RESUMEN
The kinin B1 receptor plays a critical role in the chronic phase of pain and inflammation. The development of B1 antagonists peaked in recent years but almost all promising molecules failed in clinical trials. Little is known about these molecules' mechanisms of action and additional information will be necessary to exploit the potential of the B1 receptor. With the aim of contributing to the available knowledge of the pharmacology of B1 receptors, we designed and characterized a novel class of allosteric non-peptidic inhibitors with peculiar binding characteristics. Here, we report the binding mode analysis and pharmacological characterization of a new allosteric B1 antagonist, DFL20656. We analyzed the binding of DFL20656 by single point mutagenesis and radioligand binding assays and we further characterized its pharmacology in terms of IC50, B1 receptor internalization and in vivo activity in comparison with different known B1 antagonists. We highlighted how different binding modes of DFL20656 and a Merck compound (compound 14) within the same molecular pocket can affect the biological and pharmacological properties of B1 inhibitors. DFL20656, by its peculiar binding mode, involving tight interactions with N114, efficiently induced B1 receptor internalization and evoked a long-lasting effect in an in vivo model of neuropathic pain. The pharmacological characterization of different B1 antagonists highlighted the effects of their binding modes on activity, receptor occupancy and internalization. Our results suggest that part of the failure of most B1 inhibitors could be ascribed to a lack of knowledge about target function and engagement.
Asunto(s)
Antagonistas del Receptor de Bradiquinina B1/farmacología , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Neuralgia/metabolismo , Receptor de Bradiquinina B1/química , Regulación Alostérica , Sitio Alostérico , Animales , Antagonistas del Receptor de Bradiquinina B1/química , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Humanos , Unión Proteica , Transporte de Proteínas , Receptor de Bradiquinina B1/metabolismoRESUMEN
The thyroid hormone-inactivating (TH-inactivating) enzyme type 3 iodothyronine deiodinase (D3) is an oncofetal protein that is rarely expressed in adult life but has been shown to be reactivated in the context of proliferation and neoplasms. D3 terminates TH action within the tumor microenvironment, thereby enhancing cancer cell proliferation. However, the pathological role of D3 and the contribution of TH metabolism in cancer have yet to be fully explored. Here, we describe a reciprocal regulation between TH action and the cancer-associated microRNA-21 (miR21) in basal cell carcinoma (BCC) skin tumors. We found that, besides being negatively regulated by TH at the transcriptional level, miR21 attenuates the TH signal by increasing D3 levels. The ability of miR21 to positively regulate D3 was mediated by the tumor suppressor gene GRHL3, a hitherto unrecognized D3 transcriptional inhibitor. Finally, in a BCC mouse model, keratinocyte-specific D3 depletion markedly reduced tumor growth. Together, our results establish TH action as a critical hub of multiple oncogenic pathways and provide functional and mechanistic evidence of the involvement of TH metabolism in BCC tumorigenesis. Moreover, our results identify a miR21/GRHL3/D3 axis that reduces TH in the tumor microenvironment and has potential to be targeted as a therapeutic approach to BCC.
Asunto(s)
Carcinoma Basocelular/genética , Carcinoma Basocelular/metabolismo , Proteínas Hedgehog/metabolismo , MicroARNs/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Hormonas Tiroideas/metabolismo , Animales , Carcinogénesis/genética , Carcinogénesis/metabolismo , Carcinoma Basocelular/etiología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Xenoinjertos , Humanos , Yoduro Peroxidasa/deficiencia , Yoduro Peroxidasa/genética , Yoduro Peroxidasa/metabolismo , Queratinocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Desnudos , MicroARNs/metabolismo , Transducción de Señal , Neoplasias Cutáneas/etiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas , Microambiente Tumoral/genética , Microambiente Tumoral/fisiologíaRESUMEN
The thymus is the primary organ able to support T cell ontogeny, abrogated in FOXN1(-/-) human athymia. Although evidence indicates that in animal models T lymphocytes may differentiate at extrathymic sites, whether this process is really thymus-independent has still to be clarified. In an athymic FOXN1(-/-) fetus, in which we previously described a total blockage of CD4(+) and partial blockage of CD8(+) cell development, we investigated whether intestine could play a role as extrathymic site of T-lymphopoiesis in humans. We document the presence of few extrathymically developed T lymphocytes and the presence in the intestine of CD3(+) and CD8(+), but not of CD4(+) cells, a few of them exhibiting a CD45RA(+) naïve phenotype. The expression of CD3εεpTα, RAG1 and RAG2 transcripts in the intestine and TCR gene rearrangement was also documented, thus indicating that in humans the partial T cell ontogeny occurring at extrathymic sites is a thymus- and FOXN1-independent process.
