The Research Of The Role And Related Mechanism Of FoxO3a In The Temozolomide Chemotherapeutic Resistance Of Glioma Cell

Background:Glioma is the most common primary intracranial neuroepithelial malignant tumor,which shows invasive growth with strong invasion ability and the unclear boundary between tumor and surrounding normal tissue,and also have the characteristics of rapid growth,high recurrence rate after operation and high mortality.The current standard clinical treatments for this tumor contain surgical resection,postoperative radiotherapy and adjuvant chemotherapy.Temozolomide is a kind of alkylating chemotherapeutic drug,which is the most commonly used and most effective first-line oral chemotherapeutic drug for curing glioma,which can kill tumor cells by inducing DNA double-strand breaks and promoting cell apoptosis.However,a mass of clinical data show that even the standard surgical resection,radiotherapy,and chemotherapy are applied to patients with glioma,the postoperative tumor recurrence rate and mortality rate being still high.At present,one of the known recurrence factors of glioma is the resistance of tumor cells to chemotherapeutic drugs such as temozolomide,which makes the chemotherapeutic efficacy of temozolomide weakened or even ineffective.Studies have shown that the expression level of MGMT and drug-resistant related proteins in tumor cells has important impacts on the sensitivity and resistance of tumors to temozolomide,as well as the killing effect of temozolomide on tumor cells and the prognosis of patients.Thus,studying the mechanism of resistance of glioma cells to temozolomide is of great significance and worthy for enhancing the efficacy of temozolomide and improving the prognosis of patients.FoxO3 a,a member of Fox family,is a transcriptional factor which involves and plays an important role in cell proliferation,differentiation,invasion and metastasis of tumor cells,cell apoptosis and other important cellular processes.Studies have indicated that FoxO3 a can enhance tumor cell resistance to temozolomide and inhibit apoptosis and cell death by promoting protective mitophagy of tumor cells,and consequently reducing the killing effect of temozolomide.However,the effect and role of FoxO3 a on MGMT and drug-resistant related protein expression levels in glioma cells and the related mechanism remain to be further studied.Objective:In this study,the expression levels of FoxO3 a,MGMT and drug-resistant proteins in randomly selected clinical samples of primary glioma with different pathological grades were compared and analyzed.Human glioma U87-MG cell line was used for cell experiments to explore the relationship of FoxO3 a expression level with MGMT expression level and drug-resistant related protein expression level in tumor cells,as well as the influence of FoxO3 a on drug resistance of glioma cells,to study the role of FoxO3 a in drug resistance and related mechanisms of the glioma.Methods:1.Eight clinicopathological specimens of primary gliomas of different grades that met the inclusion criteria were randomly selected,and the difference of expression levels of FoxO3 a,MGMT,P-GP and MRP-1 in primary gliomas of different grades were detected and analyzed by Western-blot technology.2.The cell experiments were performed by using human U87-MG cell line.Experimental group and contrast group were transfected with FoxO3a-targeting Si RNA and non-functional Scramble Si RNA respectively with the purpose to knock down FoxO3 a expression levels in cells and exclude other non-relative factors in the test.Cells in blank group were not transfected.3.The blank group,control group and blank group were divided into TMZ treated group and TMZ non-treated group respectively.The TMZ treated group was treated with200μmol/L TMZ for 48 hours.The cells above were then collected and lysed,and the nucleus fraction and cytoplasm were obtained by centrifugation.Western Blot analysis was used to compare and analyze the expression levels of MGMT,P-GP,MRP-1,FoxO3 a and AIF in cytoplasm of each group of cells.Meanwhile,the expression levels of FoxO3 a and AIF in the nuclei of each group were also analyzed and compared.4.The MTT assay and the Lactate Dehydrogenase release assay were performed on the 6 cell groups above to determine the activity and mortality of tumor cells.And the sensitivity of glioma cells to temozolomide in each group was analyzed and compared.Results:1.The expression levels of FoxO3 a,P-GP,MGMT and MRP-1 in tumor cells increased in accordance with the increase of tumor pathological grade during the test of randomly clinical selected primary glioma samples of different grades.2.The expression levels of FoxO3 a,P-GP,MGMT and MRP-1 in glioma cells treated with 200μmol/LTMZ were up-regulated compared with glioma cells which not be treated with TMZ.3.The results of MTT assay and Lactate Dehydrogenase release assay showed that cell sensitivity to TMZ and cell mortality of the tumor cells with FoxO3 a knocked down by transfecting with FoxO3 a targeted Si RNA were increased compared with the control and blank groups.4.The expression levels of FoxO3 a,MGMT,MRP-1 and P-GP induced by TMZ were down-regulated in the cell group with FoxO3 a knocked down by transfecting with FoxO3 a targeted Si RNA.5.TMZ-induced AIF nuclear translocation was enhanced in the cell group with FoxO3 a knocked down by transfecting with FoxO3 a targeted Si RNA.Conclusions:1.The expression levels of FoxO3 a,MGMT,P-GP and MRP-1 in tumor cells were up-regulated in accordance with the increase of pathological grade of primary glioma.2.With the stimulation of temozolomide,glioma cells can promote the up-regulation of MGMT,P-GP and MRP-1 through the up-regulation of FoxO3 a expression,thus enhancing the resistance to temozolomide.Down-regulation of FoxO3 a expression can reduce the resistance of glioma cells to temozolomide and enhance the killing effect of temozolomide on tumor cells.3.Down-regulation of FoxO3 a can promote the process of nuclear translocation of AIF induced by temozolomide.