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Les maladies du système nerveux central (SNC) comme la sclérose en plaques, les accidents vasculaires cérébraux et les maladies neurodégénératives (Alzheimer et Parkinson) entraînent une réponse inflammatoire au niveau cérébrale appelée neuroinflammation. Ce phénomène peut avoir pour conséquence la limitation de la propagation de la maladie mais aussi la réparation et la régénération des tissus touchés. La microglie, principale défense du SNC, passe à un stade activé lors de phénomènes neuroinflammatoires et va libérer de nombreux facteurs neuroprotecteurs mais aussi pro-inflammatoires. Cette dualité d’action va ainsi maintenir un cercle vicieux, pouvant conduire à la mort neuronale. Il serait donc intéressant de comprendre le mécanisme de la neuroinflammation pour diagnostiquer et traiter au mieux les pathologies du SNC. Il existe plusieurs cibles moléculaires, parmi elles se trouvent la CycloOXygénase 2 (COX-2), une enzyme qui permet la formation de prostaglandines à partir de l'acide arachidonique, qui apparaît précocement et est fortement surexprimée en cas de neuroinflammation. Cette enzyme serait donc une cible de choix pour le développement d’outils d’imagerie dans le but de diagnostiquer les pathologies dans lesquelles les processus inflammatoires centraux sont présents et ce afin d’améliorer la prise en charge du patient. La tomographie d’émission de positons (TEP) est une technique d’imagerie fonctionnelle très sensible qui permet de quantifier de manière fine les variations d’activités métaboliques ou moléculaires. Cette technique requiert l’utilisation de radiotraceurs marqués avec un émetteur béta+. / Central nervous system (CNS) disorders as multiple sclerosis, stroke and neurodegenerative diseases (Alzheimer’s and Parkinson’s) lead to inflammatory response in the brain called neuroinflammation. This phenomenon usually should result in limiting the spread of the disease but also repair and regeneration of the affected tissues. Microglia, the main defense of the SNC, which is activated during a neurodegenerative event leading to the production of many factors including neuroprotectors but also pro-inflammatories. This duality of actions will thereby maintain endless vicious circle leading to neuronal death. It would be interesting to understand the neuroinflammation mechanism to better diagnose and treat CNS diseases. There are several molecular targets, among them are the CycloOXygenase 2 (COX-2), an enzyme which allows the formation of prostaglandins from arachidonic acid, which appears early and it is significantly overexpressed in case of neuroinflammation. This enzyme is therefore a good biological target for the development of imaging tools in order to diagnose pathologies in which central inflammatory processes are present in order to improve patient care. Postiron emission tomography (PET) is a very sensitive functional imaging technique that quantifies minute variations in metabolic or molecular activities. This technique requires the use of radiotracers labeled with a beta + emitter.

Le T-DM1 est un immunoconjugué composé de l'anticorps trastuzumab qui cible HER2 lié au DM1, un agent anti-tubuline dérivé de la maytansine. Malgré son efficacité, la résistance acquise au T-DM1 a été démontré lors des tests précliniques et chez certains patients. Nous avons développé des lignées résistantes à partir de la lignée de cancer du sein MDA-MB-361 et de la lignée de cancer de l'œsophage OE-19, que nous avons exposées au T-DM1 à doses croissantes pendant une longue durée en absence ou en présence de ciclosporine A (CsA). A partir de ces conditions nous avons obtenus les lignées “TR” qui ont été exposées uniquement au T-DM1 et “TCR” qui ont été exposées au T-DM1 et CsA. Nous avons observé une augmentation de la vitesse de migration et une diminution de la force d'adhésion chez OE-19 TCR associées à une sensibilité accrue à un inhibiteur de RHOA. Aussi, la voie des prostaglandines était dérégulée chez OE-19 TR et TCR, avec une forte augmentation de l'expression de COX-2 et de prostaglandine E2 dans la lignée OE-19 TR. La sensibilité à l'aspirine, un inhibiteur des cyclooxygenases 1-2, était accrue chez les deux lignées OE-19 résistantes par rapport à la lignée parentale. En conclusion nous avons démontré que différentes voies de signalisation peuvent être impliquées dans la résistance au T-DM1. Nos résultats restent à être validés chez les patients. Nous suggérons que cibler la voie de régulation de la composition du cytosquelette ou la voie des prostaglandines pourrait permettre d'obtenir un effet thérapeutique dans le cas de cancers résistants au T-DM1 / T-DM1 is an antibody-drug conjugate composed of the monoclonal antibody trastuzumab linked to DM1, a potent tubulin binding agent. Despite its efficacy in the treatment of HER2-positive breast cancer patients, acquired resistance to T-DM1 was observed during clinical trials. In order to study resistance mechanisms to T-DM1, we developed resistance models using OE-19 (esophageal) and MDA-MB-361 (breast) cancer cell lines in the absence or presence of ciclosporin A (CsA), an inhibitor of MDR1 mediated efflux. Resistant cells selected with T-DM1 alone are named “TR” and cells selected in the presence of T-DM1 and CsA are called “TCR”. OE-19 TCR cells showed modifications in adhesion gene expression, migration and adhesion strength, combined with an increased sensitivity to a RHOA inhibitor. Also, OE-19 TR cells presented an overexpression of COX-2 associated with an increased amount of PGE2 in the supernatant. A deregulation of the genes involved in the prostaglandin pathways was found in OE-19 TR and TCR cells, associated with increased sensitivity to aspirin. In conclusion, we found two signaling pathways deregulated in cell lines resistant to T-DM1. These results need to be validated using samples from patients resistant to T-DM1. Targeting the adhesion or the prostaglandin pathway could be of benefit for patients with T-DM1 resistant cancers

FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / O cÃncer oral representa um sÃrio problema de saÃde pÃblica mundial. Entre os tumores deste sÃtio anatÃmico, os carcinomas de cÃlulas escamosas orais (CCEO) respondem por atà 94% do total. Os mecanismos moleculares envolvidos na gÃnese e desenvolvimento tumoral ainda nÃo estÃo completamente elucidados. Algumas evidÃncias tÃm sugerido a participaÃÃo viral neste processo. AlÃm disso, estes tumores ainda carecem de marcadores confiÃveis para determinar o perfil de agressividade. Neste contexto, o presente estudo teve como objetivo avaliar a expressÃo das proteÃnas p53, E-caderina, COX-2, p16, MLH1 e MYC numa sÃrie de CCEO, considerando tambÃm a marcaÃÃo citoplasmÃtica eventualmente observada para as Ãltimas trÃs proteÃnas, confrontando os resultados entre elas e com as caracterÃsticas demogrÃficas e clÃnico-patolÃgicas. AlÃm de avaliar a prevalÃncia do PapilomavÃrus Humano (HPV) e do VÃrus Epstein-Barr (EBV) na amostra e comparÃ-las com a expressÃo das referidas proteÃnas. Materiais e MÃtodos â Cem espÃcimes de CCEO, fixados em formalina e incluÃdos em blocos de parafina, foram submetidos à imunohistoquÃmica para a detecÃÃo das referidas proteÃnas e à hibridaÃÃo in situ para detecaÃà de HPV e EBV. Resultados â Foi observada associaÃÃo referente à perda de expressÃo concomitante de p16 e MLH1 (p=0,029) e na coexpressÃo de p53 e COX-2 (p=0,045). Ademais, foi verificado que a COX-2 e o MYC nuclear estavam relacionados com a marcaÃÃo citoplasmÃtica de MLH1 (p=0,060 e p=0,018; respectivamente). A anÃlise combinada dos marcadores revelou cinco grupos principais de expressÃo alterada que eram constituÃdos, em sua maioria, de tumores mais agressivos, principalmente o grupo MLH1(-)/COX-2(+)/p16(-). Os casos com marcaÃÃo citoplasmÃtica para p16, MLH1 e/ou MYC foram mais frequentes em tumores avanÃados (p=0,009) e naqueles com metÃstases em linfonodos (p=0,001). Trinta e um casos demonstraram marcaÃÃo para HPV em tecido tumoral. O EBV nÃo foi detectado em nenhum dos casos investigados, nem no tecido tumoral nem no epitÃlio nÃo neoplÃsico. O grupo HPV(+) exibiu elevada positividade para o p16 nuclear (p=0,029) e MYC cytoplasmÃtico (p=0,039), tambÃm uma maior perda de expressÃo nuclear de MLH1 (p=0,031). Houve ainda uma tendÃncia referente ao aumento da positividade de COX-2 no grupo infectado (p=0,084). ConclusÃes â As significÃncias verificadas entre p16 e MLH1 sugerem que a ausÃncia do membro do sistema de reparo de encaixe (MMR) tambÃm favoreÃa a ocorrÃncia de mutaÃÃes no gene p16, culminando na inativaÃÃo deste supressor tumoral. As associaÃÃes de COX-2 e MYC com o MLH1 de expressÃo citoplasmÃtica suscitam um mecanismo de bloqueio de entrada de MLH1 no nÃcleo. A anÃlise combinada das proteÃnas, bem como, a marcaÃÃo citoplasmÃtica de p16, MLH1 e MYC, podem representar indicadores Ãteis na avaliaÃÃo do perfil de agressividade e, provavelmente, de prognÃstico em CCEO. Acerca dos vÃrus, nossos achados sugerem que o HPV esteja envolvido em uma importante parcela de casos de CCEO e que possa promover a expressÃo de p16 nuclear, MYC citoplasmÃtico e COX-2, e suprimir a expressÃo nuclear de MLH1. Quanto ao EBV, nÃo foram detectados EBERs (EBV-encoded small RNAs) na amostra. / The oral cancer represents a serious world public health problem. The oral squamous cell carcinomas (OSCC) account for up to 94% of the tumors of this anatomic site. The molecular mechanisms involved in the genesis and progression are still not well elucidated. Some evidences have suggested the involvement of viruses in this process. Also, these tumors still lack of reliable markers to determine the aggressiveness profile. In this context, the aim of the present study was to evaluate the expression of the proteins p53, E-cadherin, COX-2, p16, MLH1 and MYC in a serie of OSCC, including the cytoplasmic staining eventually observed for the latter three proteins, confronting the results between them and with demographic and clinico-pathological features. Besides evaluating the prevalence of Human Papillomavirus (HPV) and Epstein-Barr virus (EBV) in the sample and compare them with the expression of the referred proteins. Materials and Methods â One hundred formalin-fixed paraffin-embedded OSCC specimens were submitted to immunohistochemistry for detection of the referred proteins, and to in situ hybridization for HPV and EBV detection. Results â OSCC was associated with a concomitant lack of expression of p16 and MLH1 (p=0.029) and coexpression of p53 and COX-2 (p=0.045). Additionally, COX-2 and nuclear MYC were found to be related to exclusively cytoplasmic staining of MLH1 (p=0.060 and p=0.018, respectively). The combination analyses of the markers revealed five main groups of altered protein expression, which were mostly of the more aggressive tumors, mainly the MLH1(-)/COX-2(+)/p16(-) group. The cases with cytoplasmic staining for p16, MLH1 and/or MYC were more frequent in advanced tumors (p=0.009) and in those with lymph node metastasis (p=0.001). Thirty-one cases showed staining for HPV in tumor tissue. The EBV was not detected in any case investigated, neither in the tumor tissue nor in the non-neoplastic epithelium. The HPV(+) group demonstrated high positivity for nuclear p16 (p=0,029) and cytoplasmic MYC (p=0,039), and an increase of the lack of MLH1 nuclear expression (p=0,031). There was also a trend related to the increase of the COX-2 positivity in the HPV(+) group (p=0,084). Conclusions â The significance between p16 and MLH1 suggests that the lack of this member of mismatch repair system also favors the occurrence of mutations in the p16 gene, culminating in inactivation of this tumor suppressor. The associations of COX-2 and MYC with cytoplasmic MLH1 suggest a blocking mechanism for the entry of MLH1 into the nucleus. The combined analyses of the proteins investigated, as well as the cytoplasmic staining of p16, MLH1 and MYC, may be useful in the evaluation of the aggressive profile and probably prognosis of OSCC. Regarding the viruses, our findings suggest that the HPV is involved in an important portion of OSCC cases and that may promote the expression of the nuclear p16, cytoplasmic MYC and COX-2, and suppress the nuclear expression of MLH1. About EBV, it was not detected the EBV-encoded small RNAs (EBERs) in the sample.

