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The ABCG2 transporter protein, as part of several known mechanisms involved in multidrug resistance, has the ability to transport a broad spectrum of substrates out of the cell and is, therefore, considered as a potential target to improve cancer therapies or as an approach to combat drug resistance in cancer. We have previously reported carborane-functionalized quinazoline derivatives as potent inhibitors of human ABCG2 which effectively reversed breast cancer resistance protein (BCRP)-mediated mitoxantrone resistance. In this work, we present the evaluation of our most promising carboranyl BCRP inhibitors regarding their toxicity towards ABCG2-expressing cancer cell lines (MCF-7, doxorubicin-resistant MCF-7 or MCF-7 Doxo, HT29, and SW480) and, consequently, with the co-administration of an inhibitor and therapeutic agent, their ability to increase the efficacy of therapeutics with the successful inhibition of ABCG2. The results obtained revealed synergistic effects of several inhibitors in combination with doxorubicin or cisplatin. Compounds DMQCa, DMQCc, and DMQCd showed a decrease in IC50 value in ABCB1- and ABCG2-expressing SW480 cells, suggesting a possible targeting of both transporters. In an HT29 cell line, with the highest expression of ABCG2 among the tested cell lines, using co-treatment of doxorubicin and DMQCd, the effective inhibitory concentration of the antineoplastic agent could be reduced by half. Interestingly, co-treatment of compound QCe with cisplatin, which is not an ABCG2 substrate, showed synergistic effects in MCF-7 Doxo and HT29 cells (IC50 values halved or reduced by 20%, respectively). However, a literature-known upregulation of cisplatin-effluxing ABC transporters and their effective inhibition by the carborane derivatives emerges as a possible reason.
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The presence of inflammatory mediators in the tumor microenvironment, such as cytokines, growth factors or eicosanoids, indicate cancer-related inflammatory processes. Targeting these inflammatory mediators and related signal pathways may offer a rational strategy for the treatment of cancer. This study focuses on the incorporation of metabolically stable, sterically demanding, and hydrophobic dicarba-closo-dodecaboranes (carboranes) into dual cyclooxygenase-2 (COX-2)/5-lipoxygenase (5-LO) inhibitors that are key enzymes in the biosynthesis of eicosanoids. The di-tert-butylphenol derivative tebufelone represents a selective dual COX-2/5-LO inhibitor. The incorporation of meta- or para-carborane into the tebufelone scaffold resulted in eight carborane-based tebufelone analogs that show no COX inhibition but 5-LO inhibitory activity inâ vitro. Cell viability studies on HT29 colon adenocarcinoma cells revealed that the observed antiproliferative effect of the para-carborane analogs of tebufelone is enhanced by structural modifications that include chain elongation in combination with introduction of a methylene spacer resulting in higher anticancer activity compared to tebufelone. Hence, this strategy proved to be a promising approach to design potent 5-LO inhibitors with potential application as cytostatic agents.
Asunto(s)
Adenocarcinoma , Boranos , Neoplasias del Colon , Humanos , Ciclooxigenasa 2/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Inhibidores de la Ciclooxigenasa/química , Microambiente TumoralRESUMEN
The ineffectiveness and failing of chemotherapeutic treatments are often associated with multidrug resistance (MDR). MDR is primarily linked to the overexpression of ATP-binding cassette (ABC) transporter proteins in cancer cells. ABCG2 (ATP-binding cassette subfamily G member 2, also known as the breast cancer resistance protein (BCRP)) mediates MDR by an increased drug efflux from the cancer cells. Therefore, the inhibition of ABCG2 activity during chemotherapy ought to improve the efficacy of the administered anti-cancer agents by reversing MDR or by enhancing the agents' pharmacokinetic properties. Significant efforts have been made to develop novel, powerful, selective, and non-toxic inhibitors of BCRP. However, thus far the clinical relevance of BCRP-selective MDR-reversal has been unsuccessful, due to either adverse drug reactions or significant toxicities in vivo. We here report a facile access towards carboranyl quinazoline-based inhibitors of ABCG2. We determined the influence of different methoxy-substitution patterns on the 2-phenylquinazoline scaffold in combination with the beneficial properties of an incorporated inorganic carborane moiety. A series of eight compounds was synthesized and their inhibitory effect on the ABCG2-mediated Hoechst transport was evaluated. Molecular docking studies were performed to better understand the structure-protein interactions of the novel inhibitors, exhibiting putative binding modes within the inner binding site. Further, the most potent, non-toxic compounds were investigated for their potential to reverse ABCG2-mediated mitoxantrone (MXN) resistance. Of these five evaluated compounds, N-(closo-1,7-dicarbadodecaboran(12)-9-yl)-6,7-dimethoxy-2-(3,4,5-trimethoxyphenyl)-quinazolin-4-amine (DMQCd) exhibited the strongest inhibitory effect towards ABCG2 in the lower nanomolar ranges. Additionally, DMQCd was able to reverse BCRP-mediated MDR, making it a promising candidate for further research on hybrid inorganic-organic compounds.
