Cancer, the second leading cause of death worldwide, is characterized by uncontrolled and abnormal cell growth. Even though researchers have made significant progress in its treatment over the past several decades, innovative therapeutic approaches that both improve patient survival and lessen the many debilitating side effects of conventional cancer treatments are vital. Accordingly, we first investigated the mechanism of interaction of a bimetallic complex, Ru(II)-Rh(III), with DNA. Non-covalent binding of Ru(II)-Rh(III) is strong and involves electrostatic and, potentially, groove binding interactions. Ru(II)-Rh(III) photobinds and photocleaves DNA through an O2-independent, metal-center mediated mechanism that could be beneficial in hypoxic tumors. Furthermore, the extent of covalent binding and cleavage of DNA, which inhibit PCR amplification, is dependent upon the strength of the non-covalent interactions. These results suggest that the toxicity of Ru(II)-Rh(III) could be selectively generated in tissues irradiated with light (e.g., a tumor). Secondly, we identified protein combinations selectively present in melanoma, which could be utilized in heteromultivalency. Heteromultivalent scaffolds display higher affinity towards cells that express a protein combination in comparison to those with only one of the proteins, which facilitates cell discrimination. Using an empirically-optimized threshold-based screening method and expression profiles of melanoma patients and normal tissues, we identified surface proteins and protein combinations that are selectively found in melanoma patients and not in normal tissues. After a preliminary validation process using the scientific literature, we used immunofluorescence to confirm differential expression of some of these combinations in established melanoma cell lines in comparison to immortalized keratinocytes controls. Finally, we investigated the resazurin assay, a method used for the evaluation of proliferation and cytotoxicity in more than 2,000 publications. We found that only ~14% of these utilized validated assay conditions, while ~40% failed to report essential analytical parameters needed for their replication. We evaluated assay parameters needed for accurate estimation of cell number in eight cell lines, and found that these are highly variable and independent of tissue type, growth kinetics, and energetic parameters. Furthermore, we obtained some insights into the biochemical reduction of resazurin and proposed minimum reporting standards, along with a sample protocol for assay validation. / PHD / Cancer, a group of diseases characterized by uncontrolled and abnormal cell growth, is the second-leading cause of death worldwide. Even though researchers have made significant progress in its treatment over the past several decades, innovative therapeutic approaches that both improve survival outcome and lessen the many debilitating side-effects of conventional cancer treatments are vital. First, we investigated the mechanism of interaction of a particular molecule, Ru(II)- Rh(III), with DNA. We found that Ru(II)-Rh(III) is strongly attracted to DNA due to its charge and an interaction with the indentations along its helix. Upon light activation only, Ru(II)-Rh(III) binds to and cleaves DNA without the need for molecular oxygen, which is scarce in tumors and can limit the activity of other drugs, and to an extent that is affected by the concentration of ions in the solution. Thus, the cytotoxic effect of Ru(II)-Rh(III) might be selectively activated in those tissues that are irradiated with light (e.g., a tumor). Secondly, we identified protein combinations selectively present in melanoma, which could be utilized in heteromultivalency. Heteromultivalent scaffolds bind strongly to cells that express a combination of proteins rather than one protein at a time, making them excellent candidates for delivering a payload in a selective manner. Using expression profiles of melanoma and normal tissues, we identified surface proteins and protein combinations that are selectively found in melanoma patients and not in normal tissues. After a preliminary validation process using the scientific literature, we used confirmed differences in the expression intensities of some of these combinations in melanoma cell lines in comparison to normal skin controls. Finally, we investigated the resazurin assay, a method used for the evaluation of cell growth and drug candidates in more than 2,000 publications. We found that only ~14% of these utilized validated assay conditions, while ~40% failed to report essential analytical parameters needed for their replication. We evaluated assay conditions for eight cell lines, and found that these are highly variable and independent of tissue type and some metabolic parameters. Furthermore, we obtained insights into the mechanism through which cells react with resazurin and proposed minimum reporting standards for publications, along with a protocol for assay validation.

