| Background:Glioma is the most common and aggressive type of intrinsic brain tumor,with high malignancy,very poor prognosis and high recurrence and morbidity.Temozolomide(TMZ)is the primary alkylating agent used to treat Glioblastoma multiforme(GBM).Nevertheless,a number of glioma patients are resistant to TMZ.Therefore,there is an urgent need for more effective therapeutic options.Cordycepin(COR)is a natural chemical with anti-tumor effects,although its mechanism of action is poorly understood.Several lines of evidence suggest that O6-methylguanine DNA methyltransferase(MGMT)repairs damaged DNA and contributes to drug resistance to TMZ in gliomas.The Wnt/?-catenin pathway regulates MGMT gene expression,cordycepin regulates Wnt/?-catenin pathway.However,whether cordycepin inhibits MGMT expression by downregulating the ?-catenin pathway and augmenting chemosensitivity to TMZ in glioma cells remains unclear.Objective:In this study,we used glioma cell lines and xenograft glioma models to investigate the roles of ?-catenin in cordycepin-induced MGMT inhibition and reduction of temozolomide resistance and the underlying mechanisms.Methods:In this study,cell viability was assessed using the MTT assay.The long-term toxic effects of cordycepin on glioma cell proliferation were investigated using a colony formation assay.Flow cytometry was used to clarify the glioma cell death model and cell cycle.The redox-sensitive dye DCFH-DA was used to evaluate the levels of intracellular ROS.The DTNB-GSSG reductase recycling assay kit was used to measure the intracellular total GSH.Quantitative real-time PCR was used to investigate MGMT m RNA expression in glioma cells.The role of MGMT and ?-catenin in cordycepininduced glioma cells death and reduction of temozolomide resistance was investigated by cell transfections.Western blot was used to analyze the expressional level of related proteins.Immunohistochemical staining was used to investigate the expressional level of related proteins in glioma xenograft tissue.The anti-glioma effect of cordycepin in vivo was investigated in Wistar rats with intracranial orthotopic xenograft gliomas and nude mice with subcutaneous xenograft gliomas.Results:1.The MTT assay results showed that cell viability decreased with increasing concentrations of cordycepin and with increased duration of treatment.Cordycepin had limited toxic effects on normal cells.The colony formation assay results showed that cordycepin inhibited the growth of human glioma cells.Cordycepin induced apoptosis by decreasing the expression of anti-apoptosis protein Bcl-2 and increasing the expression of the pro-apoptosis proteins Bax,cleaved PARP-1 and cleaved caspase-3,and cordycepin induced cell cycle arrest by down-regulating Cyclin B1.2.Cordycepin inhibited MGMT gene expression in glioma cells.Cordycepin had a synergistic effect with TMZ when they were used in combination in glioma cells.Overexpression of MGMT reversed the synergistic effect of cordycepin and TMZ and that cordycepin-induced reduction of TMZ resistance in glioma cells.Knockdown of ?-catenin reduced the expression of MGMT and augmented TMZ-mediated chemotherapy in glioma cells.3.Cordycepin inhibited the expression of p-GSK-3? and ?-catenin proteins in a dose-and time-dependent manner in glioma cells.Pharmacological inhibition of GSK-3? with CHIR-99021 or overexpression of ?-catenin reversed cordycepin-induced reduction of cell viability,downregulation of ?-catenin and MGMT,increase of apoptosis and reduction of TMZ resistance.4.Cordycepin induced overproduction of ROS in glioma cells.Cordycepin significantly decreased GSH in glioma cells in a dose-dependent manner.After pretreatment with CHIR-99021,the cordycepin-induced increase of intracellular ROS and decrease of intracellular GSH levels in glioma cells was markedly inhibited.Inhibition of ROS with NAC not only rescued the reduction of cell viability but also mitigated ?-catenin and MGMT inhibition and prevented glioma cell apoptosis and reversed the synergistic effect of cordycepin and TMZ.5.Cordycepin significantly inhibited the growth of glioma in vivo.Cordycepin treatment resulted in downregulation of p-GSK-3?,?-catenin and MGMT protein expression in tumor-bearing rats.Combination treatment of cordycepin and TMZ significantly prolonged median survival of tumor-bearing rats compared to the effects of single agents.Conclusions:1.Cordycepin inhibited the viability of glioma cells and augmented chemosensitivity to TMZ in glioma cells in vitro and in vivo.2.MGMT contributed to cordycepin-induced reduction of TMZ resistance in glioma cells.3.?-catenin contributed to cordycepin-induced MGMT inhibition and reduction of TMZ resistance in glioma cells.4.Overproduction of ROS contributed to cordycepin-induced ?-catenin inhibition,MGMT inhibition and reduction of TMZ resistance in glioma cells. |
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