Mechanism Of Cinobufacini Injection Against Triple-Negative Breast Cancer Through Pin1-YAP/TAZ Pathway

Triple negative breast cancer(TNBC)is the most aggressive molecular subtype of breast cancer.It is characterized by the lack of expression of progesterone receptor(PR),estrogen receptor(ER)and human epidermal growth factor receptor 2(HER2).At present,radiotherapy and chemotherapy are mainly used for treatment at home and abroad,but the toxic and side effects are obvious,and the chemotherapeutic drugs are prone to drug resistance.Therefore,there is an urgent need to use more effective and less toxic drugs to treat TNBC.Traditional Chinese medicine has unique advantages in the prevention and treatment of tumors.The traditional Chinese medicine compound contains a variety of active ingredients,which can play a synergistic effect through multiple targets and multiple ways,and the effect is mild.Cinobufacini injection(CI)is a light yellow water-soluble preparation made from the skin of Chinese giant toads.It has the characteristics of broad anti-cancer spectrum,high efficiency and low toxicity.It is clinically used for the treatment of various tumors including TNBC,but its molecular mechanism and target are still unclear.Pin1-YAP/TAZ is a hot signal pathway in the research and development of antitumor drugs in recent years.Among them,peptidylprolyl cis-trans isomerase NIMA-interacting 1(Pin1)is considered to be a new biomarker for the diagnosis of TNBC,Yes-related protein(YAP)/transcriptional co-activator with PDZ-binding motif(TAZ),as two core components of the Hippo pathway,plays a very critical role in tumorigenesis and drug resistance.Some studies have found that Pin1 can interact with YAP/TAZ in TNBC,suggesting that drugs targeting the Pin1-YAP/TAZ signaling pathway may be helpful for the clinical treatment of TNBC,suggesting that drugs targeting the Pin1-YAP/TAZ signaling pathway may be helpful for the clinical treatment of TNBC.First,the effect of cinobufacini injection on TNBC in vitro and in vivo was investigated.Animal experiments showed that cinobufacin injection can inhibit the growth of tumors in tumor-bearing mice,and compared with doxorubicin,cinobufacin injection had no effect on the body weight and organs of mice,suggesting that the drug has less toxic and side effects;The CCK8 and clone formation experiments of TNBC cells showed that Cinobufacini injection could inhibit the proliferation of human MDA-MB-231 cells and mouse 4T1 cells in a time-dose-dependent manner;The detection of cell apoptosis and cycle were performed by flow cytometry,and the results showed that cinobufacini injection could induce TNBC cell cycle arrest,arrest MDA-MB-231 cells in G2/M phase,and arrest 4T1 cells in S phase,and finally induce TNBC cells to undergo apoptosis;The results of western blot confirmed that cinobufacini injection induced cell cycle arrest and apoptosis by down-regulating the levels of CDK1,CDK2 and Bcl-2/Bax proteins.Next,clinical data mining based on the cancer genome atlas(TCGA)database showed that compared with normal tissues,Pin1 and TAZ were overexpressed in breast cancer;compared with other breast cancers,advanced breast cancer The expression levels of Pin1 and TAZ were increased in TNBC,and were most pronounced in stage 4;compared with normal tissues and other breast cancer subtypes,Pin1 and TAZ were more highly expressed in TNBC,and their expression levels were negatively correlated with survival.Although YAP expression was not highest in TNBC patients,higher YAP expression was associated with lower patient survival in TNBC patients.Using immunohistochemistry to dock the virtual data with real cases in the real world,the results showed that the positive expression rates of Pin1,YAP and TAZ in TNBC tissues were 60%,56% and 56%,respectively,compared with paracancerous tissues,in TNBC tissues The positive expression rates of Pin1 and TAZ in TNBC tissues were significantly increased(P<0.05),and YAP was increased,but there was no significant difference(P>0.05);the expressions of Pin1,YAP,and TAZ were not related to some clinicopathological features,such as tumor stage,With or without lymphatic metastasis(P>0.05).Correlation analysis showed that the expressions of Pin1 and YAP(r=0.577,P<0.05)and TAZ(r=0.645,P<0.05)in TNBC patient tissues were significantly positively correlated.In order to further clarify the correlation between Pin1 and YAP and TAZ,we used si RNA to knock down Pin1 in TNBC cells,and the expression of YAP and TAZ was significantly downregulated.The above experimental results all suggest that Pin1-YAP/TAZ is one of the core signaling pathways of TNBC.Finally,the molecular mechanism of cinobufacini injection against TNBC was further studied in vitro and in vivo.First,the RNA-seq results showed that cinobufacini injection can affect multiple signaling pathways in TNBC,and its anti-TNBC mechanism may be related to the regulation of Pin1-YAP/TAZ signaling pathway.The results of western blot showed that cinobufacini injection could not only down-regulate the expressions of Pin1,YAP and TAZ in MDA-MB-231 and 4T1 cells of TNBC cell lines,but also down-regulate the expressions of Pin1,YAP and TAZ in TNBC tumors.The injection exerts an anti-TNBC effect by inhibiting the Pin1-YAP/TAZ signaling pathway.In summary,we used in vivo and in vitro experiments to clarify the efficacy of cinobufacini injection against TNBC by inducing cell cycle arrest and apoptosis;comprehensively using the TCGA database and transcriptomic technology,we explored its potential molecules.Mechanism;for the first time,this study focused on the down-regulation of Pin1-YAP/TAZ signaling pathway by cinobufacini injection,thereby exerting an antiTNBC effect,which not only highlights the importance of Pin1-YAP/TAZ signaling pathway in the occurrence and development of TNBC,but also It provides a new strategy for traditional Chinese medicine treatment of TNBC.