Mechanism Study On The Inhibitory Effect Of Betulinic Acid Combined With Honokiol On Breast Cancer Via Hippo Pathway

Background and Objective: Breast cancer is one of the most common malignancies affecting women.Yes-associated protein(YAP)is the core effector of the Hippo signaling pathway,breast cancer is often accompanied by overexpression and nuclear translocation of YAP.It has been suggested that betulinic acid and honokiol exhibit significant anti-tumor activities on breast cancer cells,but the molecular mechanism remains to be elucidated.This study attempted to compare the inhibitory effects of the two natural compounds on breast cancer cells when administered alone and in combination,and explore whether it exerted anti-tumor activity through Hippo-YAP signaling in breast cancer.Methods: Firstly,MTT,Transwell and flow cytometry were performed to detect the combined effect of betulinic acid and honokiol on cell viability,migration and apoptosis in breast cancer.Then,the mRNA levels of YAP and its downstream related genes affected by the drug combination were examined via qRT-PCR,the protein expression levels of YAP,p-YAP and downstream target genes were detected by Western blot.Meanwhile,the changes of YAP localization in breast cancer cells were measured by immunofluorescence.Furthermore,flow cytometry,Transwell and Western blot were carried out to evaluate whether the roles of betulinic acid and honokiol were affected by the knockdown or overexpression of YAP.Finally,the binding sites of drugs and YAP were predicted by molecular docking and co-immunoprecipitation was used to verify whether the drug combination could block the interaction between YAP and TEAD.Results: The results of MTT,Transwell and flow cytometry showed that the combination of betulinic acid and honokiol could inhibit the growth and migration of breast cancer cells and promote cell apoptosis.The results of q RT-PCR and Western blot assays revealed that the m RNA and protein expression levels of YAP,CTGF and CYR61 decreased due to the drug combination.The result of the immunofluorescence assay showed that the drug combination could inhibit the nuclear translocation of YAP in the cells.Correspondingly,the total abundance of YAP protein and the amount of YAP entering the nucleus in the cells decreased,while the protein expression level of p-YAP in the cytoplasm increased.The overexpression of YAP weakened the effect of the drug combination on cell apoptosis and reversed the inhibitory effect on cell migration and the protein expression level of YAP,while the knockdown of YAP had the opposite effect.The results of molecular docking simulation showed that betulinic acid and honokiol had good binding interactions with YAP.The results of co-immunoprecipitation correspondingly confirmed that the drug combination affected the binding of YAP and TEAD.Conclusion: Our collective results revealed that betulinic acid combined with honokiol could inhibit the proliferation of breast cancer cells,inhibit cell migration and promote apoptosis through the Hippo-YAP pathway.It suggested that the combination of betulinic acid and honokiol may be a promising anti-cancer strategy for the treatment of breast cancer patients.