The Actions And Mechanisms Of Caspase-dependent Oxidative Stress In Human U251 Glioma Cells

Background and significanceGliomas is one of the most common malignant tumors of central nervous system.Infinite growth and invasion are the greatest biological feature of gliomas and the major causes for poor prognosis.Active oxygen is a by-product of normal oxygen metabolism in the body.The relationship between reactive oxygen species?ROS?and oxidative stress on gliomas is extremely complex and has not been elucidated yet.The observations of biological effects of proliferation,cycle,and apoptosis of glioma under different ROS and oxidative stress in glioma cells are of great significance to elucidate the pathogenesis of glioma and find new clinical treatment strategies.Gene chip technology and bioinformatics analysis were used to explore the gene expression of glioma cells from the whole transcription or DNA level and screening gene expression sets related to the process of gliomas.These findings could provide useful ideas and clues for studying pathogenesis and treatment of glioma.ObjectiveWe aimed to observe the effects of different ROS and oxidative stress levels on the biological effects of glioma proliferation,cycle and apoptosis in glioma cells and to explore whether H₂O₂-induced oxidative stress can inhibit proliferation and promote apoptosis of glioma cells through Caspase pathway.Besides,whether the regulation of oxidative stress and mitochondrial-Caspase pathway could be used as a potential target for glioma treatment,which is of great significance for clarifying the pathogenesis of glioma?The questions need to be elucidated in this study.We analyzed the whole gene expression pattern of glioma cells under different oxidative stress conditions.The gene expression changes of the glioma cells and screen Caspsae inhibitor-affected genes were observed in order to provide new directions for target treatment and drugs.MethodsFirst,human glioma U251 cell line was cultured in vitro and treated with different concentrations of H₂O₂.Flow cytometry was used to detect the intracellular ROS level,cell cycle and apoptosis,methyl thiazolyl tetrazolium?MTT?assay was used for the cell proliferation detection,western blot was conducetd to evaluate the apoptosis-related protein Cleaved-Caspase 3,Cleaved-PARP levels.The effect of different concentrations of H₂O₂ on the proliferation,cycle and apoptosis of glioma cells could be explored through the aforementioned assays.Next,we intervented H₂O₂-treated glioma U251 cell line with Caspase 3 inhibitor,then flow cytometry,MTT assay and western blot were used to detect all the indexes and to observe the changes of cell proliferation,cycle and apoptosis caused by Caspase inhibitor.We uses NOISeq package to screen the differentially expressed genes?DEGs?caused by Caspase 3 inhibitor under oxidative stress in glioma cells and conducted Gene Ontology?GO?analysis,Kyoto Encyclopedia of Genes and Genomes?KEGG?pathway enrichment analysis and drug enrichment analysis.Protein-protein interaction?PPI?network structure was constructed by String.The construction and analysis of sub-network module in PPI network was extracted using MCODE plug-ins in cytoscape software.Using webgestalt tool and cytoscape software,we also constructed the microRNA?miRNA?-gene and transcription factor?TF?-gene regulatory networks.Results?1?The levels of?O2.-?in glioma U251 cells treated with H₂O₂ at the concentrations of100?M,300?M and 500?M were detected by Dihydroethidium?DHE??214.33±27.14?%,?589.52±35.17?%and?643.28±31.54?%,respectively,?P<0.05?.With the increase concentration of H₂O₂,the level of?O2.-?in the cells gradually increased?P<0.05?.?2?Cell viability of glioma U251 cells treated with H₂O₂ at the concentrations of 100?M,300?M and 500?M were?82.57±5.34?%,?23.38±6.27?%and?18.21±3.11?%,respectively.The higher the H₂O₂ concentration,The lower the survival rate of glioma cells was?P<0.05?.?3?The early and late apoptosis rates of glioma cells in 100?M,300?M and 500?M groups were?6.21±0.88?%and?10.71±0.42?%,?24.19±2.46?%and?19.59±2.33?%,?31.78±3.07?%and?37.18±4.20?%respectively.The apoptosis rate was increased with the increase of H₂O₂ concentration?P<0.05?.The apoptosis rate of glioma cells in 100?M group was not significantly changed compared with in control group?P>0.05?,while the apoptosis rate of glioma cells in 300?M and 500?M group was significantly higher than that in control group?P<0.05?.?4?The percentage of cells in G1 phase,G2 phase and S phase were?28.79±3.64?%,?28.51±3.17?%and?46.67±2.16?%respectively in 100?M group;which were?21.35±1.37?%,?32.48±1.62?%and?50.69±3.73?%respectively in 300?M group and?41.54±4.78?%,?18.51±2.35?%and?43.82±2.42?