RESUMEN
OBJECTIVES: The aim of this study was to: (1) investigate the expression patterns of antimicrobial peptides (AMPs), specifically psoriasin (S100A7) and calgranulin A and B (S100A8/A9), in patients with oral lichen planus (OLP) compared to healthy individuals; (2) evaluate the oral health-related quality of life (OHrQoL) in OLP patients versus healthy controls; (3) investigate the impact of clinical severity of OLP on OHrQoL; and (4) assess the influence of AMP expression on clinical severity and OHrQoL in OLP patients. MATERIALS AND METHODS: Oral mucosal biopsies (n = 38) were collected from healthy individuals (n = 17) and patients with OLP (n = 21). Levels of AMPs (S100A7, S100A8, S100A9) and pro-inflammatory cytokines interleukin-8 (IL-8) and tumor necrosis factor alpha (TNFα) were assessed by RT-qPCR. AMP protein localization was identified by indirect immunofluorescence analysis. OHrQoL was assessed using the OHIP-G14 questionnaire, and clinical severity was evaluated with the Oral Disease Severity Score (ODSS). Correlations between OLP manifestation, OHrQoL, and AMP expression were evaluated. RESULTS: (1) S100A7 (p < 0.001), IL-8 (p < 0.001), and TNFα (p < 0.001) mRNA levels were significantly upregulated in OLP tissue compared to healthy tissue, while S100A8 (p < 0.001) and S100A9 (p < 0.001) mRNA levels were downregulated. Immunofluorescence staining revealed an enhanced expression of S100A7 and decreased protein expression of S100A9 in OLP tissue. (2) OLP patients (9.58 ± 8.32) reported significantly higher OHIP-G14 scores compared to healthy individuals (0.67 ± 0.87; p < 0.001), particularly in the categories "physical pain" (p < 0.001) and "psychological discomfort" (p = 0.025). (3,4) Clinical severity (25.21 ± 9.77) of OLP correlated positively with OHrQoL (ρ = 0.497) and psoriasin expression (ρ = 0.402). CONCLUSIONS: This study demonstrated differential expression patterns of AMPs in OLP and highlighted the correlation between the clinical manifestation of OLP and OHrQoL. Further research approaches should address the role of psoriasin in the risk of malignant transformation of OLP. CLINICAL RELEVANCE: Psoriasin is a putative biomarker to monitor disease severity including malignant transformation of OLP lesions. OHIP-G14 scores can be useful to monitor OHrQoL in OLP patients.
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Liquen Plano Oral , Proteína A7 de Unión a Calcio de la Familia S100 , Femenino , Humanos , Masculino , Biopsia , Calgranulina A/metabolismo , Estudios de Casos y Controles , Liquen Plano Oral/metabolismo , Calidad de Vida , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteína A7 de Unión a Calcio de la Familia S100/metabolismo , Proteínas S100/metabolismo , Índice de Severidad de la Enfermedad , Encuestas y Cuestionarios , Regulación hacia ArribaRESUMEN
Axonal degeneration is an early and ongoing event that causes disability and disease progression in many neurodegenerative disorders of the peripheral and central nervous systems. Chemotherapy-induced peripheral neuropathy (CIPN) is a major cause of morbidity and the main cause of dose reductions and discontinuations in cancer treatment. Preclinical evidence indicates that activation of the Wallerian-like degeneration pathway driven by sterile alpha and TIR motif containing 1 (SARM1) is responsible for axonopathy in CIPN. SARM1 is the central driver of an evolutionarily conserved programme of axonal degeneration downstream of chemical, inflammatory, mechanical or metabolic insults to the axon. SARM1 contains an intrinsic NADase enzymatic activity essential for its pro-degenerative functions, making it a compelling therapeutic target to treat neurodegeneration characterized by axonopathies of the peripheral and central nervous systems. Small molecule SARM1 inhibitors have the potential to prevent axonal degeneration in peripheral and central axonopathies and to provide a transformational disease-modifying treatment for these disorders. Using a biochemical assay for SARM1 NADase we identified a novel series of potent and selective irreversible isothiazole inhibitors of SARM1 enzymatic activity that protected rodent and human axons in vitro. In sciatic nerve axotomy, we observed that these irreversible SARM1 inhibitors decreased a rise in nerve cADPR and plasma neurofilament light chain released from injured sciatic nerves in vivo. In a mouse paclitaxel model of CIPN we determined that Sarm1 knockout mice prevented loss of axonal function, assessed by sensory nerve action potential amplitudes of the tail nerve, in a gene-dosage-dependent manner. In that CIPN model, the irreversible SARM1 inhibitors prevented loss of intraepidermal nerve fibres induced by paclitaxel and provided partial protection of axonal function assessed by sensory nerve action potential amplitude and mechanical allodynia.