Melanoma is the most deadly form of skin cancer worldwide, which causes the 75% of deaths related to skin cancer. National Cancer Institute estimated that 91,270 new case and 9,320 deaths are expected in 2018 caused by melanoma. Early detection of melanoma is the key for the treatment. The image technique to diagnose skin cancer is dermoscopy, which leads to improved diagnose accuracy compared to traditional ABCD criteria. But reading and examining dermoscopic images is a time-consuming and complex process. Therefore, computerized analysis methods of dermoscopic images have been developed to assist the visual interpretation of dermoscopic images. The automatic segmentation of skin lesion attributes is a key step in computerized analysis of dermoscopic images. The International Skin Imaging Collaboration (ISIC) hosted the 2018 Challenges to help the diagnosis of melanoma based on dermoscopic images. In this thesis, I develop a deep learning based approach to automatically segment the attributes from dermoscopic skin lesion images. The approach described in the thesis achieved the Jaccard index of 0.477 on the official test dataset, which ranked 5th place in the challenge. / Master of Science / Melanoma is the most deadly form of skin cancer worldwide, which causes the 75% of deaths related to skin cancer. Early detection of melanoma is the key for the treatment. The image technique to diagnose skin cancer is called dermoscopy. It has become increasingly conveniently to use dermoscopic device to image the skin in recent years. Dermoscopic lens are available in the market for individual customer. When coupling the dermoscopic lens with smartphones, people are be able to take dermoscopic images of their skin even at home. However, reading and examining dermoscopic images is a time-consuming and complex process. It requires specialists to examine the image, extract the features, and compare with criteria to make clinical diagnosis. The time-consuming image examination process becomes the bottleneck of fast diagnosis of melanoma. Therefore, computerized analysis methods of dermoscopic images have been developed to promote the melanoma diagnosis and to increase the survival rate and save lives eventually. The automatic segmentation of skin lesion attributes is a key step in computerized analysis of dermoscopic images. In this thesis, I developed a deep learning based approach to automatically segment the attributes from dermoscopic skin lesion images. The segmentation result from this approach won 5th place in a public competition. It has the potential to be utilized in clinic application in the future.

No / Metastatic melanoma is the leading cause of skin‑cancer related deaths and while in recent years some progress has been made with targeted therapies, there remains an urgent unmet need for novel therapeutic treatments and reliable diagnostic, prognostic and predictive biomarkers. The emergence of next generation sequencing (NGS) has seen a growing appreciation for the role played by non‑coding genomic transcripts in regulating gene expression and by extension impacting on disease progression. The long non‑coding RNAs (lncRNAs) represent the most enigmatic of these new regulatory molecules. Our understanding of how lncRNAs regulate biological functions and their importance to disease aetiology, while still limited, is rapidly improving, in particular with regards to their role in cancer. Herein we review the identification of several lncRNAs shown to impact on melanoma disease progression and discuss how these molecules are operating at the molecular level.

A proteína dermicidina (DCD) é codificada por um gene localizado na porção 12q13 do cromossomo 12, presente apenas em primatas e humanos. A proteína é secretada por células de glândulas da pele, melanócitos, neurônios e células epiteliais da mama normal. Alguns estudos inciais revelaram a participação da proteína DCD em processos oncogênicos nos carcinomas de mama, próstata e melanoma pela sua capacidade de atuar como um fator de crescimento e sobrevivência celular. Nos estudos realizados no nosso laboratório mostramos que a DCD é expressa em células normais da pele, placenta, cérebro e em vários tumores, incluindo os carcinomas de mama e melanoma maligno. Ensaios biológicos e bioquímicos mostraram que o \"knockdown\" da expressão de DCD no melanoma maligno G-361 via RNA de interferência (RNAi) diminuiu significativamente o crescimento in vitro em cultura celular e a formação de tumores em camundongos Nude. Resultados similares foram obtidos quando camundongos Nude transplantados com células de melanoma G-361 foram tratados com anticorpos policlonais de coelhos contra a proteína DCD. Para compreender melhor o papel da proteína DCD na transformação de células de melanoma G-361 foram feitos ensaios de microarranjo de DNA para identificar os genes diferencialmente expressos entre as sublinhagens pLKO (controle) e IBC-I que expressa o shRNA para o mRNA da DCD. Entre os 374 genes alterados