RESUMEN
Molecular targeted therapy using a drug that suppresses the growth and spread of cancer cells via inhibition of a specific protein is a foundation of precision medicine and treatment. High expression of the proto-oncogene Bcl-3 promotes the proliferation and metastasis of cancer cells originating from tissues such as the colon, prostate, breast, and skin. The development of novel drugs targeting Bcl-3 alone or in combination with other therapies can cure these patients or prolong their survival. As a proof of concept, in the present study, we focused on metastatic melanoma as a model system. High-throughput screening and in vitro experiments identified BCL3ANT as a lead molecule that could interfere with Bcl-3-mediated cyclin D1 expression and cell proliferation and migration in melanoma. In experimental animal models of melanoma, it was demonstrated that the use of a Bcl-3 inhibitor can influence the survival of melanoma cells. Since there are no other inhibitors against Bcl-3 in the clinical pipeline for cancer treatment, this presents a unique opportunity to develop a highly specific drug against malignant melanoma to meet an urgent clinical need.
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Melanoma , Neoplasias Cutáneas , Masculino , Animales , Humanos , Melanoma/patología , Ciclina D1/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Neoplasias Cutáneas/patología , Proliferación Celular , Línea Celular Tumoral , ApoptosisRESUMEN
The deregulation of BCL2 family proteins plays a crucial role in leukemia development. Therefore, pharmacological inhibition of this family of proteins is becoming a prevalent treatment method. However, due to the emergence of primary and acquired resistance, efficacy is compromised in clinical or preclinical settings. We developed a drug sensitivity prediction model utilizing a deep tabular learning algorithm for the assessment of venetoclax sensitivity in T-cell acute lymphoblastic leukemia (T-ALL) patient samples. Through analysis of predicted venetoclax-sensitive and resistant samples, PLK1 was identified as a cooperating partner for the BCL2-mediated antiapoptotic program. This finding was substantiated by additional data obtained through phosphoproteomics and high-throughput kinase screening. Concurrent treatment using venetoclax with PLK1-specific inhibitors and PLK1 knockdown demonstrated a greater therapeutic effect on T-ALL cell lines, patient-derived xenografts, and engrafted mice compared with using each treatment separately. Mechanistically, the attenuation of PLK1 enhanced BCL2 inhibitor sensitivity through upregulation of BCL2L13 and PMAIP1 expression. Collectively, these findings underscore the dependency of T-ALL on PLK1 and postulate a plausible regulatory mechanism.
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Leucemia-Linfoma Linfoblástico de Células T Precursoras , Animales , Humanos , Ratones , Algoritmos , Modelos Animales de Enfermedad , Proteínas Proto-Oncogénicas c-bcl-2/genética , Quinasa Tipo Polo 1RESUMEN
The bone marrow microenvironment provides indispensable factors to sustain blood production throughout life. It is also a hotspot for the progression of hematologic disorders and the most frequent site of solid tumor metastasis. Preclinical research relies on xenograft mouse models, but these models preclude the human-specific functional interactions of stem cells with their bone marrow microenvironment. Instead, human mesenchymal cells can be exploited for the in vivo engineering of humanized niches, which confer robust engraftment of human healthy and malignant blood samples. However, mesenchymal cells are associated with major reproducibility issues in tissue formation. Here, we report the fast and standardized generation of human mini-bones by a custom-designed human mesenchymal cell line. These resulting humanized ossicles (hOss) consist of fully mature bone and bone marrow structures hosting a human mesenchymal niche with retained stem cell properties. As compared to mouse bones, we demonstrate superior engraftment of human cord blood hematopoietic cells and primary acute myeloid leukemia samples and also validate hOss as a metastatic site for breast cancer cells. We further report the engraftment of neuroblastoma patient-derived xenograft cells in a humanized model, recapitulating clinically described osteolytic lesions. Collectively, our human mini-bones constitute a powerful preclinical platform to model bone-developing tumors using patient-derived materials.
