| Hepatocellular carcinoma(HCC)is still a major problem that seriously threatens human life and health.The recurrence and metastasis of liver cancer is a key problem that limits the clinical efficacy of liver cancer.Compound Phyllanthus urinaria L.(CP)is composed of Phyllanthus urinaria L,Astragalus membranaceus(Fisch)Bunge,Curcuma zedoaria(Christm)Rosc,Scutellariabarbata D.Don and Cremastraappendiculata(D.Don)Makino.It was the basic prescription carefully designed with syndrome differentiation based on the clinical manifestations of patients with pre-lesion and cancer caused by chronic hepatitis B by the Liver Disease Research Center of Shenzhen Traditional Chinese Medicine Hospital.Relevant clinical observations have been carried out and CP are mainly used in the progression of hepatitis B-associated HCC(HBV-associated HCC).However,its anti-metastatic effect and mechanism are still unclear.This study found that CP could significantly inhibit the proliferation,migration and invasion of hepatitis B-related HCC cells.The comprehensive analysis of network pharmacology and bioinformatics predicted Caveolin-1(Cav-1)as one of the main targets of CP,mediating its anti-metastatic effect.The Wnt/?-catenin pathway is one of the core mechanisms of CP against HBV-related HCC.Further experiments showed that high expression of Cav-1 could promote the metastasis of HBV-related HCC by stabilizing ?-catenin,while CP can induce Cav-1 autophagy degradation and activate Akt/Gsk3 ?-mediated ?-catenin protease pathway through activating ?-catenin ubiquitin,thereby weakening the migration promotion effect of Cav-1.In addition,we have further confirmed the anti-proliferative and metastatic effect of CP through in vivo and in vitro experiments.It was found in mouse liver cancer transplantation and zebrafish xenotransplantation models that CP could inhibit the growth and metastasis of HBV-associated HCC.In suary,our study not only emphasized the function of CP in inhibiting the metastasis of HBV-associated HCC.but also further explored the key role of Cav-1 in mediating the Akt/GSK3?/?-catenin signal axis in the regulation of advanced cancer progression.ObjectiveTo investigate the effect and mechanism of CP inhibiting the metastasis of HBV-associated HCC by regulating Akt/GSK3?/?-catenin signaling axisvia Cav-1.MethodsPart 1:The effect of CP on the proliferative phenotype of HBV-related HCC.1.Different concentrations of CP solution(0,30,60,120,240,300,480,600 ?g/ml)were used to intervene HepG2,SMMC-7721,Huh-7 cells,and CCK-8,cell counting,the clone formation experiment were used to study the effect of CP on the proliferation and clone formation of liver cancer cells.At the same time,CCK-8 method was used to detect and observe the toxic effect of CP on normal hepatocytes HL-7702.2.The pENTER-URG11 plasmid was used to transfect into HepG2 to construct stable HBV-related HCC cell HepG2-URG11.Scratch experiment and Western blot experiment were used to verify the migration ability of HepG2-HBx and HepG2-URG11 and the expression of metastasis-related proteins in HBV-related HCC.3.Different concentrations of CP solution were used to intervene HBV-related HCC cells HepG2-HBx and HepG2-URG11.CCK-8 and clone formation experiments were used to study the effect of CP on the proliferation and clone formation of HBV-related HCC cells.Part 2:The effect of CP on the metastasis ability of HBV-related HCC cells.1.Different concentrations of CP solution(0,30,60,120 ?g/ml)were used to intervene HBV-related HCC cells HepG2-HBx and HepG2-URG11.120 ?g/ml CP solution was also used to intervene two types of HBV-related HCC cells at 0h,24h,48h,72h.Scratch test,Transwell test and Western blot test were used to study the effect of CP on the migration and invasion ability of HBV-related HCC and expression of metastasis-related protein markers.Part 3:CP promoted the destabilization of ?-catenin in HBV-associated HCC.1.Different concentrations of CP solution(0,30,60,120 ?g/ml)applied to HBV-associated HCC cells,and 120 ?g/ml CP solution was to used to interfere with HBV-associated HCCcells respectively at 0h,24h,48h,72h.Nuclear and cytoplasmic separation experiment and Western blot method twere used to study the changes of total?-catenin,nuclear ?-catenin and ?-catenin protein content in cytoplasm of HBV-associated HCC cells.2.Different concentrations of CP solutions(0,30,60,120 ?g/ml)were applied to two types of HBV-associated HCC cells,and ?-catenin protein was labeled with iunofluorescencetechnology.The effect of CP on the expression level and cell location of?-catenin in HBV-associated HCC cells were observed by ultra-high resolution laser confocal microscope.3.120 ?g/ml CP solution were applied to intervene two types of HBV-associated HCC cells for 48 hours,and real-time fluorescent quantitative PCR(QPCR)was used to detect the transcription levels of ?