Made available in DSpace on 2014-07-29T15:30:41Z (GMT). No. of bitstreams: 1 DissertacaoElianeDuarteMotaIPTSP.pdf: 4130183 bytes, checksum: 334a4dd3d5b4498f488ee03e5a9d338a (MD5) Previous issue date: 2012-03-23 / BACKGROUND: Colorectal adenocarcinoma (CRA) is a worldwide distributed pathology, and lymph node metastasis is associated with a poor prognostic outcome. Angiogenesis and lymphangiogenesis are correlated with metastasis. Vascular endothelial growth factor C (VEGF-C) and Cyclooxygenase 2 (COX-2) are molecules directly involved in those processes. We investigated the possible association of the expression of VEGF-C and COX-2 with clinical/pathology features and five year survival rate. MATERIALS AND METHODS: One hundred and thirty four cases of CRA from a Cancer Reference Hospital were randomly selected. The expression of VEGF-C and COX-2 was detected by immunohistochemistry and a univariate analysis was applied to evaluate the association between their expression and clinical stages, metastasis, and/or survival. RESULTS: COX-2 expression was observed in 98.67% of the adenocarcinoma cases while VEGF-C was found in 54.48% of the cases. COX-2 was highly expressed by all clinical stages of CRA, but its expression was not associated with LN metastasis, tumor infiltration or five years survival. A correlation was observed between LN metastasis and VEGF-C positivity (p=0.02) and clinical stages of the disease. The range of VEGF-C expressions was from 34.6% to 88.9% in stages 1 through 4, respectively. CONCLUSION: The majority of the CRA cases were positive for COX-2. It was observed association between the expression of VEGF-C and LN metastasis as well as with clinical stages of the disease, although no association was found to the overall five year survival rate. / INTRODUÇÃO: O adenocarcinoma colorretal (ACC) é uma doença de distribuição mundial e a metástase para linfonodos regionais está associada com pior prognóstico. A angiogênese e a linfangiogênese estão relacionadas com metástases. O Fator de crescimento endotelial vascular C (VEGF-C) e a Ciclooxigenase 2 (COX-2) são moléculas diretamente envolvidas nestes processos. Investigamos a possível associação da expressão de VEGF-C e COX-2 com características clínico/patológicas e a sobrevida em cinco anos. MÉTODOS: Cento e trinta e quatro casos de adenocarcinoma colorretal foram selecionados randomicamente. A expressão de COX-2 e VEGF-C foi detectada por imuno-histoquímica e as possíveis associações com aspectos clínicos e patológicos investigados. RESULTADOS: A expressão de COX-2 foi positiva em 133 (98,67%) pacientes e a expressão de VEGF-C foi encontrada em 73 (54,48%) pacientes. Não foram observadas associações significativas entre a expressão de COX-2 e metástases para linfonodos, invasão tumoral e nem com a sobrevida global em cinco anos. Quando a avaliação da expressão de VEGF-C foi realizada segundo Soumaoro et al, 2006, não associação com os aspectos estudados (p>0,05) e quando a expressão foi avaliada segundo Agaki et al., 2000, houve associação com metástases para linfonodos regionais o grau histológico do tumor (p=0,01) e com o estadiamento clínico (p=0,005). CONCLUSÃO: A maioria dos casos de ACR foram positivos para COX-2. Foram observadas associações entre a expressão de VEGF-C e metástases para linfonodos, bem como com os estadios clínicos da doença, embora, não tenha sido encontrada associação com a sobrevida em cinco anos.