Asunto(s)
Leucemia Mieloide Aguda , Nicho de Células Madre , Animales , Huesos , Modelos Animales de Enfermedad , Hematopoyesis , Humanos , Ratones , Reproducibilidad de los Resultados , Microambiente TumoralRESUMEN
Hepatocellular carcinoma (HCC), the most common primary liver cancer, is usually diagnosed in its late state. Tyrosine kinase inhibitors such as sorafenib and regorafenib are one of the few treatment options approved for advanced HCC and only prolong the patient's life expectancy by a few months. Therefore, there is a need for novel effective treatments. Cyclophilins are intracellular proteins that catalyze the cis/trans isomerization of peptide bonds at proline residues. Cyclophilins are known to be overexpressed in HCC, affecting therapy resistance and cell proliferation. In the present study, we explored the potential of cyclophilin inhibitors as new therapeutic options for HCC in vitro and in vivo. Our results showed that the novel cyclophilin inhibitor, NV651, was able to significantly decrease proliferation in a diverse set of HCC cell lines. The exposure of HCC cells to NV651 caused an accumulation of cells during mitosis and consequent accumulation in the G2/M phase of the cell cycle. NV651 reduced tumor growth in vivo using an HCC xenograft model without affecting the body weights of the animals. The safety aspects of NV651 were also confirmed in primary human hepatocytes without any cytotoxic effects. Based on the results obtained in this study, we propose NV651 as a potential treatment strategy for HCC.
RESUMEN
Glioblastoma (GB) is the most common and the most aggressive form of brain tumor in adults, which currently lacks efficient treatment strategies. In this study, we investigated the therapeutic effect of function-blocking antibodies targeting integrin α10ß1 on patient-derived-GB cell lines in vitro and in vivo. The in vitro studies demonstrated significant inhibiting effects of the integrin α10 antibodies on the adhesion, migration, proliferation, and sphere formation of GB cells. In a xenograft mouse model, the effect of the antibodies on tumor growth was investigated in luciferase-labeled and subcutaneously implanted GB cells. As demonstrated by in vivo imaging analysis and caliper measurements, the integrin α10-antibodies significantly suppressed GB tumor growth compared to control antibodies. Immunohistochemical analysis of the GB tumors showed lower expression of the proliferation marker Ki67 and an increased expression of cleaved caspase-3 after treatment with integrin α10 antibodies, further supporting a therapeutic effect. Our results suggest that function-blocking antibody targeting integrin α10ß1 is a promising therapeutic strategy for the treatment of glioblastoma.
RESUMEN
Drug resistance of childhood cancer neuroblastoma is a serious clinical problem. Patients with relapsed disease have a poor prognosis despite intense treatment. In the present study, we aimed to identify chemoresistance gene expression signatures in vincristine resistant neuroblastoma cells. We found that vincristine-resistant neuroblastoma cells formed larger clones and survived under reduced serum conditions as compared with non-resistant parental cells. To identify the possible mechanisms underlying vincristine resistance in neuroblastoma cells, we investigated the expression profiles of genes known to be involved in cancer drug resistance. This specific gene expression patterns could predict the behavior of a tumor in response to chemotherapy and for predicting the prognosis of high-risk neuroblastoma patients. Our signature could help chemoresistant neuroblastoma patients in avoiding useless and harmful chemotherapy cycles.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Resistencia a Antineoplásicos/genética , Proteínas de Neoplasias/biosíntesis , Neuroblastoma/genética , Transcriptoma , Vincristina/farmacología , Antineoplásicos/farmacología , Antineoplásicos Fitogénicos/uso terapéutico , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Cisplatino/farmacología , Células Clonales , Etopósido/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas de Neoplasias/genética , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/patología , Nocodazol/farmacología , Pronóstico , Vincristina/uso terapéuticoRESUMEN
Neuroblastoma is a childhood solid tumour originating from undifferentiated neural progenitor cells of the sympathetic nervous system. Drug resistance of childhood cancer neuroblastoma is a serious clinical problem. In the present study, we aimed to identify novel drugs that can inhibit the growth and survival of chemoresistant neuroblastoma. High-throughput screening identified a small molecule, epi-enprioline that was able to induce apoptosis of vincristine-resistant neuroblastoma cells via the mitochondrial apoptotic pathway. Epi-enprioline reduced tumour growth in multiple preclinical models, including an orthotopic neuroblastoma patient-derived xenograft model in vivo. In summary, our data suggest that epi-enprioline can be considered as a lead compound for the treatment of vincristine-resistant neuroblastoma uncovering a novel strategy, which can be further explored as a treatment for drug-resistant neuroblastoma.