Asunto(s)
Factores de Transcripción Forkhead/deficiencia , Intestinos/citología , Subgrupos de Linfocitos T/patología , Timo/patología , Antígenos CD/genética , Antígenos CD/inmunología , Complejo CD3/genética , Complejo CD3/inmunología , Antígenos CD4/genética , Antígenos CD4/inmunología , Proliferación Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Feto , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Expresión Génica , Silenciador del Gen , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/inmunología , Humanos , Inmunofenotipificación , Intestinos/inmunología , Antígenos Comunes de Leucocito/genética , Antígenos Comunes de Leucocito/inmunología , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , ARN Mensajero/genética , ARN Mensajero/inmunología , Subgrupos de Linfocitos T/inmunología , Timo/anomalías , Timo/inmunologíaRESUMEN
To determine whether cell cultures maintain the cellular heterogeneity of primary tissues and may therefore be used for in vitro modeling of breast cancer subtypes, we evaluated the expression of a cell surface marker panel in breast cancer cell cultures derived from various subtypes of human breast carcinoma. We used a four-color flow cytometry strategy to immunophenotype seven human breast cancer cell cultures and four reference breast cancer cell lines. We analyzed 28 surface markers selected based on their potential to distinguish epithelial or mesenchymal lineage, to identify stem cell populations, and to mediate cell adhesion and migration. We determined their ability to form mammospheres and analyzed luminal cytokeratins CK18, CK19, and myoepithelial/basal CK5, SMA (alpha-smooth muscle actin), and vimentin expression by western blot. All cell surface markers showed a unimodal profile. Ten/28 markers were homogenously expressed. Four (CD66b, CD66c, CD165, CD324) displayed negative/low expression. Six (CD29, CD55, CD59, CD81, CD151, CD166) displayed homogenous high expression. Eighteen (CD9, CD10, CD24, CD26, CD44, CD47, CD49b, CD49f, CD54, CD61, CD90, CD105, CD133, CD164, CD184, CD200, CD227, CD326) were heterogeneously expressed. Spearman's rank test demonstrated a significant correlation (p< 0.001) between mesenchymal phenotype and breast cancer cell cultures. Breast cancer cell cultures, all CD44+, displayed concomitant high expression of only three antigens (CD10, CD54, CD90), and low expression of CD326; cell cultures formed mammospheres and expressed CK5, SMA and vimentin, and were weakly CK19-positive. We demonstrate that breast cancer cell cultures preserve inter-tumor heterogeneity and express stem/progenitor markers that can be identified, quantified and categorized by flow cytometry. Therefore, cell cultures can be used for in vitro modeling of breast cancer subtypes; immunophenotyping may mirror breast cancer heterogeneity and reveal molecular characteristics of individual tumors useful for testing target therapy.
Asunto(s)
Biomarcadores de Tumor/análisis , Neoplasias de la Mama/patología , Mioepitelioma/patología , Neoplasia Residual/patología , Fenotipo , Actinas/metabolismo , Antígenos de Neoplasias/análisis , Western Blotting , Neoplasias de la Mama/metabolismo , Antígeno CD24/metabolismo , Adhesión Celular , Movimiento Celular , Forma de la Célula , Células Epiteliales/patología , Femenino , Citometría de Flujo/métodos , Humanos , Receptores de Hialuranos/metabolismo , Inmunofenotipificación/métodos , Queratina-18/metabolismo , Mioepitelioma/metabolismo , Neoplasia Residual/metabolismo , Células Tumorales Cultivadas , Vimentina/metabolismoRESUMEN
The cancer stem cell (CSC) theory represents a breakthrough in cancer research. We characterized the protein pattern of CSCs to identify specific intracellular pathways in this subpopulation of tumor cells. We studied colon CSCs using two different colon cancer cell lines: CaCo-2 and HCT-116. Putative CSCs were separated from non-CSCs by flow cytometry using CD133 as stemness marker. Total protein extracts of CD133+ cells were then compared to protein extracts of CD133- cells by 2D DIGE. The protein spots differentially expressed in the two subpopulations of cells were analyzed by mass spectrometry. Bioinformatics analysis of the identified proteins indicated alteration of two main processes: energy metabolism and the Wnt pathway. Interestingly, we observed upregulation of the splicing factor SRp20, a newly identified target gene of the Wnt/ß-catenin pathway, and we demonstrated a direct cause-effect relationship between Wnt pathway activation and the increased SRp20 expression. Our results also show that SRp20 influences cell proliferation, which suggests it plays a role in the tumorigenicity of CD133+ cells. In conclusion, activation of the Wnt pathway in CD133+ cells and upregulation of SRp20, which is implicated in tumorigenesis, raises the possibility of a sequential series of molecular events occurring in connection with this process.
