Made available in DSpace on 2014-07-29T15:21:56Z (GMT). No. of bitstreams: 1 DISSERTACAO NADIA DO L COSTA.pdf: 215211 bytes, checksum: 98b2cf88b4766dae4e25cfa06b0970d4 (MD5) Previous issue date: 2010-02-26 / The cytotoxic T lymphocytes (CTL) and natural killer (NK) cells are more effective in fighting cancer because these cells recognize tumor cells and release cytotoxic granules rich in perforin and granzyme B (GB). The perforin form pores in tumor cells allowing the influx of GB. When inside the cell, the GB promotes tumor cell death through apoptosis. In this context, high expression of GB into the microenvironment of different types of cancers has been considered a key event for effective antitumor immunity. Therefore, the objective of this study was to identify and quantify GB+ cells, peri- and intratumoral, and its relationship with clinical prognostic parameters (size of the primary lesion, location, metastasis and survival) and microscopic (microscopic grading and cell proliferation index and apoptosis of neoplastic cells) of squamous cell carcinoma (SCC) of the oral cavity. GB+ cells were identified by the technique of immunohistochemistry, a method of polymer, in 20 samples from patients with SCC of the oral cavity that had metastasized to cervical lymph nodes and in 35 samples from patients with SCC of the oral cavity without metastasis to cervical lymph nodes. Our results showed that the density of peritumoral GB+ cells was significantly higher in the non-metastatic SCC when compared with metastatic group (p=0.03). In addition, patients with high expression of peritumoral GB had a longer survival than those with low expression of this protease (Kaplan Meier, Log Rank p=0.02). We showed also that patients with high density of peritumoral GB+ cells showed a low percentage of neoplastic cells bcl2+ (antiapoptotic protein) (P=0.004) and high density of neoplastic cells Bax+ (apoptotic protein) (p=0.031) when compared to the group of low density of peritumoral GB+ cells. In agreement with these data, patients who had a high density of peritumoral GB+ cells showed high expression of Annexin V by neoplastic cells. Additionally, the density of GB+ cells intratumoral and peritumoral was significantly higher in the T1 and T2 (39.44±7.21 and 14.61±3.60, respectively) when compared to T3 and T4 (31.03±3.76 and 11.90±1.88, respectively), however these results were not statistically significant (p>0.05). The association between the density of cells GB+ and other characteristics of SCC of the oral cavity, such as location, tumor proliferation and tumor grading was not observed. Our findings suggest that the increased of expression of GB in tumor microenvironment of SCC of the oral cavity may have beneficial effect against neoplastics cells, contributing to apoptosis of these cells, lower lymph node involvement and increased survival time of patients. / Os Linfócitos T Citotóxicos (LTC) e as células Natural Killer (NK) são as células mais efetivas no combate ao câncer, pois essas células reconhecem células tumorais e liberam grânulos citotóxicos ricos em perforina e granzima B (GB). A perforina forma poros nas células tumorais permitindo o influxo da GB. Quando no interior da célula, a GB promove a morte da célula tumoral através da apoptose. Neste contexto, a alta expressão de GB no microambiente de diferentes tipos de cânceres tem sido considerado um evento fundamental para uma efetiva imunidade antitumoral. Portanto, o objetivo do presente estudo foi identificar e quantificar células GB+, peri- e intratumoral, e sua relação com parâmetros de prognóstico clínicos (tamanho da lesão primária, localização, metástase e sobrevida) e microscópicos (gradação microscópica e índices de proliferação celular e de apoptose das células neoplásicas) do carcimoma espinocelular (CEC) de cavidade oral. As células GB+ foram identificadas pela técnica da imunoistoquímica, método do polímero, em 20 amostras de pacientes com CEC de cavidade oral que apresentaram metástase para linfonodos cervicais e em 35 amostras de pacientes com CEC de cavidade oral sem metástase para linfonodos cervicais. Nossos resultados demonstraram que a densidade de células GB+ peritumoral foi significativamente maior no grupo de CEC não metastático quando comparado com o metastático (p=0,03). Além disso, os pacientes com alta expressão de GB peritumoral apresentaram um maior tempo de sobrevida do que aqueles com baixa expressão dessa protease (Kaplan Meier, Log Rank p=0,02). Evidenciamos, também, que os pacientes com alta densidade de células GB+ peritumoral apresentaram baixa porcentagem de células neoplásicas bcl2+ (proteína anti-apoptótica) (P=0.004) e alta densidade de células neoplásicas Bax+ (proteína pró-apoptótica) (p=0,031) quando comparado ao grupo de baixa densidade de células GB+ peritumoral. Em concordância com esses dados, os pacientes que apresentaram alta densidade de células GB+ peritumoral apresentaram alta expressão de Anexina V por células neoplásicas. Adicionalmente, a densidade de células GB+ peritumoral e intratumoral foi significativamente maior no grupo T1 e T2 (39,44±7,21 e 14,61±3,60, respectivamente) quando comparado a T3 e T4 (31,03±3,76 e 11,90±1,88, respectivamente), no entanto esses resultados não foram estatisticamente significativo (p>0,05). A associação entre a densidade de células GB+ e outras características do CEC de cavidade oral, como localização, proliferação tumoral e gradação tumoral não foi observada. Nossos achados sugerem que a alta expressão da GB no microambiente do CEC de cavidade oral pode ter um efeito benéfico contra células neoplásicas, contribuindo para apoptose dessas células, baixa metástase linfonodal e maior tempo sobrevida desses pacientes

