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SYK inhibition decreases ERK1/2 and Akt phosphorylation. SYK inhibitors BAY 61-3606, R406, and PRT062607 reduce the phosphorylation of ERK1/2 and Akt in SH-SY5Y cells after a 4 or 24 h treatment (A). Control cells were treated with the corresponding vehicles (water = vehicle control 1 and DMSO = vehicle control 2). Densitometric analysis of the protein bands was performed. Phosphorylated and total protein were normalized to their respective GAPDH loading controls and the ratios between normalized p-ERK1/2 and ERK as well as normalized p-Akt and Akt were calculated. The values are displayed as mean ± SD relative to the vehicle control = 1 (B and C). The results are based on three independent experiments. One sample t-test (two-tailed) was performed to compare the vehicle (theoretical mean = 1) vs. inhibitor treatment: * p < 0.05 ** p < 0.01 *** p < 0.001.
Source publication
Neuroblastoma is a malignancy arising from the developing sympathetic nervous system and the most common and deadly cancer of infancy. New therapies are needed to improve the prognosis for high-risk patients and to reduce toxicity and late effects. Spleen tyrosine kinase (SYK) has previously been identified as a promising drug target in various inf...
Citations
... The dysregulation of FcγR signaling is associated with RA [23]. In this condition, immune complexes comprising autoantibodies and self-antigens can activate Syk signaling in immune cells, ultimately resulting in joint inflammation and tissue damage [24]. By inhibiting Syk, it becomes possible to reduce B cell activation and antibody production. ...
Rheumatoid arthritis (RA) is a chronic autoimmune disease that primarily affects the joints but can also involve other parts of the body. In 2019, an estimated 18 million people across the globe were living with rheumatoid arthritis (RA). Among these individuals, approximately 13 million experienced varying degrees of severity in their RA symptoms. Rheumatoid arthritis (RA) is a multifaceted systemic autoimmune disorder affected by both genetic predisposition and environmental factors, leading to inflammation in the synovial tissue and dysregulation of the immune system. This dysregulation is driven by abnormal cytokines and immune cells, which significantly contribute to the pathogenesis and advancement of the disease. Presently, treatments for RA mainly concentrate on relieving symptoms and achieving disease remission through the use of disease-modifying antirheumatic drugs (DMARDs). These DMARDs encompass conventional DMARDs (cDMARDs) like methotrexate and targeted DMARDs such as JAK inhibitors. Biologic DMARDs (bDMARDs) have also been used but have many limitations. This review explored the different signaling pathways crucial for RA development, as well as the small-molecule drugs that specifically target these pathways.
... Recent emerging studies have shown that Syk also contributes to tumorigenesis; however, its role in the progression of solid tumors is complex (37)(38)(39). A recent report has shown that Syk acts as a tumor promoter in neuroblastoma and Syk inhibitors potentiate the effect of chemotherapeutic drugs on NB cells in vitro (40). However, this study did not reveal the presence of Syk in immune cells and the efficacy of Syk inhibitors in suppressing NB growth in vivo. ...
... Immunofluorescence microscopy revealed the presence of SYK+ cells in the tissue sections of both MYCN-A and MYCN-NA NB human specimens and immunoreactivity of SYK in CD68+ TAMs (Figure 2A). A recent study has shown that Syk is abundantly present in both MYCN-A and MYCN-NA human NB tumors, but it is expressed explicitly in human MYCN-NA NB cell lines (40). Hence, we evaluated the protein expression of Syk in MYCN-A (SKNBE2, IMR 32) and MYCN-NA (SH-SY-5Y, SKNSH) human NB cell lines and immune cells isolated from the mice bearing NB9464 (MYCN-A) tumors. ...
... A recent study found that Syk inhibition decreases cell viability of NB tumor cells that express Syk and potentiates cytotoxicity of chemotherapy drugs in vitro (40). In contrast to this study, we found that Syk is not expressed by human and murine NB cell lines, and Syk inhibition has no or modest effect on the viability of these cells. ...