Pacientes com doença do refluxo não erosiva apresentam sintomas típicos causados pelo refluxo do conteúdo gástrico para o esôfago. Entretanto, estes pacientes não apresentam alterações de mucosa visualizadas à endoscopia. O objetivo deste estudo é caracterizar a expressão de moléculas relacionadas à junção celular (claudinas 1, 3 e 4), da proteína pró-inflamatória Cox-2, da população global de linfócitos (CD45), população de linfócitos T (CD3), linfócitos B (CD20) e natural killer (CD57) em portadores de esofagite de refluxo erosiva e não erosiva. O estudo verificou que quanto mais intensa e crônica a inflamação no epitélio escamoso esofágico, menor a expressão das proteínas juncionais (claudinas 1 e 4), não alterando a expressão da claudina 3. Em relação à Cox-2 o estudo mostra aumento de sua expressão na forma erosiva da doença. Em relação à população de linfócitos, não foi detectada diferença significativa / Patients with non-erosive reflux disease conditions show typical symptons caused by the reflux of the gastric content to the esophagus. However, these patients dont show any alteration on the mucous membrane visualized trhough endoscopy. The aim of this study is to characterize the molecule expression related to the cell junction (claudins 1 , 3 and 4)the Cox 2 pro-inflamatory protein, the general population of (CD45) lymphocytes ,the population of lymphocytes T (CD3),the B (CD20) lymphocytes and the(CD57) natural killer in patients with erosive and non-erosive reflux esophagitis. The study found that the more intense and chronic the inflamation is in the esophageal squamous epithelium, less junction protein expressios (claudins 1 and 4) were found, not altering the 3 expressions of claudin 3. Regarding Cox 2, the study shows increase in its expression in the erosive form of the disease. Regarding the population of lymphocytes, no significant difference was detected

Les stratégies actuelles de lutte contre le cancer consistent à développer de nouveaux traitements à base de molécules d’origine naturelle susceptibles de déclencher l’apoptose de cellules malignes et d’inhiber les principales voies de survie cellulaire. Le premier objectif de ces travaux porte sur les effets anti-prolifératifs et apoptotiques de l’extrait éthanolique de la plante Berberis libanotica sur les lignées érythroleucémiques humaines HEL, K562 et K562 (COX-2+) et sur son effet sur l’expression de la COX-2 dans ces lignées. Nos résultats montrent que l’extrait induit l’apoptose dans les lignées étudiées et ceci par activation de la caspase-3, le clivage de la PARP-1 et la fragmentation de l’ADN. De même, il induit la diminution de l’expression de COX-2. Nous avons démontré que les voies survie de NF-ĸB et p-AKT sont inhibées. Le deuxième objectif de ces travaux consistent à identifier la molécule présente dans cet extrait et qui est capable de déclencher ces effets anticancéreux. Nous avons démontré que la berbérine est la molécule majoritaire dans cet extrait et possède des effets apoptotiques et des effets inhibiteurs des voies de survie, ce qui est similaire aux effets de l’extrait brut.Mots clés :érythroleucémie, apoptose, berberis libanotica, berbérine, COX-2, NF-kB, p-AKT. / The first aim of this study focuses on the apoptotic effect of Berberis Libanotica on human erythroleukemia cell lines HEL, K562, and K562(COX-2+) and it is effect on the expression of COX-2. In light of the reported chemopreventive and chemosensitive effects of natural products on various tumor cells and animal models, we postulated that our Bl extract may mediate their effects through apoptosis induction with suppression of cell survival pathways. We showed that this extract induces apoptosis in eryrhtroleukemia cells by activation of the late markers of caspase-3 activation, PARP cleavage and DNA fragmentation. In the other hand, we showed that Bl extract reduced significantly expression of COX-2 by a dose-dependent manner. In regard to our results, it is clear that the simultaneous inhibition of Akt and NF-κB signalling can significantly contribute to the anticancer effects of Bl extract in human erythroleukemia cells.The second objective of this report is to elucidate wich molecule present in our extract can exert this effects. We found that berberine, the majoritory compound, can induce an apoptotic effect and inhibits the survival pathway of NF-ĸB and p-AKT similarly to the extract.Key words: erythroleukemia, apoptosis, Berberis libanotica ,berberine, COX-2, NF-ĸB, p-AKT

L'augmentation de l'incidence et de la mortalité du cancer colorectal nécessitent la découverte de nouvelles méthodes de prévention et de traitement. Les chalcones ont été identifiées comme des composés intéressants ayant des propriétés chimiopreventives et antitumorales. Dans cette étude, nous avons étudié les effets de la 3-hydroxy-3', 4, 4', 5'-tétra-méthoxy-chalcone (3-HTMC), une chalcone synthétisée par notre laboratoire, sur la prolifération, la distribution du cycle cellulaire, l'apoptose et son mécanisme d'action sur les cellules cancéreuses colorectales humaines HT-29 (COX-2 sauvage) et HCT116 (déficientes en COX-2). Nous avons montré que la 3-HTMC diminue la viabilité cellulaire d'une façon dose dépendante avec un effet antiprolifératif plus puissant sur les cellules HCT116 que les cellules HT-29. L'analyse par cytométrie en flux a révélé une accumulation des cellules en phase G2/M du cycle cellulaire pour les cellules HT-29 et un arrêt significatif en G2/M pour les cellules HCT116 avec une apparition du pic sub-G1 caractéristique de l'apoptose. Nous avons démontré que le traitement des deux lignées cellulaires par la 3-HTMC induit un processus apoptotique associé à la surexpression du récepteur de mort DR5, l'activation des caspase-8 et -3, le clivage de la PARP et la fragmentation de l'ADN. De plus, la 3-HTMC induit l'activation des voies de survie PI3K/Akt et MEK/ERK qui sont impliquées dans la résistance à l'apoptose. De