GBM Derived Gangliosides Induce T Cell Apoptosis through Activation of the Caspase Cascade Involving Both the Extrinsic and the Intrinsic Pathway.
GBM Derived Gangliosides Induce T Cell Apoptosis through Activation of the Caspase Cascade Involving Both the Extrinsic and the Intrinsic Pathway.
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DOI：
10.1371/journal.pone.0134425
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Biswas K
中科院分区：
文献类型：
--
作者：
Mahata B;Biswas S;Rayman P;Chahlavi A;Ko J;Bhattacharjee A;Li YT;Li Y;Das T;Sa G;Raychaudhuri B;Vogelbaum MA;Tannenbaum C;Finke JH;Biswas K
先前我们证明了人胶质母细胞瘤细胞系通过部分参与分泌神经节苷来诱导外周血T细胞凋亡。在这里，我们表明gbm衍生的神经节苷通过参与TNF受体和激活caspase级联诱导细胞凋亡。用GBM细胞系衍生的神经节苷脂（10-20μg/ml）培养T淋巴细胞，早在18小时就显示ROS产生增加，这表明H2DCFDA染料的摄取增加，而western blotting显示线粒体损伤，这表明Bid裂解成T -Bid和细胞色素c释放到细胞质中。48-72小时内，核泡、台盼蓝阳性和annexinV/7AAD染色显示细胞凋亡明显。gbm -神经节苷脂在T细胞中诱导效应caspase-3和两种引发剂caspase（-9和-8）的激活，而caspase-8和-9抑制剂在阻止细胞凋亡方面同样有效（60%的保护），证实了caspase在细胞凋亡过程中的作用。神经节苷脂诱导的T细胞凋亡不涉及TNF-α的产生，因为抗人TNF-α抗体不能保护T细胞免受核泡和随后的细胞死亡。然而，共聚焦显微镜显示GM2神经节苷脂与TNF受体共定位，共免疫沉淀实验显示神经节苷脂治疗后TNF受体募集死亡结构域FADD和TRADD，提示神经节苷脂与TNF受体直接相互作用。利用野生型和TNFR1 KO （TALEN介导）Jurkat细胞的共聚焦显微镜数据进一步证实了GM2和TNFR1之间的相互作用，这清楚地表明GM2和TNFR1在野生型细胞中共定位，而在TNFR1 KO克隆中不存在。因此，gbm -神经节苷脂可以通过与TNF受体相互作用，激活半胱天冬酶的外源性和内源性途径，介导T细胞凋亡。
Previously we demonstrated that human glioblastoma cell lines induce apoptosis in peripheral blood T cells through partial involvement of secreted gangliosides. Here we show that GBM-derived gangliosides induce apoptosis through involvement of the TNF receptor and activation of the caspase cascade. Culturing T lymphocytes with GBM cell line derived gangliosides (10-20μg/ml) demonstrated increased ROS production as early as 18 hrs as indicated by increased uptake of the dye H2DCFDA while western blotting demonstrated mitochondrial damage as evident by cleavage of Bid to t-Bid and by the release of cytochrome-c into the cytosol. Within 48-72 hrs apoptosis was evident by nuclear blebbing, trypan blue positivity and annexinV/7AAD staining. GBM-ganglioside induced activation of the effector caspase-3 along with both initiator caspases (-9 and -8) in T cells while both the caspase-8 and -9 inhibitors were equally effective in blocking apoptosis (60% protection) confirming the role of caspases in the apoptotic process. Ganglioside-induced T cell apoptosis did not involve production of TNF-α since anti-human TNFα antibody was unable to protect T cells from nuclear blebbing and subsequent cell death. However, confocal microscopy demonstrated co-localization of GM2 ganglioside with the TNF receptor and co-immunoprecipitation experiments showed recruitment of death domains FADD and TRADD with the TNF receptor post ganglioside treatment, suggesting direct interaction of gangliosides with the TNF receptor. Further confirmation of the interaction between GM2 and TNFR1 was obtained from confocal microscopy data with wild type and TNFR1 KO (TALEN mediated) Jurkat cells, which clearly demonstrated co-localization of GM2 and TNFR1 in the wild type cells but not in the TNFR1 KO clones. Thus, GBM-ganglioside can mediate T cell apoptosis by interacting with the TNF receptor followed by activation of both the extrinsic and the intrinsic pathway of caspases.