O carcinoma epidermoide de cabeça e pescoço, hoje considerada mundialmente uma das neoplasias mais frequentes desta região, tornou-se um problema de saúde pública, necessitando urgente de medidas a serem tomadas, a fim de melhorar a qualidade de vida dos pacientes acometidos, consequentemente aumentando a sobrevida, avaliada em cinco anos. O carcinoma epidermoide de cabeça e pescoço é uma doença complexa, e inclui vários fatores etiológicos, além de alterações moleculares, capazes de desencadear e dar continuidade a alguns eventos. No geral os carcinomas orais são tratados primeiramente com cirurgia excisional ou radioterapia individualmente, ou em combinação para os estágios mais avançados. As preparações dos extratos fermentados de Viscum album (VA), uma planta semiparasita da família das Lorantáceas, vêm sendo utilizada, principalmente em países da Europa, com resultados promissores no âmbito das terapias coadjuvantes, especialmente a medicina antroposófica. Em conjunto com as terapias convencionais, tem demonstrado uma melhora na qualidade de vida dos pacientes portadores de neoplasias malígnas. Estudos in vitro, realizados em células cancerígenas, têm demonstrado que vários tipos de VA podem apresentar citotoxicidade em células de carcinoma, sendo capazes de ativar a cascata apoptótica ou levando as células à necrose. Este estudo teve por objetivo verificar a ação de três tipos de extratos de VA (Iscador Qu Spezial, Iscador P e Iscador M), em linhagens celulares de carcinoma epidermoide de língua (SCC9 e SCC25). Para isso foi verificada a ação citotóxica do fármaco Iscador Qu Spezial, por meio do teste de viabilidade celular, para a obtenção da IC50. Em seguida foram realizados com os três fármacos: o teste de Tunel, para verificação de apoptose, Anexina V FITC/Iodeto de Propídeo, com auxílio de citômetro de fluxo, para a verificação quantitativa do índice de apoptose provocado pelos fármacos. Em seguida foi verificado o comportamento de cinco genes (Akt1, Akt2, Akt3, PTEN e Ciclina D1) por meio de análises feitas por qRT-PCR, e de seus produtos (pAkt, PTEN e Ciclina D1) pelas técnicas de Imunofluorescência e Western-Blot. Os fármacos, na concentração de 0,3 mg/mL (IC50) apresentaram resultados positivos na indução de apoptose nas células analisadas, e mostraram diferentes níveis de expressão para os genes analisados. Nos ensaios por Imunofluorescência, as proteínas exibiram diferentes localizações e comportamentos nos compartimentos celulares analisados. As análises quantitativas dessas proteínas, analisadas por Western-Blotting, mostraram diferentes níveis de expressão. Por meio deste estudo foi possível concluir que os fármacos Iscador Qu Spezial e Iscador M, possuem maior potencial citotóxico nas células das linhagens SCC9 e SCC25, quando comparados aos controles e ao fármaco Iscador P. As células da linhagem SCC9 exibiram um perfil mais resistente à ação dos fármacos analisados do que as células da linhagem SCC25. / Head and neck squamous cell carcinoma, one of the most common malignancies worldwide in this area, became a public health problem that requires urgent attitudes to be taken in order to improve the quality of life of the affected patients, increasing survival that today valued at five years. Head and neck squamous cell carcinoma is a complex disease which includes several etiologic factors and different molecular changes that may trigger and get on with some events. In general, oral carcinomas are treated primarily with surgical excision or radiotherapy alone or in combination for cases in more advanced stages. Preparations of fermented extracts from fermented Viscum album (VA), a plant from the Loranthaceae family have been used, mainly in European countries, with promising results as adjuvant therapies, especially in the Anthroposophy Medicine. In combination with conventional therapies, VA has lead to improvement in quality of life of patients with cancer. in vitro studies have demonstrated that various types of VA may have cytotoxicity in carcinoma cells being able to activate the apoptotic cascade or leading cells to necrosis. The present study aimed to verify the effect of three types of VA extracts (Iscador Qu Spezial, Iscador P and Iscador M) in squamous cell carcinoma of the tongue (SCC9 and SCC25). The cytotoxic action of Iscador Qu Spezial was verified by cell viability test obtaining the IC50. The three drugs were tested as follow: Tunnel to evaluate apoptosis, Annexin V and FITC/propidium iodide were evaluated by flow cytometry to quantify the apoptosis rate induced by the drugs. Then we verified the expression levels of five genes (Akt1, Akt2, Akt3, PTEN and Cyclin D1) by qRT-PCR and their products (pAkt, PTEN and Cyclin D1) by immunofluorescence and Western blot. The concentration of 0.3 mg/mL (IC50) of the drugs presented positive results in the induction of apoptosis and showed different expression levels for the genes analyzed. Immunofluorescence assays evidenced that the proteins exhibited different locations and behaviors in the cellular compartments analyzed. Quantitative analysis of these proteins by Western-Blot, evidenced that they presented different levels of expression. Through this study, it was concluded that Iscador Qu Spezial drugs and Iscador M have higher cytotoxic potential on SCC9 and SCC25 cell lines when compared to controls and the drug Iscador P. It could also be concluded that the SCC9 cell line was more resistant to the action of the drugs examined.

