Study On The Mechanisms Of TIGAR Regulated Radiosensitivity Of TrxR1-overexpressing Glioma Cells

Objective:The up-regulation of thioredoxin reductase-1(TrxR1)is detected in more than half of gliomas,which is significantly associated with increased malignancy grade and recurrence rate.Our previous work has proved that TP53 induced glycolysis and apoptosis regulator(TIGAR)knockdown could notably radiosensitize glioma cells.However,whether TrxRl-overexpressing glioma cells could be re-radiosensitized by TIGAR silence is still far from clear.Methods:TrxRl was subcloned into pcDNA3.1 vector,named pcDNA3.1-TrxR1.In order to establish glioma cells stably overexpressing TrxRl,after transfection,cells were treated with G418 for 1 week.Western blot was performed to investigate the protein expression levels post TrxR1 overexpression.The proliferation and invasion activity of TrxR1 overexpressing cells was measured by CCK-8 assay and Transwell assay,respectively.After irradiation,survival rates of glioma cells underwent TrxR1 overexpression and TIGAR lowexpression were determined by clonogenic survival assay.The nuclear translocation of Trx1 in irradiated glioma cells was illustrated by Western blot assay and immunofluoresence assay,meanwhile the process of DNA damage repair was also detected.ROS levels were measured by flow cytometric analysis.NADPH level was detected by NADP/NADPH quantitation kit according to the manufacture's introduction.Finally,animals were anesthetized and underwent stereotactic injections of U-87MG glioma cells co-treated with TrxR1 overexpression and TIGAR shRNA transfection.Contrast enhanced MRI scans were carried out using a 3 Tesla clinical MRI scanner with a special coil designed for small-animal imaging.In order to evaluate the radiosensitizing efficacy of TIGAR abrogation,representative sections from each specimen were immunohistochemically stained with TIGAR,Trx1 and TrxR1 antibodies.Results:(1)TrxRl overexpression enhanced the invasive activity and diminished the radiosensitivity of glioma cells.(2)TrxR1 overexpression-induced radioresistance of glioma cells was reversed by TIGAR interfering.(3)TIGAR silence inhibited IR-induced Trx1 nuclear translocation in glioma cells with TrxRl overexpression.Although TrxR1 was overexpressed,both the level of nuclear Trxl expression and the radioresistance of Trx1 mutant U-87MG glioma cells were not enhanced at all.(4)TIGAR knockdown disturbed the pro-oxidant-antioxidant balance in TrxR1-overexpressing glioma cells post IR.Whilst,TIGAR knockdown induced NADPH depletion in irradiated glioma cells no matter whether TrxRl was overexpressed or not.(5)TIGAR silencing postponed the process of DNA damage repair(DDR)in TrxR1-overexpressing glioma cells.(6)Postradiotherapy,TIGAR low-expression predicted significant longer survival time for animals suffering from TrxR1-overexpessing xenografts,suggesting TIGAR abrogation might be a promising strategy for radiosensitizing TrxRl-overexpressing glial tumours.The mechanism depended on the inhibition of Trx1 nuclear translocation induced by TIGAR knockdown.Conclusions:In conclusion,this study demonstrated the radiosensitizing effect of TIGAR knockdown on TrxRl-overexpressing glioma.TrxR1 overexpression predicted shorter survival time for animals suffering from xenografts.TIGAR knockdown could notably inhibit IR-induced Trx1 nuclear translocation both in vitro and in vivo,and prolong the survival time of nude mice bearing TrxR1-overexpressing gliomas,which supported the idea that TIGAR abrogation might be a possible adjunctive therapeutic strategy against TrxR1-overexpressing glial tumours.