Study On The Role For Autophagy-related Gene/Beclin 1 In The Apoptosis Of Malignant Glioma Cells

Astrocytoma remains the most common primary neoplasm of the central nervous system and accounts for approximately 45～55% of all brain tumors. It represents a heterogenous group of diseases with different degree of malignancy from relatively indolent pilocytic astrocytomas to highly aggressive glioblastomas. Unfortunately, current therapeutic modalities including surgical resections, chemotherapy, radiotherapy or combinations can not ensure a cure and, the molecular mechanisms underlying the initiation, maintenance and progression of astrocytomas still remain largely unclarified. Hence, identification and characterization of the regulatory molecules that involved in the astrocytoma tumorigenesis may offer important targets for treatment strategies.The human Beclin 1 gene has been mapped to a tumor-susceptibility locus on chromosome 17q21 that is monoallelically deleted in up to 75% of ovarian cancers,50% of breast cancers, and 40% of prostate cancers. The previous study also demonstrated the lower expression of Beclin 1 mRNA and protein expressions in glioblastomas. Moreover, Beclin 1-/-mutant mice die early in embryogenesis and Beclin 1+/-mutant mice suffer from a high incidence of spontaneous tumors, establishing that Beclin 1 is a critical component of mammalian autophagy and a role for autophagy in tumor suppression. Liang's group found a frequent loss of Beclin 1 protein expression in breast cancers, and suggested that tumor suppressor functions of Beclin-1 are lost in the cancer cells by loss of protein expression.The interaction between Beclin 1 and its binding partners regulates the initial steps of autophagy. The association of Beclin 1 with hVps34 and PI3k is essential for the induction of autophagy, and the autophagy-inducing activity of Beclin 1 is inhibited by over-expression of Bcl-2 family anti-apoptotic proteins including Bcl-2, Bcl-xL and Mcl-1 but not pro-apoptotic proteins. There is evidence that the orthologs of Beclin 1, hVps34 and Bcl-2 do not form a trimolecular complex, suggesting that Beclin 1 can be present in two different complexes, one that stimulates autophagy and involves an interaction with hVps34 and another one that inhibits autophagy and involves an interaction with Bcl-2. Thus, it is possible that the interaction between Beclin 1 and Bcl-2 has dual effects:one is to modulate the apoptotic pathway and the second is to inhibit autophagy. These two effects may be mutually exclusive or act together. Accordingly, upon complexation, Bcl-2 might inhibit Beclin 1's ability to participate in the autophagic process whereas Bcl-2 will be concomitantly neutralized, thereby sensitizing the cells to apoptosis. In this way, the interaction between Beclin 1 and anti-apoptotic Bcl-2 family members may affect cell fate. Taken together these observations imply that Beclin 1, in addition to its role in autophagy, may play a role in apoptosis by neutralizing the anti-apoptotic proteins in Bcl-2 family.The aim of this study is to investigate the expression of Beclin 1 in glioma tissues of different grades and glioma cell lines, and explore the influence of Beclin 1 on autophagy, apoptosis and cell proliferation in glioma cells. This study is consisted of three main parts: the first part is to investigate expression of Beclin 1 in astrocytic tumors; the second is to investigate the expression of Beclin 1, Bcl-2 family proteins and Vps34 in deferent treated glioma cell lines and detect the complexes of Beclin 1 with Bcl-2 family proteins or Vps34 in U87 cells, which over-expressed or super-expresses Beclin 1; finally, to explore whether Beclin 1 induced autophagy, altered apoptosis-related proteins and had influence on cell proliferation in glioma cells.Part I Expression of Beclin 1 in astrocytic tumorsObjective:investigate the expression of autophagy-related gene Beclin 1 in astrocytic tumors, and explore their correlations to the development of astrocytic tumors.Methods:Beclin 1 and LC3B expressions in 62 specimens of different grade astrocytic tumors were detected by immunohistochemistry and(or) western blot. The correlations of LC3B and Beclin 1 expression to the clinicopathologic and clinical characteristics of the patients were analyzed.Results:Immunohistochemistry showed a decrease of Beclin 1 expression in different grade astrocytic tumors. Western blot indicated that average optical density ratio of Beclin 1 in high-grade astrocytic tumors(grade III/IV) was lower than that in low-grade astrocytic tumors(gradeⅠ/Ⅱ, p=0.036). The expressions of LC3B-I had no significant difference in different grade astrocytic tumors. But average optical density ratio of LC3B-II in glioblastomas was lower than that in other grade astrocytic tumors(p= 0.030). The expressions of Beclinl and LC3B-Ⅱwere related to survival time, they also correlated to each other(P=0.035).Conclusion:Expressions of LC3B-Ⅱand Beclinl are down-regulated in glioblastomas. The decrease of autophagic capacity relates to the progression of astrocytic tumors. PartⅡExpression