Doxycycline-inducible expression of SPARC/Osteonectin/BM40 in MDA-MB-231 human breast cancer cells results in growth inhibition

SPARC (secreted protein acidic and rich in cysteine)/BM40/Osteonectin is a matricellular protein with multiple effects on cell behaviour. In vitro, its major known functions are anti-adhesive and anti-proliferative, and it is associated with tissue remodelling and cancer in vivo. SPARC is overexpressed in many cancers, including breast cancer, and the effects of SPARC seem to be cell type-specific. To study the effects of SPARC on breast cancer, we transfected SPARC into the MDA-MB-231 BAG, human breast cancer cell line using the Tet-On inducible system. By western analysis, we found low background levels in the MDA-MB-231 BAG and clone X parental cells, and prominent induction of SPARC protein expression after doxycycline treatment in SPARC transfected clones X5, X21, X24 and X75. Induction of SPARC expression did not affect cell morphology or adhesiveness to collagens type I and IV, but it slowed the rate of proliferation in adherent cultures. Cell cycle analysis showed that SPARC slowed the progression to S phase. Doxycycline induction of SPARC also slowed the rate of monolayer wound closure in the cultured wound healing assay. Thymidine inhibition of proliferation abrogated this effect, confirming that it was due to anti-proliferation rather than inhibition of migration. Consistent with this, we were unable to detect any differences in migration and Matrigel outgrowth analysis of doxycycline-stimulated cells. We conclude that SPARC is inhibitory to human breast cancer cell proliferation, and does not stimulate migration, in contrast to its stimulatory effects reported for melanoma (proliferation and migration) and glioma (migration) cells. Similar growth repression by SPARC has been reported for ovarian cancer cells, and this may be a common feature among carcinomas.

Epidermal growth factor promotes MDA-MB-231 breast cancer cell migration through a phosphatidylinositol 3'-kinase and phospholipase C-dependent mechanism

Epidermal growth factor receptor (EGFR) levels predict a poor outcome in human breast cancer and are most commonly associated with proliferative effects of epidermal growth factor (EGF), with little emphasis placed on motogenic responses to EGF. We found that MDA-MB-231 human breast cancer cells elicited a potent chemotactic response despite their complete lack of a proliferative response to EGF. Antagonists of EGFR ligation, the EGFR kinase, phosphatidylinositol 3'-kinase, and phospholipase C, but not the mitogen- activated protein kinases (extracellular signal-regulated protein kinase 1 and 2), blocked MDA-MB-231 chemotaxis. These findings suggest that EGF may influence human breast cancer progression via migratory pathways, the signaling for which appears to be dissociated, at least in part, from the proliferative pathways.

Pro-matrix metalloproteinase-2 transfection increases orthotopic primary growth and experimental metastasis of MDA-MB-231 human breast cancer cells in nude mice

The ability to activate pro-matrix metalloproteinase (pro-MMP)-2 via membrane type-MMP is a hallmark of human breast cancer cell lines that show increased invasiveness, suggesting that MMP-2 contributes to human breast cancer progression. To investigate this, we have stably transfected pro-MMP-2 into the human breast cancer cell line MDA-MB-231, which lacks MMP-2 expression but does express its cell surface activator, membrane type 1-MMP. Multiple clones were derived and shown to produce pro-MMP-2 and to activate it in response to concanavalin A. In vitro analysis showed that the pro-MMP-2-transfected clones exhibited an increased invasive potential in Boyden chamber and Matrigel outgrowth assays, compared with the parental cells or those transfected with vector only. When inoculated into the mammary fat pad of nude mice, each of the MMP-2-tranfected clones grew faster than each of the vector controls tested. After intracardiac inoculation into nude mice, pro-MMP-2-transfected clones showed a significant increase in the incidence of metastasis to brain, liver, bone, and kidney compared with the vector control clones but not lung. Increased tumor burden was seen in the primary site and in lung metastases, and a trend toward increased burden was seen in bone, however, no change was seen in brain, liver, or kidney. This data supports a role for MMP-2 in breast cancer progression, both in the growth of primary tumors and in their spread to distant organs. MMP-2 may be a useful target for breast cancer therapy when refinement of MMP inhibitors provides for MMP-specific agents.

