The Role Of γ-secretase Inhibitor GSI As A Glioma Radiosensitizer And Its Mechanism

Objective:Glioma is one of the most common primary intracranial tumors,with glioblastoma(GBM)being the most malignant.The current domestic methods for treating glioblastoma mainly include surgical resection of tumor combined with postoperative radiotherapy and temozolomide(TMZ)chemotherapy,and radiotherapy(RT)is the most important part of the treatment of GBM,but the efficacy is often limited due to the presence of radiation resistance in gliomas,especially in relapsed and refractory gliomas.Notch pathway is an important signaling pathway involved in the formation of radiation resistance of glioma stem cells,and y-secretase precisely cleaves notch protein to form Notch protein intracellular domain(NICD)is the most important process to initiate Notch signaling pathway.The y-secretase inhibitor GSI can function to block the Notch pathway by blocking the production of NICD.Considering the non-specificity of GSI and the almost universal expression of Notch signaling pathway in whole body tissues,our study tried to use lower doses of the y-secretase inhibitor GSI as a radiation sensitizer applying to glioma cells and glioma-bearing mice,observed whether low-dose GSI can increase the sensitivity of glioma cells and living tumor tissues to radiation and we also did the preliminary research on the possible mechanisms.Method:In cell experiments We used the glioma cell lines U251 and U87.First,we studied the effection on cell growth of different GSI concentrations in U251 and U87.We found that U251 and U87 cell growth were significantly changed when the GSI concentration exceeded a certain threshold,at the same time,the morphology of the cells changed.Then,we applied this critical concentration to U251 and U87 cells.The cells were divided into control group,pure GSI group,simple irradiation group,and radiation+GSI group.Then we observed the change of their growth and the ability to clone formation under 0,2,4,6,and 8 Gy radiation.We found that both U87 and U251,after addition of the sensitizer GSI,the growth speed and clone formation ability were inhibited,and this effect will become more pronounced as the radiation dose increases.Finally,we used Western-Blot method to analyze the changes of Notch pathway-related proteins and autophagy-related proteins in U251 and U87 cells treated with radiation with the dose of 4Gy.In mice experiments,we selected the glioma stem/group cell line SU3,SU3-5R created by our own research team.The SU3-5R is a radioresistant strain induced by SU3 after 5 times of 4Gy radiation.First,planted SU3 and SU3-5R under the skin of Bal b/c nude mice.Logarithmic growth stage tumor tissue after tumor formation,.The tumor tissue is properly treated and implanted in the right hind limb of the nude mouse.The nude mice were divided into SU3 and SU3-5R groups,and each group was divided into control group,GSI group,irradiation group,and irradiation+GSI group.Recording the growth of tumor tissue in different groups of nude mice and observed the effect of low-dose GSI as a radiosensitizer in vivo.Results:In vitro studies have shown that cell growth is not affected by simple low-dose GSI on U251 and U87.When small doses of GSI combined with ionizing radiation were applied to the glioma cell lines U251 and U87,the growth and clonality of the two glioma cell lines were significantly inhibited and the effect of this radiation sensitization will become more and more obvious as the radiation dose increases.At the same time,the results of Western-Blot analysis can be seen under 4Gy radiation,Ionizing radiation activatesd the Notch signaling pathway in glioma cells,the protein of NICD and downstream target gene HES-1 experssed more.This pathway activation was significantly inhibited after GSI treatment.At the same time,simple irradiation of glioma cells will cause an increase in autophagic death.After adding low-dose GSI as a radiation sensitizer,autophagy-related protein expression was increased and autophagic cell death was improved.Animal experimental research results show that in the SU3 group,pure ionizing radiation limited the growth rate of tumor tissue.Tumor tissue growth was more significantly restricted by the addition of the sensitizer GSI.In the SU3-5R group,tumor tissue showed insensitivity to pure ionizing radiation.This radiation resistance can be significantly improved after intraperitoneal injection of GSI and tumor tissue growth rate was significantly slower.Conclusion:Low-dose GSI in the situation of not affecting the growth of glioma cells and increasing the risk of drug-induced death in nude mice can significantly increase the radiation sensitivity of glioma when use it as a radiation sensitizer acting on glioma cell lines and tumor-bearing nude mice.