Preliminary Study On Adjuvant Application Of Sinapinic Acid To Cisplatin In The Treatment Of Hepatocellular Carcinoma With Different Differentiation Types

Objective:To observe the effect of different concentrations of sinapinic acid（Sa）combined with cisplatin（DDP）on the proliferation,migration,apoptosis and autophagy of poorly differentiated hepatoma cell HepG2 and highly differentiated hepatoma cell SMMC-7721 at different time,and to explore the theoretical basis for Sa combined with DDP in the adjuvant treatment effect of hepatoma of different differentiated type.Methods:The Sa（100～2000μmol/L）combined with DDP（5μmol/L）to treatment HepG2 cell,SMMC-7721 cell and normal hepatocyte cell HL-7702 for 24h,48 h and 72 h to evaluate the toxicity of drugs to cells.The cell viability was determined by MTT assay.Colony formation assay and flow cytometry cycle assay were used to measure the influence on proliferation.The effect of the migration was determined by transwell assay and Wound healing assay.Effects of the apoptosis was determined by microscopy,Hoechst staining and Annexin V-FITC/PI staining assay.The fluorescence staining of dansylcadaverine（MDC）was used to observed the effect on autophagy;Western blot was used to analyze the expression of apoptosis-related proteins PARP,c-PARP,Bcl-2,Bax,caspase-3,active-caspase 3and autophagy-related proteins p62,Beclin-1,Atg5 and LC3Ⅱ.Ultimately,inhibitor Z-VAD-FMK（20μmol/L）and 40μmol/L hydrochloroquine（HCQ）were used to pretreat hepatoma cells to observe the effects of Sa,DDP,and their combination on apoptosis and autophagy,as well as detected the effects of related protein expression levels.Results:Sa significantly inhibited the proliferation of HepG2 and SMMC-7721 cells by inducing G₂/M cell cycle arrest.Based on the results of MTT and colony formation assay,the synergistic activity of Sa and DDP was observed in HepG2 and SMMC-7721 cells.The results of scratch and transwell assay showed that Sa could effectively inhibit the migration of HepG2 and SMMC-7721 cells.Hoechst staining and Annexin V-FITC/PI staining were showed that Sa could induce apoptosis of hepatoma,and MDC staining showed that Sa also induce autophagy.Western Blot showed that Sa up-regulates the expression of Beclin-1,Atg5,c-PARP and reduces the expression of p62,PARP,Bcl-2/Bax to activation caspase-3 and LC3,induces apoptosis and autophagy.MTT assay showed that HCQ pretreatment can significantly enhance the cytotoxic effects of Sa combined with DDP.Especially,the expression of active-caspase-3 protein increased and the LC3Ⅱdecreased compared with before,suggesting that cytoprotective autophagy also plays a role in the autophagy induced by Sa combined with DDP.Conclusion:Sa has strong anti-cancer activity on different differentiated hepatoma cells,and has a certain effect in inhibiting the proliferation and migration of tumor cells.When DDP was used as adjuvant therapy for hepatoma,it can significantly enhance the anti-tumor activity of DDP by blocking the cell cycle in the G₂/M phase,the cell proliferation was inhibited,and induced the apoptosis of hepatoma cells by mediating the Bax/Bcl-2 signaling pathway.Meanwhile,it can also autophagy death of hepatoma cell by up-regulating LC3 protein expression.But,Inhibition the cell autophagy activity can promote the apoptosis and enhance the anti-tumor effect of drugs,which provides a theoretical basis for the combination of autophagy inhibitor,Sa and DDP for clinical tumor treatment.