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Proteínas del Dominio Armadillo/antagonistas & inhibidores , Axones/efectos de los fármacos , Proteínas del Citoesqueleto/antagonistas & inhibidores , Paclitaxel/toxicidad , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/tratamiento farmacológico , Tiazoles/uso terapéutico , Animales , Antineoplásicos Fitogénicos/toxicidad , Proteínas del Dominio Armadillo/deficiencia , Proteínas del Dominio Armadillo/genética , Axones/metabolismo , Células Cultivadas , Proteínas del Citoesqueleto/deficiencia , Proteínas del Citoesqueleto/genética , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Ratones Noqueados , Enfermedades del Sistema Nervioso Periférico/genética , Enfermedades del Sistema Nervioso Periférico/metabolismo , Tiazoles/farmacologíaRESUMEN
BACKGROUND: One key approach for anticancer therapy is drug combination. Drug combinations can help reduce doses and thereby decrease side effects. Furthermore, the likelihood of drug resistance is reduced. Distinct alterations in tumor metabolism have been described in past decades, but metabolism has yet to be targeted in clinical cancer therapy. Recently, we found evidence for synergism between dichloroacetate (DCA), a pyruvate dehydrogenase kinase inhibitor, and the HIF-1α inhibitor PX-478. In this study, we aimed to analyse this synergism in cell lines of different cancer types and to identify the underlying biochemical mechanisms. METHODS: The dose-dependent antiproliferative effects of the single drugs and their combination were assessed using SRB assays. FACS, Western blot and HPLC analyses were performed to investigate changes in reactive oxygen species levels, apoptosis and the cell cycle. Additionally, real-time metabolic analyses (Seahorse) were performed with DCA-treated MCF-7 cells. RESULTS: The combination of DCA and PX-478 produced synergistic effects in all eight cancer cell lines tested, including colorectal, lung, breast, cervical, liver and brain cancer. Reactive oxygen species generation and apoptosis played important roles in this synergism. Furthermore, cell proliferation was inhibited by the combination treatment. CONCLUSIONS: Here, we found that these tumor metabolism-targeting compounds exhibited a potent synergism across all tested cancer cell lines. Thus, we highly recommend the combination of these two compounds for progression to in vivo translational and clinical trials.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Línea Celular Tumoral/efectos de los fármacos , Ácido Dicloroacético/farmacología , Compuestos de Mostaza/farmacología , Fenilpropionatos/farmacología , Células A549 , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales/métodos , Sinergismo Farmacológico , Células HT29 , Células HeLa , Humanos , Células MCF-7 , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: Despite an improvement of prognosis in breast and colon cancer, the outcome of the metastatic disease is still severe. Microevolution of cancer cells often leads to drug resistance and tumor-recurrence. To target the driving forces of the tumor microevolution, we focused on synergistic drug combinations of selected compounds. The aim is to prevent the tumor from evolving in order to stabilize disease remission. To identify synergisms in a high number of compounds, we propose here a three-step concept that is cost efficient, independent of high-throughput machines and reliable in its predictions. METHODS: We created dose response curves using MTT- and SRB-assays with 14 different compounds in MCF-7, HT-29 and MDA-MB-231 cells. In order to efficiently screen for synergies, we developed a screening tool in which 14 drugs were combined (91 combinations) in MCF-7 and HT-29 using EC25 or less. The most promising combinations were verified by the method of Chou and Talalay. RESULTS: All 14 compounds exhibit antitumor effects on each of the three cell lines. The screening tool resulted in 19 potential synergisms detected in HT-29 (20.9%) and 27 in MCF-7 (29.7%). Seven of the top combinations were further verified over the whole dose response curve, and for five combinations a significant synergy could be confirmed. The combination Nutlin-3 (inhibition of MDM2) and PX-478 (inhibition of HIF-1α) could be confirmed for all three cell lines. The same accounts for the combination of Dichloroacetate (PDH activation) and NHI-2 (LDH-A inhibition). Our screening method proved to be an efficient tool that is reliable in its projections. CONCLUSIONS: The presented three-step concept proved to be cost- and time-efficient with respect to the resulting data. The newly found combinations show promising results in MCF-7, HT-29 and MDA-MB231 cancer cells.