Study On Mechanisms Of The Radiosensitivity Of Glioma Cells Affected By TIGAR Regulated Autophagy

Objective: TP53-induced glycolysis and apoptosis regulator(TIGAR) knockdown is proven to induce autophagy in tumor cells. Our previous data reveals that TIGAR abrogation could radiosensitize glioma cells. In this study, the effect of TIGAR-regulated autophagy on radiosensitivity of glioma cells was investigated and the mechanisms were demonstrated. Finally, to assess the relationship between TIGAR expression and the clinical features of malignant glioma patients, the expression level of TIGAR in malignant gliomas and adjacent tissues were detected.Methods: Protein levels of TIGAR and phosphated p53 in glioma cells being irradiated were determined with Western blot analysis. Real-time PCR revealed the transcriptional activity of TIGAR at different points in glioma cells suffered by ionizing radiation. TIGAR si RNA synthesized and transfected into glioma cells to make TIGAR abrogated, while TIGAR-overexpression glioma cells were made by pc DNA3.1-TIGAR transfection. Forty-eight hours post-transfection, glioma cells were irradiated and the redox state was determined. To evaluate the radiosensitivity of glioma cells suffer from TIGAR interference, clonogenic assay was performed to examine the cell survival fractions at 14 days post-IR. Immunofluoresence assay was carried out to quantify GFP-LC3 foci post-IR. Finally, by using TIGAR sh RNA lentivirus-based gene therapy, TIGAR abrogation-induced radiosensitization of glioma in vivo was illustrated by MRI. Immunohistochemistry was carried out to determine the expression level of TIGAR in malignant gliomas from the clinical patients. At the same time, the relationship between the expression of TIGAR and the clinical features such as pathological grade, radiosensitivity and prognosis were investigated.Results:(1) In wild-type p53-expressing A172 cells TIGAR expression was increased 1 h post-IR and decreased to basal level 8 h post-IR. Meanwhile, p53 was phosphated in 0.5 h post-IR and the peak time of which was 1 h post-IR. The TIGAR m RNA level was increased in 0.5 h post-IR and the peak time of which was 2 h post-IR. While there was no difference in the m RNA level of p53 among different time points post-IR. However, in mutant-type p53-expressing(M237I) T98 G cells, there seemed to be no IR-induced increase in TIGAR expression. Clonogenic assay revealed that the survival fractions of both A172 and T98 G cells treated with TIGAR si RNAs were significantly lower than that of parental cells. Clonogenic assy also revealed that TIGAR over-expression could diminish the radiosensitivity of glioma cells.(2) TIGAR interference reduced by approximately 75% cellular NADPH in irradiated glioma cells, compared with a reduction of less than 40% in cells irradiated alone. Similarly, the ratio of GSH/GSSG was significantly further decreased by TIGAR interference in cells exposed to IR.(3) Protective autophagy was induced in glioma cells by irradiation alone. While in TIGAR knock-down cells, traumatic autophagy was induced by ionizing radiation.(4) By using TIGAR sh RNA lentivirus-based gene therapy, it was proven TIGAR interference could radiosensitize glioma in vivo.(5) Finally, TIGAR expression in malignant gliomas was significantly higher than it in adjacent tissues. The expression level of TIGAR might be correlated with the pathological grade of malignant glioma, and might negatively impact the short-term effects of irradiated malignant glioma.Conclusion: The present study demonstrated that traumatic autophagy was induced by TIGAR knockdown in glioma cells being irradiated. The mechanism might be the disruption of oxidative-redox balance. TIGAR interference was also proved to radiosensitize glioma in vivo. TIGAR expression might be contribute to the pathological judgment of patients with malignant glioma, and be negatively correlated with the short-term effects of radiotherapy.