Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2006. / Vita. Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Thesis (Doctoral).--Montpellier, 1887.

Mestre -- Escola Nacional de Saude Publica, Rio de Janeiro, 2003.

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The aim of the present study was to characterize the phenomenon of resistance in putative preneoplastic hepatocyte nodules. These hyperplastic nodules are generated during the development of liver cancer in response to chemical carcinogens, and comprise a population of cells from which hepatocellular carcinoma can develop. As hepatocyte nodules grow in an environment that is otherwise toxic they possess a resistant phenotype. To understand this resistance phenomenon at the biochemical level, several phase I and II drug-metabolizing enzymes in the nodules were examined. Initial experiments were carried out in rats with nodules produced by initiation with diethylnitrosamine, followed by selection with 2-acetylaminofluorene and carbon tetrachloride. These nodules showed a large decrease in phase I enzymes and enzymic activities, such as the cytochromes P-450, cytochrome b[5], total microsomal haem, aminopyrine N-demethylase and ethoxyresorufin 0-deethylase, but glutathione and the phase II enzymes, namely, glutathione S-transferase, UDP-glucuronyl transferase, DT-diaphorase and gamma-glutamyltransferase were significantly increased. The pattern of changes of these drug-metabolizing enzymes of the nodules was similar when the nodules were produced by different initiation-promotion treatments, including diethylnitrosamine plus a choline/methionine-deficient diet, 2-acetamidofluorene plus phenobarbi-tone, or diethylnitrosamine plus orotic acid. In addition, the resistance phenotype was maintained when these nodules were transferred into the spleen of a rat not exposed to chemical carcinogens, and allowed to grow for several months, thus indicating that the newly acquired biochemical pattern in the nodules had become constitutive. Unlike the hepatic nodules generated by previous initiation-pro-motion treatments, nodules generated by the hypolipidemic agent, ciprofibrate, exhibited only a decrease in phase I components of the drug-metabolizing enzymes, with no increase in the phase II components. Similarly, hyperplastic nodules in liver mouse showed a decrease in phase I components, but no increase in phase II components. In addition to cytochrome P-450 and cytochrome b5, the total haem and two other haem containing proteins, namely, catalase and tryptophan 2,3-dioxygenase were also decreased in the nodules. A deficiency in hepatic iron, and a decrease in the activity of delta-ALA-synthetase, the first rate limiting enzyme in haem synthesis, were also apparent. Characterization of the phase II components revealed the presence of a new glutathione-S-transferase polypeptide, which has been shown to be identical to a placental form of the transferase. This polypeptide, although present to a minimal extent, or absent, in normal rat liver, is present in normal male mouse liver. Administration of lead nitrate to rats induces a biochemical pattern in the liver similar to that seen in the hepatocyte nodules, including a decrease of phase I components and an increase in phase II components of the drug-metabolizing enzymes, and the induction of the novel glutathione S-transferase. Further studies with lead nitrate may yield new insights into the mechanisms of production of the biochemical changes induced in the nodules, as this agent generates the same changes within 30 hours. Furthermore, the lead nitrate-induced changes in phase I and phase II enzymes are reversible, while the changes seen in the hyperplastic nodules are not. Thus this study has characterized one pattern of biochemical changes exhibited by the resistant phenotype of hyperplastic hepatic nodules, and a model system has been developed which induces the same changes, more rapidly and in a reversible fashion. One of the important questions yet to be answered however is the biological significance of the resistant phenotype in cancer development. Is the acquisition of resistance only important in expanding the initiated cell population to generate nodules or does it also have a more direct role in the progression of nodules to cancer? This is highly relevant to the clarification of the carcinogenic process in the liver and perhaps in other organs as well.

O objetivo deste trabalho foi avaliar a correlação entre parâmetros clínicos (TNM), graduação histopatológica, número de AgNORs por núcleo e expressão de Ki-67 na zona de invasão com o prognóstico de carcinoma espinocelular de língua. Foram selecionados dez casos de carcinoma espinocelular de língua e divididos em dois grupos: bom prognóstico (ausência de metástases á distância e/ou regionais, sobrevida livre de doença) e mau prognóstico (presença de metástases, recidiva, óbito). O material de biópsia foi submetido às técnicas de hematoxilina/eosina, de impregnação pela prata para evidenciação das NORs e de detecção imunohistoquímica do antígeno de proliferação nuclear Ki-67. Concluiu-se que T4 por si só já é um indicador de mau prognóstico e que nos tumores de grau II, a proliferação celular pode refletir o prognóstico do tumor.

