自噬相关基因Beclin 1表达对恶性胶质瘤细胞凋亡影响的实验研究
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摘要
脑胶质瘤是中枢神经系统最常见的恶性肿瘤之一,约占全部脑肿瘤的45～55%。目前,脑胶质瘤的治疗手段主要包括手术治疗、放射治疗、化学治疗和综合治疗等,尽管近年来上述治疗措施已取得长足进步,但由于胶质瘤呈浸润性生长,与周围脑组织无明显分界且易于复发,且疗效均不甚理想。新的治疗手段如基因治疗等虽有望成为治愈胶质瘤的途径之一,但是目前仍缺乏理想可靠的治疗靶标。因此寻找调控胶质瘤细胞恶性生物学行为的分子靶标,明确其在胶质瘤发病过程中的病理机制,已成为神经外科领域的研究热点之一。
自噬相关基因(autophagy-related gene, Atg)调节的细胞自噬活性下降在肿瘤的形成中具有重要作用。Beclin 1 (Atg6)是重要的自噬调节基因,主要作用是通过与Vps34/PI3K形成复合物促进自噬, Bcl-2能与Beclin 1结合抑制自噬活性。因为Beclin 1与Bcl-2家族中抑制凋亡的蛋白结合,但不能与促凋亡蛋白结合,并且研究Beclin 1的分子结构发现其具有Bcl-2家族中促凋亡蛋白共有的BH3结构,所以Beclin1可能参与凋亡的调节。Beclin 1被认为是肿瘤抑制基因,其杂合性缺失是细胞发生恶性转化的原因之一。Beclin 1抑制肿瘤的作用可能是提高肿瘤细胞自噬活性。Yue等应用Beclin 1基因敲除小鼠的胚胎干细胞,发现Beclin 1完全缺失小鼠死于胚胎早期,Beclin 1杂合性缺失小鼠虽然表型正常,但体内细胞自噬活性降低,而且自发肿瘤发生率高。Liang等将Beclin 1稳定转染MCF-7细胞株后,发现自噬空泡的数量增加,癌细胞体外增殖能力及恶性表型下降,并且在裸鼠中肿瘤形成能力降低。实验发现化疗能引发细胞自噬性死亡,而在凋亡抑制细胞中自噬诱导剂可上调Beclin 1具有治疗作用。Beclin 1抑制肿瘤不仅诱发细胞自噬性死亡,而且能停滞细胞周期,抑制细胞增殖,诱导肿瘤细胞凋亡,抑制肿瘤坏死及炎症扩散,预防细胞基因组突变。Beclin 1基因还和一些肿瘤相关信号传导通道关系密切,如PTEN通道和Rb信号传导通道都与Beclin 1不同程度相关。因此,探讨Beclin 1参与脑胶质瘤发生发展以及恶变的分子病理机制,尤其是在细胞自噬和凋亡中的作用,有助于明确胶质瘤的恶性生物学行为及机理,有望为胶质瘤的临床治疗提供新的分子靶标。
本研究拟通过观察Beclin 1在不同病理级别脑胶质瘤以及胶质瘤细胞株中的确切表达情况,并通过过表达和敲减Beclin 1在胶质瘤细胞株中的表达,研究Beclin 1在恶性胶质瘤细胞自噬尤其是凋亡方面的作用。实验共分为三个部分：第一部分,观察Beclin 1在脑星形细胞肿瘤组织中的蛋白表达水平以及与病理级别的相关性；第二部分,观察Beclin 1在U251和U87脑胶质瘤细胞株中的mRNA和蛋白表达水平及相关蛋白的表达,以及构建Beclin 1蛋白表达抑制和过表达的U87细胞株和检测相关蛋白的结合情况;第三部分,研究Beclin 1在U87细胞自噬、凋亡和增殖中的作用。
第一部分Beclin 1在星形细胞肿瘤组织中的表达
目的：本研究检测自噬相关基因Beclin 1在星形细胞肿瘤中的表达情况,探讨其与星形细胞肿瘤病理和临床表现的相关性及意义。
方法：用免疫组化和(或)免疫印迹法检测62例不同级别的星形细胞肿瘤Beclin1蛋白表达,并结合临床和病理因素进行分析。
结果：免疫组化显示Beclin 1蛋白在星形细胞肿瘤胞质中表达强弱随肿瘤级别升高而降低,在胞核中则相反。免疫印迹法检测高级别星形细胞肿瘤(Ⅲ／Ⅳ级)Beclin1平均光密度比值小于低级别肿瘤(Ⅰ／Ⅱ级,p=0.036)。LC3B-Ⅰ在不同级别的星形细胞肿瘤中表达无明显差异,但LC3B-Ⅱ在胶母细胞瘤中的平均光密度比值低于其他级别的肿瘤(p=0.030)。Beclin 1和LC3B-Ⅱ高表达者的生存率有升高趋势,两者在星形细胞肿瘤中表达正性相关(p=0.035)。
结论：Beclin 1和LC3B-Ⅱ在胶母细胞瘤中的表达下调,自噬活性的改变可能与星形细胞肿瘤的发生、发展相关。
第二部分Beclin 1在恶性胶质瘤细胞系中的表达
