The Promigratory Activity of the Matricellular Protein Galectin-3 Depends on the Activation of PI-3 Kinase

Expression of galectin-3 is associated with sarcoma progression, invasion and metastasis. Here we determined the role of extracellular galectin-3 on migration of sarcoma cells on laminin-111. Cell lines from methylcholanthrene-induced sarcomas from both wild type and galectin-3−/− mice were established. Despite the presence of similar levels of laminin-binding integrins on the cell surface, galectin-3−/− sarcoma cells were more adherent and less migratory than galectin-3+/+ sarcoma cells on laminin-111. When galectin-3 was transiently expressed in galectin-3−/− sarcoma cells, it inhibited cell adhesion and stimulated the migratory response to laminin in a carbohydrate-dependent manner. Extracellular galectin-3 led to the recruitment of SHP-2 phosphatase to focal adhesion plaques, followed by a decrease in the amount of phosphorylated FAK and phospho-paxillin in the lamellipodia of migrating cells. The promigratory activity of extracellular galectin-3 was inhibitable by wortmannin, implicating the activation of a PI-3 kinase dependent pathway in the galectin-3 triggered disruption of adhesion plaques, leading to sarcoma cell migration on laminin-111

Establishment and characterization of the malignant transformation pathways of a novel murine melanoma cell line

Ao longo dos processos de imortalização e transformação maligna, as células adquirem inúmeras alterações genéticas, que são causadas por fatores endógenos e exógenos como agentes biológicos e a geração de espécies reativas de oxigênio. Neste trabalho, uma linhagem celular espontaneamente transformada foi clonada a partir de explantes de embriões de camundongos C57bl/6. Esta linhagem mostrou-se produtora de pigmento escuro; a análise citoquímica e ultraestrutural permitiu caracterizar a linhagem como tendo origem melanocítica. A linhagem, denominada Mgal3, mostrou-se tumorigênica quando implantada no tecido subcutâneo de animais singenéicos, apresentando capacidade de disseminação linfática, dando origem a metástases em linfonodos, o que permitiu caracteriza-la como uma linhagem de melanoma murino. O processo de transformação deste melanoma caracterizou-se pela expressão de genes retrovirais endógenos, com expressão do antígeno associado a melanoma (MAA), reconhecido pelo anticorpo monoclonal MM2-9B6; ausência de mutações nos exons 5 a 8 do gene supressor de tumor TP53; e, silenciamento do gene CDKN2a, que codifica duas proteínas que atuam em redes de supressão de tumores, p16INK4a e p19ARF. A perda de expressão de pelo menos um destes produtos gênicos parece associada a mecanismos epigenéticos, uma vez que o tratamento de Mgal3 com o inibidor de DNA metiltransferase 5-Aza-2-deoxicitidina, restaurou a transcrição de pelo menos um dos transcritos do gene CDKN2a. Da mesma forma, observamos que o gene LGALS3, que codifica a lectina animal galectina-3 também é silenciado nesta linhagem, mostrando que esta molécula não está associada à manutenção desta célula transformada em condições de cultivo.A novel murine melanoma cell line named Mgal3 was generated from embryo explants. This cell line gave rise to metastatic tumors when injected subcutaneously in C57bl/6 mice. Tumor histogenesis was determined at the cytochemical (Fontana Masson staining), immunohistochemical (staining with anti-HMB45 and anti-S100) and ultrastructural levels. Mgal3 produces high amounts of retroviral C particles and was recognized by the mAb MM2-9B6, which reacts with a melanoma associated antigen derived from the envelope of the ecotropic retrovirus MelArv. No mutations were found in TP53 exons 5-8, however loss of CDKN2a expression was observed. Treatment of Mgal3 with the demethylating agent azadeoxycytidine indicated that at least one of the genes encoded at the CDKN2a locus was silenced by promoter hypermethylation. Furthermore, this cell line did not express the animal lectin, galectin-3. The galectin-3 gene promoter seemed to be hypermethylated, since treatment of Mgal3 with azadeoxycytidine led to the de novo expression of the lectin

Establishment and characterization of the malignant transformation pathways of a novel murine melanoma cell line

