The EZH2 Inhibitor GSK343 Suppresses Cell Proliferation,Invasion,and Cancer Stem-like Phenotypes And Reverses Epithelial To Mesenchymal Transition In Glioma

Research background:Enhancer of zeste homolog 2 is the catalytic unit of polycomb repressive complex 2,which epigenetically silences many genes involved in tumor-suppressive mechanisms via the trimethylation of lysine 27 of histone H3.We recently found that overexpression of EZH2 was associated with poor outcome of glioblastoma multiforme.In this study,we examined the antitumor effects of the EZH2 inhibitor GSK343 on glioma cells in vitro and in vivo.GSK343 reduced proliferation,attenuated cell motility,reversed epithelial-mesenchymal transition,and suppressed the stemness of U87 and LN229 glioma cells.Further,GSK343 inhibited histone H3K27 methylation and upregulated the expression of EZH2 target genes,thereby regulating the levels of markers involved in epithelial-mesenchymal transition and sternness.GSK343 treatment caused downregulation of EZH2,H3K27me3,and increased protein and mRNA levels of EZH2-silenced genes including E-cadherin,PTEN,and p21.GSK343 treatment also inhibited tumor growth in a nude mice model with intracranially xenotransplanted glioma.Together,our results indicate that GSK343 could be a potential drug against glioblastoma.Methods:1.Colony formation,CCK8 assay,wound-healing and transwell invasion assays were used to examined cell viability and the capacity of migration and invasion in U87 and LN229 glioma cells treated with GSK343.Then,cell flow cytometry was used to access cell cycles in glioma cells.Finally,we used tumor-forming capacity to examined the impact of GSK343 on glioma stem cells(GSCs).2.We next performed a time-course analysis to investigate H3K27 methylation following GSK343 treatment.Because EZH2-mediated methylation of H3 is based on the integrity of the PRC2 multi-subunit complex and the interaction between EZH2 and H3,the status of EZH2,EED,SUZ12,and H3 was examined.Furthermore,immunoprecipitation experiments were performed to estimate the interaction between EZH2 and H3.To examine the mechanisms underlying the reversion of malignant characteristics in cells by GSK343,we evaluated the expression levels of E-cadherin,PTEN,and p21 by western blot analysis.3.The levels of mesenchymal markers and transcription factors including N-cadherin,Vimentin,MMP2,Snail,and Slug were examined in U87 and LN229 cells.We also performed immunofluorescence analysis of H3K27me3,N-cadherin,and Vimentin levels in glioma cells.4.We examined levels of the pluripotency factors Nestin,Sox-2,and Oct-4 to determine the influence of GSK343 on the stemness of GSCs.Nude mice were first intracranially xenotransplanted with luciferase-expressing U87 glioma cells.After tumor formation,nude mice were treated with intraperitoneal injections of GSK343(10 mg/kg in 200 ?L PBS)or diluted DMSO(10 ?L in 200 ?L PBS)every other day.After the start of treatment,tumor growth was monitored by a live animal bioluminescence imaging system every week.Results:1.GSK343 suppresses cell proliferation and invasion and induces G0/G1 arrest in glioma cells.2.GSK343 inhibits histone H3K27 methylation and up-regulates the expression of EZH2 target genes.3.GSK343 reverses epithelial-mesenchymal transition in glioma cells.4.GSK343 treatment attenuates the stemness of glioma stem-like cells.5.GSK343 inhibits tumor growth in vivo.Conclusions:1.The EZH2 inhibitor GSK343 suppresses cell proliferation,invasion,and cancer stem-like phenotypes and reverses epithelial to mesenchymal transition in glioma.2.The effects of GSK343 in glioma cells are depending on the increase of EZH2 target genes via the decrease of H3K27me3 and EZH2 as well as the dissociation between EZH2 and H3.