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Antineoplásicos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Neuroblastoma/tratamiento farmacológico , Piridinas/farmacología , Animales , Antineoplásicos/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/farmacología , Ensayos de Selección de Medicamentos Antitumorales/métodos , Femenino , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Ratones , Ratones Desnudos , Bibliotecas de Moléculas Pequeñas/farmacología , Vincristina/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The ß-catenin/Wnt signaling pathway plays an important role in all stages of T cell development. Nemo-like kinase (NLK) is an evolutionary conserved serine/threonine kinase and a negative regulator of the Wnt signaling pathway. NLK can directly phosphorylate histone deacetylase 1 (HDAC1), as well as T cell factor/lymphoid enhancer-binding factor (TCF/LEF), causing subsequent repression of target gene transcription. By engineering mice lacking NLK in early stages of T cell development, we set out to characterize the role NLK plays in T cell development and found that deletion of NLK does not affect mouse health or lymphoid tissue development. Instead, these mice harbored a reduced number of single-positive (SP) CD8+ thymocytes without any defects in the SP CD4+ thymocyte population. The decrease in SP CD8+ thymocytes was not caused by a block in differentiation from double-positive CD4+CD8+ cells. Neither TCR signaling nor activation was altered in the absence of NLK. Instead, we observed a significant increase in cell death and reduced phosphorylation of LEF1 as well as HDAC1 among NLK-deleted SP CD8+ cells. Thus, NLK seems to play an important role in the survival of CD8+ thymocytes. Our data provide evidence for a new function for NLK with regard to its involvement in T cell development and supporting survival of SP CD8+ thymocytes.
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Linfocitos T CD8-positivos/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Subgrupos de Linfocitos T/fisiología , Timocitos/fisiología , Animales , Diferenciación Celular , Supervivencia Celular , Histona Desacetilasa 1/metabolismo , Activación de Linfocitos , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal , Proteínas Wnt/metabolismoRESUMEN
Inflammation is an established risk factor for colorectal cancer. We and others have shown that colorectal cancer patients with elevated cysteinyl leukotriene receptor 2 (CysLT2R) and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) levels exhibit good prognoses. However, both CysLT2R and 15-PGDH, which act as tumour suppressors, are often suppressed in colorectal cancer. We previously reported that leukotriene C4 (LTC4)-induced differentiation in colon cancer via CysLT2R signalling. Here, we investigated the involvement of Hedgehog (Hh)-GLI1 signalling, which is often hyperactivated in colorectal cancer. We found that the majority of colorectal cancer patients had high-GLI1 expression, which was negatively correlated with CysLT2R, 15-PGDH, and Mucin-2 and overall survival compared with the low-GLI1 group. LTC4-induced 15-PGDH downregulated both the mRNA and protein expression of GLI1 in a protein kinase A (PKA)-dependent manner. Interestingly, the LTC4-induced increase in differentiation markers and reduction in Wnt targets remained unaltered in GLI1-knockdown cells. The restoration of GLI1 in 15-PGDH-knockdown cells did not ameliorate the LTC4-induced effects, indicating the importance of both 15-PGDH and GLI1. LTC4-mediated reduction in the DCLK1 and LGR5 stemness markers in colonospheres was abolished in cells lacking 15-PGDH or GLI1. Both DCLK1 and LGR5 were highly increased in tumour tissue compared with the matched controls. Reduced Mucin-2 levels were observed both in zebrafish xenografts with GLI1-knockdown cells and in the cysltr2-/- colitis-associated colon cancer (CAC) mouse model. Furthermore, GLI1 expression was positively correlated with stemness and negatively correlated with differentiation in CRC patients when comparing tumour and mucosal tissues. In conclusion, restoring 15-PGDH expression via CysLT2R activation might benefit colorectal cancer patients.