Tyrosine phosphorylation mediates ConA-induced membrane type 1-matrix metalloproteinase expression and matrix metalloproteinase-2 activation in MDA-MB-231 human breast carcinoma cells

ConA-induced cell surface activation of pro-matrix metalloproteinase-2 (pro-MMP-2) by MDA-MB-231 human breast cancer cells is apparently mediated by up-regulation of membrane type 1 MMP (MT1-MMP) through transcriptional and posttranscriptional mechanisms. Here, we have explored the respective roles of cell surface clustering and protein tyrosine phosphorylation in the ConA- induction effects. Treatment with succinyl-ConA, a variant lacking significant clusterability, partially stimulated MT1-MMP mRNA and protein levels but did not induce MMP-2 activation, suggesting that clustering contributes to the transcriptional regulation by ConA but appears to be critical for the nontranscriptional component. We further found that genistein, an inhibitor of tyrosine phosphorylation, blocked ConA-induced pro-MMP-2 activation and ConA-induced MT1-MMP mRNA level in a dose-dependent manner, implicating tyrosine phosphorylation in the transcriptional aspect. This was confirmed by the dose-dependent promotion of pro-MMP-2 activation by sodium orthovanadate in the presence of suboptimal concentrations of ConA (7.5 μg/ml), with optimal effects seen at 25 μg/g orthovanadate. Genistein did not inhibit the ConA potentiation of MMP-2 activation in MCF-7 cells, in which transfected MT1-MMP is driven by a heterologous promoter, supporting the major implication of phosphotyrosine in the transcriptional component of ConA regulation. These data describe a major signaling event upstream of MT1- MMP induction by ConA and set the stage for further analysis of the nontranscriptional component.

Complex regulation of membrane-type matrix metalloproteinase expression and matrix metalloproteinase-2 activation by concanavalin A in MDA-MB-231 human breast cancer cells

Matrix Metalloproteinase-2 (MMP-2) is secreted as a zymogen, the activation of which has been associated with metastatic progression in human breast cancer (HBC). Concanavalin A (Con A) has been found to induce activation of MMP-2 in invasive HBC cell lines. Con A effects on the expression of mRNA for membrane-type matrix metalloproteinase (MT-MMP), a newly described cell surface-associated MMP, showed a close temporal correlation with induction of MMP-2 activation. It is surprising that MT-MMP mRNA is constitutively present in the uninduced MDA-MB-231 cell, despite a lack of MMP-2 activation. We have used actinomycin D to demonstrate a partial requirement for de novo gene expression in the induction of MMP-2 activation by Con A in MDA-MB-231 HBC cells. Furthermore, this transcriptional response to Con A appeared to require the continued presence of Con A for its manifestation. The nontranscriptional component of the Con A induction manifests rapidly, is quite substantial, and persists strongly despite actinomycin D abrogation of both constitutive and Con A-induced MT-MMP. Cycloheximide analyses suggest that protein synthesis may be involved in this rapid transcription-independent response. These studies suggest that Con A induces MMP-2-activation in part by up-regulation of MT-MMP expression but has a more complicated mode of action, involving additional nontranscriptional effects, which apparently require protein synthesis.

Transfection of MDA-MB-231 human breast carcinoma cells with bone sialoprotein (BSP) stimulates migration and invasion in vitro and growth of primary and secondary tumors in nude mice

We have investigated the role of bone sialoprotein (BSP), a secreted glycoprotein normally found in bone, in breast cancer progression. To explore functions for BSP in human breast cancer invasion and metastasis, the full-length BSP cDNA was transfected into the MDA-MB-231-BAG human breast cancer cell line under the control of the CMV promoter. Clones expressing BSP and vector control clones were isolated. BSP producing clones showed increased monolayer wound healing, a faster rate of stellate outgrowth in Matrigel and increased rate of invasion into a collagen matrix when compared to control clones. Clones were also examined in models of breast cancer growth and metastasis in vivo. BSP transfected clones showed an increased rate of primary tumor growth following mammary fat pad injection of nude mice. BSP transfected clones and vector control clones metastasized to soft organs and bone at a similar rate after intra-cardiac injection as determined by real-time PCR and X-ray analysis. Although these organs were targets for both BSP transfected and non-transfected cells, the size of the metastatic lesion was shown to be significantly larger for BSP expressing clones. This was determined by real-time PCR analysis for soft organs and by X-ray analysis of bone lesions. For bone this was confirmed by intra-tibial injections of cells in nude mice. We conclude that BSP acts to drive primary and secondary tumor growth of breast cancers in vivo.