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Lipoxygenases convert polyunsaturated fatty acids into biologically active metabolites such as inflammatory mediators-prostaglandins and leukotrienes. The inhibition of lipoxygenases is increasingly employed in the treatment of cancer. We evaluated the anticancer potential of two novel 5-lipoxygenase inhibitors, named CarbZDNaph and CarbZDChin, which are analogues of the commercially available inhibitor Rev-5901. The in vitro segment of this study was conducted on a mouse colorectal carcinoma cell line-CT26CL25. For an in vivo model, we induced tumors in BALB/c mice by the implantation of CT26CL25 cells, and we treated the animals with potential inhibitors. A 48 h treatment resulted in diminished cell viability. Calculated IC50 values (half-maximal inhibitory concentrations) were 25 µM, 15 µM and 30 µM for CarbZDNaph, CarbZDChin and Rev-5901, respectively. The detailed analysis of mechanism revealed an induction of caspase-dependent apoptosis and autophagy. In the presence of chloroquine, an autophagy inhibitor, we observed an increased mortality of cells, implying a cytoprotective role of autophagy. Our in vivo experiment reports tumor growth attenuation in animals treated with CarbZDChin. Compounds CarbZDNaph and Rev-5901 lacked an in vivo efficacy. The results presented in this study display a strong effect of compound CarbZDChin on malignant cell growth. Having in mind the important role of inflammation in cancer development, these results have a significant impact and are worthy of further evaluation.
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Boranos , Carcinoma , Neoplasias del Colon , Animales , Apoptosis , Autofagia , Boranos/farmacología , Caspasa 3/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Lipooxigenasas , Ratones , Ratones Endogámicos BALB CRESUMEN
12-Lipoxygenase is crucial for tumour angiogenesis. 5,6,7-Trihydroxy-2-phenyl-4H-1-benzopyran-4-one (baicalein) is a suitable inhibitor for this enzyme but is rapidly metabolised inâ vivo. Thus, an improvement of the metabolic stability is necessary to enhance the therapeutic efficiency. An emerging approach to enhance metabolic stability of carbon-based pharmaceuticals is the use of metabolically stable, non-toxic boron clusters, such as dicarba-closo-dodecaborane(12)s (carboranes) as phenyl mimetics. Therefore, the unsubstituted phenyl ring of baicalein was replaced by meta-carborane, resulting in borcalein, the carborane analogue of baicalein. This substitution resulted in a decreased inhibitory activity toward 12-lipoxygenase, but led to increased toxicity in melanoma (A375, B16, B16F10) and colon cancer cell lines (SW480, HCT116, CT26CL25) with decreased tumour selectivity in comparison to baicalein. Surprisingly, borcalein displays a different mechanism of cytotoxicity with increased intracellular production of reactive oxygen species (ROS), reactive nitrogen species (RNS) and nitric oxide (NO).
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Antineoplásicos/farmacología , Araquidonato 12-Lipooxigenasa/metabolismo , Inhibidores Enzimáticos/farmacología , Flavanonas/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Flavanonas/síntesis química , Flavanonas/química , Humanos , Ratones , Estructura Molecular , Óxido Nítrico/metabolismo , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Owing to the involvement of cyclooxygenase-2 (COX-2) in carcinogenesis, COX-2-selective inhibitors are increasingly studied for their potential cytotoxic properties. Moreover, the incorporation of carboranes in structures of established anti-inflammatory drugs can improve the potency and metabolic stability of the inhibitors. Herein, we report the synthesis of carborane-containing derivatives of rofecoxib that display remarkable cytotoxic or cytostatic activity in the micromolar range with excellent selectivity for melanoma and colon cancer cell lines over normal cells. Furthermore, it was shown that the carborane-modified derivatives of rofecoxib showed different modes of action that were dependent on the cell type.
