RESUMEN

Despite a major interest in understanding how the endothelial cell phenotype is established, the underlying molecular basis of this process is not yet fully understood. We have previously reported the generation of induced pluripotent stem cells (iPS) from human umbilical vein endothelial cells and differentiation of the resulting HiPS back to endothelial cells (Ec-Diff), as well as neural (Nn-Diff) cell lineage that contained both neurons and astrocytes. Furthermore, the identities of these cell lineages were established by gene array analysis. Here, we explored the same arrays to gain insight into the gene alteration processes that accompany the establishment of endothelial vs. non-endothelial neural cell phenotypes. We compared the expression of genes that code for transcription factors and epigenetic regulators when HiPS is differentiated into these endothelial and non-endothelial lineages. Our in silico analyses have identified cohorts of genes that are similarly up- or downregulated in both lineages, as well as those that exhibit lineage-specific alterations. Based on these results, we propose that genes that are similarly altered in both lineages participate in priming the stem cell for differentiation in a lineage-independent manner, whereas those that are differentially altered in endothelial compared to neural cells participate in a lineage-specific differentiation process. Specific GATA family members and their cofactors and epigenetic regulators (DNMT3B, PRDM14, HELLS) with a major role in regulating DNA methylation were among participants in priming HiPS for lineage-independent differentiation. In addition, we identified distinct cohorts of transcription factors and epigenetic regulators whose alterations correlated specifically with the establishment of endothelial vs. non-endothelial neural lineages.

RESUMEN

Tumor suppressor/transcription factor p53 is mutated in over 50% of all cancers. Some mutant p53 proteins have not only lost tumor suppressor activities but they also gain oncogenic functions (GOF). One of the most frequently expressed GOF p53 mutants is Arg175His (p53R175H ) with well-documented roles in cancer development and progression. Plakoglobin is a cell adhesion and signaling protein and a paralog of ß-catenin. Unlike ß-catenin that has oncogenic function through its role in the Wnt pathway, plakoglobin generally acts as a tumor/metastasis suppressor. We have shown that plakoglobin interacted with wild type and a number of p53 mutants in various carcinoma cell lines. Plakoglobin and mutant p53 interacted with the promoter and regulated the expression of several p53 target genes. Furthermore, plakoglobin interactions with p53 mutants restored their tumor suppressor/metastasis activities in vitro. GOF p53 mutants induce accumulation and oncogenic activation of ß-catenin. Previously, we showed that one mechanism by which plakoglobin may suppress tumorigenesis is by sequestering ß-catenin's oncogenic activity. Here, we examined the effects of p53R175H expression on ß-catenin accumulation and transcriptional activation and their modifications by plakoglobin coexpression. We showed that p53R175H expression in plakoglobin null cells increased total and nuclear levels of ß-catenin and its transcriptional activity. Coexpression of plakoglobin in these cells promoted ß-catenin's proteasomal degradation, and decreased its nuclear levels and transactivation. Wnt/ß-catenin targets, c-MYC and S100A4 were upregulated in p53R175H cells and were downregulated when plakoglobin was coexpressed. Plakoglobin-p53R175H cells also showed significant reduction in their migration and invasion in vitro.

Asunto(s)

Mutación , Proteína p53 Supresora de Tumor/genética , beta Catenina/genética , gamma Catenina/genética , Carcinogénesis/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Movimiento Celular/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Oncogenes/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Proteína p53 Supresora de Tumor/metabolismo , beta Catenina/metabolismo , gamma Catenina/metabolismo

RESUMEN

Plakoglobin (also known as γ-catenin) is a member of the Armadillo family of proteins and a paralog of ß-catenin. Plakoglobin is a component of both the adherens junctions and desmosomes, and therefore plays a vital role in the regulation of cell-cell adhesion. Similar to ß-catenin, plakoglobin is capable of participating in cell signaling in addition to its role in cell-cell adhesion. In this context, ß-catenin has a well-documented oncogenic potential as a component of the Wnt signaling pathway. In contrast, while some studies have suggested a tumor promoting activity of plakoglobin in a cell/malignancy specific context, it generally acts as a tumor/metastasis suppressor. How plakoglobin acts as a growth/metastasis inhibitory protein has remained, until recently, unclear. Recent evidence suggests that plakoglobin may suppress tumorigenesis and metastasis by multiple mechanisms, including the suppression of oncogenic signaling, interactions with various proteins involved in tumorigenesis and metastasis, and the regulation of the expression of genes involved in these processes. This review is primarily focused on various mechanisms by which plakoglobin may inhibit tumorigenesis and metastasis.

