Research Of Usnic Acid On PD-L1 Expression And Its Anti-cervical Cancer Mechanisms

Background: PD-1/ PD-L1 pathway is abnormally expressed in cervical cancer cells.Moreover,PD-1/PD-L1 blockade reduces the apoptosis and exhaustion of T cells and inhibits the development of malignant tumors.Usnic acid is a dibenzofuran compound originating from Usnea diffracta Vain and has anti-inflammatory,antifungal,and anticancer activities.However,the molecular mechanism of its antitumor effects has not been fully elucidated.The current study aimed to investigate the anticancer activity mechanisms of usnic acid and explored whether it mediates its effect through inhibits PD-L1 expression in cervical cancer cells.Materials and methods:(1)The underlying mechanisms of usnic acid at inhibiting PD-L1 expression were investigated by western blotting,MTT,RT-PCR,homology modeling,and molecular docking;(2)The effects of usnic acid on the function and activity of lysosomes and the translocation of PD-L1 to lysosomes were studied by Lyso Tracker Red staining,immunofluorescence,and western blotting experiments;(3)Changes in the tumor-killing activity of T-cells and the level of membrane PD-L1 were analyzed via T-cell killing assay and flow cytometry;(4)Changes in cellular proliferation and invasion were detected using colony formation,Ed U incorporation,tube formation,migration,and invasion assays;(5)Immunoprecipitation,immunofluorescence,and overexpression plasmid/si RNA transfection experiment were used to study the effects of usnic acid on STAT3 and RAS proteins levels’ interaction,and the JAK/Src/STAT3 signal pathway and RAS/RAF/MEK/ERK signaling pathway cooperatively.Results:(1)Usnic acid decreased the expression of PD-L1 protein and m RNA levels in He La cells;(2)Usnic acid induced Mi T/TFE nuclear translocation through the suppression of m TOR signaling pathways,and promoted the biogenesis of lysosomes and the translocation of PD-L1 to the lysosomes for proteolysis;(3)Usnic acid inhibited the expression of PD-L1 on the surface of He La cells and increased the cytokine levels of TNF-α and IFN-γ in the He La/T cell co-culture model,thereby enhancing the killing activity of co-cultured T cells on tumor cells;(4)Usnic acid inhibited cell proliferation,angiogenesis,migration,and invasion,respectively,by downregulating PD-L1,thereby inhibiting tumor growth;(5)Usnic acid decreased the expression of p Tyr705 STAT3 through JAK1/JAK2/Src signaling pathways;(6)Usnic acid reduced the expression of PD-L1 by inhibiting the RAS/RAF/MEK/ERK signaling pathway;(7)Usnic acid inhibited PD-L1 protein synthesis by reducing STAT3 and RAS pathways cooperatively.Conclusions: Usnic acid inhibits PD-L1 expression and tumor proliferation in human cervical cancer cell by downregulating the STAT3 and RAS signaling pathways.These studies reveal that usnic acid is an effective inhibitor of PD-L1 and our study provides novel insights into the mechanism of its anticancer targeted therapy.