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Antineoplásicos , Compuestos de Boro/química , Neoplasias del Colon/etiología , Neoplasias del Colon/patología , Inhibidores de la Ciclooxigenasa 2/síntesis química , Inhibidores de la Ciclooxigenasa 2/farmacología , Ciclooxigenasa 2/fisiología , Lactonas/síntesis química , Lactonas/farmacología , Melanoma/etiología , Melanoma/patología , Sulfonas/síntesis química , Sulfonas/farmacología , Carcinogénesis/genética , Línea Celular Tumoral , Diseño de Fármacos , Humanos , Interacciones Hidrofóbicas e HidrofílicasRESUMEN
Ketoprofen is a widely used nonsteroidal anti-inflammatory drug (NSAID) that also exhibits cytotoxic activity against various cancers. This makes ketoprofen an attractive structural lead for the development of new NSAIDs and cytotoxic agents. Recently, the incorporation of carboranes as phenyl mimetics in structures of established drugs has emerged as an attractive strategy in drug design. Herein, we report the synthesis and evaluation of four novel carborane-containing derivatives of ketoprofen, two of which are prodrug esters with an nitric oxide-releasing moiety. One of these prodrug esters exhibited high cytostatic activity against melanoma and colon cancer cell lines. The most pronounced activity was found in cell lines that are sensitive to oxidative stress, which was apparently induced by the ketoprofen analogue.
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The main focus of this study is exploring the effect and mechanism of two HIV-protease inhibitors: Ritonavir and Ritonavir-nitric oxide (Ritonavir-NO) on in vitro growth of melanoma cell lines. NO modification significantly improved the antitumor potential of Ritonavir, as the IC50 values of Ritonavir-NO were approximately two times lower than IC50 values of the parental compound. Our results showed for the first time, that both compounds induced senescence in primary and metastatic melanoma cell lines. This transformation was manifested as a change in cell morphology, enlargement of nuclei, increased cellular granulation, upregulation of ß-galactosidase activity, lipofuscin granules appearance, higher production of reactive oxygen species and persistent inhibition of proliferation. The expression of p53, as one of the key regulators of senescence, was upregulated after 48 hours of Ritonavir-NO treatment only in metastatic B16F10 cells, ranking it as a late-response event. The development of senescent phenotype was consistent with the alteration of the cytoskeleton-as we observed diminished expression of vinculin, α-actin, and ß-tubulin. Permanent inhibition of S6 protein by Ritonavir-NO, but not Ritonavir, could be responsible for a stronger antiproliferative potential of the NO-modified compound. Taken together, induction of senescent phenotype may provide an excellent platform for developing therapeutic approaches based on selective killing of senescent cells.
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Proliferación Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Inhibidores de la Proteasa del VIH/farmacología , Melanoma/tratamiento farmacológico , Ritonavir/farmacología , Actinas/biosíntesis , Línea Celular Tumoral , Humanos , Lipofuscina/metabolismo , Melanoma/patología , Especies Reactivas de Oxígeno/metabolismo , Proteínas Quinasas S6 Ribosómicas/antagonistas & inhibidores , Tubulina (Proteína)/biosíntesis , Proteína p53 Supresora de Tumor/biosíntesis , Vinculina/biosíntesis , beta-Galactosidasa/metabolismoRESUMEN
We generated a nitric oxide (NO)-releasing derivative of the anti-HIV protease inhibitor lopinavir by linking the NO moiety to the parental drug. We investigated the effects of lopinavir and its derivative lopinavir-NO on melanoma cell lines in vitro and in vivo. Lopinavir-NO exhibited a twofold stronger anticancer action than lopinavir in vitro. These results were successfully translated into syngeneic models of melanoma in vivo, where a significant reduction in tumour volume was observed only in animals treated with lopinavir-NO. Both lopinavir and lopinavir-NO inhibited cell proliferation and induced the trans-differentiation of melanoma cells to Schwann-like cells. In melanoma cancer cell lines, both lopinavir and lopinavir-NO induced morphological changes, minor apoptosis and reactive oxygen species (ROS) production. However, caspase activation and autophagy were detected only in B16 cells, indicating a cell line-specific treatment response. Lopinavir-NO released NO intracellularly, and NO neutralization restored cell viability. Treatment with lopinavir-NO induced only a transient activation of Akt and inhibition of P70S6 kinase. The results of this study identify lopinavir-NO as a promising candidate for further clinical trials in melanoma and possibly other solid tumours.
