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1.
Anticancer Res ; 39(10): 5437-5448, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31570438

RESUMO

BACKGROUND/AIM: Epithelial-mesenchymal transition (EMT) is a key multi-step process which enables cancer cells to detach from the epithelial primary tumor mass and allows them to metastasize to distant organs. We immunohistochemically analyzed the expression of the transcription factors (TWIST-1, SLUG, ZEB1, ZEB2) and components of the extracellular matrix (laminin-5, fibronectin) which influence the EMT. MATERIALS AND METHODS: Primary human breast (MDA-MB-231), colon (HT29, HCT116), ovarian (SKOV3, OVCAR3) and head and neck squamous cell carcinoma cell lines (UTSCC2, UTSCC24A) grown as xenografts were immunohistochemically analyzed in vitro and in vivo. RESULTS: A high SLUG expression was observed in every cancer entity both in vitro and in vivo. ZEB1 and ZEB2 showed a high in vivo expression especially in SKOV3 and in in vitro grown MDA-MB-231 cells. CONCLUSION: SLUG expression showed the highest expression in all cancer entities investigated. Hence, it presumably represents the master regulator of EMT in these metastatic tumor entities.


Assuntos
Biomarcadores Tumorais/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Fatores de Transcrição/metabolismo , Linhagem Celular Tumoral , Matriz Extracelular/metabolismo , Células HCT116 , Células HT29 , Humanos , Imuno-Histoquímica/métodos , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo
2.
J Oral Pathol Med ; 48(10): 943-950, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31400171

RESUMO

BACKGROUND: Osteoblast adhesion is a crucial step in osseointegration of dental implants and can be influenced by modification of implant surface or the addition of bioactive agents. Bisphosphonates affect bone turnover, attenuating bone healing in implants patients. PRP and PRF are sources of growth factors involved in osteoblast adhesion, improving subsequent bone healing. The aim of the study was to investigate the impacts of PRP and PRF on adhesion of bisphosphonate-pretreated osteoblasts on titanium implant surfaces using the cell-count wash assay, the MTT-assay as well as real-time-cell analyser assay and scanning electronic microscopy. METHODS: Titanium implants were colonised for 24 hours with osteoblasts and zolendronic acid, PRP or PRF in different combinations. Afterwards, primary osteoblast adhesion was evaluated by counting the number of attached cells using a wash-assay cell analysis. Scanning electronic microscopy was performed and evaluated semi-quantitatively to assess the influence of the different groups on the ultrastructural cell morphology, such as cell size and shape as well as length and number of filopodia. RESULTS: Zoledronic acid led to a decrease of osteoblast adherence onto implant surface. This effect was reversed by adding PRP or PRF. Scanning electronic microscopy showed that both PRP and PRF increased number and length of filopodia in adherent osteoblasts. CONCLUSIONS: Zoledronic acid decreased osteoblast adhesion on implant surfaces, and PRF as well as PRP increased primary adhesion of zoledronic acid-treated osteoblasts on implant surfaces in vitro. Therefore, PRP and PRF may improve initial bone apposition and primary healing of dental implants in patients with bisphosphonate treatment.

3.
Sci Rep ; 9(1): 8310, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31165745

RESUMO

Bisphosphonates are frequently used for the antiresorptive treatment in bone metastasis diseases or for osteoporosis. A side effect of this therapy is osteonecrosis of the jaw. This inhibits osteoclast function, but osteoblasts and fibroblasts are also negatively affected in terms of impaired proliferation. Additive local treatment with platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) promotes adhesion, proliferation and migration of cells due to high concentrations of growth factors like PDGF, TGF and IGF. The aim of the study was to investigate the effect of PRP or PRF on proliferation, migration and viability of osteoblasts and oral fibroblasts, treated with zoledronic acid (ZA). ZA treated fibroblasts and osteoblasts were exposed to PRP/PRF. Cell proliferation, migration and viability were measured using the real-time cell-analyzer assay (RTCA), the scratch assay and the MTT assay. There was a significant increase in closure of the scratch area by PRP/PRF treated osteoblasts (PRP = 40.6%, PRF = 100.0%, NC = 0.0%) as well as fibroblasts (PRP = 100.0%, PRF = 100.0%, NC = 12.7%) in comparison to the group of negative control (all p ≤ 0.05). Furthermore, the negative effect of ZA on cell migration was generally reduced in both cell lines using PRP/PRF. The viability and proliferation of cells decreased after exposure to ZA, whereas we observed an enhancement of cell viability within 24 hours by application of PRP/PRF in ZA treated cells. The negative effect of ZA on cell proliferation was especially reduced when using PRF. The use of PRF/PRP improves the behavior of ZA-treated cells, but PRF appears to have an advantage in comparison to PRP. This study demonstrates that treatment with PRF/PRP may have positive effects in the therapy of Bisphosphonate-Related Osteonecrosis of the Jaw (BRONJ).