même, la 3-HTMC induit une surexpression de la COX-2 dans les cellules HT-29 qui contribue aussi à la résistance à l'apoptose, ceci expliquant la différence de sensibilité entre les cellules HT-29 et HCT116 traitées par la 3-HTMC. Donc, malgré l'activation des voies de résistance à l'apoptose, la 3-HTMC peut être un agent chimiopréventif ou thérapeutique prometteur pour le traitement du cancer colorectal. / Increasing incidence and mortality of colorectal cancer brings the necessity to uncover new possibilities in its prevention and treatment. Chalcones have been identified as interesting compounds having chemopreventive and antitumor properties. In this study, we investigated the effects of the synthetic chalcone derivative 3-hydroxy-3',4,4',5'-tetra-methoxy-chalcone (3-HTMC) on proliferation, cell cycle distribution, apoptosis and its mechanism of action in human colorectal HT-29 (COX-2 sufficient) and HCT116 (COX-2 deficient) cancer cells. We showed that 3-HTMC decreased cell viability in a dose-dependent manner with a more potent antiproliferative effect on HCT116 than HT-29 cells. Flow cytometric analysis revealed G2/M cell cycle accumulation in HT-29 cells and significant G2/M arrest in HCT-116 cells with a subsequent apoptosis shown by appearance of Sub-G1 peak. We demonstrated that 3-HTMC treatment on both cell lines induced apoptotic process associated with overexpression of death receptor DR5, activation of caspase-8 and -3, PARP cleavage and DNA fragmentation. In addition, 3-HTMC induced activation of PI3K/Akt and MEK/ERK principal survival pathways which delay 3-HTMC-induced apoptosis in both cell lines. Furthermore, COX-2 overexpression in HT-29 cells contributes to apoptosis resistance which explains the difference of sensitivity between HT-29 and HCT116 cells to 3-HTMC treatment. Even if resistance mechanisms to apoptosis reduced chalcone antitumoral potential, our results suggest that 3-HTMC may be considered as an interesting compound for colorectal cancer therapy or chemoprevention.

Bien qu’au 11ème rang des cancers les plus fréquents et au 12ème rang des cancers les plus mortels, le mélanome reste un problème médical majeur préoccupant. En effet, cette pathologie, au stade métastatique, reste réfractaire à la chimiothérapie et aux thérapies ciblées. Un certain nombre d’arguments définissent la résistance à l’apoptose comme un point crucial dans l’échec aux traitements anti-cancéreux. Ces mécanismes de résistance et chimiorésistance spécifiques aux mélanomes, déclenchés en réponse aux traitements traditionnels, sont la conséquence d’une dérégulation des voies apoptotiques suite à l’activation des protéines anti-apoptotiques, l’inactivation des protéines pro-apoptotiques avec renforcement des signaux de survie (voies de survie PI3K/Akt, NF-κB et MAPK/ERK). Une étude effectuée sur la lignée murine de mélanome B16-F0 a introduit la mélanogenèse comme une forme de résistance à l’apoptose induite par l’acide ursolique (AU), un triterpène pentacyclique d’origine naturelle ; ainsi les cellules entrant en apoptose sont capables de déclencher une résistance qui se manifeste par une surproduction de la mélanine tout en retardant la mort cellulaire. D’autres études ont montré l’implication de la COX-2 dans un mécanisme de résitance à l’apoptose dans plusieurs types de cancers dans le but de retarder leur mort cellulaire. Dans cette optique, nous nous sommes intéressés à étudier l’implication de la mélanogenèse et de la voie COX-2/PGE2 dans la résistance à l’apoptose dans le mélanome et plus précisément dans un modèle d’apoptose induite par l’AU sur la lignée humaine de mélanome M4Beu. Par la suite, nous avons décrit une interaction probable entre ces deux voies distinctes, la mélanogenèse et la voie COX-2/PGE2. Dans un autre contexte, nous avons montré que l’AU inhibe les voies de survie PI3K/Akt et ERK1/2, ce qui favorise ses effets pro-apoptotique et anti-prolifératif. Notre étude permet de mieux explorer les mécanismes de résistance spécifiques aux mélanomes tout en suggérant l’effet bénéfique de l’AU comme adjuvant naturel aux traitements chimio-thérapeutiques traditionnels. / Despite the deployment of targeted therapies, the incidence and mortality rates of cutaneous melanoma is increasing very fast making it a pre-eminent public health threat. Previously, we had showed that B16-F0 murine melanoma cells undergoing apoptosis are able to delay their own death induced by ursolic acid (UA), a natural pentacyclic triterpenoid compound. We had demonstrated that tyrosinase and TRP-1 up-regulation in apoptotic cells and the subsequent production of melanin were implicated in an apoptosis resistance mechanism. Several resistance mechanisms to apoptosis have been characterized in melanoma such as hyperactivation of DNA repair mechanisms, drug efflux systems, and reinforcement of survival signals (PI3K/Akt, NF-κB and MAPK/ERK pathways). Otherwise, other mechanisms of apoptosis resistance involving different proteins, such as cyclooxygenase-2 (COX-2), have been described in many cancer types. In this study, we demonstrated the involvement of melanogenesis and COX-2/PGE2 pathway in resistance to UA-induced apoptosis in human M4Beu melanoma cells. Then, we established the evidence that an interaction exists between these two pathways by investigating on the one hand the effect of inhibiting melanogenesis by N-phenylthiourea (PTU) on COX-2 expression and its product PGE2, and on the other hand the effect of inhibiting COX-2 activity using NS-398 on tyrosinase expression and melanin production. Furthermore, we showed that anti-proliferative and proapoptotic effects of UA were mediated through modulation of multiple signaling pathways including Akt and ERK-1/2 proteins. Our study not only uncovers underlying molecular mechanisms of UA action in human melanoma cancer cells but also suggest its great potential as an adjuvant in treatment and cancer prevention.