Asunto(s)

Adhesión Celular , Transformación Celular Neoplásica/metabolismo , Neoplasias/metabolismo , gamma Catenina/metabolismo , Uniones Adherentes/metabolismo , Animales , Adhesión Celular/genética , Transformación Celular Neoplásica/genética , Desarrollo Embrionario/genética , Regulación de la Expresión Génica , Humanos , Complejos Multiproteicos/metabolismo , Metástasis de la Neoplasia , Neoplasias/genética , Neoplasias/patología , Células Neoplásicas Circulantes/metabolismo , Células Madre Neoplásicas/metabolismo , Unión Proteica , Transducción de Señal , Células Madre/metabolismo , gamma Catenina/genética

RESUMEN

Network-based strategies provided by systems biology are attractive tools for cancer therapy. Modulation of cancer networks by anticancer drugs may alter the response of malignant cells and/or drive network re-organization into the inhibition of cancer progression. Previously, using systems biology approach and cancer signaling networks, we identified top-5 highly expressed and connected proteins (HSP90AB1, CSNK2B, TK1, YWHAB and VIM) in the invasive MDA-MB-231 breast cancer cell line. Here, we have knocked down the expression of these proteins, individually or together using siRNAs. The transfected cell lines were assessed for in vitro cell growth, colony formation, migration and invasion relative to control transfected MDA-MB-231, the non-invasive MCF-7 breast carcinoma cell line and the non-tumoral mammary epithelial cell line MCF-10A. The knockdown of the top-5 upregulated connectivity hubs successfully inhibited the in vitro proliferation, colony formation, anchorage independence, migration and invasion in MDA-MB-231 cells; with minimal effects in the control transfected MDA-MB-231 cells or MCF-7 and MCF-10A cells. The in vitro validation of bioinformatics predictions regarding optimized multi-target selection for therapy suggests that protein expression levels together with protein-protein interaction network analysis may provide an optimized combinatorial target selection for a highly effective anti-metastatic precision therapy in triple-negative breast cancer. This approach increases the ability to identify not only druggable hubs as essential targets for cancer survival, but also interactions most susceptible to synergistic drug action. The data provided in this report constitute a preliminary step toward the personalized clinical application of our strategy to optimize the therapeutic use of anti-cancer drugs.

Asunto(s)

Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , ARN Interferente Pequeño/metabolismo , Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Técnicas Químicas Combinatorias , Biología Computacional , Progresión de la Enfermedad , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Invasividad Neoplásica , Transducción de Señal , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología

RESUMEN

We have synthesized new, biologically active mono- and di-substituted 2,3,3a,4,5,6-hexahydrocyclopenta[c]pyrazole derivatives bearing electron withdrawing groups and electron donating groups. These derivative structures were characterized by their spectral and analytical data. The newly synthesized hexahydropyrazole analogues were evaluated for their in vitro anticancer activity against breast and lung cancer cell lines using a cytotoxicity bioassay. To understand their mechanism of action, tubulin binding assays were performed which pointed to their binding to microtubules in a mode similar to but not identical to colchicine, as evidenced by their KD value evaluation. Computational docking studies also suggested binding near the colchicine binding site on tubulin. These results were further confirmed by colchicine-binding assays on the most active compounds, which indicated that they bound to tubulin near but not at the colchicine site. The moderate cytotoxic effects of these compounds may be due to the presence of electron donating groups on the para-position of the phenyl ring, along with the hexahydropyrazole core nucleus. The observed anti-cancer activity based on inhibition of microtubule formation may be helpful in designing more potent compounds with a hexahydropyrazole moiety.

Asunto(s)

Antineoplásicos/síntesis química , Pirazoles/química , Antineoplásicos/química , Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Sitios de Unión , Línea Celular Tumoral , Ciclopentanos/química , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Estructura Terciaria de Proteína , Pirazoles/síntesis química , Relación Estructura-Actividad , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/síntesis química , Moduladores de Tubulina/química , Moduladores de Tubulina/toxicidad

