An early diagnosis of melanoma is essential to reduce mortality of patients. The diagnosis is also fundamental to predict the outcome of patients and to select the most adapted treatment. Current diagnostic assessments are obtained after visual inspection of the histological section of the primary tumor. The pathologist has first to determine the malignant nature of the lesion and then to assess the potential of the lesion to form metastases. Depending on several characteristics of the primary tumor (mainly tumor thickness, ulceration and mitotic rate), the sentinel node is surgically removed and the detection of tumor cells is based on its histopathological examination. These assessments are time consuming, to some degree subjective and are particularly challenging. Among melanoma patients that are not subject to sentinel node surgery, 6.5 % will develop metastases while 20 % of patients that undergo sentinel node surgery will effectively present metastases. The search for biomarkers that can identify malignant cells, evaluate potential of invasion or help selecting a treatment is still on.<p>In this thesis we used a new and promising technique of imaging based on infrared spectroscopy to study melanoma primary tumors and metastatic lymph nodes. Infrared spectroscopy brings information on the biochemical composition of the main components of the cells. When combined with a microscope and with multivariate statistical analyses, images that are generated allow the identification of melanoma cells and stromal cells in the biopsy. We also focused on the immune infiltration as it was shown to carry an important prognosis value for melanoma patients.<p>The first part of the thesis was a prerequisite for the rest of the study. It addresses the effects of the process of fixation that tissues obtained by surgical resection undergo for their long term preservation. In chapter III, we showed that Formalin-Fixation and Paraffin-Embedding (FFPE) procedure induces small but significant modifications in the infrared spectra of cells but these are very similar for different cell lines. In turn, it preserves the potential to identify closely-related cell lines by infrared spectroscopy.<p>We thus pursued our study on primary melanomas. In chapter IV, we first developed an automatic tool capable of identifying melanoma cells and the main cells of the tumor microenvironment in tissue sections. Importantly, we built a second model that brings information on the presence of metastases on the basis of the spectral signature of the primary tumor.<p>The next chapter is dedicated to the prediction of the response of melanoma to dacarbazine, the first-line chemotherapy to treat stage IV patients. Infrared spectra of the primary tumor were shown to contain information capable of predicting whether dacarbazine will be a useful treatment.<p>In the last two chapters, we focused on lymphocytes. In chapter VI, we first demonstrated that helper and cytotoxic T cells purified from peripheral blood can be identified on the basis of their infrared signature. Then, in chapter VII we investigated metastatic lymph nodes. We created different statistical models using infrared spectra that first identified the melanoma cells invading the lymph nodes and secondly, the different subpopulations of lymphocytes (B and T cells).<p>In conclusion, we developed an automatic and reliable tool of imaging to help pathologists in the anatomopathological assessment of primary lesions and lymph nodes./Le mélanome est la forme de cancer cutané la plus mortelle, provoquant environ 80% des décès dus à un cancer de la peau. Lorsque le mélanome est localisé, la chirurgie est le principal traitement et est suffisante pour 80% des patients. A l’opposé, lorsque le mélanome primaire a formé des métastases, le cancer devient beaucoup plus difficile à traiter et la survie de ces patients diminue drastiquement. Seuls 10% des patients vont vivre 5 ans lorsqu’ils développent des métastases à distance. C’est pourquoi un diagnostic précoce est essentiel pour diminuer la mortalité causée par le mélanome. L’étape du diagnostic est également très importante pour donner un pronostic et pour planifier les traitements. Le diagnostic actuel du mélanome est basé sur l’analyse en microscopie optique de sections de la tumeur primaire. Sur base de la morphologie cellulaire et de l’architecture du tissu, cette étape permet premièrement d’identifier le caractère cancéreux de la lésion et deuxièmement d’évaluer son potentiel métastatique. Une analyse du ganglion sentinelle permet également de détecter la présence de métastases. Dans le cadre de cette thèse, nous proposons d’utiliser une nouvelle technique d’imagerie basée sur la spectroscopie infrarouge qui apporte une information complète et unique sur la biochimie de la cellule et des tissus. Les résultats présentés ici indiquent que lorsque les spectres infrarouges sont combinés à des analyses statistiques multivariées, des images sont reconstituées et révèlent les structures et les composants cellulaires majeurs présents dans les coupes de mélanomes. Une étude préliminaire a d’abord pu démontrer que la fixation au formol subies par les biopsies pour les conserver n’entrave pas l’étude de celles-ci par spectroscopie infrarouge. Nous nous sommes alors intéressés aux tumeurs primaires de mélanome et avons développé un modèle