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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

GBM derived gangliosides interact with TNFR1 to induce recruitment of FADD and TRADD leading eventually to downstream activation of caspases.Differential profiles of ganglioside isolates from GBM cell lines CCF52 and CCF4 were obtained by HPTLC using procedure described earlier, as shown in Fig 6A. ELISA assay was also used to detect GM2 and GD1a in extracted gangliosides from CCF52 cell line (Fig 6B). Wells of a 96 well ELISA plate were coated with varying concentrations of CCF52 gangliosides as indicated in the figure legend (Fig 6B). Ganglioside GM2 shed by tumors are taken up by T cells (around 35% GM2-+ve T cells) when cultured with CCF52 supernatants as shown in Fig 6C. Co-localization of GM2 (green) and TNFR1 (red) in T cells treated with CCF52 ganglioside (15μg/ml) for 24hrs was demonstrated from overlay by confocal microscopy (Fig 6D). T cells from normal volunteers were co-cultured with CCF52 gangliosides for 6, 12, 24 and 48hrs. Cells were washed, lysed and co-immunoprecipitated with anti-human TNFR1 Ab. Western immunoblot analysis confirmed that TRADD and FADD co-immunoprecipitated with TNFR1 (Fig 6E), indicating recruitment of the DISC. Representative data from one of 3 experiments is shown.
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pone.0134425.g006: GBM derived gangliosides interact with TNFR1 to induce recruitment of FADD and TRADD leading eventually to downstream activation of caspases.Differential profiles of ganglioside isolates from GBM cell lines CCF52 and CCF4 were obtained by HPTLC using procedure described earlier, as shown in Fig 6A. ELISA assay was also used to detect GM2 and GD1a in extracted gangliosides from CCF52 cell line (Fig 6B). Wells of a 96 well ELISA plate were coated with varying concentrations of CCF52 gangliosides as indicated in the figure legend (Fig 6B). Ganglioside GM2 shed by tumors are taken up by T cells (around 35% GM2-+ve T cells) when cultured with CCF52 supernatants as shown in Fig 6C. Co-localization of GM2 (green) and TNFR1 (red) in T cells treated with CCF52 ganglioside (15μg/ml) for 24hrs was demonstrated from overlay by confocal microscopy (Fig 6D). T cells from normal volunteers were co-cultured with CCF52 gangliosides for 6, 12, 24 and 48hrs. Cells were washed, lysed and co-immunoprecipitated with anti-human TNFR1 Ab. Western immunoblot analysis confirmed that TRADD and FADD co-immunoprecipitated with TNFR1 (Fig 6E), indicating recruitment of the DISC. Representative data from one of 3 experiments is shown.

Mentions: Since, ganglioside GM2 has been identified as one of the potential candidates for inducing T cell apoptosis mediated by tumor derived ganglioside [22], we used HPTLC to first determine the expression profile of individual gangliosides expressed by CCF52 and CCF4 (Fig 6A). Both of these lines expressed multiple gangliosides including GM2 and GD1a by HTPLC analysis which was confirmed by ELISA (Fig 6B) using hamster anti-human GM2 Ab and mouse anti-human GD1a Ab. When normal T lymphocytes were treated with conditioned media obtained by culturing CCF52 cell lines, ganglioside GM2 was taken up by T cells as evidenced by FITC-positive T cells (Fig 6C), indicating that gangliosides were not only shed by tumor cells but also taken up significantly (~ 35%) by T cells.


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)

GBM derived gangliosides interact with TNFR1 to induce recruitment of FADD and TRADD leading eventually to downstream activation of caspases.Differential profiles of ganglioside isolates from GBM cell lines CCF52 and CCF4 were obtained by HPTLC using procedure described earlier, as shown in Fig 6A. ELISA assay was also used to detect GM2 and GD1a in extracted gangliosides from CCF52 cell line (Fig 6B). Wells of a 96 well ELISA plate were coated with varying concentrations of CCF52 gangliosides as indicated in the figure legend (Fig 6B). Ganglioside GM2 shed by tumors are taken up by T cells (around 35% GM2-+ve T cells) when cultured with CCF52 supernatants as shown in Fig 6C. Co-localization of GM2 (green) and TNFR1 (red) in T cells treated with CCF52 ganglioside (15μg/ml) for 24hrs was demonstrated from overlay by confocal microscopy (Fig 6D). T cells from normal volunteers were co-cultured with CCF52 gangliosides for 6, 12, 24 and 48hrs. Cells were washed, lysed and co-immunoprecipitated with anti-human TNFR1 Ab. Western immunoblot analysis confirmed that TRADD and FADD co-immunoprecipitated with TNFR1 (Fig 6E), indicating recruitment of the DISC. Representative data from one of 3 experiments is shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4520498&req=5

pone.0134425.g006: GBM derived gangliosides interact with TNFR1 to induce recruitment of FADD and TRADD leading eventually to downstream activation of caspases.Differential profiles of ganglioside isolates from GBM cell lines CCF52 and CCF4 were obtained by HPTLC using procedure described earlier, as shown in Fig 6A. ELISA assay was also used to detect GM2 and GD1a in extracted gangliosides from CCF52 cell line (Fig 6B). Wells of a 96 well ELISA plate were coated with varying concentrations of CCF52 gangliosides as indicated in the figure legend (Fig 6B). Ganglioside GM2 shed by tumors are taken up by T cells (around 35% GM2-+ve T cells) when cultured with CCF52 supernatants as shown in Fig 6C. Co-localization of GM2 (green) and TNFR1 (red) in T cells treated with CCF52 ganglioside (15μg/ml) for 24hrs was demonstrated from overlay by confocal microscopy (Fig 6D). T cells from normal volunteers were co-cultured with CCF52 gangliosides for 6, 12, 24 and 48hrs. Cells were washed, lysed and co-immunoprecipitated with anti-human TNFR1 Ab. Western immunoblot analysis confirmed that TRADD and FADD co-immunoprecipitated with TNFR1 (Fig 6E), indicating recruitment of the DISC. Representative data from one of 3 experiments is shown.
Mentions: Since, ganglioside GM2 has been identified as one of the potential candidates for inducing T cell apoptosis mediated by tumor derived ganglioside [22], we used HPTLC to first determine the expression profile of individual gangliosides expressed by CCF52 and CCF4 (Fig 6A). Both of these lines expressed multiple gangliosides including GM2 and GD1a by HTPLC analysis which was confirmed by ELISA (Fig 6B) using hamster anti-human GM2 Ab and mouse anti-human GD1a Ab. When normal T lymphocytes were treated with conditioned media obtained by culturing CCF52 cell lines, ganglioside GM2 was taken up by T cells as evidenced by FITC-positive T cells (Fig 6C), indicating that gangliosides were not only shed by tumor cells but also taken up significantly (~ 35%) by T cells.

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