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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

Involvement of mitochondria in GBM ganglioside mediated caspase activation.T cells were co-cultured with GBM derived gangliosides CCF52 and CCF4, and t-Bid induction was detected as shown in Fig 3A. Reduced fluorescence of DiOC6 in T cells treated with U87 gangliosides (15μg/ml) for 48hrs, versus control cells is indicative of mitochondrial damage (Fig 3B). Evidence of mitochondrial damage was also observed in T cells exposed to GBM derived gangliosides as evidenced from mitochondrial cytochrome c release (Fig 3C). Time dependent induction of reactive oxygen species (ROS) was measured in purified T cells treated with 15μg/ml CCF52 and CCF4 derived gangliosides for 18hrs and 48hrs, as compared to media control for 48hrs, evidenced by H2DCFDA staining and flow cytometric analysis (Fig 3D) (*p<0.05 vs Media, **p<0.01 vs Media, ***p<0.01 vs Media). ROS production is represented as the mean fluorescence intensity (MFI) of at least 3 independent experiments.
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pone.0134425.g003: Involvement of mitochondria in GBM ganglioside mediated caspase activation.T cells were co-cultured with GBM derived gangliosides CCF52 and CCF4, and t-Bid induction was detected as shown in Fig 3A. Reduced fluorescence of DiOC6 in T cells treated with U87 gangliosides (15μg/ml) for 48hrs, versus control cells is indicative of mitochondrial damage (Fig 3B). Evidence of mitochondrial damage was also observed in T cells exposed to GBM derived gangliosides as evidenced from mitochondrial cytochrome c release (Fig 3C). Time dependent induction of reactive oxygen species (ROS) was measured in purified T cells treated with 15μg/ml CCF52 and CCF4 derived gangliosides for 18hrs and 48hrs, as compared to media control for 48hrs, evidenced by H2DCFDA staining and flow cytometric analysis (Fig 3D) (*p<0.05 vs Media, **p<0.01 vs Media, ***p<0.01 vs Media). ROS production is represented as the mean fluorescence intensity (MFI) of at least 3 independent experiments.

Mentions: Data presented in the previous section of this manuscript demonstrated time dependent activation of caspase 8 (Fig 2). Since, apoptosis signals can be amplified through caspase-8 dependent release of cytochrome c from the mitochondria through formation of t-Bid [41] ultimately leading to caspase-9 activation, experiments were performed to see whether GBM derived gangliosides induce formation of t-Bid. Western blot analysis demonstrates time dependent induction of t-Bid (17KDa) in T cells treated with GBM gangliosides (Fig 3A). CCF52 mediated induction of t-Bid peaks at 48hrs, while CCF4 mediated t-Bid induction occurs much earlier at 24hrs (Fig 3A). Since, t-Bid induction is usually associated with a change in mitochondrial permeability potential (MPT), we tested whether GBM gangliosides caused disruption of MPT. As seen in Fig 3B, treatment of T cells with U87 ganglioside results in mitochondrial damage through an induction of MPT as observed by the decreased fluorescence of DiOC6 dye in U87 treated T cells versus the control (Fig 3B), indicating a change in the mitochondrial permeability. Evidence of mitochondrial damage was further supported by western blot analysis of mitochondrial lysates from T cells cultured with GBM gangliosides which shows release of cytochrome c from the mitochondria (Fig 3C) of T cells exposed to GBM derived gangliosides but not media. As observed in Fig 3D, incubation of T cells with both GBM derived gangliosides caused significant induction of ROS formation, which tripled with CCF52 or doubled with CCF4 ganglioside within 18hrs. Interestingly, however, although ROS formation kept on increasing thereafter with CCF4 ganglioside, it dropped significantly with CCF52 ganglioside by 48hrs (Fig 3D).


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)

Involvement of mitochondria in GBM ganglioside mediated caspase activation.T cells were co-cultured with GBM derived gangliosides CCF52 and CCF4, and t-Bid induction was detected as shown in Fig 3A. Reduced fluorescence of DiOC6 in T cells treated with U87 gangliosides (15μg/ml) for 48hrs, versus control cells is indicative of mitochondrial damage (Fig 3B). Evidence of mitochondrial damage was also observed in T cells exposed to GBM derived gangliosides as evidenced from mitochondrial cytochrome c release (Fig 3C). Time dependent induction of reactive oxygen species (ROS) was measured in purified T cells treated with 15μg/ml CCF52 and CCF4 derived gangliosides for 18hrs and 48hrs, as compared to media control for 48hrs, evidenced by H2DCFDA staining and flow cytometric analysis (Fig 3D) (*p<0.05 vs Media, **p<0.01 vs Media, ***p<0.01 vs Media). ROS production is represented as the mean fluorescence intensity (MFI) of at least 3 independent experiments.
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Related In: Results  -  Collection

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pone.0134425.g003: Involvement of mitochondria in GBM ganglioside mediated caspase activation.T cells were co-cultured with GBM derived gangliosides CCF52 and CCF4, and t-Bid induction was detected as shown in Fig 3A. Reduced fluorescence of DiOC6 in T cells treated with U87 gangliosides (15μg/ml) for 48hrs, versus control cells is indicative of mitochondrial damage (Fig 3B). Evidence of mitochondrial damage was also observed in T cells exposed to GBM derived gangliosides as evidenced from mitochondrial cytochrome c release (Fig 3C). Time dependent induction of reactive oxygen species (ROS) was measured in purified T cells treated with 15μg/ml CCF52 and CCF4 derived gangliosides for 18hrs and 48hrs, as compared to media control for 48hrs, evidenced by H2DCFDA staining and flow cytometric analysis (Fig 3D) (*p<0.05 vs Media, **p<0.01 vs Media, ***p<0.01 vs Media). ROS production is represented as the mean fluorescence intensity (MFI) of at least 3 independent experiments.
Mentions: Data presented in the previous section of this manuscript demonstrated time dependent activation of caspase 8 (Fig 2). Since, apoptosis signals can be amplified through caspase-8 dependent release of cytochrome c from the mitochondria through formation of t-Bid [41] ultimately leading to caspase-9 activation, experiments were performed to see whether GBM derived gangliosides induce formation of t-Bid. Western blot analysis demonstrates time dependent induction of t-Bid (17KDa) in T cells treated with GBM gangliosides (Fig 3A). CCF52 mediated induction of t-Bid peaks at 48hrs, while CCF4 mediated t-Bid induction occurs much earlier at 24hrs (Fig 3A). Since, t-Bid induction is usually associated with a change in mitochondrial permeability potential (MPT), we tested whether GBM gangliosides caused disruption of MPT. As seen in Fig 3B, treatment of T cells with U87 ganglioside results in mitochondrial damage through an induction of MPT as observed by the decreased fluorescence of DiOC6 dye in U87 treated T cells versus the control (Fig 3B), indicating a change in the mitochondrial permeability. Evidence of mitochondrial damage was further supported by western blot analysis of mitochondrial lysates from T cells cultured with GBM gangliosides which shows release of cytochrome c from the mitochondria (Fig 3C) of T cells exposed to GBM derived gangliosides but not media. As observed in Fig 3D, incubation of T cells with both GBM derived gangliosides caused significant induction of ROS formation, which tripled with CCF52 or doubled with CCF4 ganglioside within 18hrs. Interestingly, however, although ROS formation kept on increasing thereafter with CCF4 ganglioside, it dropped significantly with CCF52 ganglioside by 48hrs (Fig 3D).

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