4.
J Craniomaxillofac Surg ; 47(2): 365-372, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30578012

RESUMO

The aim of this study was to investigate the influence of different incubation methods on the growth factor content of lysates of platelet-rich fibrin (PRF), advanced-platelet-rich fibrin (A-PRF) and platelet-rich plasma (PRP) products. A comparison of related studies suggests that the method of sample preparation has a significant influence on growth factor content. There are few reports on the comparison of non-Ca2+-activated PRP, Ca2+-activated PRP, A-PRF, and PRF, along with a lack of information on the release of PDGF-BB, TGF-ß1, and VEGF among the different incubation methods. The lysate preparation was made of non-Ca2+-activated PRP, Ca2+-activated PRP, PRF, and A-PRF, using a room-temperature, 37 °C, or freeze-thaw-freeze incubation method. Afterwards the VEGF, PDGF-BB, and TGF-ß1 content was investigated by running ELISA tests. Growth factor levels were significantly increased in the non-Ca2+-activated PRP with freeze-thaw-freeze incubation, and in the PRF preparation there was a significant disadvantage to using room temperature incubation for releasing growth factors. In conclusion, the freeze-thaw-freeze method is sufficient for releasing growth factors, and calcium activation is not necessary. Finally, the study demonstrates the possibility of preparing PRP products from platelet concentrates, so that preoperative blood sampling might not be required.


Assuntos
Plaquetas/metabolismo , Cálcio/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Fibrina Rica em Plaquetas/metabolismo , Plasma Rico em Plaquetas/metabolismo , Becaplermina/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Fator de Crescimento Derivado de Plaquetas/metabolismo , Plasma Rico em Plaquetas/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
5.
Eur J Pharmacol ; 835: 140-146, 2018 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-30081034

RESUMO

Chronic rhinosinusitis with nasal polyps (CRSwNP) represents a benign neoplasm of the nasal mucosa, which leads to a decreased breathing capacity and reduced olfaction. The pathogenesis and the molecular mechanisms driving nasal polyps are not very well known. GSK-3 is involved in the regulation of various biosynthetic pathways and various kinases are able to regulate the GSK-3. Therefore, we investigated the effect of the monoterpene oxide 1,8-cineol on the regulation of the Wnt/ß-catenin signaling pathway with its central regulator protein GSK-3 in vitro. We determined GSK-3 expression and phosphorylation as well as the expression of negative regulators (Akt and SGK) and downstream activation of ß-catenin in nasal polyps of patients with CRSwNP by immunohistochemistry and Western blot experiments. In this study we demonstrated for the first time, that 1,8-cineol acts as a potential inhibitor of the Wnt/ß-catenin signaling pathway, by affecting the inhibitory phosphorylation of GSK-3, which is the key regulator of the ß-catenin activity. Our data provide novel insights in the regulatory networks responsible for the progression of CRSwNP and furthermore represent a new mechanism of 1,8-cineol activity, which may lead to novel treatment approaches to this natural drug.

6.
Oncotarget ; 9(45): 27630-27644, 2018 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-29963225

RESUMO

Background: Head and neck squamous cell cancer (HNSCC) is one of the most common tumors worldwide and there is an enormous need for innovative therapy approaches. Several recent studies suggest tumor entity specific roles of glycogen synthase kinase 3 (GSK3) in different human cancers, acting as tumor suppressor or as tumor promoter. Here we describe the role of GSK3 with respect to different parameters within HNSCC progression. Methods: Base line expression and activity profiles of p-GSK3α/ß (Ser21/9) and p-GSK3α/ß (Tyr279/216) were analyzed by immunohistochemistry and western blotting. Four different permanent HNSCC cell lines were exposed to the potent GSK3α/ß inhibitor SB 216763. Cell viability was controlled via the MTT test. Cell migration was quantified with the Real Time Cell Analyzer (RCTA) xCELLigence. Regulation of the epithelial-mesenchymal transition (EMT) was measured with the Human Epithelial to Mesenchymal Transition (EMT) RT2 Profiler™ PCR Array and scratch assays. Taqman probes were used to detect the specific gene expression profiles of inflammatory cytokines Interleukin IL1ß, IL6, IL8, IL10, TNFα and IFNß. Results: Exposure of permanent HNSCC cell lines to the specific GSK3α/ß inhibitor SB 216763 leads to significant growth inhibition, inhibition of migration and decreased levels of active GSK3α/ß in a dose dependent manner.Exposure of HNSCC lines to SB 216763 also resulted in a markable shift of EMT markers and functional EMT dysregulation. Functionally GSK3 differentially mediates the expression of TLR4- and TLR3-induced inflammatory cytokines in HNSCC, whereas no effect of SB 216763 on the NFkB activity was noticed. Conclusion: GSK3α/ß plays a crucial role in a variety of regulatory networks for HNSCC cancer progression as it drives proliferation or migration and thus GSK3 could serve as an interesting target for clinical drug development.