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-10-09T12:38:26Z No. of bitstreams: 2 Dissertação - Maisa Ribeiro - 2015.pdf: 1621645 bytes, checksum: 2b3a1e65f1e53b4a1264cf3febb2c630 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-10-09T12:41:15Z (GMT) No. of bitstreams: 2 Dissertação - Maisa Ribeiro - 2015.pdf: 1621645 bytes, checksum: 2b3a1e65f1e53b4a1264cf3febb2c630 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-10-09T12:41:15Z (GMT). No. of bitstreams: 2 Dissertação - Maisa Ribeiro - 2015.pdf: 1621645 bytes, checksum: 2b3a1e65f1e53b4a1264cf3febb2c630 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-02-19 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / In the lesions carcinomatous, low oxygen tension plays a crucial step in the self-renewal, metastatic potential, and therapy resistance of cancers. To adapt to the hypoxic microenvironment, neoplastic cells activate hypoxia-induced factor-1 alpha (HIF-1 alpha), which may mediates invasion and metastasis. In addition, the human THY-1 (CD90) cell surface protein mediates cell adhesion expressed in stem cells, and seens to drive tumor development in some malignant tumors. The present study investigates HIF-1 alpha (n=98) and CD90 (n=97) expression in oral squamous cell carcinoma (OSCC) and metastatic lymph nodes (n=24), the intratumoral region and the invasive front, by immunohistochemistry. Furthermore, clinicopathological data revised from the medical records. In superficial OSCCs, most tumor cells overexpressed HIF-1 alpha, whereas was restricted in the intratumoral region in invasive conventional SCCs. Interestingly, metastatic lymph nodes (91.7%, p=0.001), and intratumoral regions of its corresponding primary tumors (83.3%, p<0.001) were invaded by HIF-1 alpha-positive neoplastic cells. Overall survival was poor in patients with nodal involvement. CD90 was expressed mostly in microvessels and granulocyte cells similar to mast cells. These cells expressed CD90 mostly in the peritumoral region of invasive SCC (p<0.001). Microvessels CD90 positive were higher in the intratumoral region (p=0.032). Interesting, mast cell and microvessels positively correlated in OSCC (p=0.006; r²=0.077). In conclusion, hypoxic environment may facilitate regional metastasis and serve as a potential diagnostic and prognostic marker in OSCC primary tumors. Microvessels CD90 positive seems to promote tumor growth except in BSCC. Mast cell may occur via CD90 for tumor progression. / Nas lesões carcinomatosas a baixa tensão de oxigênio desempenha um passo crucial para a auto-renovação, potencial metastático, e resistência à terapia no câncer. Para se adaptar ao ambiente hipóxico, células neoplásicas ativam o fator induzido por hipóxia-1 alfa (HIF-1 alfa), que pode facilitar a invasão e metástase. Além disso, o THY-1 (CD90) humano, uma proteína de superfície celular expressa em células estaminais, medeia a adesão celular, e parece promover o desenvolvimento em alguns tumores malignos. O presente estudo analisou a expressão das proteínas HIF-1 alfa (n = 98) e CD90 (n = 97) no carcinoma espinocelular de boca (CEC de boca) e linfonodos metastáticos (n=24), na região intratumoral e no fronte de invasão, por meio de imunoistoquímica. Além disso os dados clinicopatológicos foram revisados a partir dos prontuários médicos e a sobrevida foi analisada. No CEC microinvasivo, a maioria das células tumorais apresentaram superexpressão do HIF-1 alfa, enquanto que no CEC invasivo a superexpressão foi restrita na região intratumoral. Verificou-se que em linfonodos metastáticos (91,7%, p = 0,001), e regiões intratumorais dos seus tumores primários correspondentes (83,3%, p <0,001) houve forte expressão do HIF-1 alfa em células neoplásicas. A sobrevida global foi pior em pacientes com metástase regional. A proteína CD90 foi expressa principalmente em microvasos e células de granulócitos semelhantes aos mastócitos. Estas células expressaram CD90 principalmente na região fronte de invasão do CEC invasivo (p<0,001). A média de microvasos CD90 positivo foi maior na região intratumoral (p=0,032). Interessantemente, mastócitos e microvasos foram positivamente correlacionados no CEC de boca (p=0,006; r²=0,077). Em conclusão, o ambiente hipóxico pode facilitar metástases regionais e funcionar como um potencial marcador de diagnóstico e prognóstico em tumores primários do CEC de boca. Os microvasos CD90 positivo parecem promover o crescimento do tumor, exceto no carcinoma escamoso basalóide (CEB). Os mastócitos ativados via CD90 podem contribuir com a progressão do tumor.

Os genes homeobox atuam como reguladores da morfogênese e diferenciação celular embrionária, portanto, é evidente a possibilidade de sua expressão anormal estar presente na progressão de tumores. Estudos preliminares em nosso laboratório verificaram a participação de alguns genes homeobox em carcinoma epidermóide de boca (CEB). Este trabalho teve como objetivo avaliar a amplificação, expressão e o perfil de metilação dos genes HOXA5, HOXA7, HOXA9, HOXB5, HOXB13, HOXC12 em linhagem celular derivadas de CEB e tecidos frescos tumoral e não-tumoral. Além disso, verificar o efeito da desmetilação na expressão gênica de linhagens celulares que apresentaram genes 100% metilados e a participação das enzimas responsáveis pela metilação do DNA, bem como a expressão das DNAmetiltransferases (DNMT) nos tumores. A análise de amplificação do DNA e expressão de mRNA foi realizada por qRT-PCR. O perfil de metilação foi avaliada pelo sistema PCR Array e a análise proteica das DNMT1, DNMT3a, DNMT3b e HOXA9 foi verificada por meio de reações imunohistoquímicas. As linhagens celulares SCC4 e SCC9 foram utilizadas para análise de desmetilação com 5-aza-2\'-deoxicitidina e a linhagem SCC4 para avaliar os efeitos do aumento de expressão do HOXA9, na proliferação celular por imunocitoquimica para Ki67, migração celular por transwell e apoptose pela avaliação de células positivas no ensaio de TUNEL. Na comparação entre os grupos, o gene HOXA5 apresentou-se amplificado na margem em relação ao tumor; o HOXA9 apresentou nível de metilação aumentada no tumor; o HOXB5 com amplificação maior na margem, com nível de expressão do mRNA aumentada nos tumores, e nível de metilação do tumor maior em relação a margem, sendo correlacionada com menor sobrevida; HOXB13 se apresentou amplificado no tumor em relação a margem, e com nível de metilação maior nos tumores e, HOXC12 com níveis de metilação maior nos tumores em relação a margem. É