Ao longo dos processos de imortalização e transformação maligna, as células adquirem inúmeras alterações genéticas, que são causadas por fatores endógenos e exógenos como agentes biológicos e a geração de espécies reativas de oxigênio. Neste trabalho, uma linhagem celular espontaneamente transformada foi clonada a partir de explantes de embriões de camundongos C57bl/6. Esta linhagem mostrou-se produtora de pigmento escuro; a análise citoquímica e ultraestrutural permitiu caracterizar a linhagem como tendo origem melanocítica. A linhagem, denominada Mgal3, mostrou-se tumorigênica quando implantada no tecido subcutâneo de animais singenéicos, apresentando capacidade de disseminação linfática, dando origem a metástases em linfonodos, o que permitiu caracteriza-la como uma linhagem de melanoma murino. O processo de transformação deste melanoma caracterizou-se pela expressão de genes retrovirais endógenos, com expressão do antígeno associado a melanoma (MAA), reconhecido pelo anticorpo monoclonal MM2-9B6; ausência de mutações nos exons 5 a 8 do gene supressor de tumor TP53; e, silenciamento do gene CDKN2a, que codifica duas proteínas que atuam em redes de supressão de tumores, p16INK4a e p19ARF. A perda de expressão de pelo menos um destes produtos gênicos parece associada a mecanismos epigenéticos, uma vez que o tratamento de Mgal3 com o inibidor de DNA metiltransferase 5-Aza-2-deoxicitidina, restaurou a transcrição de pelo menos um dos transcritos do gene CDKN2a. Da mesma forma, observamos que o gene LGALS3, que codifica a lectina animal galectina-3 também é silenciado nesta linhagem, mostrando que esta molécula não está associada à manutenção desta célula transformada em condições de cultivo.A novel murine melanoma cell line named Mgal3 was generated from embryo explants. This cell line gave rise to metastatic tumors when injected subcutaneously in C57bl/6 mice. Tumor histogenesis was determined at the cytochemical (Fontana Masson staining), immunohistochemical (staining with anti-HMB45 and anti-S100) and ultrastructural levels. Mgal3 produces high amounts of retroviral C particles and was recognized by the mAb MM2-9B6, which reacts with a melanoma associated antigen derived from the envelope of the ecotropic retrovirus MelArv. No mutations were found in TP53 exons 5-8, however loss of CDKN2a expression was observed. Treatment of Mgal3 with the demethylating agent azadeoxycytidine indicated that at least one of the genes encoded at the CDKN2a locus was silenced by promoter hypermethylation. Furthermore, this cell line did not express the animal lectin, galectin-3. The galectin-3 gene promoter seemed to be hypermethylated, since treatment of Mgal3 with azadeoxycytidine led to the de novo expression of the lectin

Establishment and characterization of the malignant transformation pathways of a novel murine melanoma cell line