of Beclin 1 in U87 glioma cell linesObjective:To investigate the expression of Beclin 1, Bcl-2 family proteins and Vps34 in deferent treated glioma cell lines and detect the complexes of Beclin 1 with Bcl-2 family proteins or Vps34 in U87 cells, which over-expressed or super-expresses Beclin 1.Methods:Beclin 1 expressions were altered in U87 and U87 cells by introducing a Beclin 1 expression vector (pcDNA3.1-Bec) or a siRNA targeted to Beclin 1 gene (pSUPER-Bec). The expression level of Beclin 1 mRNA was evaluated by real-time PCR in U87 and U251 glioma cell lines. The protein expressions of Beclin 1, Bcl-2 family members and Vps34 were measured by western blot. Furthermore, the complexes of Beclin 1 with Bcl-2 family proteins or Vps34 were detected in immunoprecipitates.Results:Beclin 1siRNA and Beclin 1 expression vector transfect more effectively in U87 cells than in U251 cells. The mRNA or protein expressions of Beclin 1, demonstrated by RT-PCR or western blot, were decreased in U251 and U87 glioma cells versus that in normal glial cell line HEB (P<0.05). Campared with the normal brain tissues, the proteins of Bcl-2 family members were elevated in U87 cells. There was no significant difference between the Vps34 expression in those two glioma cell lines and that in the normal brain tissues. The Beclin 1-Vps34 complexes were detected in all cell lines. Over-expressed Beclin 1 in U87 cells, Beclin 1 was detected in immunoprecipitates prepared with anti-Bcl-2 antibody or anti-Bcl-xL antibody. Bcl-2 and Bcl-xL were detected in immunoprecipitates prepared with anti-Beclin 1 antibody. In addition, neither Beclin 1-Bax complex nor Beclin 1-Bak complex was detected. On the other hand, the complex of Beclin 1 and Bcl-2 family protein was hardly detected in other transfectants or untreated cells.Conclusion:Beclin 1 is down-regulated, but Bcl-2 family membersin are up-regulated in U87 cells. In U87 cells, Beclin 1 can bend to Vps34, but Beclin 1 bends to Bcl-2 alike proteins only when Beclin 1 was over-expressed. Beclin may take part in regulation of autophagy and apoptosis by the binding of Beclin 1 to Bcl-2 or Vps34.PartⅢOver-expression of Beclin 1 augments apoptosis in U87 glioma cells Objective:To explore whether Beclin 1 induced autophagy, altered apoptosis-related proteins and had influence on cell proliferation in U87 glioma cells.Methods:Beclin 1 expressions were altered in U87 cells by introducing a Beclin 1 expression vector (pcDNA3.1-Bec) or a siRNA targeted to Beclin 1 gene (pSUPER-Bec). After 48h of transfection, the expression levels of LC3 and p62 Protein were measured by western blot. Cell apoptosis was analyzed by Hoechst 33258 stain and flow cytometry, and cell proliferative activity was assessed by 3H-TdR assay. Immunofluorescene labelling assay was employed to investigate the celluar location of cytochrome c in U87 cells. The level of cytochrome c in cytoplasm and in mitochondria was evaluated by western blot. The active caspase3/8/9 were also was assessed by western blot.Results:The lanes of Western blotting showed that expression of LC3-Ⅱwas slightly increased in PCDNA3.1-Bec transfectants compared with vector transfectants or untreated cells, whereas LC3-Ⅱexpression was dramatically decreased in pSUPER-Bec transfectants. Conversely, the level of p62 was lower in pcDNA3.1-Bec transfectants but higher in pSUPER-Bec transfectants than vector transfectants or scramble RNA transfectants. An obviously increased rate of apoptosis was observed in flow cytometry (FCM) assay in pcDNA3.1-Bec transfectants compared with other transfectants or untreated cells. The result was consistent with trituum—labelled thymidine (3H-TdR) detecting for cell proliferation. pcDNA3.1-Bec transfectants showed lower cell proliferation than other transfectants or untreated cells. The fluorescence staining of cytochrome c appeared confined in mitochondria in vector transfectants, while it was diffused throughout the entire cytoplasm in pcDNA3.1-Bec transfectants. Western blot showed that cytosolic localization of cytochrome c was significantly increased, but also confirmed the amount of mitochondrial cytochrome c was obviously decreased in pcDNA3.1-Bec transfectants compared with that in other transfectants or untreated cells. Western blot also showed that no clear difference in the increase of caspase-8 activity was seen between pcDNA3.1-Bec transfectants and other transfectants, however, not only increase of caspase-3 activity but also increase of caspase-9 activity was greater in pcDNA3.1-Bec transfectants than in other transfectants.Conclusion:Overall, silencing of Beclin 1 demonstrated decrease of autophagic capacity, but had little effect on apoptosis and cell proliferation.Over-expression of Beclin 1 in U87 cells augmented autophagic capacity and apoptosis but reduced cell proliferation. Beclin 1 induces apoptosis via binding to Bcl-2 and Bcl-xL, followed by release of cytochrome c into the cytosol and activation of caspases3/9. Beclin 1 may play an important role in fine tuning autophagy and apoptosis through interaction with Bcl-2 and Bcl-xL.