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Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Inhibidores de la Proteasa del VIH/farmacología , Lopinavir/farmacología , Melanoma/tratamiento farmacológico , Óxido Nítrico/metabolismo , Animales , Autofagia , Hipersensibilidad a las Drogas , Femenino , Humanos , Técnicas In Vitro , Melanoma/metabolismo , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Especies Reactivas de Oxígeno/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most common way of treating inflammatory disorders. Their widespread use helped reveal their other modes of action as pharmaceuticals, such as a profound effect on various cancers. Celecoxib has proven to be a very prominent member of this group with cytostatic activities. On the other hand, the highly dynamic field of drug design is constantly searching for new ways of modifying known structures to obtain more powerful and less harmful drugs. A very interesting development is the implementation of carboranes in pharmacologically active structures, mostly as phenyl mimetics. Herein we report the synthesis of three carborane-containing derivatives of the COX-2-selective NSAID celecoxib. The new compounds proved to have promising cytostatic potential against various melanoma and colorectal adenocarcinoma cell lines. Inhibited proliferation accompanied by caspase-independent apoptotic cell death was found to be the main cause of decreased cell viability upon treatment with the most efficient celecoxib analogue, 3 b (4-[5-(1,7-dicarba-closo-dodecaboranyl)-3-trifluoromethyl-1H-pyrazol-1-yl]-1-methylsulfonylbenzene).
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Antineoplásicos/farmacología , Celecoxib/farmacología , Inhibidores de la Ciclooxigenasa 2/farmacología , Citostáticos/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Celecoxib/síntesis química , Celecoxib/química , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa 2/síntesis química , Inhibidores de la Ciclooxigenasa 2/química , Citostáticos/síntesis química , Citostáticos/química , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Melanoma/tratamiento farmacológico , Melanoma/patología , Ratones , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Cytological analysis of fine-needle aspiration (FNA) is the first step in evaluation of patients with nodular thyroid disease with the primary goal to exclude thyroid malignancy. Its improvement by combining cytology with molecular markers is still a matter of investigation. In this study, 2 molecular markers were used: caveolin-1 and epidermal growth factor receptor (EGFR), along with the well-established genetic marker BRAF V600E mutation. We set out to determine the expression signatures of EGFR and caveolin-1 in patients with thyroid malignancy as well as to determine their possible association with disease severity. In FNA biopsy samples (n=186), immunocytochemical expression of caveolin-1 and BRAF V600E mutation coincided with malignancy. The patients were sorted according to 3 parameters: final histopathological diagnosis, caveolin-1 expression, and BRAF V600E mutation status before measurement of EGFR mRNA expression. EGFR upregulation was detected in the group of patients with malignant diagnosis, no caveolin-1 expression, and wild-type BRAF. Spearman rank correlation yielded a statistically significant negative correlation of EGFR and caveolin-1. Double immunofluorescence confirmed colocalization and inverse expression of EGFR and caveolin-1. Our data demonstrated that EGFR overexpression is associated with malignancy but not with tumor aggressiveness. Furthermore, high-caveolin-1/low-EGFR cases were associated with an advanced pT status and had a greater degree of neoplastic infiltration than low-caveolin-1/high-EGFR cases. Combining caveolin-1 and BRAF V600E with EGFR might help in recognizing more aggressive thyroid lesions in a pool of relatively indolent tumors in FNA biopsies and thus be useful for early risk stratification of thyroid cancer patients.
