Diss. (sammanfattning) Stockholm : Karol. Inst. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.

Theses (Ph. D.)--Marshall University, 2007. / Title from document title page. Includes abstract. Includes vitae. Document formatted into pages: contains xviii, 167 pages. Bibliography: p. 144-164.

Orientador : Profª. Drª. Carolina Camargo de Oliveira / Coorientador : Prof. Dr. Edvaldo da Silva Trindade / Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 24/03/2016 / Inclui referências : f. 74-83 / Resumo: O melanoma compreende a maior causa de morte por cancer de pele, pois e altamente refratario as terapias existentes. Celulas tumorais apresentam mutacoes somaticas responsaveis pela ativacao constitutiva de vias de sinalizacao que normalmente seriam ativadas pela interacao de fatores de crescimento com seus receptores. Uma vez ativas em celulas de melanoma, essas vias levam a proliferacao continua das celulas e, consequentemente, a progressao da doenca ao promover escape da morte celular, expressao de oncogenes, invasividade, angiogenese e metastase. Alem disso, essas celulas apresentam alteracoes em diversas moleculas de adesao a outras celulas e a matriz extracelular, o que contribui para seu fenotipo agressivo e invasivo. As celulas cancerosas, apesar de suas caracteristicas fenotipicas adquiridas por alteracoes geneticas e epigeneticas, nao agem sozinhas no desenvolvimento da doenca. Os fibroblastos associados ao cancer estao envolvidos em todos os processos que levam as transformacoes fisiologicas que permitem as celulas cancerosas tornarem-se malignas, como a producao de moleculas de matriz extracelular e de proteinas de remodelacao dessa matriz, fornecendo sinais para a sobrevivencia, invasao e proliferacao das celulas cancerosas. Terapias para pacientes portadores de cancer, que utilizam altas diluicoes de compostos naturais, apresentam evidencias de eficacia, levando a melhoria do bem-estar. Nosso grupo de pesquisa tem obtido resultados interessantes com complexos naturais altamente diluidos, denominados M1 ou M8, em especial em melanoma, com estudos in vitro e in vivo. Dessa forma, esse trabalho teve como objetivo entender de que forma M1 ou M8 atuam sobre os mecanismos moleculares de celulas de melanoma murino in vitro, resultando na reducao de suas caracteristicas metastaticas, bem como avaliar se induzem algum tipo de alteracao no processo de comunicacao entre essas celulas cancerosas e fibroblastos. Os resultados obtidos mostraram que o tratamento com M1 foi capaz de reduzir a marcacao das celulas B16-F10 para N-caderina e aumentar para CD54, enquanto M8 reduziu as marcacoes para N-caderina e tambem CD44. Ambos tratamentos nao alteraram a marcacao para NG2, LAMP-1 e ƒÀ-catenina, bem como a ativacao de AKT. Ambos tratamentos com M1 ou M8 reduziram a capacidade das celulas B16-F10 de formar colonias a partir de si. Quando as celulas B16-F10 foram cocultivadas com fibroblastos Balb/3T3 nao houve alteracao na marcacao para CD44 e ƒ¿-SMA apos ambos os tratamentos. M8 foi capaz de inibir a sintese/liberacao de acido hialuronico no sobrenadante da cocultura, bem como a marcacao para N-caderina no extrato celular. Tanto M1 quanto M8 nao causam alteracoes nos padroes de proliferacao e viabilidade quando aplicados sobre os fibroblastos isolados. A atividade da MMP-2 secretada no sobrenadante das culturas nao foi alterada para B16-F10, porem mostrou-se reduzida na cocultura. Com esse trabalho foi possivel demonstrar que os tratamentos com M1 e com M8, alem de seguros possuem atividade seletiva modulando as caracteristicas tumorais, levando-as a um fenotipo de menor malignidade. Palavras-chave: B16-F10. Altas diluicoes. Moleculas de adesao. Comunicacao celular. Microambiente tumoral. / Abstract: Melanoma comprises the leading cause of death from skin cancer, especially because it is highly refractory to existing therapies. Tumor cells exhibit somatic mutations responsible for constitutive activation of signaling pathways that would normally be activated by the interaction of growth factors with their receptors. Once active in melanoma cells, these pathways lead to continued cells proliferation and, therefore, the disease progression by promoting cell death scape, oncogene expression, cell survival, invasiveness, angiogenesis and metastasis. Moreover, these cells present changes in several adhesion molecules to other cells and to the extracellular matrix that contributes to their aggressive and invasive phenotype. Cancer cells, despite their phenotypic characteristics acquired by genetic and epigenetic alterations, do not act alone in the development of the disease. Cancer-associated fibroblasts are involved in all the processes leading to physiological changes that allow cancer cells to become malignant, such as the production of extracellular matrix molecules, and its remodeling, providing survival signals, and promoting cancer cells invasion and proliferation. Therapies for cancer patients using high dilutions of natural compounds present evidence of efficacy leading to improved welfare. Our research group has obtained interesting in vitro and in vivo results with highly diluted natural complexes, called M1 and M8, particularly in melanoma studies. Thus, this study aimed to understand how M1 and M8 act on the molecular mechanisms of murine melanoma cells in vitro, resulting in reduced metastatic characteristics and also assess whether they induce changes in the communication process between these cancerous cells and fibroblasts. The results showed that treatment with M1 was able to reduce B16-F10 cells labeling for N-cadherin and increase to CD54, while M8 reduced N-cadherin and CD44 labeling. No alterations were observed on NG2, LAMP-1, ?-catenin, and AKT activation; on the other hand, B16-F10 colony formation capacity was decreased by both treatments. When B16-F10 cells were co-cultured with Balb/3T3 fibroblasts there were no changes in CD44 and ?-SMA labeling after both treatments. M8 was able to inhibit hyaluronic acid synthesis/release by cells to coculture supernatant, as well as N-cadherin cell expression. Fibroblasts proliferation and viability were not altered by any treatment. The activity of MMP-2 secreted into the cultures supernatant was not changed for B16-F10 alone but was reduced in coculture with fibroblasts. The current work demonstrated that treatments with M1 and M8 are safe and have selective activity modulating tumor characteristics, leading to a less malignant phenotype. Keywords: B16-F10. High dilutions. Adhesion molecules. Cell communication. Tumor microenvironment.

