Study On The Mechanisms Of The Radiosensitivity Of Glioma Cells Affected By TIGAR Regulated Nuclear Translocation Of TRX1

Objective: TP53-induced glycolysis and apoptosis regulator (TIGAR) knockdownis proven to radiosensitize glioma cells, but the mechanisms are not fully understood.Thioredoxin-1(TRX1) is a redox-sensitive oxidoreductase, which plays critical roles inDNA damage signal transduction via nuclear translocation in irradiated cells. Becausethe TRX1-dependent DNAdamage signaling pathway relies on NADPH to maintain thereduced state of TRX1, and TIGAR functions to increase NADPH generation underoxidative stress, in this study, the role of TRX1in TIGAR abrogation-inducedradiosensitization was investigated. Primary astrocytes were used in order to elaboratethe functions of TIGAR in irradiated normal human glial cells. Finally, human xenograftorthotopic models were used to prove whether TIGAR interference could radiosensitizeglioma in vivo.Methods: Protein levels of TIGAR and p53in glioma cells being irradiated weredetermined with Western blot analysis. Real-time PCR revealed the transcriptionalactivity of TIGAR and TP53at different points in glioma cells suffered by ionizingradiation. TIGAR siRNA and pcDNA3.1-TIGAR were synthesized and transfected intoglioma cells to make TIGAR abrogated or over-expressed. Forty-eight hourspost-transfection, glioma cells were irradiated and the reactive oxygen species (ROS)was determined by flow cytometry. Glioma cells over-expressing wild-type (WT) ormutant-type (MT) TRX1were estabolished by transfecting pcDNA3.1-WT-TRX1orpcDNA3.1-MT-TRX1and by G418selection. To evaluate the radiosensitivity of gliomacells suffer by TIGAR interference, clonogenic assay was performed to examine the cellsurvival fractions at14days post-IR, and immunofluoresence assay was carried out toquantify γ-H2AX foci in0.5-12h post-IR. To elaborate the mechanism of TIGARknockdown-induced radiosensitization of glioma cells, IR-induced TRX1nucleartranslocation was determined by Western blot. Redox Western blot indicated the redox state of cytoplasmic and nuclear TRX1in irradiated glioma cells. Finally, by usingTIGAR shRNA lentivirus-based gene therapy, TIGAR abrogation-inducedradiosensitization of glioma in vivo was illustrated by MRI.Results:(1) In wild-type p53-expressing A172cells TIGAR expression wasincreased1h post-IR and decreased to basal level8h post-IR. Meanwhile, p53expression was increased within0.5h post-IR, which was earlier than TIGARupregulation. However, in mutant-type p53-expressing (M237I) T98G cells, thereseemed to be no IR-induced increase in p53and TIGAR expression. Clonogenic assayrevealed that the survival fractions of both A172and T98G cells treated with TIGARsiRNAs were significantly lower than that of parental cells.(2) ROS generation wassignificantly increased by TIGAR abrogation in irradiated glioma cells. Meanwhile,TIGAR interference reduced by approximately75%cellular NADPH in irradiatedglioma cells, compared with a reduction of less than40%in cells irradiated alone.Similarly, the ratio of GSH/GSSG was significantly further decreased by TIGARinterference in cells exposed to IR. In addition, both NADPH level and the ratio ofGSH/GSSG rebounded by TIGAR overexpression. Western blot analysis proved thatTRX1was imported into nuclei in irradiated cells. The peak time of TRX1nucleartranslocation was approximately2h post-IR. However, IR-induced nucleartranslocation of TRX1was strikingly hindered by TIGAR interference.(3) Interestingly,TIGAR silencing-induced blockage of TRX1nuclear translocation could be abolishedby WT-TRX1overexpression. Redox Western blot indicated that in irradiated A172cells, the levels of cytoplasmic oxidized TRX1were raised within0.5h post-IR andreturned to their original reducing state within8h post-IR. TIGAR knockdown coulddelay the reduction process of oxidized cytoplasmic TRX1. Cytoplasmic TRX1remained oxidized at8h post-IR by TIGAR interference. However, WT-TRX1overexpression preserved the reducing state of TRX1in cells treated with TIGARsiRNA. The alteration in redox state of nuclear TRX1in response to IR showed thesame pattern as that of cytoplasmic TRX1. Clonogenic survival assay determined thatTIGAR interference-induced radiosensitization of glioma cells was notably diminishedby WT-TRX1overexpression. Data also revealed that the combination of MT-TRX1overexpression and TIGAR interference could not further radiosensitize glioma cellsany more. Importantly, TIGAR overexpression also could not rescue MT-TRX1overexpression-induced radiosensitization, indicating that TRX1nuclear translocation might play a major role in TIGAR-regulated radiosensitivity of glioma cells.(4) Byusing immunofluoresence assay, it was shown that γ-H2AX foci disappeared within4hpost-IR in control A172cells. However, the γ-H2AX foci remained even at12h post-IRin TIGAR low-expressing cells, indicating the delayed DNA damage repair (DDR)process. The nuclear translocation of TRX1was observed with a peak time of2hpost-IR in cells overexpressing scramble siRNA. However, in TIGAR-silenced A172cells, there seemed to be no TRX1transported into the nuclei. InWT-TRX1-overexpressing A172cells, both TIGAR-silencing-induced delay of theDDR process and blockage of TRX1nuclear translocation were abolished.(5)Clonogenic survival assay showed that there was no difference in radiosensitivityamong primary astrocytes over-expressing scramble siRNA, pcDNA3.1, TIGAR siRNA,and pcDNA3.1-TIGAR. Although IR-induced TRX1nuclear translocation was observedin primary astrocytes, Western blot assay showed that neither IR-induced TRX1nucleartranslocation nor the reduction process of oxidized TRX1was hindered by TIGARknockdown in primary astrocytes. Data also showed that in primary astrocytes theoxidative levels of both cytoplasmic and nuclear TRX1induced by IR wereconsiderably lower than those in glioma cells.(6) By using TIGAR shRNAlentivirus-based gene therapy, it was proven TIGAR interference could radiosensitizeglioma in vivo.Conclusion: The present study demonstrated that TIGAR knockdown significantlynot only inhibits IR-induced nuclear translocation of TRX1in glioma cells butconsiderably defers the reduction process of oxidized TRX1in irradiated glioma cells.Both the blockage of TRX1nuclear translocation and radiosensitization induced byTIGAR interfering are abolished by wild-type TRX1overexpression. TIGARabrogation considerably radiosensitizes glioma cells but has no effect on radiosensitivityof primary astrocytes. Finally, TIGAR interference might radiosensitize glioma in vivo.