Parthenolide Down-Regulates PD-L1 Expression In A549 Cells By Inhibiting HIF-1A And RAS

Background: In non-small cell lung cancer(NSCLC),due to the high expression of programmed cell death ligand 1(PD-L1),immunosuppression will occur,resulting in inactivation of T cells,which in turn leads to tumor cell survival.At the same time,the high expression of PD-L1 also promotes the proliferation and development of lung cancer cells in vivo.Parthenolide is a sesquiterpene lactone monomer compound extracted from traditional Chinese herbal medicines,parthenium and tansy,which possesses potent anti-inflammatory and analgesic activity.Moreover,parthenolide also exhibits obvious anti-cancer activity,but its specific anti-cancer mechanism is still unclear.The purpose of this study is to clarify whether parthenolide can exert its antitumor effect by regulating PD-L1 protein in non-small cell lung cancer cells.Methods:(1)MTT assay,molecular docking experiment,RT-PCR experiment,western blotting experiment,and cell surface staining experiment were used to investigate the effects of parthenolide on PD-L1 protein and RNA expression in A549 cells,and analyze the effect of PD-L1 on the surface of A549 cell membrane.(2)The effect of parthenolide on the metabolism of PD-L1 in A549 cells was studied by using western blot experiments,ubiquitination experiments,and Lyso Tracker Red lysosomal staining experiments.(3)The effects of parthenolide on the interaction between HIF-1α and RAS and their signaling pathways in A549 cells were explored by molecular docking experiments,western blotting experiments,co-immunoprecipitation experiments,plasmid transfection and immunofluorescence experiments.(4)A549/T cell co-culture model experiments,surface staining experiments and enzyme-linked immunosorbent assay experiments were used to analyze the effects of parthenolide on the activity of T cells.(5)The effects of parthenolide on tumor cell development and endothelial cell angiogenesis were detected by western blotting,cell colony formation,wound healing,Matrigel transwell invasion and tube formation experiments.Results:(1)Parthenolide inhibited the expression of PD-L1 in the A549 cells.(2)Parthenolide reduced the synthesis of PD-L1 and activates the lysosomal pathway to promote PD-L1 degradation.(3)Parthenolide inhibited the expression of HIF-1αprotein in A549 cells by down-regulating the m TOR/p70S6K/e IF4 E signal transduction pathway.(4)Parthenolide inhibits the expression of RAS/RAF/MEK/MAPK-ERK signal transduction pathway in A549 cells.(5)Parthenolide down-regulated the expression of PD-L1 by synergistically inhibiting HIF-1α and RAS.(6)Parthenolide restored the killing ability of cytotoxic T cells.(7)Parthenolide down-regulated the expression of PD-L1 and inhibited tumor cell development and endothelial cell angiogenesis.Conclusion: Parthenolide inhibits the synthesis and expression of PD-L1 by inhibiting the HIF-1α and RAS signaling pathways and interactions.And parthenolide promotes the biological activity of lysosomes and accelerates the degradation of PD-L1 protein through Mi T/TFEs-related proteins.Parthenolide enhances the activity of T lymphocytes by inhibiting the expression of PD-L1 on the surface of tumor cells,which restores the killing ability of tumor cells.Parthenolide inhibits tumor proliferation and angiogenesis by inhibiting the expression of PD-L1.This research provides a new theoretical idea and basis for the development of parthenolide-related PD-L1 small molecule inhibitors.