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Neuroblastoma is the most common paediatric cancer type. Patients diagnosed with high-risk neuroblastoma have poor prognosis and occasionally tumours relapse. As a result, novel treatment strategies are needed for relapse and refractory neuroblastoma patients. Here, we found that high expression of Mps1 kinase (mitotic kinase Monopolar Spindle 1) was associated with relapse-free neuroblastoma patient outcomes and poor overall survival. Silencing and inhibition of Mps1 in neuroblastoma or PDX-derived cells promoted cell apoptosis via the caspase-dependent mitochondrial apoptotic pathway. The mechanism of cell death upon Mps1 inhibition was dependent on the polyploidization/aneuploidization of the cells before undergoing mitotic catastrophe. Furthermore, tumour growth retardation was confirmed in a xenograft mouse model after Mps1-inhibitor treatment. Altogether, these results suggest that Mps1 expression and inhibition can be considered as a novel prognostic marker as well as a therapeutic strategy for the treatment of high-risk neuroblastoma patients.
Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Mitosis , Neuroblastoma/enzimología , Neuroblastoma/patología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Humanos , Ratones , Mitocondrias/metabolismo , Poliploidía , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BRCA1-associated protein 1 (BAP1) is a nuclear deubiquitinating enzyme that is associated with multiprotein complexes that regulate key cellular pathways, including cell cycle, cellular differentiation, cell death, and the DNA damage response. In this study, we found that the reduced expression of BAP1 pro6motes the survival of neuroblastoma cells, and restoring the levels of BAP1 in these cells facilitated a delay in S and G2/M phase of the cell cycle, as well as cell apoptosis. The mechanism that BAP1 induces cell death is mediated via an interaction with 14-3-3 protein. The association between BAP1 and 14-3-3 protein releases the apoptotic inducer protein Bax from 14-3-3 and promotes cell death through the intrinsic apoptosis pathway. Xenograft studies confirmed that the expression of BAP1 reduces tumor growth and progression in vivo by lowering the levels of pro-survival factors such as Bcl-2, which in turn diminish the survival potential of the tumor cells. Patient data analyses confirmed the finding that the high-BAP1 mRNA expression correlates with a better clinical outcome. In summary, our study uncovers a new mechanism for BAP1 in the regulation of cell apoptosis in neuroblastoma cells.
Asunto(s)
Proteínas 14-3-3/metabolismo , Apoptosis , Ciclo Celular , Regulación Neoplásica de la Expresión Génica , Neuroblastoma/metabolismo , Proteínas Supresoras de Tumor/biosíntesis , Ubiquitina Tiolesterasa/biosíntesis , Proteínas 14-3-3/genética , Animales , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones Desnudos , Neuroblastoma/genética , Neuroblastoma/patología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Supresoras de Tumor/genética , Ubiquitina Tiolesterasa/genética , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
The Wnt signaling pathway is essential in regulating various cellular processes. Different mechanisms of inhibition for Wnt signaling have been proposed. Besides ß-catenin degradation through the proteasome, nemo-like kinase (NLK) is another molecule that is known to negatively regulate Wnt signaling. However, the mechanism by which NLK mediates the inhibition of Wnt signaling was not known. In the present study, we used primary embryonic fibroblast cells isolated from NLK-deficient mice and showed that these cells proliferate faster and have a shorter cell cycle than wild-type cells. In NLK-knockout cells, we observed sustained interaction between Lef1 and ß-catenin, leading to elevated luciferase reporter of ß-catenin/Lef1-mediated transcriptional activation. The mechanism for the reduced ß-catenin/Lef1 promoter activation was explained by phosphorylation of HDAC1 at serine 421 via NLK. The phosphorylation of HDAC1 was achieved only in the presence of wild-type NLK because a catalytically inactive mutant of NLK was unable to phosphorylate HDAC1 and reduced the luciferase reporter of ß-catenin/Lef1-mediated transcriptional activation. This result suggests that NLK and HDAC1 together negatively regulate Wnt signaling, which is vital in preventing aberrant proliferation of nontransformed primary fibroblast cells.