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias del Colon/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Neoplasias del Colon/patología , Ácido Dicloroacético/farmacología , Ácido Dicloroacético/uso terapéutico , Ensayos de Selección de Medicamentos Antitumorales/métodos , Sinergismo Farmacológico , Femenino , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Imidazoles/farmacología , Imidazoles/uso terapéutico , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Compuestos de Mostaza/farmacología , Compuestos de Mostaza/uso terapéutico , Fenilpropionatos/farmacología , Fenilpropionatos/uso terapéutico , Piperazinas/farmacología , Piperazinas/uso terapéutico , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Complejo Piruvato Deshidrogenasa/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Neuroinflammation is often associated with pathological changes in the function of the blood-brain barrier (BBB) caused by disassembly of tight and adherens junctions that under physiological conditions are important for the maintenance of the BBB integrity. Consequently, in inflammation the BBB becomes dysfunctional, facilitating leukocyte traversal of the barrier and accumulation of immune cells within the brain. The extracellular matrix (ECM) also contributes to BBB integrity but the significance of the main ECM receptors, the ß1 integrins also expressed on endothelial cells, is less well understood. To evaluate whether ß1 integrin function is affected during inflammation and impacts barrier function, we used a transformed human brain microvascular endothelial cell (THBMEC)-based Interleukin 1ß (IL-1ß)-induced inflammatory in vitro BBB model. We demonstrate that IL-1ß increases cell-matrix adhesion and induces a redistribution of active ß1 integrins to the basal surface. In particular, binding of α5ß1 integrin to its ligand fibronectin is enhanced and α5ß1 integrin-dependent signalling is upregulated. Additionally, localisation of the tight junction protein claudin-5 is altered. Blockade of the α5ß1 integrin reduces the IL-1ß-induced transendothelial migration of peripheral blood mononuclear cells (PBMCs). These data imply that IL-1ß-induced inflammation not only destabilizes tight junctions but also increases α5ß1 integrin-dependent cell-matrix adhesion to fibronectin.
Asunto(s)
Encéfalo/irrigación sanguínea , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Integrina alfa5beta1/metabolismo , Interleucina-1beta/farmacología , Leucocitos Mononucleares/fisiología , Migración Transendotelial y Transepitelial , Barrera Hematoencefálica , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Endotelio Vascular/enzimología , Fibronectinas/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Humanos , Integrina alfa5/metabolismo , Integrina alfa5beta1/antagonistas & inhibidores , Integrina beta1/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación hacia ArribaRESUMEN
Soluble guanylyl cyclase (sGC) plays an important role in cardiovascular function and catalyzes formation of cGMP. sGC is activated by nitric oxide and allosteric stimulators and activators. However, despite its therapeutic relevance, the regulatory mechanisms of sGC are still incompletely understood. A major reason for this situation is that no crystal structures of active sGC have been resolved so far. An important step toward this goal is the identification of high-affinity ligands that stabilize an sGC conformation resembling the active, "fully closed" state. Therefore, we examined inhibition of rat sGCα1ß1 by 38 purine- and pyrimidine-nucleotides with 2,4,6,-trinitrophenyl and (N-methyl)anthraniloyl substitutions at the ribosyl moiety and compared the data with that for the structurally related membranous adenylyl cyclases (mACs) 1, 2, 5 and the purified mAC catalytic subunits VC1:IIC2. TNP-GTP [2',3'-O-(2,4,6-trinitrophenyl)-GTP] was the most potent sGCα1ß1 inhibitor (Ki, 10.7 nM), followed by 2'-MANT-3'-dATP [2'-O-(N-methylanthraniloyl)-3'-deoxy-ATP] (Ki, 16.7 nM). Docking studies on an sGCαcat/sGCßcat model derived from the inactive heterodimeric crystal structure of the catalytic domains point to similar interactions of (M)ANT- and TNP-nucleotides with sGCα1ß1 and mAC VC1:IIC2. Reasonable binding modes of 2'-MANT-3'-dATP and bis-(M)ANT-nucleotides at sGC α1ß1 require a 3'-endo ribosyl conformation (versus 3'-exo in 3'-MANT-2'-dATP). Overall, inhibitory potencies of nucleotides at sGCα1ß1 versus mACs 1, 2, 5 correlated poorly. Collectively, we identified highly potent sGCα1ß1 inhibitors that may be useful for future crystallographic and fluorescence spectroscopy studies. Moreover, it may become possible to develop mAC inhibitors with selectivity relative to sGC.