目的：研究Beclin 1、Bcl-2家族蛋白和Vps34在胶质瘤细胞系U251和U87中的表达和蛋白之间的结合情况；研究Beclin 1在U87细胞株经过表达或RNA干扰后与Bcl-2家族蛋白和Vps34结合情况。
方法：用脂质体法将表达Beclin 1 siRNA和Beclin 1基因质粒分别转染U87细胞株,鉴定转染效率和细胞Beclin 1蛋白表达。应用real-time PCR检测Beclin 1在胶质瘤细胞系U251和U87中的mRNA表达水平,应用免疫印迹方法研究Beclin 1、Bcl-2家族蛋白和Vps34在胶质瘤细胞系中的蛋白表达水平,应用免疫共沉淀检测]Beclin 1与Bcl-2家族蛋白和Vps34之间的结合情况。
结果：Beclin 1 siRNA和Beclin 1基因表达的质粒在转染进入U87细胞后抑制和过表达效果较好。RT-PCR和免疫印迹结果提示Beclin 1 mRNA和蛋白表达水平在胶质瘤细胞系U251和U87中明显下调。免疫印迹提示Bcl-2家族蛋白在U251和U87中明显上调(P<0.05)。Beclin 1和Vps34复合物能在各处理组U87中发现；在各对照组和RNA干扰后的U87中检测免疫共沉淀未发现Beclin 1分别与Bcl-2、Bcl-xL形成复合物,而在过表达Beclin 1的U87中检测出Beclin 1分别与Bcl-2、Bcl-xL形成复合物。
结论：Beclin 1在所有被检测的胶质瘤细胞系中,其mRNA和蛋白表达水平明显低于正常胶质细胞,且Bcl-2家族蛋白在U87细胞中表达增高。U87中Beclin 1与Vps34能形成复合物,在Beclin 1过表达时Beclin 1才与Bcl-2类似蛋白形成复合物。不同处理组之间Beclin 1蛋白结合变化可能是Beclin 1参与调节白噬和凋亡作用的机制。
第三部分Beclin 1表达对胶质瘤细胞自噬、凋亡和增殖的作用
目的：研究过表达或抑制Beclin 1对U87胶质瘤细胞株白噬、凋亡和增殖的影响;以及Beclin 1在凋亡通路中的具体作用。
方法：利用第二部分中U87细胞株的不同处理组培养48小时,采用免疫印迹检测LC3、p62在各组的表达；用Hoechst 33258染色及流式细胞仪测定各组凋亡细胞比例;氚标胸腺嘧啶脱氧核苷(trituum—labelled thymidine,3H-TdR)掺入检测各组细胞增殖率。用免疫荧光和免疫印迹检测各组细胞色素C的分布和表达情况；用免疫印迹检测活性caspase3/8/9表达；用免疫荧光检测Beclinl和caspase3在细胞中的表达。
结果：LC3-Ⅱ在过表达组呈高表达；在抑制组呈低表达。P62在过表达组呈低表达；抑制组呈高表达。Hoechst 33258染色及流式细胞仪测定显示过表达组凋亡细胞比例增高；其他各组无明显变化。3H-TdR法显示过表达组细胞增殖减缓,其他各组无明显变化。免疫荧光显示过表达组细胞色素C弥散分布于细胞中；其他各组细胞色素C分布比较集中且仅限于胞质。免疫印迹发现过表达组胞质中细胞色素C有表达,其他各组未见明显表达。免疫印迹检测显示过表达组活性caspase3/9表达增高,caspase8在各组间无明显差别。免疫荧光显示Beclin 1和caspase3在过表达Beclin 1的U87细胞中的表达增加,并有相关性。
结论：在Beclin 1抑制组中细胞自噬活性下降,凋亡活性无明显变化,细胞增殖无明显变化。而过表达组白噬活性增强,凋亡活性增强,细胞增殖减缓。过表达组胞质中细胞色素C表达增高,caspase3/9活性增高。结合第二部分结果(Beclin 1过表达时Beclin 1才与Bcl-2类似蛋白形成复合物),我们认为Beclin 1通过与Bcl-2和Bcl-xL结合,引起细胞色素C从线粒体释放进入胞质,激活caspases3/9,从而促进细胞凋亡。
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 tumors
Objective: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 lines
Objective: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.
