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1.
Int J Mol Sci ; 25(12)2024 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-38928033

RESUMO

Bladder cancer (BC) is the 12th most commonly diagnosed cancer worldwide. Although there are several well-established molecular and immunological classifications, prognostic and predictive markers for tumor cells and immune cells are still needed. Using a tissue microarray, we analyzed the expression of the chemokine CC motif ligand 5 (CCL5) by immunohistochemistry (IHC) in 175 muscle-invasive BC samples. The application of a single cutoff for the staining status of tumor cells (TCs; positive vs. negative) and immune cells (ICs; positive vs. negative) revealed 75 patients (42.9%) and 123 patients (70.3%) with CCL5-positive TCs or ICs, respectively. IHC results were associated with prognostic and predictive data. Multivariate Cox regression analysis revealed that positive CCL5 staining in TCs was associated with significantly shorter disease-specific survival (DSS; RR = 1.51; p = 0.047), but CCL5-negative ICs were associated with significantly shorter overall survival (OS; RR = 1.66; p = 0.005), DSS (RR = 2.02; p = 0.001) and recurrence-free survival (RFS; RR = 1.94; p = 0.002). Adjuvant chemotherapy was favorable for patients with CCL5-negative ICs for OS (RR = 0.30; p = 0.006), DSS (RR = 0.36; p = 0.022) and RFS (RR = 0.41; p = 0.046) but not for patients with CCL5-positive ICs, except in the subgroup of N1 + N2 patients, where it was associated with better OS. We suggest that CCL5 expression can be a prognostic and predictive marker for muscle-invasive bladder cancer patients.


Assuntos
Biomarcadores Tumorais , Quimiocina CCL5 , Neoplasias da Bexiga Urinária , Humanos , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/mortalidade , Quimiocina CCL5/metabolismo , Quimiocina CCL5/genética , Masculino , Feminino , Idoso , Prognóstico , Pessoa de Meia-Idade , Biomarcadores Tumorais/metabolismo , Invasividade Neoplásica , Idoso de 80 Anos ou mais , Adulto , Imuno-Histoquímica
2.
J Proteome Res ; 19(11): 4389-4392, 2020 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-32568543

RESUMO

Mass spectrometry (MS) can deliver valuable diagnostic data that complement genomic information and allow us to increase our current knowledge of the COVID-19 disease caused by the SARS-CoV-2 virus. We developed a simple, MS-based method to specifically detect SARS-CoV-2 proteins from gargle solution samples of COVID-19 patients. The protocol consists of an acetone precipitation and tryptic digestion of proteins contained within the gargle solution, followed by a targeted MS analysis. Our methodology identifies unique peptides originating from SARS-CoV-2 nucleoprotein. Building on these promising initial results, faster MS protocols can now be developed as routine diagnostic tools for COVID-19 patients. Data are available via ProteomeXchange with identifier PXD019423.


Assuntos
Betacoronavirus/química , Infecções por Coronavirus/diagnóstico , Espectrometria de Massas/métodos , Boca/virologia , Pneumonia Viral/diagnóstico , COVID-19 , Teste para COVID-19 , Cromatografia Líquida de Alta Pressão , Técnicas de Laboratório Clínico , Infecções por Coronavirus/virologia , Humanos , Nucleoproteínas/análise , Nucleoproteínas/química , Pandemias , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Pneumonia Viral/virologia , SARS-CoV-2 , Proteínas Virais/análise , Proteínas Virais/química
3.
Cancer Immunol Immunother ; 66(5): 565-571, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28184970

RESUMO

The human leukocyte antigen (HLA) system is a major part of the human immune system and has an impact on tumor initiation, tumor progression, and immunosurveillance. Renal cell carcinoma tumors are considered to be immunogenic. Therefore, we studied the allele frequencies of four gene loci (HLA-A, -B, -C, and HLA-DR) in a cohort of German renal cell carcinoma (RCC) patients and in healthy controls. HLA-A-C were determined using serological methods, whereas HLA-C12, C14, C16, C18, and HLA-DR were characterized through the use of standard molecular biological methods. The occurrence of the HLA-C*12 allele was significantly increased in German RCC patients compared with healthy controls (P < 0.005; Fisher's exact test), whereas the occurrence of the HLA-DRB1*04 allele was significantly reduced in RCC patients compared with healthy controls (P < 0.05; Fisher's exact test). However, the presence of allele HLA-C*12 was not significantly associated with 10 year overall survival. We suggest that the frequency of HLA alleles can affect development of RCC and could add knowledge as predictive marker for future immunotherapies.