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Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Caveolina 1/análisis , Receptores ErbB/genética , Neoplasias de la Tiroides/química , Neoplasias de la Tiroides/genética , Biopsia con Aguja Fina , Análisis Mutacional de ADN , Receptores ErbB/análisis , Técnica del Anticuerpo Fluorescente , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Mutación , Estadificación de Neoplasias , Fenotipo , Valor Predictivo de las Pruebas , Proteínas Proto-Oncogénicas B-raf/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias de la Tiroides/patologíaRESUMEN
BACKGROUND: Fine-needle aspiration (FNA) has been employed for many years for examining thyroid nodules, and the cytology of aspirates is the primary determinant for whether thyroidectomy is indicated. Fifteen to thirty percent of thyroid nodules, not being clearly benign or malignant, fall into an indeterminate category. The main goals of molecular diagnostics for thyroid nodules are to prevent unnecessary surgery in patients with benign nodules and to stop patients with malignant nodules from being subjected to repeated operations. METHODS: This study was designed to evaluate the diagnostic utility of 4 markers in thyroid FNA cytology via testing for the BRAF V600E mutation and the expression of microRNA-221, microRNA-222, and galectin-3 protein in FNA samples with indeterminate cytology. RESULTS: A predictor model distinguishing benign samples from malignant samples on the basis of the 4 aforementioned markers was formulated. This decision model provided a sensitivity of 73.5%, a specificity of 89.8%, and a diagnostic accuracy of 75.7%. The positive predictive value was 80.6%, and the negative predictive value was 85.5%; this suggested that the prediction had good reliability. CONCLUSIONS: One hundred twenty FNA samples were examined, and 62 nodules were classified as benign with the proposed diagnostic algorithm. This resulted in a reduction of the initial 120 patients to 58 and thus decreased by half the number of persons undergoing surgery.
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Biomarcadores de Tumor/metabolismo , Transformación Celular Neoplásica/patología , Galectina 3/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Neoplasias de la Tiroides/patología , Nódulo Tiroideo/patología , Adulto , Área Bajo la Curva , Biopsia con Aguja Fina/métodos , Citodiagnóstico/métodos , Diagnóstico Diferencial , Femenino , Galectina 3/genética , Humanos , Inmunohistoquímica , Masculino , MicroARNs/genética , Persona de Mediana Edad , Proteínas Proto-Oncogénicas B-raf/genética , Medición de Riesgo , Sensibilidad y Especificidad , Neoplasias de la Tiroides/genética , Nódulo Tiroideo/genéticaRESUMEN
Thyroid cancer comprises a heterogeneous group of lesions with great diversity of biological behaviour. Markers which could help clinicians to identify high-risk patients for tailored optimization of clinical management are of crucial importance. HBME-1 protein level was analysed immunohistochemically using routinely prepared archival tissue sections of a broad range of papillary thyroid carcinoma (PTC) variants and in corresponding lymph node metastases (LNM). The results were evaluated in comparison with clinicopathological features of PTC. Positive immunoreaction was noticed in most classical (83/92; 90.2 %), follicular (60/71; 84.5 %) and trabecular (4/5; 80.0 %) variants of PTC. All cases of macrofollicular, Warthin-like and diffuse sclerosing PTC variants were HBME-1 positive (4/4, 3/3, 2/2; 100 % respectively). Tall cell and solid PTC variants showed diversity of staining (2/3; 66.67 % and 13/23; 56.52 % respectively), while PTCs with mixed histological pattern containing insular areas were mainly weakly positive (2/5; 40.0 %). A single case of clear cell PTC variant showed no reaction. Moreover, all matched metastatic PTC into lymph nodes (LNM) were HBME-1 positive (17/17; 100 %) and expressed HBME-1 in a similar pattern to the matched primary tumour. We also found a statistically significant association between high HBME-1 expression and the presence of lymph node metastasis, advanced pT status and pTNM stage (P < 0.05), but only a tendency for association with extrathyroidal invasion of the tumour (P = 0.058). Therefore, we recommend using immunoexpression of HBME-1 as useful mean to increase the likelihood of detecting most PTC variants and to predict some unfavourable clinical parameters in these patients.