Background
Neuroblastoma (NB) is considered an immunologically cold tumor and is usually less responsive to immune checkpoint blockade (ICB). Tumor-associated macrophages (TAMs) are highly infiltrated in NB tumors and promote immune escape and resistance to ICB. Hence therapeutic strategies targeting immunosuppressive TAMs can improve responses to ICB in NB. We recently discovered that spleen tyrosine kinase (Syk) reprograms TAMs toward an immunostimulatory phenotype and enhances T-cell responses in the lung adenocarcinoma model. Here we investigated if Syk is an immune-oncology target in NB and tested whether a novel immunotherapeutic approach utilizing Syk inhibitor together with radiation and ICB could provide a durable anti-tumor immune response in an MYCN amplified murine model of NB.
Methods
Myeloid Syk KO mice and syngeneic MYCN-amplified cell lines were used to elucidate the effect of myeloid Syk on the NB tumor microenvironment (TME). In addition, the effect of Syk inhibitor, R788, on anti-tumor immunity alone or in combination with anti-PDL1 mAb and radiation was also determined in murine NB models. The underlying mechanism of action of this novel therapeutic combination was also investigated.
Results
Herein, we report that Syk is a marker of NB-associated macrophages and plays a crucial role in promoting immunosuppression in the NB TME. We found that the blockade of Syk in NB-bearing mice markedly impairs tumor growth. This effect is facilitated by macrophages that become immunogenic in the absence of Syk, skewing the suppressive TME towards immunostimulation and activating anti-tumor immune responses. Moreover, combining FDA-approved Syk inhibitor, R788 (fostamatinib) along with anti-PDL1 mAb provides a synergistic effect leading to complete tumor regression and durable anti-tumor immunity in mice bearing small tumors (50 mm³) but not larger tumors (250 mm³). However, combining radiation to R788 and anti-PDL1 mAb prolongs the survival of mice bearing large NB9464 tumors.
Conclusion
Collectively, our findings demonstrate the central role of macrophage Syk in NB progression and demonstrate that Syk blockade can “reeducate” TAMs towards immunostimulatory phenotype, leading to enhanced T cell responses. These findings further support the clinical evaluation of fostamatinib alone or with radiation and ICB, as a novel therapeutic intervention in neuroblastoma.
... To explore the role of CCT6A in OS, we analyzed the expression of CCT6A messenger ribonucleic acid (mRNA) in OS tissues and cell lines using the R2 online genomics analysis and visualization application [26][27][28]. We also assessed the role of CCT6A in cell proliferation and apoptosis in OS cell lines after transfection with CCT6A small interfering RNA (siRNA), by observation of cell confluence and colony formation, cell counting, cell-counting kit (CCK-8) assay, propidium iodide (PI) staining, and annexin V staining. ...
... The mRNA level of CCT6A in OS tissue (GEO ID: gse14359) and cells (GEO ID: gse11414) were analyzed using the R2 Genomics Analysis and Visualization Platform (http://r2.amc.nl), which is a web-based genomics analysis and visualization application widely used to analyze the expression of genes in diseases, including cancer [26][27][28]. ...
... Using the R2 platform [26][27][28], we analyzed the mRNA expression of CCT6A in OS tissue and cells. As shown in Fig 1A, there was significantly greater mRNA expression of CCT6A in OS tissue than in non-neoplastic primary osteoblasts (p <0.05). ...
We assessed the role of the protein-coding gene chaperonin-containing TCP1 subunit 6A (CCT6A) in osteosarcoma, as this is currently unknown. Using data from the R2 online genomic analysis and visualization application, we found that CCT6A messenger ribonucleic acid (RNA) expression is increased in osteosarcoma tissue and cells. Transfection of CCT6A small interfering RNA into cultured osteosarcoma cells revealed that CCT6A knockdown attenuates cell growth, cell viability, cell survival, and induced apoptosis and cell cycle progression at the G0/G1 phases. Moreover, CCT6A knockdown downregulated phospho-protein kinase B (p-Akt), cyclinD1 and B-cell lymphoma-2, whereas upregulated Bcl-2-associated X-protein expression. Thus, CCT6A knockdown inhibits cell proliferation, induces cell apoptosis, and suppresses the Akt pathway.