statistique, à partir des spectres infrarouges, identifiant automatiquement les cellules de mélanome dans le tissu ainsi que les autres cellules du microenvironnement de la tumeur. Dans ce chapitre, nous avons également créé un autre modèle statistique capable de prédire le potentiel métastatique de la tumeur primaire en se basant sur sa signature spectrale. Nous avons ensuite mis en évidence une signature spectrale corrélée à la réponse à la dacarbazine chez des patients traités pour leurs métastases. Nous avons également montré que des sous-populations de lymphocytes purifiées d’échantillons sanguins pouvaient être identifiées sur base de leur spectre. Cette capacité de la spectroscopie infrarouge à distinguer les différents types de lymphocytes a ensuite été démontrée pour les lymphocytes infiltrant les métastases. Finalement, nous avons mis en évidence l’utilité de cette technique d’imagerie pour la détection de métastases ganglionnaires.<p> / Doctorat en sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

Made available in DSpace on 2014-07-29T15:16:34Z (GMT). No. of bitstreams: 1 Mestrado Paula.pdf: 2508093 bytes, checksum: eaf6dc0cbce3c8dd66202b6bce9a4939 (MD5) Previous issue date: 2008-05-21 / The thiosemicarbazones are chemical compounds of considerable scientific interest due to their important biological properties, such as antitumoral, antibacterial, antiviral, and antiprotozoal, among others. The studied compounds have the natural monoterpene camphene in the R4 position and the benzaldehyde in R2, with the substitution in the ring for different (R) groups, making this work unique. In this way, thirteen different compounds were derived from the benzaldehyde camphene thiosemicarbazone: camphene isotyiocyanate, camphene thiosemicarbazide, benzaldehyde camphene thiosemicarbazone, p-metyl benzaldehyde camphene thiosemicarbazone, p-methoxy benzaldehyde camphene thiosemicarbazone, o-chloro benzaldehyde camphene thiosecarbazone, m-chloro benzaldehyde camphene thiosemicarbazone, p-chloro-benzaldehyde camphene thiosemicarbazone, o-nitrobenzaldehyde camphene thiosemicarbazone, m-nitro- benzaldehyde camphene thiosemicarbazone, p-nitrobenzaldehyde camphene thiosemicarbazone, p-dimetylamino benzaldehyde camphene thiosemicarbazone and p-hydroxy benzaldehyde camphene thiosemicarbazone. The experiments were conducted in vitro using human melanoma cells (SK-Mel-37), because the melanoma is the most serious type of skin cancer due to its high possibility to metastasize and its resistance to the induction of apoptosis by antineoplastic drugs. We first performed a visual screening to observe if there was detachment of more than 85% of the total number of cells, after treatment with the sole concentration of 100 &#956;M. Thus, six compounds were selected: benzaldehyde camphene thiosemicarbazone, m-chloro benzaldehyde camphene thiosemicarbazone, m-nitro-benzaldehyde camphene thiosemicarbazone, p-dimetylamino benzaldehyde camphene thiosemicarbazone, p-hydroxy benzaldehyde camphene thiosemicarbazone e p-methoxy benzaldehyde camphene thiosemicarbazone. The morphological analysis of the treated cells, by inverted phase contrast microscope and Giemsa stain, showed intense cellular vacuolization and nuclear fragmentation. In the cellular viability test, through the MTT colorimetric assay, the compounds showed IC50 (inhibitory concentration for 50% of the cells) values between 12, 84 &#956;M and 32, 18 &#956;M, which are bellow of those described in the literature for compounds with antineoplastic action. The analysis of nuclear fragmentation by fluorescence microscopy was performed by the incorporation of propidium iodide, and it was observed, at different time points, a progressive increase of cellular damage and a pronounced nuclear fragmentation after the cellular detachment. The analysis of the DNA fragmentation by agarose gel electrophoresis revealed that the unequivocal cytotoxic action of the compounds occurred by apoptosis. Our results showed that six compounds derived from the benzaldehyde camphene thiosemicarbazones had cytotoxic activity in human melanoma cells (SK-Mel-37) in vitro, and constitute potential candidates for future analysis aiming its therapeutic application. / As tiossemicarbazonas são compostos de considerável interesse científico devido às suas importantes propriedades biológicas, tais como antitumoral, antimicrobiana, antiviral, antiprotozoária, dentre outras. Os compostos estudados apresentam o monoterpeno natural canfeno na posição R4 e o benzaldeído em R2, com substituição no anel por diferentes grupos (R), o que confere ao presente trabalho um caráter inédito. Dessa forma, foram apresentados para este estudo onze diferentes compostos derivados de benzaldeído canfeno tiossemicarbazona: benzaldeído canfeno tiossecarbazona, p-metil benzaldeído canfeno tiossemicarbazona, p-metóxi benzaldeído canfeno tiossemicarbazona, o-cloro benzaldeído canfeno tiossemicarbazona, m-cloro benzaldeído canfeno tiossemicarbazona, p-cloro benzaldeído canfeno tiossemicarbazona, onitro benzaldeído canfeno tiossemicarbazona, m-nitro benzaldeído canfeno tiossemicarbazona, p-nitro benzaldeído canfeno tiossemicarbazona, p-dimetilamino benzaldeído canfeno tiossemicarbazona e p-hidróxi benzaldeído canfeno tiossemicarbazona, além de isotiocianato canfeno e canfeno tiossemicarbazida. Os experimentos foram