| Triple negative breast cancer(TNBC)has the strongest drug resistance,highest relapse rate and the most importantly poor overall survival because of aggressive metastasis.Developing novel low-toxic therapeutic strategies to overcome TNBC is urgently needed.Golgi Membrane Protein 1(GOLPH2)has been proposed as a potential target for cancer therapy because GOLPH2 overexpression in many solid tumors correlates to tumor growth and metastasis and leads to unfavorable survival.Various approaches including siRNA interference and antibody targeting have been attempted,but until now GOLPH2 has remained an undruggable target.Numerous natural anti-tumoral plant substances have been identified while their possible function on GOLPH2 has never been revealed.Our present study aims to find plant extracts which can affect the expression of GOLPH2 and inhibit TNBC cell migration,and explore its possible cellular mechanisms.The thesis includes three aspects as shown below:First,the natural candidates that can down-regulate the expression of GOLPH2 and inhibit the migration of TNBC MDA-MB-231 cells were screened.EGCG was found to have the most potent inhibition effect on GOLPH2 expression among three tested possible migration-inhibiting natural substances,Epigallocatechin gallate(EGCG),Betulinic acid(BA)and Lupeol,by Semi-quantitative PCR and qRT-PCR.Wound healing and transwell analyses comfirmed that EGCG could inhibit the migration of MDA-MB-231 cells in a dose-dependent manner.Second,whether EGCG could regulate MDA-MB-231 metastasis through GOLPH2 was verified.qRT-PCR and Western Blot assay showed that EGCG treatment down-regulated the expression of GOLPH2 in both mRNA and protein levels.Wound healing and transwell analyses indicated that GOLPH2 supressed the migration of GOLPH2 knocking-down stable transfected MDA-MB-231 cell lines,and EGCG treatment further dramaticly reduced cell migration.It demostreated that EGCG may inhibit the migration of MDA-MB-231 cells by down-regulating GOLPH2.At last,the possible mechanism how EGCG inhibits of MDA-MB-231 cells migration through affecting GOLPH2 expression was explored.Network pharmacology prediction and molecular docking indicated that EGCG may inhibit MDA-MB-23 1 cells’ progression via HGF(hepatocyte growth factor)/HGFR(hepatocyte growth factor receptor)/β-catenin signal pathway.HGF/HGFR pathway have been known to be able to activate downstream signaling pathways through inducing the phosphorylation of GSK-3β and AKT and the nuclear translocation of β-catenin.c-Myc,a downstream effector of β-catenin,is recently reported to have the activity of regulating GOLPH2 expression.Combining above points,we hypothesized that EGCG might inhibit MDA-MB-231 cells migration through HGF/HGFR/AKT/GSK-3β/β-catenin/c-Myc/GOLPH2 signaling pathway.To confirm the hypothesis,MDA-MB-231 cells were treated with EGCG and/or HGF,then the expression of related proteins were detected by western blot analyses.The results suggested that HGF could induce the expression of GOLPH2,accompanying with the phosphorylation of AKT and GSK-3β,the nuclear translocation of β-catenin and the overexpression of c-Myc,whereas HGF treatment together with EGCG inhibited HGF-induced the expression and phosphorylation of related proteins,as well as the nuclear translocation of β-catenin.These findings demonstreated that EGCG could indeed inhibit HGF-induced activation of downstream signals(β-catenin,c-Myc and GOLPH2),leading to the decline of migration capacities in MDA-MB-231 cells.In summary,this study revealed a GOLPH2 downregulation natural product--EGCG for the first time and shed a new way for cancer therapy against GOLPH2 target.Also this study verified HGF could induce the expression of GOLPH2,while EGCG inhibited HGF-induced overexpression of GOLPH2.Moreover,this study proposed a novel molecular mechanism of EGCG on anti-migration in MDA-MB-231 cells,that is,EGCG inhibits TNBC cell migration through HGF-induced AKT/GSK-3β/β-catenin/c-Myc pathway mediated GOLPH2 expression down-regulation. |
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