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Nucleotídeos da adenina e adenosina estão envolvidos em uma variedade de processos patofisiológicos em diferentes células. O metabolismo extracelular destes nucleotídeos gerando nucleosídeos tem uma importante função regulatória no controle da homeostase, principalmente por regularem a agregação plaquetária, via receptores purinérgicos P2. Além disso, uma associação destes nucletídeos e nucleosídeo em processos neoplásicos tem sido sugerida. Enquanto ATP tem demonstrado exibir atividade anticâncer em uma grande variedade de células animais e humanas, estudos sugerem que adenosina possui ações promotoras de tumor. Muitos estudos também demonstram que antiinflamatórios não esteroidais, como o ácido acetil salicílico (AAS), podem proteger contra o desenvolvimento do câncer. As ações induzidas pela sinalização purinérgica são reguladas pelas ectonucleotidases, que incluem membros da família das ectonucleosídeo trifosfato difosfoidrolase (E-NTPDase) e ecto-nucleotídeo pirofosfatases/fosfodiesterases (E-NPPs), e ecto-5’-nucleotidase (ecto-5’N). Assim, no presente estudo nós investigamos o efeito do AAS na hidrólise do ATP até adenosina, bem como estudamos as características bioquímicas e expressão das ectonucleotidases no tumor de W256. A hidrólise dos nucleotídeos da adenina por plaquetas de ratos foi significativamente inibida por AAS, tanto para ATP quanto para ADP, mas não houve alteração na hidrólise do AMP. Em ratos submetidos ao tumor de W256, a hidrólise dos nucleotídeos da adenina em plaquetas e soro, obtidos 6, 10 e 15 dias após a indução subcutânea do tumor, foi significativamente reduzida. Em células tumorais de W256 (forma ascítica do tumor), a análise cinética indicou que várias ectonucleotidases estão envolvidas nessa cascata enzimática. Quanto às propriedades bioquímicas das E-NTPDases e ecto-5'-nucleotidase, para a hidrólise de ATP, ADP e AMP, o pH ótimo ficou entre 6,5-8,0 e também foi observado um requerimento de cátions divalentes (Mg2+ > Ca2+). Uma significativa inibição na hidrólise do ATP e ADP foi observada em presença de altas concentrações de azida sódica e de 0,5 mM de cloreto de gadolínio. Ainda, a análise de mRNA por PCR identificou a presença das NTPDases 2 e 5, e também da ecto-5'-nucleotidase nas células tumorais de W256. Com o objetivo de investigar as enzimas envolvidas no catabolismo dos nucleotídeos da adenina durante o crescimento do tumor, nós avaliamos a expressão destas enzimas bem como o padrão de degradação dos nucleotídeos extracelulares no tumor de W256, 6, 10 e 15 dias após a indução subcutânea. Os mRNAs de todas as ectonucleotidases estudadas (NTPDase 1, 2, 3 5 e 6 e ecto- 5’-nucleotidase), foram identificados por RT-PCR. A análise quantitativa, realizada por real-time PCR, apresentou como genes dominantes expressos durante o crescimento do tumor, as NTPDases 1 e 2 e ecto-5’-nucleotidase. O padrão de hidrólise do ATP extracelular determinado por HPLC, embora com alguma diferença entre os tempos estudados, se manteve similar. As células do tumor de W256 rapidamente hidrolisaram ATP levando à formação transitória de ADP, que foi completamente hidrolisado a AMP. A participação das NPPS 1, 2 e 3 na forma ascítica do tumor e VI durante o desenvolvimento do tumor subcutâneo de W256 também foi investigada. Nas duas formas do tumor o gene dominante foi o da enzima NPP3. Considerando que o ATP é reconhecido como um composto citotóxico em células tumorais, e que adenosina tem sido considerada promotora de tumor, é possível sugerir que, na circulação, a substancial redução na hidrólise dos nucleotídeos da adenina em plaquetas e soro após a indução do tumor pode representar um mecanismo de proteção contra o desenvolvimento tumoral. Por outro lado, nas células tumorais de W256, a elevada expressão de enzimas potencialmente envolvidas na hidrólise do ATP e do AMP, bem como a rápida hidrólise do ATP, pode representar um mecanismo que facilita a proliferação e invasão do tumor. / Adenine and adenosine nucleotides are involved in a variety of pathophysiological process in different cells. The extracellular metabolism of these nucleotides creating nucleosides has an important regulatory function in controlling the