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Histona Desacetilasa 1/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Animales , Ciclo Celular , Fibroblastos , Histona Desacetilasa 1/fisiología , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Ratones Noqueados , Proteínas Quinasas Activadas por Mitógenos/fisiología , Fosforilación , Cultivo Primario de Células , Proteínas Serina-Treonina Quinasas , Transducción de Señal/fisiología , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/genética , Vía de Señalización Wnt/fisiología , beta Catenina/metabolismoRESUMEN
The role of inflammatory lipid-mediators in tumor progression is well recognized in colorectal cancer; however, if this includes promotion of cancer-initiating cells remains unclear. We show that the inflammatory lipid-mediators leukotriene D4 and prostaglandin E2 increased the Aldehyde dehydrogenase (ALDH(+) ) population, the colony formation capacity, and tumor growth in a xenograft model of colon cancer. The ALDH(+) cells showed significant resistance to irradiation and 5-fluorouracil treatment that could be further augmented by these lipid-mediators, occurring in parallel with increased target gene expression. Our data emphasize a role for tumor microenvironment derived inflammatory lipid-mediators to favor cancer stem cells-like characteristics and thus promote tumor progression.
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Neoplasias del Colon/genética , Dinoprostona/genética , Inflamación/genética , Leucotrieno D4/genética , Lípidos/genética , Células Madre Neoplásicas/metabolismo , Animales , Células CACO-2 , Línea Celular Tumoral , Neoplasias del Colon/patología , Progresión de la Enfermedad , Resistencia a Antineoplásicos/genética , Femenino , Fluorouracilo/farmacología , Células HCT116 , Células HT29 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células Madre Neoplásicas/patología , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The expression of the inflammatory G-protein coupled receptor CysLT1R has been shown to be upregulated in colon cancer patients and associated with poor prognosis. The present study investigated the correlation between CysLT1R and colon cancer development in vivo using CysLT1R antagonists (ZM198,615 or Montelukast) and the nude mouse xenograft model. Two drug administration regimens were established. The first regimen was established to investigate the importance of CysLT1R in tumor initiation. Nude mice were inoculated with 50 µM CysLT1R antagonist-pretreated HCT-116 colon cancer cells and received continued treatment (5 mg/kg/day, intraperitoneally). The second regimen aimed to address the role of CysLT1R in tumor progression. Nude mice were inoculated with non-pretreated HCT-116 cells and did not receive CysLT1R antagonist treatment until recordable tumor appearance. Both regimens resulted in significantly reduced tumor size, attributed to changes in proliferation and apoptosis as determined by reduced Ki-67 levels and increased levels of p21(WAF/Cip1) (P<0.01), cleaved caspase 3, and the caspase-cleaved product of cytokeratin 18. Decreased levels of VEGF (P<0.01) and reduced vessel size (P<0.05) were also observed, the latter only in the ZM198,615-pretreatment group. Furthermore, we performed a series of in vitro studies using the colon cancer cell line HCT-116 and CysLT1R antagonists. In addition to significant reductions in cell proliferation, adhesion and colony formation, we observed induction of cell cycle arrest and apoptosis in a dose-dependent manner. The ability of Montelukast to inhibit growth of human colon cancer xenograft was further validated by using two additional colon cancer cell lines, SW-480 and HT-29. Our results demonstrate that CysLT1R antagonists inhibit growth of colon cancer xenografts primarily by reducing proliferation and inducing apoptosis of the tumor cells.