pelo silenciamento, encontramos 162 com expressão aumentada e 212 com a expressão reduzida. Os estudos de bioinformática pelo software MetaCore mostraram que o silenciamento do gene DNA modula as vias canônicas e redes de sinalização mediadas pelo receptores e ligantes da família BAFF/APRIL que contralam a ativação do fator de transcrição NF-kB, bem como para histonas envolvidas na remodelação da cromatina. Os níveis de expressão de mRNA de 9 genes de interesse foram validados por ensaios de RT-qPCR. Em uma segunda fase do estudo, foram analisadas as proteínas presentes em extratos nuclares dos clones pLKO e IBC-I de melanoma maligno G-361 por espectrometria de massas. Nos extratos proteicos da sublinhagem pLKO foram identificadas 74 proteínas nucleares, enquanto que na sublinhagem IBC-I foram identificadas 31 proteínas. Um grupo de 21 proteínas foi identificado em ambas sublinhagens. Estudos de bioinformática pelo programa STRING revelou que 14 das proteínas identificadas na sublinagem G-361-pLKO faziam interações diretas ou indiretas com a DCD. A rede formada por estas proteínas tem como centro a proteína p53, uma proteína chave na regulação do programa de morte celular e sobrevivência ao estresse oxidativo. Por outro lado, notou-se que a maioria das proteínas identificadas no extrato nuclear da sublinhagem IBC-I é da família das histonas e que poderiam atuar em complexos de remodelação da cromatina nas células G-361-IBC-I. Nossos resultados nos possibilitaram sugerir que futuros estudos sobre a expressão das histonas e suas modificações pós-traducionais poderão ajudar a desvendar o possível papel da DCD na regulação epigenética do melanoma e em outros tipos de cânceres / Dermcidin (DCD) is a human gene mapped to chromosome 12q13 region, only identified in primates and humans, and normally expressed in the eccrine glands of skin and brain. Several studies have confirmed that DCD-derived peptides contribute to innate and immune surveillance and in the oncogenic processes of breast, prostate and skin cancers, as revealed by its role as a growth factor and cell survival. We have further explored DCD function and its tumorigenic potential on skin melanocytes by specifically knocking down its expression in G-361 malignant melanoma cells via expressing constitutively short hairpin RNA against DCD mRNA. Biological and biochemical assays showed that the \"knockdown\" in the expression of DCD in G-361-pLKO control clone and a G-361-IBC-I clone expressing constitutively short hairpin RNA against DCD mRNA decreased significantly the in vitro growth in cell culture and tumor formation in nude mice. Similar results were obtained treating nude mice bearing G-361 melanoma xenografts with rabbit polyclonal antibodies against DCD protein. Here, we present a DNA microarray-based study that identified the genes that are up- and down-regulated in a G-361-pLKO control clone and a G-361-IBC-I clone expressing constitutively short hairpin RNA against DCD mRNA. A total of 372 genes differentially expressed were identified; being 162 genes up-regulated and 212 genes down-regulated. Bioinformatic studies showed that DCD gene silencing modulates canonical pathways and signaling networks mediated APRIL/BAFF receptors and ligands and NF-kB signaling pathway as well as chromatin remodeling mediated by histone family. The mRNA expression levels of 9 genes of interest were validated by RT-qPCR assays. Next we analyzed the proteins present in nuclear extracts from G-361- pLKO and G-361-IBC-I clones by mass spectrometry. We identified 74 proteins in the G-361-pLKO clone and 31 proteins in the G-361-IBC-I. A group of 21 proteins was identified in both sublineages. Bioinformatics analyses by STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) platform showed that a small portion of the proteins identified only in G-361- pLKO cells was predicted to interact directly with DCD. The network formed by these proteins is centered in the p53 protein, a key regulator of survival and cell death program in response to DNA damage and oxidative stress. On the other hand, this network was completed abrogated using the nuclear protein from G-361-IBC-I because of absence of DCD protein. Since most of the proteins identified in nuclear extracts are of the histone family, it is likely that they are acting in the chromatin-remodeling complexes which are important to remodel nucleosomes of the G-361-IBC-I cells. Our results allowed us to suggest that future studies on the expression of histones and their posttranslational modifications may help to unravel the possible role of DCD in the epigenetic regulation of melanoma and other cancers