Asunto(s)
Guanilato Ciclasa/antagonistas & inhibidores , Nitrocompuestos/química , Nucleótidos de Purina/química , Nucleótidos de Pirimidina/química , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , ortoaminobenzoatos/química , Inhibidores de Adenilato Ciclasa , Adenilil Ciclasas/química , Animales , Guanilato Ciclasa/química , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación Proteica , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/química , Ratas , Receptores Citoplasmáticos y Nucleares/química , Proteínas Recombinantes/química , Guanilil Ciclasa Soluble , Relación Estructura-ActividadRESUMEN
Intact HEK293 cells and B103 neuroblastoma cells possess high basal concentrations of the established second messengers cAMP and cGMP and of the emerging second messengers cCMP and cUMP. We asked the question which nucleotidyl cyclase accounts for the high basal cNMP concentrations. Activators and inhibitors of soluble guanylyl cyclase had no major effects on cNMPs, and the activator of membranous adenylyl cyclase forskolin increased only cAMP. Addition of bicarbonate to medium increased, whereas removal of bicarbonate decreased levels of all four cNMPs. The inhibitor of soluble adenylyl cyclase, 2-(1H-benzo[d]imidazol-2-ylthio)-N'-(5-bromo-2-hydroxybenzylidene) propanehydrazide (KH7), reduced bicarbonate-stimulated cNMPs. In conclusion, bicarbonate-stimulated soluble adenylyl cyclase plays an important role in the regulation of basal cellular cNMP levels, most notably cCMP and cUMP.
Asunto(s)
Adenilil Ciclasas/metabolismo , CMP Cíclico/metabolismo , Neuroblastoma/metabolismo , Nucleótidos Cíclicos/metabolismo , Uridina Monofosfato/metabolismo , Bencimidazoles/farmacología , Bicarbonatos/farmacología , Línea Celular Tumoral , Colforsina/farmacología , Inhibidores Enzimáticos/farmacología , Guanilato Ciclasa/antagonistas & inhibidores , Guanilato Ciclasa/metabolismo , Células HEK293/efectos de los fármacos , Células HEK293/metabolismo , Humanos , Hidrazinas/farmacología , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/metabolismo , Guanilil Ciclasa SolubleRESUMEN
Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and generates the second messenger cyclic GMP (cGMP). Recently, purified sGC α1ß1 has been shown to additionally generate the cyclic pyrimidine nucleotides cCMP and cUMP. However, since cyclic pyrimidine nucleotide formation occurred only the presence of Mn(2+) but not Mg(2+), the physiological relevance of these in vitro findings remained unclear. Therefore, we studied cyclic nucleotide formation in intact cells. We observed NO-dependent cCMP- and cUMP formation in intact HEK293 cells overexpressing sGC α1ß1 and in RFL-6 rat fibroblasts endogenously expressing sGC, using HPLC-tandem mass spectrometry. The identity of cCMP and cUMP was unambiguously confirmed by HPLC-time-of-flight mass spectrometry. Our data indicate that cCMP and cUMP play second messenger roles and that Mn(2+) is a physiological sGC cofactor.
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Guanilato Ciclasa/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Línea Celular , AMP Cíclico/biosíntesis , CMP Cíclico/biosíntesis , GMP Cíclico/biosíntesis , Guanilato Ciclasa/genética , Células HEK293 , Humanos , Manganeso/metabolismo , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/farmacología , Nitroprusiato/farmacología , Nucleótidos Cíclicos/biosíntesis , Ratas , Receptores Citoplasmáticos y Nucleares/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sistemas de Mensajero Secundario , Guanilil Ciclasa Soluble , Transfección , Uridina Monofosfato/biosíntesisRESUMEN
Integrin α1ß1 is an adhesion receptor that binds to collagen and laminin. It regulates cell adhesion, cytoskeletal organization, and migration. The cytoplasmic tail of the α1 subunit consists of 15 amino acids and contains six positively charged lysine residues. In this study, we present evidence that the α1 integrin cytoplasmic tail (α1CT) directly associates with phosphoinositides, preferentially with phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). Since the association was disrupted by calcium, magnesium and phosphate ions, this interaction appears to be in ionic nature. Here, the peptide-lipid interaction was driven by the conserved KIGFFKR motif. The exchange of both two potential phospholipid-binding lysines for glycines in the KIGFFKR motif increased α1ß1 integrin-specific adhesion and F-actin cytoskeleton formation compared to cells expressing the unmodified α1 subunit, whereas only mutation of the second lysine at position 1171 increased levels of constitutively active α1ß1 integrins on the cell surface. In addition, enhanced focal adhesion formation and increased phosphorylation of focal adhesion kinase, but decreased phosphorylation of AKT was observed in these cells. We conclude that the KIGFFKR motif, and in particular lysine1171 is involved in the dynamic regulation of α1ß1 integrin activity and that the interaction of α1CT with phosphoinositides may contribute to this process.