Assuntos
Carcinoma de Células Renais/genética , Carcinoma de Células Renais/imunologia , Frequência do Gene/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe I/genética , Neoplasias Renais/genética , Neoplasias Renais/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Alemanha , Antígenos HLA-A/genética , Antígenos HLA-A/imunologia , Antígenos HLA-B/genética , Antígenos HLA-B/imunologia , Antígenos HLA-C/genética , Antígenos HLA-C/imunologia , Antígenos HLA-DR/genética , Antígenos HLA-DR/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico
4.
Int J Mol Sci ; 18(12)2017 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-29215551

RESUMO

The capillary morphogenesis gene 2 (CMG2), also known as the anthrax toxin receptor 2 (ANTXR2), is a transmembrane protein putatively involved in extracellular matrix (ECM) adhesion and tissue remodeling. CMG2 promotes endothelial cell proliferation and exhibits angiogenic properties. Its downregulation is associated with a worsened survival of breast carcinoma patients. Aim of this study was to analyze the CMG2 mRNA and protein expression in soft tissue sarcoma and their association with patient outcome. CMG2 mRNA was measured in 121 tumor samples of soft tissue sarcoma patients using quantitative real-time PCR. CMG2 protein was evaluated in 52 tumor samples by ELISA. CMG2 mRNA was significantly correlated with the corresponding CMG2 protein expression (rs = 0.31; p = 0.027). CMG2 mRNA expression was associated with the mRNA expressions of several ECM and tissue remodeling enzymes, among them CD26 and components of the uPA system. Low CMG2 mRNA expression was correlated with a worsened patients' disease-specific survival in Kaplan-Meier analyses (mean patient survival was 25 vs. 96 months; p = 0.013), especially in high-stage tumors. A decreased CMG2 expression is a negative prognostic factor for soft tissue sarcoma patients. CMG2 may be an interesting candidate gene for the further exploration of soft tissue sarcoma genesis and progression.


Assuntos
Biomarcadores Tumorais/metabolismo , Receptores de Peptídeos/metabolismo , Sarcoma/metabolismo , Neoplasias de Tecidos Moles/metabolismo , Biomarcadores Tumorais/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Peptídeos/genética , Sarcoma/patologia , Neoplasias de Tecidos Moles/patologia , Análise de Sobrevida
5.
Biochim Biophys Acta ; 1852(1): 146-55, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25446989

RESUMO

Recently, Aß peptide variants with an N-terminal truncation and pyroglutamate modification were identified and shown to be highly neurotoxic and prone to aggregation. This modification of Aß is catalyzed by glutaminyl cyclase (QC) and pharmacological inhibition of QC diminishes Aß deposition and accompanying gliosis and ameliorates memory impairment in transgenic mouse models of Alzheimer's disease (AD). QC expression was initially described in the hypothalamus, where thyrotropin-releasing hormone (TRH) is one of its physiological substrates. In addition to its hormonal role, a novel neuroprotective function of TRH following excitotoxicity and Aß-mediated neurotoxicity has been reported in the hippocampus. Functionally matching this finding, we recently demonstrated QC expression by hippocampal interneurons in mouse brain. Here, we detected neuronal co-expression of QC and TRH in the hippocampus of young adult wild type mice using double immunofluorescence labeling. This provides evidence for TRH being a physiological QC substrate in hippocampus. Additionally, in neocortex of aged but not of young mice transgenic for amyloid precursor protein an increase of QC mRNA levels was found compared to wild type littermates. This phenomenon was not observed in hippocampus, which is later affected by Aß pathology. However, in hippocampus of transgenic - but not of wild type mice - a correlation between QC and TRH mRNA levels was revealed. This co-regulation of the enzyme QC and its substrate TRH was reflected by a co-induction of both proteins in reactive astrocytes in proximity of Aß deposits. Also, in primary mouse astrocytes a co-induction of QC and TRH was demonstrated upon Aß stimulation.


Assuntos
Aminoaciltransferases/metabolismo , Astrócitos/enzimologia , Hipocampo/enzimologia , Neurônios/enzimologia , Hormônio Liberador de Tireotropina/metabolismo , Aminoaciltransferases/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Sequência de Bases , Primers do DNA , Hipocampo/citologia , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Especificidade por Substrato , Hormônio Liberador de Tireotropina/genética
6.
Biol Chem ; 397(1): 45-55, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26351917

RESUMO

Secretory peptides and proteins are frequently modified by pyroglutamic acid (pE, pGlu) at their N-terminus. This modification is catalyzed by the glutaminyl cyclases QC and isoQC. Here, we decipher the roles of the isoenzymes by characterization of IsoQC-/- mice. These mice show a significant reduction of glutaminyl cyclase activity in brain and peripheral tissue, suggesting ubiquitous expression of the isoQC enzyme. An assay of substrate conversion in vivo reveals impaired generation of the pGlu-modified C-C chemokine ligand 2 (CCL2, MCP-1) in isoQC-/- mice. The pGlu-formation was also impaired in primary neurons, which express significant levels of QC. Interestingly, however, the formation of the neuropeptide hormone thyrotropin-releasing hormone (TRH), assessed by immunohistochemistry and hormonal analysis of hypothalamic-pituitary-thyroid axis, was not affected in isoQC-/-, which contrasts to QC-/-. Thus, the results reveal differential functions of isoQC and QC in the formation of the pGlu-peptides CCL2 and TRH. Substrates requiring extensive prohormone processing in secretory granules, such as TRH, are primarily converted by QC. In contrast, protein substrates such as CCL2 appear to be primarily converted by isoQC. The results provide a new example, how subtle differences in subcellular localization of enzymes and substrate precursor maturation might influence pGlu-product formation.