Os pacientes com melanomas, em geral, apresentam extrema quimiorresistência e prognóstico ruim, com taxa de sobrevida de aproximadamente seis meses; portanto, novas estratégias terapêuticas são necessárias. As células deste tipo de tumor acumulam alterações na expressão gênica que contribuem para a proliferação descontrolada, evasão de senescência e inibição de morte celular em múltiplas rotas intracelulares. No Capítulo 1, foi explorado o controle epigenético do supressor tumoral RECK. Esse gene é largamente expresso em tecidos normais, com correlação de sua expressão com melhor prognóstico. Em tumores, RECK é inibido, incluindo em melanoma. Neste estudo, sua inibição por hipermetilação do promotor e remodelamento da cromatina por HDACs não foi o único fator inibitório nas linhagens de melanoma analisadas. Mesmo após a utilização a remoção de marcas epigenéticas associadas ao silenciamento gênico, a expressão proteica não pôde ser recuperada nas linhagens utilizadas neste trabalho. No Capítulo 2, isolou-se subpopulações clonais ao acaso a fim de modelar a heterogeneidade tumoral intrínseca. Neste modelo experimental, caracterizamos a presença de dois perfis tumorais subclonais: uma mais proliferativa, invasiva e sensível a vemurafenibe; e outra menos proliferativa, mais resistente e com maior expressão de RECK e fatores ligados a EMT. Nossos resultados, em conjunto, mostraram que as linhagens celulares parentais utilizadas apresentam diferenças entre si quanto à viabilidade celular, indução de apoptose e fosforilação de ERK após o tratamento com vemurafenibe. Segundo o nosso modelo, a resistência intrínseca ao vemurafenibe está presente e reflete a heterogeneidade do tumor inicial. Com estes resultados, pretendemos contribuir para o conhecimento sobre a composição clonal presente na heterogeneidade tumoral, além de contribuir para a função de RECK na biologia do melanoma. / Patients with melanoma often present extreme chemoresistance and poor prognosis, with survival rates of approximately six months; therefore, new therapeutic strategies are necessary. Melanoma cells accumulate genotypic changes that contribute to uncontrolled proliferation, evasion of senescence and cell death inhibition in multiple cellular pathways. In Chapter 1, we have explored the epigenetic control of the tumor suppressor RECK. This gene is widely expressed in normal tissues, with correlation of its expression with better prognosis. In tumors, RECK is inhibited, including melanoma. Here, RECK inhibition by promoter hypermethylation and chromatin remodeling by HDACs was not the only inhibiting factor in the melanoma cell lines analyzed. Even after removing the epigenetic marks associated to gene expression silencing, the protein expression could not be recovered in the cell lines used in this study. In Chapter 2, it has been isolated several tumor clonal subpopulations in order to modulate the intrinsic tumor heterogeneity. In this experimental model, we have characterized the presence of two tumor subclonal profiles: a more proliferative, invasive and sensitive to Vemurafenib; and other less proliferative, less sensitive to Vemurafenib and presenting more expression of RECK and factors associated to EMT. Our results together have showed the parental cell lines used here differed from each other in terms of cell viability, induction of apoptosis, and ERK phosphorylation after treatment with Vemurafenib. According to our model, the intrinsic resistance to Vemurafenib is present and reflects the heterogeneity of the initial tumor. With these results, we intend to contribute to the knowledge of the tumor clonal subpopulations composition in tumor heterogeneity, and contributes to the RECK function in melanoma biology.

Nos últimos anos, o desenvolvimento da computação tem auxiliado profissionais da saúde no tratamento, prevenção e diagnóstico de doenças. Um dos grandes desafios do campo tem sido o desenvolvimento de métodos para a discriminação do câncer de pele do tipo melanoma maligno em relação à outras lesões benignas. Para tal, pesquisadores usam técnicas de processamento e análise de imagens, explorando métricas baseadas na regra ABCD, para o desenvolvimento de métodos de diagnóstico de melanoma através de imagens. Enquanto diversos estudos abordam a coloração e textura do melanoma, um tratamento sistemático da irregularidade dos melanomas ainda não foi relatado. O presente trabalho traz um estudo dos fatores que influenciam a discriminação de lesões melanocíticas malignas e benignas, tomando como referência a assimetria das lesões e as irregularidades contidas em sua borda. Foram coletadas 143 imagens de casos clínicos de melanoma maligno, nevos regulares e nevos atípicos. Após tratamentos iniciais das imagens e posterior segmentação das lesões, extraiu-se 52 métricas referentes ao propósito do trabalho. A visualização da projeção LDA das três categorias revelou boa discriminação entre as categorias melanoma com relação as demais, reforçando a premissa original da acentuada irregularidade dos melanomas. Este resultado foi confirmado pela validação cruzada da projeção, com acertos da ordem de 75% para o grupo melanoma e 54% e 40% para os respectivos grupos nevo regular e nevo atípico. Deste resultado prevê-se uma das aplicações do sistema, na discriminação geral entre melanoma dos demais nevos. Para tal, uniu-se