Asunto(s)
Antígenos CD/metabolismo , Neoplasias del Colon/metabolismo , Glicoproteínas/metabolismo , Péptidos/metabolismo , Proteínas de Unión al ARN/metabolismo , Vía de Señalización Wnt/fisiología , Antígeno AC133 , Antígenos CD/química , Western Blotting , Células CACO-2 , Procesos de Crecimiento Celular/genética , Neoplasias del Colon/química , Neoplasias del Colon/patología , Electroforesis en Gel Bidimensional , Citometría de Flujo , Silenciador del Gen , Glicoproteínas/química , Células HCT116 , Humanos , Células Madre Neoplásicas/química , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Péptidos/química , ARN Interferente Pequeño , Proteínas de Unión al ARN/genética , Reproducibilidad de los Resultados , Factores de Empalme Serina-Arginina , Regulación hacia ArribaRESUMEN
Fe65 is a binding partner of the Alzheimer's beta-amyloid precursor protein APP. The possible involvement of this protein in the cellular response to DNA damage was suggested by the observation that Fe65 null mice are more sensitive to genotoxic stress than WT counterpart. Fe65 associated with chromatin under basal conditions and its involvement in DNA damage repair requires this association. A known partner of Fe65 is the histone acetyltransferase Tip60. Considering the crucial role of Tip60 in DNA repair, we explored the hypothesis that the phenotype of Fe65 null cells depended on its interaction with Tip60. We demonstrated that Fe65 knockdown impaired recruitment of Tip60-TRRAP complex to DNA double strand breaks and decreased histone H4 acetylation. Accordingly, the efficiency of DNA repair was decreased upon Fe65 suppression. To explore whether APP has a role in this mechanism, we analyzed a Fe65 mutant unable to bind to APP. This mutant failed to rescue the phenotypes of Fe65 null cells; furthermore, APP/APLP2 suppression results in the impairment of recruitment of Tip60-TRRAP complex to DNA double strand breaks, decreased histone H4 acetylation and repair efficiency. On these bases, we propose that Fe65 and its interaction with APP play an important role in the response to DNA damage by assisting the recruitment of Tip60-TRRAP to DNA damage sites.
