The Role Of Bcl-2 In Autophagy And Apoptosis Induced By Oxidative Stress In Glioma Cells

Gliomas are by far the most common primary brain tumors in adults. Malignant gliomas are resistant to various proapoptotic therapies,such as radiotherapy and conventional chemotherapy,glioma cells are also resistant to the conventional proapoptotic cancer therapeutics. Therefore, effective treatment of malignant gliomas may rely on the development of novel strategies for inducing nonapoptotic cell death,such as autophagic cell death or cell death through mitotic catastrophe,which has been recently described as alternative death pathways.recent reports suggest a role for ROS as signaling molecules in autophagy, high levels of ROS can oxidize cell lipids, proteins and DNA, causing dysfunction of mitochondria, lysosomes and other organelles.Various defense mechanisms can protect cells against oxidative stress,including the degradation and recycling of damaged cell proteins and organelles by autophagy.H2O2 is active oxygen and intermediate product of oxidation in vivo.it is easy to penetrate cytoplasmic membrane and product cellular toxicity.Atg4,an essential protease in the autophagic pathway, has been identified as a direct target for oxidation by H2O2. However, these pathways remain to be elucidated. In this study, we have demonstrated that H2O2 induces apoptosis and autophagy in U251 glioma cells and construct Bcl-2 Overexpression and Bcl-2 gene-specific small interfering RNA(siRNA)to observe and approach that the role and mechanism of Bcl-2 in H2O2 induces apoptosis and autophagy ,meanwhile discuss the relation about apoptosis and autophagy. And their significance in therapy cancer.A variety of physiological death signals, as well as pathological cellular insults, trigger the genetically programmed pathway of apoptosis.in mitochondria pathway of apoptosis. A major checkpoint in the common portion of this pathway is the ratio of pro-apoptotic(BAX) to anti-apoptotic(BCL-2) members.Downstream of this checkpoint are two major execution programs: the caspase pathway and mitochondria dysfunction. Mitochondrial dysfunction includes a change in the mitochondrial membrane potential , production of reactive oxygen species(ROS), opening of the permeability transition pore(PTP),and the release of the intermembrane space protein,cytochrome c(Cyt c). Released cytochrome c activates Apaf-1, which in turnactivates a downstream caspase program.Apoptosis and autophagy are both tightly regulated biological processes that play a central role in tissue homeostasis, development,and disease.The antiapoptotic protein,Bcl-2,interacts with the evolutionarily conserved autophagy protein, Beclin 1. However, little is known about the functional significance of this interaction. Autophagy is an evolutionarily conserved pathway that involves the sequestration and delivery of cytoplasmic material to the lysosome, where it is degraded and recycled. In theory, autophagy may help promote cell survival,either by purging the cell of damaged organelles, toxic metabolites, and intracellular pathogens or by generating the intracellular building blocks required to maintain vital functions during nutrient-limiting conditions. However, in theory,autophagy may also promote cell death through excessive self-digestion and degradation of essential cellular constituents. Despite recent advances in understanding its molecular mechanisms and biological functions, it is unclear whether autophagy acts fundamentally as a cell survival or cell death pathway—or both.The mechanisms responsible for autophagy are still not very clear.The interaction between the antiapoptotic protein, Bcl-2,and the autophagy protein, Beclin 1, represents a potentially important point of convergence of the apoptotic and autophagic machinery. Beclin 1,the mammalian ortholog of yeast Atg6/Vps30, was first identified in a yeast two-hybrid screen as a Bcl-2-interacting protein and is a haplo insufficient tumor-suppressor gene that is frequently monoallelically deleted in human sporadic breast, ovarian, and prostate cancer . Beclin 1 is part of a Class III PI3K complex that participates in autophagosome formation, mediating the localization of other autophagy proteins to the preautophagosomal membrane.The signaling pathway composed of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), and mammalian target of rapamycin (mTOR) plays a central role in the regulation of cell proliferation, differentiation, and survival.The class I PI3K /Akt pathway which is constitutively activated in glioma cells,is known to play an important role in cell survival (inhibition of apoptosis) and has been linked to various human cancers. Disruption of the PI3K/Akt pathway, culminating in inhibition of Akt, has been found to be associated with autophagy induced by a variety of anti-neoplastic agents in cancer cells. Studies have indicated that Bcl-2 can be a strict mediator downstream of PI3K/Akt signaling, and that Akt contributes to the positive regulation of mTOR signaling pathway, which can inhibit cell autophagic activity.In HT-29 cells, the inhibitory effects of Bcl-2 are associated with disruption of the Beclin 1/hVps34 complexes, which are thought to be essential for early stages of autophagosomal formation Upon the induction of autophagy, phosphatidylethanolamine