Navegando por Palavras-chave "Melanoma murino"
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- ItemAcesso aberto (Open Access)Além da atividade proteolítica das metaloproteases: atividade imunomoduladora da Thimet oligopeptidase (TOP) no tratamento do melanoma murino B16F10Nex2(Universidade Federal de São Paulo (UNIFESP), 2017-02-28) De Amat Herbozo, Carolina Cecilia [UNIFESP]; Rodrigues, Elaine Guadelupe [UNIFESP]; http://lattes.cnpq.br/6913514130496062; http://lattes.cnpq.br/9647501450376151; Universidade Federal de São Paulo (UNIFESP)Previous studies in our laboratory have shown that two bacterial metalloproteases, oligopeptidase A (OpdA) and arazyme, have antitumor activity. This property was independent of their proteolytic activity, and it was mediated by an immunomodulatory effect, involving the activation of antigen presenting cells (APCs). Those results could indicate that OpdA and arazyme were recognized by APCs because of their bacterial origin or otherwise, the immunomodulatory ability was a characteristic of metalloproteases in general, regardless of their origin. The aim of the present study was to evaluate the immunomodulatory and antitumor activity of thimet oligopeptidase (TOP), a murine metallopeptidase, in the B16F10-Nex2 murine melanoma model. Expression and purification of the recombinant version of TOP (rTOP) were optimized, and active and heat-inactivated forms were analyzed. rTOP did not show direct cytotoxic activity in vitro on tumor cells. In vivo assays showed that rTOP has a dose-dependent antitumor activity only in the metastatic, but not in the subcutaneous model. The antitumor effect was independent of the rTOP proteolytic activity, and dependent on a functional adaptive immune response, since it was abolished in immunodeficient animals. Cytokine analysis of serum samples and ex vivo restimulated spleen and lymph node cells revealed that a tumor-specific Th1 response was favored in rTOP treated animals. The active form of the metalloprotease stimulated an increase of IFN-γ, whereas the heat-inactivated form inhibited IL-10 production. Murine bone marrow-derived macrophages and dendritic cells (BMDMs and BMDCs, respectively), were activated in the presence of both active and inactive rTOP, resulting in increased NO production, IL-12p40, TNF-α and IL-10 secretion, and expression of the costimulatory molecules CD80, CD86 and CD40 in BMDCs. Assays in the presence of polymyxin B and with proteinase K-degraded metaloprotease demonstrated that the immunomodulatory activity of rTOP on APCs is not due to the residual LPS of the recombinant preparation. Assays with cells obtained from knockout mice or using specific antagonists/inhibitors determined that the activation of BMDCs is dependent on the presence and function of the TLR4 receptor and the MyD88 and TRIF adapters, and participation of the JNK, p38 and MAP/ERK kinases. In addition, the MyD88 pathway was essential for the antitumor effect of rTOP in the metastatic melanoma model in vivo. In conclusion, rTOP exhibits antitumor activity in the murine metastatic melanoma model mediated by an immunomodulatory effect, and the activation of APCs by rTOP requires the same receptor and signaling pathways as the bacterial metalloproteases studied before.
- ItemAcesso aberto (Open Access)Estudo do papel do óxido nítrico na ativação da GTPase Rac1 em células endoteliais e de melanoma murino(Universidade Federal de São Paulo (UNIFESP), 2015-11-25) Borges, Roberta Eller [UNIFESP]; Monteiro, Hugo Pequeno [UNIFESP]; Batista, Wagner Luiz [UNIFESP]; http://lattes.cnpq.br/4373797404389169; http://lattes.cnpq.br/6154759166234850; http://lattes.cnpq.br/1300439181330301; Universidade Federal de São Paulo (UNIFESP)As proteínas Rac pertencem à família Rho-GTPases que controlam uma ampla variedade de processos celulares como, por exemplo, a formação de fibras de estresse e de adesão focal, a diferenciação celular, migração celular e apoptose. Elas atuam como chaves moleculares, e quando ativadas se encontram associadas ao nucleotídeo GTP e nesta conformação interagem com outras proteínas sinalizadoras participando ativamente dos processos de transdução de sinais originários de vários estímulos extracelulares. Muitos estudos mostraram que a família Rho-GTPases desempenha um papel importante em processos como a migração celular e produção de espécies reativas de oxigênio (ERO). Além disso, também é conhecido que a GTPase Rac-l, está envolvida na migração de células endoteliais associada a angiogênese com as ERO promovendo estes eventos (L. Moldovan et ai, 2006). A GTPase Rac-l também participa na migração de células tumorais relacionada a processos de invasão e de metástase. Entretanto, pouco se sabe sobre o papel do óxido nítrico (NO) nestes processos. Assim, o objetivo deste trabalho foi avaliar a participação do NO na regulação da atividade de Rac-l emcélulas endoteliais de aorta de coelho (RAEC), bem como em modelo de melanoma murino. Inicialmente foi observado que tanto NO derivado de estimulação por bradicinina (BK) como o S-nitrosotiol nitrosoglutationa (GSNO) foram capazes de ativar Rac-l em células RAEC. Este processo levou a igração das células e mostrou-se dependente da S-nitrosilação de Ras, com participação de PI3K. Nas células tumorais B16FIO, foram isoladas duas sublinhagens com capacidades metastáticas polares, sendo a sublinhagem Nex8H (células metastáticas), e a sublinhagem NexlOC (mimetiza tumor primário). Observamos que a ativação de Rac- 1 está envolvida na inibição da migração e invasão celular do melanoma murino metastático Nex8H, quando a produção endógena de NO foi inibida. Curiosamente, o 1 resultado inverso foi observado em células NexlOC (não metastáticas). Esses resultados mostram que a GTPase Rac-I desempenha papéis antagônicos frente a um mesmo estímulo dependendo do estágio de desenvolvimento em que se encontra a célula tumoral, e os mesmos pcidem contribuir para melhor compreender os mecanismos que envolvem a relação entre NO, Rac-I e processos de invasão e migração celular.
