Study On The Effects Of LiCl On Proliferation And Migration Of Gliomas Harboring IDH2Mutant

Gliomas are the most common type of human brain tumors, with the characteristics of high incidence, recurrence rate and mortality rate and low cure rate. These tumors have been classified as grade Ⅰ to grade Ⅳ on the basis of histopathological and clinical criteria established by the World Health Organization (WHO), the grade Ⅳ glioma known as glioblastoma multiforme (GBM) is the most prevalent and malignant primary brain tumor in adults. Isocitrate dehydrogenase (IDH) is a family of enzymes, the human genome has five IDH genes coding for three distinct IDH enzymes:IDH1, IDH2and IDH3, IDH enzymes can catalyze the oxidative decarboxylation of isocitrate (ICT) into α-ketoglutarate (α-KG) and NADPH. IDH1(R132) is somatically mutated predominantly in low grade glioma and secondary glioblastomas, however, the frequencies of mutations in IDH2in these gliomas are low. Both IDH1and IDH2mutations are a single amino acid substitution. IDH1mutations lead to simultaneous loss and gain of activities in the production of α-ketoglutarate (α-KG) and2-hydroxyglutarate (2-HG), respectively. In response to IDH1mutations, the stabilization of HIF-1α is elevated, which contributes to tumor growth.Tumor cell microenvironment is the key problem in the field of cancer research. An accurate understanding of the biological mechanisms of tumor growth and migration is beneficial to provide theoretical and experimental basis for targeted drug.design and screening. In the present study, the effect of IDH2R172G on rat C6glioma cells migration was analyzed at the cellular level and the role of HIF-la pathway in the glioma cancer harboring IDH2mutant was discussed. pEGFP-N1, pEGP-N1-IDH2R172G and pEGFP-N1-IDH2were transfected into C6cells. The expression level of HIF-1α, Cyclin D1, VEGF and M2-PK was analyzed in the western blot assay. At the same time, the secretion level of MMP-2,9was tested in gelatin zymography assay. Compared to the EGFP and IDH2treatment groups, the stability of HIF-1α in IDH2R172G treatment group was increased and the expression level of Cyclin D1, VEGF, MMP-2,9and M2-PK was elevated, which indicated that the low level of α-KG attributed to IDH2R172G increased the stabilization of HIF-1α, then HIF-1α improved the expression of Cyclin D1, VEGF. The activity of pro-MMP-2,9was elevated in the IDH2R172G treatment groups compared to that in the IDH2and EGFP treatment groups, which demonstrated that mutant IDH2could also elevate the secretion levels of MMP-2and MMP-9by stimulating HIF-1α pathway. Furthermore, IDH2R172G displayed a higher activity in promoting cell migration than IDH2did, and MMP-2and MMP-9were critical for C6cells metastasis in this aspect.Lithium remains one of the most important drugs for the treatment and prophylaxis of bipolar mood disorders and depression. It inhibits the activity of inositol monophosphatase and glycogen synthase kinase-3(GSK3). GSK-3is a multifunctional serine-threonine protein kinase found in all eukaryotes that regulates diverse processes, including metabolism, cell division and death. In this study, LiCl and LY294002were used to regulate the phosphorylation of GSK-3β, and its effect on C6cells proliferation was analyzed. LY294002can inhibit the PI3K/Akt signaling pathway by inhibiting the phosphorylation of Akt and then decreasing the phosphorylation levels of GSK-3β. LY294002could decrease the phosphorylation of GSK-3βin the western blot assay. However, LiCl(?)couldn’t increase GSK-3β phosphorylation anymore after C6cells were treated with LY294002. LY294002inhibited the proliferative activity of C6cells in the MTT test, and the inhibition rate in pEGFP-N1-IDH2treatment group was higher than that in pEGFP-N1group and pEGFP-N1-IDH2R172G group. However LiCl had no effect on the proliferative activity of C6cells after treated with LY294002.Subsequently, the effect of LiCl on the migration and proliferation of C6cells harboring IDH2mutant was analyzed. Here, we found that the inhibition effect induced by LiCl on cell proliferation had no obvious difference whether C6glioma cells harboring IDH2mutant or not, although IDH2mutant increased the stability of HIF-1a. Further, GSK-3β would be phosphorylated at Ser9and its activity was inhibited when C6cells were treated by LiCl. In addition, the degree of phosphorylation in IDH2R172G treatment group was lower than that in the control and IDH2treatment groups. At the same time, the accumulation ofβ-catenin in cell nucleus was decreased. Although over-activation of Wnt/β-catenin signaling was highlighted in the progression of multiple tumor cell lines, little was known about its role in glioma development. In theory, LiCl-induced inactivation of GSK-3β could activate the Wnt/β-catenin pathway, which induced the expression of MMP-2and9. However, we found that, although the β-catenin and HIF-1a induced the secretion level of pro-MMP-2and pro-MMP-9as well as the conversion from pro-MMP-2to its active form were improved in C6cells harboring IDH2mutant, the migration potential of LiC1treated C6cells harboring the IDH2and its mutant treatment was comparable. These results indicated that LiCl could decrease the proliferation and migration potential of C6glioma cells harboring IDH2mutant.Our research attempted to reveal the role of LiCl on the regulation of signaling pathway in glioma cells, disclose the molecular mechanism of its inhibition effect on glioma cells proliferation and migration. These results serve as an important complement to existing research on glioma therapy.