Asunto(s)
Roturas del ADN , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Proteínas del Tejido Nervioso/fisiología , Proteínas Nucleares/fisiología , Acetilación , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Reparación del ADN , Lisina Acetiltransferasa 5 , Ratones , Proteínas Nucleares/metabolismo , Nexinas de Proteasas , Transporte de Proteínas , Receptores de Superficie Celular/metabolismo , TransactivadoresRESUMEN
BACKGROUND: Through negative regulation of gene expression, microRNAs (miRNAs) can function in cancers as oncosuppressors, and they can show altered expression in various tumor types. Here we have investigated medulloblastoma tumors (MBs), which arise from an early impairment of developmental processes in the cerebellum, where Notch signaling is involved in many cell-fate-determining stages. MBs occur bimodally, with the peak incidence seen between 3-4 years and 8-9 years of age, although it can also occur in adults. Notch regulates a subset of the MB cells that have stem-cell-like properties and can promote tumor growth. On the basis of this evidence, we hypothesized that miRNAs targeting the Notch pathway can regulated these phenomena, and can be used in anti-cancer therapies. METHODOLOGY/PRINCIPAL FINDINGS: In a screening of MB cell lines, the miRNA miR-199b-5p was seen to be a regulator of the Notch pathway through its targeting of the transcription factor HES1. Down-regulation of HES1 expression by miR-199b-5p negatively regulates the proliferation rate and anchorage-independent growth of MB cells. MiR-199b-5p over-expression blocks expression of several cancer stem-cell genes, impairs the engrafting potential of MB cells in the cerebellum of athymic/nude mice, and of particular interest, decreases the MB stem-cell-like (CD133+) subpopulation of cells. In our analysis of 61 patients with MB, the expression of miR-199b-5p in the non-metastatic cases was significantly higher than in the metastatic cases (P = 0.001). Correlation with survival for these patients with high levels of miR-199b expression showed a positive trend to better overall survival than for the low-expressing patients. These data showing the down-regulation of miR-199b-5p in metastatic MBs suggest a potential silencing mechanism through epigenetic or genetic alterations. Upon induction of de-methylation using 5-aza-deoxycytidine, lower miR-199b-5p expression was seen in a panel of MB cell lines, supported an epigenetic mechanism of regulation. Furthermore, two cell lines (Med8a and UW228) showed significant up-regulation of miR-199b-5p upon treatment. Infection with MB cells in an induced xenograft model in the mouse cerebellum and the use of an adenovirus carrying miR-199b-5p indicate a clinical benefit through this negative influence of miR-199b-5p on tumor growth and on the subset of MB stem-cell-like cells, providing further proof of concept. CONCLUSIONS/SIGNIFICANCE: Despite advances in our understanding of the pathogenesis of MB, one-third of these patients remain incurable and current treatments can significantly damage long-term survivors. Here we show that miR-199b-5p expression correlates with metastasis spread, identifying a new molecular marker for a poor-risk class in patients with MB. We further show that in a xenograft model, MB tumor burden can be reduced, indicating the use of miR199b-5p as an adjuvant therapy after surgery, in combination with radiation and chemotherapy, for the improvement of anti-cancer MB therapies and patient quality of life. To date, this is the first report that expression of a miRNA can deplete the tumor stem cells, indicating an interesting therapeutic approach for the targeting of these cells in brain tumors.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Regulación hacia Abajo/genética , Proteínas de Homeodominio/genética , Meduloblastoma/patología , Meduloblastoma/terapia , MicroARNs/fisiología , Células Madre Neoplásicas/patología , Animales , Proliferación Celular , Humanos , Meduloblastoma/etiología , Ratones , MicroARNs/administración & dosificación , MicroARNs/uso terapéutico , Metástasis de la Neoplasia , Receptores Notch/metabolismo , Factor de Transcripción HES-1 , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVES: Therapeutic results in advanced chronic lymphocytic leukemia (CLL) are still unsatisfactory in terms of complete remission achievement and duration, in spite of the extensive use of purine analogs. The objective of this study was to describe the clinical characteristics and treatment results from a series of 32 patients managed with a therapeutic program based on the combination of fludarabine and cyclophosphamide (CTX). METHODS: Thirty-two patients (median age 63 yr, range 42-75 yr) with newly diagnosed (47%) or refractory-relapsed (53%) CLL were programmed to receive six courses of a 3-d combination of fludarabine at 30 mg/m2/d plus CTX at 300 mg/m2/d. Refractory-relapsed patients had previously received different chemotherapy lines from 1 to 5. RESULTS: Fourteen of 32 (44%) patients achieved a complete remission, 16 (50%) obtained partial remission and two (6%) failed to respond. The CR rate was higher in untreated patients; in particular, CR was achieved in nine of 15 (60%) newly diagnosed cases as opposed to five of 17 (29%) among pretreated patients. Toxicity was caused by myelosuppression and/or infections in most cases. After a median follow-up of 24 months (range 8-48 months), 20 of 32 patients (62%) are alive, and 14 of 32 (44%) are free from progression. Median overall survival and median time to progression were 35 and 25 months, respectively. CONCLUSION: The combination of fludarabine with CTX is effective in advanced CLL with acceptable toxicity, either as first-line therapy or in refractory-relapsed patients. In particular, a considerable rate of complete remission can be achieved in untreated patients. Myelosuppression represents the major side-effect.