O carcinoma epidermoide de cabeça e pescoço, hoje considerada mundialmente uma das neoplasias mais frequentes desta região, tornou-se um problema de saúde pública, necessitando urgente de medidas a serem tomadas, a fim de melhorar a qualidade de vida dos pacientes acometidos, consequentemente aumentando a sobrevida, avaliada em cinco anos. O carcinoma epidermoide de cabeça e pescoço é uma doença complexa, e inclui vários fatores etiológicos, além de alterações moleculares, capazes de desencadear e dar continuidade a alguns eventos. No geral os carcinomas orais são tratados primeiramente com cirurgia excisional ou radioterapia individualmente, ou em combinação para os estágios mais avançados. As preparações dos extratos fermentados de Viscum album (VA), uma planta semiparasita da família das Lorantáceas, vêm sendo utilizada, principalmente em países da Europa, com resultados promissores no âmbito das terapias coadjuvantes, especialmente a medicina antroposófica. Em conjunto com as terapias convencionais, tem demonstrado uma melhora na qualidade de vida dos pacientes portadores de neoplasias malígnas. Estudos in vitro, realizados em células cancerígenas, têm demonstrado que vários tipos de VA podem apresentar citotoxicidade em células de carcinoma, sendo capazes de ativar a cascata apoptótica ou levando as células à necrose. Este estudo teve por objetivo verificar a ação de três tipos de extratos de VA (Iscador Qu Spezial, Iscador P e Iscador M), em linhagens celulares de carcinoma epidermoide de língua (SCC9 e SCC25). Para isso foi verificada a ação citotóxica do fármaco Iscador Qu Spezial, por meio do teste de viabilidade celular, para a obtenção da IC50. Em seguida foram realizados com os três fármacos: o teste de Tunel, para verificação de apoptose, Anexina V FITC/Iodeto de Propídeo, com auxílio de citômetro de fluxo, para a verificação quantitativa do índice de apoptose provocado pelos fármacos. Em seguida foi verificado o comportamento de cinco genes (Akt1, Akt2, Akt3, PTEN e Ciclina D1) por meio de análises feitas por qRT-PCR, e de seus produtos (pAkt, PTEN e Ciclina D1) pelas técnicas de Imunofluorescência e Western-Blot. Os fármacos, na concentração de 0,3 mg/mL (IC50) apresentaram resultados positivos na indução de apoptose nas células analisadas, e mostraram diferentes níveis de expressão para os genes analisados. Nos ensaios por Imunofluorescência, as proteínas exibiram diferentes localizações e comportamentos nos compartimentos celulares analisados. As análises quantitativas dessas proteínas, analisadas por Western-Blotting, mostraram diferentes níveis de expressão. Por meio deste estudo foi possível concluir que os fármacos Iscador Qu Spezial e Iscador M, possuem maior potencial citotóxico nas células das linhagens SCC9 e SCC25, quando comparados aos controles e ao fármaco Iscador P. As células da linhagem SCC9 exibiram um perfil mais resistente à ação dos fármacos analisados do que as células da linhagem SCC25. / Head and neck squamous cell carcinoma, one of the most common malignancies worldwide in this area, became a public health problem that requires urgent attitudes to be taken in order to improve the quality of life of the affected patients, increasing survival that today valued at five years. Head and neck squamous cell carcinoma is a complex disease which includes several etiologic factors and different molecular changes that may trigger and get on with some events. In general, oral carcinomas are treated primarily with surgical excision or radiotherapy alone or in combination for cases in more advanced stages. Preparations of fermented extracts from fermented Viscum album (VA), a plant from the Loranthaceae family have been used, mainly in European countries, with promising results as adjuvant therapies, especially in the Anthroposophy Medicine. In combination with conventional therapies, VA has lead to improvement in quality of life of patients with cancer. in vitro studies have demonstrated that various types of VA may have cytotoxicity in carcinoma cells being able to activate the apoptotic cascade or leading cells to necrosis. The present study aimed to verify the effect of three types of VA extracts (Iscador Qu Spezial, Iscador P and Iscador M) in squamous cell carcinoma of the tongue (SCC9 and SCC25). The cytotoxic action of Iscador Qu Spezial was verified by cell viability test obtaining the IC50. The three drugs were tested as follow: Tunnel to evaluate apoptosis, Annexin V and FITC/propidium iodide were evaluated by flow cytometry to quantify the apoptosis rate induced by the drugs. Then we verified the expression levels of five genes (Akt1, Akt2, Akt3, PTEN and Cyclin D1) by qRT-PCR and their products (pAkt, PTEN and Cyclin D1) by immunofluorescence and Western blot. The concentration of 0.3 mg/mL (IC50) of the drugs presented positive results in the induction of apoptosis and showed different expression levels for the genes analyzed. Immunofluorescence assays evidenced that the proteins exhibited different locations and behaviors in the cellular compartments analyzed. Quantitative analysis of these proteins by Western-Blot, evidenced that they presented different levels of expression. Through this study, it was concluded that Iscador Qu Spezial drugs and Iscador M have higher cytotoxic potential on SCC9 and SCC25 cell lines when compared to controls and the drug Iscador P. It could also be concluded that the SCC9 cell line was more resistant to the action of the drugs examined.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A LesÃo Central de CÃlulas Gigantes dos maxilares (LCCG) à intra-Ãssea, nÃo tem predileÃÃo por sexo, classifica-se em agressivas e nÃo-agressivas, histologicamente consistem tecido fi-broso e celularizado fusiforme associado a cÃlulas gigantes multinucleadas (CGM), focos de hemorragia e neovascularizaÃÃo, tendo na cirurgia seu habitual tratamento. Novas abordagens terapÃuticas foram propostas, sendo a principal delas o uso de corticÃides intralesionais. Este trabalho analisa retrospectivamente 21 pacientes portadores de LCCG que foram tratados por hexacetonido de triancinolona intralesional, atravÃs do seguinte protocolo: injeÃÃo de hexace-tonido de triancinolona 20mg/ml diluÃdo na soluÃÃo anestÃsica de lidocaÃna 2%/epinefrina 1:200.000 numa proporÃÃo de 1:1; infiltrando 1ml de soluÃÃo para cada 1cm3 de lesÃo, totali-zando 06 aplicaÃÃes em intervalos quinzenais. Estabeleceu-se 04 critÃrios clÃnicos para classi-ficar a resposta ao tratamento: 1- estabilizaÃÃo ou regressÃo clÃnica da lesÃo 2- ausÃncia de sintomas 3- aumento da densidade nos controles radiogrÃficos 4- aumento da resistÃncia a infiltraÃÃo intralesional da droga, bem como, fez-se uma anÃlise imunohistoquÃmica quanto à expressÃo dos Receptores de corticÃides (GCR) e Calcitonina (CTR), Cox-2, proteÃna p16 e amplificaÃÃo gÃnica da Ciclina D1 por CISH, comparando quanto a agressividade e a resposta terapÃutica a corticoterapia intralesional. Dos 21 pacientes incluÃdos neste estudo, 11 eram homens e 10 mulheres, 09 tinham lesÃo em maxila, 12 em mandÃbula. Dez eram lesÃes agres-sivas e 11 nÃo-agressivas, 15 (71,4%) apresentaram uma boa resposta ao tratamento, 04(19%) moderada e 02(9,1%) negativa. Das 11 nÃo agressivas, 10(90,9%) apresentaram boa resposta e 01 (9,1%) resposta moderada, das 10 agressivas 05(50%), 03(30%) e 02(20%) apresentaram boa, moderada e negativa resposta respectivamente, nenhuma apresentou recidiva apÃs o tra-tamento, com preservaÃÃo que variou entre 04 a 08 anos. Os achados histopatolÃgicos mos-traram uma reduÃÃo da densidade e do tamanho das CG, e um estroma fibro-colagenoso das lesÃes. Dentre os marcadores pesquisados, apenas GCR em CG antes do tratamento mostrou significÃncia estatÃstica (p<0,004) com relaÃÃo a uma boa resposta terapÃutica. O CTR ex-pressou-se em cÃlulas gigantes e mononucleares de forma variada. A p16 apresentou-se