ABSTRACT
BACKGROUND AND PURPOSE: Second mitochondria-derived activator of caspase (SMAC)-mimetics are a new class of targeted drugs that specifically induce apoptotic cancer cell death and block pro-survival signaling by antagonizing selected members of the inhibitor of apoptosis protein (IAP) family. MATERIAL AND METHODS: The present study was designed to investigate the radiosensitizing effect and optimal sequence of administration of the novel SMAC-mimetic Debio 1143 in vitro and in vivo. Apoptosis, alteration of DNA damage repair (DDR), and tumor necrosis factor-alpha (TNF-α) signaling were examined. RESULTS: In vitro, Debio 1143 displayed anti-proliferative activity and enhanced intrinsic radiation sensitivity in 5/6 head and neck squamous cell carcinoma (HNSCC) cell lines in a synergistic manner. In vivo, Debio 1143 dose-dependently radio-sensitized FaDu and SQ20B xenografts, resulting in complete tumor regression in 8/10 FaDu-xenografted mice at the high dose level. At the molecular level, Debio 1143 combined with radiotherapy (RT) induced enhancement of caspase-3 activity, increase in Annexin V-positive cells and karyopyknosis, and increase in TNF-α mRNA levels. Finally, in a neutralization experiment using a TNF-α-blocking antibody and a caspase inhibitor, it was shown that the radiosensitizing effect of Debio 1143 is mediated by caspases and TNF-α. CONCLUSIONS: These results demonstrate that the novel SMAC-mimetic Debio 1143 is a radiosensitizing agent that is worthy of further investigation in clinical trials in combination with radiotherapy.
Subject(s)
Antineoplastic Agents/pharmacology , Azocines/pharmacology , Benzhydryl Compounds/pharmacology , Carcinoma, Squamous Cell/therapy , Head and Neck Neoplasms/therapy , Radiation-Sensitizing Agents/pharmacology , Tumor Necrosis Factor-alpha/physiology , Animals , Apoptosis/drug effects , Apoptosis Regulatory Proteins , Caspase 3/metabolism , Cell Death/drug effects , Cell Line, Tumor , Chemoradiotherapy/methods , Female , Humans , Inhibitor of Apoptosis Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/pharmacology , Mice, Inbred Strains , Mitochondrial Proteins/pharmacology , Neoplasm Transplantation , Signal Transduction/drug effects , Squamous Cell Carcinoma of Head and Neck , Transplantation, Heterologous , Xenograft Model Antitumor Assays/methodsABSTRACT
Growth arrest-specific gene 6 (Gas6) is expressed in antigen-presenting cells and endothelial cells (ECs) but not in T cells. When wild-type (WT) or Gas6(-/-) mice received allogeneic non-T cell-depleted bone marrow cells, hepatic graft-versus-host disease (GVHD) was alleviated in Gas6(-/-) recipients regardless of donor genotype, but not in WT recipients. T-cell infiltration was more prominent and diffuse in WT than in Gas6(-/-) recipients' liver. When mice received 0.5 x 10(6) allogeneic T cells with T cell-depleted allogeneic bone marrow, clinical signs indicated that GVHD was less severe in Gas6(-/-) than in WT recipients, as shown by a significant improvement of the survival and reduced liver GVHD. These data demonstrate that donor cells were not involved in the protection mechanism. In addition, lack of Gas6 in antigen-presenting cells did not affect WT or Gas6(-/-) T-cell proliferation. We therefore assessed the response of WT or Gas6(-/-) ECs to tumor necrosis factor-alpha. Lymphocyte transmigration was less extensive through Gas6(-/-) than WT ECs and was not accompanied by increases in adhesion molecule levels. Thus, the lack of Gas6 in ECs impaired donor T-cell transmigration into the liver, providing a rationale for considering Gas6 pathway as a potential nonimmunosuppressive target to minimize GVHD in patients receiving allogeneic hematopoietic stem cell transplantation.