7.
Oncotarget ; 9(44): 27460-27470, 2018 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-29937998

RESUMO

Cancer immunotherapy has been revolutionised by drugs that enhance the ability of the immune system to detect and fight tumors. Immune checkpoint therapies that target the programmed death-1 receptor (PD-1), or its ligand (PD-L1) have shown unprecedented rates of durable clinical responses in patients with various cancer types. However, there is still a large fraction of patients that do not respond to checkpoint inhibitors, and the challenge remains to find cellular and molecular cues that could predict which patients would benefit from these therapies. Using a series of qualitative and quantitative methods we show here that PBMCs and platelets from smokers and patients with head and neck squamous cell carcinoma (HNSCC) or lung cancer express and up-regulate PD-L1 independently of tumor stage. Furthermore, treatment with Atezolizumab, a fully humanised monoclonal antibody against PD-L1, in 4 patients with lung cancer caused a decrease in PD-L1 expression in platelets, which was restored over 20 days. Altogether, our findings reveal the expression of the main therapeutic target in current checkpoint therapies in human platelets and highlight their potential as biomarkers to predict successful therapeutic outcomes.

8.
Nitric Oxide ; 78: 89-94, 2018 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-29885366

RESUMO

INTRODUCTION: Chronic rhinosinusitis with nasal polyps (CRSwNP) is a significant health problem, but the pathogenesis remains unclear to date. Nitric oxide (NO) has known airway modulating functions. Therefore, we investigated nitric oxide production to determine the role of eNOS in nasal polyps, with additional analysis of the effect of the monoterpene oxide 1,8-cineol on the possible regulation of eNOS signaling and thus NO production. METHODS: We determined eNOS expression, as well as regulatory and effector proteins like NOSTRIN and CASP8, using whole genome microarray, immunohistochemistry and western blot. To evaluate the influence of 1,8-cineol on eNOS signaling, we examined tissue samples of nasal polyps of patients with CRSwNP incubated with 100 µM 1,8-cineol using quantitative real-time PCR, western blot and phosphorylation arrays. RESULTS: Microarray analysis revealed an increased gene expression of eNOS (1.40-fold) as well as a decreased gene expression of NOSTRIN (0.53-fold) and CASP8 (0.44-fold) in nasal polyps. At the protein level, we detected 2.3-fold higher protein expression of eNOS and significant higher phosphorylation levels of eNOS in nasal polyps (19.7-fold, p ≤ 0.001) compared to inferior turbinates. Additionally, 1,8-cineol did not influence NOSTRIN and CASP8, but decreased the eNOS phosphorylation significantly (p ≤ 0.05). DISCUSSION: Our study demonstrated for the first time that nasal polyps exhibit an increased phosphorylation of eNOS, which could be important for vascular permeability and the associated edema and elevated inflammation. Additionally, we detected that 1,8-cineol affects the eNOS phosphorylation significantly and thus its activation. This could be important to handle the elevated inflammation and edema formation by regulating the vascular permeability.

9.
Front Immunol ; 9: 680, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29686675

RESUMO

Anti-neutrophil cytoplasmic autoantibodies (ANCA) targeting proteinase 3 (PR3) and myeloperoxidase expressed by innate immune cells (neutrophils and monocytes) are salient diagnostic and pathogenic features of small vessel vasculitis, comprising granulomatosis with polyangiitis (GPA), microscopic polyangiitis, and eosinophilic GPA. Genetic studies suggest that ANCA-associated vasculitides (AAV) constitute separate diseases, which share common immunological and pathological features, but are otherwise heterogeneous. The successful therapeutic use of anti-CD20 antibodies emphasizes the prominent role of ANCA and possibly other autoantibodies in the pathogenesis of AAV. However, to elucidate causal effects in AAV, a better understanding of the complex interplay leading to the emergence of B lymphocytes that produce pathogenic ANCA remains a challenge. Different scenarios seem possible; e.g., the break of tolerance induced by a shift from non-pathogenic toward pathogenic autoantigen epitopes in inflamed tissue. This review gives a brief overview on current knowledge about genetic and epigenetic factors, barrier dysfunction and chronic non-resolving inflammation, necro-inflammatory auto-amplification of cellular death and inflammation, altered autoantigen presentation, alternative complement pathway activation, alterations within peripheral and inflamed tissue-residing T- and B-cell populations, ectopic lymphoid tissue neoformation, the characterization of PR3-specific T-cells, properties of ANCA, links between autoimmune disease and infection-triggered pathology, and animal models in AAV.