os grupos benignos em uma única categoria para a validação cruzada, gerando cálculos de sensibilidade e especificidade da ordem de 90% e 73% respectivamente. Outro importante resultado foi a comparação desses valores com as informações sobre o diâmetro das lesões. Conclui-se que ambas medidas (borda e tamanho) são igualmente relevantes no diagnóstico do melanoma, reflexo da própria patologia do melanoma, na qual acentuam-se ambas características em relação as demais lesões. Porém, a junção de ambas informações num único processamento não melhora a qualidade do diagnóstico, que nos permite prever que possa ser mais vantajoso proceder duas validações distintas com pesos iguais para o diagnóstico final. Por fim, um teste realizado com clínicos gerais e especialistas em melanoma revelou uma importante contribuição do método no auxílio de triagens ambulatoriais de casos suspeitos, principalmente para médicos com baixa ou nenhuma experiência em diagnóstico de melanoma. / Recent development in computer science have helped health professionals in the heath area in the treatment, prevention and diagnostic of illnesses. One of the leading challenges in this field has been the development of methods for the skin cancer discrimination between the types of malignant melanoma in relation to other benign lesions. For this, researchers have been using techniques of image processing and analysis, exploring metrics based on the ABCD rule, to the development of methods of diagnostic of melanoma through images. While plenty of studies are about the color and texture of the melanoma, a systematic treatment of the irregularity of the melanoma has not been reported yet. The present work presents a study of the factors which influence the discrimination among the malignant melanocytic and benign lesions, having as a reference the asymmetry of the lesions and the irregularities in their edges. It was collected 143 images of clinic cases of malignant melanoma, regular nevus and atypical nevus. After initial treatment of the images, followed by a segmentation of the lesions, it was extracted 52 metrics referent to the purpose of this study. The visualization of the projection LDA in three categories revealed a good discrimination among the categories of melanoma in relation to the others, reinforcing the original premise of the melanomas´ sharp irregularity. This result was confirmed by the cross-validation of the projection, with successes of 75 % to the melanoma group and 54 %-40 % to the regular nevus group and atypical nevus group, respectively. This result predicts one of the applications of the system in the general discrimination between the melanoma and the other nevus. In this regard, it was joined together the benign groups in one category to the cross-validation, generating calculations of sensibility and specificity of 90 % and 73 % respectively. Another important result was the comparison among these metrics with the information about the diameter of the lesions. It was concluded that both measurements (edges and size) are equally relevant in the diagnostic of the melanoma, reflection of the melanoma´s own pathology, in which both characteristics are enhanced in relation to the other lesions. However, the combination of both measurement in only one processing does not improve the quality of the diagnostic, which allows the prediction that it can be more advantageous to produce two distinct validations with the same weighs to the final diagnostic. To sum up, an accomplished test with doctors who are general practioner and specialists in melanoma revealed a significant contribution in the method to help in the triage of medical consultations in suspicious cases, mainly to doctors with little or no experience in the diagnostic of melanoma.

Evidências sugerem que o fator de crescimento de fibroblasto 2 (FGF2), produzido por melanomas, possui importante papel no crescimento tumoral, angiogênese e metástase. Assim, o uso de anticorpo monoclonal (mAb) que reconhece e bloqueia a atividade de FGF2 é uma abordagem a ser considerada em oncologia. O propósito desse estudo foi avaliar a aplicação diagnóstica e terapêutica de um novo anticorpo anti-FGF2, 3F12E7 IgG1, em melanoma experimental B16-F10. Para isso, camundongos C57Bl/6 foram implantados subcutaneamente (ou intravenosamente, para ensaios de metástase) com células de melanoma murino B16-F10 (5x105 células/animal). Quando tumores alcançaram 3-4 mm de diâmetro (ou 24 h pós-inóculo de células B16-F10, no caso de ensaios de metástase), camundongos foram tratados com anti-FGF2 3F12E7 IgG. Animais controle receberam igual volume do veículo ou quantidade de anticorpo controle de isotipo. Grupos: animais tratados com (1) anti-FGF2 3F12E7 IgG1; (2) ligante de CEA IgG1 (controle de isotipo); e (3) veículo. O tratamento dos camundongos portadores de tumor com anti-FGF2 IgG resultou, comparado com os controles salina e de isotipo, em uma redução no número de focos metastáticos nos pulmões (ANOVA, p < 0,05), em