... In the present study, primary cultured microglial cells treated with cerebral cortex extract (CCE) were used to mimic the TBI model in vitro. In addition, an in vivo controlled cortical impact (CCI) mouse model was used to evaluate the effects of the Syk inhibitor BAY61-3606 (BAY) on microglial activation and phenotypic transformation after TBI (27). BAY (C 20 H 20 Cl 20 N 6 O 6 ) is a potent selective Syk inhibitor that competes with ATP, which has a Ki and IC 50 of 7.5 and 10 nM, respectively (28). ...
Neuroinflammatory processes mediated by microglial activation and subsequent neuronal damage are the hallmarks of traumatic brain injury (TBI). As an inhibitor of the macrophage‑inducible C‑type lectin (Mincle)/spleen tyrosine kinase (Syk) signaling pathway, BAY61‑3606 (BAY) has previously demonstrated anti‑inflammatory effects on some pathological processes, such as acute kidney injury, by suppressing the inflammatory macrophage response. In the present study, the potential effects of BAY on microglial phenotype and neuroinflammation after TBI were investigated. BAY (3 mg/kg) was first administered into mice by intraperitoneal injection after TBI induction in vivo and microglia were also treated with BAY (2 µM) in vitro. The levels of inflammatory factors in microglia were assessed using reverse transcription‑quantitative PCR and ELISA. Cortical neuron, myelin sheath, astrocyte and cerebrovascular endothelial cell markers were detected using immunofluorescence. The levels of components of the Mincle/Syk/NF‑κB signaling pathway [Mincle, phosphorylated (p)‑Syk and NF‑κB], in addition to proteins associated with inflammation (ASC, caspase‑1, TNF‑α, IL‑1β and IL‑6), apoptosis (Bax and Bim) and tight junctions (Claudin‑5), were measured via western blotting and ELISA. Migration and chemotaxis of microglial cells were evaluated using Transwell and agarose spot assays. Neurological functions of the mice were determined in vivo using the modified neurological severity scoring system and a Morris water maze. The results of the present study revealed that the expression levels of proteins in the Mincle/Syk/NF‑κB signaling pathway (including Mincle, p‑Syk and p‑NF‑κB), inflammatory cytokines (TNF‑α, IL‑1β and IL‑6), proteins involved in inflammation (ASC and caspase‑1), apoptotic markers (Bax and Bim) and the tight junction protein Claudin‑5 were significantly altered post‑TBI. BAY treatment reversed these effects in both the cerebral cortex extract‑induced cell model and the controlled cortical impact mouse model. BAY was also revealed to suppress activation of the microglial proinflammatory phenotype and microglial migration. In addition, BAY effectively attenuated TBI‑induced neurovascular unit damage and neurological function deficits. Taken together, these findings provided evidence that BAY may inhibit the Mincle/Syk/NF‑κB signaling pathway in microglia; this in turn could attenuate microglia‑mediated neuroinflammation and improve neurological deficits following TBI.
... DNA was eluted in 50 µL, and 5 µL was used in PCR. To test DNA quality, APRT PCR was carried out as previously described [42]. ...
Polyomaviruses infect many species, including humans. So far, 15 polyomaviruses have been described in humans, but it remains to be established whether all of these are genuine human polyomaviruses. The most recent polyomavirus to be detected in a person is Quebec polyomavirus (QPyV), which was identified in a metagenomic analysis of a stool sample from an 85-year-old hospitalized man. We used PCR to investigate the presence of QPyV DNA in urine samples from systemic lupus erythematosus (SLE) patients (67 patients; 135 samples), multiple sclerosis patients (n = 35), HIV-positive patients (n = 66) and pregnant women (n = 65). Moreover, cerebrospinal fluid from patients with suspected neurological diseases (n = 63), nasopharyngeal aspirates from patients (n = 80) with respiratory symptoms and plasma samples from HIV-positive patients (n = 65) were examined. QPyV DNA was found in urine from 11 (16.4%), 10 (15.4%) and 5 (14.3%) SLE patients, pregnant women, and multiple sclerosis patients, respectively. No QPyV DNA could be detected in the other samples. Alignment with the only available QPyV sequence in the GenBank revealed amino acid substitutions in the HI-loop of capsid protein VP1 in 6/28 of the isolates. Our results show that QPyV viruria can occur, but whether it may cause clinical symptoms in the patients remains to be determined.