Assuntos
Aminoaciltransferases/metabolismo , Administração Oral , Aminoaciltransferases/deficiência , Animais , Células Cultivadas , Glucose/administração & dosagem , Teste de Tolerância a Glucose , Inflamação/induzido quimicamente , Inflamação/metabolismo , Isoenzimas/metabolismo , Lipopolissacarídeos/administração & dosagem , Camundongos , Camundongos Endogâmicos C3H , Camundongos Knockout , Ácido Pirrolidonocarboxílico/metabolismo , Especificidade por Substrato
7.
Biochim Biophys Acta ; 1843(5): 965-75, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24487064

RESUMO

Interleukin-6 is one of the most prominent triggers of inflammatory processes. We have shown recently that heteroarylketones (HAKs) interfere with stimulated interleukin-6 expression in astrocytes by suppression of STAT3 phosphorylation at serine 727. Surprisingly, this effect is not based on the inhibition of STAT3-relevant kinases. Therefore, we here used the structurally modified HAK compound biotin-HAK-3 in a reverse chemical approach to identify the relevant molecular target in UV-mediated cross-linking experiments. Employing streptavidin-specific 2D-immunoblotting followed by mass spectrometry we identified nine proteins putatively interacting with biotin-HAK-3. After co-immunoprecipitation, co-immunofluorescence, surface plasmon resonance analyses and RNAi-mediated knock-down, the eukaryotic elongation factor 1A1 (eEF1A1) was verified as the relevant target of HAK bioactivity. eEF1A1 forms complexes with STAT3 and PKCδ, which are crucial for STAT3(S727) phosphorylation and for NF-κB/STAT3-enhanced interleukin-6 expression. Furthermore, the intracellular HAK accumulation is strongly dependent on eEF1A1 expression. Taken together, the results reveal a novel molecular mechanism for a non-canonical role of eEF1A1 in signal transduction via direct modulation of kinase-dependent phosphorylation events.


Assuntos
Interleucina-6/metabolismo , Fator 1 de Elongação de Peptídeos/fisiologia , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Humanos , Cetonas/farmacologia , Fosforilação , Reação em Cadeia da Polimerase em Tempo Real , Fator de Transcrição STAT3/metabolismo , Ressonância de Plasmônio de Superfície
8.
Acta Neuropathol ; 129(4): 565-83, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25666182

RESUMO

The brains of Alzheimer's disease (AD) patients are characterized by deposits of Abeta peptides and by accompanying chronic inflammation. Here, we provide evidence that the enzyme isoglutaminyl cyclase (isoQC) is a novel factor contributing to both aspects of AD pathology. Two putative substrates of isoQC, N-truncated Abeta peptides and the monocyte chemoattractant chemokine CCL2, undergo isoQC-catalyzed pyroglutamate (pGlu) modification. This triggers Abeta aggregation and facilitates the biological activity of CCL2, which collectively results in the formation of high molecular weight Abeta aggregates, glial cell activation, neuroinflammation and neuronal cell death. In mouse brain, we found isoQC to be neuron-specifically expressed in neocortical, hippocampal and subcortical structures, localized to the endoplasmic reticulum and Golgi apparatus as well as co-expressed with its substrate CCL2. In aged APP transgenic Tg2576 mice, both isoQC and CCL2 mRNA levels are up-regulated and isoQC and CCL2 proteins were found to be co-induced in Abeta plaque-associated reactive astrocytes. Also, in mouse primary astrocyte culture, a simultaneous up-regulation of isoQC and CCL2 expression was revealed upon Abeta and pGlu-Abeta stimulation. In brains of AD patients, the expression of isoQC and CCL2 mRNA and protein is up-regulated compared to controls and correlates with pGlu-Abeta load and with the decline in mini-mental state examination. Our observations provide evidence for a dual involvement of isoQC in AD pathogenesis by catalysis of pGlu-Abeta and pGlu-CCL2 formation which mutually stimulate inflammatory events and affect cognition. We conclude that isoQC inhibition may target both major pathological events in the development of AD.


Assuntos
Doença de Alzheimer/patologia , Aminoaciltransferases/metabolismo , Encéfalo/metabolismo , Quimiocina CCL2/metabolismo , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Aminoaciltransferases/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Encéfalo/patologia , Células Cultivadas , Quimiocina CCL2/genética , Modelos Animais de Doenças , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fragmentos de Peptídeos/metabolismo , Fatores de Tempo , Regulação para Cima/genética
9.
Int J Cancer ; 135(9): 2096-106, 2014 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-24676787