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Biomarcadores de Tumor/metabolismo , Carcinoma Papilar/metabolismo , Carcinoma Papilar/secundario , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patología , Adolescente , Adulto , Anciano , Niño , Femenino , Estudios de Seguimiento , Humanos , Técnicas para Inmunoenzimas , Metástasis Linfática , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Factores de Riesgo , Adulto JovenRESUMEN
AIM: To determine whether matrix metalloproteinase-9 (MMP-9) may be a useful adjunctive tool for predicting unfavorable biological behavior of papillary thyroid carcinoma (PTC) by evaluating the expression profile and proteolytic activity of MMP-9 in PTC by different techniques and correlating the findings with clinicopathological prognostic factors. METHODS: Immunohistochemical localization of MMP-9 was analyzed with antibodies specific for either total or active MMP-9. Activation ratios of MMP-9 were calculated by quantifying gel zymography bands. Enzymatic activity of MMP-9 was localized by in situ zymography after inhibiting MMP-2 activity. RESULTS: Immunostaining of total and active MMP-9 was observed in tumor tissue and occasionally in non-neoplastic epithelium. Only active MMP-9 was significantly associated with extrathyroid invasion, lymph-node metastasis, and the degree of tumor infiltration (P<0.001, P=0.004, and P<0.001, respectively). Gelatin zymography revealed a correlation between the MMP-9 activation ratio and nodal involvement, extrathyroid invasion, and the degree of tumor infiltration. In situ zymography showed that gelatinases exerted their activity in tumor parenchymal and stromal cells. Moreover, after application of MMP-2 inhibitor, the remaining gelatinase activity, corresponding to MMP-9, was highest in cancers with the most advanced degree of tumor infiltration. CONCLUSIONS: This is the first report suggesting that the evaluation of active MMP-9 by immunohistochemistry and determination of its activation ratio by gelatin zymography may be a useful adjunct to the known clinicopathological factors in predicting tumor behavior. Most important, in situ zimography with an MMP-2 inhibitor for the first time demonstrated a strong impact of MMP-9 activity on the degree of tumor infiltration during PTC progression.
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Carcinoma/enzimología , Carcinoma/patología , Metaloproteinasa 9 de la Matriz/metabolismo , Neoplasias de la Tiroides/enzimología , Neoplasias de la Tiroides/patología , Adulto , Carcinoma Papilar , Activación Enzimática , Femenino , Humanos , Inmunohistoquímica , Metástasis Linfática , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Cáncer Papilar TiroideoRESUMEN
OBJECTIVE: We aimed to investigate the role of caveolin-1 in papillary thyroid carcinoma pathogenesis. STUDY DESIGN: Case series with chart review. SETTING: Institute for the Application of Nuclear Energy. SUBJECTS AND METHODS: We evaluated the expression of caveolin-1 in papillary thyroid carcinoma (PTC) by Western blot (WB) and compared the findings with immunohistochemical (IHC) expression of both epithelial and stromal caveolin-1 on the corresponding histological specimens. The results were related to clinicopathological features and BRAF mutation status. RESULTS: Caveolin-1 expression was found in malignant thyroid epithelium and more abundantly in tumor stroma but varied in both compartments within and between PTC subtypes. Caveolin-1 expression in the epithelium was more intense in classical PTC than in the other histological types. On the contrary, stromal caveolin-1 expression was stronger in the follicular, solid, and trabecular PTC variants than in classical PTC. Trends for down-regulation of caveolin-1 expression in epithelium and up-regulation in stroma from the classical via follicular to the solid variant were observed. The relation of WB and IHC results with clinicopathological parameters showed lower caveolin-1 tissue content in BRAF mutated tumors (P < .05), a positive correlation of epithelial caveolin-1 expression with lymph node metastasis (P < .05), and a negative association of stromal caveolin-1 expression with the degree of neoplastic infiltration and BRAF status. CONCLUSION: Altered expression of caveolin-1 in the thyroid epithelial and stromal compartments may be involved in the pathogenesis of PTC. The potential clinical significance of caveolin-1 expression, as well as its relation to BRAF mutation status, deserves further investigation.
