ITRAQ Based Proteomics Analysis Of Anti-prolifereation Mechanism Of Asiatic Acid On Human SMMC-7721 Hepatocellular Carcinoma Cells

Objective:This research aim to study the influence of Asiatic acid on protein expression profile in human hepatocellular carcinoma SMMC-7721 cells,and to perform bioinformatics analysis of differential proteins.Including GO function annotation,KEGG pathway enrichment and protein-protein interaction network for differential proteins were used to explore the potential function and mechanism of Asiatic acid induced anti-proliferation effect on human hepatocellular carcinoma SMMC-7721 cells.Methods:1.MTT assay was used to detect the anti-proliferation effect of Asiatic acid with different concentration?20,30,40,50,60?M?on SMMC-7721 cells for24 h.The optimal concentration,namely 50% inhibitive concentration(IC₅₀),was selected for following experiments.2.i TRAQ labeling combined with nanoliter liquid chromatography-tandem mass spectrometry?nano lc-ms/ms?was used to obtain the protein spectrum data before and after Asiatic acid treatment,and Proteome Discoverer was employed to screen differential protein protein expression profile.Bioinformatics analysis including GO annotation,KEGG pathway enrichiment and protein-protein interaction network established by Omicsbean online software were applied to explore the function of differently expressed proteins and the potential mechanism of in vitro anti-proliferation effect of Asiatic acid to SMMC-7721 cells.3.Western blot assay was used to verify some differentially expressed proteins,including mitochondrial apoptotic related proteins in apoptosis enrichment pathway?Bax,Cyt C,Caspase-3 and Cleaved Caspase-3?and autophagy related proteins in m TOR-4EBP1 autophagy pathway?LC3-I/II,m TOR,p-m TOR,4EBP1 and p-4EBP1?.4.Based on the western blot result that Asiatic acid could significantly cause changes in the expression of apoptosis and autophagy related proteins,Hoechst 33342/PI apoptotic cells staining and MDC autophagic vacuoles staining were employed to further confirm the feature of apoptosis and autophagy.And Asiatic acid combined with autophagy inhibitor 3-MA treatment was used to determine the inhibition effect that Asiatic acid induced autophagy on the proliferation to SMMC-7721 cells.Results:1.Treated with Asiatic acid for 24 h could dose-dependently inhibit the proliferation of SMMC-7721 cells with the IC₅₀ at 37.31?M.Therefore,40?M of Asiatic acid was selected in this research for the following experiments and proteomics analysis2.Compared with control group,i TRAQ quantitative proteomics results indicated that there were 495 proteins up-regulated and 141 proteins down-regulated after treated with 40?M Asiatic acid for 24 h in SMMC-7721 cells.Bioinformatics analysis revealed that these differentially expressed proteins were mainly enriched in metabolism pathway,including Oxidative phosphorylation pathway,Glycolysis/Gluconeogenesis pathway and Fructose and mannose metabolism pathway.After further analysis,it was found that Asiatic acid may affect metabolism by inducing mitochondrial apoptosis and inducing autophagy by negatively regulating the m TOR-4EBP1 pathway.3.Western blot results showed that 40?M Asiatic acid could up-regulate the protein expression of Bax,Cyt C,Cleaved Caspase-3,4EBP1 and LC3-II,and down-regulate the expression of Caspase-3,p-m TOR,p-4EBP1.These results were consistent with proteomics analysis and revealed that Asiatic acid could induce apoptosis and autophagy in SMMC-7721 cells.4.Hoechst 33342/PI staining and MDC staining further verified that Asiatic acid could induce apoptosis and autophagic vacuole in SMMC-7721 cells.And when Asiatic acid combined with autophagy inhibitor 3-MA,the anti-proliferation effect of Asiatic acid could be partly blocked.These results suggested that apoptosis and autophagy both play an important role in the anti-tumor effect of Asiatic acid.Conclusion:1.Asiatic acid could dose-dependently inhibit the proliferation of human hepatocellular carcinoma SMMC-7721 cells in vitro,which was mainly related to the inhibition of protein expressions in metabolism pathways and may be regulated by apoptosis and autophagy process.2.Asiatic could inhibit the proliferation of SMMC-7721 cells in vitro by affecting mitochondrial apoptosis related proteins expression and inducing apoptosis.Meanwhile,AA also could affect the autophagy process related proteins expression in m TOR-4EBP1 pathway and induce excessive autophagy.