-catnenin downstream transcription factors,including cell proliferation-related transcription factors Cyclin D1,Epithelial mesenchymal transformation(EMT)related factors TFs Snail,Slug,ZEB1 and ZEB2.4.Proteasome inhibitor(Mg 132 10 ?M)and protein synthesis inhibitor(Cycloheximide,CHX 10 ?M)alone or in combination with CP solution(120 ?g/ml)were used to interfere with HBV-associated HCC cell HepG2-HBx.Effect of CP on ?-catenin protein synthesis and protease pathway degradation in HepG2-HBx cells were observe by Western Blotting experiment.5.Different concentrations of CP solution(0,30,60,120 ?g/ml)were applied to two types of HBV-associated HCC cells,while 120 ?g/ml CP solution was used to intervene hepatitis B-related liver cancer cells at 0h,24h,At 48h,72h.The effects of CP on the expression levels of P-GSK-3?,GSK-3?,P-AKT and AKT in HBV-associated HCC cells were observed by Western Blotting experiment;GSK-3? inhibitor LiC1(20 ?M)or The AKT inhibitor LY294002(LY,25 ?M)combined with CP solution(120 ?g/ml)were applied to HBV-associated HCC cells.The effect of CP on the expression of ?-catenin protein in HBV-associated HCC cells was observed by Western Blotting.Part 4:Establishment network of CP ingredient-CP target-HBV-associated HCC.1.The online databases TCMSP and TCMID were used to search the active ingredients of each component of CP.Candidate compounds with the criteria of OB?30%and DL?0.18 were screened;Cytoscape software(version 3.2.1)was used to visualize the component-target network of each medicinal material.2.NCBI GEO database was used to obtain the genes of HBV-related HCC;GEo2R software was used to identify differentially expressed genes of HBV-related HCC;Venn diagram was used to obtain targets of CP acting on HBV-related HCC;String database and Cytoscape software were used to construct protein-proteins Interaction Network(PPI).3.The DAVID database was used to perform enrichment analysis on the target genes of HBV-related HCC.The enrichment items include biological process(BP)enrichment,molecular function(MF)enrichment,and cellular components(CC)enrichment and KEGG pathway.Part 5:Study on the role of CP in regulating the autophagic degradation of Cav-1 in HBV-associated HCC cells.1.Different concentrations of CP solution(0,30,60,120?g/ml)were applied to two types of HBV-associated HCC cells Immunofluorescence technology was used to label Cav-1 protein.The effect of CP on the expression level and cell localization of Cav-1 in HBV-associated HCC cells was observed with ultra-high resolution laser confocal microscope.2.Different concentrations of CP solution(0,30,60,120?g/ml)were applied to two kinds of HBV-associated HCC cells.120 ?g/ml CP solution was used to interfere with HBV-associated HCC cells respectively at 0h,24h,48h,72h.Western blot was used to study the changes of Cav-1 protein content in HBV-associated HCC cells.3.In order to further explore the intervention pattern of CP on Cav-1,the protein degradation inhibitor Mg 132(10 ?M)and the autophagy inhibitor CQ(30 ?M)combined with CP(120 ?g/ml)were were applied to HepG2-HBx cells.Western Blotting experiment was used to detect the changes of Cav-1 protein content in HBV-associated HCC cells.4.HepG2-HBx cells were treated with 120 ?g/ml CP solution.Immunofluorescence double staining technique was used to label lysosomal markers-lysosomal-associated membrane protein 1 antibody LAMP1 and Cav-1.Ultra-high resolution laser confocal microscope was usde to observe the effect of CP on the autophagic degradation of Cav-1 in HepG2-HBx cells.Part 6:CP activates Akt/Gsk3?-mediated ?-catenin proteasome degradation pathway by inhibiting Cav-1.1.LipoFiterTM liposome transfection reagent was used to transfect pcDNA 3.1(C)-CAV1 plasmid into HepG2-HBx and HepG2-URG11 cells to construct a HBV-associated HCC cell line with stable and high expression of CAV-1.X-tremeGENE siRNA Transfection reagents transiently was used to transfect the pcDNA 3.1(+)-?-catenin plasmid into the HepG2-HBx-Cav-1 cell line to construct HepG2-HBx-Cav-1 cell line with low ?-catenin expression.2.120 ?g/ml CP solution was applied to HepG2-HBx-Cav-1 and control cell lines.Scratch experiment was used to observe the effect of CP on the metastasis ability of HepG2-HBx-Cav-1 cell.3.120 ?g/ml CP solution was used to intervene with HepG2-HBx-Cav-1 and control cell lines.Western Blotting experiment was used to detect the effects of CP on the expression levels of metastasis-related proteins Vimentin,E-cadherin and N-cadherin protein.4.120 ?g/ml CP solution was applied to HepG2-HBx-Cav-1 and control cell lines.Western Blotting experiment was used to detect the relationship between CP and Akt/Gsk3?/?-catenin signal axis related proteins.5.120 ?g/ml CP solution were applied to HepG2-HBx-Cav-1 cell line.The effects of CP on ?-catenin in HepG2-HBx-Cav-1 were observed via immunoprecipitation and ubiquitination experiments.6.CP solution(120 ?g/ml)combined with GSK-3? inhibitor LiC1(20 ?M)or AKT inhibitor LY294002(LY,25 ?M)were applied to HepG2-HBx-Cav-1 cell line.The Western Blotting experiment was used to observe the effect of CP on the expression levels of?