ABSTRACT
Bladder cancer and head and neck squamous cell carcinoma (HNSCC) are frequent but lack efficient therapies especially in advanced disease. Almost no studies on mTOR function and inhibition in these tumor entities have been reported. We examined the gene and protein expression levels of mTOR and its activated form (pmTOR) in three human bladder carcinoma cell lines (RT-4, T24, EJ28) and three HNSCC cell lines (PCI-1, PCI-13, BHY). Furthermore, the consequences of mTOR inhibition by mTOR-specific siRNAs and the mTOR inhibitor temsirolimus were analysed in vitro using immunohistochemical Ki-67 staining, mTOR and pmTOR western blot analysis, MTT assay, as well as cell cycle analysis with flow cytometry. Especially pmTOR protein expression levels showed marked differences between cell lines. siRNA transfection was associated with dose-dependent target protein reduction but not proliferation inhibition or apoptosis. On the contrary, temsirolimus significantly reduced cell viability and induced apoptosis and cell cycle arrest. According to these data, bladder cancer and HNSCC are promising tumor entities for mTOR inhibition with temsirolimus.
Subject(s)
Carcinoma, Squamous Cell/drug therapy , Head and Neck Neoplasms/drug therapy , Protein Kinase Inhibitors/therapeutic use , TOR Serine-Threonine Kinases/antagonists & inhibitors , Urinary Bladder Neoplasms/drug therapy , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Proliferation/drug effects , Drug Evaluation, Preclinical , Gene Expression Regulation, Neoplastic/drug effects , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/pathology , Humans , Protein Kinase Inhibitors/pharmacology , RNA, Small Interfering/pharmacology , Sirolimus/analogs & derivatives , Sirolimus/pharmacology , Sirolimus/therapeutic use , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Tumor Cells, Cultured , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/pathologyABSTRACT
Natural killer (NK) cells play a crucial role in innate immunity as effectors against tumor cells and pathogen-infected cells. Our data show for the first time that NK cells produce high levels of cytokines interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in response to stimulation with the artificial RNA analogue Poly I:C without additional cytokines or contact to other types of immune cells. An incubation period of 48 h is necessary to induce cytokine release by Poly I:C. These data suggest Poly I:C as a competent direct activator and immunomodulator of NK cell functions.
Subject(s)
Interferon Inducers/pharmacology , Interferon-gamma/metabolism , Interleukin-6/metabolism , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Poly I-C/pharmacology , Tumor Necrosis Factor-alpha/metabolism , Adolescent , Adult , Aged , DEAD-box RNA Helicases/metabolism , Female , Gene Expression Regulation/drug effects , Humans , Interferon-Induced Helicase, IFIH1 , Male , Middle Aged , Toll-Like Receptor 3/metabolismABSTRACT
BACKGROUND: Natural killer (NK) cells play a crucial role in innate immunity as effectors against tumor cells and pathogen-infected cells. Human NK cells can be subdivided into two functional subsets, the immunoregulatory CD56(bright) NK cells and the cytotoxic CD56(dim) NK cells. NK-mediated host defence against tumor cells is strongly impaired in patients with head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: NK cells were isolated from peripheral blood of 70 HNSCC patients and 22 healthy donors by magnetic bead separation and subsequently analyzed using flow cytometric as well as immunohistochemical methods. RESULTS: In this work we demonstrate that the population of circulating immunoregulatory CD56(bright) NK cells is lower in the peripheral blood of patients with HNSCC as compared with that in healthy donors, regardless of the individual tumor stage or tumor type. CONCLUSION: These data underline the complex network of HNSCC-mediated immunomodulation as part of the immune escape mechanisms of malignant head and neck tumors.