realizados in vitro utilizando-se células de melanoma humano (SKMel- 37), visto que o melanoma é o tipo mais grave de câncer de pele devido à sua alta possibilidade de metástase e resistência à indução de apoptose por drogas antineoplásicas. Inicialmente, realizamos um screening visual observando-se o desprendimento de mais de 85% do total das células, após tratamento com a concentração única de 100 &#956;M. Dessa análise, foram selecionados seis compostos: benzaldeído canfeno tiossemicarbazona, mcloro- benzaldeído canfeno tiossemicarbazona, m-nitro-benzaldeído canfeno tiossemicarbazona, p-dimetilamino-benzaldeído canfeno tiossemicarbazona, p-hidróxibenzaldeído canfeno tiossemicarbazona e p-metóxi benzaldeído canfeno tiossemicarbazona. A análise morfológica das células tratadas e coradas com Giemsa e observadas através de microscópio óptico invertido em contraste de fase demonstrou intensa vacuolização celular e fragmentação nuclear. Na análise da viabilidade celular, através do ensaio colorimétrico do MTT, os compostos apresentaram valores de IC50 (Concentração Inibitória para 50% das células) entre 12,84 &#956;M e 32,18 &#956;M, valores que se encontram abaixo do descrito na literatura para compostos com ação antineoplásica. A análise da fragmentação nuclear através de microscopia de fluorescência foi realizada pela incorporação de iodeto de propídio. Observamos, em diferentes tempos, o aumento progressivo dos danos celulares e a fragmentação nuclear pronunciada após o desprendimento das células. A análise de fragmentação de DNA por eletroforese em gel de agarose revelou de maneira inequívoca que a ação citotóxica observada ocorre por apoptose. Os resultados obtidos demonstraram que seis compostos derivados do benzaldeído canfeno tiossemicarbazonas apresentaram atividade citotóxica em células de melanoma humano (SK-Mel-37) in vitro, e são candidatos promissores para as análises futuras visando à sua aplicação terapêutica.

La sensibilité des cellules de mélanomes aux molécules de thérapies ciblées dépend du microenvironnement tumoral (interactions cellule-cellule et cellule-matrice extracellulaire). Les systèmes tridimensionnels (3D) de culture in vitro reflètent mieux l’architecture structurelle native des tissus et sont attrayants pour l’étude des interactions cellulaires. Nous avons développé et comparé plusieurs modèles de mélanome métastatique : les cellules de mélanomes (SK-MEL-28 et SK-MEL-3, mutées BRAF V600E et SK-MEL-2, BRAF sauvages) cultivées en monocouche (2D) et co-cultivées en 3D sur des équivalents de derme avec des fibroblastes, afin de mieux comprendre les facteurs modulant la sensibilité cellulaire à un inhibiteur de BRAF (BRAFi, Vémurafenib) et au Vémurafenib associé à un inhibiteur de MEK (MEKi, Cobimetinib). La sensibilité cellulaire aux traitements a été évaluée sous différents aspects : prolifération cellulaire (numération cellulaire, incorporation d'EdU, test MTS), analyse des voies de signalisation MAPK et PKB / Akt (Western-blot), apoptose (TUNEL), libération de cytokines et de facteurs de croissance (ELISA) et histologie (modèles 3D). Un effet cytostatique de BRAFi a été observé sur les cellules SK-MEL-28 et SK-MEL-3 cultivées dans les modèles 2D et 3D. La lignée cellulaire SK-MEL-2 était résistante au BRAFi lorsqu'elle a été cultivée en monocouche, mais sensible lorsqu'elle a été co-cultivée avec des fibroblastes incorporés dans une matrice de collagène de type I. Les milieux conditionnés par les fibroblastes 3D (équivalents de derme) ont sensibilisé les cellules SK-MEL-2 (2D) au BRAFi. L'analyse des surnageants de culture cellulaire a révélé que les équivalents de derme libéraient certains facteurs solubles (IL-6, IL-8, HGF, TGF-β) : ces sécrétions ont été modifiées au cours du traitement par Vémurafenib. La combinaison du traitement avec MEKi a renforcé l'action du Vémurafenib sur les cellules de mélanomes métastatiques tout en diminuant la capacité de prolifération des fibroblastes. Des populations de cellules contenant des cellules de mélanomes ou des fibroblastes associés au cancer (CAFs) ont été isolées à partir d'une biopsie de métastase cutanée provenant d'une patiente atteinte d'un mélanome métastatique. Ces cellules ont permis de réaliser des modèles de mélanome métastatique patient-spécifique afin d’étudier in vitro la sensibilité des cellules de la patiente aux traitements dans un microenvironnement tumoral (sécrétion paracrine de cellules stromales et matrice de collagène). Ces modèles prédictifs 3D patient-spécifique pourront être utilisés pour déterminer des stratégies de thérapies personnalisées, ainsi que pour comprendre les phénomènes de résistance des cellules de mélanomes aux traitements. / Melanoma cell sensitivity to targeted therapy molecules is dependent on the tumor microenvironment (cell-cell and cell-extracellular matrix interactions). Three dimensional (3D) in vitro cell culture systems better reflect the native structural architecture of tissues and are attractive to investigate cellular interactions. We have developed and compared several metastatic melanoma models: melanoma cells (SK-MEL-28 and SK-MEL-3, BRAF V600E mutant and SK-MEL-2 BRAF wt) cultured as a monolayer (2D) and co-cultured on 3D dermal equivalents with fibroblasts to better unravel factors modulating cell sensitivity to a BRAF inhibitor (BRAFi, Vemurafenib) and a BRAFi combined