interessante, que os mesmos genes que tiveram níveis de metilação aumentados nos tumores em relação a margem, também estavam 100% metilados nas linhagens celulares SCC4 e SCC9 e tiveram sua expressão restaurada após o tratamento com 5-aza-2´-deoxicitina. Na avaliação do nível de expressão das DNMTs nos tumores, a DNMT3b apresentou-se com níveis aumentados em relação a DNMT1 e DNMT3a, e quando avaliado em nível proteico, a DNMT3a pode ser correlacionada com melhor sobrevida. O aumento da expressão do gene HOXA9 mostrou diminuição da migração celular, porém não alterou a proliferação e apoptose celular. A expressão proteica não apresentou correlação com parâmetros clínicos. Em conclusão, os resultados mostram que os genes homeobox estudados estão pouco metilados nas linhagens celulares e em tecidos de CEB. A amplificação desses genes não é um evento frequente. O gene HOXA9 é pouco expresso no tumor, e o aumento da sua expressão em linhagens celulares diminui a migração celular. / Homeobox genes are important as morphogenetic and embrionary cellular differentiation regulators, therefore there is basis for their abnormal expression in tumor progression. Preliminary studies in our laboratory have shown that homeobox genes are dysregulated in oral squamous cell carcinoma (OSCC). This study evaluates the genomic amplification, mRNA expression and methylation status of HOXA5, HOXA7, HOXA9, HOXB5, HOXB13, HOXC12 in squamous cell carcinoma derived cell lines, and fresh tumor tissue. Also, analyzes the demethylation effect in gene expression of cell lines with genes showing 100% methylation, and the participation of enzymes responsible for DNA methylation, DNAmetiltransferases (DNMT), in gene and protein expression in tumors. DNA amplification and mRNA expression was analyzed by qRT-PCR. The methylation profile was evaluated by PCR Array System and DNMT1, DNMT3a, DNMT3b and HOXA9 protein expression was verified by immunohistochemistry. SCC4 and SCC9 cell lines were submitted to 5-aza-2\'-deoxycytidine for demethylation analysis. SCC4 cell lineage was analyzed by immunocytochemistry for Ki67, cell migration by transwell and apoptosis by TUNEL test after increased expression of HOXA9. HOXA5 gene was amplified in the adjacent margin when compared with the tumor; HOXA9 showed increased level of methylation in tumor; HOXB5 showed amplification in the margin, increased mRNA expression in tumors, increased methylation level and was correlated with decreased survival; HOXB13 was amplified in tumor samples when compared to the margins and also higher methylation level in tumors; and HOXC12 also showed increased methylation levels in tumors when compared to margin. Interestingly, the same genes with increased methylation levels in tumors, were also 100% methylated in cell lines SCC4 and SCC9. The expression of these gens was restored after treatment with 5-aza-2\'-deoxycytidine. DNMT3b presented higher levels of protein expression relative to DNMT1 and DNMT3a. DNMT3a protein expression was correlated with improved survival. SCC4 cells overexpressing HOXA9 gene showed decreased cell migration, with no effect on cell proliferation and apoptosis. HOXA9 protein expression was not correlated with clinical parameters. In conclusion, the results shows that homeobox genes are methylated in some OSCC cell lines and tissues. Amplification of these genes is not a frequent event. HOXA9 gene has low expression in OSCC, and when overexpressed in cell lines decreases cell migration.

Quatro décadas de pesquisas mostraram que muitos mecanismos inflamatórios estão intrinsecamente ligados ao desenvolvimento e manutenção do câncer, e ainda, que as citocinas inflamatórias exercem papel primordial nessa relação. Os anti-inflamatórios não esteroides (AINEs) podem reduzir o desenvolvimento neoplásico por afetar a produção de citocinas inflamatórias pelas células neoplásicas. No entanto, até o momento não foi bem definido se o tratamento com AINEs é capaz de modular a expressão de citocinas inflamatórias por células do carcinoma epidermoide oral (CEO). O objetivo deste trabalho foi avaliar a expressão de citocinas inflamatórias em linhagens celulares de CEO após tratamento com ácido acetilsalicílico (AAS) e celecoxibe (CLX). Foi realizado screening da expressão de 84 citocinas e quimiocinas, através de PCR array, das linhagens SCC4, 9 e 25 tratadas com doses de AAS e CLX próximas às concentrações plasmáticas dos fármacos em humanos. Os resultados mostraram que AAS e CLX modularam a expressão de citocinas e que as linhagens responderam de maneira diferente aos tratamentos. Observou-se aumento de expressão de citocinas pró-inflamatórias como a IL-1?, IL-8 e TNF na SCC9 e 25, assim como diminuição de expressão de ACKR4 e CXCL10 na SCC4 e 9. / Four decades of research have shown that many inflammatory mechanisms are intrinsically linked to the development and maintenance of cancer and that inflammatory cytokines play pivotal role in this association. Non-steroidal anti-inflammatory drugs (NSAIDs) can reduce neoplastic growth on affecting the production of inflammatory cytokines by the neoplastic cells. So far, it is not well established if the treatment with NSAIDs can modulate the expression of inflammatory cytokines by OSCC cells. The objective of this study was to evaluate the expression of inflammatory cytokines by OSCC cell lines after treatment with acetylsalicylic acid (ASA) and celecoxib (CLX). Eighty-four cytokines and chemokines mRNA expression were screened by PCR array on SCC4, 9 and 25 cell lines treated with ASA and CLX at plasma concentrations in humans. The results showed that ASA and CLX modulate the expression of cytokines with all cell lines responding differently to the treatments. Increased expression of proinflammatory cytokines such as IL-1?, IL-8 and TNF in SCC9 and 25, and reduced expression of ACKR4 and CXCL10 in SCC4 and 9, was observed. Thus it follows that the treatments of lines SCC4, SCC9 and SCC25 with ASA and CLX at the next plasma concentrations in humans are able to modulate the gene expression of inflammatory cytokines.

No description available.

