Irigenin Inhibits Glioblastoma Progression Through Suppressing YAP/β-catenin Signaling

Background:Glioblastoma multiform（GBM）is the most common primary malignant tumor in the brain.It is extremely aggressive and diverse,with a 5-year survival rate of only about 5%.Currently,very little research has been done on GBM.The treatment of GBM is also severely limited by the blood-brain barrier（BBB）and the suppression of the immune system.Patients often require a combination of treatments including surgery,radiation and chemotherapy,but the results are poor.TMZ,as a first-line chemotherapy drug for GBM,which is always found to be resistant after treatment,and the high recurrence rate also makes the curative effect very limited.Therefore,exploring drugs with good efficacy and low toxicity to treat GBM will be urgent to improve patients’survival and prognosis.As an isoflavone compound,irigenin has potential anti-GBM activity,but the specific efficacy and mechanism need to be further investigated.Objective:So far,the role of irigenin in GBM and its mechanism are still unclear.Therefore,this study will focus on the role of irigenin in GBM and the regulatory mechanism of YAP/β-catenin on GBM.Methods:（1）C6 and DBTRG cell lines were used as experimental subjects to detect the activity of GBM cells by cell viability assay kit（CCK8）,and the effect of irigenin on the proliferation of GBM cells was detected by the colony formation assay and immunofluorescence staining of PH3 cells.（2）The effect of irigenin on the migration of GBM cells was detected by wound healing test and transwell test;the m RNA expression of MMP-2 and MMP-9 was detected by real-time fluorescence quantitative PCR.（3）Fluorescent dye propidium iodide（PI）staining,flow cytometry,and Cleaved-Caspase3 cell immunofluorescence staining were used to detect the effects of irigenin on the cell cycle and apoptosis of GBM cells.WB was used to detect the expression levels of apoptosis-related factors as well as cell cycle proteins in GBM cells of irigenin.（4）WB and immunofluorescence were used to detect changes in protein expression levels of YAP/β-catenin pathway-related factors under the influence of irigenin.（5）The interactions of irigenin with YAP andβ-catenin were investigated by molecular docking,including the analysis of the mode of action between them.（6）YAP overexpression plasmid was transfected into C6 cells to obtain stable expression cell line.The important role of YAP in the inhibition of GBM by irigenin was studied by CCK8 test,wound healing test,transwell test,PH3 cell immunofluorescence staining and WB.（7）In the model of GBM orthotopic transplantation,the three-dimensional bioluminescence imaging system in small animals and immunohistochemical staining were used to study the effect of irigenin on GBM in vivo.Results:（1）Set different drug concentration gradients,and the CCK8 experiment detected the IC₅₀ value at 24 and 48 hours.The results showed that irigenin could inhibit the proliferation of GBM cells in dosage and time-dependent manner.PH3immunofluorescence staining,and colony formation assay also showed that the irigenin under the IC₅₀concentration significantly inhibited the proliferation of GBM cells.（2）Flow cytometric assay and WB results showed that irigenin could triggered GBM cell cycle arrest at G2/M.（3）Irigenin induced apoptosis of GBM cells by flow cytometric assay,WB,Cleaved-Caspase3 cell immunofluorescence staining,PI staining results.（4）The wound healing experiment and the transwell experiment showed that under the influence of the drug IC₅₀ concentration,the migration capacity of GBM cells was reduced compared with the control group.The results of q PCR experiments showed that the m RNA expression of MMP-2 and MMP-9 were down-regulated,which was also associated with reduced migration ability.（5）YAP cell immunofluorescence staining and WB results showed that irigenin reduced the expression of YAP in GBM cells.At the same time,the WB results also showed that the protein expressions of the YAP channel-related signal molecules were also changed.（6）β-catenin cell immunofluorescence staining and WB results showed that irigenin down-regulated the expression ofβ-catenin in GBM cells.（7）CCK8 experiments,wound healing experiments,transwell,PH3 and WB results showed that overexpression of YAP partially reversed the anti-GBM effect of irigenin,confirming that YAP was the key target of irigenin-induced GBM cell death,which would cause the protein expression change ofβ-catenin.（8）The results of molecular docking also further proved that irigenin has a strong affinity with YAP andβ-catenin.（9）The three-dimensional bioluminescence imaging system in small animals and immunohistochemical staining after orthotopic transplantation model of GBM cells in nude mice indicated that irigenin were used to play the role of anti-GBM by inhibiting YAP/β-catenin signal.Conclusion:In this study,we have provided evidence that irigenin inhibited the proliferation of GBM cells in a dose-dependent manner by several assays in vitro.Subsequently,we found that irigenin arrested cell cycle at G2/M phase and induced apoptosis of GBM cells in vitro.In addition,irigenin inhibited the migration of GBM cells.Mechanically,we found that irigenin treatment decreased the expression of YAP（yes-associated protein）,suppressedβ-catenin signaling.Furthermore,overexpression of YAP partially restored the anti-tumor effects of irigenin on GBM cells in vitro.Finally,we found that irigenin inhibited the growth of tumor in GBM xenograft mice model through inactivation of YAP.Taken together,these results suggested that irigenin exerts its anticancer effects on GBM via inhibiting YAP/β-catenin signaling,which may provide a new strategy for the treatment of GBM.