Study On The Mechanism Of Inhibition Of Metastasis And Invasion Of Hepatocellular Carcinoma Cells By Xanthoangelol

Objective: The purpose of this study was to examine the anti-hepatocellular Carcinoma(HCC)efficacy of the chalcone compound Xanthoangelol(XAG)and to explore the potential targets and mechanisms of XAG in HCC.Research Methods: Cell experiment: We trade with different concentration of XAG(0,10 and 20 μM)and different time(24 and 48 h)to the Bel 7402,SMMC 7721,Hep 3b and Huh 7 cells.Cell activity was detected by cck-8 assay,apoptosis rate was detected by flow cytometry,cell migration and invasion were detected by Transwell assay,expressions of apoptosis-related proteins and differential proteins were detected by Western Blotting assay,and the effect of XAG on mitochondrial membrane potential(MMP)was investigated by jc-1 staining.Animal experiment: The xenotransplantation model of HCC was constructed by injecting SMMC 7721 cells into the left abdomen of mice to evaluate the anti-tumor activity of XAG in vivo.The apoptosis of cells in tumor tissues was detected by TUNEL method,and the toxic and side effects of XAG on lung,liver,spleen,kidney and heart tissues were observed.The expression of CCNB1,CCNB2,CDC20,MCM2,MCM3,MCM4,MCM5,MCM6,MCM7 and PLK1 proteins in tumor tissues and adjacent tissues of mice were detected by immunohistochemistry(IHC).The xenotransplantation model of liver cancer with lung metastasis was constructed by injecting Huh7 cells into tail vein to evaluate the effect of XAG on lung metastasis.The pathological changes of lung tissue sections were detected by HE staining.Clinical tissue sample experiment: From June 2016 to February 2018,cancer tissues and adjacent tissues of 21 HCC patients were collected.The expression of CCNB1,CCNB2,CDC20,MCM2,MCM3,MCM4,MCM5,MCM6,MCM7 and PLK1 proteins in cancer and adjacent tissues were detected by IHC.Proteomics experiment: The protein changes of SMMC7721 and Huh 7 treated by XAG were detected by proteomics and nanometer mass spectroscopy-chromatogram,and were verified by Western Blotting and PCR.With the help of the data information of HCC patients in the TCGA database,the different proteins detected in the above experiment were verified again.Results: HCC cells were incubated for 24 hours and 48 hours in an environment containing XAG(0,10,and 20 μM),which was found to effectively inhibit the growth of HCC cells and induce endogenous apoptosis.No significant differences in cell viability were detected after XAG(0,5 and 10 μM)incubation for 24 hours and 48 hours,but XAG(5 and 10 μM)significantly inhibited the epithelial-mesenchymal transition(EMT)and the migration and invasion of HCC cells.Intraperitoneal injection(40 and 80mg/kg)in mice showed that XAG significantly inhibited tumor growth and lung metastasis without acute injury to lung,liver,spleen,kidney,or heart tissue.The proteomic results showed that 130 proteins were up-regulated and 167 proteins were down-regulated.GO analysis,KEGG analysis and PPI network plots showed that the differential proteins were mainly concentrated in biological processes such as cell cycle and DNA replication.Immunohistochemical staining was used to determine the expression of 10 candidate genes in the HCC and normal adjacent tissues of 21 patients.The results of kaplan-meier survival analysis were consistent with the results of proteomics.Immunohistochemical staining was used to determine the expression of 10 candidate genes in HCC tissues and normal para-carcinoma tissues of hepatocellular carcinoma xenograft mice.Discussion: In vivo and in vitro experiments,we found that XAG can effectively inhibit the growth,invasion and migration of HCC cells,EMT and induce endogenous apoptosis of HCC cells.XAG had no acute injury to lung,liver,spleen,kidney and heart tissues in mice,indicating that XAG had significant anti-tumor effect in vivo and weak toxic and side effects on mice.Proteomic results showed that the differential proteins were mainly concentrated in biological processes such as cell cycle and DNA replication.Therefore,we speculate that XAG may play an anti-tumor role by acting on the cell cycle and is expected to be used in combination with other anti-tumor drugs to reduce the resistance of HCC cells.