CDC73, also known as HRPT2, is a tumour suppressor gene whose expression is lost or downregulated in parathyroid, renal, breast, uterine and gastric cancers. However, the reports regarding the role of CDC73 in oral squamous cell carcinoma (OSCC) are lacking. As part of the Paf1 complex, it remains associated with ribonucleic acid (RNA) polymerase II and is involved in transcript site selection, transcriptional elongation, histone H2B ubiquitination, histone H3 methylation, poly(A) length control and, coupling of transcriptional and posttranscriptional events. It has been reported to negatively regulate cellularproliferation by targeting oncogenes CCND1 (cyclin D1) and MYC (c-Myc). Moreover, it has also been indicated to inhibitβ-catenin-mediated transcription. Taken together, these findings strongly suggest that it contributes to the expression of genes whose products have an important role in the suppression of tumor development and cell death. In this study, we have attempted to study the transcriptional and posttranscriptional regulation of CDC73 and its role in OSCC. The main findings of the present study are listed below. 1. To begin with, the expression analysis of CDC73 was performed both at the RNA and the protein levels by qRT-PCR and IHC, respectively. As expected, a majority of the OSCC samples showed downregulation of CDC73 both at the RNA and the protein levels compared to their normal oral tissues. 2. Loss-of-heterozygosity (LOH), mutation and promoter methylation are the hallmarks of a tumor suppressor gene (TSG). Therefore, to characterize CDC73 as a TSG in OSCC and to look into the mechanisms that could be the cause of CDC73 downregulation in OSCC, LOH, mutation and promoter methylation of CDC73 were studied. The results showed that LOH, mutation and promoter methylation are not the major causes of CDC73 downregulation in OSCC. 3. To identify the alternate mechanisms as the cause of CDC73 downregulation in OSCC, a combination of bioinformatics and molecular approaches were used. The results showed that the upregulation of an inhibitory transcription factor WT1 (Wilms tumor protein 1) and an oncogenic microRNA-155 are the major causes of its downregulation in OSCC. 4. The luciferase reporter assay of SCC131 cells co-transfected with a WT1 construct and a CDC73 promoter construct showed that WT1 over expression represses CDC73 expression in a dose-dependent manner. 5. Due to the presence of zinc fingers in its C-terminal half, WT1 has been found to be a potent transcriptional regulator of genes. Therefore, to determine if WT1 down regulates CDC73 via binding its promoter, the chromatin immunoprecipitation (ChIP) assay was performed. The results showed the binding of WT1 to the CDC73 promoter in vivo. Binding of WT1 to the CDC73 promoter was further confirmed in vitro by the electrophoretic mobility shift assay (EMSA). 6. The 5-aza-2’-deoxycytidine (AZA) treatment of SCC131 cells led to upregulation of WT1 with a concomitant downregulation of CDC73. The COBRA technique demonstrated that the upregulation of WT1 upon the 5-AZA treatment was due to its promoter methylation. 7. To determine if the WT1-mediated reduction of CDC73 expression has a functional relevance in cell growth and proliferation, we knocked down CDC73 expression by transient over expression of WT1 in SCC131 cells and quantitated cell proliferation by the MTT assay. As expected, the results demonstrated that the reduced CDC73 level was associated with an increased cell proliferation. Cotransfection of CDC73 with WT1 in SCC131 cells attenuated the pro-oncogenic effect of WT1 by apoptosis induction. 8. After validating CDC73 as the target of WT1 by bioinformatics and in vitro assays, we quantitated the expression levels of WT1 and CDC73 by qRT-PCR in OSCC samples and their matched normal oral tissue samples. The results showed an inverse correlation between the expression levels of WT1 and CDC73 in a majority of the samples. To exclude the possibility of alternate mechanisms as the cause of CDC73 downregulation in OSCC, we selected a subset of OSCC samples with downregulated level of CDC73 and analysed them for LOH at the CDC73 locus and promoter methylation. Further, some of these OSCC samples were also analyzed for mutations in CDC73. The results showed that these OSCC samples did not have LOH, promoter methylation or any mutation, again validating the fact that CDC73 is a biological target of oncogenic WT1, and the transcriptional repression of CDC73 by WT1 could be a major mechanism for CDC73 downregulation in OSCC. 9. Recent studies have shown that a growing class of noncoding RNAs called microRNAs (miRNAs) is involved in posttranscriptional regulation of genes. There is a growing body of literature supporting the potential role of miRNAs in tumorigenesis. The importance of CDC73 in orchestration of several cellular functions and its role in tumorigenesis make it an attractive candidate for miRNA-mediated regulation of cell growth and proliferation. Using bioinformatics approaches, we identified an oncogenic microRNA-155 (miR-155) that could posttranscriptionally regulate CDC73 expression. 10. Consistent with its oncogenic role, miR-155 was found dramatically upregulated in OSCC samples and was found to be another mechanism for downregulation of CDC73 in a panel of human cell lines and a subset of OSCC samples in the absence of LOH, mutations and promoter methylation. 11. miRNAs regulate posttranscriptional gene expression generally via binding to their cognate sites in the 3’UTR. Therefore, a luciferase reporter construct was made by cloning the 3’UTR of CDC73 downstream to the luciferase reporter gene and the reporter assay was performed. Our experiments clearly indicated that the mature miR-155 regulates CDC73 expression by interacting with its 3’UTR in a site specific manner. 12 Ectopic expression of miR-155 in HEK293 cells dramatically reduced CDC73 levels, enhanced cell viability and decreased apoptosis. Conversely, the delivery of a miR-155 antagonist (antagomir-155) to KB cells over expression miR-155 resulted in increased CDC73 level, decreased cell viability, increased apoptosis and marked regression of engrafts in nude mice. Cotransfection of miR-155 with CDC73 in HEK293 cells abrogated its pro-oncogenic effect. Reduced cell proliferation and increased apoptosis of KB cells were dependent on the presence or absence of the 3’UTR in CDC73. In nutshell, the knockdown of CDC73 expression due to over expression of WT1 and miR-155 not only adds a novelty to the list of mechanisms responsible for its downregulation in different tumors, but the restoration of CDC73 levels by the use of inhibitors to WT1 and antagomir-155 may also have an important role in therapeutic intervention of cancers, including OSCC.

