE staining showed the results that compared with the model group,AA 50 mg/Kg group and AA 100 mg/Kg group could promote human hepatoma tumor cells apoptosis apparently:with the apoptosis-like morphological changing and the number of cells decreasing;TUNEL assay showed that,compared with the model group,tumor cell apoptosis rate increased significantly in the group of AA 50mg/Kg and 100mg/Kg;IHC results showed that AA significantly up-regulated the expression level of Bax,Cyt-C and Caspase-3（P<0.05 or P<0.01）,down-regulated the expression level of Bcl-2（P<0.01）,whereas had no influence on the expression of P53 compared with those of the model group.Western blot results showed that AA could up-regulated the protein expression levels of Bax,Cyt-C and Cleaved Caspase-3,（P<0.05 or P<0.01）,down-regulated the expression level of Bcl-2 and Caspase-3（P<0.01 or P<0.05）,but had no influence on the expression of P53compared with those of the model group.Conclusion:AA inhibits the proliferation of human hepatoma SMMC-7721 cells in vivo and vitro,an effect that may be related to the AA-inducted mitochondrial apoptosis in a P53-indepandent manner.