Study On The Molecular Mechanism Of 1,3-dicaffeoylquinic Acid Inhibiting The Proliferation And Metastasis Of Breast Cancer Cells

The treatment of breast cancer is the most difficult one.Chemotherapy and radiotherapy have been the most widely used as traditional therapies.However,due to the limitations of traditional chemotherapy and radiotherapy,it has serious side effects on the human body and is not conducive to the recovery of patients.Drug resistance,which has caused treatment failure in some cases.14-3-3τ plays a vital role in the tumorigenesis and metastasis of breast cancer and can be used as a new drug target protein.Dicaffeoylquinic acid(DCQAs)is a natural product with antioxidant,antimicrobial and anti-inflammatory activity.Some studies have shown that certain dicaffeoylquinic acid has a good inhibitory effect on many types of tumors such as gastric cancer,colorectal cancer,melanoma,breast cancer.The development and exploration of the pharmacodynamic effects of dicaffeoylquinic acid will become another hot topic in the development of new drugs.In this study,we investigated the anticancer effect of DCQAs on breast cancer cells MCF-7 and MDA-MB-231 cell lines and the mechanism of action on triple negative breast cancer(TNBC).First,we used biological methods and Surface Plasmon Resonance(SPR)to analyze and screen out 1,3-DCQA and 1,5-DCQA,that can bind to 14-3-3τ and have significant inhibitory effects on breast cancer cells.Then using MTT method,soft agar clone formation experiment,Transwell cell migration and flow cytometry analysis,we found that 1,3-DCQA and 1,5-DCQA can significantly inhibit the proliferation,colony formation and metastasis,induce apoptosis of breast cancer cells;Subsequently,we selected 1,3-DCQA which has the best effect on breast cancer cell inhibition.By analyzing the over expression and knockdown assay of 14-3-3τ in breast cancer cells,we confirmed that 14-3-3τ is one of the 1,3-DCQA target proteins.By sequencing the eukaryotic transcriptome analysis of MDA-MB-231 cells treated with 1,3-DCQA(96 μM,48 h),we determined that 1,3-DCQA binds to 14-3-3τ affect MDA-MB-231 cells mainly through PI3K-Akt,Jak-STAT,and cAMP pathways.Then we found 16 genes overlapping in the three pathways: Creb5,Csf3,Crebbp,Ep300,Ifnar2,Il2 rb,Il3ra,Il6,Osmr,Pik3 cg,Pik3r1,Prlr,Jak2,Bad,Sos1,Sos2 and performed qRT-PCR the verification shows that 1,3-DCQA regulates these 15 genes by combining with 14-3-3τ,among which Il2 rb,Bad,Pik3 cg are significantly up-regulated,Sos1,Sos2,Ifnar2,Csf3,Osmr,Jak2,Il6,Creb5,Pik3r1,Ep300,Prlr,Crebbp are significantly down-regulated,which in turn affects the biological function of breast cancer cells.In addition,we also verified the transcription levels of 15 target genes at 96 μM for 9 h and 24 h,and obtained a dynamic change process,that is,Bad was significantly up-regulated from 9 h to 48 h,and Il2 rb was up-regulated after 24 h.Pik3 cg was up-regulated at 48 h.In addition,Crebbp was up-regulated only at 9 h,and later all were down-regulated,while the remaining 11 genes were significantly down-regulated after 9 h.Flow cytometry results and Western Blot analysis of cell death markers showed that 1,3-DCQA induced breast cancer cell apoptosis and cell pyroptosis with increasing compound concentration.Western Blot analysis of the seven isoforms of the 14-3-3 protein family shows that 1,3-DCQA also regulates the remaining isoforms to varying degrees,in addition to affecting 14-3-3τ.The tumor marker Western Blot analysis showed that 1,3-DCQA significantly reduced the phosphorylation level of key molecules through 14-3-3τ,and reduced the metabolic level of breast cancer cells.Through the analysis and verification of transcriptomics sequencing results,we obtained that the molecular mechanism of 1,3-DCQA inhibiting the proliferation and metastasis of breast cancer cells is as follows: 1,3-DCQA binds to 14-3-3τ and down-regulates JAK,AKT,Raf-1,and mTOR in breast cancer cells,inhibit the protein synthesis and cell proliferation of breast cancer cells;up-regulation BAD to induces apoptosis of breast cancer cells based on the Caspase pathway.In this study,we explored the effects of 1,3-DCQA on the proliferation and metastasis of breast cancer cells by using various experimental methods.The results suggest that 1,3-DCQA can be used as a lead compound against the proliferation and metastasis of breast cancer cells,which can provide a theoretical basis for the development of new targeted drugs for the treatment of TNBC.