O carcinoma epidermóide bucal (CEC) é uma neoplasia maligna com alta morbidade e mortalidade e de difícil tratamento. O tratamento convencional para o CEC inclui cirurgia e radioterapia, seguida ou não de quimioterapia. Apesar de serem amplamente difundidos, esses tratamentos podem ser ineficazes para alguns CECs resistentes. A terapia fotodinâmica (PDT) oncológica tem sido utilizada para o tratamento adjuvante do CEC bucal, principalmente nos casos menos invasivos e que necessitam de redução do tumor para a ressecção cirúrgica. Contudo, semelhantemente aos tratamentos convencionais, a PDT pode também induzir o aparecimento de populações celulares resistentes, fato já descrito para carcinoma cutâneo, adenocarcinoma de cólon e adenocarcinoma mamário. A hipótese de que células de CEC bucal possam desenvolver resistência à PDT ainda não foi testada. Portanto, o objetivo deste trabalho foi verificar se células de CEC bucal (SCC9) desenvolvem resistência a ciclos repetidos de PDT mediada pelo ácido 5- aminolevulínico (5-ALA-PDT) e avaliar se nesse processo ocorre modificação da expressão de marcadores relacionados a sobrevivência celular (NF?B, Bcl-2, iNOS, mTOR e Akt). Foi utilizada linhagem de células de CEC bucal (SCC9), submetida às seguintes condições: 1) Controle - células cultivadas sem nenhum tratamento; 2) ALA - células incubadas com 5-ALA (1mM durante 4 horas); 3) LED - tratadas com iluminação LED (630nm, 5,86J/cm2, 22,5J, 150mW, 150s); 4) PDT - tratadas com 5- ALA-PDT, com os protocolos do grupo ALA e LED combinados, gerando dose letal de 90%. Inicialmente foi realizado somente um ciclo de PDT, sendo avaliada a viabilidade celular em todos os grupos após 24, 48, 72 e 120h da irradiação. Também foi realizado ensaio de detecção da fragmentação de DNA (TUNEL) e análise por imunofluorescência da expressão das proteínas NF?B, Bcl-2, iNOS, pmTOR e pAkt nas células viáveis. Como resultado desse primeiro tratamento com 5-ALA-PDT, observou-se que as células sobreviventes ao tratamento apresentaram intensa marcação para pmTOR e exibiram potencial de crescimento durante o período analisado. Após esses ensaios, as células que sobreviveram a essa primeira sessão foram coletadas, replaqueadas e novamente cultivadas, sendo então submetidas a novo ciclo de 5-ALA-PDT. Esse processo foi realizado 5 vezes, variando-se a intensidade de irradiação à medida que se observava aumento na viabilidade celular. As populações celulares que exibiram viabilidade 1,5 vezes maior do que a detectada no primeiro ciclo PDT foram consideradas resistentes ao tratamento. Os mesmos marcadores analisados no primeiro ciclo de PDT foram novamente avaliados nas populações resistentes. Foram obtidas quatro populações celulares resistentes, com viabilidade de até 4,6 vezes maior do que a do primeiro ciclo de PDT e irradiação com LED que variou de 5,86 a 9,38J/cm2. A população mais resistente apresentou ainda menor intensidade de protoporfirina IX, maior capacidade de migração e modificação na morfologia nuclear. As populações resistentes testadas exibiram aumento na expressão de pNF?B, iNOS, pmTOR e pAkt, mas não da proteína anti-apoptótica Bcl- 2. Ensaio in vivo foi também conduzido em ratos, nos quais CEC bucal foi quimicamente induzido e tratado ou não com 5-ALA-PDT. Houve intensa expressão imuno-histoquímica das proteínas pNF?B, Bcl-2, iNOS, pmTOR e pAkt em relação ao controle não tratado, nas células adjacentes à área de necrose provocada pela PDT. Concluiu-se que as células de CEC bucal tratadas com 5-ALA-PDT a uma dose de 90% de letalidade desenvolveram viabilidade crescente após ciclos repetidos do tratamento, bem como exibiram superexpressão de proteínas relacionadas à sobrevivência celular, tanto in vitro quanto in vivo. Esses fatos, aliados à maior capacidade de migração, sugerem a aquisição de fenótipo de resistência à 5-ALAPDT. Esse aspecto deve ser cuidadosamente considerado no momento da instituição dessa terapia para os CECs bucais. / Oral squamous cell carcinoma (SCC) is a malignant tumor with high morbidity and mortality rates, and it is difficult to treat. Conventional treatment for oral SCCs includes surgery and radiotherapy that may be followed by chemotherapy. Although these treatments are widely used, they are ineffective against some resistant tumors. Oncologic photodynamic therapy (PDT) has been used as an adjuvant treatment for oral SCCs, especially in less invasive cases that require tumor reduction before surgical resection. However, like conventional treatments, PDT can induce the occurrence of resistant cell populations such as cutaneous carcinomas and colon and breast adenocarcinomas. The hypothesis that oral SCCs develop resistance to PDT has not yet been tested. Therefore, the aims of this study were to investigate whether oral SCCs (SCC9) develop resistance to several cycles of 5-aminolevulinic acidmediated PDT (5-ALA-PDT) and to determine whether the expression of markers associated with cell survival (NF?B, Bcl-2, iNOS, mTOR, and Akt) is altered during this process. An oral SCC (SCC9) cell line was used, which was subjected to the following conditions: 1) Control: cultured without any treatment; 2) ALA: incubated with 5-ALA (1 mM for 4 h); 3) LED: treated with LED light (630 nm, 5.86 J/cm2, 22.5 J, 150 mW, 150 s); and 4) PDT: treated with 5-ALA-PDT (with the protocols of the ALA and LED groups combined) generating a lethal dose of 90%. Initially, only one cycle of PDT was administered, and cell viability was determined in all groups 24, 48, 72, and 120 h after irradiation. Subsequently, the DNA fragmentation detection assay (TUNEL) and immunofluorescence analysis of the expression of proteins NF?B, Bcl-2, iNOS, pmTOR, and pAkt were performed on viable cells. The fraction of cells that survived the first treatment with 5-ALA-PDT exhibited intense staining for pmTOR and growth potential during the testing period. After these assays, the cells that survived the first cycle were collected, plated, and cultured and were subjected to another cycle of 5- ALA-PDT. This process was repeated five times at various irradiation intensities, and cell viability gradually increased. The cell populations that exhibited 1.5-times higher viability than that detected after the first PDT cycle were considered to be resistant to treatment. The markers analyzed after the first PDT cycle were again assessed in the resistant populations. Four resistant cell populations were obtained with a viability of up to 4.6-times higher than that of the first PDT cycle and LED light treatment, which varied between 5.86 and 9.38J/cm2. The most resistant population exhibited lower intensity of protoporphyrin IX, higher migration capacity, and changes in nuclear morphology. The resistant populations tested showed increased expression of pNF?B, iNOS, pmTOR, and pAkt, but not of the anti-apoptotic Bcl-2 protein. Moreover, an in vivo assay was conducted in rats; oral SCCs were chemically induced and treated with 5-ALA-PDT. The intensity of the immunohistochemical expression of proteins pNF?B, Bcl-2, iNOS, pmTOR, and pAkt in cells adjacent to the area with necrosis caused by PDT was higher than that observed in the untreated control. In conclusion, oral SCCs treated with 5-ALA-PDT at a lethal dose of 90% exhibited increasing viability after several treatment cycles and overexpression of proteins associated with cell survival both in vitro and in vivo. These results, together with the higher migration capacity, suggest the acquisition of the phenotype of resistance to 5-ALA-PDT. This aspect should be carefully considered when initiating this therapy for oral SCCs.