is covalently linked to the cytosolic protein LC3-I to yield LC3-II, which then associates with the autophagosome. This conversion is commonly used as a marker for autophagy .The purpose of this study was to determine whether H₂O₂ can induce programmed cell death in human glioma U251 cells to example the relationship between autophagy and apoptosis in this process, and to identify the signaling pathways that might be involved.Methods:(1) Cell culture(2) The Bcl-2 cDNA was amplified by PCR and then construct Anti-apoptosis protern Bcl-2 Overexpression vectors and detect their Overexpression and silencing effects by RT-PCR,Western Blot. (3) To detect the mechanisms of H₂O₂-induced glioma U251 cells injured. Expression of Bax,Bcl-2,cytC and Caspase-3 mRNA and protern detect by RT-PCR and Western Blot.and also detect expression of Beclin 1,Akt,FKHR,mTOR,p70S6K,LC 3 protern.(4) MDC, Hoechst 33342 and acridine orange staining were used to detect the autophagic vacuoles and cell apoptotic chromatin condensation by Confocal microscopy。autophagic vacuoles and cell apoptotic chromatin condensation in U251 cells treated with H₂O₂ by transmission electron microscopy.(5) To detect cell vitality and Expression of Mfn1, Mfn2 ,Opa1mRNA of fusion gene and Expression of Fis1, Drp1,MTP18 mRNA of fision gene by MTT and RT-PCR. Expression of Beclin 1,Akt,FKHR,mTOR,p70S6K,LC 3 protein by Western blot. The loss ofΔΨm was measured with Rhodamine123. autophagic vacuoles and cell apoptotic chromatin condensation were detected by Confocal microscopy.The generation of ROS was measured with the ROS-detecting fluorescent dye DCFH-DA.(6) To detect relationship to apoptosis and autophagic cell death though 3-MA and Z-VAD were used in H₂O₂-induced glial cells by Flowcytometry analysis and Immunofluorescent.Results:(1) MTT,Hoechst 33342 dyeing,western blot indicated H₂O₂ induced dose-dependent cell death in malignant glioma U251 cells,Cell vitality decrease, chromatic agglutination。the Bcl-2 proteins expression decreased significantly. the ratio of Bax/Bcl-2 mRNA and protein increased obviously. Caspase-3 signal activation and cytC release.RT-PCR result indicated H₂O₂ induced disruption of mitochondrial dynamics.(2) At the same time to detect the autophagy induced by H₂O₂, we have found The expression of autophagy-associated Beclin 1 protein was up-regulated and significant reduction of Akt, FKHR, mTOR and p70S6K phosphorylation. MDC staining can be used to detect autophagic vacuoles. compared with the control, autophagosomes were increased in U251 cells after exposed to H₂O₂ treatment.AO stain indicated red fluorescence increase. (3) We have constructed successfully a recombinant Overexpression plasmid of Bcl-2 ,and have also confirmed by restrictive enzyme digestion and DNA sequencing. RT-PCR and Western Blot analysis revealed a strongly decreased level of Bcl-2 mRNA, And Bcl-2 mRNA and protern do increased strongly in U251 cells transfected with the pEGFP-C1-Bcl-2 compared with the negative control.(4) flow cytometry and RT-PCR result indicated overexpression of Bcl-2 inhibited Cell vitality decrease and the generation of ROS and also inhibition the loss ofΔΨm induced by H₂O₂.(5) Co-immunoprecipitation of Beclin 1 and Bcl-2 in non-transfected U251 cells and U251 cells transfected with the overexpressed Bcl-2 vector indicate Bcl-2 co-immunoprecipitated with more endogenous Beclin 1 than in control U251 cells inhibiting autophagy though the class III PI3K/Beclin 1 pathway .on other hand, Bcl-2 inhibiting autophagy though decreased Akt,mTOR proteins expression. MDC staining indicated cell acidification and formation of autophagic vacuoles.(6) MTT assay indicated that 3-MA or Z-VAD-FMK alone had no effect on cell viability.MDC and AO staining indicated 3-MA treatment enhanced the autophagy induced by H₂O₂. MTT and Annexin V-FITC/PI staining indicated Inhibition autophagy by 3-MA accelerates the apoptosis induced by H₂O₂. the addition of Z-VAD-FMK increased cell viability of H₂O₂ treated cell but viability was still decreased compared with the control group,but the addition of Z-VAD-FMK have no effect with autophagy.Conclusions:(1) H₂O₂ can induced glioma U251 cells apoptosis, and mitochondria signal include Bax/Bcl-2,Caspase-3 and cytC participated in the regulation of apoptosis. H₂O₂ induces loss of mitochondrial membrane potential and disruption of mitochondrial dynamics.(2) H₂O₂ can induced glioma U251 cells autophagy though both Beclin 1 and Akt/mTOR signaling.(3) we have identified that successfully constructed a recombinant overexpression Bcl-2 plasmid and also successfully constructed U251-Bcl-2 stable cells.(4) Bcl-2 can partial inhibited ROS accumulation and decrease mitochondria membrane potential stablized mitochondria.at the same time, Bcl-2 can inhibited autophagy induced by H₂O₂ though both Beclin 1 and Akt/mTOR signaling.(5) Inhibition autophagy by 3-MA accelerates the apoptosis induced by H₂O₂.we presume autophagy induced by H₂O₂ in glioma U251 cells could protect cells from injure.In glioma U251 cells injure induced by H₂O₂, autophagy can portect cells though supplying energy, and rivalry oxidative stress injure, thereby infer that autophagy suppressant and anticancer drugs combination application may become new and effective strategy to tumor therapy.