ex-pressa em 30% da amostra, COX2 nÃo apresentou expressÃo na lesÃo e 33% da amostra apre-sentou amplificaÃÃo gÃnica da ciclina D1. NÃo mostraram significÃncia estatÃstica nem quanto à agressividade, nem quanto resposta ao tratamento, nenhum dos marcadores, exceto o GCR. O estudo mostrou que a corticoterapia intralesional à efetiva e segura para o tratamento das LCCG, com tendÃncia a melhor resposta nas lesÃes nÃo-agressivas do que nas agressivas. Mostrou ainda que a marcaÃÃo para GCR em CG demonstrou ser um parÃmetro confiÃvel para prever a resposta à terapÃutica com a corticoterapia intralesional e que 33% das LCCG tÃm comportamento neoplÃsico pela amplificaÃÃo gÃnica da ciclina D1. / Central Giant Cells Lesion (CGCL) of the jaws is an intra-bone lesion with no predilection for sex and clinically divided into aggressive and non-aggressive subtypes. Histological, it shows as fibrous tissue with fusiform cells, as well as multinucleated giant cells (GC) clusters, he-morrhagic foci and neovascularization. Surgery is the regular treatment option. As new the-rapeutic approaches have been proposed, intralesional glucocorticoid injection is the main option. This paper assesses retrospectively 21 patients presenting CGCL, treated with intrale-sional triamcinolone hexacetonide by using the following protocol: intralesional injection of triamcinolone hexacetonide 20mg/mL, diluted in a solution of lidocain 2% plus epinephrine 1:200000, at a 1:1 proportion; 1mL of this final solution for each 1cm3 of lesion volume was the injected, with a total of 06 injections, one in every 15 days. Four clinical criteria were sta-bilished to evaluate treatment outcome: 1- Clinical regression or stabilization of the lesion; 2- Absence of symptoms; 3- Raising in density on radiographic controls; 4-Increased resistence when injecting the drug intralesionally. It was also performed immunohistochemical assess-ment for glucocorticoid receptor (GCR) expression, calcitonin receptor (CTR) expression, COX-2 expression, p16 expression and Ciclin D1 gene amplification by CISH, making com-parisons related to aggressivity and to therapeutic outcome. Eleven out of 21 patients of this study were women, and 10 were men. Nine of the patients had lesion located in the maxilla, 12 in the mandible. Ten patients showed aggressive lesions and 11 non-aggressive lesions. Fifteen patients showed good treatment outcome, four patients showed moderate outcome, and two patients showed negative answer to the treatment. Among the 11 patients with non-aggressive lesions, ten showed good outcome and the other, moderate outcome. Among the ten aggressive lesions, five patients showed good outcome, three patients showed moderate outcome and the remaining two patients showed negative answer to the treatment. None of them showed reicidive in a four to eight years follow-up period. Morphologic analysis found positive correlation between volume density of GC/mm2 and lesion aggressiveness, as well as significant reduction in number of GC/mm2 after treatment. Among the markers, only GCR in GC showed statistical relevance associated to the treatment. CTR was espresse in GC and in mononuclear cells in a varying way; p16 was expressed in 30% of the sample; COX-2 was not expressed at all in lesion samples and 33% of the sample showed gene amplification in Ciclin D1. None of the markers showed any statistical significant difference related to aggres-siveness nor to treatment outcome, except for GCR. The study showed the feasibility of the adopted treatment, with tendency to better outcomes in non-aggressive lesion, if compared to the aggressive ones. It also showed evidence pointing to GCR expression in GC as a reliable parameter to predict therapeutic responsiveness to glucocorticoids; and it showed that 33% of CGCL have neoplastic behaviour by Ciclin D1 gene amplification.

Diversos estudos epidemiológicos mostram que nutrientes e outros compostos bioativos presentes nos alimentos (CBA) apresentam atividade quimiopreventiva contra o câncer. Assim, destaca-se o estudo dos isoprenóides devido a sua ação promissora tanto na prevenção quanto na terapia do câncer. Todavia, apesar dessas evidências, pouco se sabe a respeito da ação dessas substâncias nos processos de proliferação celular e apoptose in vivo. Assim, 141 ratos Wistar foram tratados durante duas semanas consecutivas com farnesol (grupo FR, 25 mg/100 g de peso corporal) ou óleo de milho (grupo OM; controle, 0,25 mL/100 g de peso corporal) e sacrificados em diferentes momentos após a hepatectomia parcial (HP; 0 h, 30 min, 2 h, 4 h, 8 h, 12 h, 18 h e 24 h). Os parâmetros hepáticos analisados foram a proliferação celular (núcleos marcados para PCNA/mm2), apoptose (corpúsculos apoptóticos [CA\'s] por mm2) e expressão de p65, ciclina D1 (\"western blot\") e HMG-CoA redutase (\"dot-blot\"). Os animais tratados com o isoprenóide, assim como o grupo controle, apresentaram reduzida taxa de proliferação celular até 8h após a cirurgia. No entanto, a partir desse momento, o grupo FR passou a apresentar taxa de proliferação celular inferior ao grupo OM, diferença esta que atingiu significância estatística (p<0,05) 24h após a HP. Com relação a apoptose, animais tratados com FR apresentaram maior número de CA\'s (p<0,05) do que o grupo OM 30 min após a HP. Já em relação à ação do FR em âmbito molecular, houve uma redução de 40% e 50% na expressão de p65 e ciclina D1 30min e 24h após a HP, respectivamente, embora essas diferenças não tenham atingido significância estatística (p>0,05). Além disso, animais tratados com o isoprenóide apresentaram maior (p<0,05) expressão do gene que codifica para HMG-CoA redutase 2 h e 12 h após a cirurgia. Assim, tanto a inibição da proliferação celular quanto a indução de apoptose podem ser reflexo das alterações da expressão hepática dos genes para HMG-CoA redutase, p65 e ciclina D1 por parte do isoprenóide. / Epidemiological data have shown that nutrients and others bioactive compounds in food have chemopreventive activities against cancer. Among these compounds, isoprenoids are suggested either as a chemopreventive or chemotherapy agents. However, despite these evidences, studies focused on the isoprenoids activities on cell proliferation and apoptosis in vivo are rare. Thus, the effect of the 15-carbon isoprenoid farnesol on liver regeneration after partial hepatectomy was evaluated. Wistar rats were treated for two consecutive weeks with farnesol (FR group, 25 mg/100 g body weight) or corn oil (OM group, control, 0,25 mL/100 g body weight) and killed at different time points after partial hepatectomy (HP; 0 h, 30 min, 2 h, 4 h, 8 h, 12 h, 18 h and 24 h). Still, hepatic cell proliferation (PCNA lebeled nuclei), apoptosis (quantification of apoptotic bodies), p65 and cyclin D1 protein expression (western blot) and HMG-CoA reductase mRNA expression (dot blot) were also evaluated. Comparing to OM group, farnesol treatment significantly inhibited (p<0,05) hepatic cell proliferation 24 h after HP. Regarding apoptosis, also compared to controls, farnesol treated rats presented more (p<0,05) apoptotic bodies at 30 min. Besides, there were a suggestion of a higher number of apoptotic bodies 2 and 12 hours after HP in FR group comparing to OM group. According to western blot analysis, comparing to controls, this 15-carbon isoprenoid reduced 40% and 50% p65 and cyclin D1 hepatic protein expression, 30 min and 24 h after partial hepatectomy, respectively, although the differences did not also reach the statistical significance. Furthermore, farnesol treated rats had higher (p<0,05) HMG-CoA reductase mRNA levels than controls 2 h and 12 h after the surgery. Theses data suggest that the alterations on p65, cyclin D1 and HMG¬-CoA reductase gene expression observed in FR group might be associated with the inhibition of cell proliferation and the induction of apoptosis by farnesol.