O carcinoma epidermóide oral (CEO) tem alta incidência e mortalidade no mundo, sendo frequentes casos de recidivas em função da resistência aos tratamentos convencionais. Essa resistência tem sido associada à presença de células tronco tumorais (CTT). A terapia fotodinâmica (PDT) tem sido utilizada nos casos de desordens orais potencialmente malignas (DOPM) e CEOs superficiais, com relativo índice de sucesso. Contudo, são relatados casos de recidivas, que sugerem certa resistência à PDT. O objetivo do presente trabalho foi avaliar se células de DOPM desenvolvem resistência à PDT mediada pelo ácido 5-aminolevulínico (5-ALA), tal qual ocorre com células de CEO expostas ao mesmo tratamento, bem como verificar se as células displásicas e neoplásicas orais exibem um fenótipo sugestivo de células tronco tumoral. Foram cultivadas células epiteliais displásicas orais (linhagem DOK) e células de CEO de língua (linhagem SCC9), divididas nos seguintes grupos experimentais: Grupo Controle - células sem tratamento; Grupo ALA - células tratadas com 5-ALA, com concentração e tempo de incubação igual ao do grupo PDT; Grupo LED - células tratadas com LED, com dosimetria igual ao do grupo PDT; Grupo PDT - células tratadas com PDT mediada pelo 5-ALA, subdivididas em duas gerações: geração 1 - células expostas a somente um ciclo de PDT; geração 4 - células expostas a quatro ciclos de PDT e que exibiram resistência (maior viabilidade) à dose inicial aplicada para a geração 1. Para esses grupos, foram realizados ensaios de formação de colônias e de invasão, bem como citometria de fluxo para verificar a expressão de CD44, ESA e p75NTR. Observou-se que todos os grupos expostos à PDT contendo células da linhagem DOK exibiram resistência à dose inicial de PDT, porém em intensidade menor àquela observada para a linhagem SCC9. As células resistentes à PDT de ambas as linhagens exibiram maior potencial de formação de colônias, mas somente a primeira geração de células exibiu maior potencial de invasão. Na análise de citometria de fluxo, observou-se que as células resistentes à PDT de ambas as linhagens exibiram superexpressão de CD44high/ESAhigh. A linhagem SCC9 exibiu ainda maior frequência de células CD44high/ESAlow e CD44high/p75NTR+, sugerindo a presença de uma subpopulação com alto potencial de invasão e resistente aos tratamentos convencionais. Não houve diferenças significativas na frequência de células ESAhigh/p75NTR+ em ambas as linhagens. Concluiu-se que as células displásicas orais desenvolvem resistência à PDT com intensidade menor se comparada às células SCC9. Em ambas as linhagens, as células resistentes exibiram um fenótipo sugestivo de células tronco tumorais. Esse fato deve ser considerado ao se instituir a PDT mediada pelo 5-ALA em lesões displásicas e neoplásicas orais que demandam múltiplos ciclos de tratamento. / Oral squamous cell carcinoma (SCC) has a high incidence and mortality in the world. There are frequent cases of relapses due to its resistance to conventional treatments. This resistance has been associated with the presence of cancer stem cells (CSC). Photodynamic therapy (PDT) has been used in cases of potentially malignant oral lesions (PMOL) and superficial SCC\'s, with a relative success rate. However, relapses are reported, which suggest some resistance to PDT. The aim of the present study was to evaluate whether PMOL cells develop resistance to PDT mediated by the 5- aminolevulinic acid (5-ALA), as occurs with SCC cells exposed to the same treatment, as well as to verify if dysplastic and oral neoplastic cells exhibit a suggestive phenotype of CSC. Oral dysplastic epithelial cells (DOK lineage) and CSC cells of the tongue (SCC9 lineage) were divided into the following experimental groups: Control Group - cells with no treatment; Group ALA - 5-ALA treated cells, with concentration and incubation time equal to that of the PDT group; LED group - LED light treated cells, with dosimetry equal to that of the PDT group; PDT group - cells treated with PDT mediated by the 5-ALA, subdivided into two generations: Generation 1 - cells exposed to only one PDT cycle; Generation 4 - cells exposed to four PDT cycles and exhibiting resistance (greater viability) to the initial dose applied for Generation 1. Colony forming and invasion assays, as well as flow cytometry to verify the expression of CD44, ESA and p75NTR were performed to the experimental groups. It was observed that all groups exposed to PDT containing DOK lineage cells exhibited resistance to the initial dose of PDT, but at a lower intensity than those observed for the SCC9 lineage. PDTresistant cells from both lineages exhibited increased potential for colony formation, but only the first generation of cells exhibited increased invasion potential. In flow cytometric analysis, PDT-resistant cells from both lineages exhibited overexpression of CD44high / ESAhigh. SCC9 lineage also exhibited higher frequency of CD44high / ESAlow and CD44high / p75NTR+ cells, suggesting the presence of a subpopulation with high invasion potential and resistant to conventional treatments. There were no significant differences in the frequency of ESAhigh / p75NTR+ cells in both lineages. It was concluded that the oral dysplastic cells develop resistance to PDT, with lower intensity when compared to SCC9 cells. In both lineages, the resistant cells exhibited a phenotype suggestive of CSC. This fact should be considered when establishing PDT mediated by the 5-ALA in oral dysplastic and neoplastic lesions that require multiple cycles of treatment.