The ability to identify melanoma patients with progressive disease is central to efficient management. The challenge therefore, is to develop prognostic markers and techniques which will allow the identification of those patients whom, at the time of primary tumor diagnosis, already have micrometastases (occult or clinically undetectable metastases). The use of the reverse transcription-PCR (RT-PCR) technique for the detection of circulating melanoma cells (CMCs) is potentially a powerful tool for identifying those patients at risk for developing metastases. The first aim of this study was to develop a more sensitive, reproducible, cost effective and clinically applicable assay and to eliminate the problem of false positives. A combined RT-PCR assay for tyrosinase mRNA, a marker specific for melanoma cells, was developed and tested. It was shown that the assay can reproducibly detect a single, viable melanoma cell in 10-15 ml of peripheral blood. Furthermore, a simple but effective procedure was developed to prevent carryover contamination. It was found that the chance of obtaining normal melanocyte contamination with the needle prick during blood sampling was only 2% and that illegitimate transcription does not contribute to sporadic false positives. The second aim of this study was to determine whether the early detection of CMCs is of any clinical value to monitor melanoma progression. Peripheral blood samples from 143 patients with primary melanoma (PM) were analysed by RT-PCR for the presence of tyrosinase mRNA. Seven percent (10/143) of the patients with PM had detectable CMCs. The percentage of PCR-positive patients was higher for stage II patients (9.0%) compared to stage I (5.3%) but the difference was not significant. A significantly higher percentage (P < 0.05) PCR-positive patients were found to have tumors greater than 1.5 mm thick and with ulceration present. Although this finding supports the notion that tumor thickness and ulceration are the two most significant prognostic factors, it was not possible at this stage, to link this directly to a poor prognosis since the majority of the PCR-positive patients have not yet (within four years) developed metastatic disease. However, the data does indicate that cells from tumors greater than 1.5 mm thick and with ulceration have a greater propensity to enter the circulation but that these cells do not necessarily have the ability to establish metastases. The results suggest that the detection rate of 9% for patients with stage II disease is much lower than would be expected, since 23.9% (16/67) of the stage II patients subsequently developed metastases. Of these 16 patients, only one was PCR-positive, one week before the metastases became clinically evident. Thus, the current technique fails to predict the likelihood of developing metastatic disease (P = 0.3485). The other nine PCR-positive patients had not yet developed metastases after a median follow-up period of four years. It is concluded that the current technique for the detection of CMCs is of limited clinical value to predict the likelihood of metastasis in patients with PM. It is suggested that other anatomic compartments, such as sentinel lymph nodes, should be explored for the identification of patients at risk for developing metastases. The third aim of this study was to determine whether high or low plasma levels and/or activity of plasminogen activator inhibitor type 1 (PAI1) correlate with the presence of metastatic disease in patients with melanoma. PAI1 is considered to be the main regulator of fibrinolytic activity in blood and has been identified as a key enzyme in the metastasis and vascularization of solid tumors. A unique enzyme-linked immunosorbant assay was developed to measure both the total amount of PAI1 in plasma as well as the active fraction of the inhibitor. This novel assay was then used to analyse and compare the plasmatic PAI1 levels and activity of a group of patients with advanced melanoma (AM) with a group of patients with primary disease and a control population. There was no statistical difference in the total plasmatic PAI1 levels between the controls and patients with PM and AM (P = 0.6199). In contrast, there was a significant difference in the active fraction of PAI1 between the controls and patients with PM or AM (P = 0.0076). A value of less than 44% active PAI1 was shown to be clinically meaningful by linear discriminant analysis. This means that a melanoma patient with a plasmatic PAI1 activity value less than 44% will have a 50% chance of harbouring metastases. Of the patients with PM, 19% had PAI1 activity values less than 44%, which strongly supports further investigations to determine whether plasmatic PAI1 activity levels might be predictive of metastatic disease. The false positive rate was 2.6%. It is speculated that this reduction in the active fraction of PAI1 for patients with AM might be attributed to tumor-derived tissue plasminogen activator and/or other melanoma-derived proteases or factors. The last section of this study describes several monoclonal antibodies (Mabs) that were developed against PAI1 in order to obtain useful reagents to study the regulatory functions of PAI1 in the metastasis and vascularization of solid tumors. The baculovirus expression system was used to express human PAI1 in insect cells and the crude infected cell population was used as the immunogen in mice. This approach was followed since the Escherichia coli-derived recombinant molecule elicited a poor immune response. A unique panel of anti-PAI1 Mabs was developed that were characterized with regard to their use for immunoblotting, immunofluorescence and immunocytochemistry. One of these antibodies blocked the binding of PAI1 to vitronectin and inhibited the activity of the inhibitor. Finally, two of these Mabs turned out to be extremely valuable and were used to develop a novel microtiter plate assay for measuring the active fraction of PAI1 in biological fluids by making use of Mabs against different epitopes of PAI1.

