Unravelling The Cytotoxicity Of C₂N Nanosheets And The Underlying Mechanism

Purpose:Isocitrate dehydrogenases(IDH)are important enzymes in the tricarboxylic acid cycle.They catalyze the oxidative decarboxylation of isocitrate to a-ketoglutarate(?-KG)to produce NADPH and carbon dioxide.IDH1 mutation transforms the catalytic pocket of the enzyme and lead to a neomorphic ability to produce 2-hydroxyglutarate(2HG)while oxidizing NADPH to NADP+.2HG accumulation competitively inhibits a-KG-dependent dioxygenases,including histone lysine demethylases and DNA demethylases.IDH1 mutations have been suggested as an important early event in tumorigenesis and clinical studies have shown that patients with IDH 1 mutations responsed better to chemoradiotherapy,IDH1 mutations are well-represented in WHO class ? and ? gliomas and secondary glioblastomas as a good prognostic marker.However,the mechanism is still unclear.We intended to explore the effects and mechanisms of IDH1 R132H mutation on the radiosensitivity of glioma cells.Methods:Firstly,The IDH1 R132H overexpression plasmid was constructed and transfected into glioma cells.The cck8 cell proliferation assay was used to detect the effect of IDH1 R132H mutation on the proliferation of glioma cells.Clonal formation assay to detected clone survival fraction of IDH 1 R132H and vector glioma cells after 0,2,4,6,and 8 Gy irradiation.Immunofluoresence assay was carried out to quantify?-H2AX foci of control group and IDH1 R132H group in 4 h and 12 h post-IR,and observed the effect of IDH1 R132H on DNA double-strand break repair of glioma cells.Cell cycle experiments were performed to observe the effect of IDH1 R132H on cell cycle arrest of glioma cells induced by ionizing radiation.The effect of IDH 1 R132H on the expression of TIGAR was detected by Western blot.Finally,the mechanism of IDHI R132H mutation on expression of TIGAR protein was detected by ChIP assay.Results:The IDHI R132H overexpression plasmid was successfully constructed.Cell proliferation experiments showed that IDH1 R132H mutation reduced the proliferation of glioma cells.Clonogenic assay revealed that the survival fractions of U87and T98G cells Transfected with IDH1 R132H were significantly lower than that transfected with vector.The immunofluorescence experiments showed that the IDH1 mutation delayed the disappearance of ?-H2AX foci,and the number of ?-H2AX foci in the mutant group at 4 h and 12 h after X-ray irradiation was higher than that in the control group.So IDH1 R132H mutation can delay the process of DNA damage repair.Cell cycle experiments showed that IDH1 R132H mutant were able to delay the cycle arrest caused by ionizing radiation.In control group and IDH1 R132H group,12 hours after 4Gy irradiation,the G2/M phase of the IDHI R132H group was twice as high as that of the vector group.Western blot analysis showed that IDH1 R132H mutation could down-regulate the expression of TIGAR protein,and AGI-5198,a specific inhibitor of IDH1 R132H,could reverse this down-regulation.The results of the Chip experiment indicated that the IDH1 R132H mutation can down-regulate TIGAR protein expression by up-regulatirtg the TIGAR promoter histone H3K9me3 methylation.IDHI mutation inhibited DSB repair caused by ionizing radiation,down-regulate the expression of TIGAR,and increase the radiosensitivity of glioma cells.Conclusion:In conclusion,this study demonstrated that IDH1 R132H mutation reduced the proliferation of glioma cells,and the survival fraction of IDH1 R132H mutant glioma cells was lower than that of wild-type cells upon ionizing radiation exposure.IDH1 R132H can inhibit DSB repair and mutant cells were more sensitive to ionizing radiation,possibly by down-regulating TIGAR expression via epigenetic modification.