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Colon/efectos de los fármacos , Colon/patología , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Antagonistas de Leucotrieno/uso terapéutico , Receptores de Leucotrienos/metabolismo , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Colon/metabolismo , Neoplasias del Colon/metabolismo , Femenino , Células HCT116 , Humanos , Ratones , Ratones DesnudosRESUMEN
Tumour-associated macrophages (TAMs) of the M2 phenotype are present in the stroma of many tumours and are frequently associated with the progression of several types of cancer. We investigated the role of M2 macrophages in colon cancer progression and found that human colon cancer tissue had elevated numbers of CD68(+) (macrophage marker) cells and CD206(+) (M2 macrophage marker) cells and increased CD47 expression. To explore potential interplay between colon cancer cells and M2 macrophages, we differentiated the monocyte cell line THP-1 into M1 and M2 macrophages (CD206(high) and Th2 cytokine-secreting cells), respectively. M2 macrophages migrated faster than M1 macrophages towards SW480-conditioned medium. Similarly, M2 macrophage-conditioned medium induced SW480 cell migration and CD47 expression. Factors released by macrophages were involved in this induction. In addition, SW480 cells migrated faster when co-cultured with M2 macrophages. Inhibition of CD47 with blocking antibodies or siRNA significantly reduced the migration of SW480 cells in the presence of M2 macrophages. This effect was further decreased via blocking antibodies against the CD47 ligand signal-regulatory protein α (SIRPα). Additionally, cancer cells also secreted significant levels of IL-10, thereby promoting M2 macrophage differentiation. These findings indicate that a TAM-enriched tumour microenvironment promotes colon cancer cell migration and metastasis.
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Antígeno CD47/inmunología , Comunicación Celular/inmunología , Movimiento Celular/inmunología , Neoplasias del Colon/inmunología , Mediadores de Inflamación/metabolismo , Macrófagos/inmunología , Animales , Diferenciación Celular/inmunología , Procesos de Crecimiento Celular/inmunología , Línea Celular Tumoral , Neoplasias del Colon/patología , Femenino , Células HCT116 , Xenoinjertos , Humanos , Inmunohistoquímica , Macrófagos/patología , Ratones , Ratones Endogámicos BALB C , Ratones DesnudosRESUMEN
BACKGROUND: Leukotriene D(4) (LTD(4)) belongs to the bioactive lipid group known as eicosanoids and has implications in pathological processes such as inflammation and cancer. Leukotriene D(4) exerts its effects mainly through two different G-protein-coupled receptors, CysLT(1) and CysLT(2). The high affinity LTD(4) receptor CysLT(1)R exhibits tumor-promoting properties by triggering cell proliferation, survival, and migration in intestinal epithelial cells. In addition, increased expression and nuclear localization of CysLT(1)R correlates with a poorer prognosis for patients with colon cancer. METHODOLOGY/PRINCIPAL FINDINGS: Using a proximity ligation assay and immunoprecipitation, this study showed that endogenous CysLT(1)R formed heterodimers with its counter-receptor CysLT(2)R under basal conditions and that LTD(4) triggers reduced dimerization of CysLTRs in intestinal epithelial cells. This effect was dependent upon a parallel LTD(4)-induced increase in CysLT(1)R tyrosine phosphorylation. Leukotriene D(4) also led to elevated internalization of CysLT(1)Rs from the plasma membrane and a simultaneous increase at the nucleus. Using sucrose, a clathrin endocytic inhibitor, dominant-negative constructs, and siRNA against arrestin-3, we suggest that a clathrin-, arrestin-3, and Rab-5-dependent process mediated the internalization of CysLT(1)R. Altering the CysLT(1)R internalization process at either the clathrin or the arrestin-3 stage led to disruption of LTD(4)-induced Erk1/2 activation and up-regulation of COX-2 mRNA levels. CONCLUSIONS/SIGNIFICANCE: Our data suggests that upon ligand activation, CysLT(1)R is tyrosine-phosphorylated and released from heterodimers with CysLT(2)R and, subsequently, internalizes from the plasma membrane to the nuclear membrane in a clathrin-, arrestin-3-, and Rab-5-dependent manner, thus, enabling Erk1/2 signaling and downstream transcription of the COX-2 gene.
