Fraxinellone Inhibits Programmed Cell Death-Ligand 1 Expression By Downregulating STAT3 And HIF-1?

Background:Dictamnus dasycarpus is a traditional Chinese medicine that has been commonly used in the treatment of cancer.Fraxinellone is a natural product isolated from the root bark of the D.dasycarpus plant,which has been shown to exhibit neuroprotective and anti-inflammatory activities.However,fraxinellone inhibits tumor growth molecular mechanisms remain unknown.Aim of the study:We investigated the effect of fraxinellone on PD-L1 expression in lung cancer cells.To elucidate the potential anti-tumor mechanism of fraxinellone.Materials and methods:1.We tested the effect of fraxinellone on PD-L1 expression by performing flow cytometry,western blot,and RT-PCR assays.2.We investigated whether fraxinellone affected STAT3 protein levels by molecular docking,luciferase reporter,immunofluorescence,and western blot assays.We performed western blot assay to detect the effect of fraxinellone on the PD-L1 expression mediated by STAT3 in A549 cells.3.We investigated whether fraxinellone affected HIF-1? protein levels by molecular docking,Luciferase reporter,immunofluorescence,and western blot assays.We performed western blot assay to detect the effect of fraxinellone on the PD-L1 expression mediated by HIF-1? in A549 cells.4.We next investigated the effects of fraxinellone on HIF-1? protein synthesis and degradation by western blot and immunoprecipitation assays.We performed western blot assay to examine the effect of fraxinellone on mTOR/p70S6K/eIF4E and MAPK signaling pathways.5.We tested the effect of fraxinellone on cells proliferation by performing western blot,colony formation,EdU,and flow cytometry assays.6.Western blot,tube formation,migration,invasion assays were performed to examine the effect of fraxinellone on angiogenesis.7.We further examined effect of fraxinellone on human lung cancer in xenograft model in vivo.Results:1.Flow cytometry,western blot,and RT-PCR assays demonstrated that fraxinellone significantly inhibited PD-L1 protein expression in various cells.2.Molecular docking,luciferase reporter,immunofluorescence,and western blot assays showed that fraxinellone inhibited activation of STAT3 via the JAK1,JAK2,and Src pathways.Western blot assay showed that fraxinellone inhibited PD-L1 expression by reducing STAT3 in A549 cells.3.Molecular docking,luciferase reporter,immunofluorescence,and western blot assays showed that fraxinellone inhibited HIF-1? protein expression in various tumor cells.Western blot assay showed that fraxinellone inhibited PD-L1 expression by reducing HIF-1? in A549 cells.4.Western blotting and immunoprecipitation assays demonstrated that fraxinellone inhibited the protein synthesis of HIF-1? but not its degradation.It was subsequently shown that fraxinellone inhibited HIF-1? protein synthesis via the mTOR/p70S6K/eIF4E and MAPK pathways in A549 cells.5.Immunoprecipitation and western blot assays showed that fraxinellone inhibited PD-L1 expression by reducing STAT3 and HIF-1?cooperatively.Western blot,colony formation,EdU,and flow cytometry assays demonstrated that fraxinellone inhibited cells proliferation by suppressing PD-L1.6.Western blot,tube formation,migration,and invasion assays showed that fraxinellone inhibited angiogenesis by suppressing PD-L1.7.In vivo experiments confirmed that fraxinellone treatment caused significant inhibition of tumor growth in a xenografted tumor model.Conclusion:We concluded that fraxinellone inhibits PD-L1 expression by downregulating the STAT3 and HIF-1? signaling pathways,subsequently inhibiting proliferation and angiogenesis in cancer cells.These studies reveal previously unknown characteristics of fraxinellone and provide new perspectives into the mechanism of cancer inhibition of the compound.