with a MEK inhibitor (MEKi, Cobimetinib). Cell sensitivity to treatments was evaluated under various aspects: cell proliferation (cell counting, EdU incorporation, MTS assay), MAPK and PKB/Akt signaling pathway analysis (Western-blotting), apoptosis (TUNEL), cytokine and growth factor release (ELISA) and histology (3D models). A cytostatic effect of BRAFi was observed on SK-MEL-28 and SK-MEL-3 cells in both models. SK-MEL-2 cell line was clearly resistant to BRAFi when cultured as a monolayer but not when co-cultured with 3D fibroblasts embedded in a type I collagen matrix. Conditioned media provided by 3D fibroblasts (dermal equivalents) underlined 2D SK-MEL-2 sensitivity to BRAFi. Cell culture supernatant analysis revealed that dermal equivalents released some soluble factors (IL-6, IL-8, HGF, TGF-β): these secretions were modified during vemurafenib treatment. The combination of treatment with MEKi enhances the action of Vemurafenib on metastatic melanoma cells while decreasing the proliferation capacity of fibroblasts. Cell populations containing melanoma cells or fibroblasts associated with cancer (CAFs) were isolated from a cutaneous metastasis biopsy of a patient with metastatic melanoma. These cells allowed the realization of patient-specific models of metastatic melanoma in order to study in vitro the sensitivity of the patient’s melanoma cells to treatments in a tumor microenvironment (paracrine secretion of stromal cells and collagen matrix). These 3D predictive patient-specific models could be used to determine personalized therapy strategies, as well as to understand the resistance phenomena of melanoma cells to treatments

In the present investigation peripheral blood lymphocytes from patients with inflammatory bowel disease (IBD) and different cancer states were treated with various agents and compared with lymphocytes from healthy control individuals (HCI) treated in the same way and measured in the Comet assay. For inflammatory bowel disease, patient's responses in IBD patients treated with H2O2 were higher than in HCI and Crohn's patients (CD) were found to have higher responses than Ulcerative colitis (UC) patients. The responses for all IBD and HCI were all reduced in the presence of chaga mushroom extract which behaved in an antioxidant manner. A second group of IBD patients were treated with the heterocyclic amine (food mutagen), IQ and H2O2 and responses were reduced in the presence of the flavonoids, quercetin and epicatechin and compared with HCI similarity treated. In all cells responses were reduced with flavonoids and again CD had higher responses than the UC patients and IBD patients higher than HCI. The responses with CD and UC were that confirmed in two independent studies with IBD, one with chaga mushroom extract and the other with flavonoids. Peripheral lymphocytes from malignant melanoma and suspected melanoma patients and colon cancer and polyposis patients were compared to the lymphocytes from HCI and treated with UVA. There were differential sensitivities when measured in the micronucleus and Comet assays. The cancer patients had higher responses than those in the precancerous states and they in turn were higher than responses in HCI. In all the studies, untreated baseline DNA damage values were also higher in IBD and cancer patients and pre-cancerous patients than HCIs. This would suggest that baseline frequencies of different diseases compared to controls could be an important biomarker in the diagnosis of pre-cancers and early stage cancers. Also peripheral lymphocytes are a useful surrogate for cancers and pre-cancerous disease states since, blood is present in all organs and tissues and DNA is basically the same in all cells.

The aim of the study was to evaluate the expression of immunosuppressive (FOXP3, NKG2A), and cytotoxic (perforin) or cytotoxic / immunomodulating (IFNγ) T-cell properties representing biomarkers in the peripheral blood CD8h T-cell population of patients with advanced renal cell carcinoma (RCC) or high risk cutaneous melanoma and healthy controls by multicolour flow cytometry. Determination of the percentage of functionally competing T-cell subsets (especially immunosuppressive) in the CD8hCD57+ T-cell subpopulation in future may serve as one of parameters enabling to assess the overall status of antitumor immune response and select cancer patients most suitable for antitumor immunotherapy while dismissing those to whom it would be ineffective or even harmful. / Darbo tikslas buvo įvertinti imunosupresines (FOXP3, NKG2A), citotoksines (perforin) bei citotoksines / imunomoduliuojančias (IFNγ) savybes atspindinčių žymenų raiškos skirtumus išplitusiu inkstų vėžiu ar didelės rizikos odos melanoma sergančių pacientų periferinio kraujo CD8h T limfocitų populiacijoje, lyginant su kontroline grupe. Skirtingas T limfocitų savybes atspindinčių žymenų raiška buvo tiriama tėkmės citometrijos būdu. Nustatyta, kad inkstų vėžiu ar odos melanoma sergančių pacientų periferiniame kraujyje Įvairių subpopuliacijų (ypač imunosupresinės) nuošimčio nustatymas CD8hCD57+ T limfocitų populiacijoje ateityje gali būti naudingas klinikinėje praktikoje, individualizuojant priešnavikinę imunoterapiją ir selektyviai parenkant tik tuos pacientus, kuriems imuninės sistemos aktyvinimas sukeltų navikinių ląstelių naikinimą, o ne dar labiau gilintų imunosupresiją.