Ao longo dos processos de imortalização e transformação maligna, as células adquirem inúmeras alterações genéticas, que são causadas por fatores endógenos e exógenos como agentes biológicos e a geração de espécies reativas de oxigênio. Neste trabalho, uma linhagem celular espontaneamente transformada foi clonada a partir de explantes de embriões de camundongos C57bl/6. Esta linhagem mostrou-se produtora de pigmento escuro; a análise citoquímica e ultraestrutural permitiu caracterizar a linhagem como tendo origem melanocítica. A linhagem, denominada Mgal3, mostrou-se tumorigênica quando implantada no tecido subcutâneo de animais singenéicos, apresentando capacidade de disseminação linfática, dando origem a metástases em linfonodos, o que permitiu caracteriza-la como uma linhagem de melanoma murino. O processo de transformação deste melanoma caracterizou-se pela expressão de genes retrovirais endógenos, com expressão do antígeno associado a melanoma (MAA), reconhecido pelo anticorpo monoclonal MM2-9B6; ausência de mutações nos exons 5 a 8 do gene supressor de tumor TP53; e, silenciamento do gene CDKN2a, que codifica duas proteínas que atuam em redes de supressão de tumores, p16INK4a e p19ARF. A perda de expressão de pelo menos um destes produtos gênicos parece associada a mecanismos epigenéticos, uma vez que o tratamento de Mgal3 com o inibidor de DNA metiltransferase 5-Aza-2-deoxicitidina, restaurou a transcrição de pelo menos um dos transcritos do gene CDKN2a. Da mesma forma, observamos que o gene LGALS3, que codifica a lectina animal galectina-3 também é silenciado nesta linhagem, mostrando que esta molécula não está associada à manutenção desta célula transformada em condições de cultivo.A novel murine melanoma cell line named Mgal3 was generated from embryo explants. This cell line gave rise to metastatic tumors when injected subcutaneously in C57bl/6 mice. Tumor histogenesis was determined at the cytochemical (Fontana Masson staining), immunohistochemical (staining with anti-HMB45 and anti-S100) and ultrastructural levels. Mgal3 produces high amounts of retroviral C particles and was recognized by the mAb MM2-9B6, which reacts with a melanoma associated antigen derived from the envelope of the ecotropic retrovirus MelArv. No mutations were found in TP53 exons 5-8, however loss of CDKN2a expression was observed. Treatment of Mgal3 with the demethylating agent azadeoxycytidine indicated that at least one of the genes encoded at the CDKN2a locus was silenced by promoter hypermethylation. Furthermore, this cell line did not express the animal lectin, galectin-3. The galectin-3 gene promoter seemed to be hypermethylated, since treatment of Mgal3 with azadeoxycytidine led to the de novo expression of the lectin

Potential of [11C](R)-PK11195 PET Imaging for Evaluating Tumor Inflammation: A Murine Mammary Tumor Model

Background: Breast tumor inflammation is an immunological process that occurs mainly by mediation of Tumor-Associated Macrophages (TAM). Aiming for a specific measurement of tumor inflammation, the current study evaluated the potential of Positron Emission Tomography (PET) imaging with [11C](R)-PK11195 to evaluate tumor inflammation in a mammary tumor animal model. Methods: Female Balb/C mice were inoculated with 4T1 cells. The PET imaging with [11C](R)-PK11195 and [18F]FDG was acquired 3 days, 1 week, and 2 weeks after cell inoculation. Results: The [11C](R)-PK11195 tumor uptake increased from 3 days to 1 week, and decreased at 2 weeks after cell inoculation, as opposed to the [18F]FDG uptake, which showed a slight decrease in uptake at 1 week and increased uptake at 2 weeks. In the control group, no significant differences occurred in tracer uptake over time. Tumor uptake of both radiopharmaceuticals is more expressed in tumor edge regions, with greater intensity at 2 weeks, as demonstrated by [11C](R)-PK11195 autoradiography and immunofluorescence with TSPO antibodies and CD86 pro-inflammatory phenotype. Conclusion: The [11C](R)-PK11195 was able to identify heterogeneous tumor inflammation in a murine model of breast cancer and the uptake varied according to tumor size. Together with the glycolytic marker [18F]FDG, molecular imaging with [11C](R)-PK11195 may provide a better characterization of inflammatory responses in cancer

Protease-Based Subunit Vaccine in Mice Boosts BCG Protection against Mycobacterium tuberculosis

The significant number of people with latent and active tuberculosis infection requires further efforts to develop new vaccines or improve the Bacillus Calmette-Guérin (BCG), which is the only approved vaccine against this disease. In this study, we developed a recombinant fusion protein (PEPf) containing high-density immunodominant epitope sequences from Rv0125, Rv2467, and Rv2672 Mycobacterium tuberculosis (Mtb) proteases that proved immunogenic and used it to develop a recombinant BCG vaccine expressing the fusion protein. After challenging using Mtb, a specific immune response was recalled, resulting in a reduced lung bacterial load with similar protective capabilities to BCG. Thus BCG PEPf failed to increase the protection conferred by BCG. The PEPf was combined with Advax4 adjuvant and tested as a subunit vaccine using a prime-boost strategy. PEPf + Advax4 significantly improved protection after Mtb challenge, with a reduction in bacterial load in the lungs. Our results confirm that Mtb proteases can be used to develop vaccines against tuberculosis and that the use of the recombinant PEPf subunit protein following a prime-boost regimen is a promising strategy to improve BCG immunity

