title: GBM Derived Gangliosides Induce T Cell Apoptosis through Activation of the Caspase Cascade Involving Both the Extrinsic and the Intrinsic Pathway.
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.
authors.affiliation: Division of Molecular Medicine, Bose Institute, Kolkata, India.
authors.forename: Barun
authors.initials: B
authors.lastname: Mahata
authors.affiliation: Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, United States of America.
authors.forename: Soumika
authors.initials: S
authors.lastname: Biswas
authors.affiliation: Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, United States of America.
authors.forename: Patricia
authors.initials: P
authors.lastname: Rayman
authors.affiliation: Spine and Brain Institute, St. Vincent Medical Center, Jacksonville, Florida, United States of America.
authors.forename: Ali
authors.initials: A
authors.lastname: Chahlavi
authors.affiliation: Pathology Institute, Cleveland Clinic, Cleveland, United States of America.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Ko
authors.affiliation: Department of Biotechnology, National Institute of Technology, Durgapur, India.
authors.forename: Ashish
authors.initials: A
authors.lastname: Bhattacharjee
authors.affiliation: Department of Biochemistry and Molecular Biology, Tulane University School of Medicine, New Orleans, United States of America.
authors.forename: Yu-Teh
authors.initials: YT
authors.lastname: Li
authors.affiliation: Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, United States of America.
authors.forename: Yuntao
authors.initials: Y
authors.lastname: Li
authors.affiliation: Division of Molecular Medicine, Bose Institute, Kolkata, India; Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, United States of America.
authors.forename: Tanya
authors.initials: T
authors.lastname: Das
authors.affiliation: Division of Molecular Medicine, Bose Institute, Kolkata, India; Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, United States of America.
authors.forename: Gaurisankar
authors.initials: G
authors.lastname: Sa
authors.affiliation: Brain Tumor and Neuro-oncology Center in the Neurological Institute, Cleveland Clinic, Cleveland, United States of America.
authors.forename: Baisakhi
authors.initials: B
authors.lastname: Raychaudhuri
authors.affiliation: Brain Tumor and Neuro-oncology Center in the Neurological Institute, Cleveland Clinic, Cleveland, United States of America.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Vogelbaum
authors.affiliation: Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, United States of America.
authors.forename: Charles
authors.initials: C
authors.lastname: Tannenbaum
authors.affiliation: Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, United States of America.
authors.forename: James H
authors.initials: JH
authors.lastname: Finke
authors.affiliation: Division of Molecular Medicine, Bose Institute, Kolkata, India.
authors.forename: Kaushik
authors.initials: K
authors.lastname: Biswas
mesh_terms: D017209:Apoptosis / Q000502:physiology*; D020169:Caspases / Q000502:physiology*; D045744:Cell Line, Tumor; D005678:G(M2) Ganglioside / Q000502:physiology*; D055786:Gene Knockout Techniques; D005909:Glioblastoma / Q000378:metabolism / Q000503:physiopathology*; D006801:Humans; D047468:Immunoprecipitation; D019169:Jurkat Cells / Q000502:physiology; D018613:Microscopy, Confocal; D017382:Reactive Oxygen Species / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*; D013601:T-Lymphocytes / Q000502:physiology*
chemical_list: D017382:Reactive Oxygen Species; D018124:Receptors, Tumor Necrosis Factor; D005678:G(M2) Ganglioside; D020169:Caspases