O carcinoma epidermóide bucal (CEC) é uma neoplasia maligna com alta morbidade e mortalidade e de difícil tratamento. O tratamento convencional para o CEC inclui cirurgia e radioterapia, seguida ou não de quimioterapia. Apesar de serem amplamente difundidos, esses tratamentos podem ser ineficazes para alguns CECs resistentes. A terapia fotodinâmica (PDT) oncológica tem sido utilizada para o tratamento adjuvante do CEC bucal, principalmente nos casos menos invasivos e que necessitam de redução do tumor para a ressecção cirúrgica. Contudo, semelhantemente aos tratamentos convencionais, a PDT pode também induzir o aparecimento de populações celulares resistentes, fato já descrito para carcinoma cutâneo, adenocarcinoma de cólon e adenocarcinoma mamário. A hipótese de que células de CEC bucal possam desenvolver resistência à PDT ainda não foi testada. Portanto, o objetivo deste trabalho foi verificar se células de CEC bucal (SCC9) desenvolvem resistência a ciclos repetidos de PDT mediada pelo ácido 5- aminolevulínico (5-ALA-PDT) e avaliar se nesse processo ocorre modificação da expressão de marcadores relacionados a sobrevivência celular (NF?B, Bcl-2, iNOS, mTOR e Akt). Foi utilizada linhagem de células de CEC bucal (SCC9), submetida às seguintes condições: 1) Controle - células cultivadas sem nenhum tratamento; 2) ALA - células incubadas com 5-ALA (1mM durante 4 horas); 3) LED - tratadas com iluminação LED (630nm, 5,86J/cm2, 22,5J, 150mW, 150s); 4) PDT - tratadas com 5- ALA-PDT, com os protocolos do grupo ALA e LED combinados, gerando dose letal de 90%. Inicialmente foi realizado somente um ciclo de PDT, sendo avaliada a viabilidade celular em todos os grupos após 24, 48, 72 e 120h da irradiação. Também foi realizado ensaio de detecção da fragmentação de DNA (TUNEL) e análise por imunofluorescência da expressão das proteínas NF?B, Bcl-2, iNOS, pmTOR e pAkt nas células viáveis. Como resultado desse primeiro tratamento com 5-ALA-PDT, observou-se que as células sobreviventes ao tratamento apresentaram intensa marcação para pmTOR e exibiram potencial de crescimento durante o período analisado. Após esses ensaios, as células que sobreviveram a essa primeira sessão foram coletadas, replaqueadas e novamente cultivadas, sendo então submetidas a novo ciclo de 5-ALA-PDT. Esse processo foi realizado 5 vezes, variando-se a intensidade de irradiação à medida que se observava aumento na viabilidade celular. As populações celulares que exibiram viabilidade 1,5 vezes maior do que a detectada no primeiro ciclo PDT foram consideradas resistentes ao tratamento. Os mesmos marcadores analisados no primeiro ciclo de PDT foram novamente avaliados nas populações resistentes. Foram obtidas quatro populações celulares resistentes, com viabilidade de até 4,6 vezes maior do que a do primeiro ciclo de PDT e irradiação com LED que variou de 5,86 a 9,38J/cm2. A população mais resistente apresentou ainda menor intensidade de protoporfirina IX, maior capacidade de migração e modificação na morfologia nuclear. As populações resistentes testadas exibiram aumento na expressão de pNF?B, iNOS, pmTOR e pAkt, mas não da proteína anti-apoptótica Bcl- 2. Ensaio in vivo foi também conduzido em ratos, nos quais CEC bucal foi quimicamente induzido e tratado ou não com 5-ALA-PDT. Houve intensa expressão imuno-histoquímica das proteínas pNF?B, Bcl-2, iNOS, pmTOR e pAkt em relação ao controle não tratado, nas células adjacentes à área de necrose provocada pela PDT. Concluiu-se que as células de CEC bucal tratadas com 5-ALA-PDT a uma dose de 90% de letalidade desenvolveram viabilidade crescente após ciclos repetidos do tratamento, bem como exibiram superexpressão de proteínas relacionadas à sobrevivência celular, tanto in vitro quanto in vivo. Esses fatos, aliados à maior capacidade de migração, sugerem a aquisição de fenótipo de resistência à 5-ALAPDT. Esse aspecto deve ser cuidadosamente considerado no momento da instituição dessa terapia para os CECs bucais. / Oral squamous cell carcinoma (SCC) is a malignant tumor with high morbidity and mortality rates, and it is difficult to treat. Conventional treatment for oral SCCs includes surgery and radiotherapy that may be followed by chemotherapy. Although these treatments are widely used, they are ineffective against some resistant tumors. Oncologic photodynamic therapy (PDT) has been used as an adjuvant treatment for oral SCCs, especially in less invasive cases that require tumor reduction before surgical resection. However, like conventional treatments, PDT can induce the occurrence of resistant cell populations such as cutaneous carcinomas and colon and breast adenocarcinomas. The hypothesis that oral SCCs develop resistance to PDT has not yet been tested. Therefore, the aims of this study were to investigate whether oral SCCs (SCC9) develop resistance to several cycles of 5-aminolevulinic acidmediated PDT (5-ALA-PDT) and to determine whether the expression of markers associated with cell survival (NF?B, Bcl-2, iNOS, mTOR, and Akt) is altered during this process. An oral SCC (SCC9) cell line was used, which was subjected to the following conditions: 1) Control: cultured without any treatment; 2) ALA: incubated with 5-ALA (1 mM for 4 h); 3) LED: treated with LED light (630 nm, 5.86 J/cm2, 22.5 J, 150 mW, 150 s); and 4) PDT: treated with 5-ALA-PDT (with the protocols of the ALA and LED groups combined) generating a lethal dose of 90%. Initially, only one cycle of PDT was administered, and cell viability was determined in all groups 24, 48, 72, and 120 h