Elevated cyclic AMP suppresses ConA-induced MT1-MMP expression in MDA- MB-231 human breast cancer cells

We have previously reported that induction of MMP-2 activation by Concanavalin A (ConA) in MDA-MB-231 human breast cancer cells involves both transcriptional and post-transcriptional mechanisms, and that the continuous presence of ConA is required for MMP-2 activation (Yu et al. Cancer Res, 55, 3272-7, 1995). In an effort to identify signal transduction pathways which may either contribute to or modulate this mechanism, we found that three different cAMP-inducing agents, cholera toxin (CT), forskolin (FSK), and 3- isobutyl-1-methylxanthine (IBMX) partially inhibited ConA-induced MT1-MMP expression and MMP-2 activation in MDA-MB-231 cells. Combinations of CT or FSK with IBMX exhibited additive effects on reduction of MT1-MMP mRNA expression and MMP-2 activation. Agents which increase cAMP levels appeared to target transcriptional aspects of ConA induction, reducing MT1-MMP mRNA and protein in parallel with the reduced MMP-2 activation. In the absence of ConA, down-regulation of constitutive production of MT1-MMP mRNA and protein was observed, indicating that cAMP acts independently of ConA. These observations may help to elucidate factors regulating MT1-MMP expression, which may be pivotal to the elaboration of invasive machinery on the cell surface.

Cytotoxic and pro-apoptotic effects of Abrus precatorius L. on human metastatic breast cancer cell line, MDA-MB-231

Abrus precatorius is highly regarded as a universal panacea in the herbal medicine with diverse pharmacological activity spectra. This experimental study on the mechanism of the anticancer activity of A. precatorius leaf extracts, may offer new evidence for A. precatorius in the treatment of breast cancer in clinical practice. Cell death was determined by using MTT assay. Further analyses were carried out by doing DNA laddering, PARP cleavage, FACS, semi-quantitative RT-PCR and detection of cellular reactive oxygen species (ROS) by DCFDA assay. A. precatorius showed very striking inhibition on MDA-MB-231 cells. MTT assay showed more than 75 % inhibition of the cells and treated cells indicated visible laddering pattern with thick compact band. PARP cleavage produced 89 kDa cleavage product which was associated with apoptosis. Flow cytometer exhibited a sub-G0/G1 peak as an indicative of apoptosis. mRNA expression level of apoptosis-related genes p21 and p53 was markedly increased in cells treated with the extract as compared to control. The up-regulation of p21 and p53 may be the molecular mechanisms by which A. precatorius extract which induces apoptosis. An increase in the concentration of A. precatorius extract does not generate ROS, instead it reduces ROS formation in MDA-MB-231 cells, as evident from the shift in fluorescence below untreated control. This is the first report showing that A. precatorius leaf extract exhibits a growth inhibitory effect by induction of apoptosis in MDA-MB-231 cells. Our results contribute towards validation of the A. precatorius extract as a potentially effective chemopreventive or therapeutic agent against breast cancer.

Composición química de la biomasa verde y amonificada pasto Gamba (Andropogon gayanus, Kunth), cv CIAT-612, en inicio de floración, Santa Rosa, Sabana Grande, Managua, Nicaragua