Metformin-induced chemosensitization to cisplatin depends on P53 status and is inhibited by Jarid1b overexpression in non-small cell lung cancer cells

International audienceMetformin has been tested as an anti-cancer therapy with potential to improve conventional chemotherapy. However, in some cases, metformin fails to sensitize tumors to chemotherapy. Here we test if the presence of P53 could predict the activity of metformin as an adjuvant for cisplatin-based therapy in non-small cell lung cancer (NSCLC). A549, HCC 827 (TP53 WT), H1299, and H358 (TP53 null) cell lines were used in this study. A549 cells were pre-treated with a sub-lethal dose of cisplatin to induce chemoresistance. The effects of metformin were tested both in vitro and in vivo and related to the ability of cells to accumulate Jarid1b, a histone demethylase involved in cisplatin resistance in different cancers. Metformin sensitized A549 and HCC 827 cells (but not H1299 and H358 cells) to cisplatin in a P53-dependent manner, changing its subcellular localization to the mitochondria. Treatment with a sub-lethal dose of cisplatin increased Jarid1b expression, yet downregulated P53 levels, protecting A549Res cells from metformin-induced chemosensitization to cisplatin and favored a glycolytic phenotype. Treatment with FL3, a synthetic flavagline, sensitized A549Res cells to cisplatin. In conclusion, metformin could potentially be used as an adjuvant for cisplatin-based therapy in NSCLC cells if wild type P53 is present

Multimodal Tracking of Hematopoietic Stem Cells from Young and Old Mice Labeled with Magnetic–Fluorescent Nanoparticles and Their Grafting by Bioluminescence in a Bone Marrow Transplant Model

This study proposes an innovative way to evaluate the homing and tracking of hematopoietic stem cells from young and old mice labeled with SPIONNIRF-Rh conjugated with two types of fluorophores (NIRF and Rhodamine), and their grafting by bioluminescence (BLI) in a bone marrow transplant (BMT) model. In an in vitro study, we isolated bone marrow mononuclear cells (BM-MNC) from young and old mice, and analyzed the physical–chemical characteristics of SPIONNIRF-Rh, their internalization, cell viability, and the iron quantification by NIRF, ICP-MS, and MRI. The in vivo study was performed in a BMT model to evaluate the homing, tracking, and grafting of young and old BM-MNC labeled with SPIONNIRF-Rh by NIRF and BLI, as well as the hematological reconstitution for 120 days. 5FU influenced the number of cells isolated mainly in young cells. SPIONNIRF-Rh had adequate characteristics for efficient internalization into BM-MNC. The iron load quantification by NIRF, ICP-MS, and MRI was in the order of 104 SPIONNIRF-Rh/BM-MNC. In the in vivo study, the acute NIRF evaluation showed higher signal intensity in the spinal cord and abdominal region, and the BLI evaluation allowed follow-up (11–120 days), achieving a peak of intensity at 30 days, which remained stable around 108 photons/s until the end. The hematologic evaluation showed similar behavior until 30 days and the histological results confirm that iron is present in almost all tissue evaluated. Our results on BM-MNC homing and tracking in the BMT model did not show a difference in migration or grafting of cells from young or old mice, with the hemogram analysis trending to differentiation towards the myeloid lineage in mice that received cells from old animals. The cell homing by NIRF and long term cell follow-up by BLI highlighted the relevance of the multimodal nanoparticles and combined techniques for evaluation

Dinâmica populacional de Aedes aegypti (L) em área urbana de alta incidência de dengue Population dynamics of Aedes aegypti (L) in an urban area with high incidence of dengue

Aspectos da dinâmica populacional de Aedes aegypti foram investigados a partir de coletas no decorrer de dois anos na área urbana de Uberlândia, MG. A dinâmica populacional do mosquito foi influenciada por fatores físicos como temperatura e pluviosidade. Altas densidades larvais também influenciaram no desenvolvimento do mosquito.<br>Aspects of the population dynamics of Aedes aegypti were investigated from collections over a two-year period in the urban area of Uberlândia, State of Minas Gerais. The population dynamics of this mosquito were influenced by physical factors such as temperature and rainfall. High larval densities also had an influence on mosquito development