ensaios de metástase experimental, bem como em uma menor taxa de crescimento de tumores subcutâneos (n=7/grupo). Esse resultado é acompanhado por uma redução na densidade vascular do tumor, conforme determinado por imunomarcação para CD34 ou CD31. A captação tumoral de anti-FGF2 3F12E7 IgG foi avaliada por métodos de medicina nuclear, usando esse anticorpo radiomarcado com tecnécio-99m. Estudos SPECT/CT in vivo e de biodistribuição ex vivo revelaram que 99mTc-anti-FGF2 3F12E7 IgG pode atingir eficientemente tumores subcutâneos e metastáticos de B16-F10. Assim, esses dados sugerem que anti-FGF2 3F12E7 IgG pode ser uma estratégia antitumoral promissora para melanoma, bem como uma potencial ferramenta de imagem a ser explorada, atuando como um possível traçador para rastrear tumores FGF2-positivos e mapear esse estímulo angiogênico no microambiente tumoral. Aprovado pelo comitê de ética (CAPPesq): número 0942/09 / Compelling evidence suggests that fibroblast growth factor 2 (FGF2), produced by melanomas, plays important role in tumor growth, angiogenesis and metastasis. Therefore, the use of a monoclonal antibody (mAb) that recognizes and blocks FGF2 activity is seen as an approach to be considered in oncology. The purpose of this study was to evaluate the diagnostic and therapeutic application of a new anti-FGF2 antibody, 3F12E7 IgG1, in experimental melanoma B16-F10. For this, C57Bl/6 mice were subcutaneously (or intravenously, for experimental metastasis assay) implanted with murine melanoma B16-F10 cells (5x105 cells/animal). When tumors reached 3-4 mm in diameter (or 24 h after B16-F10 cells injection, in the case of metastasis assay), mice started receiving anti-FGF2 3F12E7 IgG. Control mice received equal volume of vehicle or isotype control IgG amount. Groups: (1) anti-FGF2 3F12E7 IgG1-treated, (2) CEA-binding IgG1-treated (isotype control) and (3) vehicle-treated mice. The treatment of tumor-bearing mice with anti-FGF2 IgG, compared with saline and isotype controls, led to a reduction in the number of metastatic foci in the lungs (ANOVA test, p < 0.05), in experimental metastasis assays, as well as to a lower subcutaneous tumor growth rate (n=7 per group). This result is accompanied by a reduction in the tumor vascular density, as determined by CD34 or CD31 staining. The anti-FGF2 3F12E7 IgG tumor uptake was evaluated by nuclear medicine approaches, using this antibody radiolabeled with technetium-99m. In vivo SPECT/CT and ex vivo biodistribution studies reveled that 99mTc-anti-FGF2 IgG could efficiently achieved B16-F10 subcutaneous and metastatic tumors. Thus, these data suggest that the anti-FGF2 3F12E7 IgG may be a promising antitumor strategy for melanoma, as well as a potential imaging tool to be explored, working as a possible tracer to identify FGF2-positive tumors and map this angiogenic stimulus in the tumor microenvironment. Ethics committee (CAPPesq) approval number 0942/09

O Melanoma é um tipo de neoplasia que se origina de melanócitos normalmente presentes na epiderme. Uma das características do melanoma é a capacidade de adquirir resistência a terapias. As células de melanoma podem aumentar a liberação de vesículas extracelulares (VEs) em resposta ao tratamento com quimioterápicos. A cisplatina (CDDP) e a temozolomida (TMZ) são drogas utilizadas para o tratamento de tumores. Ambas as drogas formam adutos no DNA, mas as vias de sinalização que deflagram a morte celular são distintas. O objetivo desse estudo é investigar a morte celular da linhagem B16-F10 na presença de VEs oriundas de células B16-F10 tratadas com cisplatina CDDP ou TMZ. Inicialmente as VEs oriundas de células de melanoma murino, B16-F10, tratadas com CDDP ou TMZ e seus controles, foram isoladas por ultracentrifugações sucessivas. Para os experimentos in vitro, as células foram tratadas com as drogas em combinação com as respectivas VEs. As amostras foram realizados avaliações de ciclo celular e de morte e ensaio clonogênico. Para os experimentos in vivo, as células B16-F10 foram pré-tratadas com VEs, e posteriormente, as células foram inoculadas via subcutânea em camundongos C57BL/6 e os tumores foram mensurados diariamente. Em nosso estudo concluimos que a metodologia do isolamento de VEs é eficiente. Além disso, observamos que o tratamento com CDDP ou TMZ aumenta a liberação de VEs por células tumorais. Apesar do resultado contraditorio, as VEs liberadas por células tumorais tratadas com quimioterápicos aumentam a capacidade de sobrevivência das células de melanoma in vitro. VEs oriundas de células de melanoma não participam inicialmente da sensibilização à morte de células tumorais causada pelas mesmas drogas, mas a longo prazo, as VEs oriundas de células tratadas com a TMZ podem conferir uma resposta celular de sobrevivência às células tumorais in vitro. In vivo, o resultado é inconclusivo, uma vez que para confirmar se as VEs fazem parte da adaptação tumoral conferindo fenômenos de sobrevivência celular in vivo, é necessário avaliar em outros