RESUMEN

Aberrant expression of cadherins and catenins plays pivotal roles in ovarian cancer development and progression. Plakoglobin (PG, γ-catenin) is a paralog of ß-catenin with dual adhesive and signaling functions. While ß-catenin has known oncogenic function, PG generally acts as a tumor/metastasis suppressor. We recently showed that PG interacted with p53 and that its growth/metastasis inhibitory function may be mediated by this interaction. Very little is known about the role of PG in ovarian cancer. Here, we investigated the in vitro tumor/metastasis suppressor effects of PG in ovarian cancer cell lines with mutant p53 expression and different cadherin profiles. We showed that the N-cadherin expressing and E-cadherin and PG deficient ES-2 cells were highly migratory and invasive, whereas OV-90 cells that express E-cadherin, PG and very little/no N-cadherin were not. Exogenous expression of PG or E-cadherin or N-cadherin knockdown in ES-2 cells (ES-2-E-cad, ES-2-PG and ES-2-shN-cad) significantly reduced their migration and invasion. Also, PG expression or N-cadherin knockdown significantly decreased ES-2 cells growth. Furthermore, PG interacted with both cadherins and with wild type and mutant p53 in normal ovarian and ES-2-PG cell lines, respectively.

Asunto(s)

Antígenos CD/metabolismo , Cadherinas/metabolismo , Desmoplaquinas/metabolismo , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Proteína p53 Supresora de Tumor/metabolismo , Antígenos CD/genética , Cadherinas/antagonistas & inhibidores , Cadherinas/genética , Línea Celular Tumoral , Movimiento Celular/genética , Movimiento Celular/fisiología , Proliferación Celular/genética , Proliferación Celular/fisiología , Desmoplaquinas/genética , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Invasividad Neoplásica/genética , Invasividad Neoplásica/fisiopatología , Neoplasias Ováricas/genética , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , gamma Catenina

RESUMEN

Plakoglobin (PG) is a paralog of ß-catenin with similar adhesive, but contrasting signalling functions. Although ß-catenin has well-known oncogenic function, PG generally acts as a tumor/metastasis suppressor by mechanisms that are just beginning to be deciphered. Previously, we showed that PG interacted with wild type (WT) and a number of mutant p53s, and that its tumor/metastasis suppressor activity may be mediated, at least partially, by this interaction. Here, carcinoma cell lines deficient in both p53 and PG (H1299), or expressing mutant p53 in the absence of PG (SCC9), were transfected with expression constructs encoding WT and different fragments and deletions of p53 and PG, individually or in pairs. Transfectants were characterized for their in vitro growth, migratory and invasive properties and for mapping the interacting domain of p53 and PG. We showed that when coexpressed, p53-WT and PG-WT cooperated to decrease growth, and acted synergistically to significantly reduce cell migration and invasion. The DNA-binding domain of p53 and C-terminal domain of PG mediated p53/PG interaction, and furthermore, the C-terminus of PG played a central role in the inhibition of invasion in association with p53.

Asunto(s)

Carcinoma/patología , Movimiento Celular/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Carcinoma/metabolismo , Línea Celular Tumoral , Humanos , Invasividad Neoplásica/patología , gamma Catenina/metabolismo

RESUMEN

Plakoglobin (γ-catenin) is a homolog of ß-catenin with dual adhesive and signaling functions. Plakoglobin participates in cell-cell adhesion as a component of the adherens junction and desmosomes whereas its signaling function is mediated by its interactions with various intracellular protein partners. To determine the role of plakoglobin during tumorigenesis and metastasis, we expressed plakoglobin in the human tongue squamous cell carcinoma (SCC9) cells and compared the mRNA profiles of parental SCC9 cells and their plakoglobin-expressing transfectants (SCC9-PG). We observed that the mRNA levels of SATB1, the oncogenic chromatin remodeling factor, were decreased approximately 3-fold in SCC9-PG cells compared to parental SCC9 cells. Here, we showed that plakoglobin decreased levels of SATB1 mRNA and protein in SCC9-PG cells and that plakoglobin and p53 associated with the SATB1 promoter. Plakoglobin expression also resulted in decreased SATB1 promoter activity. These results were confirmed following plakoglobin expression in the very low plakoglobin expressing and invasive mammary carcinoma cell line MDA-MB-231 cells (MDA-231-PG). In addition, knockdown of endogenous plakoglobin in the non-invasive mammary carcinoma MCF-7 cells (MCF-7-shPG) resulted in increased SATB1 mRNA and protein. Plakoglobin expression also resulted in increased mRNA and protein levels of the metastasis suppressor Nm23-H1, a SATB1 target gene. Furthermore, the levels of various SATB1 target genes involved in tumorigenesis and metastasis were altered in MCF-7-shPG cells relative to parental MCF-7 cells. Finally, plakoglobin expression resulted in decreased in vitro proliferation, migration and invasion in different carcinoma cell lines. Together with the results of our previous studies, the data suggests that plakoglobin suppresses tumorigenesis and metastasis through the regulation of genes involved in these processes.

