ABSTRACT
The success of cancer immunotherapy relies on the induction of an immunoprotective response targeting tumor antigens (TAs) presented on MHC-I molecules. We demonstrated that the splicing inhibitor isoginkgetin and its water-soluble and non-toxic derivative IP2 act at the production stage of the pioneer translation products (PTPs). We showed that IP2 increases PTP-derived antigen presentation in cancer cells in vitro and impairs tumor growth in vivo. IP2 action is long-lasting and dependent on the CD8+ T cell response against TAs. We observed that the antigen repertoire displayed on MHC-I molecules at the surface of MCA205 fibrosarcoma is modified upon treatment with IP2. In particular, IP2 enhances the presentation of an exon-derived epitope from the tumor suppressor nischarin. The combination of IP2 with a peptide vaccine targeting the nischarin-derived epitope showed a synergistic antitumor effect in vivo. These findings identify the spliceosome as a druggable target for the development of epitope-based immunotherapies.
Subject(s)
Adaptive Immunity/drug effects , Antigens, Neoplasm/metabolism , Antineoplastic Agents, Phytogenic/pharmacology , Biflavonoids/pharmacology , Cancer Vaccines/pharmacology , Fibrosarcoma/drug therapy , Lymphocytes, Tumor-Infiltrating/drug effects , T-Lymphocytes/drug effects , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Fibrosarcoma/immunology , Fibrosarcoma/metabolism , Fibrosarcoma/pathology , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class I/metabolism , Imidazoline Receptors/immunology , Imidazoline Receptors/metabolism , Lymphocyte Activation/drug effects , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Mice , Mice, Inbred C57BL , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Tumor Burden/drug effects , Tumor MicroenvironmentABSTRACT
Specific antibodies are essential in the study of receptor protein. Gene matching shows that Nischarin (NISCH) is a mouse homologue of human imidazoline receptor antisera-selective (IRAS) protein, a viable candidate for imidazoline I-1 receptor. However, selectivity of this antibody against imidazoline I-2 or imidazoline I-3 receptors remained obscure. At first, an intracerebroventricular (ICV) injection of anti-NISCH antibody blocked the blood pressure lowering action of rilmenidine (I-1 receptor agonist) in spontaneous hypertensive rat (SHR). However, the same injection of anti-NISCH antibody showed no effect in SHR treated with clonidine (α2 agonist). In order to clarify the selectivity of anti-NISCH antibody for each subtype of imidazoline receptors, this anti-NISCH antibody was subjected to the lysate of organs isolated from Wistar rats including cortex, hippocampus, cerebellum, and brain stem as central nervous tissues, and heart, liver, pancreas, skeletal muscle, kidney, prostate, and bladder as peripheral tissues. The results show that anti-NISCH antibody positively reacted with all tissues including heart, pancreas, skeletal muscle, kidney and bladder by Western blot analysis. Also, the blotting spots for anti-NISCH antibody show a concentration-dependent manner. Moreover, anti-NISCH antibody blocked the action of glucose uptake induced by 2-BFI (I-2 receptor agonist) in L6 cells. Taken together, the obtained data suggest that anti-NISCH antibody can be used not only for imidazoline I-1 receptor but also for I-2 and I-3 subtypes in immunoassays.
Subject(s)
Antibodies/immunology , Imidazoline Receptors/analysis , Animals , Antibodies/analysis , Antibody Specificity , Biological Transport/drug effects , Blood Pressure/drug effects , Glucose/metabolism , Humans , Imidazoline Receptors/immunology , Immunoassay , Intracellular Signaling Peptides and Proteins/analysis , Intracellular Signaling Peptides and Proteins/immunology , Male , Rats , Rats, Inbred SHRABSTRACT
Imidazoline receptors were first proposed by Bousquet et al., when they studied antihypertensive effect of clonidine. A strong candidate for I1R, known as imidazoline receptor antisera-selected protein (IRAS), has been cloned from human hippocampus. We reported that IRAS mediated agmatine-induced inhibition of opioid dependence in morphine-dependent cells. To elucidate the functional and structure properties of I1R, we developed the newly monoclonal antibody against the N-terminal hIRAS region including the PX domain (10-120aa) through immunization of BALB/c mice with the NusA-IRAS fusion protein containing an IRAS N-terminal (10-120aa). Stable hybridoma cell lines were established and monoclonal antibodies specifically recognized full-length IRAS proteins in their native state by immunoblotting and immunoprecipitation. Monoclonal antibodies stained in a predominantly punctate cytoplasmic pattern when applied to IRAS-transfected HEK293 cells by indirect immunofluorescence assays and demonstrated excellent reactivity in flow immunocytometry. These monoclonal antibodies will provide powerful reagents for the further investigation of hIRAS protein functions.
