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GBM Derived Gangliosides Induce T Cell Apoptosis through Activation of the Caspase Cascade Involving Both the Extrinsic and the Intrinsic Pathway.

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 - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Medicine, Bose Institute, Kolkata, India.

ABSTRACT
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 (10-20 μg/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.

No MeSH data available.


Related in: MedlinePlus

Human apoptosis proteome profiler array demonstrates ganglioside induced activation of pro-apoptotic and downregulation of anti-apoptotic proteins.Differential expression of pro- and anti-apoptotic proteins were examined in T cells cultured with CCF52 ganglioside (15μg/ml) for 48hrs using a human proteome profiler array kit (R&D Biosystems) as represented in Fig 4A and 4B. Fig 4C shows the entire apoptosis proteome profile array of T cells in presence or absence of CCF52 ganglioside. Data is representative of a single experiment out of two experiments done.
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pone.0134425.g004: Human apoptosis proteome profiler array demonstrates ganglioside induced activation of pro-apoptotic and downregulation of anti-apoptotic proteins.Differential expression of pro- and anti-apoptotic proteins were examined in T cells cultured with CCF52 ganglioside (15μg/ml) for 48hrs using a human proteome profiler array kit (R&D Biosystems) as represented in Fig 4A and 4B. Fig 4C shows the entire apoptosis proteome profile array of T cells in presence or absence of CCF52 ganglioside. Data is representative of a single experiment out of two experiments done.

Mentions: To get a global view of differential expression of pro- and anti-apoptotic proteins in T cells in response to GBM gangliosides, normal donor T cells were cultured with CCF52 ganglioside (15μg/ml) for 48hrs. Lysates were used to profile differential expression levels of 56 proteins involved in apoptosis, using a human apoptosis proteome profiler array kit (R&D Systems). Analysis of the membranes indicate significant changes in the expression levels of several different proteins involved in apoptotic machinery, as shown in Fig 4C. As expected caspase-3 and cytochrome-c were found to be upregulated significantly in CCF52 treated T cells versus the control cells (Fig 4A). There were also increased Bad and FADD expression (Fig 4A) while the expression of the anti-apoptotic proteins, cIAP-1 and survivin (Fig 4B) was diminished in T cells incubated with gangliosides. Thus, GBM derived gangliosides promote T cell death not only by inducing pro-apoptotic proteins, but in the process, they decrease the levels of select anti-apoptotic proteins as well.


GBM Derived Gangliosides Induce T Cell Apoptosis through Activation of the Caspase Cascade Involving Both the Extrinsic and the Intrinsic Pathway.

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 - PLoS ONE (2015)

Human apoptosis proteome profiler array demonstrates ganglioside induced activation of pro-apoptotic and downregulation of anti-apoptotic proteins.Differential expression of pro- and anti-apoptotic proteins were examined in T cells cultured with CCF52 ganglioside (15μg/ml) for 48hrs using a human proteome profiler array kit (R&D Biosystems) as represented in Fig 4A and 4B. Fig 4C shows the entire apoptosis proteome profile array of T cells in presence or absence of CCF52 ganglioside. Data is representative of a single experiment out of two experiments done.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520498&req=5

pone.0134425.g004: Human apoptosis proteome profiler array demonstrates ganglioside induced activation of pro-apoptotic and downregulation of anti-apoptotic proteins.Differential expression of pro- and anti-apoptotic proteins were examined in T cells cultured with CCF52 ganglioside (15μg/ml) for 48hrs using a human proteome profiler array kit (R&D Biosystems) as represented in Fig 4A and 4B. Fig 4C shows the entire apoptosis proteome profile array of T cells in presence or absence of CCF52 ganglioside. Data is representative of a single experiment out of two experiments done.
Mentions: To get a global view of differential expression of pro- and anti-apoptotic proteins in T cells in response to GBM gangliosides, normal donor T cells were cultured with CCF52 ganglioside (15μg/ml) for 48hrs. Lysates were used to profile differential expression levels of 56 proteins involved in apoptosis, using a human apoptosis proteome profiler array kit (R&D Systems). Analysis of the membranes indicate significant changes in the expression levels of several different proteins involved in apoptotic machinery, as shown in Fig 4C. As expected caspase-3 and cytochrome-c were found to be upregulated significantly in CCF52 treated T cells versus the control cells (Fig 4A). There were also increased Bad and FADD expression (Fig 4A) while the expression of the anti-apoptotic proteins, cIAP-1 and survivin (Fig 4B) was diminished in T cells incubated with gangliosides. Thus, GBM derived gangliosides promote T cell death not only by inducing pro-apoptotic proteins, but in the process, they decrease the levels of select anti-apoptotic proteins as well.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Medicine, Bose Institute, Kolkata, India.

ABSTRACT
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 (10-20 μg/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.

No MeSH data available.


Related in: MedlinePlus