Darbo tikslas buvo įvertinti imunosupresines (FOXP3, NKG2A), citotoksines (perforin) bei citotoksines / imunomoduliuojančias (IFNγ) savybes atspindinčių žymenų raiškos skirtumus išplitusiu inkstų vėžiu ar didelės rizikos odos melanoma sergančių pacientų periferinio kraujo CD8h T limfocitų populiacijoje, lyginant su kontroline grupe. Skirtingas T limfocitų savybes atspindinčių žymenų raiška buvo tiriama tėkmės citometrijos būdu. Nustatyta, kad inkstų vėžiu ar odos melanoma sergančių pacientų periferiniame kraujyje Įvairių subpopuliacijų (ypač imunosupresinės) nuošimčio nustatymas CD8hCD57+ T limfocitų populiacijoje ateityje gali būti naudingas klinikinėje praktikoje, individualizuojant priešnavikinę imunoterapiją ir selektyviai parenkant tik tuos pacientus, kuriems imuninės sistemos aktyvinimas sukeltų navikinių ląstelių naikinimą, o ne dar labiau gilintų imunosupresiją. / The aim of the study was to evaluate the expression of immunosuppressive (FOXP3, NKG2A), and cytotoxic (perforin) or cytotoxic / immunomodulating (IFNγ) T-cell properties representing biomarkers in the peripheral blood CD8h T-cell population of patients with advanced renal cell carcinoma (RCC) or high risk cutaneous melanoma and healthy controls by multicolour flow cytometry. Determination of the percentage of functionally competing T-cell subsets (especially immunosuppressive) in the CD8hCD57+ T-cell subpopulation in future may serve as one of parameters enabling to assess the overall status of antitumor immune response and select cancer patients most suitable for antitumor immunotherapy while dismissing those to whom it would be ineffective or even harmful.

Apesar de vários avanços no combate ao câncer, a incidência do câncer de pele tipo melanoma e a mortalidade relacionada a essa doença tem aumentado. Considerando-se que a radiação ultravioleta (UV) é o principal agente causador de diversos danos à pele, inclusive o câncer de pele, é de grande importância medir de forma adequada as propriedades fotoprotetoras dos filtros solares. Diante dos problemas provocados pelo uso de filtros solares, as pesquisas têm se voltado no sentido de encontrar produtos naturais com propriedades antioxidantes visto que já foi demonstrado que muitos desses agentes naturais possuem efeitos anticarcinogênico, e antimutagênico. Assim, um dos objetivos desse trabalho foi a avaliação de três formulações fotoprotetoras comerciais por meio de parâmetros bioquímicos não comumente utilizados, mas que refletem o efeito dessas formulações no sistema antioxidante natural da pele e também no processo inflamatório induzido pela radiação UV. Esses efeitos foram mensurados in vivo em camundongos sem pelos, utilizando-se como marcadores o antioxidante não enzimático glutationa reduzida (GSH), enzima antioxidante superóxido dismutase (SOD), a enzima mieloperoxidase (MPO) e as citocinas IL-1? e TNF-?. Outro importante objetivo desse estudo foi avaliar o efeito de um extrato de soja biotransformado (ESB) pelo fungo A. awamori em células de melanoma humano das linhagens 451LU e A375, altamente metastáticas, e estudar os mecanismos de ação pelos quais o extrato induz a morte celular por apoptose nestas células e também avaliar o potencial desse extrato para a prevenção e tratamento do câncer de pele. Os resultados mostraram que com relação ao processo inflamatório induzido pela radiação UVB, verificou-se que o FPS, apesar de avaliar somente o eritema, é um bom indicador do nível de proteção da pele, uma vez que todas as formulações apresentaram um potencial protetor contra o aumento da atividade da MPO e liberação das citocinas pró-inflamatórias IL-1? e TNF-?. No entanto, as formulações não forneceram proteção contra a depleção do antioxidante endógeno GSH e, apesar de possuírem mesmo FPS 15 essas formulações apresentaram diferentes níveis de proteção com relação à redução da atividade da enzima SOD induzida pela radiação UV. Os resultados referentes ao estudo do ESB mostraram que o tratamento das células de melanoma altamente invasivas com o extrato resultou em inibição do crescimento/viabiliade das células associado com a indução de apoptose. As análises revelaram que o ESB resultou em indução da clivagem de PARP e ativação das caspases-3, -7 e -8; aumento da expressão de TNF-R2 e da expressão