Asunto(s)
Integrina alfa1 , Proteínas Proto-Oncogénicas c-akt , Integrina alfa1/química , Fosfatidilinositoles , Lisina , Adhesión Celular/genéticaRESUMEN
Oral inflammatory diseases are highly prevalent in the worldwide population. Topical treatment of inflammation is challenging due to dilution effects of saliva and crevicular fluid. Thus, there is a great medical need to develop smart anti-inflammatory drug delivery systems for mucosa treatment. We compared two promising anti-inflammatory dendritic poly(glycerol-caprolactone) sulfate (dPGS-PCL) polymers for their applicability to the oral mucosa. Using an ex vivo porcine tissue model, cell monolayers, and full-thickness 3D oral mucosal organoids, the polymers were evaluated for muco-adhesion, penetration, and anti-inflammatory properties. The biodegradable dPGS-PCL97 polymers adhered to and penetrated the masticatory mucosa within seconds. No effects on metabolic activity and cell proliferation were found. dPGS-PCL97 revealed a significant downregulation of pro-inflammatory cytokines with a clear preference for IL-8 in cell monolayers and mucosal organoids. Thus, dPGS-PCL97 exhibits excellent properties for topical anti-inflammatory therapy, suggesting new therapeutic avenues in the treatment of oral inflammatory diseases.
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Interleucina-8 , Mucosa Bucal , Animales , Porcinos , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Polímeros/farmacología , Sistemas de Liberación de MedicamentosRESUMEN
The inactivation of multi resistant pathogens is an important clinical need. One approach is UV-C irradiation, which was previously not possible in vivo due to cytotoxicity. Recently, far UV-C irradiation at λ < 240 nm was successfully used on skin with negligible damage. A potential application site is the nasal vestibule, where MRSA accumulates and cannot be treated using antiseptics. We irradiated 3D mucosa models and excised human mucosa with 222 and 233 nm far UV-C in comparison to 254 nm and broadband UV-B. Eradication efficiency was evaluated by counting colony forming units; irritation potential was evaluated by hen's egg-chorioallantoic membrane assay and trans epithelial electrical resistance; cell viability was assessed by MTT. DNA damage and cell protective mechanisms were evaluated immunohistopathologically. On mucosa models, MRSA reduced by ≈ 5 log10 for 60 mJ/cm2 irradiation at 233 nm. A slightly increased cell viability was observed after 24 h. Lower doses showed lower irritation potential than the positive controls or commercial mouthwash, while 80 mJ/cm2 had strong irritation potential. DNA damage occurred only superficially and decreased after 24 h. On excised human mucosa, < 10% of keratinocytes were affected after 150 mJ/cm2 222 nm or 60 mJ/cm2 233 nm.
Asunto(s)
Infección Hospitalaria , Mucosa Bucal , Humanos , Animales , Femenino , Pollos , Daño del ADN , Piel , Rayos UltravioletaRESUMEN
A synthetic route for adhesive core-multishell (CMS) nanocarriers for application to the oral mucosa was established using mussel-inspired catechol moieties. The three CMS nanocarriers with 8%, 13%, and 20% catechol functionalization were evaluated for loading capacity using Nile red, showing an overall loading of 1 wt%. The ability of Nile red loaded and functionalized nanocarriers to bind to a moist mucosal surface was tested in two complementary adhesion assays under static and dynamic conditions using monolayers of differentiated gingival keratinocytes. Adhesion properties of functionalized nanocarriers were compared to the adhesion of the non-functionalized nanocarrier. In both assays, the CMS nanocarrier functionalized with 8% catechol exhibited the strongest adhesion compared to its catechol-free counterpart and the CMS nanocarriers functionalized with 13% and 20% catechol.