RESUMO

Chemokines are involved in both the negative and positive regulation of inflammatory processes, angiogenesis and cancer/cancer stem cell proliferation as well as the chemoattraction of tumor cells to metastatic sites. The aim of this study was to measure the mRNA expression levels of three chemokines, CCL2, CCL7 and CX3CL1, in soft tissue sarcomas (STSs) and to assess the correlations between these levels as well as their correlations with clinicopathological data and the disease-specific survival of STS patients. The mRNA levels of CCL2, CCL7 and CX3CL1 were analyzed in tumor tissues from 126 STS patients using qPCR. Low mRNA expression of CCL2 and CX3CL1 was significantly correlated with a worse prognosis (RR = 1.98; p = 0.019 and RR = 2.10; p = 0.014; multivariate Cox's regression analysis). A combined low expression of CCL2 and CX3CL1 was associated with a significantly increased risk of tumor-related death as compared to patients with high expression levels of both chemokines (RR = 3.08; p = 0.003). A gender-specific multivariate analysis revealed that female STS patients with low CX3CL1 mRNA expression had a 3.46-fold increased risk of death (p = 0.004). Low expression of both CCL2 and CX3CL1 mRNAs resulted in an additive 5.37-fold increased risk of tumor-related death (p = 0.003) as compared to those with high expression of both parameters in female patients. In conclusion, this is the first study to show a significant correlation between combined low expression of CCL2 and CX3CL1 and a poor prognosis for STS patients, particularly in female patients.


Assuntos
Biomarcadores Tumorais/metabolismo , Quimiocina CCL2/metabolismo , Quimiocina CX3CL1/metabolismo , Sarcoma/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Biomarcadores Tumorais/genética , Western Blotting , Proliferação de Células , Quimiocina CCL2/genética , Quimiocina CX3CL1/genética , Feminino , Seguimentos , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sarcoma/genética , Sarcoma/mortalidade , Fatores Sexuais , Taxa de Sobrevida , Células Tumorais Cultivadas , Adulto Jovem
10.
Int J Exp Pathol ; 94(3): 217-25, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23560443

RESUMO

Inflammation is an integral part of non-alcoholic fatty liver disease (NAFLD), the most prevalent form of hepatic pathology found in the general population. In this context, recently we have examined the potential role of Glutaminyl Cyclases (QC and isoQC), and their inhibitors, in the maturation of chemokines, for example, monocyte chemoattractant protein 1 (MCP-1, CCL2), to generate their bioactive conformation. Catalysis by isoQC leads to the formation of an N-terminal pyroglutamate residue protecting CCL2 against degradation by aminopeptidases. This is of importance because truncated forms possess a reduced potential to attract immune cells. Since liver inflammation is characterized by the up-regulation of different chemokine pathways, and within this CCL2 is known to be a prominent example, we hypothesised that application of QC/isoQC inhibitors may alleviate liver inflammation by destabilizing CCL2. Therefore, we investigated the role of QC/isoQC inhibition, in comparison with the angiotensin receptor blocker Telmisartan, during development of pathology in a mouse model of non-alcoholic fatty liver disease. Application of a QC/isoQC inhibitor led to a significant reduction in circulating alanine aminotransferase and NAFLD activity score accompanied by an inhibitory effect on hepatocyte ballooning. Further analysis revealed a specific reduction of inflammation by decreasing the number of F4/80-positive macrophages, which is in agreement with the proposed CCL2-related mechanism of action of QC/isoQC inhibitors. Finally, QC/isoQC inhibitor application attenuated liver fibrosis as characterized by reduced collagen deposition in the liver parenchyma. Thus in conclusion, QC/isoQC inhibitors are a promising novel class of anti-non-alcoholic steatohepatitis drugs which have a comparable disease-modifying effect to that of Telmisartan, which is probably mediated via specific interference with a comparable monocyte/macrophage infiltration that occurs under inflammatory conditions.


Assuntos
Aminoaciltransferases/antagonistas & inibidores , Quimiocina CCL2/metabolismo , Inibidores Enzimáticos/farmacologia , Fígado Gorduroso , Hepatite , Aminoaciltransferases/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Benzimidazóis/farmacologia , Benzoatos/farmacologia , Linhagem Celular Tumoral , Quimiocina CCL2/imunologia , Modelos Animais de Doenças , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/enzimologia , Fígado Gorduroso/imunologia , Hepatite/tratamento farmacológico , Hepatite/enzimologia , Hepatite/imunologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Leucemia Monocítica Aguda/patologia , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/citologia , Hepatopatia Gordurosa não Alcoólica , Telmisartan
11.
Stem Cell Res ; 69: 103072, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37001364

RESUMO

Late-onset Alzheimer disease (LOAD) is the most frequent neurodegenerative disease, and the APOE ε4 allele is the most prominent risk factor for LOAD. Four human induced pluripotent stem cell (iPSC) lines MLUi007-J, MLUi008-B, MLUi009-A, and MLUi010-B were generated from LOAD patients and healthy matched donors by reprogramming of B-lymphoblastoid cells (B-LCLs) with episomal plasmids. The application of B-LCLs holds a great promise to model LOAD and other diseases because they can easily be generated from primary peripheral blood mononuclear cells (PBMCs) by infection with the Epstein-Barr virus (EBV).


