Research Report On Anti-tumor Pharmacodynamics Of Formononetin From Astragalus Membranaceus

Background:Astragalus membranaceus is a traditional Chinese medicine that regulates blood sugar levels,suppresses inflammation,protects the liver,and enhances immunity.In addition,A.membranaceus is also widely used in diet therapy and is a well-known health tonic.Formononetin is a natural product isolated from A.membranaceus that has multiple biological functions,including anti-cancer activity.However,the mechanism by which formononetin inhibits tumor growth is not fully understood.Aim of the study:We mainly discussed the effect of formononetin on the expression of PD-L1 in cervical cancer cells.To clarify the potential mechanism of formononetin against tumors.To provide an effective basis for the full development and utilization of Astragalus on cancer prevention.Materials and methods:(1)Molecular docking experiments were used simulate the interaction between formononetin and PD-L1,MYC or STAT3;(2)We explored the effect of formononetin on PD-L1 expression by flow cytometry,RT-PCR and Western blot;(3)We investigated whether formononetin is involved in the synthesis and degradation of PD-L1 through protein immunoprecipitation;(4)We proved that formononetin through inhibition MYC and STAT3 protein synthesis to reduce the expression of PD-L1 by Western blotting;(5)We proved that formononetin promotes the degradation of PD-L1 through increasing lysosome excretion by western blotting and immunofluorescence;(6)We explored the effect of formononetin on the activity of T lymphocytes by T cell killing assays;(7)We detected the effect of formononetin on cell proliferation and apoptosis through cell Colony experiments,Edu experiment and apoptosis experiment;(8)We tested the effect of formononetin on cell invasion,migration and angiogenesis through chamber invasion,scratch and tube formation experiment;(9)We further examined the effect of Astragalus extract and formononetin on cervical cancer by xenograft model in vivo.Results:(1)Astragalus extract inhibited the protein expression of PD-L1,and formononetin played an important role;(2)Formononetin inhibits the activation and interaction of STAT3 and MYC signalings,consequently inhibits PD-L1 expression;(3)Formononetin promoted PD-L1 protein degradation via TFEB and TFE3-mediated lysosome biogenesis;(4)Formononetin could enhance the activity of cytotoxic T lymphocytes(CTLs)and restore ability to kill tumor cells in a co-culture system of T cells and tumor cells;(5)Formononetin inhibited cell proliferation,tube formation,cell migration,and promoted tumor cell apoptosis by suppressing PD-L1;(5)Astragalus extract and formononetin inhibited tumor growth in a xenograft model.Conclusion:(1)Formononetin inhibited the expression of PD-LI in human cervical cancer cells through STAT3 and MYC signaling pathway;(2)Formononetin promoted PD-L1 protein degradation via lysosome biogenesis.These studies reveal the anti-tumor potential of formononetin,which are helpful for further research and development of effective PD-L1 inhibitors for cervical cancer.