Asunto(s)

Neoplasias de la Mama/metabolismo , Movimiento Celular , Proliferación Celular , Desmoplaquinas/biosíntesis , Regulación Neoplásica de la Expresión Génica , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Desmoplaquinas/genética , Regulación hacia Abajo , Femenino , Humanos , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Proteínas de Neoplasias/genética , gamma Catenina

RESUMEN

Molecular classification of breast cancer is based, in part, on the presence or absence of amplification of the human epidermal growth factor receptor 2 (ERBB2) gene, which leads to HER2 protein overproduction. While the presence of the overexpressed HER2 protein is a necessary precondition for sensitivity to anti-HER2 therapies, many patients develop resistance. Thus, identification of the downstream effectors of this pathway will help in understanding mechanism(s) of chemoresistance and further, the identified molecules themselves may have the potential to be used as therapeutic targets. In this work, we studied the proteomic changes that accompany the HER2 gene amplification to identify potential new therapeutic targets and biomarkers. We analyzed bio-triplicate proteome samples extracted from wild-type MCF-7 human breast cancer cells and their isogenic stably overexpressing HER2 (amplified) transfectants. In total, 2455 unique proteins were quantified with 1278 of them differentially expressed in HER2 normal and HER2 overexpressing MCF-7 cells. Select biomarker candidates of particular interest were validated by western blotting, and evaluated for clinical relevance by the immunohistochemical assessment of protein abundance in breast tumor biopsies. HER2 transfection produced marked changes in proteins related to multiple aspects of cancer biology, and the identified expression patterns were recapitulated in the clinical samples. BIOLOGICAL SIGNIFICANCE: Breast cancer is a major cause of death in women. Molecular classification of breast cancer is based, in part, on the presence or absence of amplification of the human epidermal growth factor receptor 2 (ERBB2) gene, which leads to HER2 protein overproduction that triggers intracellular signaling events that drive proliferation, invasion, metastases, and resistance to apoptosis. While the presence of the overexpressed HER2 gene product, HER2 protein, is a necessary precondition for sensitivity to the therapeutic monoclonal antibody trastuzumab, the downstream effects of HER2 protein overexpression are incompletely understood. In this work, we applied quantitative proteomics to identify proteomic changes accompanying ERBB2 gene amplification. The significance of this work includes 1) identification of new biomarkers associated with the HER2 phenotype, 2) measurement of the magnitude of the proteomic changes triggered by the amplification of this single gene, and 3) better understanding of the downstream biological changes triggered by HER2 overexpression.

Asunto(s)

Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteoma/metabolismo , Receptor ErbB-2/metabolismo , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Proliferación Celular , Biología Computacional , Femenino , Amplificación de Genes , Perfilación de la Expresión Génica , Genes erbB-2 , Humanos , Células MCF-7 , Proteómica

RESUMEN

Plakoglobin (γ-catenin), a constituent of the adherens junction and desmosomes, has signaling capabilities typically associated with tumor/metastasis suppression through mechanisms that remain undefined. To determine the role of plakoglobin during tumorigenesis and metastasis, we expressed plakoglobin in human tongue squamous cell carcinoma (SCC9) cells and compared the mRNA profiles of parental SCC9 cells and their plakoglobin-expressing transfectants (SCC9-PG). We detected several p53-target genes whose levels were altered upon plakoglobin expression. In this study, we identified the p53 regulated tumor suppressor 14-3-3σ as a direct plakoglobin-p53 target gene. Coimmunoprecipitation experiments revealed that plakoglobin and p53 interact, and chromatin immunoprecipitation and electrophoretic mobility shift assays revealed that plakoglobin and p53 associate with the 14-3-3σ promoter. Furthermore, luciferase reporter assays showed that p53 transcriptional activity is increased in the presence of plakoglobin. Finally, knockdown of plakoglobin in MCF-7 cells followed by luciferase assays confirmed that p53 transcriptional activity is enhanced in the presence of plakoglobin. Our data suggest that plakoglobin regulates gene expression in conjunction with p53 and that plakoglobin may regulate p53 transcriptional activity, which may account, in part, for the tumor/metastasis suppressor activity of plakoglobin.