... The signaling pathways by which Syk exerts its anti-proliferative and anti-invasive effects in epithelial cells remain unknown, and undoubtedly differ from the ones in hematopoietic cells where Syk appears to be pro-proliferative and pro-survival [29]. It is crucial to understand the mechanisms underlying this dual role because Syk kinase inhibitors might potentiate the effect of certain chemotherapeutic drugs in vitro [30] and they are being clinically evaluated but "their use might be inappropriate for people with a family history of breast cancer" [31]. Using a quantitative SILAC-based phosphoproteomic approach to compare mammary cell lines with different Syk expression or catalytic activity [32] we identified potential Syk substrate proteins involved in cell-cell adhesion (E-Cdh, α-Ctn) and epithelial polarity (occludin, Scrib, Dlg, ZO3, claudin3, InaDL, MAGUK5, and Lin7C). ...
While first discovered in immunoreceptor signaling, the Syk protein kinase behaves as a tumor and metastasis suppressor in epithelial cells. Its reduced expression in breast and other carcinomas is correlated with decreased survival and increased metastasis risk, but its action mechanism remains largely unknown. Using phosphoproteomics we found that Syk phosphorylated E-cadherin and α-, β-, and p120-catenins on multiple tyrosine residues that concentrate at intercellular junctions. Increased Syk expression and activation enhanced E-cadherin/catenin phosphorylation, promoting their association and complex stability. In human breast cancer cells, Syk stimulated intercellular aggregation, E-cadherin recruitment and retention at adherens junctions, and promoted epithelial integrity, whereas it inhibited cell migration and invasion. Opposite effects were obtained with Syk knockdown or non-phosphorylatable mutant E-cadherin expression. Mechanistically, Syk stimulated the interaction of the E-cadherin/catenin complex with zonula occludens proteins and the actin cytoskeleton. Conditional Syk knockout in the lactating mouse mammary gland perturbed alveologenesis and disrupted E-cadherin localization at adherens junctions, corroborating the observations in cells. Hence, Syk is involved in the maintenance of the epithelial integrity of the mammary gland via the phosphorylation and stabilization of the E-cadherin/catenin adherens junction complex, thereby inhibiting cell migration and malignant tumor invasion.
O Neuroblastoma (NB) é uma neoplasia do sistema nervoso simpático, e o segundo tumor sólido maligno extracraniano mais comum da infância. Na terapia antineoplásica, complicações orais podem ser observadas, dentre elas a mucosite oral (MO). Trata-se de uma inflamação aguda da mucosa, proveniente da toxicidade dos quimioterápicos. Este relato de caso enfatiza o manejo da MO, bem como sua influência na condição sistêmica e qualidade de vida. Paciente do sexo feminino, nove anos, apresentou recidiva de NB metastático, após tratamento de primeira linha. Admitida na unidade hospitalar para tratamento oncológico na enfermaria de pediatria oncológica, queixando-se de dor intensa em cavidade bucal e orofaringe, associada a pancitopenia severa febril. Ao exame físico apresentava disfagia e déficit ponderal grave, que debilitava a deglutição da própria saliva. O exame intraoral revelou lesões de MO grau 3, segundo a Organização Mundial de Saúde (OMS), em lábios, gengiva anterior e orofaringe. O tratamento consistiu em remoção de debris local, higiene da cavidade bucal com clorexidina 0,12% e utilizando haste flexível de algodão estéril tipo “Swab”, visando controle microbiano local, diariamente. Além disso, foi aplicado no leito eritematoso e hemorrágico fotobiomodulação (660 nm, 50 mW, 2 J/cm2, 90 segundos) pontualmente nas áreas do leito da lesão e em varredura na região de orofaringe (sessões intercaladas). Foi prescrito acetato de racealfatocoferol (vitamina E) para ação antioxidante e hidratação dos lábios. Concomitante a mucosite, a paciente apresentou pancitopenia febril severa, sendo necessário uso de cefepima 150 mg/kg/dia, com coleta prévia de hemoculturas, fator estimulador de colônias de granulócitos, Fluconazol, hidratação e suporte nutricional. As hemoculturas foram negativas. As consequências da mucosite contribuíram para desnutrição e piora da qualidade de vida. Conclui-se que a intervenção odontológica em interdisciplinaridade, possibilitou o restabelecimento físico e emocional, possibilitando melhor uma qualidade de vida da paciente.