after irradiation. Subsequently, the DNA fragmentation detection assay (TUNEL) and immunofluorescence analysis of the expression of proteins NF?B, Bcl-2, iNOS, pmTOR, and pAkt were performed on viable cells. The fraction of cells that survived the first treatment with 5-ALA-PDT exhibited intense staining for pmTOR and growth potential during the testing period. After these assays, the cells that survived the first cycle were collected, plated, and cultured and were subjected to another cycle of 5- ALA-PDT. This process was repeated five times at various irradiation intensities, and cell viability gradually increased. The cell populations that exhibited 1.5-times higher viability than that detected after the first PDT cycle were considered to be resistant to treatment. The markers analyzed after the first PDT cycle were again assessed in the resistant populations. Four resistant cell populations were obtained with a viability of up to 4.6-times higher than that of the first PDT cycle and LED light treatment, which varied between 5.86 and 9.38J/cm2. The most resistant population exhibited lower intensity of protoporphyrin IX, higher migration capacity, and changes in nuclear morphology. The resistant populations tested showed increased expression of pNF?B, iNOS, pmTOR, and pAkt, but not of the anti-apoptotic Bcl-2 protein. Moreover, an in vivo assay was conducted in rats; oral SCCs were chemically induced and treated with 5-ALA-PDT. The intensity of the immunohistochemical expression of proteins pNF?B, Bcl-2, iNOS, pmTOR, and pAkt in cells adjacent to the area with necrosis caused by PDT was higher than that observed in the untreated control. In conclusion, oral SCCs treated with 5-ALA-PDT at a lethal dose of 90% exhibited increasing viability after several treatment cycles and overexpression of proteins associated with cell survival both in vitro and in vivo. These results, together with the higher migration capacity, suggest the acquisition of the phenotype of resistance to 5-ALA-PDT. This aspect should be carefully considered when initiating this therapy for oral SCCs.

Made available in DSpace on 2014-12-17T15:32:22Z (GMT). No. of bitstreams: 1 KaruzaMAP.pdf: 544780 bytes, checksum: e0fadeed7d2d1ec7568970935306d05c (MD5) Previous issue date: 2006-02-22 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The Human Papillomavirus (HPV) has been strongly implicated on development of some cases of oral squamous cell carcinoma (OSCC). However, the immunological system somehow reacts against the presence of this virus. Among the cells involved on such mechanism of defense detaches the Langerhans cells (LC), which are responsible for processing and presenting antigens. The purpose of this study was to evaluate the immunohistochemical reactivity for Langerhans cells between HPV positive and HPV negative OSCC, as well as, the relation of the immunoreactivity for this cells and the histological grading of malignancy proposed by Bryne (1998) and modified by Miranda (2002). Additionally, HPV infection was evaluated in relation to sex, age, lesion localization and histological grading of malignancy. In the total, 27 cases of OSSC were evaluated, 09 of them HPV positive and 18 HPV negative. Anti S-100 antibody was utilized for the immunohistochemical labelling, followed by the counting of LCs in 5 highpower fields (400x). No statistically significant difference was verified between the variables sex, age, lesion localization, histological grading of malignancy and HPV presence in OSSC. There was neither association between the immunohistochemical labeling for LCs (S-100+) and HPV infection nor correlation between the quantity of LCs labeled and the histological grading of malignancy of OSSC. The results suggest that despite the absence of statistically significant difference, the presence of HPV in such cases of OSCC can alter the immunological system, particularly the Langerhans cells / O Papilomav?rus Humano (HPV) tem sido implicado fortemente no desenvolvimento de alguns carcinomas epiderm?ides orais (CEOs). Contudo, o sistema imunol?gico reage de alguma forma ? presen?a desse v?rus. Dentre as c?lulas envolvidas nesse mecanismo de defesa, destaca-se a c?lula de Langerhans (CL), por serem c?lulas processadoras e apresentadoras de ant?genos. O objetivo desse estudo foi avaliar a marca??o imuno-histoqu?mica das c?lulas de Langerhans entre os casos de CEOs HPV positivos e negativos, bem como a rela??o da imunomarca??o dessas c?lulas e a grada??o histol?gica de malignidade proposta por Bryne (1998) e modificada por Miranda (2002). Adicionalmente, a infec??o pelo HPV foi estudada com rela??o ao sexo, idade, localiza??o da les?o e a grada??o histol?gica de malignidade. Foram analisados 27 casos de CEOs, sendo 09 destes HPV positivos e 18 casos negativos. Para a marca??o imuno-histoqu?mica utilizou-se o anticorpo anti S-100, sendo as CLs quantificadas em 5 campos de maior aumento (400x). A an?lise estat?stica revelou n?o existir rela??o das vari?veis, sexo, idade, localiza??o da les?o e grada??o histol?gica, com a presen?a do HPV nos CEOs estudados. N?o existiu associa??o entre a marca??o imuno-histoqu?mica das CLs(S-100+) e a infec??o pelo HPV, e tamb?m n?o houve correla??o entre as CLs imunomarcadas e a grada??o histol?gica nos casos de CEOs analisados. Diante desses resultados, pode-se sugerir que mesmo n?o havendo diferen?a significativa, a presen?a do HPV nos casos de carcinoma epiderm?ide oral pode alterar o sistema imune, particularmente as c?lulas de Langerhans