Made available in DSpace on 2016-05-02T13:54:42Z (GMT). No. of bitstreams: 1 DISSERTACAO COMPLETA ERICA SILVA BIANCHETTI.pdf: 401322 bytes, checksum: d859a5d12a9b16ecd28ee0fecfa86d09 (MD5) Previous issue date: 2006-06-01 / Conselho Nacional de Desenvolvimento Cientifico e Tecnologico / Etoricoxib is a new medicine expected to lead the next generation of selective inhibitors of COX-2 Presently the focus of many clinical assays it is a potent fast-action second generation coxib and the most selective of all The purpose of this study was to evaluate the interference of different nutritional diets in the anti-inflammatory and analgesic actions of etoricoxib and also the interactions of this NSAID plus different diets and their gastric and hematological side effects The following assays were carried out a) paw edema induced by carrageenin b) granuloma test c) dermatitis induced by croton oil d) vascular permeability in rats e) writhing test in mice f) formalin test g) gastric ulcers by stress and h) evaluation of the hematological parameters after sub-chronic treatment With regard to edema by carrageenin the etoricoxib-treated group showed a maximum peak of edema 49.04% (1.061 +- 0.1886) the other groups showed the following percentages of inhibiton etoricoxib + hyperproteic diet 30.2% (1.4537 +- 0.0955) etoricoxib + hyperlipidic diet 35.96% (64.04 +- 0.0578) etoricoxib + hyperglicidic diet 35.35% (1.346 +-0.0423) etoricoxib + standardized diet 33% (1.2968+-0.047) all of them in relation to the control group (2.0825+-0.1886) and the results were statistically significant (p < 0.01) However when the groups treated with etoricoxib associated to different diets were compared there was no statistically significant difference In the granuloma test the daily oral administration of 1 mg/kg of etoricoxib during 6 days significantly (p < 0.01) inhibited the formation of granulomatous tissue 57.02% (153.2 +- 21.908) the other groups showed the following percentages of inhibition etoricoxib + hyperlipidic diet 59% (144.98 +- 9.632) etoricoxib + hyperproteic diet 47.5% (185.575 +- 26.043) etoricoxib + hyperglicidic diet 38.5% (217.4 +- 21.318) etoricoxib + standardized diet 47.13% (166.583 2 +- 2.229) all of them in relation to the control group (353.475 +- 37.692) In the croton oil-induced dermatitis the edema of the control group had 10.33 mg The treatment with etoricoxib (1 mg/kg) associated with different nutritional diets showed inhibition of the edema but not significantly when compared to the control group The inhibition percentages were etoricoxib-treated group 7.86% (9.525 +- 1.345) etoricoxib + hyperproteic diet 31.43% (7.0875 +- 1.160) etoricoxib + hyperlipidic diet 35.6% (6.6625 +- 1.523) etoricoxib + hyperglicidic diet 39.5% (6.2571 +- 1.362) etoricoxib + standardized diet 30.7% (7.1875 +- 1.130) *p < 0.05 (Student s t test) when compared to the control group (10.3375 +- 1.462) In the vascular permeability by histamine etoricoxib (1 mg/kg) etoricoxib + hyperproteic diet etoricoxib + hyperlipidic diet etoricoxib + hyperglicidic diet and etoricoxib + standardized diet exhibited the following inhibition percentages 5.29% -31.4% -31.3% 4.05% and 15.82 respectively These results were not significant when compared to the control group (527.862 +- 66.869) In the writhing test the administration of etoricoxib (1mg/kg) inhibited the algogenic process in 9.32% (49.86 +- 4.166) When associated with different nutritional diets the inhibition percentages were hyperproteic diet 29.27% (38.9 +- 6.166) hyperlipidic diet 11.36% (48.75 +- 5.384) hyperglicidic diet 9.81% (49.6 +- 6.775) standardized diet -7.3% (59 +- 4.946) In the formalin test both in the acute and late phase all the treatments caused significant (p < 0.05) inhibitions of the hyperalgesic process etoricoxib (1mg/kg) 47.74% (62.71 +- 8.462) etoricoxib + hyperproteic diet 74.64% (30.428 +- 5.163) etoricoxib + hyperglicidic diet 68.61% (37.67 +- 5.308) etoricoxib + hyperlipidic diet 46.46% (64.25 +- 5.662) etoricoxib + standardized diet 68.2% (38.17 +- 5.528) when compared to the control group (120 +- 5.021) In the late phase the percentages of inhibition were 84.4% (10.142 +- 2.98) for etoricoxib 82.65% (11.28 +- 2.705) for etoricoxib + hyperproteic diet 66.16% (22 +- 11.781) for etoricoxib + hyperlipidic diet 98.72% (0.18 +- 0.0) for etoricoxib + hyperglicidic diet and 99.74% (0.16 +- 0.1667) for etoricoxib + standardized diet in comparison with the control group (65 +- 4.167) In the test of stress-induced ulcer the animals treated with etoricoxib (1mg/kg) + standardized diet showed the highest lesion index when compared to the other groups The lowest lesion index was shown by the group treated with etoricoxib + hyperlipidic diet whose significance was p < 0.01 (Student s t test) when compared to the control group The hematological parameters in the groups treated with etoricoxib (1mg/kg) + hyperlipidic diet (19.637 +- 3.879) and etoricoxib + hyperglicidic diet (19.3 +- 4.562) showed statistically significant differences in the hematocrit (HCT) in relation to the group treated only with etoricoxib (40.5375 +- 2.410) for p < 0.01 (Student s t test) There was a significant difference in the group treated with etoricoxib + hyperglicidic diet (8.9 +- 1.940) in relation to the group treated with etoricoxib (19.9 +- 2.134) (Student s t test) In the erythrocyte dosage the group etoricoxib + hyperglicidic diet (3.82125 +- 0.893) showed a significant difference when compared to the group treated with etoricoxib (9.4037 +- 1.027) (p < 0.01 Student s t test) No statistically significant differences were observed in the other hematological parameters The evaluation of the ponderal development of the animals treated with etoricoxib (1mg/kg) and etoricoxib assiciated with different kinds of nutritional diets showed no significant differences between the treated and control groups however the group treated with etoricoxib + hyperglicidic diet revealed lower ponderal development than the other groups With regard to diuresis there were variations in all the groups For water and feed consumption variations were practically similar in all the experimental groups The weight of the organs from different groups of animals treated with etoricoxib (1mg/kg) and etoricoxib associated with different nutritional diets showed no significant differences when compared to the control group The mean weight of the organs are within the normal parameters for rats The results suggest that a) the association of etoricoxib to different kinds of diets did not change the anti-inflammatory effect in the present assays b) the association of different types of diets potentialized the analgesic effect mainly when associated to hyperproteic diet for peripheral pain and hyperglicidic diet for central pain c) the association of etoricoxib to hyperglicidic diet decreases the gastric lesion index d) the use of etoricoxib alone did not interfere with the hematological parameters e) the association of etoricoxib to hyperglicidic diet interfered with the hemoglobin and erythrocyte index f) the treatment of the sub-chronic phase (30 days) with etoricoxib alone and etoricoxib associated to different nutritional diets caused no changes on the ponderal development diuresis and water and feed consumption / O etoricoxib (Arcóxia®) um medicamento novo posicionado para liderar a próxima geração de inibidores seletivos de COX-2 é um coxib de segunda geração potente e de ação rápida sendo motivo atualmente de muitos ensaios clínicos É o mais seletivo de COX-2 de todos os coxibs O objetivo deste estudo foi estudar em modelos in vivo a interferência sobre a atividade antiinflamatória e analgésica do etoricoxib antiinflamatório inibidor seletivo de COX-2 associado a diferentes tipos de dietas nutricionais observando as possíveis interações entre este AINE com as dietas empregadas e observar possíveis efeitos adversos ao nível gástrico e hematológico com o uso da associação do etoricoxib com as dietas nutricionais Para tanto foram utilizados os seguintes ensaios a) edema de pata por carragenina b) teste do granuloma c) dermatite induzida por óleo de croton d) permeabilidade vascular em ratos e) contorções em camundongos f) teste da formalina g) úlcera por estresse e h) avaliação dos parâmetros hematológicos após tratamento sub-crônico No edema por carragenina o grupo tratado com etoricoxib produziu inibição no pico máximo do edema de 49,04% (1.061 +- 0.1886) no tratado com etoricoxib + dieta hiperprotéica a inibição foi de 30,2% (1.4537 +- 0.0955) no tratado com etoricoxib + dieta hiperlipídica a inibição foi de 35,96% (64.04 +- 0.0578) no tratado com etoricoxib + dieta hiperglicidica a inibição foi de 35,36% (1.346 +- 0.0423) e no grupo tratado com etoricoxib + dieta padrão a inibição foi de 33% (1.3968 +- 0.047) todos em relação ao grupo controle (2.0825 +- 0.1886) apresentando resultados significativos estatisticamente (p < 0,01) Entretanto quando comparados entre si os grupos tratados com etoricoxib associado às diferentes dietas não houve diferença estatística significativa No teste do granuloma a administração diária de 1 mg/kg/v.o de etoricoxib durante 6 dias inibiu de forma significativa a formação do tecido granulomatoso (p < 0,01) em 57,02% (153.2 +- 21.908) e 59% (144.98 +- 9.632) pelo grupo tratado com etoricoxib + dieta hiperlipídica respectivamente no grupo tratado com etoricoxib + dieta hiperprotéica a inibição foi de 47,5% (185.575 +- 26.043) no tratado com etoricoxib + dieta hiperglicídica a inibição foi de 38,5% (217.4 +- 21.318) e no tratado com etoricoxib + dieta padrão foi 47,13% (166.583 2 +- 2.229) todas em relação ao grupo controle (353.475 +- 37.692) Na dermatite por óleo de cróton o edema no grupo controle foi de 10,33 mg Neste experimento observou-se que o tratamento dos animais com etoricoxib (1mg/kg) associado aos diferentes tipos de dietas nutricionais apresentou inibição do processo edematogênico mas não de forma significativa quando comparado com o grupo controle Para o grupo tratado com etoricoxib a inibição foi de 7,86% (9.525 +- 1.354) etoricoxib + dieta