modelos celulares e animais / Melanoma is a neoplasm derived from melanocytes normally present in the skin specifically in the epidermis. One of the malignancies of melanoma is the ability to acquire chemoresistance. Cisplatin (CDDP) and temozolomide (TMZ) are drugs used for the treatment of tumors. Both drugs can form alkylating adducts in DNA, however, the pathways that trigger cell death are distinct. Tumor cells, including melanoma, may increase the release of extracellular vesicles (EVs) in response to chemotherapeutic treatment. The aim of this study is to investigate the cell death phenomenon in B16-F10 cell line in presence of EVs derived from chemotherapeutic-treated B16-F10 cells. For in vitro experiments, the cells were treated with CDDP or TMZ in combination with EVs from chemotherapictreated samples. For in vivo experiments, B16-F10 cells were exposed to EVs and inoculated subcutaneously in C57BL/6 mice. The growth was measured daily. In this work, we established and characterized VEs released by melanoma cells treated with chemotherapics and we established chemotherapics treatments to isolate EVs for next EVs isolation. Our results showed that CDDP or TMZ treatment increase the release of EVs by tumor cells. The EVs released by melanoma cells after CDDP or TMZ treatment seem to increase the survival capacity of melanoma cells. Thus, we concluded that EVs derived from melanoma cells do not participate in the cell death sensitization induced by CDDP or TMZ. However, EVs derived from TMZ treated cells may offer a survival effect to tumor cells in vitro a long term. In vivo, The result is inconclusive since to confirm how VEs are part of the tumor adaptation conferring cellular survival phenomena in vivo, it is necessary to evaluate in other cellular and animal models

O melanoma é o mais agressivo tumor da pele, cuja resistência aos tratamentos quimioterápicos tem promovido a crescente utilização de imunoquimioterapia, como é o caso da utilização de agonistas dos receptores Toll-Like (TLRs). Nesse contexto, os compostos abreviados por P-MAPA e seu sintético estrutural MRB-CFI-1 com reconhecidas propriedades antitumorais e imunológicas, são fortes candidatos na terapia e prevenção desse tipo de câncer. Esse estudo visa determinar o potencial anticâncer do P-MAPA e de MRB-CFI-1 contra o melanoma murino em consequência ao padrão de resposta microambiental semelhante ao de morte imunogênica, em regimes de tratamento terapêutico ou vacinal, na vigência de quimioterapia com cisplatina e/ou em associação com antígenos de células tumorais totais. Após avaliação In vivo do crescimento de tumores B16F10 implantados em modelos murinos selvagem e nocaute para o gene Myd88, na vigência ou não do tratamento com cisplatina e/ou P-MAPA, nossos resultados mostraram que o P-MAPA apresentou atividade pró-tumoral e antagonizou a ação da cisplatina em inibir o crescimento dos tumores, de forma dependente de Myd88. Além disso, através de análises qualitativa e quantitativa pelo software ImageJ em fotomicrografias de secções tumorais coradas histologicamente, observamos que o P-MAPA promoveu mudanças microambientais nos tumores que podem impactar negativamente em seu desempenho. Como monoterapia em esquema de vacinação com lisado tumoral total em combinação com quimioterapia, o P-MAPA em dose baixa falhou em suprimir o crescimento de tumores B16F10, mas o seu sintético MRB-CFI-1 foi capaz de prevenir o crescimento desse tipo de melanoma num regime de vacinação profilática. Apesar do sucesso terapêutico desse imunomodulador em diversos modelos de câncer e de doenças infecciosas, o P-MAPA não foi eficaz em produz respostas microambientais contra o melanoma murino, dados esses que limitam a aplicabilidade clínica do composto. De outro modo, o composto fosfato inorgânico MRB-CFI-1 foi protetivo em retardar o aparecimento desse tipo de doença. Assim, o presente estudo foi importante por ampliar o entendimento funcional do P-MAPA numa abordagem imunoquimioterápica em modelos biológicos de tumores de melanoma, e representa uma importante mudança na utilização de constituintes individuais similares ao P-MAPA que sejam mais eficazes, de fácil obtenção, e de produção controlada e garantida / Melanoma is the most aggressive skin cancer, whose resistance to chemotherapeutic treatments has promoted the increasing use of immunochemotherapy, as is the case for the use of Toll-Like receptor agonists (TLRs). In this context, the compounds abbreviated by P-MAPA and its structural synthetic MRB-CFI-1 with recognized antitumor and immunological properties are strong candidates in the therapy and prevention of this type of cancer. This study aims to determine the anti-cancer potential of P-MAPA and MRB-CFI-1 against murine melanoma as a consequence of the microenvironmental response pattern similar to that of immunogenic death in therapeutic or vaccine treatment regimens when using chemotherapy with cisplatin alone or in combination with whole tumor cell antigens. After