Studies On Pyrvinium Pamoate Enhancing The Glioblastoma Sensitivity To Temozolomide Through Regulating ?-catenin

Background: Glioma is the most common primary intracranial malignant tumor in human beings.At present,the overall therapeutic effect of glioma is still poor.In recent years,significant progress has been made in surgical treatment,radiotherapy and chemotherapy of glioma.At the same time,new treatment methods have emerged,such as tumor electric field treatment and immunotherapy,but the clinical efficacy of the above treatment is not satisfactory.Therefore,there is an urgent need for more effective glioma treatment models or drugs.Temozolomide(TMZ)is the first choice for glioma chemotherapy.However,the insensitivity and drug resistance of glioma to TMZ are important reasons affecting the clinical efficacy.Therefore,increasing the sensitivity of TMZ and overcoming drug resistance are the hot spots to improve the therapeutic effect of glioma.The high expression of MGMT and the low methylation level in the promoter region are considered to be important factors for TMZ insensitivity and drug resistance.MGMT is an important target for TMZ sensitization and improvement of drug resistance.Studies have shown that Wnt/?-Catenin pathway regulates the expression of MGMT and participates in the sensitization of TMZ chemotherapy.In recent years,the antitumor effect of pyrvinium has attracted attention,and its mechanism includes the inhibition of Wnt/?-catenin pathway.However,it has not been reported that can pyrvinium inhibit Wnt/?-catenin pathway,regulate MGMT expression or enhance the sensitivity of GBM cells to TMZ.Objective: In this study,we used a variety of glioma cell lines and glioma in situ models in Balb/c nude mice to study the effect of pyrvinium on glioma TMZ sensitivity and its possible molecular mechanism.Methods: In this study,the effect of pyrvinium on cell viability was detected by MTT assay.The effect of drugs on the proliferation of glioma cells was detected by cell cloning experiment.The effects of drugs on the death mode and cell cycle of glioma cells were detected by flow cytometry.Western blot and immunofluorescence were used to detect the changes of related proteins in cells and tissues.The expression level of MGMT m RNA in glioma cells was detected by real-time quantitative PCR.Cell transfection experiment to study MGMT and ?-catenin in drug-induced glioma cell death and temozolomide sensitization.The Wnt pathway was detected by Topflash/Fopflash Luciferase Report experiment.We constructed MGMT promoter reporter gene vector and detected the effect of drugs on the vector.In vivo experiment was used to detect the effect of drugs on glioma in nude mice.TUNEL assay was used to detect the apoptosis of tumor cells in tumor tissues.Results:(1)MTT showed that pyrvinium significantly decreased the survival rate of glioma cells in a concentration and time-dependent manner.Cell cloning experiment showed that pyrvinium inhibited the clone formation of glioma cells.Flow cytometry showed that pyrvinium could significantly promote apoptosis and block cell cycle.Western blot showed that pyrvinium down regulated the protein levels of survivin,caspase 9,cyclin D1 and cyclin B1 in glioma cells,and up regulated the protein levels of Bax,cleaved caspase-3 and cleaved PARP-1.(2)Pyrvinium down regulated the transcription and protein levels of MGMT in a concentration dependent manner.The combination of temozolomide and pyrvinium has synergistic effect on glioma cells.Overexpression of MGMT partially antagonized the inhibition of Temozolomide Combined with pyrvinium on the viability of glioma cells.Knock-down ?-catenin can inhibit the expression level of MGMT and increase the sensitivity of glioma cells to TMZ.(3)Western blot and immunofluorescence showed that pyrvinium reduced the content of in nucleus and cytoplasm ?-Catenin protein level;knock-down ?-Catenin can inhibit the expression level of MGMT and increase the sensitivity of glioma cells to TMZ.Overexpression of ?-catenin can reverse the MGMT inhibition and TMZ sensitization induced by pyrvinium.Pyrvinium inhibited the luciferase activity of MGMT promoter reporter gene vectors of three lengths in a concentration dependent manner.(4)Western blot showed that pyrvinium increased the level of S33/37/T41 phosphorylation in glioma cell lines in a concentration dependent manner.There was no significant trend change in the GSK3? Protein level and phosphorylation level of GSK3? Y216 site.However,the phosphorylation level of GSK3? S9 site decreased with the concentration of pyrvinium.Western blot showed that pyrvinium significantly reduced the level of phosphorylated Akt.Glioma cells pretreated with chir-99021(GSK3? inhibitor)then added with pyrvinium,the down-regulation of ?-catenin was partially inhibited.Pyrvinium decreased the phosphorylation level of ?-catenin ser552 site,(5)In the in situ model of glioma in nude mice,pyrvinium inhibited the growth of glioma and prolonged the median survival time in vivo.Western blot showed that compared with PBS group,the phosphorylation level of Akt,Phosphorylation level of GSK3? S9 site,?-catenin and MGMT protein levels were down regulated in the pyrvinium treatment group.TUNEL test showed that compared with PBS group,apoptosis in tumor tissues and cells in pyrvinium treatment group increased.Pyrvinium and temozolomide have synergistic anti-glioma effect in nude mice in vivo.Conclusion:(1)In vivo and in vitro experiments showed that pyrvinium could inhibit the viability of glioma.Pyrvinium induced apoptosis and cell cycle arrest of glioma cells.(2)Pyrvinium increases the sensitivity of glioma cells to temozolomide by inhibiting the expression of MGMT.(3)Pyrvinium inhibits Wnt/?-catenin pathway and regulates MGMT expression,thereby increasing the sensitivity of temozolomide.(4)Pyrvinium inhibited Wnt pathway by increasing the ?-catenin protein degradation and influencing the distribution of ?-catenin in cells.