Mechanism Of Pyrvinium Pamoate Inhibiting Glioblastoma Invasion By Interfering With GLI1 Nuclear Translocation

Purpose:To clarify the anti-tumor effect of PP in GBM,investigate the effect of PP on the migration and invasion of GBM cells,and clarify the role and mechanism of GLI1 in PP changing the migration and invasion of GBM cells.Methods:1.Culture GBM cell lines U87 MG and T98 G,the CCK8 experiment was conducted to detect the toxic effect of PP on GBM cells,calculate the IC50,and determine the concentration and action time for subsequent experiments.2.The effect of PP on the proliferation of GBM cells was detected by cloning experiment.3.Tunel fluorescence staining was used to detect the effect of PP on the apoptotic ability of GBM cells,and Green Nuc Caspase-3 activity detection kit and Western Blot were used to detect the change of Caspase-3 content under the action of PP.4.The effects of PP on the migration and invasion of GBM cells were detected by wound healing test and in vitro cell invasion test（Transwell）.The protein contents of Twist1,Snail,Slug,MMP2,and MMP9 were detected by Western Blot.5.TGF-β was added to promote the EMT of GBM cells,and Western Blot was used to test the effect of PP on the migration and invasion ability of GBM cells under the above conditions.6.Immunofluorescence is used to detect the content and distribution changes of GLI1 in GBM cells treated with PP,to extract nuclear and cytoplasmic proteins,respectively.Western Blot is used to detect the corresponding content changes of GLI1.7.SiRNA was used to knock down the expression of GLI1 in U87 MG,and a GLI1 overexpressed T98 G cell line was constructed,screened and identified.After PP was added,Western Blot was used to detect the changes of protein contents such as Twist1,Snail,Slug,MMP2 and MMP9.Results:1.The toxic effect of PP on GBM cells is time-dependent and concentration-dependent.With the increase of drug concentration and the action time of drugs,the cell survival rate is lower.2.PP can inhibit the proliferation of GBM cells and promote their apoptosis.With the increase of drug concentration,the contents of Tunel fluorescence,Green Nuc fluorescence and Cleaved-Caspase3 protein also increase.3.The results of wound healing test and Transwell test indicated that the migration and invasion of GBM cells were significantly inhibited by PP,and the protein contents of Twist1,Snail,Slug,MMP2 and MMP9 were decreased accordingly.After the addition of TGF-β,PP could still inhibit the migration and invasion of GBM cells.4.Immunofluorescence staining showed that after PP was added,the content of GLI1 in the nucleus of GBM cells was decreased,while that in the cytoplasm was increased.Nuclear and cytoplasmic proteins were extracted separately,and Western Blot test results also revealed this change.5.GLI1 is relatively highly expressed in U87 MG and relatively low in T98 MG.After the GLI1 in U87 MG cell line was knocked down by siRNA,the changes of protein content such as Twist1,Snail,Slug,MMP2 and MMP9 were consistent with the treatment with PP.However,in T98 G cell line which utilizes lentivirus to overexpress GLI1,the overexpression of GLI1 can partially resist the inhibition effect of PP on GBM cells.Conclusion:1.PP can inhibit the proliferation of GBM cells and induce apoptosis of GBM cells.2.PP inhibits the migration and invasion of GBM cells by interfering with the nuclear translocation of GLI1 and preventing GLI1 from entering the nucleus,which can also be exerted under the condition of TGF-β induction.