%respectively in the control group.With the increase of H₂O₂,cells in G1 phase decreased and cells in G2 phase increased significantly?P<0.05?,incicating that H₂O₂ could cause G2 phase arrest in glioma cells.?5?The protein expression of Cleaved-Caspase 3 in H₂O₂-treated glioma cells was significantly decreased and the expression of Cleaved-PARP protein was significantly increased?P<0.05?,which showed a dose-dependent changes with the concentration of H₂O₂.?6?The rate of early and late apoptosis of H₂O₂+Caspase 3 inhibitor group were?4.95±0.89?%and?8.32±1.22?%respectively,which were significantly lower than H₂O₂ group[?23.94±2.34?%and?17.55±2.78?%]?P<0.05?and control group??6.71±0.69?%and?11.35±1.32?%?and control group without any intervention.The apoptosis rate of H₂O₂group was the highest,followed by the control group.The apoptosis rate of H₂O₂+Caspase3 inhibitor group was the lowest.Caspase 3 inhibitor significantly decreased the apoptosis of H₂O₂-induced glioma cells.?7?The percentage of cells in G1,G2 and S phase of H₂O₂+Caspase 3 inhibitor group were 36.05%,16.91%and 47.03%respectively,which were 19.88%,33.64%and 46.48%in H₂O₂ group and 41.15%,14.19%and 44.66%in the control group.There were no significant differences in S phase cellsamong groups?P>0.05?.The cells in G1 phase in H₂O₂+Caspase 3 inhibitor group were significantly increased compared with that of H₂O₂ group?P<0.05?,while the cells in G2 phase were significantly lower than those in H₂O₂ group?P<0.05?,indicating caspase inhibitor reversed H₂O₂-induced G2 arrest.?8?The level of intracellular?O2.-?in H₂O₂+Caspase 3 inhibitor group was?488.76±25.73?%,which was significantly lower than H₂O₂ group?728.17±33.65?%?P<0.05?and higher that the control?100.16±8.24?%?P<0.05?.The Caspase 3 inhibitor could reduce ROS levels in H₂O₂-treated glioma cells.?9?A total of 105 DEGs associated with proliferation of glioma cells induced by Caspase 3 inhibitor under oxidative stress and 127 other specific DEGs were screened out.?10?GO analysis showed that the function of DEGs related to proliferation was mainly enriched in cellular component morphogenesis,including FIS1,ATOH1,CCK,EZR,SSBP1and CTNNB1,intracellular receptor-mediated signaling pathway,including DNAJA1,CALR and CTNNB1,mitochondrial morphogenesis,including DNAJA1,CALR and CTNNB1,cellular macromolecular complexes,including KIF2B,TUBB2A,SNRNP200,SNRPB and CALR,the organelle fission pathway,including KIF2B,FIS1,TUBB2A and DCTN3.Enrichment results through the KEGG pathway showed that 18 pathways met the threshold requirements and were significantly enriched,which mainly involved RhoA activity and RhoA signaling pathway,RAC1 activity and RAC1 signaling pathway,RNA metabolism,protein metabolism,mRNA metabolism,androgen receptor,diabetes pathway,viral infection,cell protein cleavage,mRNA splicing-small channel,insulin synthesis and processing,integrin and apoptosis.Three miRNAs?miR-185,miR-7 and miR-485-5P?were filtered out and six target genes were combined to form 8 regulatory pairs.Besides,a total of 20 TFs including ETS2,NFKAPPAB,NFKB,AP1,CREL,OCT1,STAT5A,PAX3,et al were filtered out and 8 target genes were combined to form 38 regulatory pairs.?11?The other 127 functional annotations of specific DEGs were mainly enriched in negative regulation of protein binding,including CTHRC1,HERPUD1,transcriptional regulation of RNA polymerase II promoter,including ZBTB32 and NRARP,skeletal muscle cell differentiation,including NUPR1,LEMD2 and ANKRD1,alveolar development,including ASXL1 and NKX2-1 and phospholipid metabolism which included TAZ,KX2-1 and LA2G4B.The KEGG pathway enrichment results showed that the four pathways met the threshold requirements and were significantly enriched,which mainly included glycosaminoglycan biosynthesis,oxidative phosphorylation,and Staphylococcus aureus infection.The constructed PPI network included 82 nodes and 209 interaction pairs.The sub-network module included 19 nodes and 50 interaction pairs.A total of 17 miRNAs?miR-192,miR-196,miR-215,LET-7,mi R-299 etc.?were screened out,forming 105regulatory pairs with 26 target genes and 12 TFs?STAT5,CEBPBP,AX6,PPAR,IRF,PPARG,CHX10,USF,CREBP1,E4BP4,HLF,PAX4?and 26 target genes constituted 51regulatory pairs.ConclusionOxidative stress inhibited proliferation,arrests cell cycle progression and induces apoptosis death of human U251 glioma cells through a mitochondrial-Cassase-dependent pathway.Caspase 3 inhibitor cause changes of gene expression profile of glioma cells under oxidative stress.RPL5,PSMC5,RABGAp-TBC1D25 and C1s may play a key role in the pathogenesis of glioma,which provides new directions and strategies for future researches on the targeted treatment of gliomas.