Effects And Mechanisms Of 1,2:5,6-dianhydrogalactitol(DAG) On Proliferation,Apoptosis,Invation And Angiogenesis In Glioma Cells

[Objective]To investigate the effects of DAG on cell proliferation and apoptosis in glioma cells and explore the underlying mechanisms,aim to provide reference for further research and clinical application of DAG.[Methods]1.Cell proliferation and apoptosis assay:CCK-8 and single cell colony formation assay were performed to detect the glioma BT325 cells viability at different doses and times,in order to evaluate the effects of DAG on cell viability and cell proliferation.Cell apoptosis were detected by Hoechst 33342staining and transmission electron microscope methods respectively,with which apoptotic nuclear and?Ltrostructure morphological changes were observed.2.Influence on Ca²⁺,mitochondrial membrane potential and DNA in gliomacells:The content of Ca²⁺in the cells was analyzed by using Fluo-3AM fluoresent dyes.The mitochondrial membrane potential was detected by using Rhodamine 123 fluoresence probe kit.Both of the results were recorded by confocal imaging with confocal microscope under 488nm laser excitation.DNA damage of glioma cells caused by DAG was analyzed by single cell gel electrophoresis.3.Mechanisms of cell proliferation and apoptosis:The m RNA expression of gene P53,Caspase-8,Fas,Caspase-4,CHOP,JNK,GRP78 and GRP94 were analyzed by real-time quantitative PCR(RT-PCR)assay.Cell immunohistochemistry and Western-Blot methods were performed to analyze the expressions of Caspase-8,Fas related to death receptor pathway,and Caspase-4,CHOP,JNK related to endoplasmic retic?Lum stress(ERS)pathway.[Results]1.DAG inhibited the glioma cells prolifration and induced cell apoptosis:The results of CCK-8 and colony formation assay showed that DAG inhibited the viability and proliferation of glioma BT325 cells in a dose-time-dependent manners.Comoared with control group,cell viability and proliferation were significantly lower.The concentration of inhibited cell viability(IC₅₀)was108.81?g·m L^-1for 24h,26.61?g·m L^-1and 11.38?g·m L^-1for 48 h and 72 h respectively.The results of nuclear morphological changes of BT325 cells with Hoechst 33342 staining showed that after 48h treatment of DAG,the number of cells decreased,and the nuclear condensed and chromatin gathered at edge or splintered.By transmission electron microscopy,the Ultrostructure of glioma U251 cells were observed that the cells became small,membrane became incomplete or foaming,organelle structure were not clear,endoplasmic retic?Lum dilated,vacuolization in mitochondria with ridge blurred,chromatin condensed,apoptotic bodies appeared.These results indicated that DAG exerted3.DAG significantly enhanced Ca²⁺concentration and decreased mitochondrial transmembrane potential in human glioma cells teated with Fluo-3AM dye and Rhodamine 123 probe respevtively.The results indicated that DAG-induced apoptosis of glioma cells might associated with ERS triggered and damage of mitochondrial membrane integrity that caused the dissipation of mitochondrial transmembrane potential.The results of single cell gel electrophoresis showed that DAG damaged the DNA of human glioma cells,and therefore induced apoptosis and inhibited cell growth.4.RT-PCR results showed that m RNA expression of P53,Caspase-8,Fas,Caspase-4,CHOP,GRP78,GRP94 upregulated,while JNK was downregulated after treated with DAG for 72h.According to the results of cell immunohistochemistry and Western-Blot assay,Caspase-8 and Fas,Caspase-4and CHOP were up-regulated,while JNK1+JNK2 was down-regulated at protein levels.These results indicated that DAG inhibited cell proliferation and induced apoptosis in glioma cells might via the death receptor pathway,also the ERS pathway.[Conclusions]1.DAG inhibits proliferation and induce apoptosis in glioma cells.2.DAG induces apopotosis of glioma cells are associated with changes of Ca²⁺concentration and dissipation of dissipation of mitochondrial transmembrane potential,also associated with DNA damage.3.DAG inhibits glioma cells proliferation and induces apoptosis via the death receptor pathway,also via endoplasmic reticu Lum pathway.[Objective]To investigate the effects of DAG on invasion and angiogenesis in human glioma cells,and explore the underlying mechanisms,inorder to provide theoretical and experimental reference for the clinical application of DAG.[Methods]1.The effects of DAG on migration and invasion of human glioma cells was evaluated by scratch,Transwell migration and invasion assay respectively.2.HUVECs tube formation assay in vitro was used to evaluated the effects of DAG on angiogenesis of human glioma cells.3.VEGF and FGF-2 in glioma cells culture supernatant were determined by ELISA assay in ordre to evaluate the effects of DAG on expression level of vascular endothelial growth factor in human glioma cells.4.RT-PCR,Cell immunohistochemistry and Western-Blot assay were performed to investegate the influence of DAG on expression level of MMP-2,PTEN and Notch1 in human glioma cells.5.Zebrafish model was used to evaluate the effect of DAG on anti angiogenesis.The target of anti angiogenesis activity of DAG may be explored by computer Docking software.[Results]1.The results of Scratch,Transwell migration and invasion assay showed that DAG inhibited glioma cells migration in dose-and time-dependent manners,and significantly inhibit cell invasion and angiogenesis in vitro.2.VEGF and FGF-2 in glioma cells culture supernatant treated with DAG after 24 h were reduced significantly in dose-dependent manner,and were significantly lower than that of control group,according to the results of ELISA assay.3.According to RT-PCR,immunohistochemistry and Western-Blot results,MMP-2 and Notch1 were downregulated,while PTEN was upregulated at m RNA expression and protein level.4.DAG significantly inhibited the formation of mesenteric vessels in zebrafish embryos,and the anti angiogenesis action was in a dose-dependent manner.5.Molecules docking experiment reprented that DAG could dock with VEGFR-2 by hydrogen bonds,that might be the target for DAG anti angiogenesis.Conclusions:1.DAG inhibits the invasion,migration and angiogenesis of human glioma cells in vitro.2.DAG inhibits glioma cells invasion,migration and angiogenesis by promoting the expression of PTEN,reducing VEGF and FGF-2 expression,also by downregulating the protein level of MMP-2.3.DAG inhibits glioma cells invasion and angiogenesis may be associated with Notch1 pathway,but still need to be investigated.4.DAG exhibits significant anti angiogenesis action.The anti angiogenesis action of DAG may be associated with it dock with VEGFR-2 by hydrogen bonds and reduce VEGFR-2 expression.