Effect And Mechanism Of PARP Inhibitor Niraparib On Radiosensitivity Of Glioma

Glioblastoma（GBM）is the most common primary intracranial tumor,although treatment of Stupp significantly prolonged progression-free survival（PFS）and overall Survival（OS）of GBM,the 5-year Survival rate is still below 10%.Poor prognosis of GBM is closely related to resistance to radiotherapy and chemotherapy,few drugs were used to treat GBM in clinical guidelines,and tumor cells were prone to drug resistance.PARP inhibitors could cause synthetic lethality in tumor cells with homologous recombination deficiency via DNA damage repair,and couled also inhibite the proliferation of tumor cells with intact homologous recombination repair ability by affected ribosome biosynthesis.There are no reports on PARP inhibitor Niraparib combine with radiotherapy for GBM.In our study,we found that Niraparib could increased radiosensitivity of A172 and U87 cells,had a radiosensitizing effect in GBM.The radiosensitization mechanism of Niraparib in GBM cell lines had been defined preliminary,and provided a theoretical basis for clinical treatment of GBM.Objective:The purpose of our study is to determine whether PARP inhibitor Niraparib can increased radiosensitivity of GBM cell lines and to explore the mechanism of Niraparib as a radiosensitizer.Methods:（1）The mechanism of PARP inhibitor monotherapy in glioma was analyzed by bioinformatics analysis,and the signaling pathways involved in PARP inhibitor combine with radiotherapy in GBM were predicted;（2）CCK-8 was used to determine the inhibitory effect of Niraparib on GBM cell lines,and the optimum concentration and duration of Niraparib were obtained;（3）Clonogenic assay was used to determine radiosensitizing effect of Niraparib in GBM cell lines.Flow cytometry was used to research the effect of Niraparib combine with radiotherapy on cell cycle and apoptosis of GBM cell lines;（4）Based on the results of bioinformatic analysis,DDX21 was presumed to be a key molecule in the radiosensitization of Niraparib in GBM.Western blot,CCK-8 and immunohistochemistry were used to investigate the effect of DDX21 in GBM;（5）Western blot,immunofluorescence and clonogenic assay were used to investigate the effect of DDX21 on the radiosensitization of Niraparib in GBM,and to further research the mechanism of DDX21 in the radiosensitization of Niraparib.Result:（1）Genes that affected drug susceptibility to PARP inhibitors in glioma were involved in ribosome biosynthesis pathway and mitosis pathway,radiotherapy resistance of glioma was negatively correlate with homologous recombination repair,ribosome biosynthesis and cell cycle;（2）Niraparib inhibited the proliferation of GBM cell lines（A172,U251 and U87）;The optimumand duration of Niraparib was 48h,the IC₅₀values of Niraparib treated for 48h in A172,U251 and U87 cells were 10.44±3.31μM,18.91±2.95μM and 31.69±2.84μM;（3）Niraparib had radiosensization effects in A172 and U87 cells of p53 wild-type,but had no effect on radiosensitization in U251 cell of p53 mutant.Niraparib increased radiosensitivity through G2/M phase arrest in A172 and U87 cellls;（4）DDX21 was differentially expressed in GBM cell lines and paraffin-embedded tissues from GBM patient’s tumor.DDX21 was not expressed in A172 cells and was highly expressed in U251 and U87 cells,and knockdown of DDX21 in U87 cells,can inhibited U87 cell proliferation.The expression of DDX21 was positively correlated with the expression of Ki67 in paraffin-embedded tissues from GBM patient’s tumor tissue（r=0.428,p=0.029）;（5）In U87 cells,Niraparib combined with irradiation didn’t affect the protein expression of DDX21,but it could lead to the redistribution of DDX21 from the nucleolus to the nucleoplasm.Knockdown of DDX21 in U87 cells with high expression of DDX21 resulted radiotherapy resistance,and Niraparib still has radiosensitization effect in U87 cells after DDX21 knockdown.Conclusion:Niraparib had radiosensitizing effect on HR repair intact GBM cell lines（A172 and U87）.Niraparib affected ribosome biosynthesis via redistribution of DDX21 from the nucleolus to the nucleoplasm,caused G2/M phase arrest,thus increased the radiosensitivity.