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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

Induction of T cell apoptosis by GBM derived gangliosides is mediated through caspase activation.Lysates from T cells treated with GBM derived gangliosides for 72hrs were resolved in a 12% SDS-PAGE and western immunoblot was performed to detect expression of caspases, as shown in Fig 2A. Fig 2B shows graphical representation demonstrating time dependent induction of caspases in purified T cells in response to CCF52 gangliosides for 18hrs, 48hrs & 72hrs as measured by staining the cells with fluorochrome labeled inhibitors of caspases (FLICA) (*p<0.05 vs Media, **p<0.01 vs Media, ***p<0.001 vs Media). Cells were acquired on a FACS-calibur multivariable flow cytometer and % caspase +ve T cells were analyzed using CellQuest 3.3 software. Representative density plot showing time dependent induction of caspases in T cells is shown in Fig 2C. Pre-treatment of T cells with inhibitors of caspases-3, -8, -9 (at 50μM each) and a pan caspase inhibitor (at 12.5μM) 2hrs prior to ganglioside (15μg/ml) treatment, significantly protected T cells from CCF52 ganglioside induced T cell death as evident by trypan blue exclusion in Fig 2D (*p<0.05 vs Media; **p<0.01 vs CCF52 ganglioside-15μg/ml; ***p<0.001 vs CCF52 ganglioside-15μg/ml), and by microscopic analysis of nuclear blebbing in Fig 2D (**p<0.01 vs Media; **p<0.01 vs CCF52 ganglioside-15μg/ml) as shown in Fig 2D. Data represents mean of at least 3 independent experiments unless mentioned otherwise.
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pone.0134425.g002: Induction of T cell apoptosis by GBM derived gangliosides is mediated through caspase activation.Lysates from T cells treated with GBM derived gangliosides for 72hrs were resolved in a 12% SDS-PAGE and western immunoblot was performed to detect expression of caspases, as shown in Fig 2A. Fig 2B shows graphical representation demonstrating time dependent induction of caspases in purified T cells in response to CCF52 gangliosides for 18hrs, 48hrs & 72hrs as measured by staining the cells with fluorochrome labeled inhibitors of caspases (FLICA) (*p<0.05 vs Media, **p<0.01 vs Media, ***p<0.001 vs Media). Cells were acquired on a FACS-calibur multivariable flow cytometer and % caspase +ve T cells were analyzed using CellQuest 3.3 software. Representative density plot showing time dependent induction of caspases in T cells is shown in Fig 2C. Pre-treatment of T cells with inhibitors of caspases-3, -8, -9 (at 50μM each) and a pan caspase inhibitor (at 12.5μM) 2hrs prior to ganglioside (15μg/ml) treatment, significantly protected T cells from CCF52 ganglioside induced T cell death as evident by trypan blue exclusion in Fig 2D (*p<0.05 vs Media; **p<0.01 vs CCF52 ganglioside-15μg/ml; ***p<0.001 vs CCF52 ganglioside-15μg/ml), and by microscopic analysis of nuclear blebbing in Fig 2D (**p<0.01 vs Media; **p<0.01 vs CCF52 ganglioside-15μg/ml) as shown in Fig 2D. Data represents mean of at least 3 independent experiments unless mentioned otherwise.

Mentions: To understand the signaling events involved in ganglioside mediated T cell death, caspase activation was determined by immunoblotting using antibodies to caspase-3, caspase-8 and -9 following incubation of isolated T cells with gangliosides derived from 3 distinct GBM lines (72 hrs). Induction of the effector caspase-3 and caspase-8 (Fig 2A) was demonstrated by increased expression of the corresponding cleaved products while caspase-9 was also found to be activated as evident by decreased expression of its pro-form (Fig 2A). β-actin was used as the endogenous control with no change noted. The time frame (18, 48 and 72hrs) for activation of different caspases in T lymphocytes treated or not with CCF52 ganglioside was assessed by staining with fluorochrome labeled inhibitors of caspases (FLICA) followed by flow cytometric analysis as shown graphically in Fig 2B. Gangliosides induced activation of caspases-3, -8 and -9 by 18hrs, however, careful analysis of the representative density plot shows that although, caspase 8 activity reached a maximum at 48hrs, caspase-3 and -9 activity reached maximum at 72hrs (Fig 2C). Inhibitors specific to caspases-3 (Z-DEVD-FMK), -8 (Z-IETD-FMK) or -9 (Z-LEHD-FMK) and a pan caspase inhibitor (Z-VAD-FMK) when added to T cells 3hrs prior to addition of CCF52 ganglioside caused significant reduction of T cell death (~60%) when compared to that of untreated cells, as evidenced from trypan blue assay as well as nuclear blebbing, suggesting that ganglioside induced caspase activation and subsequent T cell apoptosis likely involves both receptor mediated (caspase-8) and mitochondrial (caspase-9) pathway of caspase activation (Fig 2D).