Orientador: Rebeca de Souza Azevedo / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-27T23:04:36Z (GMT). No. of bitstreams: 1 Dutra_SabrinaNogueira_D.pdf: 4132202 bytes, checksum: 8d692501f423535818a567ec7a79ed11 (MD5) Previous issue date: 2015 / Resumo: A queilite actínica (QA) é uma desordem potencialmente maligna dos lábios, resultado da exposição crônica e excessiva ao raios ultravioleta, e que pode evoluir para um carcinoma de células escamosas de lábio. A via de sinalização Wnt/?-catenina atua em genes relacionados ao ciclo e a proliferação celular e está envolvida no desenvolvimento e progressão tumoral, e algumas de suas moléculas já foram identificadas em QA. O objetivo deste estudo foi correlacionar as características histopatológicas da QA com dois sistemas de classificação para gradação histopatológica, o da OMS e do sistema binário; e avaliar a participação de marcadores da via de sinalização Wnt/?-catenina por meio de reação imunoistoquímica contra os anticorpos Wnt1, Wnt5a, ?-catenina, axina, APC e ciclina D1 em casos de QA, e relacionar esta expressão com os dois sistemas de classificação. Os resultados mostraram correlação das características histopatológicas com o aumento da displasia epitelial de forma mais evidente pelo sistema binário do que pela classificação da OMS; positividade de Wnt 1, potente ativador da via canônica, em 96,7% dos casos; marcação anormal citoplasmática com ou sem marcação nuclear de ?-catenina em 81,9% dos casos; positividade de ciclina D1 em 75,4 % dos casos; negatividade de Wnt 5a, que possui potencial inibidor da via e pode contribuir indiretamente para ativação da via canônica; positividade de APC e axina em 96,7% e 95% dos casos de QA. Além disso, os anticorpos Wnt1, ciclina D1, APC e axina apresentaram aumento do índice de marcação imunoistoquímica de acordo com o aumento do grau de displasia epitelial de forma progressiva pelo sistema binário. Pode-se, assim, sugerir uma possível relação destas proteínas da via de sinalização Wnt/?-catenina com o desenvolvimento e a progressão da QA e que o uso do sistema binário de classificação de gradação histopatológica em QA apresentou melhor correlação com as características histopatológicas e com os índices de marcação imunoistoquímica da via de sinalização Wnt/?-catenina do que o sistema de classificação da OMS / Abstract: Actinic cheilitis (AC) is a potentially malignant disorder of the lips, resulting from chronic and excessive exposure to ultraviolet radiation, which can develop into a lip squamous cell carcinoma. Wnt/?-catenin signaling pathway acts on genes related to cell cycle and proliferation and is involved in tumor development and progression, and some of its molecules have already been identified in AC. Thus, the aim of this study was to correlate the histopathological features of AC with two grading histopathological systems, the WHO and the binary system; and to evaluate Wnt/?-catenin signaling pathway involvement by means of immunohistochemistry reaction against Wnt1, Wnt5a, ?-catenin, axin, APC and cyclin D1 in AC, and to relate this expression with the two histopathological grading systems. The results showed a correlation between the histopathologic features with the increased epithelial dysplasia mainly by using the binary system than using the WHO classification; Wnt 1 positivity, a potent canonical pathway activator, in 96.7% of the cases; abnormal ?-catenin cytoplasmic staining with or without nuclear staining in 81.9% of cases; cyclin D1 positivity in 75.4% of cases; Wnt 5a negativity, which has a potential for an inhibiting the pathway and may indirectly contribute to the activation of canonical pathway; APC and axin positivity in 96.7% and 95% of AC. In addition, Wnt1, cyclin D1, APC and axin showed a progressive increased immunohistochemical staining index according to the increasing degree of epithelial dysplasia by the use of the binary system. Therefore, we were able to suggest a possible relationship of these Wnt/?-catenin signaling pathway proteins with the AC development and progression and that the use of binary grading system in the histopathological classification of AC showed a better correlation with the histopathological features and the immunohistochemical staining indexes of Wnt/?-catenin signaling pathway than the use of WHO grading system / Doutorado / Patologia / Doutora em Estomatopatologia

La sénescence est considérée comme un mécanisme de suppression tumorale puisque les cellules potentiellement dangereuses, activent leurs protéines de sauvegarde pour arrêter leur prolifération. Les protéines de sauvegarde telles que RB et p53 sont activées suite à différents stress comme des dommages à l’ADN, le raccourcissement des télomères ou l’induction oncogénique. Les cellules sénescentes restent métaboliquement actives, subissent des modifications dans leur expression génique, et sécrètent des cytokines et des chimiokines qui ont des effets paracrines pro-oncogéniques, mais peuvent également contribuer à la stabilité de l’arrêt du cycle cellulaire dans la sénescence de façon autocrine. Une des particularités du phénotype sénescent est la dégradation sélective des protéines dépendante de l’ubiquitination et du protéasome. Parmi les cibles de dégradation se trouvent des protéines impliquées dans la biogenèse du ribosome, ainsi que celles d’autres voies cellulaires requises pour la croissance de cellules cancéreuses. Ceci est lié à un stress nucléolaire qui affecte la biogenèse du ribosome, menant à l’accumulation, dans le nucléoplasme ou le nucléole, de protéines ribosomiques. Ce comportement suggère que les ribosomes des cellules sénescentes seraient structurellement différents. Par conséquent, ceci pourrait entrainer des effets sur leurs capacités à réguler l’initiation, l’élongation et/ou la terminaison de la traduction des ARN messagers (ARNm). Par ailleurs, la déplétion de certaines protéines impliquées dans la ribogenèse, ainsi que la surexpression de