Assuntos
Doença de Alzheimer , Infecções por Vírus Epstein-Barr , Células-Tronco Pluripotentes Induzidas , Doenças Neurodegenerativas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Apolipoproteína E4/genética , Apolipoproteína E4/metabolismo , Apolipoproteína E3 , Leucócitos Mononucleares , Doenças Neurodegenerativas/metabolismo , Infecções por Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4 , Envelhecimento
12.
J Clin Med ; 11(12)2022 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-35743381

RESUMO

In the tissue donation field, to prevent pathogen transmission, all donors are screened by postmortem swabs for SARS-CoV-2 using qRT-PCR. Corneas from donors who tested positive for SARS-CoV-2 were subjected to further investigations. Corneal transplants and culture medium from positive donors were cultured under appropriate safety conditions for further analyses. Cornea tissue samples, including sclera/limbus/cornea, and culture media were taken at different time points for testing for SARS-CoV-2 using qRT-PCR, immunohistochemistry (IHC) and subgenomic RNA (sgRNA) analysis. Between January and May 2021, in four donors with initial negative premortem rapid tests, SARS-CoV-2 was detected post-mortem using qRT-PCR. In these cases, SARS-CoV-2 was observed at the beginning of cultivation in both tissue and culture medium using qRT-PCR and IHC. The virus was mainly localized in the limbus epithelial cells, with a stable detection level. Premortem rapid tests are potentially insufficient to exclude SARS-CoV-2 infection in corneal donors. While, for SARS-CoV-2, the risk of infection via transplants is considered low, a residual risk remains for presymptomatic new infections. However, our investigations provide the first indications that, with organ cultures, the risk of virus transmission is minimized due to the longer minimum culture period.

13.
J Neuroinflammation ; 8: 86, 2011 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-21801384

RESUMO

BACKGROUND: Elevated brain levels of the pleiotropic cytokine interleukin-6, which is mainly secreted from activated local astrocytes, contribute to pathological events including neuroinflammation and neurodegeneration. Thus, inhibition of pathological IL-6 expression provides a rationale strategy for targeting the onset or further progression of neurological disorders including Alzheimer's disease, multiple sclerosis, Parkinson's disease and traumatic brain injury. The purpose of this study was to identify and to characterize new potent inhibitors of astrocytic IL-6 expression for further therapeutic development of novel anti-inflammatory and neuroprotective drugs. METHODS: Oncostatin M (OSM)-treated human glioma U343 cells were used as model for induction of astrocytic IL-6 expression. This model was characterized by immunoblotting, siRNA technique, ELISA and qRT-PCR and used to screen low molecular weight compound libraries for IL-6-lowering effects. To validate bioactive compounds identified from library screens, bacterial lipopolysaccharide was used to induce IL-6 expression in cultivated primary astrocytes and in mice in vivo. To dissect underlying molecular mechanisms, protein extracts from OSM-treated U343 cells were analyzed by phospho-specific immunoblotting and immunocytochemistry as well as by co-immunoprecipitation. RESULTS: OSM-treatment (100 ng/ml; 24 h) led to 30-fold increase of IL-6 secretion from U343 cells. The temporal profile of IL-6 mRNA induction displayed a biphasic induction pattern with peak synthesis at 1 h (6.5-fold) and 16 h (5.5-fold) post stimulation. IL-6 protein release did not show that biphasic pattern and was detected as early as 3 h post stimulation reaching a maximum at 24 h. The screen of compound libraries identified a set of heteroarylketones (HAKs) as potent inhibitors of IL-6 secretion. HAK compounds affected the second peak in IL-6 mRNA synthesis, whereas the first peak was insensitive to HAK treatment. HAK compounds also suppressed lipopolysaccharide-induced IL-6 expression in primary murine astrocytes as well as in brain and plasma samples from lipopolysaccharide-treated mice. Finally, HAK compounds were demonstrated to specifically suppress the OSM-induced phosphorylation of STAT3 at serine 727 and the physical interaction of pSTAT3S727 with p65. CONCLUSION: Heteroarylketone compounds are potent inhibitors of IL-6 expression in vitro and in vivo and may represent a new class of potent anti-inflammatory and neuroprotective drugs.


Assuntos
Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Interleucina-6/metabolismo , Cetonas/farmacologia , NF-kappa B/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Astrócitos/citologia , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Interleucina-6/genética , Cetonas/química , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Estrutura Molecular , NF-kappa B/genética , Oncostatina M/farmacologia , Distribuição Aleatória , Ratos , Fator de Transcrição STAT3/genética
14.
Mol Hum Reprod ; 17(2): 115-26, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20876610