Asunto(s)

Proteínas 14-3-3/genética , Carcinogénesis/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteína p53 Supresora de Tumor/metabolismo , gamma Catenina/metabolismo , Carcinogénesis/genética , Femenino , Perfilación de la Expresión Génica , Humanos , Células MCF-7 , Regiones Promotoras Genéticas/genética , Unión Proteica , ARN Interferente Pequeño/genética , Activación Transcripcional/genética , Transgenes/genética , gamma Catenina/genética

RESUMEN

Plakoglobin (γ-catenin) is a member of the Armadillo family of proteins and a homolog of ß-catenin. As a component of both the adherens junctions and desmosomes, plakoglobin plays a pivotal role in the regulation of cell-cell adhesion. Furthermore, similar to ß-catenin, plakoglobin is capable of participating in cell signaling. However, unlike ß-catenin that has well-documented oncogenic potential through its involvement in the Wnt signaling pathway, plakoglobin generally acts as a tumor/metastasis suppressor. The exact roles that plakoglobin plays during tumorigenesis and metastasis are not clear; however, recent evidence suggests that it may regulate gene expression, cell proliferation, apoptosis, invasion, and migration. In this paper, we describe plakoglobin, its discovery and characterization, its role in regulating cell-cell adhesion, and its signaling capabilities in regulation of tumorigenesis and metastasis.

RESUMEN

The complexity of breast cancer biology makes it challenging to analyze large datasets of clinicopathologic and molecular attributes, toward identifying the key prognostic features and producing systems capable of predicting which patients are likely to relapse. We applied machine-learning techniques to analyze a set of well-characterized primary breast cancers, which specified the abundance and localization of various junctional proteins. We hypothesized that disruption of junctional complexes would lead to the cytoplasmic/nuclear redistribution of the protein components and their potential interactions with growth-regulating molecules, which would promote relapse, and that machine-learning techniques could use the subcellular locations of these proteins, together with standard clinicopathological data, to produce an efficient prognostic classifier. We used immunohistochemistry to assess the expression and subcellular distribution of six junctional proteins, in addition to a panel of eight standard clinical features and concentrations of four "growth-regulating" proteins, to produce a database involving 36 features, over 66 primary invasive ductal breast carcinomas. A machine-learning system was applied to this clinicopathologic dataset to produce a decision-tree classifier that could predict whether a novel breast cancer patient would relapse. We show that this decision-tree classifier, which incorporates a combination of only four features (nuclear alpha- and beta-catenin levels, the total level of PTEN and the number of involved axillary lymph nodes), is able to correctly classify patient outcomes essentially 80% of the time. Further, this classifier is significantly better than classifiers based on any subgroup of these 36 features. This study demonstrates that autonomous machine-learning techniques are able to generate simple and efficient decision-tree prognostic classifiers from a wide variety of clinical, pathologic and biomarker data, and unlike other analytic methods, suggest testable biologic relationships among explicitly identified key variables. The decision-tree classifier resulting from these analytic methods is sufficiently simple and should be widely applicable to a spectrum of clinical cancer settings. Further, the subcellular distribution of junctional proteins, which influences growth regulatory pathways involved in locoregional and metastatic relapse of breast cancer, helped to identify which patients would relapse while their total concentration did not. This emphasizes the need to evaluate the subcellular distribution of junctional proteins in assessing their contribution to tumor progression.

Asunto(s)

Biomarcadores de Tumor/análisis , Neoplasias de la Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Conexinas/metabolismo , Recurrencia Local de Neoplasia/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/patología , Árboles de Decisión , Femenino , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Recurrencia Local de Neoplasia/patología , Pronóstico

RESUMEN

We recently showed that estrogen withdrawal from the ERalpha(+), high Bcl-2-expressing breast carcinoma cells (MCF-7B) reduced Bcl-2 protein levels while increasing cell-cell adhesion, and junction formation. Here we compared these cells with the ERalpha(+) and low Bcl-2-expressing MCF-7 cells and with the normal mammary epithelial cell line MCF-10-2A not expressing ERalpha or Bcl-2. All cell lines expressed normal HER2. Antiestrogen (Tamoxifen and ICI 182,780) treatment increased Bcl-2 levels in both MCF-7 and -7B cells and led to the formation of acinar structures. This treatment led to the dissociation of junctions and redistribution of junctional components to the cytoplasm in MCF-10-2A and -7 cells, while in MCF-7B cells junctional proteins redistributed to membranes. Antiestrogen treatment decreased PI3K/Akt activation and increased ERK activation regardless of ERalpha status. IGF-1R was inactivated in the antiestrogen-treated MCF-7 cells while it was activated in MCF-7B cells. Our data show that Tamoxifen and ICI 182,780 can induce growth inhibitory effects via the sustained activation/inactivation of signaling pathways that regulate cell survival, cell death and differentiation in the absence of ERalpha. Furthermore, Bcl-2 overexpression may alter the functional interactions among these pathways in response to antiestrogens, which also may provide a potential explanation for the observation that Bcl-2 overexpressing tumors have a better prognosis.