Se llevó a cabo un estudio con el objetivo de determinar el mejoramiento de la composición química de la biomasa verde y tratada con Urea como proceso de amonificación. El pasto utilizado fue el Gamba (Andropogon gayanus Kunth), cv CIAT-621 el cual se encontraba en etapa fenológica de pansoneo. El muestreo se realizó durante el mes de noviembre del año 2009 en la Finca Santa Rosa, Universidad Nacional Agraria. Managua, Nicaragua. Los tratamiento consistieron en cuatro niveles de aplicación de Urea; 0, 1, 3 y 5 % en base al forraje verde a tratar, diluido en 0.5 lt de agua, y almacenados en bolsas de polietileno durante 21 días a temperatura ambiente. El diseño utilizado fue un DCA (Diseño completo al Azar) con tres repeticiones. Las variables de estudio para cada tratamiento fueron, porcentajes de; materia seca, proteína cruda, fibra ácido detergente (FAD), fibra neutro detergente (FND), calcio y fósforo. Se realizaron análisis de varianza (ANDEVA) y separaciones de medias, usando Duncan (P<0.05). Para el análisis estadístico las variables codificadas en porcentajes se transformaron, según, arco seno 2 p (Arco seno de dos veces la raíz cuadrada de la proporción). Los resultados encontrados demuestran diferencias significativas (P ≤ 0.05) para las variables Proteína Cruda (PC), Fibra Neutro y Ácido Detergente (FND, FAD), Calcio y Fósforo. La PC varío de 2.22 % a 6.07 % para 0 % y 5 % de Urea respectivamente, mientras la FND disminuyó de 78.47 % a 73.16 % para 0 y 3 % respectivamente. La FAD disminuyo de 52.46 % a 47.72 % para 0 y 3 % de inclusión de Urea. Los minerales evaluados presentaron una tendencia inversa con los tratamientos de Urea, Incrementándose el Ca desde el tratamiento testigo(0.65 %) hasta 1.24 % para 0 y 5 % de Urea. El Fósforo disminuyo de 0.21 % para el testigo a 0.14 % para 1 % de Urea.. Se concluye que el tratamiento de 3 % de Urea es el más recomendado para la amonificación de forraje verde en etapa fonológica de inicio de floración del Andropogon gayanus Kunth y que la tecnología de amonificación en verde ejerce un efecto positivo en el mejoramiento de la calidad del forraje.

Diário da Constituinte [gravação de vídeo] : [programa n. 231]

O Líder do PDS, Deputado Amaral Neto defende a inclusão da pena de morte no substitutivo. Parlamentaristas e presidencialistas continuam divididos na Comissão de Sistematização da Assembleia Nacional Constituinte (ANC). O Senador João Menezes (PFL-PA) considera que, no momento, o presidencialismo é a melhor escolha, mas a opção parlamentarista poderá ser discutida e colocada em prática mais tarde. O Deputado Paulo Marques (PFL-PE) defende o parlamentarismo misto. O Senador Leite Chaves (PMDB-PR) declara que o presidencialismo é um sistema vencido, só adotado em países ultrapassados, como os da América Latina. O Deputado Eduardo Bonfim (PMDB-AM) é a favor do parlamentarismo já, com eleições diretas em 1988. O Deputado Flávio Rocha (PL-RN) informa que só o presidencialismo pode suprir o anseio popular de escolher, pela via direta, o chefe do Poder Executivo. Na Assembleia Nacional Constituinte (ANC) aumentam as críticas às posições assumidas pelo governo a favor do presidencialismo. O Deputado Gerson Perez (PDS-PR) afirma que o Presidente deveria interferir de forma mais discreta. O Deputado Luiz Roberto Ponte (PMDB-RS) declara que o Presidente pode externar sua opinião, mas quando sentir uma nítida tendência favorável a determinado regime, ele deverá ser o instrumento de viabilização desse sistema, acatando a decisão do parlamento. Para o Deputado Álvaro Antônio (PMDB-MG) esses problemas devem ficar restritos ao Congresso Nacional. O Líder do PDT, Deputado Bocaiuva Cunha considera que a opinião dos constituintes sobre o sistema de governo tem evoluído e que o presidencialismo está absolutamente vitorioso. O Relator Bernardo Cabral (PMDB-AM) aceita a inclusão no substitutivo da jornada de quarenta horas semanais. Para o Deputado José Geraldo (PMDB-MG) a jornada de trabalho é uma reivindicação válida e deve constar no corpo permanente do texto constitucional. O Deputado Luiz Soyer (PMDB-GO) considera que o assunto deve ser tratado por lei ordinária, não na Constituição. O Deputado José Luiz de Sá (PL-RS) afirma que a jornada de trabalho deve ser discutida entre o sindicato e o patrão.

Audiências públicas como instrumento de democracia deliberativa: estudo de caso do PL nº 612/07, lei das sacolas

Monografia (especialização) – Curso de Política e Representação Parlamentar, Câmara dos Deputados, Centro de Formação, Treinamento e Aperfeiçoamento (Cefor), 2013.

C 231 INST-D 2013. 393 Productos del ejercicio de capacitación a indígenas desplazados a Bogotá.

9 fotografías a color.

Livres I-XXIV du Digeste, avec l'apparat. - A la fin, lettre d'Innocent III à Jean-sans-Terre (1. XI, ep. 231).

Célestins.