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)

Induction of T cell apoptosis by GBM derived gangliosides is mediated through caspase activation.Lysates from T cells treated with GBM derived gangliosides for 72hrs were resolved in a 12% SDS-PAGE and western immunoblot was performed to detect expression of caspases, as shown in Fig 2A. Fig 2B shows graphical representation demonstrating time dependent induction of caspases in purified T cells in response to CCF52 gangliosides for 18hrs, 48hrs & 72hrs as measured by staining the cells with fluorochrome labeled inhibitors of caspases (FLICA) (*p<0.05 vs Media, **p<0.01 vs Media, ***p<0.001 vs Media). Cells were acquired on a FACS-calibur multivariable flow cytometer and % caspase +ve T cells were analyzed using CellQuest 3.3 software. Representative density plot showing time dependent induction of caspases in T cells is shown in Fig 2C. Pre-treatment of T cells with inhibitors of caspases-3, -8, -9 (at 50μM each) and a pan caspase inhibitor (at 12.5μM) 2hrs prior to ganglioside (15μg/ml) treatment, significantly protected T cells from CCF52 ganglioside induced T cell death as evident by trypan blue exclusion in Fig 2D (*p<0.05 vs Media; **p<0.01 vs CCF52 ganglioside-15μg/ml; ***p<0.001 vs CCF52 ganglioside-15μg/ml), and by microscopic analysis of nuclear blebbing in Fig 2D (**p<0.01 vs Media; **p<0.01 vs CCF52 ganglioside-15μg/ml) as shown in Fig 2D. Data represents mean of at least 3 independent experiments unless mentioned otherwise.
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pone.0134425.g002: Induction of T cell apoptosis by GBM derived gangliosides is mediated through caspase activation.Lysates from T cells treated with GBM derived gangliosides for 72hrs were resolved in a 12% SDS-PAGE and western immunoblot was performed to detect expression of caspases, as shown in Fig 2A. Fig 2B shows graphical representation demonstrating time dependent induction of caspases in purified T cells in response to CCF52 gangliosides for 18hrs, 48hrs & 72hrs as measured by staining the cells with fluorochrome labeled inhibitors of caspases (FLICA) (*p<0.05 vs Media, **p<0.01 vs Media, ***p<0.001 vs Media). Cells were acquired on a FACS-calibur multivariable flow cytometer and % caspase +ve T cells were analyzed using CellQuest 3.3 software. Representative density plot showing time dependent induction of caspases in T cells is shown in Fig 2C. Pre-treatment of T cells with inhibitors of caspases-3, -8, -9 (at 50μM each) and a pan caspase inhibitor (at 12.5μM) 2hrs prior to ganglioside (15μg/ml) treatment, significantly protected T cells from CCF52 ganglioside induced T cell death as evident by trypan blue exclusion in Fig 2D (*p<0.05 vs Media; **p<0.01 vs CCF52 ganglioside-15μg/ml; ***p<0.001 vs CCF52 ganglioside-15μg/ml), and by microscopic analysis of nuclear blebbing in Fig 2D (**p<0.01 vs Media; **p<0.01 vs CCF52 ganglioside-15μg/ml) as shown in Fig 2D. Data represents mean of at least 3 independent experiments unless mentioned otherwise.
Mentions: To understand the signaling events involved in ganglioside mediated T cell death, caspase activation was determined by immunoblotting using antibodies to caspase-3, caspase-8 and -9 following incubation of isolated T cells with gangliosides derived from 3 distinct GBM lines (72 hrs). Induction of the effector caspase-3 and caspase-8 (Fig 2A) was demonstrated by increased expression of the corresponding cleaved products while caspase-9 was also found to be activated as evident by decreased expression of its pro-form (Fig 2A). β-actin was used as the endogenous control with no change noted. The time frame (18, 48 and 72hrs) for activation of different caspases in T lymphocytes treated or not with CCF52 ganglioside was assessed by staining with fluorochrome labeled inhibitors of caspases (FLICA) followed by flow cytometric analysis as shown graphically in Fig 2B. Gangliosides induced activation of caspases-3, -8 and -9 by 18hrs, however, careful analysis of the representative density plot shows that although, caspase 8 activity reached a maximum at 48hrs, caspase-3 and -9 activity reached maximum at 72hrs (Fig 2C). Inhibitors specific to caspases-3 (Z-DEVD-FMK), -8 (Z-IETD-FMK) or -9 (Z-LEHD-FMK) and a pan caspase inhibitor (Z-VAD-FMK) when added to T cells 3hrs prior to addition of CCF52 ganglioside caused significant reduction of T cell death (~60%) when compared to that of untreated cells, as evidenced from trypan blue assay as well as nuclear blebbing, suggesting that ganglioside induced caspase activation and subsequent T cell apoptosis likely involves both receptor mediated (caspase-8) and mitochondrial (caspase-9) pathway of caspase activation (Fig 2D).

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