Asunto(s)

Neoplasias de la Mama/metabolismo , Estradiol/análogos & derivados , Proteínas Proto-Oncogénicas c-bcl-2/efectos de los fármacos , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Transducción de Señal/efectos de los fármacos , Tamoxifeno/farmacología , Western Blotting , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Conexinas/efectos de los fármacos , Estradiol/farmacología , Receptor alfa de Estrógeno/efectos de los fármacos , Receptor alfa de Estrógeno/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Fulvestrant , Humanos , Fosfatidilinositol 3-Quinasas/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptor IGF Tipo 1/efectos de los fármacos , Receptor IGF Tipo 1/metabolismo

RESUMEN

Plakoglobin (Pg) and beta-catenin are homologous proteins that function in cell-cell adhesion and signaling. The cadherin-associated form of these proteins mediates adhesion, whereas the cytosolic/nuclear form has a signaling role. Despite their interactions with common cellular partners, beta-catenin has a well-documented oncogenic potential while Pg has a less characterized tumor suppressor activity. We showed previously that Pg overexpression in Pg-deficient SCC9 cells (SCC9-Pg-WT) induced Bcl-2 expression and inhibited apoptosis. To assess the exact role of Pg in Bcl-2 expression, we generated and characterized SCC9 transfectants expressing Pg with a restricted cytoplasmic (Pg-NES) or nuclear (Pg-NLS) distribution. We show that Bcl-2 was expressed regardless of Pg localization, although its level was substantially lower in SCC9-Pg-NLS cells. Bcl-2 expression coincided with increased nuclear beta-catenin levels (Pg-NES) or a decrease in the level of total and nuclear beta-catenin associated with N-cadherin and alpha-catenin (Pg-WT and -NLS) cells. Bcl-2 expression also was induced in SCC9 cells overexpressing beta-catenin. In contrast, SCC9 cells expressing mutant Pg proteins, unable to interact with N-cadherin and alpha-catenin, had noticeably lower Bcl-2 levels. Our data suggest that Bcl-2 expression is induced by beta-catenin and modulated by Pg. We show that the inhibition of beta-catenin-dependent TCF transactivation had no effect on Bcl-2 levels, suggesting that induction of Bcl-2 expression by beta-catenin and its modulation by Pg may involve factors other than, or in addition, to, TCF. These results provide a possible mechanism for the tumor suppressor activity of Pg via its role as a regulator of the oncogenic potential beta-catenin.

Asunto(s)

Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , beta Catenina/farmacología , gamma Catenina/metabolismo , Apoptosis , Adhesión Celular , Línea Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Humanos , Transducción de Señal , Factores de Transcripción TCF/metabolismo , Activación Transcripcional , Transfección , beta Catenina/metabolismo

RESUMEN

A strategy based on isotope labeling of peptides and liquid chromatography matrix-assisted laser desorption ionization mass spectrometry (LC-MALDI MS) has been employed to accurately quantify and confidently identify differentially expressed proteins between an E-cadherin-deficient human carcinoma cell line (SCC9) and its transfectants expressing E-cadherin (SCC9-E). Proteins extracted from each cell line were tryptically digested and the resultant peptides were labeled individually with either d(0)- or d(2)-formaldehyde. The labeled peptides were combined and the peptide mixture was separated and fractionated by a strong cation exchange (SCX) column. Peptides from each SCX fraction were further separated by a microbore reversed-phase (RP) LC column. The effluents were then directly spotted onto a MALDI target using a heated droplet LC-MALDI interface. After mixing with a MALDI matrix, individual sample spots were analyzed by MALDI quadrupole time-of-flight MS, using an initial MS scan to quantify the dimethyl labeled peptide pairs. MS/MS analysis was then carried out on the peptide pairs having relative peak intensity changes of greater than 2-fold. The MS/MS spectra were subjected to database searching for protein identification. The search results were further confirmed by comparing the MS/MS spectra of the peptide pairs. Using this strategy, we detected and compared relative peak intensity changes of 5480 peptide pairs. Among them, 320 peptide pairs showed changes of greater than 2-fold. MS/MS analysis of these changing pairs led to the identification of 49 differentially expressed proteins between the parental SCC9 cells and SCC9-E transfectants. These proteins were determined to be involved in different pathways regulating cytoskeletal organization, cell adhesion, epithelial polarity, and cell proliferation. The changes in protein expression were consistent with increased cell-cell and cell-matrix adhesion and decreased proliferation in SCC9-E cells, in line with E-cadherin tumor suppressor activity. Finally, the accuracy of the MS quantification and subcellular localization for 6 differentially expressed proteins were validated by immunoblotting and immunofluorescence assays.