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Carcinoma Papilar/metabolismo , Carcinoma/metabolismo , Caveolina 1/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Neoplasias de la Tiroides/metabolismo , Adulto , Western Blotting , Carcinoma/genética , Carcinoma/patología , Carcinoma Papilar/genética , Carcinoma Papilar/patología , Epitelio/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Mutación , Cáncer Papilar Tiroideo , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patologíaRESUMEN
We evaluated caveolin-1 expression in the human thyroid neoplasia spectrum with the aim of examining differences in expression as detected by two anti-caveolin-1 antibodies, and secondly, to investigate the association of caveolin-1 expression levels with aggressive papillary thyroid carcinoma (PTC). Immunohistochemical staining using sc894 or AV09019 antibodies revealed that caveolin-1 was generally overexpressed in the PTC group as a whole (classical and follicular variant) when compared to peritumoral tissue (PT), while it was not detected in about half of follicular thyroid carcinoma (FTC) and majority of follicular adenomas (FTA). Caveolin-1 expression decreased in the following order: clPTC, fvPTC, FTC, PT and FTA. The diagnostic accuracy of AV09019 was better than that of sc894 for discriminating: FTA from FTC, FTA or FTC from the follicular variant of PTC, total PTC from nonmalignant tissue, and malignant tumors from nonmalignant tissue. Spearman's analysis revealed positive correlations of caveolin-1 expression and extrathyroidal invasion (p< 0.05) in PTC for both antibodies. Additionally, AV09019 antibody correlated caveolin-1 upregulation with pathological T status. To conclude, as an immunohistochemical marker AV09019 antibody performed better than sc894 in distinguishing certain histotypes of thyroid tumors. In addition, increased expression of caveolin-1 may be considered as an indicator of papillary carcinoma progression.
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Anticuerpos/inmunología , Biomarcadores de Tumor/análisis , Carcinoma/química , Caveolina 1/análisis , Neoplasias de la Tiroides/química , Biomarcadores de Tumor/inmunología , Carcinoma/clasificación , Carcinoma Papilar , Estudios de Casos y Controles , Caveolina 1/inmunología , Humanos , Inmunohistoquímica/métodos , Cáncer Papilar Tiroideo , Neoplasias de la Tiroides/clasificaciónRESUMEN
Combined analyses of gene networks and DNA sequence variation can provide new insights into the aetiology of common diseases that may not be apparent from genome-wide association studies alone. Recent advances in rat genomics are facilitating systems-genetics approaches. Here we report the use of integrated genome-wide approaches across seven rat tissues to identify gene networks and the loci underlying their regulation. We defined an interferon regulatory factor 7 (IRF7)-driven inflammatory network (IDIN) enriched for viral response genes, which represents a molecular biomarker for macrophages and which was regulated in multiple tissues by a locus on rat chromosome 15q25. We show that Epstein-Barr virus induced gene 2 (Ebi2, also known as Gpr183), which lies at this locus and controls B lymphocyte migration, is expressed in macrophages and regulates the IDIN. The human orthologous locus on chromosome 13q32 controlled the human equivalent of the IDIN, which was conserved in monocytes. IDIN genes were more likely to associate with susceptibility to type 1 diabetes (T1D)-a macrophage-associated autoimmune disease-than randomly selected immune response genes (P = 8.85 × 10(-6)). The human locus controlling the IDIN was associated with the risk of T1D at single nucleotide polymorphism rs9585056 (P = 7.0 × 10(-10); odds ratio, 1.15), which was one of five single nucleotide polymorphisms in this region associated with EBI2 (GPR183) expression. These data implicate IRF7 network genes and their regulatory locus in the pathogenesis of T1D.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Sitios Genéticos/genética , Predisposición Genética a la Enfermedad/genética , Inmunidad Innata/genética , Virus/inmunología , Animales , Secuencia de Bases , Cromosomas Humanos Par 13/genética , Cromosomas de los Mamíferos/genética , Diabetes Mellitus Tipo 1/inmunología , Redes Reguladoras de Genes/genética , Estudio de Asociación del Genoma Completo , Humanos , Inflamación/genética , Inflamación/inmunología , Factor 7 Regulador del Interferón/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Especificidad de Órganos , Polimorfismo de Nucleótido Simple/genética , Sitios de Carácter Cuantitativo/genética , Ratas , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Human cytomegalovirus (HCMV) establishes a life-long persistent infection. HCMV infection could be associated with chronic inflammatory diseases, such as cardiovascular disease and atherosclerosis. Here we observed that in HCMV (AD-169) pre-exposed human umbilical vein endothelial cells (HUVEC), thrombin-induced expression of IL-1alpha and M-CSF is markedly enhanced compared to the un-exposed cells. Study of the expression of thrombin receptor genes in HUVEC showed that HCMV triggered a time- and concentration-dependent expression of the thrombin receptors PAR1, PAR3 and PAR4 at the mRNA level. Induction of PAR1 and PAR3 mRNA expression is due to transcriptional activation of their promoters as shown by gene reporter assay. Furthermore, the virus induced expression of PAR1 and PAR3 but not PAR4 proteins, as analyzed by Western immunoblotting. However, flow cytometric analysis revealed that only PAR3, expressed at very low level in control HUVEC, is induced at the surface during the exposure to the virus. Our data suggest that although exposure to HCMV induces a minor increase of cell-surface receptors expression, it does make endothelial cells more responsive to additional thrombin stimulation.