de TRAIL e do receptor DR4. Além disso, o tratamento das células de melanoma com o extrato ESB aumentou a fosforilação e ativação de IKK, degradação de I?B? e translocação de p65/NF?B para o núcleo. Apesar de ser geralmente aceito que a ativação do NF-?B seja responsável pela resistência à apoptose, neste estudo foi demonstrado que a estimulação da via do NF-?B é necessária pela indução da apoptose mediada pelo extrato ESB. Finalmente, conclui-se que as formulações fotoprotetoras devem ser avaliadas de forma mais profunda, empregando-se diferentes métodos a fim de se garantir formulações mais eficazes tanto contra os danos induzidos tanto pela radiação UVB quanto pela radiação UVA. Além disso, esses estudos identificaram uma atividade anticâncer do extrato ESB que é altamente relevante para a quimioprevenção/quimioterapia contra o câncer de pele tipo melanoma. / Despite several advances in fighting cancer, the incidence of melanoma type skin cancer and the mortality related to this disease have increased. Considering that ultraviolet radiation (UV) is the main causative agent of various skin damages, including skin cancer, it is of great importance to adequately measure the photoprotective properties of sunscreens. Considering the problems caused by the use of sunscreens, researches have been focused towards finding natural products with antioxidant properties as it has been shown that many of these natural agents possess anticarcinogenic and antimutagenic effects. Thus, an objective of this study was to evaluate three marketed photoprotective formulations through biochemical parameters not commonly used, but which reflect the effect of these formulations on the skin\'s natural antioxidant system and also in the inflammatory process induced by UV radiation. These effects were measured in vivo in hairless mice, employing as markers reduced glutathione (GSH), a non-enzymatic antioxidant; superoxide dismutase (SOD), an antioxidant enzyme; myeloperoxidase (MPO) enzyme and IL-1? and TNF-? cytokines. Another important objective of this study was to evaluate the effect of a biotransformed soy extract (BSE) by the A. awamori fungus against 451LU and A375 human melanoma cell strains, highly metastatic, and to study the action mechanisms by which the extract induces cell death by apoptosis in these cells; in addition it was intended to evaluate the potential of this extract for the prevention and treatment of skin cancer. The results demonstrated that regarding the inflammatory process induced by UVB irradiation, it was found that the FPS, although only assessing the erythema, is a good indicator of the level of skin protection, since all formulations showed a protector potential against the increased MPO activity and the release of IL-1? and TNF-? pro-inflammatory cytokines. Nevertheless, the formulations did not provide protection against the depletion of the GSH endogenous antioxidant and, despite having the same nominal SPF (SPF=15), these formulations showed different levels of protection with respect to the reduction of SOD activity induced by UV radiation. The results of the BSE study demonstrated that the treatment of highly invasive melanoma cells with the extract resulted in the cell growth/viability inhibition associated with the induction of apoptosis. The analysis revealed that BSE resulted in induction of PARP cleavage and activation of caspase-3, -7 and -8, increased expression of TNF-R2 and expression of TRAIL and DR4 receptor. Furthermore, the treatment of melanoma cells with BSE extract increased phosphorylation and activation of IKK, degradation of I?B? and translocation of p65/NF?B to the nucleus. Although it is generally accepted that the activation of NF-kB is responsible for resistance to apoptosis, this study demonstrated that the stimulation of NF-?B is required for the induction of apoptosis mediated by BSE extract. Finally, it is concluded that the photoprotective formulations should be more deeply evaluated, using different methods in order to ensure more effective formulations against damages induced both by UVB radiation and UVA radiation. In addition, these studies identified an anticancer activity of the BSE extract that is highly relevant to chemoprevention/chemotherapy against melanoma type skin cancer.