Subject(s)
Graft vs Host Disease/prevention & control , Hematopoietic Stem Cell Transplantation/adverse effects , Intercellular Signaling Peptides and Proteins/deficiency , Liver/immunology , Animals , Cell Separation , Chemotaxis, Leukocyte/genetics , Chemotaxis, Leukocyte/immunology , Endothelial Cells/metabolism , Flow Cytometry , Graft vs Host Disease/genetics , Graft vs Host Disease/immunology , Immunohistochemistry , Intercellular Signaling Peptides and Proteins/genetics , Liver/pathology , Lymphocyte Activation/immunology , Lymphocyte Culture Test, Mixed , Mice , Mice, Knockout , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes/cytology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Transplantation, HomologousABSTRACT
Chemokines have recently been postulated to have important functions in the central nervous system (CNS) in addition to their principal role of directional migration of leukocytes. In particular, it has been shown that chemokines may play a role in the regulation of oligodendrocyte biology. Here, we have chosen to study the role of certain chemokines in regulating myelination. We have used the murine oligodendrocyte precursor-like cell line, Oli-neu, and primary mixed cortical cultures as experimental systems to assess their activities on oligodendrocyte precursor proliferation and developmental in vitro myelination. GRO-alpha, IL-8, SDF-1alpha and RANTES dose-dependently increased proliferation of this mouse A2B5 precursor-like cell line, while MCP-1 did not. Furthermore, the CXC chemokines GRO-alpha, IL-8 and SDF-1alpha stimulated myelin basic protein synthesis in a dose-dependent manner in primary myelinating cultures and enhanced myelin segment formation in this system, while the CC chemokines MCP-1 and RANTES did not. We also demonstrate that the receptor for SDF-1alpha, CXCR4, is expressed in mixed cortical cultures by PDGFalphaR positive oligodendrocyte precursors (OLPs) as well as by Oli-neu cells. SDF-1alpha induced proliferation in primary mixed cultures and the Oli-neu cell line was mediated through this receptor. We propose, therefore, that CXC chemokines and in particular SDF-1alpha regulates CNS myelination via their effects on cells of the oligodendrocyte lineage, specifically stimulation of OLP proliferation.
Subject(s)
Chemokines/pharmacology , Oligodendroglia/drug effects , Stem Cells/drug effects , Analysis of Variance , Animals , Antigens/metabolism , Cell Count/methods , Cell Proliferation/drug effects , Cells, Cultured , Cerebral Cortex/cytology , Dose-Response Relationship, Drug , Drug Interactions , Embryo, Mammalian , Enzyme-Linked Immunosorbent Assay/methods , Gene Expression/drug effects , Glial Fibrillary Acidic Protein/metabolism , Immunohistochemistry/methods , Insulin-Like Growth Factor I/pharmacology , Ki-67 Antigen/metabolism , Leukocyte L1 Antigen Complex/metabolism , Mice , Microtubule-Associated Proteins/metabolism , Myelin Basic Protein/metabolism , Myelin Basic Protein/pharmacology , Proteoglycans/metabolism , Receptor, Platelet-Derived Growth Factor alpha/metabolism , Receptors, CXCR4/metabolism , Synaptosomal-Associated Protein 25/metabolism , Time FactorsABSTRACT
We have used in vitro oligodendrocyte differentiation and the in vivo remyelination model, the cuprizone model, to identify genes regulating oligodendrocyte function and remyelination. One of the genes we identified, osteopontin (opn), is a secreted glycoprotein with cytokine-like, chemotactic, and anti-apoptotic properties that contains an Arg-Gly-Asp (RGD) cell adhesion motif-mediating interactions with several integrins. Both microglia and astrocytes in demyelinating brain regions of cuprizone-fed mice expressed OPN protein. Recombinant OPN protein produced in a baculovirus expression system induced proliferation of both the rat CG-4 and the mouse Oli-neu oligodendrocyte precursor (OLP)-like cell lines in a dose-dependent manner. In addition, recombinant OPN treatment stimulated both myelin basic protein (MBP) synthesis and myelin sheath formation in mixed cortical cultures from embryonic mouse brain, an in vitro primary culture model of myelination. Interestingly, myelinating mixed cultures prepared from OPN(-/-) mice contained significantly less MBP compared to wild-type cultures after 17 days in culture. We propose that in the central nervous system, OPN may act as a novel regulator of myelination and remyelination.
