Inhibition Of TCTP Enhances Radiosensitivity Of Human Glioma Cells Through Activation Of Autophagy

Glioma is the most common malignant tumor of the central nervous system,which accounts for 31.1% of the incidence of primary brain tumors in China.Glioblastoma(GBM,Glioblastoma Mutiforme)is the most malignant glioma in astrocytic tumors,accounting for about 30% of gliomas.GBM tends to grow invasively and is not possible to distinguish with normal brain tissue clearly.In light of the poor surgical results,postoperative radiotherapy combined with temozolomide adjuvant chemotherapy is still the standard GBM treatment program.Despite the rapid development of glioma radiotherapy in recent years,the overall prognosis remains unsatisfactory due to the radiotherapy resistance.According to statistics,the current median survival of GBM patients in China is only 14.4 months.Studies have shown that the death of glioma cells is mainly through autophagy rather than apoptosis,and therefore,to improve the sensitivity of glioma radiotherapy through exploring the intervention of autophagy in GBM cell effectively has become a hot topic nowadays.TCTP is a highly conserved,ubiquitous protein and has been identified to involve in processes such as transcription,protein synthesis,cell cycle regulation,cytoskeleton,immune response,development and cancer.Besides,we and others identified translationally controlled tumor protein(TCTP)as a clinical prognostic factor in glioma and validated the role of TCTP in the regulation of proliferation and migration ability of glioma cells.Although there is no direct evidence that TCTP involves in the regulation of autophagy in glioma cells,studies have shown that TCTP can regulate the activity of mTORC1 through AMPK or some other pathways in hypoxia or diabetic conditions.Besides,it can also act as a guanylate exchange factor to activate m TORC1.Therefore,this study aims to investigate whether TCTP involves in the regulation of autophagy in irradiated glioma cells and its effect on radiotherapy sensitivity of GBM cells.The change of autophagy flux in irradiated glioma cells was monitored by AVO staining,autophagy specific staining,mRFP-GFP-LC3 adenovirus transfection,scanning electron microscopy and Western blot.Then,to evaluate the effect of TCTP on autophagy,TCTP was inhibited pharmacologically or genetically.Besides,the migration ability was analyzed through wound healing and transwell methods and then,we adopted MTT analysis,colony-forming assays and xenograft model in nude mice to investigate the role of TCTP on cell proliferation.First,through searching the TCGA database and analyzing data from 609 cases of GBM patients,we found that TPT1 mutation(mainly deletion)significantly prolonged the median survival and disease-free survival time of GBM patients.And the mutation could increase the average expression of TP53 and PTEN and decrease the expression of EGFR.Second,5-7 days after irradiation compared with the normal group,GBM cells irradiated by 6Gy X-ray showed more red fluorescence with AVO staining and more blue bright spots with autophagy specific staining and more autophagosomes observed through scanning electron microscopy.Third,pharmacological or genetical inhibition of TCTP activates autophagy 48 hours after irradiation.Then,as for the investigation of mechanisms on autophagy regulation by TCTP,we demonstrated that TCTP directly binds to Beclin 1 complex for the first time and this interaction inhibited the interaction between Beclin 1 and Vps34.Besides,it was shown that inhibition of TCTP attenuated mTORC1 activity through the activation of ATM-AMPK pathway.Moreover,we screened out two available TCTP inhibitors: Thioridazine(TDZ)and Dihydroartemisinin(DHA),which can freely penetrated blood brain barrier and demonstrated their effects on autophagy induction and radiosensitizing both in vivo and vitro.We,therefore,highlighted TCTP as a novel radiosensitizer for GBM patients.