RESUMEN
OBJECTIVE: New alkylphospholipids (APLs) that are structurally derived from the platelet-activating factor (PAF) are promising candidates for anticancer treatment. After incorporation into cell membranes, APLs are able to interfere with a wide variety of key enzymes implicated in cell growth, motility, invasion, and apoptosis. In addition to the prototype 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (edelfosine), we presented a novel group of APLs, the glycosidated phospholipids that efficiently inhibit cell proliferation. Two members of this group, Ino-C2-PAF and Glc-PAF, display high efficacy and low cytotoxicity in immortalized nontumorigenic skin keratinocyte cell line, HaCaT. This study investigated the impact of APLs on the transcription of the whole genome. MATERIALS AND METHODS: Using Agilent complementary DNA microarray technology, we compared global gene expression profiles of HaCaT cells treated with edelfosine, Ino-C2-PAF, or Glc-PAF with the profile of control cells. RESULTS: We found that Ino-C2-PAF has the strongest influence on gene expression in comparison with edelfosine and Glc-PAF. Gene Ontology analysis showed that differentially expressed transcripts regulated by the three APLs are mainly implicated in lipid metabolism, lipid biosynthesis, cell differentiation, cell development, and ion homeostasis. Nevertheless, the most remarkable finding is represented by the ability of Ino-C2-PAF to downregulate a broad spectrum of genes associated with the regulation of the innate and acquired immune response and of genes linked to inflammation. CONCLUSION: These results identify Ino-C2-PAF as the most effective APL used in this study. Therefore, Ino-C2-PAF might be a promising compound for further studies that concentrate on the inhibition of inflammatory responses.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias/genética , Factor de Activación Plaquetaria/análogos & derivados , Factor de Activación Plaquetaria/farmacología , Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Membrana Celular , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Perfilación de la Expresión Génica , Humanos , Inmunidad Innata/efectos de los fármacos , Queratinocitos/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , Éteres Fosfolípidos/farmacología , Transcripción Genética/efectos de los fármacosRESUMEN
Apoptosis is modulated by extrinsic and intrinsic signaling pathways through the formation of the death receptor-mediated death-inducing signaling complex (DISC) and the mitochondrial-derived apoptosome, respectively. Ino-C2-PAF, a novel synthetic phospholipid shows impressive antiproliferative and apoptosis-inducing activity. Little is known about the signaling pathway through which it stimulates apoptosis. Here, we show that this drug induces apoptosis through proteins of the death receptor pathway, which leads to an activation of the intrinsic apoptotic pathway. Apoptosis induced by Ino-C2-PAF and its glucosidated derivate, Glc-PAF, was dependent on the DISC components FADD and caspase-8. This can be inhibited in FADD--/-- and caspase-8--/-- cells, in which the breakdown of the mitochondrial membrane potential, release of cytochrome c and activation of caspase-9, -8 and -3 do not occur. In addition, the overexpression of crmA, c-Flip or dominant negative FADD as well as treatment with the caspase-8 inhibitor z-IETD-fmk protected against Ino-C2-PAF-induced apoptosis. Apoptosis proceeds in the absence of CD95/Fas-ligand expression and is independent of blockade of a putative death-ligand/receptor interaction. Furthermore, apoptosis cannot be inhibited in CD95/Fas--/-- Jurkat cells. Expression of Bcl-2 in either the mitochondria or the endoplasmic reticulum (ER) strongly inhibited Ino-C2-PAF- and Glc-PAF-induced apoptosis. In conclusion, Ino-C2-PAF and Glc-PAF trigger a CD95/Fas ligand- and receptor-independent atypical DISC that relies on the intrinsic apoptotic pathway via the ER and the mitochondria.
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Apoptosis/efectos de los fármacos , Caspasa 8/metabolismo , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Mitocondrias/metabolismo , Neoplasias/fisiopatología , Fosfolípidos/farmacología , Transducción de Señal , Caspasa 8/genética , Activación Enzimática/efectos de los fármacos , Proteína de Dominio de Muerte Asociada a Fas/genética , Glicosilación , Humanos , Células Jurkat , Mitocondrias/genética , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Fosfolípidos/síntesis química , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
Periodontitis is one of the most common oral diseases worldwide and is caused by a variety of interactions between oral bacteria and the host. Here, pathogens induce inflammatory host responses that cause the secretion of proinflammatory cytokines such as IL-1ß, IL-6, and IL-8 by oral epithelial cells. In various systems, it has been shown that inflammation compromises physical barriers, which enables bacteria to invade the tissue. In this study, we investigated the barrier properties of the oral mucosa under physiological and inflamed conditions. For this purpose, we assessed the influence of IL-1ß on the transepithelial electrical resistance and in particular on tight junctions in vitro in human stratified squamous epithelium models. Indirect immunofluorescence and western blot analyses were performed to investigate localization and expression of tight junction proteins in primary gingival cells, immortalized gingival cells and native gingiva. Furthermore, the TEER of gingival keratinocytes was assessed. The results showed that IL-1ß led to strengthening of the gingival keratinocyte barrier. This was demonstrated by an increase in TEER, the upregulation of TJ proteins, and an increase in the formation of TJ strands. The IL-1ß-mediated upregulation of occludin was prevented by the NF-κB inhibitor BAY 11-7085. These observations provide insights into host responses in the early stages of periodontal disease and offer information about TJ formation in human gingival epithelial cells under physiological and inflammatory conditions. Comprehensive knowledge of the physical barrier during inflammation may help in developing strategies to effectively target the inflammatory barrier to improve the bioavailability of drugs for the treatment of periodontitis.