RESUMO

The human endometrium is a cyclically regenerating organ under the influence of ovarian steroid hormones. Disturbances in this highly coordinated regulation of endometrial proliferation and differentiation may result in infertility and diseases such as endometriosis and endometrial cancer. Environmental toxins belonging to the group of polyhalogenated aromatic hydrocarbons (PAHs) are lipophilic xenobiotics, which accumulate in biological systems. PAHs have been implicated in the etiology of uterine pathologies, including infertility, endometriosis and endometrial cancer. However, suitable cellular models of the endometrium are lacking and the molecular mechanism of PAH action in the endometrium is not fully understood. In this study, we have characterized a previously established immortalized human telomerase reverse transcriptase (hTERT) endometrial epithelial cell (hTERT-EEC) model as a responsive in vitro cell model to investigate the cellular and molecular mechanisms of selected environmentally relevant PAH in human EECs. We show that dioxin-type PAHs activate the endogenous arylhydrocarbon receptor (AhR) signaling pathway in hTERT-EEC in a time-, concentration- and congener-specific manner and that the induction of AhR target genes is modulated by estrogen. Strikingly, AhR activation did not interfere with estrogenic actions in these EECs. Independent of their ability to bind to AhR, the PAHs investigated here increased cell migration by hTERT-EEC. Furthermore, we have identified several candidates by proteomic analysis, which are involved in heat shock responses and protein modification and turnover. Our data suggest that AhR-activating environmental pollutants directly alter endometrial cell stress responses and metabolism independent of estrogenic actions.


Assuntos
Dioxinas/farmacologia , Endométrio/metabolismo , Poluentes Ambientais/farmacologia , Células Epiteliais/metabolismo , Hidrocarbonetos Halogenados/farmacologia , Receptores de Hidrocarboneto Arílico/agonistas , Western Blotting , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Dioxinas/toxicidade , Poluentes Ambientais/toxicidade , Estradiol/metabolismo , Estradiol/farmacologia , Moduladores de Receptor Estrogênico/metabolismo , Estrogênios/metabolismo , Estrogênios/farmacologia , Feminino , Humanos , Hidrocarbonetos Halogenados/toxicidade , Reação em Cadeia da Polimerase , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Estresse Fisiológico/efeitos dos fármacos , Telomerase
15.
Cells ; 10(1)2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33467469

RESUMO

Bladder cancer (BCa) is the tenth most commonly diagnosed malignant cancer worldwide. Although adjuvant chemotherapy following radical cystectomy is a common therapy for muscle invasive bladder cancer patients, no applicable biomarkers exist to predict which patients will benefit from chemotherapy. In this study, we examined three immune cell markers, the chemokine CC motif ligand 2 (CCL2), the pan macrophage marker cluster of differentiation 68 (CD68) and the M2 macrophage marker cluster of differentiation 163 (CD163), using immunohistochemistry to determine their predictive value for the chemotherapy response in different nodal stage (pN0 vs. pN1 + 2) and tumor stage subgroups (pT2 vs. pT3 + 4). The prognosis was studied in terms of the overall survival (OS), disease-specific survival (DSS), and recurrence-free-survival (RFS) in 168 muscle invasive BCa patients. Chemotherapy was associated with a poorer prognosis in patients with a higher expression of the immune markers CCL2 (RFS), CD68 (DSS and RFS), and CD163 (DSS and RFS) in the N0 group and with poorer survival in patients with a higher expression of the immune markers CCL2 (OS, DSS, and RFS), CD68 (OS, DSS, and RFS), and CD163 (OS, DSS, and RFS) in the pT2 group when compared with treatments without chemotherapy. In contrast, chemotherapy was associated with a better prognosis in patients with a low expression of the immune markers CCL2 (DSS and RFS), CD68 (OS, DSS, and RFS), and CD163 (OS) in the N1 + 2 group. In addition, chemotherapy was associated with improved survival in patients with a low expression of the immune marker CD68 (OS and DSS) and there was a trend for a better prognosis in patients with a low expression of CD163 (OS) in the pT3 + 4 group compared to patients not treated with chemotherapy. Interestingly, CD68 appeared to be the most applicable immune marker to stratify patients by the outcome of chemotherapy in the nodal stage and tumor stage groups. Overall, we suggest that, in addition to the clinical factors of tumor stage and nodal stage, it is also meaningful to consider the abundance of immune cells, such as macrophages, to better predict the response to chemotherapy for BCa patients after radical treatment.


Assuntos
Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/terapia , Urotélio/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Biomarcadores/metabolismo , Quimiocina CCL2/metabolismo , Quimioterapia Adjuvante , Cistectomia , Intervalo Livre de Doença , Humanos , Sistema Imunitário , Estimativa de Kaplan-Meier , Macrófagos/metabolismo , Terapia Neoadjuvante , Recidiva Local de Neoplasia , Valor Preditivo dos Testes , Prognóstico , Modelos de Riscos Proporcionais , Receptores de Superfície Celular/metabolismo , Análise Serial de Tecidos , Resultado do Tratamento , Neoplasias da Bexiga Urinária/mortalidade , Urotélio/cirurgia
16.
Bioorg Med Chem ; 18(6): 2173-2177, 2010 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-20176490

RESUMO

Inhibition of acetylcholinesterase (AChE) and therefore prevention of acetylcholine degradation is one of the most accepted therapy opportunities for Alzheimer s disease (AD), today. Due to lack of selectivity of AChE inhibitor drugs on the market, AD-patients suffer from side effects like nausea or vomiting. In the present study the isolation of two alkaloids, infractopicrin (1) and 10-hydroxy-infractopicrin (2), from Cortinarius infractus Berk. (Cortinariaceae) is presented. Both compounds show AChE-inhibiting activity and possess a higher selectivity than galanthamine. Docking studies show that lacking pi-pi-interactions in butyrylcholinesterase (BChE) are responsible for selectivity. Studies on other AD pathology related targets show an inhibitory effect of both compounds on self-aggregation of Abeta-peptides but not on AChE induced Abeta-peptide aggregation. Low cytotoxicity as well as calculated pharmacokinetic data suggest that the natural products could be useful candidates for further drug development.


