Study On The Effect And Mechanism Of Dichloromethane Extract Of Fresh Fritillaria On The Metastasis Of Triple-negative Breast Cancer

Background:Breast cancer is one of the most common malignant tumors amony women,with increasing incidence constantly.The triple negative breast cancer(TNBC)subtype is characterized by the absent expression of estrogen receptors(ER),progesterone receptors(PR),and human epidermal growth factor receptor-2(HER-2)in the tumor.This subtype has a higher rate of recurrence and metastasis and a poorer prognosis..Objective:The present study aims to evaluate the efficacy,especially the anti-metastatic effects of the dichloromethane extract of Tubeimu(ETBM)on TNBC orthotopic mouse models and cell lines.Futhermore,we want to explore the possible mechanisms with modern scientific methods.Methods:In in vivo studies,we applied ortho topic mice models with green fluorescent protein(GFP)transfected TNBC tumor.With real-time imaging,we were able to observe tumor growth and metastasis with minimal damage to mice.In in vitro studies,we utilized IncuCyte(?)Zoom real-time imaging system and cristal violet to evaluate the anti-proliferation effect of ETBM.Through Transwell migration and invasion assay,as well as wound-healing assay,we tested the impact on cell mobility by ETBM.Then we delivered digital gene expression sequencing(DGE)to explore the differential gene expression affected by ETBM treatment.Furthermore,we conducted KEGG pathway clustering to examine the possible influenced biological process by ETBM.To confirm the results in DGE,we performed qRT-PCR,western blot and immunohistochemistry staining(IHC)in cell and animal samples.We futher examined the phosphorelation of the integrin-downstream pathway,for instance,focal adhesion kinase(FAK),Src family kinases(SFKs)and protein kinase B(AKT).Finally,by utilizing cBioPortal and TCGA platform,we detected the gene mutation and aberrant expression of ETBM targets in human breast cancer samples to better understand the clinical value of our study.Results:Our study found that ETBM has affirmative inhibitory effect on the growth of orthotopic TNBC tumors,with reasonable inhibitory effects on lung and lymph node metastasis in ETBM high-dose group.The incidence of lung metastasis was 90%in the control group,60%in the low-dose group(ETBM-L),and 30%in the ETBM-H.Compared with the control group,the high-dose ETBM group can significantly reduce the incidence of lung metastasis.Although the effect of suppressing orthotopic tumor volume in ETBM groug was not as good as the cytotoxic drug paclitaxel,the fluorescence protein intensity in Taxol group increased,suggesting vigorating tumor activity.Moreover,mortality rate in all ETBM groups is 0%,while Taxol group showed 10%mortality during treatment.Through in vitro experiments,it was found that ETBM has a proliferation-inhibitory effect on a variety of TNBC cell lines,including MDA-MB-231,MDA-MB-468 and SUM-149,with an IC50 of 45-90 ?g/ml.However,it exhibited a weaker inhibitory effect on hormone-receptor-positive breast cancer cell line,MCF-7,with an IC50 of 475.8 ?g/ml.Interestingly,it could significantly suppress the invasion,migration and wounding repair of MDA-MB-231 cells.With DGE sequencing,we found that ETBM was able to regulate many genes at the transcriptional level,such as multiple integrins(ITG),Rho GTPase activating protein 5(ARHGAP5),vascular endothelial growth.factors-A(VEGF-A),insulin-like growth factor 1 receptor(IGF-1R),platelet-derived growth factor beta(Platelet-derived growth factor)Receptors,PDGF-R?),etc..According to KEGG pathway enrichment analysis,it is presumed that ETBM could reduce cell motility by regulating the integrin family and its downstream pathways.Furthermore,qRT-PCR,western blot and IHC staining verified that ETBM can lower the expression of ITGB1,ITGB8,ARHGAP5 and other integrin family genes in both cell and tumor samples.Also,down-regulated activation of downstream kinases,such as FAK,Src and AKT,was observed.Through the use of the cBioPortal data platform,we explored desiring mutations in human breast cancer samples from multiple databases.It was shown that many genes in this study have certain mutations in breast cancer patients.At the meanwhile,mRNA and protein expression data of breast cancer samples was extracted from the TCGA.It was demonstrated that ETBM targets exhibit abnormal expression in human breast cancer patients,underlining the clinical significance of this study.Conclusion:ETBM can inhibit the tumor growth and metastasis of TNBC.The possible mechanism is that it could suppress the expression of ITGB1,ITGB8 and ARHGAP5,which leads to weakened activation of several important kinases,including FAK,Src and AKT.