自噬相关基因(autophagy-related gene, Atg)调节的细胞自噬活性下降在肿瘤的形成中具有重要作用。Beclin 1 (Atg6)是重要的自噬调节基因,主要作用是通过与Vps34/PI3K形成复合物促进自噬, Bcl-2能与Beclin 1结合抑制自噬活性。因为Beclin 1与Bcl-2家族中抑制凋亡的蛋白结合,但不能与促凋亡蛋白结合,并且研究Beclin 1的分子结构发现其具有Bcl-2家族中促凋亡蛋白共有的BH3结构,所以Beclin1可能参与凋亡的调节。Beclin 1被认为是肿瘤抑制基因,其杂合性缺失是细胞发生恶性转化的原因之一。Beclin 1抑制肿瘤的作用可能是提高肿瘤细胞自噬活性。Yue等应用Beclin 1基因敲除小鼠的胚胎干细胞,发现Beclin 1完全缺失小鼠死于胚胎早期,Beclin 1杂合性缺失小鼠虽然表型正常,但体内细胞自噬活性降低,而且自发肿瘤发生率高。Liang等将Beclin 1稳定转染MCF-7细胞株后,发现自噬空泡的数量增加,癌细胞体外增殖能力及恶性表型下降,并且在裸鼠中肿瘤形成能力降低。实验发现化疗能引发细胞自噬性死亡,而在凋亡抑制细胞中自噬诱导剂可上调Beclin 1具有治疗作用。Beclin 1抑制肿瘤不仅诱发细胞自噬性死亡,而且能停滞细胞周期,抑制细胞增殖,诱导肿瘤细胞凋亡,抑制肿瘤坏死及炎症扩散,预防细胞基因组突变。Beclin 1基因还和一些肿瘤相关信号传导通道关系密切,如PTEN通道和Rb信号传导通道都与Beclin 1不同程度相关。因此,探讨Beclin 1参与脑胶质瘤发生发展以及恶变的分子病理机制,尤其是在细胞自噬和凋亡中的作用,有助于明确胶质瘤的恶性生物学行为及机理,有望为胶质瘤的临床治疗提供新的分子靶标。
本研究拟通过观察Beclin 1在不同病理级别脑胶质瘤以及胶质瘤细胞株中的确切表达情况,并通过过表达和敲减Beclin 1在胶质瘤细胞株中的表达,研究Beclin 1在恶性胶质瘤细胞自噬尤其是凋亡方面的作用。实验共分为三个部分：第一部分,观察Beclin 1在脑星形细胞肿瘤组织中的蛋白表达水平以及与病理级别的相关性；第二部分,观察Beclin 1在U251和U87脑胶质瘤细胞株中的mRNA和蛋白表达水平及相关蛋白的表达,以及构建Beclin 1蛋白表达抑制和过表达的U87细胞株和检测相关蛋白的结合情况;第三部分,研究Beclin 1在U87细胞自噬、凋亡和增殖中的作用。
第一部分Beclin 1在星形细胞肿瘤组织中的表达
目的：本研究检测自噬相关基因Beclin 1在星形细胞肿瘤中的表达情况,探讨其与星形细胞肿瘤病理和临床表现的相关性及意义。
方法：用免疫组化和(或)免疫印迹法检测62例不同级别的星形细胞肿瘤Beclin1蛋白表达,并结合临床和病理因素进行分析。
结果：免疫组化显示Beclin 1蛋白在星形细胞肿瘤胞质中表达强弱随肿瘤级别升高而降低,在胞核中则相反。免疫印迹法检测高级别星形细胞肿瘤(Ⅲ／Ⅳ级)Beclin1平均光密度比值小于低级别肿瘤(Ⅰ／Ⅱ级,p=0.036)。LC3B-Ⅰ在不同级别的星形细胞肿瘤中表达无明显差异,但LC3B-Ⅱ在胶母细胞瘤中的平均光密度比值低于其他级别的肿瘤(p=0.030)。Beclin 1和LC3B-Ⅱ高表达者的生存率有升高趋势,两者在星形细胞肿瘤中表达正性相关(p=0.035)。
结论：Beclin 1和LC3B-Ⅱ在胶母细胞瘤中的表达下调,自噬活性的改变可能与星形细胞肿瘤的发生、发展相关。
第二部分Beclin 1在恶性胶质瘤细胞系中的表达
目的：研究Beclin 1、Bcl-2家族蛋白和Vps34在胶质瘤细胞系U251和U87中的表达和蛋白之间的结合情况；研究Beclin 1在U87细胞株经过表达或RNA干扰后与Bcl-2家族蛋白和Vps34结合情况。
方法：用脂质体法将表达Beclin 1 siRNA和Beclin 1基因质粒分别转染U87细胞株,鉴定转染效率和细胞Beclin 1蛋白表达。应用real-time PCR检测Beclin 1在胶质瘤细胞系U251和U87中的mRNA表达水平,应用免疫印迹方法研究Beclin 1、Bcl-2家族蛋白和Vps34在胶质瘤细胞系中的蛋白表达水平,应用免疫共沉淀检测]Beclin 1与Bcl-2家族蛋白和Vps34之间的结合情况。
结果：Beclin 1 siRNA和Beclin 1基因表达的质粒在转染进入U87细胞后抑制和过表达效果较好。RT-PCR和免疫印迹结果提示Beclin 1 mRNA和蛋白表达水平在胶质瘤细胞系U251和U87中明显下调。免疫印迹提示Bcl-2家族蛋白在U251和U87中明显上调(P<0.05)。Beclin 1和Vps34复合物能在各处理组U87中发现；在各对照组和RNA干扰后的U87中检测免疫共沉淀未发现Beclin 1分别与Bcl-2、Bcl-xL形成复合物,而在过表达Beclin 1的U87中检测出Beclin 1分别与Bcl-2、Bcl-xL形成复合物。