10.
Oncol Lett ; 15(3): 3985-3990, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29456743

RESUMO

It has been shown that head and neck squamous cell carcinoma (HNSCC) are infiltrated by plasmacytoid dendritic cells (pDCs). The HNSCC TH2 biased microenvironment leads to strong alterations of the cellular functions of pDC and thus impairs the initiation and function of adequate immune responses. In this work we comprehensively analyzed the capacity of CpG-oligonucleotides to activate interferon (IFN)-α secretion of human pDC in the presence of HNSCC. IFN-α secretion was measured using the ELISA Technique. Class A CpG dinucleotide 2216 was used in different concentrations and time frames to stimulate the IFN-α production of human pDC from peripheral blood in the absence and presence of the HNSCC microenvironment. To elucidate single components that might induce the reduction of IFN-α secretion, pDC were exposed to different concentrations of HNSCC relevant cytokines such as IL-6, IL-8 and IL-10. In accordance to former experiments we found that HNSCC micro milieu severely depresses up to 75% of IFN-α secretion capacity of pDCs, if the stimulating Class A CpG 2216 is added to the culture. Preincubation of HNSCC supernatant leads to unrestorable reduction of IFN-α secretion in pDC and can not be restored by CpG 2216. Incubation of pDCs with single cytokines relevant for cancer progression within the HNSCC micro milieu show that IL-6 or IL-8 have no influence on the IFN-α secretion in pDCs, whereas IL-10 massively impairs the secretion in a dose dependent manner. This effect can be potentiated by synergistic incubation with IL-6 and can be abrogated by blocking antibodies to the IL-10 receptor. Interestingly, incubation with IL-10 is not the only factor that impairs the IFN-α secretion, as incubation with the whole HNSCC supernatant is even more effective in reducing the secretion, implying that additional factors play a role. We conclude that restoration of HNSCC induced TH2 bias could be improved by the inhibition of immune cell cytokine receptors in addition to immunostimulating approaches with CpG motifs.

11.
Cell Mol Life Sci ; 75(2): 323-334, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28849249

RESUMO

Colorectal cancer (CRC) is one of the most frequent malignancies in the Western world. Early tumor detection and intervention are important determinants on CRC patient survival. During early tumor proliferation, dissemination and angiogenesis, platelets store and segregate proteins actively and selectively. Hence, the platelet proteome is a potential source of biomarkers denoting early malignancy. By comparing protein profiles of platelets between healthy volunteers (n = 12) and patients with early- (n = 7) and late-stage (n = 5) CRCs using multiplex fluorescence two-dimensional gel electrophoresis (2D-DIGE), we aimed at identifying differentially regulated proteins within platelets. By inter-group comparisons, 94 differentially expressed protein spots were detected (p < 0.05) between healthy controls and patients with early- and late-stage CRCs and revealed distinct separations between all three groups in principal component analyses. 54 proteins of interest were identified by mass spectrometry and resulted in high-ranked Ingenuity Pathway Analysis networks associated with Cellular function and maintenance, Cellular assembly and organization, Developmental disorder and Organismal injury and abnormalities (p < 0.0001 to p = 0.0495). Target proteins were validated by multiplex fluorescence-based Western blot analyses using an additional, independent cohort of platelet protein samples [healthy controls (n = 15), early-stage CRCs (n = 15), late-stage CRCs (n = 15)]. Two proteins-clusterin and glutathione synthetase (GSH-S)-featured high impact and were subsequently validated in this independent clinical cohort distinguishing healthy controls from patients with early- and late-stage CRCs. Thus, the potential of clusterin and GSH-S as platelet biomarkers for early detection of CRC could improve existing screening modalities in clinical application and should be confirmed in a prospective multicenter trial.