Asunto(s)
Encía/metabolismo , Interleucina-1beta/farmacología , Queratinocitos/efectos de los fármacos , Uniones Estrechas/metabolismo , Línea Celular , Encía/citología , Humanos , Queratinocitos/metabolismo , FN-kappa B/metabolismo , Ocludina/metabolismoRESUMEN
PURPOSE: Previous studies have identified alkyl-phospholipids as promising compounds for cancer therapy by targeting constituents of the cell membrane and different signaling pathways. We previously showed that the alkylphospholipid Inositol-C2-PAF inhibits the proliferation and migration of immortalized keratinocytes and the squamous carcinoma-derived cell line SCC-25. Here, we investigated the effect of this compound on growth and motility as well as its mode of action in mammary carcinoma-derived cell lines. METHODS: Using BrdU incorporation and haptotactic cell migration assays, we assessed the effects of Inositol-C2-PAF on MCF-7 and MBA-MB-231 cell proliferation and migration. The phosphorylation status of signaling molecules was investigated by Western blotting as well as indirect immunofluorescence analysis and capillary isoelectric focusing. RESULTS: We found that Inositol-C2-PAF inhibited the growth as well as the migration in MCF-7 and MBA-MB-231 cells. Furthermore, we found that this compound inhibited phosphorylation of the protein kinase Akt at serine residue 473, but had no impact on phosphorylation at threonine 308. Phosphorylation of other kinases, such as Erk1/2, FAK and Src, which are targeted by Inositol-C2-PAF in other cells, remained unaffected by the compound in the mammary carcinoma-derived cell lines tested. In MCF-7 cells, we found that IGF-1-induced growth, as well as phosphorylation of AktS473, mTOR and the tumor suppressor pRB, was inhibited in the presence of Inositol-C2-PAF. Moreover, we found that in these cells IGF-1 had no impact on migration and did not seem to be linked to full Akt activity. Therefore, MCF-7 cell migration appears to be inhibited by Ino-C2-PAF in an Akt-independent manner. CONCLUSION: The antagonistic effects of Inositol-C2-PAF on cell migration and proliferation are indicative for its potential for breast cancer therapy, alone or in combination with other cytostatic drugs.
Asunto(s)
Neoplasias de la Mama/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Inositol/análogos & derivados , Factor de Activación Plaquetaria/análogos & derivados , Antineoplásicos/farmacología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Femenino , Humanos , Inositol/farmacología , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Células MCF-7 , Factor de Activación Plaquetaria/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
The blood-brain barrier (BBB) provides a dynamic and complex interface consisting of endothelial cells, pericytes and astrocytes, which are embedded in a collagen and fibronectin-rich basement membrane. This complex structure restricts the diffusion of small hydrophilic solutes and macromolecules as well as the transmigration of leukocytes into the brain. It has been shown that carbonyl stress followed by the formation of advanced glycation endproducts (AGE=glycation) interfere with the BBB integrity and function. Here, we present data that carbonyl stress induced by methylglyoxal leads to glycation of endothelial cells and the basement membrane, which interferes with the barrier-function and with the expression of RAGE, occludin and ZO-1. Furthermore, methylglyoxal induced carbonyl stress promotes the expression of the pro-inflammatory interleukins IL-6 and IL-8. In summary, this study provides new insights into the relationship between AGE formation by carbonyl stress and brain microvascular endothelial barrier dysfunction.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Células Endoteliales/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Antígenos de Neoplasias/metabolismo , Barrera Hematoencefálica/patología , Células Cultivadas , Células Endoteliales/patología , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Ocludina/metabolismo , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