结论：Beclin 1在所有被检测的胶质瘤细胞系中,其mRNA和蛋白表达水平明显低于正常胶质细胞,且Bcl-2家族蛋白在U87细胞中表达增高。U87中Beclin 1与Vps34能形成复合物,在Beclin 1过表达时Beclin 1才与Bcl-2类似蛋白形成复合物。不同处理组之间Beclin 1蛋白结合变化可能是Beclin 1参与调节白噬和凋亡作用的机制。
第三部分Beclin 1表达对胶质瘤细胞自噬、凋亡和增殖的作用
目的：研究过表达或抑制Beclin 1对U87胶质瘤细胞株白噬、凋亡和增殖的影响;以及Beclin 1在凋亡通路中的具体作用。
方法：利用第二部分中U87细胞株的不同处理组培养48小时,采用免疫印迹检测LC3、p62在各组的表达；用Hoechst 33258染色及流式细胞仪测定各组凋亡细胞比例;氚标胸腺嘧啶脱氧核苷(trituum—labelled thymidine,3H-TdR)掺入检测各组细胞增殖率。用免疫荧光和免疫印迹检测各组细胞色素C的分布和表达情况；用免疫印迹检测活性caspase3/8/9表达；用免疫荧光检测Beclinl和caspase3在细胞中的表达。
结果：LC3-Ⅱ在过表达组呈高表达；在抑制组呈低表达。P62在过表达组呈低表达；抑制组呈高表达。Hoechst 33258染色及流式细胞仪测定显示过表达组凋亡细胞比例增高；其他各组无明显变化。3H-TdR法显示过表达组细胞增殖减缓,其他各组无明显变化。免疫荧光显示过表达组细胞色素C弥散分布于细胞中；其他各组细胞色素C分布比较集中且仅限于胞质。免疫印迹发现过表达组胞质中细胞色素C有表达,其他各组未见明显表达。免疫印迹检测显示过表达组活性caspase3/9表达增高,caspase8在各组间无明显差别。免疫荧光显示Beclin 1和caspase3在过表达Beclin 1的U87细胞中的表达增加,并有相关性。
结论：在Beclin 1抑制组中细胞自噬活性下降,凋亡活性无明显变化,细胞增殖无明显变化。而过表达组白噬活性增强,凋亡活性增强,细胞增殖减缓。过表达组胞质中细胞色素C表达增高,caspase3/9活性增高。结合第二部分结果(Beclin 1过表达时Beclin 1才与Bcl-2类似蛋白形成复合物),我们认为Beclin 1通过与Bcl-2和Bcl-xL结合,引起细胞色素C从线粒体释放进入胞质,激活caspases3/9,从而促进细胞凋亡。
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 tumors
Objective: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 lines
Objective: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.
引文
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9 Yue Z, Jin S, Yang C, Levine AJ, Heintz N. Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor. Proc Natl Acad Sci U S A[J].2003 Dec 9; 100(25):15077-82.
10 Zeng X, Overmeyer JH, Maltese WA. Functional specificity of the mammalian Beclin-Vps34 PI 3-kinase complex in macroautophagy versus endocytosis and lysosomal enzyme trafficking. J Cell Sci[J].2006 Jan 15;119(Pt 2):259-70.
11 Kihara A, Kabeya Y, Ohsumi Y, Yoshimori T. Beclin-phosphatidylinositol 3-kinase complex functions at the trans-Golgi network. EMBO Rep[J].2001 Apr;2(4):330-5.
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