Assuntos
Plaquetas/metabolismo , Clusterina/metabolismo , Neoplasias Colorretais/metabolismo , Glutationa Sintase/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/diagnóstico , Detecção Precoce de Câncer , Eletroforese em Gel Bidimensional , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mapas de Interação de Proteínas , Proteoma/metabolismo
12.
Oncol Rep ; 38(6): 3693-3701, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29039574

RESUMO

Transforming growth factor (TGF)-ß promotes epithelial-mesenchymal transition and cell invasion of cancer cells in part through the small GTPase RAC1. Since RAC1 can signal through reactive oxygen species (ROS), we probed the role of the ROS-producing NADPH oxidase (NOX) and p38 mitogen-activated protein kinase (MAPK) in mediating TGF-ß1/RAC1-driven random cell migration (chemokinesis). Although the NOX isoforms NOX2, 4, 5, 6, and RAC1 were readily detectable by RT-PCR in pancreatic ductal adenocarcinoma (PDAC)-derived Panc1 and Colo357 cells, only NOX4 and RAC1 were expressed at higher levels comparable to those in peripheral blood monocytes. TGF-ß1 treatment resulted in upregulation of NOX4 (and NOX2) and rapid intracellular production of ROS. To analyze whether RAC1 functions through NOX and ROS to promote cell motility, we performed real-time cell migration assays with xCELLigence® technology in the presence of the ROS scavenger N-acetyl-L-cysteine (NAC) and various NOX inhibitors. NAC, the NOX4 inhibitor diphenylene iodonium or small interfering RNA (siRNA) to NOX4, and the NOX2 inhibitor apocynin all suppressed TGF-ß1-induced chemokinesis of Panc1 and Colo357 cells as did various inhibitors of RAC1 used as control. In addition, we showed that blocking NOX4 or RAC1 function abrogated phosphorylation of p38 MAPK signaling by TGF-ß1 and that inhibition of p38 MAPK reduced TGF-ß1-induced random cell migration, while ectopic expression of a kinase-active version of the p38 activating kinase MKK6 was able to partially rescue the decline in migration after RAC1 inhibition. Our data suggest that TGF-ß1-induced chemokinesis in PDAC cells is mediated through a RAC1/NOX4/ROS/p38 MAPK cascade.


Assuntos
NADPH Oxidase 4/genética , Neoplasias Pancreáticas/genética , Fator de Crescimento Transformador beta1/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas rac1 de Ligação ao GTP/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Neoplasias Pancreáticas/patologia , Fosforilação , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genética
13.
Front Oncol ; 7: 92, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28589081

RESUMO

OBJECTIVES: Head and neck squamous cell carcinoma (HNSCC) is one of the most common tumors worldwide. The high mortality rates have not changed during the last three decades, and thus there is an enormous need for innovative therapy approaches. Several recent studies suggest an important role of the Wnt/ß-catenin signaling pathway in the tumorigenesis of HNSCC. We analyzed the effect of the monoterpene oxide 1,8-cineol on the regulation of the Wnt/ß-catenin signaling pathway and the cellular progression of different HNSCC cell lines. METHODS: Permanent HNSCC cell lines were exposed to varying concentrations and times of 1,8-cineol. Regulation and activity profiles of the Wnt/ß-catenin signaling cascade were analyzed using Western hybridization experiments, MTT assays, real-time PCR-based epithelial to mesenchymal transition array, and immunohistochemistry. RESULTS: Exposure of different cell lines to 1,8-cineol treatment resulted in a dose-dependent inhibition of proliferation and a decreased activity of the WNT/ß-catenin pathway. We can show the inhibition of glycogen synthase kinase 3 (GSK-3)α/ß (Ser-9/21) as well as a corresponding decreased endolysosomal localization, leading to a decreased ß-catenin activity. Furthermore, we can show that exposure to cineol functionally results in a reduced expression of WNT11. CONCLUSION: In this work, we demonstrate for the first time that 1,8-cineol acts as an inhibitor of the Wnt/ß-catenin activity in HNSCC via a decreased inhibition of GSK-3, which lead to reduced levels of WNT11 and a dose-dependent decrease of the cellular progression. Our data represent a new mechanism of 1,8-cineol activity, which may lead to novel molecular targets and treatment approaches of this natural drug.