O melanoma maligno é a forma mais agressiva dos tumores cutâneos. Sendo o responsável por mais de 75% das mortes relativas á este tipo de câncer. O principal quimioterápico utilizado no tratamento do melanoma é a dacarbazina (DTIC), entretanto, as taxas de resposta são insatisfatórias. O composto DM-1 é um análogo estrutural da curcumina, e por esta razão possui propriedades biológicas semelhantes, como agente antiproliferativo e próapoptótico. A terapia de captura de nêutrons por boro (BNCT) atua por meio da deposição do isótopo 10Boro nas células tumorais e após a irradiação de nêutrons térmicos há produção de partículas alfa e lítio que destroem a célula. Neste trabalho estudou-se o mecanismo de ação destas três terapias, DTIC, DM-1 e BNCT no tratamento do melanoma e seus efeitos em células normais in vitro com a finalidade de obtenção de modalidades terapêuticas diferentes para o tratamento desta neoplasia. A IC50 foi obtida pela metodologia de MTT, além da análise da progressão do ciclo celular e marcadores de morte celular por citometria de fluxo. O composto DM-1 e a BNCT apresentaram efeito citotóxico seletivo para as linhagens de melanoma, com alta produção de radicais livres peroxidados. Nas mesmas condições, estes efeitos foram mínimos em células normais, diferente do tratamento com DTIC. Houve diminuição da proporção de matriz extracelular e colágeno solúvel sintetizado em células de melanoma tratadas com DM-1, BNCT e DTIC, entretanto, o quimioterápico ocasionou isoladamente diminuição também em células normais. O potencial elétrico mitocondrial das células de melanoma foi diminuído nos três protocolos de tratamento, assim como houve aumento na quantidade de DNA fragmentado. Este efeito não foi encontrado em células normais tratadas com DM-1 e BNCT. O composto DM-1 foi capaz de induzir apoptose via intrínseca e extrínseca, avaliado pela Anexina V e por marcadores de cinética e de morte celular. A terapia de BNCT induziu apoptose e necrose, indicando que esta terapia atua por diferentes vias em cada linhagem celular. BNCT e DM-1 induziram aumento na expressão dos marcados próapoptóticos, como Bax, citocromo c, caspase 3 e 8 clivadas, além de diminuir os valores na expressão de ciclina D1 e Ki-67, relacionados com a progressão do ciclo celular e proliferação. O quimioterápico DTIC apresentou alguns indícios de apoptose em células de melanoma, mas seus efeitos em células normais foram extensivos, ocasionando morte e parada do ciclo celular em melanócitos, células endoteliais e fibroblastos. O composto DM-1 apresentou formação de corpos apoptóticos, modificações no citoesqueleto e clivagem de caspase 9 e Parp em linhagens de melanoma humano. Desta forma, o composto DM-1 e a BNCT mostraram-se ferramentas terapêuticas mais eficazes no controle da progressão e no aumento da morte celular em células de melanoma. O poder efetivo da terapia de BNCT e do composto DM-1 faz com que a possibilidade de terapias combinatórias tenha resultados extremamente favoráveis na modulação da resposta proliferativa desses tumores. / Malignant melanoma is the most aggressive skin cancer. It is responsible for more than 75% of deaths. The main and most active chemotherapy in the melanoma treatment is represented by dacarbazine (DTIC), however, response rates are disappointing. The DM-1 compound is a curcumin structural analogue and it has similar biological properties, such as an antiproliferative and pro-apoptotic agent. Boron Neutron Capture Therapy (BNCT) works through the deposition of the isotope 10Boron in tumor cells, with subsequent irradiation of thermal neutrons, which produce alpha particles and lithium that destroy the cell. In this study, the action mechanism of these three therapies, DTIC, DM-1 and BNCT in the melanoma treatment and its effects in vitro on normal cells were studied in order to obtain different therapeutic modalities for cancer treatment. The IC50 was obtained by MTT method, besides the analysis of cell cycle progression and cell death markers by flow cytometry. The DM-1 and BNCT showed selective cytotoxic in melanoma cell lines, with high of free radicals production. In the same conditions, these effects were minimal in normal cells, unlike the treatment with DTIC. There was a decrease in the proportion of extracellular matrix and soluble collagen synthesized in melanoma cells treated with DM-1, BNCT and DTIC, however, only DTIC also resulted in decreased in normal cells. The mitochondrial electrical potential of melanoma cells was decreased in the three treatment protocols, as there was an increase in the amount of fragmented DNA. This effect was not found in normal cells treated with DM-1 and BNCT. The compound DM-1 was able to induce apoptosis by the intrinsic and extrinsic pathways, as assessed by Annexin V, cell death and kinetic markers. BNCT induced apoptosis and necrosis, indicating that this therapy acts through different pathways in each cell line. DM-1 and BNCT induced an increase of pro-apoptotic markers, such as Bax, cytochrome c, cleaved caspase 3 and 8 expression, and they reduced cyclin D1 and Ki-67, expression related to the progression of the cell cycle and proliferation. The DTIC has shown some signs of apoptosis in melanoma cells, but its effect on normal cells were extensive, causing death and cell cycle arrest in melanocytes, fibroblasts and endothelial cells. The DM-1 showed apoptotic bodies formation, cytoskeleton changes and caspase 9 and Parp cleavage in human melanoma cell lines. Thus, the DM-1 and BNCT showed as therapeutic tools more with high effectiveness in controlling the cell cycle progression and cell death increase in melanoma cells. The effectiveness of BNCT and DM-1 makes the possibility of combinatorial therapies, with extremely favorable results in the modulation of the proliferative response of these tumors.