protéines ribosomiques telles que RPS14/uS11 amènent à la sénescence. Malgré le stress nucléolaire et les défauts de ribogenèse associés à la sénescence, les cellules sénescentes présentent des niveaux de translecture du codon d’arrêt très diminué, suggérant l’existence de défauts de production de protéines allongées en C-terminal. Nous émettons l’hypothèse que les défauts de la ribogenèse affecteraient la fonction des protéines ribosomiques et des ribosomes. Cette perturbation aurait un impact sur le rôle de suppresseur tumoral de la sénescence. Le premier objectif de cette thèse consiste à démontrer le rôle de RPL22/eL22 en tant que régulateur du cycle cellulaire et inducteur de la sénescence. Le deuxième but est de démontrer que, malgré la perturbation nucléolaire, les ribosomes des fibroblastes sénescents reconnaissent les codons d’arrêt de façon plus efficace que les ribosomes des cellules transformées, ou des cellules normales en prolifération. Nous avons démontré que le phénotype de sénescence peut être induit quand l’expression de RPL22/eL22 est augmentée. RPL22/eL22 s’accumule principalement dans le nucléole, de manière différente de RPS14/uS11, dont l’accumulation est nucléoplasmique. En effectuant des essais kinases in vitro, nous avons montré que RPL22/eL22, tout comme RPS14/uS11, peuvent interagir et inhiber le complexe CDK4-Cycline D1 afin d’activer la voie de RB et établir l’arrêt du cycle cellulaire et la sénescence. Afin de démontrer la fidélité de la terminaison de la traduction dans les cellules sénescentes, nous avons utilisé un système de rapporteurs de luciférases, pour détecter les erreurs de translecture ainsi que pour avoir un contrôle interne du système. L’inactivation de la voie du suppresseur tumoral RB par surexpression de CDK4 ou de l’oncoprotéine virale E7, nous a permis d’observer l’augmentation de la translecture dans les cellules sénescentes. Tandis que l’activation de la voie de suppression tumorale RB, à l’aide du suppresseur de tumeur PML, de la surexpression de RPL22/eL22 et de RPS14/uS11, ainsi que de l’utilisation de Palbociclib (PD-0332991), un inhibiteur des kinases CDK4/6, a montré une réduction des erreurs de translecture. Ces résultats indiquent une nouvelle fonction des protéines du ribosome en tant que suppresseurs de tumeur, permettant d’inhiber les erreurs de translecture du codon d’arrêt de façon dépendante de la voie de RB. Ces travaux suggèrent que de petites molécules ou peptides pourraient simuler les fonctions inhibitrices de ces protéines ribosomiques afin de traiter certains cancers où la voie de RB est activable. / Senescence is considered a mechanism for tumor suppression since potentially dangerous cells activate their protective proteins to stop their proliferation. Safeguard proteins such as RB and p53 are activated as a result of stress such as DNA damage, telomere shortening or oncogenic induction. Senescent cells are metabolically active, they undergo changes in their gene expression and secrete cytokines and chemokines with pro-oncogenic paracrine effects, but which can also contribute to the stability of the senescent cell cycle arrest in an autocrine way. One of the peculiarities of the senescent phenotype is the selective ubiquitination and proteasome dependent-degradation of proteins involved in ribosome biogenesis and other cellular pathways required for cancer cell growth, leading to the accumulation, in the nucleoplasm or nucleolus, of ribosomal proteins. This behavior suggests that the ribosomes of senescent cells are structurally different. Therefore, this could have effects on their ability to regulate the initiation, elongation and/or translation termination of messenger RNAs (mRNAs). Moreover, the depletion of some proteins involved in ribogenesis, as well as the overexpression of ribosomal proteins such as RPS14/uS11 lead to senescence. Despite nucleolar stress and ribogenesis defects associated to senescence, global translation does not seem to be affected in senescence. Strikingly, senescent cells have reduced translational readthrough suggesting that they have defects in the production of C-terminal extended proteins. We hypothesize that defects in ribogenesis would affect the function of ribosomal proteins and ribosomes influencing the tumor suppressor role of senescence. The first aim of this thesis is to demonstrate the role of RPL22/eL22 as a regulator of the cell cycle and senescence inducer. The second aim of this thesis is to demonstrate that, despite the nucleolar disruption, the ribosomes of senescent fibroblasts recognize stop codons more efficiently than ribosomes from transformed cells, but also than ribosomes from proliferating normal cells. We found that the senescent phenotype can be induced by enhancing the expression of RPL22/eL22. RPL22/eL22 accumulates mainly in the nucleolus, unlike RPS14/uS11, whose accumulation is nucleoplasmic. By performing an in vitro kinase assay, we showed that RPL22/eL22, just like RPS14/uS11, can interact and inhibit the CDK4-Cyclin D1 complex in order to activate the RB pathway and establish cellular arrest and senescence. To assess translation termination accuracy in senescent cells, we used a system of luciferase reporters to measure the fidelity of translation termination. Inactivation of the RB tumor suppressor pathway using CDK4 or the viral oncoprotein E7 also increased readthrough in senescent cells while overexpression of PML, a tumor suppressor that activates the RB pathway, overexpression of RPL22/eL22 and RPS14/uS11, as well as the use of Palbociclib (PD-0332991), a CDK4/6 inhibitor, reduce readthrough errors. These results indicate a novel function of ribosomal proteins as tumor suppressors, making it possible to inhibit translational readthrough errors, in a RB-dependent pathway. This work suggests that small molecules or peptides could mimic the inhibitory functions of these ribosomal proteins in order to treat cancers where the RB pathway is activatable.