Thesis (DHlthSc) -- Bond University, 2005. / "A thesis submitted to Bond University in fulfillment of the requirements for the degree of Doctor of Health Sciences"-- t.p. Bibliography: pages 379-442. Also available via the World Wide Web.

Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted.

Orientador: Edgard Graner / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-19T14:46:05Z (GMT). No. of bitstreams: 1 Seguin_Fabiana_D.pdf: 2599001 bytes, checksum: 8bbd7d749380b6b37b00e01c2eec7482 (MD5) Previous issue date: 2011 / Resumo: A enzima ácido graxo sintase (FASN), cuja expressão e atividade estão elevadas em várias neoplasias malignas humanas, é responsável pela síntese endógena de ácidos graxos saturados de cadeia longa e, consequentemente, pela a produção de fosfolipídios das membranas celulares. A inibição de FASN com orlistat (Xenical), uma droga anti-obesidade, é descrita como tendo propriedades anti-neoplásicas nos cânceres de próstata, mama, bem como no melanoma. Este composto parece desempenhar também um papel anti-angiogênico, uma vez que foi descrito como inibidor da proliferação de células endoteliais e da neovascularização em ensaio ex vivo. Em trabalho recente, realizado por nosso grupo de pesquisa, foi demonstrado que o tratamento de camundongos portadores de melanomas intraperitoneais com orlistat reduziu em cerca de 50% o número de metástases para linfonodos mediastínicos. Em outro estudo, também realizado por nosso grupo, foi observado que a inibição da atividade de FASN pode ter um papel sobre a angiogênese induzida por melanomas experimentais, pois a densidade de vasos sanguíneos ao redor destes tumores foi significantemente reduzida pelo tratamento com orlistat. Considerando as evidências sugerindo um papel biológico relevante de FASN na disseminação metastática de melanomas, o presente trabalho de pesquisa teve como objetivo principal investigar as consequências da inibição da atividade de FASN em cultura de células endoteliais de vasos sanguíneos, através da análise do ciclo celular e das taxas de apoptose. A expressão dos diversos fatores da família VEGF foi avaliada por RT-PCR quantitativo em células de melanoma e carcinoma espinocelular bucal. Os resultados obtidos mostram que orlistat e cerulenina inibem a viabilidade, induzem a apoptose e reduzem in vitro a formação de vasos sanguíneos pelas células endoteliais RAEC. No entanto, o mesmo tratamento não reduz a viabilidade e nem reduzem a formação de vasos sanguíneos in vitro pelas células endoteliais HUVEC. A inibição de FASN nas células neoplásicas SK-MEL-25 e SCC-9 aumenta a expressão de RNAs mensageiros para VEGFA e que o meio condicionado por estas células com orlistat reduz a formação in vitro de vasos sanguíneos e a proliferação das células HUVEC. Nossos resultados sugerem que FASN parece ser importante para a sobrevivência das células RAEC e que a inibição de FASN modula a expressão das isoformas de VEGFA / Abstract: Fatty acid synthase (FASN) is the anabolic enzyme with high expression and activity in several human malignancies, which is responsible for the endogenous synthesis of saturated fatty acids and consequently of the phospholipids present in cell membranes. Inhibition of FASN with orlistat (Xenical), an anti-obesity drug, is described as having anti-neoplastic properties in breast and prostate cancers as well as in melanoma. An anti-angiogenic role, has been attributed to this drug, since it inhibits the proliferation of endothelial cells and the neovascularization in ex vivo both assays. In recent studies performed by our group, it was demonstrated that the treatment of mice bearing intraperitoneal melanoma with orlistat reduced by 50% the number of metastases to mediastinal lymph nodes. In another study, also conducted by our group, we observed that the inhibition of FASN reduces the angiogenesis induced by experimental melanomas, since the density of blood vessels around tumors was significantly decreased by the treatment with orlistat. In the present work investigated the consequences of the treatment with FASN inhibitors the proliferation and apoptosis cultured endothelial cells. The expression of the VEGF family of growth factors were assessed by quantitative RT-PCR in both melanoma and squamous cell carcinoma cells. The results show that orlistat and cerulenin inhibit the viability and induce apoptosis and reduce the formation of blood vessels by RAEC cells in vitro. However, the same treatment does not reduce the viability and not reduce the formation of blood vessels by HUVEC cells in vitro. The inhibition of FASN in neoplastic cells SK-MEL-25 and SCC-9 increases the expression of mRNAs for VEGF and the conditioned medium by these cells with orlistat reduces the formation of blood vessels in vitro and proliferation of HUVEC cells. Together, the experiments show that FASN seems to be important for the survival of RAEC cells and FASN inhibition modulates the expression of VEGF isoforms / Doutorado / Patologia / Doutor em Estomatopatologia