Asunto(s)
Citomegalovirus/patogenicidad , Células Endoteliales/virología , Receptores de Trombina/metabolismo , Trombina/metabolismo , Venas Umbilicales/virología , Western Blotting , Células Cultivadas , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Citometría de Flujo , Humanos , Mediadores de Inflamación/metabolismo , Interleucina-1beta/metabolismo , Factor Estimulante de Colonias de Macrófagos/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Receptor PAR-1/metabolismo , Receptores de Trombina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Activación Transcripcional , Venas Umbilicales/citología , Venas Umbilicales/inmunología , Venas Umbilicales/metabolismo , Regulación hacia ArribaRESUMEN
Protease-activated receptors (PARs) are a family of G protein-coupled receptors that are activated by serine protease-mediated proteolytic cleavage of their extracellular domain. We have previously characterized the expression and function of PARs in human monocytes and macrophages, yet information about PARs in dendritic cells (DC) is scarce. Monocyte-derived immature DC do not express PARs. Upon maturation with LPS, but not with TNF-alpha or CD40 ligand, DC express PAR1 and PAR3, but not PAR2 or PAR4. Stimulation of DC with the serine protease thrombin or PAR1-activating peptide elicits actin polymerization and concentration-dependent chemotactic responses in LPS-, but not in TNF-alpha-matured DC. The thrombin-induced migration is a true chemotaxis with only negligible chemokinesis. Stimulation of PARs with thrombin or the respective receptor-activating peptides activates ERK1/2 and Rho kinase as well as subsequent phosphorylation of the regulatory myosin L chain 2. The ERK1/2- and Rho kinase 1-mediated phosphorylation of myosin L chain 2 was indispensable for the PAR-mediated chemotaxis as shown by pharmacological inhibitors. Additionally, thrombin stimulated the Rho-dependent release of the CC chemokine CCL18/pulmonary and activation-regulated chemokine, which induces chemotaxis of lymphocytes and immature DC as well as fibroblast proliferation. The colocalization of CD83(+) DC with CCL18 in human atherosclerotic plaques revealed by immunofluorescence microscopy combined with the presence of functionally active thrombin receptors on mature DC point to a previously unrecognized functional role of thrombin in DC biology. The thrombin-induced stimulation of mature DC may be of particular relevance in atherosclerotic lesions, which harbor all components of this novel mechanism.
Asunto(s)
Quimiocinas CC/metabolismo , Quimiotaxis , Células Dendríticas/inmunología , Receptores de Trombina/metabolismo , Trombina/metabolismo , Actinas/metabolismo , Miosinas Cardíacas/metabolismo , Quimiocinas CC/inmunología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Lipopolisacáridos/inmunología , Cadenas Ligeras de Miosina/metabolismo , Receptor PAR-1/metabolismo , Receptores de Trombina/inmunología , Trombina/inmunología , Quinasas Asociadas a rho/metabolismoRESUMEN
We aimed to identify genetic variants associated with heart failure by using a rat model of the human disease. We performed invasive cardiac hemodynamic measurements in F2 crosses between spontaneously hypertensive heart failure (SHHF) rats and reference strains. We combined linkage analyses with genome-wide expression profiling and identified Ephx2 as a heart failure susceptibility gene in SHHF rats. Specifically, we found that cis variation at Ephx2 segregated with heart failure and with increased transcript expression, protein expression and enzyme activity, leading to a more rapid hydrolysis of cardioprotective epoxyeicosatrienoic acids. To confirm our results, we tested the role of Ephx2 in heart failure using knockout mice. Ephx2 gene ablation protected from pressure overload-induced heart failure and cardiac arrhythmias. We further demonstrated differential regulation of EPHX2 in human heart failure, suggesting a cross-species role for Ephx2 in this complex disease.