14.
Arch Immunol Ther Exp (Warsz) ; 65(5): 431-443, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28280847

RESUMO

Chronic rhinosinusitis with nasal polyps is considered a subgroup of chronic rhinosinusitis and a significant health problem, but the pathogenesis remains unclear to date. Therefore, we investigated the stemness to determine the role of stem cells in nasal polyps, with additional analysis of the neuronal differentiation potential of nasal polyp cells. We determined gene and protein expression profiles of stem cells in nasal polyp tissues, using whole genome microarray, quantitative real-time PCR (qPCR), immunohistochemistry, and flow cytometry. To evaluate the neuronal differentiation potential of nasal polyp cells, we used an efficient xenogeneic co-culture model with unsliced adult rat brain biopsies, followed by qPCR, immunohistochemistry, and growth factor antibody arrays. During gene expression analysis and immunohistochemistry, we were able to detect different stem cell markers, like Oct-4, Sox2, Klf4, c-Myc, ABCG2, Nanog, CD133, and Nestin, which confirmed the existence of stem cell like cells within nasal polyps. In addition, co-culture experiments give evidence for a guided differentiation into the neuronal lineage by overexpression of Nestin, Neurofilament, and GM-CSF. Our study demonstrated the expression of stem cell-related markers in nasal polyps. Furthermore, we characterized, for the first time, the stemness and neuronal differentiation potential of nasal polyp cells. These results gave new insights into the pathogenesis of nasal polyps and its therapeutic effectiveness could represent a promising strategy in the future.


Assuntos
Autorrenovação Celular , Pólipos Nasais/imunologia , Rinite/imunologia , Sinusite/imunologia , Nicho de Células-Tronco/fisiologia , Células-Tronco/fisiologia , Adulto , Idoso , Animais , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Doença Crônica , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Filamentos Intermediários/genética , Filamentos Intermediários/metabolismo , Masculino , Pessoa de Meia-Idade , Pólipos Nasais/diagnóstico , Nestina/genética , Nestina/metabolismo , Neurogênese , Fator 3 de Transcrição de Octâmero/metabolismo , Ratos , Ratos Sprague-Dawley , Rinite/diagnóstico , Sinusite/diagnóstico , Transcriptoma
15.
Am J Respir Cell Mol Biol ; 56(5): 575-584, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28059551

RESUMO

The signaling pathways that sustain the disease process of chronic rhinosinusitis with nasal polyps (CRSwNP) remain poorly understood. We sought to determine the expression levels of Wnt signaling genes in CRSwNP and to study the role of the Wnt pathway in inflammation and epithelial remodeling in the nasal mucosa. Microarrays and real time-quantitative polymerase chain reaction comparing gene expression in matched NPs and inferior turbinates revealed that WNT2B, WNT3A, WNT4, WNT7A, WNT7B, and FZD2 were up-regulated and that FZD1, LRP5, LRP6, and WIF1 were down-regulated in NPs. Immunolabeling showed robust expression of Wnt ligands, nuclear ß-catenin, and Axin-2 in NP tissue, suggesting that Wnt/ß-catenin signaling is activated in NPs. We used primary human nasal epithelial cell (HNEpC) cultures to test the functional consequences of Wnt pathway activation. Monolayer HNEpCs treated with recombinant human WNT (rhWNT) 3A, but not with rhWNT4, had altered epithelial morphology and decreased adhesion, without loss of viability. We found that neither rhWNT3A nor rhWNT4 treatment induced proliferation. The expression and release of inflammatory cytokines IL-6 and granulocyte-macrophage colony-stimulating factor were increased after rhWNT3A exposure of HNEpCs. When differentiated at an air-liquid interface, rhWNT3A- and WNT agonist-, but not rhWNT4-treated HNEpCs, had abnormal epithelial architecture, failed to undergo motile ciliogenesis, and had defective noncanonical Wnt (planar cell polarity) signaling. On the basis of these results, we propose a model in which Wnt/ß-catenin signaling sustains mucosal inflammation and leads to a spectrum of changes consistent with those seen during epithelial remodeling in NPs.


Assuntos
Pólipos Nasais/complicações , Pólipos Nasais/metabolismo , Rinite/complicações , Rinite/metabolismo , Sinusite/complicações , Sinusite/metabolismo , Via de Sinalização Wnt , Doença Crônica , Cílios/efeitos dos fármacos , Cílios/metabolismo , Sistemas de Computação , Citocinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Pólipos Nasais/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Rinite/patologia , Sinusite/patologia , Conchas Nasais/patologia , Proteínas Wnt/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo
16.
Arch Immunol Ther Exp (Warsz) ; 65(2): 157-173, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27393708