In vivo evaluation of the growth of B16F10 tumors implanted in wild-type and Myd88 gene knockout mice, under treatment or not with cisplatin and / or P-MAPA, our results showed that P-MAPA showed pro-tumor activity and antagonized the action of cisplatin in inhibiting the growth of tumors in a Myd88-dependent manner. In addition, using qualitative and quantitative analysis by ImageJ software in histological images of tumor sections, we observed that P-MAPA promoted microenvironmental changes in tumors that may negatively impact its performance. As monotherapy in vaccination schedule with total tumor lysate in combination with chemotherapy, low dose P-MAPA failed to suppress the growth of B16F10 tumors, but its synthetic MRB-CFI-1 was able to prevent the growth of this type of melanoma in prophylactic vaccination regimen. Despite the therapeutic success of this immunomodulator in various cancer models and infectious diseases, P-MAPA has not been effective in producing microenvironmental responses against murine melanoma, data that limit the clinical applicability of the compound. Otherwise, the inorganic phosphate compound MRB-CFI-1 was protective in delaying the onset of this type of disease. Thus, the present study was important because it broadened the functional understanding of P-MAPA in an immuno-chemotherapeutic approach in biological models of melanoma tumors and represents an important change in the use of individual constituents similar to P-MAPA that are more efficient, easily obtainable, and controlled and guaranteed production

Neste estudo foram avaliados os efeitos antitumorais da fosfoetanolamina sintética (FS) em modelos de tumores experimentais e as vias de sinalizações envolvidas nesta atividade. In vitro a FS foi citotóxica para as linhagens de células tumorais de melanoma humano, SK-MEL-28, carcinoma renal murino RENCA e para as células do carcinoma de pulmão de não pequenas células NSCLC. Alterações ultraestruturais como a condensação da cromatina, formação de blubes de membranas e mitocôndrias eletrodensas foi acompanhada pela redução do potencial elétrico mitocondrial. Aumento da ativação da caspase 3 e 8 e da expressão da proteína Bax, além da redução da CDK9 e CDK4/6, também foram observados. A redução da migração e proliferação das células endoteliais HUVEC induzida pela FS foi associada com a modulação da expressão do VEGF-A. resultando na inibição in vitro da formação do tubo endotelial. In vivo a FS foi capaz de inibir o crescimento dos tumores sólidos do melanoma e do carcinoma renal murino de forma superior aos quimioterápicos Dacarbazina (DITC) e ao Sunitinib. No modelo de metástase pulmonar utilizando as células RENCA, a FS reduziu o número de nódulos metastáticos pulmonares e aumentou a taxa de sobrevida dos animais. Os efeitos terapêuticos da FS também foram avaliados no modelo de leucemia promielocítica aguda (LPA) transplantada em camundongos NOD/Scid. O tratamento com a FS reduziu o número de blastos periféricos e infiltrados na medula óssea e nos parênquimas, esplênico e hepático. De forma superior a Daunorrubicina (DA) e ao Ácido all-trans retinóico (ATRA), a FS induziu apoptose nos clones malignos que expressão CD34+, bem como nas células CD117+/Gr-1+. Este conjunto de resultados mostra que a FS é um composto promissor na terapêutica contra neoplasias / In this study, we evaluated the antitumor effects of synthetic phosphoethanolamine (FS) in an experimental tumor model and the signaling pathways involved in this activity. In vitro, FS was cytotoxic to tumor cell lines of human melanoma, SK- MEL-28, renal carcinoma murine, Renca, and for non-small cell lung cancer, NSCLC. Ultrastructural changes such as chromatin condensation, blubes formation on membranes and electrodense mitochondria were accompanied by a reduction of the mitochondrial electric potential. Increased activation of caspase-3, 8 and Bax protein expression, in addition to a reduction of CDK9 and CDK4/6 levels, was also observed. The reduction of migration and proliferation of endothelial cells, HUVEC, induced by FS was associated with the modulation of VEGF-A-expression, resulting in the in vitro inhibition of tubulogenesis. In vivo, FS was able to inhibit the growth of solid tumors of melanoma and renal carcinoma more potently than Dacarbazine (DITC) and Sunitinib. In the lung metastasis model, using RENCA cells, FS reduced the number of metastatic lung nodules and increased the survival rate of the animals. The therapeutic effects of FS were also evaluated in the model of acute promyelocytic leukemia transplanted in mice NOD/Scid. The treatment with FS reduced the number of peripheral blasts and infiltrates in the blood marrow, splenic and hepatic parenchyma. Fs was more potent than Daunorrubicine (DA) and all-trans retinoic acid (ATRA) in the induction of apoptosis in the malignant clones expressing CD34+ as well as CD117+/Gr-1+ cells. Taken together, theses results show that FS is a promising compound for the therapy against tumors