La sénescence est un processus caractérisé par un arrêt stable du cycle cellulaire. Ce mécanisme peut être induit en réponse à de nombreux stress, comme l’activation d’un oncogène, le raccourcissement des télomères ou bien le traitement avec des composés génotoxiques. Cette réponse cellulaire est considérée comme une barrière antitumorale limitant la prolifération des cellules exposées au risque de transformation. La mise en place de la sénescence dépend de profonds changements au niveau moléculaire, dont l’activation d’un programme de dégradation sélective des protéines. Cette dégradation de protéines associée à la sénescence (SAPD) peut expliquer plusieurs caractéristiques des cellules sénescentes, notamment la présence de défauts dans la voie de synthèse des ribosomes (SARD). Ces derniers sont liés à un stress nucléolaire qui mène à l’accumulation de certaines protéines ribosomiques dans le noyau, où elles peuvent effectuer des fonctions indépendantes de leur rôle structurale dans les ribosomes. Parmi ces protéines ribosomiques, RPS14/uS11 peut s’accumuler dans le nucléoplasme et réguler le cycle cellulaire en inhibant CDK4. Ces mécanismes de régulation post-traductionnelle -le SAPD ainsi que les conséquences des SARD- contribuent de manière importante au phénotype sénescent. Nous avons émis l’hypothèse que la caractérisation des effecteurs dans ces voies pourrait mener à l’identification de nouvelles protéines importantes pour la sénescence et la suppression tumorale. Dans un premier temps, nous avons évalué le rôle de la protéine ribosomique RPL22/eL22 dans le cycle cellulaire et la sénescence. Tout comme RPS14, RPL22 a été identifié dans l’analyse de l’interactome de CDK4 lors de la sénescence induite par la perte du facteur de la ribogenèse RSL1D1. Nous avons pensé que RPL22 pourrait agir de manière similaire à RPS14 et ainsi effectuer des fonctions extra-ribosomiques impliquées dans la régulation du cycle cellulaire. Dans le premier article présenté dans cette thèse, nous montrons que la surexpression de RPL22 dans des fibroblastes humains induit un phénotype sénescent et que RPL22 peut lier et inhiber CDK4 afin d’activer la voie de RB. Ensemble, ces données indiquent un rôle suppressif de RPL22 dans le cycle cellulaire. En second lieu, nous nous sommes penchés sur la caractérisation des effecteurs du programme de dégradation sélective de protéines associé à la sénescence. Ce programme est mené à terme par le système ubiquitine-protéasome, un mécanisme finement régulé par différents types de protéines. Parmi celles-ci, les E3 ubiquitine ligases définissent la spécificité de ce système en interagissant avec les substrats à dégrader. Nous avons donc pensé que certaines E3 ubiquitine ligases spécifiques pourraient être importantes pour le mécanisme de dégradation protéique associé à la sénescence. Afin d’identifier celles-ci, nous avons effectué un criblage de shARN ciblant des gènes d’E3 ubiquitine ligases dans le contexte de la sénescence induite par les oncogènes. Ceci a mené à l’identification d’ASB14 comme un acteur important de la sénescence. Dans le deuxième article de cette thèse, nous montrons que la perte d’ASB14 produit un contournement de la sénescence induite par l’oncogène RAS dans plusieurs modèles cellulaires. ASB14 est une protéine peu caractérisée et nous avons généré des anticorps afin d’analyser son expression. Nous montrons ensuite qu’ASB14 s’exprime fortement dans le pancréas sain, tandis que ses niveaux diminuent dans les tumeurs pancréatiques. Enfin, nous avons identifié les partenaires d’interaction d’ASB14 dans le contexte de la sénescence induite par l’oncogène RAS. Globalement, les travaux présentés dans cette thèse nous ont permis d’identifier deux nouvelles protéines impliquées dans la sénescence cellulaire : la protéine ribosomique RPL22 et l’E3 ubiquitine ligase ASB14. Ces deux protéines contribuent à la régulation post-traductionnelle du phénotype sénescent. D’un côté, RPL22 peut inhiber l’activité de CDK4 afin d’activer la voie de RB et ainsi réguler le cycle cellulaire. D’une autre part, ASB14 est importante pour le maintien du phénotype sénescent et semble avoir un rôle dans la suppression tumorale du pancréas. Nos résultats suggèrent que RPL22 et ASB14 sont importants pour la sénescence et la suppression tumorale. / Cellular senescence is characterized by a stable cell cycle arrest. This process can be induced by a variety of cellular stresses, including oncogene activation, telomere shortening and genotoxic treatments. In fact, senescence is considered an antitumor barrier that prevents cellular transformation. Senescence is associated with widespread molecular changes, including the activation of a selective protein degradation program. This senescence-associated protein degradation (SAPD) could regulate some senescence-associated phenotypes, including the senescence-associated ribosome biogenesis defects (SARD). Senescence-associated ribosome biogenesis defects are linked to a nuclear accumulation of some ribosomal proteins such as RPS14/uS11 capable of carrying out extra-ribosomal functions. In particular, RPS14 can inhibit CDK4 and mediate senescence. Thus, we hypothesize that the proteins implicated in these pathways -SAPD and SARD- could be important for senescence and tumor suppression. First, we evaluated the ability of the ribosomal protein L22 (RPL22/eL22) to regulate cellular senescence and cell cycle progression. RPL22, as RPS14, was identified as a binding partner for CDK4 in senescent cells induced by depleting the ribosome biogenesis factor RSL1D1. Hence, we though that RPL22 could act in a manner similar to RPS14. In chapter two, we show that RPL22 overexpression induces a senescent phenotype in human fibroblasts. In addition, we show that RPL22 can interact with CDK4 inhibiting its activity and stimulating the RB tumor suppressor pathway. Taken together, these results indicate a suppressive role of RPL22 in cell cycle progression. Next, we focused on the characterization of SAPD effectors. This mechanism is mediated by the ubiquitin-proteasome system which is tightly regulated by E3 ubiquitin ligases. Thus, we thought that specific E3 ubiquitin ligases could be important for SAPD and for senescence. In order to discover E3 ubiquitin ligases that contribute to senescence, we performed an unbiased screening using shRNA libraries in Ras-induced senescent cells. This led to the identification of ASB14 as an important mediator of senescence. In chapter three, we show that ASB14 depletion leads to a bypass of Ras-induced senescence. ASB14 is a poorly characterized E3 ligase, and we generated antibodies in order to analyze its expression levels. We show that ASB14 is highly expressed in the normal pancreas whereas its expression is reduced in pancreatic cancer tissues. Finally, we uncovered the interactome of ASB14 in Ras-induced senescent cells. Overall, we have discovered two new senescence mediators: ribosomal protein L22 and E3 ubiquitin ligase ASB14. These proteins are implicated in the post-translational regulation of the senescent phenotype. RPL22 acts as a CDK4 inhibitor to activate RB pathway and regulate cell cycle arrest and ASB14 is an important mediator of senescence maintenance. Taken together, our results suggest that RPL22 and ASB14 are important for cellular senescence and tumor suppression.