Assuntos
Acetilcolinesterase/metabolismo , Agaricales/química , Inibidores da Colinesterase/isolamento & purificação , Inibidores da Colinesterase/farmacologia , Carpóforos/química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Acetilcolinesterase/química , Animais , Bovinos , Inibidores da Colinesterase/química , Eritrócitos/enzimologia , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/isolamento & purificação , Estrutura Molecular , Estereoisomerismo , Relação Estrutura-Atividade
17.
J Immunol ; 181(2): 1120-7, 2008 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-18606664

RESUMO

Chemokines mediate the recruitment of leukocytes to the sites of inflammation. N-terminal truncation of chemokines by the protease dipeptidyl peptidase IV (DPPIV) potentially restricts their activity during inflammatory processes such as allergic reactions, but direct evidence in vivo is very rare. After demonstrating that N-terminal truncation of the chemokine CCL11/eotaxin by DPPIV results in a loss of CCR3-mediated intracellular calcium mobilization and CCR3 internalization in human eosinophils, we focused on the in vivo role of CCL11 and provide direct evidence for specific kinetic and rate-determining effects by DPPIV-like enzymatic activity on CCL11-mediated responses of eosinophils. Namely, it is demonstrated that i.v. administration of CCL11 in wild-type F344 rats leads to mobilization of eosinophils into the blood, peaking at 30 min. This mobilization is significantly increased in DPPIV-deficient F344 rats. Intradermal administration of CCL11 is followed by a dose-dependent recruitment of eosinophils into the skin and is significantly more effective in DPPIV-deficient F344 mutants as well as after pharmacological inhibition of DPPIV. Interestingly, CCL11 application leads to an up-regulation of DPPIV, which is not associated with negative feedback inhibition via DPPIV-cleaved CCL11((3-74)). These findings demonstrate regulatory effects of DPPIV for the recruitment of eosinophils. Furthermore, they illustrate that inhibitors of DPPIV have the potential to interfere with chemokine-mediated effects in vivo including but not limited to allergy.


Assuntos
Quimiocina CCL11/imunologia , Quimiotaxia de Leucócito , Dipeptidil Peptidase 4/metabolismo , Eosinófilos/imunologia , Animais , Quimiocina CCL11/metabolismo , Dipeptidil Peptidase 4/imunologia , Inibidores da Dipeptidil Peptidase IV , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Eosinófilos/metabolismo , Humanos , Isoleucina/análogos & derivados , Isoleucina/farmacologia , Ratos , Ratos Endogâmicos F344 , Ratos Mutantes , Receptores CCR3/metabolismo , Pele/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Tiazóis/farmacologia
18.
Cancers (Basel) ; 12(5)2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32429318

RESUMO

Bladder cancer (BCa) is the ninth most commonly diagnosed cancer worldwide. Although there are several well-established molecular and immunological classifications, markers for tumor cells and immune cells that are associated with prognosis are still needed. The chemokine CC motif ligand 2 (CCL2) could be such a marker. We analyzed the expression of CCL2 by immunohistochemistry (IHC) in 168 muscle invasive BCa samples using a tissue microarray. Application of a single cut-off for the staining status of tumor cells (TCs; positive vs. negative) and immune cells (ICs; ≤6% of ICs vs. >6% of ICs) revealed 57 cases (33.9%) and 70 cases (41.7%) with CCL2-positive TCs or ICs, respectively. IHC results were correlated with clinicopathological and survival data. Positive CCL2 staining in TCs was associated with shorter overall survival (OS), disease-specific survival (DSS), and relapse-free survival (RFS) (p = 0.004, p = 0.036, and p = 0.047; log rank test) and appeared to be an independent prognostic factor for OS (RR = 1.70; p = 0.007; multivariate Cox's regression analysis). In contrast, positive CCL2 staining in the ICs was associated with longer OS, DSS, and RFS (p = 0.032, p = 0.001, and p = 0.001; log rank test) and appeared to be an independent prognostic factor for DSS (RR = 1.77; p = 0.031; multivariate Cox's regression analysis). Most interestingly, after separating the patients according to their lymph node status (N0 vs. N1+2), CCL2 staining in the ICs was differentially associated with prognosis. In the N0 group, CCL2 positivity in the ICs was a positive independent prognostic factor for OS (RR = 1.99; p = 0.014), DSS (RR = 3.17; p = 0.002), and RFS (RR = 3.10; p = 0.002), whereas in the N1+2 group, CCL2 positivity was a negative independent factor for OS (RR = 3.44; p = 0.019)) and RFS (RR = 4.47; p = 0.010; all multivariate Cox's regression analyses). In summary, CCL2 positivity in TCs is a negative prognostic factor for OS, and CCL2 can mark ICs that are differentially associated with prognosis depending on the nodal stage of BCa patients. Therefore, CCL2 staining of TCs and ICs is suggested as a prognostic biomarker for BCa patients.