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Células Epiteliales/citología , Intestinos/citología , Receptores de Leucotrienos/metabolismo , Tirosina/química , Arrestinas/química , Células CACO-2 , Clatrina/química , Cisteína/química , Dimerización , Humanos , Ligandos , Fosforilación , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al GTP rab5/químicaRESUMEN
Mast cells (MCs) are multifunctional effectors of the immune system that are distributed in many tissues, often in close association with the basement membrane of blood vessels, epithelium and nerves. Laminins (LMs), a family of large alphabetagamma heterotrimeric proteins, are major components of basement membrane that strongly promote cell adhesion and migration. In this study, we investigated the role of LM isoforms and their integrin receptors in human MC biology in vitro. In functional assays, alpha3-(LM-332) and alpha5-(LM-511) LMs, but not alpha1-(LM-111), alpha2-(LM-211), or alpha4-(LM-411) LMs, readily promoted adhesion and migration of cultured MCs. These activities were strongly enhanced by various stimuli. alpha3-LM was also able to costimulate IL-8 production. Among LM-binding integrins, MCs expressed alpha(3)beta(1), but not alpha(6)beta(1), alpha(7)beta(1), or alpha(6)beta(4), integrins. Blocking Abs to alpha(3)beta(1) integrin caused inhibition of both cell adhesion and migration on alpha3- and alpha5-LMs. Immunohistochemical studies on skin showed that MCs colocalized with epithelial and vascular basement membranes that expressed alpha3- and alpha5-LMs and that MCs expressed alpha(3) integrin but not alpha(6) integrin(s). These results demonstrate a role for alpha3- and alpha5-LMs and their alpha(3)beta(1) integrin receptor in MC biology. This may explain the intimate structural and functional interactions that MCs have with specific basement membranes.
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Membrana Basal/irrigación sanguínea , Membrana Basal/metabolismo , Movimiento Celular , Sangre Fetal/citología , Sangre Fetal/metabolismo , Integrina alfa3beta1/fisiología , Laminina/fisiología , Mastocitos/metabolismo , Anciano , Anciano de 80 o más Años , Membrana Basal/inmunología , Adhesión Celular/inmunología , Línea Celular , Movimiento Celular/inmunología , Femenino , Sangre Fetal/inmunología , Humanos , Integrina alfa3beta1/sangre , Laminina/sangre , Masculino , Mastocitos/citología , Mastocitos/inmunología , Persona de Mediana Edad , Isoformas de Proteínas/sangre , Isoformas de Proteínas/fisiologíaRESUMEN
Providing humoral immunity, antibody-secreting plasma cells and their immediate precursors, the plasmablasts, are generated in systemic and mucosal immune reactions. Despite their key role in maintaining immunity and immunopathology, little is known about their homeostasis. Here we show that plasmablasts and plasma cells are always detectable in human blood at low frequency in any unimmunized donor. In this steady state, 80% of plasmablasts and plasma cells express immunoglobulin A (IgA). Expression of a functional mucosal chemokine receptor, C-C motif receptor 10 (CCR10) and the adhesion molecule beta(7) integrin suggests that these cells come from mucosal immune reactions and can return to mucosal tissue. These blood-borne, CCR10(+) plasmablasts also are attracted by CXCL12. Approximately 40% of plasma cells in human bone marrow are IgA(+), nonmigratory, and express beta(7) integrin and CCR10, suggesting a substantial contribution of mucosal plasma cells to bone marrow resident, long-lived plasma cells. Six to 8 days after parenteral tetanus/diphtheria vaccination, intracellular IgG(+) cells appear in blood, both CD62L(+), beta(7) integrin(-), dividing, vaccine-specific, migratory plasmablasts and nondividing, nonmigratory, CD62L(-) plasma cells of different specificities. Systemic vaccination does not impact on peripheral IgA(+) plasmablast numbers, indicating that mucosal and systemic humoral immune responses are regulated independent of each other.
