Effects Of Caffeic Acid Para-nitro Phenethly Ester On Growth And Metastasis Of Triple-negative Breast Cancer

Triple-negative breast cancer(TNBC)is a heterogeneous disease that has the characteristics of high degree of malignancy,strong invasion and rapid distant metastasis.Metastasis is the main cause to death of TNBC and the processs of metastasis is very complex that accompanied translational abnormal proteins that they have been became molecular targets for tumor treatment.Over activated epidermal growth factor receptor(EGFR)had been found in TNBC,and it as an important representative of these proteins that can activate multiple signaling pathways in tumor,which they play important roles in promoting tumor cell proliferation,invasion and angiogenesis.Therefore,biopharmaceutical analysis of endogenous active proteins expression of EGFR relative proteins in TNBC have important significances for the evaluating development of TNBC and the monitoring of drug therapeutic effects.Previous studies have found that caffeic acid p-nitro-phenethyl ester(CAPE-p NO2)can promote cancer cell apoptosis and inhibit tumor growth through p53 signaling pathway in colon cancer,and regulate apoptosis-related proteins in cervical cancer.The effects on migaration and invasion in cancers have not been addressed.This study was based on TNBC.A series of cell experiments including MTT,wound healing,Transwell,colony formation and adhesion assays were used to analyze the effects of CAPE-p NO2 on MDA-MB-231 cell viability,migration,invasion,colony and adhesion to Matrigel.MDA-MB-231 cell xenograft mice were constructed and tumor weight,tumor volume,tumor growth volume curves,HE and TUNEL staining were used to evaluate the effects on metastasis,growth and apoptosis of TNBC by CAPE-p NO2.And expression of related proteins in the EGFR/STAT3/Akt pathway in vitro and in vivo after treatment was analyzed by Western blot,and to explore the molecular biological characteristics of migration and invasion.Cell experiments: MTT assay demonstrated that low dosages(2.5,5 and 10 ?M)of CAPE-p NO2 exhibited no significant effect on the viability of MDA-MB-231 cell line,while cell viability was dose-dependently inhibited at higher doses(20,40 and 80 ?M).The results of wound healing showed that width of the wound of CAPE-p NO2 was narrower than the control,implying the migration ability of the cells was attenuated.The cells in the lower layer of the Transwell chamber were lesser than the control,and it suggested that CAPE-p NO2 could significantly inhibit the migration and invasion of tumour cells.Plate colony showed that CAPE-p NO2 could reduce the number of colony spots and inhibit the growth of spots,indicating CAPE-p NO2 decreased the cells colony formation ability,and the results of adhesion assay indicating that CAPE-p NO2 declined the adhesion ability of cells.Moreover,the results of Western blot showed that CAPE-p NO2 significantly down-regulated p-EGFR,p-STAT3 and p-Akt.MMP-2,MMP-9,Survivin and VEGFA were also down-regulated.The levels of EMT-related proteins(N-cadherin,Vimentin and E-cadherin)were also regulated to varying degrees.Xenograft mice experiments: Xenograft TNBC mice as the research objects that were successfully constructed using female BALB/c nude mice by subcutaneous injection of MDA-MB-231 cells.CAPE-p NO2 was intraperitoneally injected once a day for 38 days,and then the xenograft mice were sacrificed.The results of tumor weight,tumor volume and tumor growth volume curve showed that CAPE-p NO2 can significantly inhibit tumor growth.The results of HE and TUNEL staining showed that CAPE-p NO2 could promote obviously tumor cells apoptosis in tumors tissues.Histopathological analysis by HE staining showed that CAPE-p NO2 decreased the number of the pulmonary and splenic metastatic tumor cells.The results of IHC demonstrated that the VEGFA and Ki-67 proteins expression were also decreased in tumor tissues.And,the results of Western blot showed that CAPE-p NO2 significantly down-regulated showed that CAPE-p NO2 significantly down-regulated p-EGFR,EGFR,p-STAT3 and p-Akt.MMP-2,MMP-9,Survivin and VEGFA were also down-regulated.The levels of EMT-related proteins(N-cadherin,Snail,Vimentin and E-cadherin)were also regulated to varying degrees.In conclusion,these results demonstrated that CAPE-p NO2 had obvious effects on endogenous metastatic and growth-associated proteins expression of EGFR/STAT3/Akt pathway in vivo and in vitro,and it mainly reflected that CAPE-p NO2 significantly inhibited metastasis and growth of TNBC.