Two new analogues derived from the platelet activating factor (PAF), containing glucosamine instead of the acetyl group, were synthesized, and their effect on the human keratinocyte cell line HaCaT was evaluated with respect to cytotoxicity, proliferation, adhesion, and migration. Starting with (R)-1,2-isopropylideneglycerol (3), the glycosylation acceptor 1-O-octadecyl-3-O-tert-butyldimethylsilyl-sn-glycerol (6) was synthesized in three steps. Glycosylation of 6 with the already known O-(3,4,6-tri-O-acetyl-2-deoxy-2-dimethylmaleimido-beta-D-glycopyranosyl)trichloracetimidate gave 1-O-octadecyl-2-O-(3',4',6'-tri-O-acetyl-2'-deoxy-2'-dimethylmaleimido-beta-D-glucopyranosyl)-3-O-tert-butyldimethylsilyl-sn-glycerol (7). After removing the (tert-butyldimethyl)silyl (TBDMS) group with FeCl3x6H2O, phosphoryl choline was introduced, yielding [1-O-octadecyl-2-O-(2'-deoxy-2'-dimethylmaleimido-beta-D-glucopyranosyl)-sn-glycero(3)]phosphorylcholine (2) (glucosimide-PAF). pH controlled cleavage of the amino protection group gave [1-O-octadecyl-2-O-(2'-deoxy-2'-amino-beta-D-glucopyranosyl)-sn-glycero(3)]phosphorylcholine hydrochloride (1) (glucosamine-PAF). 2 inhibited proliferation of HaCaT cells by 26% at nontoxic concentrations, while 1 increased the proliferation rate by 30% at low concentrations. At higher concentrations, both compounds showed cytotoxic properties with LD50 = 30 micromol/L (1) and LD50 = 5-6 micromol/L (2). Both 1 and 2 were potent promoters of cell adhesion and migration of HaCaT cells.
Asunto(s)
Antineoplásicos/síntesis química , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Glucosamina/análogos & derivados , Glucosamina/síntesis química , Glicerofosfolípidos/síntesis química , Glucolípidos/síntesis química , Antineoplásicos/farmacología , Antineoplásicos/toxicidad , Línea Celular , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Glucosamina/farmacología , Glucosamina/toxicidad , Glicerofosfolípidos/farmacología , Glicerofosfolípidos/toxicidad , Glucolípidos/farmacología , Glucolípidos/toxicidad , Humanos , Relación Estructura-ActividadRESUMEN
Sialylation of glycoproteins and glycolipids plays an important role during development, regeneration and pathogenesis of several diseases. The physiological precursor of all sialic acids is N-acetyl- D-mannosamine. The N-acyl side chain of sialic acid can be modified by exposure of cells to synthetic N-acyl-modified D-mannosamines. In a new experimental approach cells were cultivated in the presence of N-propanoyl- D-mannosamine. This unnatural precursor of sialic acid is taken up by cells and efficiently metabolized to the respective N-acyl-modified neuraminic acid in vitro and in vivo. Here we report on the biological consequences of the incorporation of the unnatural N-propanoylneuraminic acid into glycoconjugates of HL60 cells. Biochemical engineering of the acyl side chain of neuraminic acids activates beta(1)-integrins (VLA4 or VLA5), resulting in an increased adhesion of HL60 cells to fibronectin.
Asunto(s)
Adhesión Celular/efectos de los fármacos , Fibronectinas/metabolismo , Integrina beta1/metabolismo , Ácidos Siálicos/farmacología , Western Blotting , Adhesión Celular/fisiología , Técnicas de Cultivo de Célula , Glicoproteínas/metabolismo , Células HL-60 , Humanos , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/farmacologíaRESUMEN
Sialylation of glycoconjugates is essential for mammalian cells. Sialic acid is synthesized in the cytosol from N-acetylmannosamine by several consecutive steps. Using N-propanoylmannosamine, a novel precursor of sialic acid, we are able to incorporate unnatural sialic acids with a prolonged N-acyl side chain (e.g., N-propanoylneuraminic acid) into glycoconjugates taking advance of the cellular sialylation machinery. Here, we report that unnatural sialylation of HL60-cells leads to an increased release of intracellular calcium after application of thapsigargin, an inhibitor of SERCA Ca2+-ATPases. Furthermore, this increased intracellular calcium concentration leads to an increased adhesion to fibronectin. Finally, we observed an increase of the lectin galectin-3, a marker of monocytic differentiation of HL60-cells.