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Diferenciación Celular/inmunología , Inmunidad Mucosa/fisiología , Células Plasmáticas/fisiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Vacuna contra Difteria y Tétanos/farmacología , Humanos , Inmunidad Mucosa/efectos de los fármacos , Inmunoglobulina A/metabolismo , Cadenas beta de Integrinas/metabolismo , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/fisiología , Persona de Mediana Edad , Células Plasmáticas/citología , Células Plasmáticas/efectos de los fármacos , Células Plasmáticas/metabolismo , Receptores CCR10/metabolismo , Vacunación , Adulto JovenRESUMEN
The tooth pulp has a dense sensory innervation which, upon stimulation, conveys sensory signals perceived as pain. This innervation, which originates from the trigeminal ganglion, is established through a series of regulated steps during development, and represents an interesting example of tissue targeting by pain-specific nerves. We have investigated various potentially neurotrophic and neurorepulsive influences during this process. The dental papilla/pulp appears to secrete neurite growth inhibitory molecular factors at early stages, which prevent nerve fibers from entering the tissue at what appears to be inappropriate timepoints. Later, a shift from repulsive to attractive factors apparently takes place, and nerve fibers then enter the tooth. When nerve fibers have invaded the dental mesenchyme, a complicated interplay of secreted and membrane-bound factors probably directs the nerve terminals to appropriate sites. Laminin-8 (alpha4beta1gamma1, Lm-411), which is produced by pulpal cells, emerges as an important candidate molecule in this context. Insights into the interactions between the dental pulp nerve fibers and their environment may become important in the search for novel ways to ameliorate pain in the tooth, as well as at other sites.
Asunto(s)
Pulpa Dental/inervación , Proteínas de la Matriz Extracelular/fisiología , Factores de Crecimiento Nervioso/metabolismo , Neuritas/fisiología , Nociceptores/crecimiento & desarrollo , Animales , Pulpa Dental/metabolismo , Humanos , Terminaciones Nerviosas/crecimiento & desarrollo , Transducción de Señal/fisiología , Diente/inervación , Diente/metabolismoRESUMEN
Following vascular injury, basement membrane (BM) components of the blood vessels are exposed to circulating cells and may contribute to hemostasis and/or thrombosis. Laminins 8 (LN-8) (alpha4beta1gamma1) and 10 (LN-10) (alpha5beta1gamma1) are major laminin isoforms of the endothelial BM, and LN-8 is also secreted by activated platelets. In the present study, we demonstrate synthesis of alpha5-laminins LN-10 and LN-11 (alpha5beta2gamma1) by megakaryocytic cells, and intracellular expression of these laminin isoforms in blood platelets. In contrast to platelet LN alpha4 chain that had an apparent molecular weight of 180 kDa and associated mostly to LNbeta1 chain, platelet LNalpha5 consisted of 300/350 kDa polypeptides and associated mainly to LNbeta2. Both alpha4- and alpha5-laminins were secreted by platelets following stimulation. When compared to recombinant human (rh) LN-8, rhLN-10 was much more adhesive to platelets, though adhesion to both proteins was largely mediated via alpha6beta1 integrin. In spite of their adhesive properties, rhLN-8 and rhLN-10 induced neither P-selectin expression nor cell aggregation, two signs of platelet activation. This study demonstrates synthesis/expression of heterotrimeric alpha5-laminins in hematopoietic/blood cells, and provides evidence for the adhesive, but not activating, role of endothelial laminin isoforms in platelet biology.