Study On The Effect Of IDH2Mutation On Gliomas Proliferation And Sensitivity To Chemotherapy

Gliomas are the most common type of human brain tumors and can be classified based on clinical and pathological criteria into Class I～IV. The grade IV glioma, commonly known as glioblastoma multiforme (GBM), is the most malignant type among all types of human tumors and has stronge invasiveness. It is very difficult to clear gliomas by complete resections, which have high incidence rate, recurrence rate, death rate and low cure rate, but the efficacy of radiotherapy and chemotherapy after surgery in clinical practice is still poor. Tumorigenesis is inseparable from gene mutation and metabolic disorder, so the gene therapy for gliomas has wide development prospects.Isocitrate dehydrogenase (IDH), including three catalytic isozymes such as IDH1, IDH2, and IDH3in humans, plays an important role in glucose metabolism by catalyzing the oxidative decarboxylation of isocitrate (ICT) into a-ketoglutarate (a-KG) and NADPH. IDH I mutations often occurred in WHO grade II and III astrocytomas and oligodendrogliomas and in glioblastomas that developed from these lower-grade lesions, whereas tumors without mutations in IDH I often had mutations in IDH2that had lower mutation frequency than IDH1. Both IDH1and IDH2mutations are a single amino acid substitution, such as R132and R172respectively.Both IDH1and IDH2are encoded in the nucleus and function as homodimers. IDH1mutant weakened enzymatic activity by forming heterodimers, meanwhile, mutated IDH1had a gain of function enabling IDH1to convert a-KG and NADPH into R(-)-2-hydroxyglutarate [R(-)-2HG] and NADP’. Therefore, the gain and loss of mutant IDH1function decreased the a-KG and NADPH production. α-KG initiates oxygen dependent degradation of hypoxia-inducible factor subunit (HIF-1α). Thus, decreased cytoplasmic level of a-KG increased level of HIF-1α and strongly induced the expression of HIF-1α target genes, such as glucose transporter1(Glutl), vascular endothelial growth factor (VEGF), and phosphoglycerate kinase (PGK1),which were critical to tumor growth. However, low level of cytoplasmic NADPH, which resulted in increasing oxidative stress, might sensitize glioblastoma to irradiation and chemotherapy.Although IDH2had low mutation frequency, the function study on IDH2mutations was not ignored. In this study, we taked C6as experimental glial cells, which were induced by weekly intravenous injections of N,N’-nitroso-methylurea in rats. Firstly, after amplifying and analyzing the full length IDH1and IDH2in C6cells, we demonstrated that IDH1and IDH2mutations did not occur in C6cells, which suggested that these mutations were not required for the development of glioblastoma induced by N,N’-nitroso-methylurea. Thus, we performed artificially site-directed mutagenesis of IDH2, and then two recombinant plasmids, pEGFP-N1-IDH2and pEGFP-N1-IDH2R172G, were constructed and transfected into C6cells. By using an Olympus FV1000laser scanning confocal microscope, we observed that heterologous protein expressed gradually12h post-transfection, and the expressions increased from24to36h. However, fluorescence decreased at48h. Then, the activity of IDH2was analyzed through the reduction of NADP1to NADPH, which is measured at25℃by spectrophotometry at340nm for5min. The result showed that exogenous expression of wild-type IDH2significantly increased the production of NADPH, which was attributed to elevated level of the enzyme. However, the gain and loss of IDH2mutant were responsible for the fact that exogenous expression of IDH2R172G reduced the generation of NADPH.When investigated the expression level of tumor growth-related factors in the western blot test, including HIF-1α, Cyclin D1and COX-2, we found that the expression level of these factors were elevated, which suggested that the low level of α-KG attributed to IDH2mutation increased the stabilization of HIF-la, and that HIF-la improved the expression of Cyclin D1and COX-2. Subsequently, we determined the effect of IDH2mutation on tumor growth by MTT assay and found that the inhibition rate of pEGFP-N1-IDH2R172G group was lower than that of pEGFP-N1group and pEGFP-N1-IDH2group, which was markedly detected from36h to48h. The assay indicated that mutant IDH2promoted the growth of the rat C6gliomas.The relative quantity of GSH was analyzed by spectrophotometry at412nm. The result showed that the relative quantity of GSH of pEGFP-N1-IDH2R172G group was lower than that of pEGFP-N1-IDH2group, which suggested that decreased NADPH production due to the gain and loss of mutant IDH2function cut down the level of GSH. Low level of cytoplasmic GSH may increase oxidative stress and then sensitize glioblastoma to chemotherapy.Subsequently, we investigated that the sensitivity of C6cells transfected with the recombinant plasmids to chemotherapy when we treated C6cells with different antitumor drugs. Interestingly, we found that the inhibition rate of pEGFP-N1-IDH2group was higher than that of pEGFP-N1group and pEGFP-N1-IDH2R172G group and the inhibition rates of pEGFP-N1group and pEGFP-N1-IDH2R172G group had no significant difference, which was ubiquitous among the five drugs at any dosage. We speculated that the expression of heterogenous protein IDH2wild-EGFP elevated the levels of a-KG, decreased the stabilization of HIF-la, and inhibited the growth of C6cells finally. The effects of heterogenous proteins IDH2Mutant-EGFP expression on C6cells proliferation were divided into two aspects:(i) it reduced the generation of NADPH, which impaired reduction of glutathione (GSH) and affected the thioredoxin system, might sensitize C6cells to chemotherapy;(ii) it decreased the level of a-KG, elevated the stabilization of HIF-la, and improved the growth of C6cells. Therefore, we conjectured that the paradoxical effects finally resulted in the fact that the inhibition rates of pEGFP-N1group and pEGFP-N1-IDH2R172G group had no significant difference, and were always lower than that of pEGFP-N1-IDH2group. In the subject, we analyzed IDH2mutant activity and the effects of IDH2mutation on C6cells proliferation and tumor growth-related factors, and explored the possible mechanism of glioma cells sensitivity to chemotherapy due to IDH2mutation. It is benefit to not only understand the biological mechanism of development and advancement of tumors, but also identify new targets of antitumor drugs and means of preventing cancers.