Asunto(s)

Cadherinas/metabolismo , Marcaje Isotópico/métodos , Espectrometría de Masas/métodos , Secuencia de Aminoácidos , Cadherinas/genética , Línea Celular Tumoral , Humanos , Datos de Secuencia Molecular , Reproducibilidad de los Resultados

RESUMEN

Endothelin receptors B (Ednrb) are involved in the development of the enteric and melanocytic lineages, which originate from neural crest cells (NCCs). In mice, trunk NCCs and their derivatives express only one Ednrb. In quail, trunk NCCs express two Ednrb: Ednrb and Ednrb2. Quail Ednrb is expressed in NCCs migrating along the ventral pathway, which gives rise to the peripheral nervous system, including enteric ganglia. Ednrb2 is upregulated in NCCs before these cells enter the dorsolateral pathway. The NCCs migrating along the dorsolateral pathway are melanocyte precursors. We analyzed the in vitro differentiation and in ovo migration of mouse embryonic stem (ES) cells expressing and not expressing Ednrb2. We generated a series of transfected ES cell lines expressing Ednrb2. This receptor, like Ednrb, oriented genuine ES cells towards melanocyte lineage differentiation in vitro. The in ovo migration of Ednrb2-expressing ES cells was massively oriented towards the dorsolateral pathway, unlike that of WT or Ednrb-expressing ES cells. Thus, Ednrb2 is involved in melanoblast differentiation and migration.

Asunto(s)

Movimiento Celular/fisiología , Melanocitos/fisiología , Cresta Neural/embriología , Cresta Neural/metabolismo , Receptor de Endotelina B/fisiología , Animales , Diferenciación Celular/fisiología , Línea Celular , Expresión Génica , Melanocitos/citología , Ratones , Cresta Neural/citología , Receptor de Endotelina B/genética , Receptor de Endotelina B/metabolismo , Células Madre/citología , Células Madre/metabolismo

RESUMEN

Synthesis of adenine derivative of triphosphono-gamma-(Z)-ethylidene-2,3-dimethoxybutenolide 4 was accomplished by treatment of phosphonate 3 with 5-phosphoribosyl 1-pyrophosphate in the presence of 5-phosphoribosyl 1-pyrophosphate synthetase. It was found that triphosphonate 4 functions as an irreversible stoichiometric inactivator of the Escherichia coli ribonucleoside diphosphate reductase (RDPR). Triphosphonate 4 exhibited potent inhibitory activity against murine leukemias (L1210 and P388), breast carcinoma (MCF7), and human T-lymphoblasts (Molt4/C8 and CEM/0) cell lines. Paclitaxel ester derivatives of adenine-containing triphosphono-gamma-(Z)-ethylidene-2,3-dimethoxybutenolide 8-10 were also synthesized. Like triphosphonate 4, compound 8 exhibited inhibitory property toward RDPR. It also induced microtubule assembly similar to paclitaxel (5). The structure of the chlorodiester linker in 8 was found to account for this dual property. After treatment of MCF7 cells with compounds 4, 5, and 8, fluorescence microscope examination demonstrated the presence of nucleus shrinkage or segmentation. Bifunctional prodrug 8 exhibited higher lipophilicity than 4 and higher water-solubility than 5. Pro-dual-drug 8 exhibited more pronounced anticancer activity relative to that of the triphosphonate 4 and paclitaxel (5). In contrast, compound 9, resulting from the linkage of triphosphonate 4 and paclitaxel (5) through a diester unit, was only found to function as a highly water-soluble prodrug for paclitaxel (5). It induced microtubule assembly in vitro, but did not show inhibitory property toward RDPR. On the other hand, compound 10, an aggregate of triphosphonate 4 and paclitaxel (5), neither functioned as an inhibitor of RDPR nor exhibited microtubule assembly stimulating activity in vitro.