O melanoma cutâneo é a neoplasia de pele de maior mortalidade. A imprevisibilidade de sua evolução é uma de suas características principais, o tratamento do tumor primário é, atualmente, de pouca morbidade e, na doença disseminada, as opções terapêuticas são pouco eficazes. É fundamental a pesquisa de marcadores tumorais que permitam a previsão da evolução, melhor compreensão da patogênese do melanoma e possibilitem a descoberta de alvos moleculares. Nesse contexto, estudos genéticos mostraram a importância da regulação do ciclo celular, especialmente a passagem da fase G1-S. Importantes fatores envolvidos compõem a cascata da proteína Rb (p16, ciclina D1, CDK4 e pRb) e da proteína p53 (p53 e p21). Objetivo: verificar a frequência da expressão de p16, ciclina D1,CDK4, pRb, p53 e p21 em melanomas cutâneos de cabeça, pescoço e tronco e sua relação com prognóstico. Métodos: Estudo retrospectivo envolvendo 46 pacientes (sendo 67,3% homens, idade média 57,7 ± 15,8 anos) com melanoma cutâneo de cabeça, pescoço e tronco que foram tratados pela mesma equipe com seguimento mínimo de dois anos. Foram estudados fatores clínicos (topografia do tumor primário, tempo de seguimento, ocorrência de metástases e óbito relacionado), histopatológicos (tipo histológico, índice de Clark, índice de Breslow) e análise imuno-histoquímica pela técnica de micro-array para as proteínas reguladoras do ciclo celular p16, ciclina D1, CDK4, pRb, p53 e p21. Resultados: Houve proporção igualitária entre as topografias (23 casos em tronco, 23 em cabeça e pescoço). Treze pacientes com Clark I (29,5%), cinco com II (11,3%), 16 com III (36,5%), 10 com IV (22,7%) e nenhum com Clark V. A média das medidas de Breslow foi 0,96 (DP=1,01). O seguimento médio foi de 77 meses (DP=47). Oito dos 46 pacientes (17,3%) tiveram evolução desfavorável, com seis óbitos relacionados. A idade foi mais elevada no grupo com evolução desfavorável (p=0,04). Houve expressão de p16 em 80%, ciclina D1 em 58,9%, CDK4 em 43,5%, pRb em 58,5%, p53 em 53,6% e p21 em 52,3% dos melanomas. Em análise univariada, a expressão do p21 foi relacionada com evolução desfavorável (p=0,04), o que não foi observado com a expressão dos outros marcadores (p>0,05). Conclusão: A expressão da proteína p21 nos melanomas cutâneos de cabeça, pescoço e tronco foi relacionada com evolução desfavorável, o que não ocorreu com outros fatores envolvidos na regulação do ciclo celular / Melanoma is the most lethal skin cancer. The outcome of melanoma is not predictable in most cases. Although the treatment for the primary tumor is well tolerated, there are no effective therapeutic options in disseminated disease. Efforts are being made in the search for tumoral markers that may predict outcome, increase the comprehension of melanoma pathogenesis, and may also help the search for molecular targets. In this issue, genetic studies concerning the regulation of cell cycle, including the G1-S checkpoint, are important. The retinoblastoma protein (pRb) pathway (p16, cyclin D1, CDK4 and pRb) and the p53 pathway (p53 and p21) are part of this regulation. Objectives: to verify the expression of p16, cyclin D1, CDK4, pRb, p53 and p21 in head, neck and trunk melanomas, and its correlation with prognosis. Method: Retrospective study approved by institution ethics committee. Fourtysix head, neck and trunk melanoma patients (67.3% men, mean age 57.7±15.8) treated by a single surgeon with minimum 2-years follow-up were enrolled. Clinical factors (primary tumor location, follow-up period, metastasis or related deaths), pathologic (histological subtype, Clark and Breslow index) and microarray immunohystochemical analysis of the cell cycle proteins p16, cyclin D1, CDK4, pRb, p53 and p21. Results: Location of the primary tumor was equal for head/neck and trunk (50% each). Thirteen patients were classified as Clark I (29.5%), five as Clark II (11.3%), 16 as Clark III (36.5%), 10 as IV (22.7%), none as Clark V. Mean Breslow measure was 0.96±1.01. Mean follow-up was 77±47 months. Eight patients (17.3%) had bad outcome, with six related deaths. Patients with worse outcome had a higher mean age at diagnosis (p=0,04). Expression of p16 was positive in 80%. Cyclin D1 was positive in 58.9%. CDK4 was positive in 43.5%. pRb was positive in 58.5%. p53 was positive in 53.6%. p21 was positive in 52.3%. Univariated analysis showed that p21 expression was related to worse outcome (p=0,04), while the other markers were not (p>0,05). Conclusion: p21 expression in head, neck and trunk melanomas was related to worse outcome. Expression of the other cell cycle regulators proteins was not

Ependimomas são tumores gliais raros. Podem ser encontrados em qualquer localização do sistema nervoso central e, apesar de histologia similar, parecem apresentar alterações genômicas distintas. As variáveis clínicas são intercorrelacionadas e, geralmente, incapazes de predizer o curso da doença. O objetivo do presente estudo foi analisar a expressão aumentada de genes e proteínas em ependimomas e correlacionar com dados clínicos dos pacientes. Foram estudados casos de pacientes com ependimoma submetidos à ressecção cirúrgica no Hospital das Clínicas, Universidade de São Paulo, no período entre 1996 e 2011 (33 amostras de tecido congelado para análise de expressão gênica por PCR quantitativo em tempo real e 149 amostras com tecido incluído em parafina, correspondentes a 121 casos devido a recidivas, para análise de proteína por imuno-histoquímica de tissue microarrays). As reações de imuno-histoquímica foram analisadas semiquantitativamente e graduadas com um índice de marcação calculado pelo produto da porcentagem de núcleos marcados pela intensidade de marcação. Oitenta e um casos eram adultos (média de 27,2 anos). Havia 60 casos intracranianos e 61 intramedulares, dos quais 10 eram mixopapilares, 92 grau II e 19 grau III. Ressecção completa foi possível em 62% dos casos e recidiva foi confirmada em 41,1%. Observou-se menor tempo para recidiva em crianças e tumores intracranianos, supratentoriais (p < 0,001 em ambos), histologia anaplásica e ressecções incompletas (p < 0,05 em ambos). Os seguintes genes foram selecionados em dados públicos de SAGE e literatura: ARMC3, CCND1, CHST5, DNALI1, FGFRL1, GNA13, IGF2, MSX1, NOTCH1 e RSPH3. ARMC3, RSHL3, CHST5 e DNALI1 apresentaram maiores níveis de expressão em ependimomas intramedulares (p < 0,05), e FGFRL1, NOTCH1 e CCND1 nos casos supratentoriais (p < 0,01). IGF2 apresentou maiores níveis de expressão em crianças e CHST5 em adultos (p < 0,05 em ambos). Foram observados maiores níveis de expressão de FGFRL1 (p < 0,05), CCND1 e IGF2 (p < 0,01 em ambos) em casos com histologia anaplásica. Nenhum dos genes analisados apresentou impacto no tempo livre de progressão ou na sobrevida. A expressão da proteína codificada por CCND1, ciclina D1, também foi avaliada por imuno-histoquímica, por ser uma proteína com expressão aumentada em diversos tipos de neoplasias e não ter ainda um valor prognóstico bem estabelecido em ependimomas. Houve correlação entre expressão de ciclina D1 a nível de mRNA e da proteína (p < 0,0001). As correlações entre ciclina D1 e histologia anaplásica e localização supratentorial foram confirmadas pela análise proteica (p < 0,0001 em ambos). Adicionalmente, foi também observada maior expressão de ciclina D1 em pacientes mais jovens (p < 0,01). A maior expressão de ciclina D1 em