O desenvolvimento de novas estratégias terapêuticas ao melanoma é de particular importância devido à sua baixa resposta aos tratamentos tradicionais. No presente trabalho, utilizamos modelo de melanoma murino experimental para estudarmos os efeitos da fosfoetanolamina (PEA) sintética sobre o desenvolvimento deste tumor. Nossos resultados demonstram que o fosfomonoéster apresentou efeito inibidor da proliferação de células da linhagem B16F10 in vitro, induzindo apoptose após estimulação por 24 a 72h. In vivo, o tratamento (via oral) de animais portadores de melanoma com diferentes doses de PEA (10, 20 e 40mg/Kg), durante 10 ou 20 dias consecutivos, resultou em volumes tumorais pelo menos 70% menores que o de animais controle e diferenças macroscópicas consideráveis. PEA induziu, de maneira dose-dependente, aumento da apoptose e diminuição da proliferação de células tumorais. O tratamento resultou em alterações hematológicas como aumento do número de plaquetas, eritrócitos e leucócitos. Dentre os leucócitos, observou-se uma maior proporção de linfócitos e monócitos após 10 e 20 dias de tratamento, respectivamente. Em adição, PEA induziu uma maior produção da citocina pró-inflamatória IL-6 e das citocinas anti-inflamatórias IL-10 e TGF- e menores níveis da citocina pró-inflamatória IFN-. Os níveis de IL-1, IL-12p70 e IL-17 não foram alterados com o tratamento. Nossos resultados demonstram um papel inibidor da PEA sobre a progressão do melanoma, contribuindo para um melhor entendimento de sua atividade anti-tumoral. / The low responsiveness of melanoma to traditional treatments together with its increasing incidence makes the development of new therapeutic strategies against this type of cancer extremely important. In this study, we used a murine melanoma model to evaluate the effects of synthetic phosphoethanolamine (PEA) on the development of this tumor. In vitro, PEA had an inhibitory effect on the proliferation of B16F10 cells, inducing apoptosis after 24 to 72h stimulation. In vivo, oral treatment of melanoma-bearing animals with different doses of PEA (10, 20 e 40mg/Kg) during 10 or 20 consecutive days resulted in reduced tumor volumes (at least 70% compared to the control) and in expressive macroscopic differences. PEA also induced a dose-dependent increase of apoptosis and decrease in tumor cell proliferation. The treatment also resulted in hematological changes, such as increased numbers of platelets, erythrocytes and leukocytes. Among leukocytes, we observed a higher proportion of lymphocytes and monocytes after 10 and 20 days of treatment, respectively. In addition, PEA induced higher levels of the pro-inflammatory cytokine IL-6 and of the anti-inflammatory cytokines IL-10 and TGF-, and it also induced a lower production of the pro-inflammatory cytokine IFN-. No differences were observed in the levels of IL-1, TNF-, IL-12p70 and IL-17 upon treatment. Our results demonstrate an inhibitory role of PEA in the development of melanoma, contributing to a better understanding of its antitumoral activity.