RESUMO

The pathogenesis of chronic rhinosinusitis (CRS) remains unclear to date. The tissue remodeling in nasal polyps may be the result of inflammatory mediators and may involve epithelial-mesenchymal transition (EMT) and EMT-associated features such as cell motility in nasal epithelial cells (NECs). We determined whether NEC in nasal polyps of CRS already display features of EMT in vivo or respond with EMT to growth factor stimulation in vitro. Nasal polyp tissues expressed both epithelial and mesenchymal markers. Primary NEC from inferior turbinates and nasal polyps responded to the EMT-inducing agents transforming growth factor (TGF)-ß1 and epidermal growth factor (EGF) with different expression patterns of EMT markers (E-cadherin, N-cadherin, Snail, Slug, Twist), however, only NEC from nasal polyps were susceptible to TGF-ß1 and EGF-dependent cell migration. Our data suggest that a partial EMT is associated with the pathogenesis of nasal polyps in CRS patients. Furthermore, we show for the first time that epithelial cells from both nasal polyps and inferior turbinates were able to undergo an EMT-like process following exposure to TGF-ß1 or EGF in vitro but that only NEC from nasal polyps responded with enhanced cell motility. Our data suggest that NEC from CRS patients have undergo partial EMT and that this process may be involved in the pathogenesis of CRS.


Assuntos
Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Pólipos Nasais/metabolismo , Sinusite/metabolismo , Conchas Nasais/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Movimento Celular , Fator de Crescimento Epidérmico/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Inflamação , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Fator de Crescimento Transformador beta1/metabolismo , Adulto Jovem
17.
J Cancer Res Clin Oncol ; 142(6): 1261-71, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27038158

RESUMO

PURPOSE: Paclitaxel is an effective chemotherapeutic agent against various human tumors inducing apoptosis via binding to ß-tubulin of microtubules and arresting cells mainly in the G2/M phase of the cell cycle. However, the underlying specific molecular mechanisms of paclitaxel on head and neck squamous cell carcinoma (HNSCC) have not been identified yet. METHODS: The apoptotic effects and mechanisms of paclitaxel on different permanent HPV-negative HNSCC cell lines (UT-SCC-24A, UT-SCC-24B, UT-SCC-60A and UT-SCC-60B) were determined by flow cytometry assays, polymerase chain reaction analysis, immunofluorescence-based assays and sequencing studies. RESULTS: Paclitaxel induced a G2/M arrest in HNSCC cell lines followed by an increased amount of apoptotic cells. Moreover, the activation of caspase 8, caspase 10 and caspase 3, and the loss of the mitochondrial outer membrane potential could be observed, whereas an activation of caspase 9 could barely be detected. The efficient activation of caspase 9 was not affected by altered methylation patterns. Our results can show that the promoter region of apoptotic protease activating factor 1 (Apaf-1) was not methylated in the HNSCC cell lines. By sequencing analysis two isoforms of caspase 9, the pro-apoptotic caspase 9 and the anti-apoptotic caspase 9b were identified. The anti-apoptotic caspase 9b is missing the catalytic site and acts as an endogenous inhibitor of apoptosis by blocking the binding of caspase 9 to Apaf-1 to form the apoptosome. CONCLUSION: Our data indicate the presence of anti-apoptotic caspase 9b in HNSCC, which may serve as a promising target to increase chemotherapeutic apoptosis induction.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma de Células Escamosas/patologia , Caspase 9/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Paclitaxel/farmacologia , Processamento Alternativo , Fator Apoptótico 1 Ativador de Proteases/genética , Carcinoma de Células Escamosas/enzimologia , Caspase 9/genética , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Metilação de DNA , Ativação Enzimática , Neoplasias de Cabeça e Pescoço/enzimologia , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Regiões Promotoras Genéticas , Carcinoma de Células Escamosas de Cabeça e Pescoço , Estaurosporina/farmacologia
18.
Am J Rhinol Allergy ; 29(3): 182-7, 2015 May-Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25975249

RESUMO

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is a recurrent, benign, extensively proliferating disease that is triggered by inflammation. The signaling pathways in sinusitis and the regulation by intracellular signaling peptides and proteins are not fully understood. Signal transducer and activator of transcription (STAT) 5a and STAT5b are two closely related phosphokinases involved in the regulation of diverse cellular functions, including proliferation and apoptosis. OBJECTIVE: The objective of the study was to investigate the expression, activation, and distribution of STAT5 Transcription factor in CRSwNP. METHODS: We studied these transcription factors in tissue samples of nasal polyps and inferior turbinates from a total of 35 patients with CRSwNP and compared them with healthy nasal mucosa. The samples were analyzed by using a DNA microarray, quantitative real-time polymerase chain reaction, a protein array, immunoblot, immunoprecipitation and immunohistochemistry. RESULTS: We found equivalent overall expression of STAT5a in all tissue types. We observed an increase in the expression of STAT5b protein in both polyps and turbinates of patients with CRSwNP. In addition, STAT5b, but not STAT5a, was activated by phosphorylation in nasal polyps. Phosphorylated STAT5a/b was not detectable in the epithelium of turbinates from either patients with CRSwNP or patients with healthy mucosa, but it was clearly expressed in the epithelium of nasal polyps. CONCLUSION: Analysis of these data indicates distinct expression and activation of STAT5a and STAT5b in nasal polyps, particularly the activation of STAT5b. It is possible that STAT5b may contribute to the development of nasal polyps.