19.
Mol Cell Biol ; 25(10): 3923-33, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15870267

RESUMO

Epigenetic inactivation of the RASSF1A tumor suppressor by CpG island methylation was frequently detected in cancer. However, the mechanisms of this aberrant DNA methylation are unknown. In the RASSF1A promoter, we characterized four Sp1 sites, which are frequently methylated in cancer. We examined the functional relationship between DNA methylation, histone modification, Sp1 binding, and RASSF1A expression in proliferating human mammary epithelial cells. With increasing passages, the transcription of RASSF1A was dramatically silenced. This inactivation was associated with deacetylation and lysine 9 trimethylation of histone H3 and an impaired binding of Sp1 at the RASSF1A promoter. In mammary epithelial cells that had overcome a stress-associated senescence barrier, a spreading of DNA methylation in the CpG island promoter was observed. When the RASSF1A-silenced cells were treated with inhibitors of DNA methyltransferase and histone deacetylase, binding of Sp1 and expression of RASSF1A reoccurred. In summary, we observed that histone H3 deacetylation and H3 lysine 9 trimethylation occur in the same time window as gene inactivation and precede DNA methylation. Our data suggest that in epithelial cells, histone inactivation may trigger de novo DNA methylation of the RASSF1A promoter and this system may serve as a model for CpG island inactivation of tumor suppressor genes.


Assuntos
Cromatina/genética , Cromatina/metabolismo , Metilação de DNA , Inativação Gênica , Regiões Promotoras Genéticas/genética , Proteínas Supressoras de Tumor/genética , Linhagem Celular Tumoral , Senescência Celular/genética , Ilhas de CpG/genética , DNA/genética , DNA/metabolismo , Humanos , Sequências Reguladoras de Ácido Nucleico/genética , Fator de Transcrição Sp1/metabolismo , Sulfitos/metabolismo
20.
Oncol Rep ; 19(6): 1485-91, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18497954

RESUMO

Autotaxin (ATX/NPP2) shows a nucleotide pyrophosphatase/phosphodiesterase and lysophospholipase D (lysoPLD) activity and is a member of a family of structurally-related mammalian ecto-nucleotide pyrophosphate/phosphodiesterases (E-NPP1-3). ATX is unique among E-NPP as it is secreted and not membrane-bound as are NPP1 and -3. The ATX gene activity is significantly higher in undifferentiated anaplastic (UTC) as compared to follicular (FTC) and papillary thyroid carcinomas (PTC) or goiter tissues. ATX also enhances the motility of thyroid tumor cells. We bio-engineered stable transfectants of the human thyroid carcinoma cell line FTC-238 expressing either bioactively-secreted (sATX) or membrane-anchored ATX (mATX) to identify the biological functions of ATX which critically depend on the E-NPP member being secreted and provide insight into the effects of high local ATX concentrations and cellular responses. An increased cell motility was exclusively observed with FTC-238 sATX transfectants, whereas membrane-anchored ATX appeared to impair motility. We identified IL-1beta as an upstream suppressor of ATX expression in FTC-238, ATX-mediated motility in FTC-238 and stable transfectants, with IL-1beta having the strongest motility-suppressive effect on FTC-238 sATX clones. sATX and mATX strongly increased the anchorage-independent colony formation of FTC-238 but the size and number of colonies formed in the soft agar were significantly smaller in FTC-238 mATX versus the FTC-238 sATX clones. The cancer-testis antigen BAGE was identified as a novel target gene of ATX in FTC-238. Transcript levels for BAGE were 6-fold higher in FTC-238 mATX versus sATX clones. Increased BAGE transcript levels were also detected in tissues of patients with UTC versus FTC, PTC or goiter tissues. In summary, enhanced tumor cell motility and tumorigenic capacity critically depended on sATX in thyroid carcinoma cells. Irrespective of its compartmentalization, the cancer-testis antigen BAGE was identified as a novel target gene of ATX in FTC-238 and a potential new tissue marker in UTC tissues, which we had previously shown to express high levels of ATX.


Assuntos
Adenocarcinoma Folicular/genética , Carcinoma Papilar/genética , Movimento Celular , Regulação Neoplásica da Expressão Gênica/fisiologia , Complexos Multienzimáticos/genética , Fosfodiesterase I/genética , Pirofosfatases/genética , Neoplasias da Glândula Tireoide/genética , Adenocarcinoma Folicular/patologia , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Carcinoma Papilar/patologia , Adesão Celular , Membrana Celular/metabolismo , Humanos , Diester Fosfórico Hidrolases , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias da Glândula Tireoide/patologia , Transfecção , Células Tumorais Cultivadas
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