Subject(s)
CD56 Antigen/metabolism , Head and Neck Neoplasms/blood , Head and Neck Neoplasms/pathology , Killer Cells, Natural/pathology , Adolescent , Adult , Aged , Case-Control Studies , Female , Flow Cytometry , Humans , Male , Microscopy, Fluorescence , Middle Aged , Neoplasm Staging , Young AdultABSTRACT
Toll-like receptors (TLRs) are the archetypal pattern recognition receptors that have emerged as key mediators of immune functions. Activation of TLRs is a first line of defense of the immune system, leading to the activation and recruitment of different types of immune cells to sites of infection and malignant cell growth. Cells of human cancers, such as head and neck squamous cell carcinoma (HNSCC), are known to develop numerous molecular strategies to escape from efficient antitumor immune responses. It is supposed that tumor production of various immunosuppressive mediators contributes to impaired and altered immune functions in cancer patients. The molecular mechanisms responsible for these immunomodulatory processes and the biosynthesis of the immunosuppressive HNSCC microenvironment remain mostly unknown. Recently, different studies have shown that tumor cells are able to modulate the expression and function of TLR proteins in different kinds of immune cells. In this review, we present recent progress in these studies on the modulation of TLR expression and signaling through malignant head and neck cancer.
Subject(s)
Carcinoma, Squamous Cell/immunology , Dendritic Cells/immunology , Head and Neck Neoplasms/immunology , Toll-Like Receptors/immunology , Tumor Escape/immunology , Carcinoma, Squamous Cell/metabolism , Cytokines/immunology , Cytokines/metabolism , Dendritic Cells/metabolism , Head and Neck Neoplasms/metabolism , Humans , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Lymphocyte Activation/immunology , Oligodeoxyribonucleotides/immunology , Oligodeoxyribonucleotides/metabolism , RNA, Small Interfering/genetics , Signal Transduction/immunology , Toll-Like Receptors/metabolismABSTRACT
Natural killer (NK) cells play a crucial role in innate immunity as effectors against tumor and pathogen-infected cells. NK-mediated host defense against tumor cells is strongly impaired in patients with head and neck squamous cell carcinoma (HNSCC). Tumor secretion of various immune suppressive mediators contributes to massively compromised immune functions. Herein we demonstrate that NK cell cytotoxicity against tumor cells of HNSCC can be efficiently stimulated by single-stranded immunostimulatory RNA (ss-isRNA). Stimulation with ss-isRNA results in an increased production of interferon-gamma and effector proteins perforin and granzyme B. Our investigations revealed that supernatants of permanent HNSCC cell lines negatively affect the ss-isRNA-triggered stimulation of cytolytic NK cell functions. Stimulation of cytotoxicity requires toll-like receptor 7 (TLR7). Increased expression of NK cell TLR7 was shown in response to ss-isRNA. These results suggest ss-isRNA as a potential immunostimulatory tool of human NK cells against HNSCC.
Subject(s)
Adjuvants, Immunologic , Carcinoma, Squamous Cell/therapy , Head and Neck Neoplasms/therapy , Immunity, Innate , Killer Cells, Natural/immunology , RNA/immunology , Toll-Like Receptor 7/metabolism , Adolescent , Adult , Aged , Carcinoma, Squamous Cell/enzymology , Carcinoma, Squamous Cell/immunology , Cell Line, Tumor , Cells, Cultured , Culture Media, Conditioned/metabolism , Cytotoxicity, Immunologic , Female , Granzymes/metabolism , Head and Neck Neoplasms/enzymology , Head and Neck Neoplasms/immunology , Humans , Interferon-gamma/metabolism , Killer Cells, Natural/enzymology , Male , Middle Aged , Perforin/metabolism , Up-Regulation , Young AdultABSTRACT
Natural killer (NK) cells play a dominant role in the network of innate immunity. Via Toll-like receptor 3 (TLR3), NK cells can be efficiently stimulated by double-stranded (ds)RNA. In head and neck squamous cell carcinoma (HNSCC), NK cells seem to be strongly impaired, but the true mechanisms of immune escape are not sufficiently known to date. It is obvious that the microenvironment of head and neck cancer results in strongly affected immune functions. NK cells play a major role in the local immune response of HNSCC. In this study we showed that TLR3 is predominantly expressed on the cell surface of native NK cells and becomes rapidly internalized in response to the HNSCC microenvironment. These findings represent a novel potential immune escape mechanism of head and neck cancer. The internalization of TLR3 in response to HNSCC was also observed in fibroblasts expressing heterologous TLR3 protein. Specific stimulation of NK cell TLR3 with its ligand polyinosinic-polycytidylic acid (Poly I:C) impairs the internalization of this Toll-like receptor and leads to activated NK cells within the HNSCC microenvironment.