- ItemAcesso aberto (Open Access)Influência do ambiente hiperglicêmico no desenvolvimento do melanoma e do adenocarcinoma de mama murinos(Universidade Federal de São Paulo (UNIFESP), 2017-09-11) Sellani, Tarciso Almeida [UNIFESP]; Rodrigues, Elaine Guadelupe [UNIFESP]; Simon, Karin Argenti [UNIFESP]; http://lattes.cnpq.br/4340953066734170; http://lattes.cnpq.br/6913514130496062; http://lattes.cnpq.br/7623361320092941; Universidade Federal de São Paulo (UNIFESP)Epidemiological studies reveal a strong correlation between diabetes and increased risk for development of some neoplasias. Diabetes and cancer have in common several risk factors such as age, obesity, diet and physical inactivity, and biological mechanisms that possibly associate both diseases include hyperinsulinemia, hyperglycemia and inflammation. However, there are no experimental evidences corroborating this association. The objective of this study was to evaluate the effect of hyperglycemia on the development of 4T1 murine mammary adenocarcinoma and B16F10-Nex2 murine melanoma in vitro and in vivo. Tumor cells 4T1 and B16F10-Nex2 were inoculated in streptozotocin-induced hyperglycemic BALB/c and C57BL/6 mice, respectively. Primary 4T1 tumor development and metastatic lung nodules were increased in hyperglycemic animals, which also showed decreased survival, and mice treatment with metformin, a hypoglycemic drug, decreased tumor development. There were no significative differences in vitro in proliferation, migration, and in nitric oxide, cytokine and proangiogenic molecules production by 4T1 cells cultivated in high glucose concentrations. Normo or hyperglycemic immunodeficient animals inoculated with 4T1 cells showed no differences on primary or metastatic tumor development, indicating that adaptive immune system components are responsible for increased tumor growth. Hyperglycemic animals inoculated with 4T1 cells showed increased IL-10 concentration and T regulatory lymphocyte percentage in tumor microenvironment, as well as increased IL-10 and tumor-specific antibodies in serum. T-CD4+or T-CD8+lymphocytes depletion reduced tumor development in this model. Hyperglycemic C57Bl/6 mice inoculated subcutaneously with B16F10-Nex2 cells showed increased tumor growth, decreased survival, and metformin-treated animals showed decreased tumor development. B16F10-Nex2 cells cultivated in high glucose concentrations in vitro showed an increased IL-10 secretion, responsible for a proangiogenic effect in vitro. Hyperglycemic immunodeficient animals were inoculated with B16F10-Nex2 cells, and only TLR4-deficient mice showed tumor development similar to their normoglycemic pairs, suggesting that TLR4-expressing cells are responsible for the increased tumor growth. Nitric oxide-producing macrophage are augmented in primary tumors of hyperglycemic C57Bl/6 mice, the depletion of macrophages led to a decreased tumor development, and the transfer of macrophages from hyperglycemic C57BL/6 to hyperglycemic TLR4-deficient mice induced an increased tumor growth in the last animals. Hyperglycemic C57BL/6 mice treated with L-NAME, a nitric oxide synthase isoforms inhibitor, showed a decreased tumor development and increased survival, indicating a macrophage and nitric oxide participation in the increased tumor development of B16F10-Nex2 cells in hyperglycemic mice. Our results suggest that there is an association between hyperglycemia and tumor development, but the mechanism involved in this association depends on the tumor analyzed. In the 4T1 murine breast adenocarcinoma model, the increased tumor development in hyperglycemic mice occurs due to the participation of the adaptive immune response of the host, while in the murine melanoma model B16F10-Nex2 the increased growth of the tumor in hyperglycemic mice depends on the nitric oxide produced by tumor infiltrating macrophages, together with increased IL-10 production by B16F10-Nex2 cells.