Assuntos
Pólipos Nasais/metabolismo , Rinite/metabolismo , Fator de Transcrição STAT5/metabolismo , Sinusite/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Expressão Gênica , Humanos , Imunoprecipitação , Fosforilação , Análise Serial de Tecidos , Ativação Transcricional/fisiologia , Conchas Nasais/metabolismo
19.
Int J Nanomedicine ; 9: 5025-40, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25378928

RESUMO

BACKGROUND: As a tomographic imaging technology, magnetic particle imaging (MPI) allows high spatial resolution and sensitivity, and the possibility to create real-time images by determining the spatial distribution of magnetic particles. To ensure a prospective biosafe application of UL-D (University of Luebeck-Dextran coated superparamagnetic nanoparticles), we evaluated the biocompatibility of superparamagnetic iron oxide nanoparticles (SPIONs), their impact on biological properties, and their cellular uptake using head and neck squamous cancer cells (HNSCCs). METHODS: SPIONs that met specific MPI requirements were synthesized as tracers. Labeling and uptake efficiency were analyzed by hematoxylin and eosin staining and magnetic particle spectrometry. Flow cytometry, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays, and real-time cell analyzer assays were used to investigate apoptosis, proliferation, and the cytokine response of SPION-labeled cells. The production of reactive oxygen species (ROS) was determined using a fluorescent dye. Experimental results were compared to the contrast agent Resovist(®), a standard agent used in MPI. RESULTS: UL-D nanoparticles and Resovist particles were taken up in vitro by HNSCCs via unspecific phagocytosis followed by cytosolic accumulation. To evaluate toxicity, flow cytometry analysis was performed; results showed that dose- and time-dependent administration of Resovist induced apoptosis whereas cell viability of UL-D-labeled cells was not altered. We observed decreased cell proliferation in response to increased SPION concentrations. An intracellular production of ROS could not be detected, suggesting that the particles did not cause oxidative stress. Tumor necrosis factor alpha (TNF-α) and interleukins IL-6, IL-8, and IL-1ß were measured to distinguish inflammatory responses. Only the primary tumor cell line labeled with >0.5 mM Resovist showed a significant increase in IL-1ß secretion. CONCLUSION: Our data suggest that UL-D SPIONs are a promising tracer material for use in innovative tumor cell analysis in MPI.


Assuntos
Diagnóstico por Imagem/métodos , Neoplasias de Cabeça e Pescoço/metabolismo , Nanopartículas de Magnetita/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citocinas/análise , Citocinas/metabolismo , Humanos , Nanopartículas de Magnetita/toxicidade , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo
20.
J Immunol Res ; 2014: 959854, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24995349

RESUMO

Chronic rhinosinusitis with nasal polyps (CRSwNP) in Caucasians is a chronic Th2 inflammatory disease of the nasal and paranasal mucosa and the recruitment of leukocytes to the site of inflammation is poorly understood. We studied mRNA and protein expression profiles of adhesion molecules in nasal polyp and associated inferior turbinate tissues using molecular, biochemical, and immunohistological methods. Analysis showed a strongly decreased E-selectin expression in nasal polyps with a significant difference between eosinophil and neutrophil counts in nasal polyps and balanced counts in inferior turbinates. E-selectin expression is known to be downregulated in a Th2 milieu and has an essential role in immunosurveillance by locally activating neutrophil arrest and migratory function. A downregulation of E-selectin may come along with an immune imbalance in Caucasian nasal polyps due to a significant inhibition of neutrophil recruitment. Therefore, we suggest that an upregulation of E-selectin and the associated influx of neutrophils may play a significant role in the resolution of inflammation as well as for the pathophysiology of nasal polyps of Caucasian chronic rhinosinusitis patients.


Assuntos
Selectina E/genética , Grupo com Ancestrais do Continente Europeu , Regulação da Expressão Gênica , Pólipos Nasais/etiologia , Rinite/complicações , Sinusite/complicações , Adulto , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Doença Crônica , Regulação para Baixo , Selectina E/metabolismo , Eosinófilos , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Pólipos Nasais/sangue , Pólipos Nasais/patologia , Neutrófilos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , RNA Mensageiro/genética , Reprodutibilidade dos Testes
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