-catenin,Cav-1,P-GSK-3?,GSK-3?,P-AKT and AKT protein in HBV-associated HCC cells.7.CP solution(120 ?g/ml)were applied to HepG2-HBx-Cav-1 cell line with ?-catenin downregulation.Scratch experiment were used to observe the migration ability of HepG2-HBx-Cav-1 cell line with ?-catenin downregulation after CP interferes.Part 7:CP inhibited proliferation and migration of HBV-associated HCC in vivo and ex vivo.1.HepG2-HBx and HepG2-null cell lines were subcutaneously injected into the right liver area of BALB/C nude mice to construct nude mice models with HCC and HBV-associated HCC.Different concentrations of CP solution(0,300,625 mg/ml)were administrated.The changes in tumor size was observed;HE staining was used to observe the number of lung metastases in nude mice.2.The HepG2-HBx cells were implanted into zebrafish abdomen by microinjection technology to construct a zebrafish hepatitis B-related liver cancer xenograft model.Different concentrations of CP solutions(0,60,120 ?g/ml)were applied to zebrafish.Dil staining was used to observe the effects of CP on tumor proliferation and metastasis;HE staining was used to further observe the changes in tumor size.Immunohistochemical experiments were used to observe the expression of Cav-1,?-catenin,vimentin and E-cadherin proteins in zebrafish tumors.ResultPart 1:CP inhibited the proliferation of HCC cells and HBV-associated HCC cells.1.CP significantly inhibited the viability and clonogenic ability of HepG2,SMMC-7721,Huh-7 with a time and dose-dependent manner.2.CP has no obvious side effects on common liver cell HL-7702.3.The HepG2-URG11 cell line was successfully constructed;HepG2-HBx and HepG2-URG11 cell lines have stronger proliferation and migration capabilities than ordinary liver cancer cell lines.EMT-related protein markers have changed significantly.Part 2:CP inhibited the migration and invasion ability of HBV-associated HCC.1.CP intervention inhibited the wound healing ability and invasive ability of HBV-associated HCC cells.2.CP affected the expression of EMT-related proteins,manifested by the decrease of Vimentin,N-cadherin and the increase of E-cadherin.Part 3:CP promoted the destabilization of ?-catenin in HBV-associated HCC.1.CP decreased the protein expression of ?-catenin in HepG2-HBx and HepG2-URG11 cells.2.CP inhibited nuclear translocation of ?-catenin in HepG2-HBx and HepG2-URG11cells.3.CP promoted the degradation of ?-cateni in HepG2-HBx through the proteasome degradation pathway4.CP promotes ?-catenin degradation through Akt/GSK3? signaling pathwayPart 4:Establishment network of CP ingredient-CP target-HBV-associated HCC.1.A total of 76 potential active substances in each component of CP were screened from the database.2.181 CP targets for HBV-associated HCC were obtained through intersection analysis,of which CAV-1 is one of the core targets of CP.3.Enrichment analysis suggested that the target of CP was related to a variety of cell biological functions,and the wnt pathway may be involved in the inhibitory effect of CP on HBV-associated HCC.Part 5:CP promotes autophagic degradation of Cav-1 in HBV-associated HCC cells.1.CP decreased the expression of Cav-1 protein in HBV-associated HCC cells in a time-and dose-dependent manner.2.CP activated the autophagy of HBV-associated HCCcells,and promoted the degradation of Cav-1 through the autophagic degradation pathway.Part 6:CP activates Akt/Gsk3?-mediated ?-catenin proteasome degradation pathway by inhibiting Cav-1.1.CP reversed the promoting effect of Cav-1 on the migration ability of HBV-associated HCC cells.2.CP reversed the regulatory effect of Cav-1 on the metastasis-related proteins of HBV-associated HCC cells.3.CP reversed the regulation of Cav-1 on Akt/Gsk3?/?-catenin signal axis of HBV-associated HCCcells.4.CP reversed the influence of Cav-1 on the distribution of ?-catenin in HBV-associated HCC cells.5.Overexpression of Cav-1 reversed the degradation of ?-catenin induced by CP,leading to the accumulation of ?-catenin.6.CP promoted the ubiquitination and accumulation of ?-catenin in HBV-associated HCC cells.7.?-catenin knockdown reversed the promotion effect of Cav-1 on the migration ability of HBV-associated HCC cells.Part 7:CP inhibited proliferation and migration of HBV-associated HCC in vivo and ex vivo.1.CP significantly inhibited the growth of tumors and the formation of lung metastases in nude mice.2.CP effectively inhibited the growth and spread of tumors in zebrafish xenograft models and significantly decreased the protein expression level of Cav-1,?-catenin,vimentin and E-cadherin in tumors.ConclusionTumor recurrence and metastasis are the main problems that limits the clinical efficacy of liver cancer.CP could regulate the Akt/GSK3?/?-catenin signal axis through Cav-1 to inhibit the metastasis of HBV-associated HCC. |
PDF Full Text Request