Asunto(s)

Adenina/análogos & derivados , Antineoplásicos/síntesis química , Diseño de Fármacos , Paclitaxel/análogos & derivados , 4-Butirolactona/análogos & derivados , Adenina/síntesis química , Adenina/farmacología , Animales , Antineoplásicos/farmacología , División Celular/efectos de los fármacos , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Difosfatos/química , Ensayos de Selección de Medicamentos Antitumorales , Furanos/química , Humanos , Microtúbulos/efectos de los fármacos , Organofosfonatos/química , Paclitaxel/química , Paclitaxel/farmacología , Profármacos/síntesis química , Profármacos/farmacología , Ribonucleósido Difosfato Reductasa/antagonistas & inhibidores , Solubilidad , Relación Estructura-Actividad

RESUMEN

Bcl-2, a member of the apoptosis-regulating family of proteins confers a survival advantage on cells by inhibiting apoptosis. Bcl-2 expression is estrogen-responsive and high in various tumors. Overexpression of Bcl-2 has been associated with the loss of contact inhibition, unregulated growth and foci formation in culture. In this study, we have examined the effects of bcl-2 overexpression and expression on cell-cell adhesion in MCF-7 and MDCK epithelial cell lines respectively. Overexpression of Bcl-2 in estrogen receptor-positive MCF-7 mammary carcinoma cells led to decreased cell surface E-cadherin and the disruption of junctional complexes concurrent with intracellular redistribution of their components. Particularly noticeable, was the partial nuclear localization of the tight junction-associated protein ZO-1 which coincided with upregulation of ErbB2. The expression of this EGF co-receptor is regulated by the ZO-1-associated transcription factor ZONAB. Growth in estrogen-depleted media led to downregulation of Bcl-2 expression and upregulation and membrane localization of all junctional proteins. Similar disruption in junctions, accompanied by decreased transepithelial resistance, was observed when Bcl-2 was expressed in MDCK cells. These results strongly suggest that Bcl-2 expression decreases the level of functional E-cadherin thereby interfering with junction formation. The inhibition of junction formation decreases cell-cell adhesion leading to the loss of contact inhibition, which, in vivo, can lead to unregulated growth and tumorigenesis.

Asunto(s)

Cadherinas/metabolismo , Adhesión Celular/fisiología , Uniones Intercelulares/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Apoptosis/fisiología , Cateninas , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al ADN/metabolismo , Perros , Regulación hacia Abajo/fisiología , Receptores ErbB/metabolismo , Estrógenos/metabolismo , Humanos , Uniones Intercelulares/ultraestructura , Proteínas de la Membrana , Fosfoproteínas/metabolismo , Unión Proteica , Receptor ErbB-2/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteína de la Zonula Occludens-1 , beta Catenina , Catenina delta

RESUMEN

A novel cephalosporin derivative of monohydroguaiaretic acid (cephem-M(3)N, 7) was synthesized and found to possess anticancer activity against human leukemia (K562), breast carcinoma (MCF7), human lung cancer (A549), human colon cancer (Colo205) and pancreatic cancer cells (Capan2 and MiaPaCa2). A tumor targeting fusion protein (dsFv3-beta-lactamase) was also used in conjunction with cephem-based M(3)N 7 and its potency toward K562, MCF7, A549, Colo205, Capan2, and MiaPaCa2 was found to approach that of the free M(3)N (4). In the presence of dsFv3-beta-lactamase, tumor cells were found to be much more susceptible to conjugate 7 than normal human embryonic lung (HEL) cells and normal fibroblasts (Hef522). These notions provide a new approach to the use of nordihydroguaiaretic acid (NDGA) and its derivatives for antitumor therapy.

Asunto(s)

Antineoplásicos/síntesis química , Inmunoconjugados/farmacología , Profármacos/síntesis química , beta-Lactamasas/farmacología , Anticuerpos Antineoplásicos , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , División Celular/efectos de los fármacos , Cefalosporinas , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Humanos , Hidrólisis/efectos de los fármacos , Inmunoconjugados/metabolismo , Masoprocol , Profármacos/metabolismo , Profármacos/farmacología , Células Tumorales Cultivadas , beta-Lactamasas/inmunología , beta-Lactamasas/metabolismo