hiperprotéica foi de 31,43% (7.0875 +- 1.160) etoricoxib + dieta hiperlipídica foi de 35,6% (6.6625 +- 1.523) etoricoxib + dieta hiperglicídica foi de 39,5% (6.2571 +- 1.362) e etoricoxib + dieta padrão foi de 30.7% (7.1875 +- 1.130) *p < 0.05 ( teste t de Student) quando comparado ao grupo controle (10.3375 +- 1.462) Na permeabilidade vascular por histamina o etoricoxib (1mg/kg) etoricoxib + dieta hiperprotéica etoricoxib + dieta hiperlipídica etoricoxib + dieta hiperglicídica e etoricoxib + dieta padrão apresentaram inibições de 5,29% -18.4% -31.3% 4,05% e 15,82% respectivamente não sendo significativas quando comparados ao grupo controle (527.862 +- 66.869) No teste de contorções a administração de etoricoxib (1mg/kg) produziu 9,32% (49.86 +- 4.166) de inibição do processo algogênico e quando associado as diferentes dietas nutricionais a inibição foi de dieta hiperprotéica 29,27% (38.9 +- 6.166) dieta hiperlipídica 11,36% (48.75 +- 5.384) hiperglicídica 9,81% (49.6 +- 6.775) e dieta padrão 7,3% (59 +- 4.946) No teste da formalina tanto na fase aguda quanto na fase tardia todos os tratamentos produziram inibições significativas do processo hiperalgésico (p < 0.05) cujas percentagens de inibições foram de 47,74% para etoricoxib (1mg/kg) (62.71 +- 8.462) 74,64% para etoricoxib (1mg/kg) + dieta hiperprotéica (30.428 +- 5.163) 68,61% para etoricoxib (1mg/kg) + dieta hiperglicídica (37.67 +- 5.308) 46,46% para etoricoxib (1mg/kg) + dieta hiperlipídica (64.25 +- 5.662) e 68,2% para etoricoxib (1mg/kg) + dieta padrão (38.17 +- 5.528) quando comparados ao grupo controle (120 +- 5.021) Na fase tardia as percentagens de inibições foram de 84,4% (10.142 +- 2.98) para etoricoxib 82,65% (11.28 +- 2.705) para etoricoxib + dieta hiperprotéica 66,16% (22 +- 11.781) para etoricoxib + dieta hiperlipídica 98,72% (0,18 +- 0.0) para etoricoxib + dieta hiperglicídica e 99,74% (0.16 +- 0.1667) para etoricoxib + dieta padrão em comparação com o grupo controle (65 +- 4.167) No teste de úlcera por estresse observou-se que o tratamento dos animais com etoricoxib (1mg/kg) + dieta padrão foi o grupo que apresentou maior índice de lesão comparado aos outros grupos de tratamentos Já o grupo tratado com etoricoxib (1mg/kg) + dieta hiperglicídica foi o que apresentou menor índice de lesão cuja significância quando comparado ao grupo controle foi de p < 0.01 (teste t de Student) Os parâmetros hematológicos nos grupos tratados com etoricoxib (1mg/kg) + dieta hiperlipídica (19.637 +- 3.879) e etoricoxib + dieta hiperglicídica (19.3 +- 4.562) apresentaram diferenças estatisticamente significativas para o hematócrito (HCT) em relação ao grupo tratado apenas com etoricoxib (40.5375 +- 2.410) para p < 0.01 (teste t de student) Quanto à hemoglobina (HGB) foi significativa a diferença para o grupo tratado com etoricoxib + dieta hiperglicídica (8.9 +- 1.940) em relação ao grupo tratado com etoricoxib (19.9 +- 2.134) (p < 0.05, teste t de Student) Na dosagem de hemácias o grupo etoricoxib + dieta hiperglicídica (3.82125+- 0.893) apresentou diferença significativa quando comparada com o grupo tratado com etoricoxib (9.4037 +- 1.027) (p < 0.01, teste t de Student) Em relação aos outros parâmetros hematológicos não foram observadas diferenças significativas estatisticamente Na avaliação do desenvolvimento ponderal dos animais tratados com etoricoxib (1mg/kg) e etoricoxib associado aos diferentes tipos de dietas nutricionais não foram observadas diferenças significativas entre os grupos tratados e o controle entretanto o grupo tratado com etoricoxib + dieta hiperglicidica apresentou desenvolvimento ponderal menor que os outros grupos Em relação à diurese pôde-se observar ocorrência de variações em todos os grupos Para os consumos de água e ração houve variações praticamente semelhantes em todos os grupos experimentais Quanto ao peso dos órgãos dos diferentes grupos de animais tratados com etoricoxib (1mg/kg) e etoricoxib associado a diferentes dietas nutricionais não apresentaram diferenças significativas quando comparados ao grupo controle O peso médio dos órgãos encontram-se dentro dos parâmetros normais para a espécie animal (ratos) A partir dos resultados obtidos pode-se sugerir que a) A associação do etoricoxib aos diferentes tipos de dietas empregadas não alterou o efeito antiinflamatório nos ensaios empregados b) A associação do etoricoxib aos diferentes tipos de dietas empregadas potencializou o efeito analgésico principalmente quando associado à dieta hiperprotéica para dor periférica e dieta hiperglicídica para dor central c) A associação do etoricoxib à dieta hiperglicídica diminui o índice de lesão gástrica d) O tratamento com etoricoxib isolado não interferiu sobre os parâmetros hematológicos avaliados e) A associação do etoricoxib à dieta hiperglicídica provocou interferência sobre a taxa de hemoglobina e hemácias f) O tratamento em fase sub-crônica (30 dias) com etoricoxib e etoricoxib associado às diferentes dietas nutricionais não produziu alterações sobre o desenvolvimento ponderal diurese consumo de água e ração