tumores com localização supratentorial foi independente do grau histológico e da idade do paciente. Recidiva foi mais frequente em casos com maiores níveis de expressão de ciclina D1 (p < 0,05), embora uma maior correlação com tempo livre de progressão foi observada apenas em casos com ressecção completa (p < 0,001). Os ependimomas apresentaram expressão gênica diferencial de acordo com idade, localização do tumor e grau histológico nesse estudo. A determinação dos níveis da expressão de ciclina D1 pode ser útil para guiar o seguimento e tratamento dos casos supratentoriais com ressecções completas / Ependymomas are rare glial cell-derived tumors. They can be found in any central nervous system localization and despite the histological similarity, they seem to display distinct genomic abnormalities. Clinical variables are intercorrelated and they are usually unable to predict the disease course. We aimed to analyze increased gene and protein expression in ependymomas and to correlate with patients\' clinical data. We studied patients with ependymoma submitted to surgical resections at Hospital das Clinicas, University of São Paulo, from 1996 to 2011 (33 fresh-frozen samples for gene expression analysis by quantitative real-time PCR and 149 formalin-fixed, paraffin-embedded samples, relative to 121 patients due to relapses, for protein analysis by tissue microarray immunohistochemistry). Immunohistochemical reactions were analyzed semi-quantitatively and scored with a labeling index (LI) calculated as the product of the percentage of the positively stained nuclei by the intensity of staining. Eighty-one cases were adults (mean 27.2 years). There were 60 intracranial and 61 spinal cases, of which 10 tumors were myxopapillary, 92 were grade II and 19 were grade III. Gross total resection was achieved in 62% of cases and relapse was confirmed in 41.4% of cases. We observed a shorther time to relapse in children and supratentorial intracranial tumor localization (p<0.001 for both), anaplastic histology and incomplete resections (p<0.05 for both). The following genes were selected based on public SAGE database and literature: ARMC3, CCND1, CHST5, DNALI1, FGFRL1, GNA13, IGF2, MSX1, NOTCH1 and RSPH3. ARMC3, RSHL3, CHST5 and DNALI1 presented higher expression levels in intramedullary ependymomas (p < 0.05) and FGFRL1, NOTCH1 and CCND1 in supratentorial cases (p < 0.01). IGF2 presented higher expression levels in pediatric cases and CHST5 in adults cases (p < 0.05 in both). Higher expression levels of FGFRLI1 (p < 0.05), CCND1 and IGF2 (p < 0.01 for both) were observed in anaplastic histology cases. None of the genes impacted in progression free survival or overall survival of patients. The expression of protein codified by CCND1, cyclin D1, was also evaluated by immunohistochemistry, because its overexpression has been related with several types of neoplasias and its prognostic value has not yet been fully established in ependymomas. There was a correlation of cyclin D1 expression at mRNA and protein levels (p < 0.0001). Correlations between cyclin D1 and anaplastic histology and supratentorial localization were confirmed by protein analyses (p < 0.0001 for both parameters). Additionally, high expression of cyclin D1 was observed in younger patients (p < 0.01). The higher cyclin D1 expression in supratentorial tumor localization was independent of histological grade and age of patient. Relapse was more frequent in cases with higher cyclin D1 expression levels (p < 0.05) although correlation with progression free survival was just observed in gross total resection cases (p < 0.001). Ependymomas presented differential gene expression according to age, tumor localization and histological grade in our study. Determination of cyclin D1 expression levels may be useful to guide follow-up and treatment in supratentorial cases with gross total resection

Breast cancer is the second leading cause of death due to cancer in women. A number of effective therapeutic strategies have been implemented in clinics to cope with the disease yet recurrent disease and toxicity reduce their effectiveness. Hence, there is a need to identify and develop more effective therapies with reduced toxic side effects to improve overall survival rates. This dissertation investigates the mechanisms of action of two natural forms of vitamin E and a cholesterol lowering drug, simvastatin, as a therapeutic strategy in human breast cancer cells. Vitamin E in nature consists of eight distinct forms which are fat soluble small lipids. Until recently, vitamin E was known as a potent antioxidant but emerging work suggests they may be resourceful agents in managing a number of chronic diseases including cancer. Anticancer properties of vitamin E have been identified to be limited to the γ- and δ- forms of both tocopherols and tocotrienols. Gamma-tocopherol ([gamma]T) and gamma-tocotrienol ([gamma]T3) have both already been identified to induce death receptor 5 (DR5) mediated apoptosis in breast cancer cells. Studies here show that similar to [gamma]T3, [gamma]T induced DR5 activation is mediated by c-Jun N-terminal kinase/C/EBP homologous protein (JNK/CHOP) proapoptotic axis which in part contributed to [gamma]T mediated dowregulation of c-FLIP, Bcl-2 and Survivin. Also, both agents activate de novo ceramide synthesis pathway which induces JNK/CHOP/DR5 proapoptotic axis and downregulates antiapoptotic factors FLICE inhibitory protein (c-FLIP), B-cell lymphoma 2 (Bcl-2) and Survivin leading to apoptosis. Simvastatin (SVA) has been identified to display pleiotropic effects including anticancer effects but mechanisms responsible for these actions have yet to be fully understood. In this dissertation, it was observed that simvastatin induced apoptosis in human breast cancer cells via activation of JNK/CHOP/DR5 proapoptotic axis and down regulation of antiapoptotic factors c-FLIP and Survivin which are in part dependent on JNK/CHOP/DR5 axis. The anticancer effects mediated by simvastatin can be reversed by exogenously added mevalonate and geranylgeranyl pyrophosphate (GGPP), implicating the blockage of mevalonate as a key event. Furthermore, work has been done to understand the factors responsible for drug resistance and identify therapeutic strategies to counteract the same. It was observed that development of drug resistance was associated with an increase in the percentage of tumor initiating cells (TICs) in both tamoxifen and Adriamycin resistant cells compared to their parental counterparts which was accompanied by an increase in phosphorylated form of Signal transducer and activator of transcription 3 (Stat3) proteins as well as its downstream mediators c-Myc, cyclin D1, Bcl-xL and Survivin. Inhibition of Stat3 demonstrated that Stat3 and its downstream mediators play an important role in regulation of TICs in drug resistant breast cancer. Moreover, SVA, [gamma]T3 and combination of SVA+[gamma]T3 has been observed to target TICs in drug resistant human breast cancer cells and downregulate Stat3 as well as its downstream mediators making it an attractive agent to overcome drug resistance. From the data presented here, the mechanisms responsible for the anticancer actions of [gamma]T, [gamma]T3 and SVA have been better understood, providing the necessary rationale to test these agents by themselves or in combination in pre-clinical models. / text