| Objective1.To assess the effects of oral Traditional Chinese medicine(TCM)for preventing recurrence and metastasis in postoperative triple negative breast cancer(TNBC)(Meta-analysis).2.In this study,mass spectrometry and network pharmacology was used to explore the mechanism of clinical experience formula-GuBen Decoction(GBF)for intervention of TNBC.3.Based on the results of mass spectrometry and network pharmacology,we futher explore the mechanism of GBF in inhibiting the invasion and metastasis of TNBC based on phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)pathway and epithelial-mesenchymal transition(EMT).Methods1.Meta-analysisWe searched in Pubmed,EMBASE,Web of Scienese,Cochrane Library,CHKD,CBM,CNKI,VIP,and Wangfang data databases for studies published before October 2018.Random effects meta-analysis was conducted about the effects of oral TCM for preventing recurrence and metastasis in postoperative TNBC.Patients were after or during chemotherapy/radiotherapy,and the TNM were Ⅰ-Ⅲ stage.Oral Chinese medicine or Chinese patent medicines was used for experimental group,while the control group was only followed up.The observation outcomes were 3-year disease-free survival(DFS)rate,3-year recurrence-free survival(RFS)rate and 3-year distant metastasis-free survival(DMFS)rate.DFS was defined as the time from surgery to recurrence or metastasis.2.Mass spectrometry combined with network pharmacologyLiquid chromatography coupled to tandem mass spectroscopy(LC-MS/MS)was used to analyze phytochemical composition of GBF’s lyophilized powders.Then SMILE structures of GBF’s lyophilized powders were obtained according to the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)and PubChem database.Using the term "triple-negative breast cancer" to search "TTD,""Drugbank," "OMIM," "KEGG," "PharmGkb;" "GeneCard"databases,and using"basal-like breast carcinoma or triple-negative breast neoplasms" to search the DisGeNet database,we combined the databases,and removed duplicates,finally got the disease targets.Using VENNY 2.1 software(http://bioinfogp.cnb.csic.es/tools/venny/index.html),the intersection of targets between GBF and TNBC was obtained.The network of "GBF phytochemical composition-target-TNBC" was constructed by using Cytoscape software.Protein-protein interaction(PPI)network of intersected targets between GBF and TNBC were constructed by the STRING database(https://string-db.org/).GO and KEGG pathway enrichment analyses were performed using the DAVID tool(http://david.abcc.ncifcrf.gov).3.Experimental StudyThe human triple negative breast cancer cell line MDA-MB-231 was used in this study.GBF and capecitabine were used as pharmacological interventions.MTT assay was used to detect the inhibition rate of GBF at different time periods and different concentrations on cell proliferation.Then we determined the 48-hour group as further experimental groups and dosage.Experimental groups were divided into blank control group(Control group),capecitabine group(CAP group),GBF high-dose group(H-GBF group),GBF medium-dose group(M-GBF group),GBF low-dose group(L-GBF group).Clone formation assays detected cell proliferation ability after medication intervention.Flow cytometry detected the effects of different drugs on cell cycle changes.Cell invasion and migration ability were evaluated by scratch-healing test and transwell test.Western blotting was performed to measure the protein expression levels of EMT-related markers(E-cadherin,N-cadherin,Twist and Slug)and Akt,PI3K after medication.Results1.Total of 10 randomized controlled trials were included in the meta-analysis,which included 785 patients with stage Ⅰ-Ⅲ triple-negative breast invasive ductal carcinoma.Results showed that oral TCM can significantly reduced the 3-year RFS rate and DMFS rate[RR=0.44,95%CI(0.34,0.56)],and increased the 3-year DFS rate[RR=1.38,95%CI(1.25,1.52)],compared with the routine follow-up review control group after postoperative radiotherapy and chemotherapy.2.LC-MS/MS analysis showed that the main chemical components of GBF were lobetyolin,ganoderic acid B,calycosin-7-glucoside,saikosaponin A,ononin,solasonine,Verticine,Calycosin,ursolic acid,oleanolic acid,formononetin,and Paclitaxel.GBF’s chemical components had 575 potential targets.There were 2256 targets for TNBC.Intersected genes between GBF and TNBC were 236.PPI network illustrated that MAPK1,MAPK3,sarcoma gene(SRC),PIK3CA,PIK3R1,Aktl,STAT3 may be the potential core targets of GBF for the treatment of TNBC.GO analysis displayed that the top 5 enrichment related to biological processes(BP)contained protein phosphorylation,protein autophosphorylation,peptidyl-serine phosphorylation,response to drug,and peptidyl-tyrosine phosphorylation.The cellular component(CC)included cytosol,nucleoplasm,nucleus,plasma membrane,and cytoplasm.Protein kinase activity,ATP binding,protein serine/threonine kinase activity,protein binding,and enzyme binding were related to molecular function(MF).KEGG pathway enrichment analysis got 161 pathways.According to gene count,the top significant 20 pathways were displayed,which included PI3K-Akt signaling pathway,proteoglycans in cancer,microRNAs in cancer,MAPK signaling pathway,human papillomavirus infection,focal adhesion,human cytomegalovirus infection,Hepatitis B,Ras signaling pathway,EGFR tyrosine kinase inhibitor resistance.Among them,the PI3K-Akt signaling pathway enrichment was significant and the number of targets was the first.3.Experiment showed that GBF inhibited the proliferation of MDA-MB-231 cells in a time-and concentration-dependent manner.According to the results of previous experiment,we used 25%,15%,and 10%(132p,g/ml,79μg/ml,53μg/ml of IC50 in the 48-hour group as the drug concentration of H-GBF,M-GBF,and L-GBF group.Clone formation assays displayed that H-GBF and M-GBF groups all had inhibitory effects on MDA-MB-231 cells’clone formation.Flow cytometry cell cycle experiments showed that GBF induced G0/G1 cell cycle block.Transwell test showed that GBF inhibited the invasion of MDA-MB-231 cells.And compared with CAP group,the inhibitory effect of H-GBF group was more obvious.Scratch-healing test demonstrated that H-GBF group significantly reduced the healing area of cell scratches,which was more effective than CAP group.Western blotting showed that GBF upregulated the expression of E-cadherin,and downregulated the expression of N-cadherin,Twist,and Slug.Moreover,GBF also downregulated the expression of Akt,p-Akt,and p-PI3K.Conclusion1.For postoperative TNBC patients,oral TCM as maintenance strategy can reduce 3-year RFS and DMFS rate and prolong 3-year DFS rate.As the potential treatment strategy for postoperative TNBC patients,oral TCM need more high quality trials with large samples and longer following-up.2.LC-MS/MS analysis obtained 12 main chemical components of GBF.Further network pharmacology analysis showed that MAPK1,MAPK3,SRC,PIK3CA,PIK3R1,Akt1,STAT3 were the potential core targets of GBF for the treatment of TNBC.The targets of GBF’s chemical components were distributed in different metabolic links.GBF plays a therapeutic role in TNBC through multi-component,multi-target,multi-channel mutual adjustment.And PI3K/Akt signaling pathway plays a major role.3.In MDA-MB-231 cells,GBF can inhibit cell proliferation,invasion and migration,and can caused G/G1 cell cycle block.In addition,132μg/ml H-GBF group inhibits invasion and migration more significantly.The mechanism of GBF in inhibiting the proliferation,invasion and migration of MDA-MB-231 cells may be related to the regulation of PI3K/Akt pathway and the inhibition of EMT. |
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