Role And Molecular Mechanism Of GSK3β Increases Temozolomide Resistance By Enhance Autophagy In Human Glioma Cells

[Objective]Glioma is the most common type of primary tumors in central nervous system.After surgical excision combined with chemotherapy the prognosis of high grade gliomas is not very well,and easy to relapse and get drug resistance.Therefore,finding the key molecule which is associated with the occurrence and development of glioma for the treatment of glioma is necessary.GSK3β is a multifunctional Ser/Thr kinase,which is expressed in all tissues,and is particularly enriched expression in brain.It is now known that GSK-3β plays a central role in a variety of signaling pathways,such as the Wnt/β-catenin、PI3K/AKT/mTOR、NF-κB、MAPK pathways,and is involved in a wide range of cellular processes,ranging from glycogen metabolism to cell cycle regulation and proliferation.Autophagy is the process of"self-eating",which can degrade proteins and organelles depending on a lysosomal pathway.It has also been reported that GSK-3β can modulate autophagy in human oral squamous cell carcinoma,breast cancer cells and prostate cancer cells.However,whether GSK-3β can affect the autophagy in human glioma cells largely remains unknown.However,the role of GSK3β in glioma development is focus on proliferation,invasion and apoptosis for the present.In this study,we overexpression the GSK-3β in glioma,applied in vivo and in vitro experiment to elucidate the specific role and the underlying mechanism of GSK3β in regulating autophagy of glioma.Furthermore,we investigated whether overexpressing Y216A-GSK-3β or it induced autophagy can effect on cell function.[Methods](1)Human glioma cell lines U87 and U251 were transfected with adenovirus carrying GSK3β,S9A-GSK-3β and Y216A-GSK-3β to build GSK3β,p-GSK-3β(Tyr 216,active)and p-GSK-3(3(Ser9,inactive)overexpressed cells,respectively.(2)Using immunofluorescence,Western blot and electron microscopy to detect the autophagy level of glioma cell before and after GSK3β overexpression.(3)We explore the underlying mechanism of GSK3β in regulating glioma autophagy at the molecular level,and also detect GSK3(3 target autophagy-related gene levels in both U87 and U251 cells.(4)To investigate the role of GSK3β induced autophagy in cell proliferation,apoptosis and cell cycle,we performed the Cell Counting Kit-8(CCK-8)and flow cytometry.We also performed the CCK-8 and colony-forming unit assays to investigate the role of GSK3β induced autophagy in temozolomide resistant.(5)Stable transfected U87 and U251cells were injected subcutaneously into flanks of the nude mice.The relative autophagy responses and GSK3β target gene levels were assayed.[Results](1)In establishment of GSK3β overexpression glioma cell lines,in comparison to the Ctrl and GFP group,the autophagy levels of GSK3β group were significantly increased.Overexpression of GSK3β increases both p-GSK-3β(Tyr 216,active)and p-GSK-3P(Ser9,inactive).(2)GSK3β induced autophagy is attributed to increase of p-GSK3β(Ser9)rather than p-GSK3β(Tyr 216).(3)Overexpression of Y216A-GSK-3β increases the p-GSK3β(Ser9),and increased p-AMPK(Thr172)and p-ULK1(Ser 555).In order to validate that AMPK-ULK1 pathway is involved in p-GSK3β(Ser9)induced autophagy,we analyzed the effect of AMPKa siRNA on autophagy in Y216A-GSK-3β transfected cells.P-AMPK,p-ULK1 and LC3-Ⅱaccumulation significantly reduce after AMPKa knockdown.(4)In vitro experiment,ectopic expression p-GSK-3β(Ser9)did not affect cells apoptosis,cell cycle and cell proliferation of human glioma,but increased the resistant to temozolomide(TMZ).After inhibiting autophagy by 3-MA,temozolomide resistant was reversed.(5)In xenograft experiment,the sizes volume and weight of tumors from Y216A+TMZ group were much larger than those from the GFP+TMZ group.The sizes volume and weight of tumors from Y216A+3-MA+TMZ group was significant small than those from the Y216A+TMZ group.Immunohistochemical results showed that LC3II of Y216A group was significantly increased compared with GFP group.[Conclusion](1)p-GSK-3β(Ser9)can increased autophagy flux in human glioma cells.The mechanism of this phenomenon is attributed to AMPK-ULK1 pathway active.(2)p-GSK-3β(Ser9)induced autophagy is a protective autophagy,which can cause glioma cells resistant to Temozolomide.Moreover,this resistant can be reversed by autophagy inhibitor 3-MA.These findings suggest the potential utility of GSK3β and it induced autophagy for glioma treatment.