No presente trabalho investigamos a relevância dos mediadores lipídicos (Leucotrienos) gerados pela enzima 5-Lipoxigenase (5-LO) na susceptibilidade ou resistência de camundongos ao Melanoma experimental com células tumorais B16-F0, utilizando como modelo camundongos produtores de leucotrienos (129_WT) e camundongos geneticamente deficientes \"knockout\" de 5-LO (129_5-LO KO). Primeiramente, verificamos que leucócitos peritoneais provenientes de animais WT implantados com melanoma B16-F0, apresentam aumento da expressão do gene para 5-LO (Alox5). Nossos resultados mostram que animais 5-LO KO, deficientes de 5-LO são mais eficientes no controle da progressão do tumor e apresentam significativo aumento na sobrevivência, quando comparados a animais WT, produtores de 5-LO. A nossa análise do perfil imunológico em células esplênicas indicam que a maior eficiência dos camundongos 5-LO KO no controle do crescimento de células tumorais B16-F0 estariam associados à presença numérica aumentada de neutrófilos (Gr-1+), células apresentadoras de antígeno (I-Ab+) majoritariamente CD19+CD80+ e esplenócitos capacitados para produção de altos níveis de citocinas pró-inflamatórias/efetoras como a IL-6, TNF?, IFN-? e baixos níveis de citocinas regulatórias como IL-10, 15 dias pós-implantação do tumor; a rápida geração da resposta imune polarizada para produção elevada de citocinas Th1 (IFN-?), mas não, citocinas Th2 (IL-10) e presença de maiores números de linfócitos T CD4+ e CD8+ efetoras, expressando o fenótipo CD44high ou CD44highCD62Llow. Ainda, verificamos que a deficiência genética da 5-LO ou a inibição da 5-LO pelo MK886 em células LAK, aumenta significativamente sua atividade citotóxica em células do melanoma B16-F0. Nossos resultados em conjunto, indicam que leucotrienos gerados pela enzima 5-LO, modulam negativamente a geração de resposta imune protetora em camundongos para o Melanoma B16-F0. / In the present work we examine the contribution of 5-lipoxigenase-derived lipid mediators during experimental melanoma (B16-F0) in 5-LO gene knockout (KO) mice and wild-type (WT) mice. The 5-LO KO mice presented delayed tumor growth, lesser tumor volume and delayed mortality. The greater resistance of 5-LO KO mice correlated with the following: High splenic Gr-1+ leukocytes counts, High and dominant presence of splenic IAb+CD19+CD80+ antigen-presenting cells counts and capacity of spleen cell to produce high levels of IL-6, TNF-?, IFN-? and lower levels of IL-10 early after tumor cells implantation; rapid T-cell polarization to secret high quantities of Th1 type cytokine IFN-? and low quantities of Th2 type cytokine IL-10; rapid generation and greater numbers of CD4+ and CD8+ activated T cells expressing CD45RB or CD44 markers; and also CD4+ and CD8+ CD44high or CD44highCD62Llow effector T cells. Herein, IL-2 induced splenic LAK cells from 5-LO KO mice, compared with splenic LAK cells from WT mice, were more efficient at killing B16-F0 melanoma cells. The increased B16-F0 melanoma cells killing activity were also found by treatment of splenic LAK cells from WT mice with a 5-LO activity inhibitor, MK886. Our findings suggest that 5-LO deficiency altered antigen-presenting cells profile, IFN-? and IL-10 production during skin cancer disease favoring the generation of protective immune responses and also provide evidence that 5-LO-derived LTs negatively affect the host survival during experimental B16-F0 melanoma.

Poucos tratamentos têm efeito sobre melanomas metastáticos, mas novas drogas vêm sendo estudadas para combater esta doença. Este trabalho avaliou efeito de ácido cinâmico (AC) em melanócitos (NGM) e células de melanoma (HT144) sobre alterações no ciclo, morte, comunicação e mobilidade celular e morfologia nuclear e investigou efeito de proteção à radiação ultravioleta (UV). AC inibiu crescimento de HT144 em relação a NGM. Essa inibição deve ocorrer devido a dano no DNA que levou à inibição da fase S, formação de aberrações nucleares e indução de morte. AC induziu formação de micronúcleos nas células NGM. O citoesqueleto foi reorganizado após AC o que diminuiu a mobilidade celular. Apesar de não induzir comunicação entre as células HT144 o AC aumentou níveis citoplasmáticos de cx43 em células de hepatocarcinoma, o que contribui para a morte celular. Isto sugere maior citotoxicidade de AC em células HT144 em comparação com NGM. Porém, AC não protegeu as células dos efeitos de UV e sua genotoxicidade em células NGM indica que sua atividade depende de mais estudos. / Consumption of vegetables could decrease the risk of malignancies. Cinnamic acid (CA), commonly found in plants have been studied because their antitumor activitie. The present work aimed to evaluate effect of CA in melanocytes (NGM) and melanoma cells (HT-144) by focusing the cell cycle, death, mobility and communication and formation of nuclear aberration and to investigate potential of protection against UV radiation. CA inhibited cell growth of melanoma cells compared to melanocytes, probably due to DNA damage leading to DNA synthesis inhibition, induction of nuclear aberrations and apoptosis. High concentration of CA induced micronuclei in NGM cells. We observed cytoskeleton reorganization that decreased cell mobility in both cells. We have not observed communication in HT144 after treatment, but CA increased expression of cx43 in hepatocarcinoma cells, probably leading to apoptosis. CA did not showed protection effects against ultraviolet radiation and its genotoxic effects in NGM cells indicates that its mechanism of action must be further investigated.