homeostasis, mainly for regulate the platelets aggregation, via purinergic P2 receptors. Besides this, an association of these nucleotides and nucleoside in neoplasics process has been suggested. While ATP has shown to exhibit anticancer activity in a great variety of animals and human cells, studies suggest that adenosine presents tumor-promoting actions. Many studies have also demonstrated that nonsteroidal anti-inflamatory, such as acetylsalicylic acid (ASA) can protect against the cancer development. The induced actions from purinergic signalization are ruled by the ectonucleotidases, that include members of the ectonucleoside triphosphate diphosphohydrolases (E-NTPDases) and ecto-nucleotide pyrophosphatases/phosphodiesterases (E-NPPs) families, and ecto-5’-nucleotidase (ecto-5’N). Then, in the present study we have investigated the effect of the ASA on the ATP hydrolysis to adenosine, such as studied the biochemical characteristics and expression of the ectonucleotidases in W256 tumor. The hydrolysis of the adenine nucleotides by rat platelets was significantly inhibited by ASA, both for ATP and ADP, but there was no alteration on AMP hydrolysis. In rats submitted to W256 tumor, hydrolysis of the adenine nucleotides by platelets and serum, obtained 6, 10 and 15 days after tumor’s subcutaneous induction, was significantly reduced. In W256 tumor cells (ascitic form of tumor), the kinetic analysis indicated that various ectonucleotidases are involved in this enzymatic cascade. Regarding the biochemistry properties of E-NTPDases and ecto-5'-nucleotidase, for the ATP, ADP e AMP hydrolysis, the optimum pH was reached among 6,5-8,0 and it was also observed a requirement of divalent cations (Mg2+ > Ca2+). A significant inhibition on ATP and ADP hydrolysis was observed in the presence of high concentrations of sodium azide and 0.5 mM of gadolinium chloride. Also, the mRNA by PCR analysis identified the presence of NTPDases 2 and 5, and also the ecto-5'-nucleotidase in W256 tumor cells. With the purpose of investigating the enzymes involved in the catabolism of adenine nucleotides during the tumor’s growth, we evaluated the expression of these enzymes as well as the pattern degradation of extracellular nucleotides in W256 tumor, 6, 10 and 15 days after the tumor’s subcutaneous induction. The mRNAs of all ectonucleotidases studied (NTPDase 1, 2, 3 5 e 6 and ecto-5’-nucleotidase) were identified by RT-PCR. The quantitative analysis, done by real-time PCR, presented as dominant genes expressed during the tumor’s growth, the NTPDases 1 and 2 and ecto-5’-nucleotidase. The pattern of ATP extracellular hydrolysis determined by HPLC, despite a small difference between the times studied, kept similar. The W256 tumor’s cells quickly hydrolyzed ATP, leading to the transitory formation of ADP, which was completely hydrolyzed to AMP. Considering that ATP is recognized as a citotoxic compound in tumor cells, and that adenosine has been considered tumor’s promoter, it is possible to suggest that, in the circulation, the substantial VIII reduction on adenine nucleotides hydrolysis in platelets and serum after tumor’s induction may represent a mechanism of protection against the tumor’s development. The participation of NPPs 1, 2 and 3 in the ascitic form of tumor and during the subcutaneous W256 tumor’s growth was also investigated. In the two forms of the tumor, the dominant gene expressed was the NPP3 enzyme. On the other hand, in W256 tumor cells, the high expression of enzymes potentially involved in ATP and ADP hydrolysis, as well as the fast ATP hydrolysis, may represent a mechanism that facilitate the proliferation and invasion of tumor.

O tratamento intra-arterial por meio de quimioembolização intra-arterial (TACE) ou embolização intra-arterial (TAE) promove aumento da sobrevida em pacientes com carcinoma hepatocelular irressecável. Não há consenso sobre a melhor forma de tratamento ou sobre o melhor agente embolizante a ser utilizado. A síndrome pós-embolização (SPE) está presente na maioria dos casos após o procedimento, sendo raros os estudos que abordam essa condição como objetivo principal. Não há estudos comparando dois agentes embolizantes quanto aos sintomas relacionados à SPE. O presente estudo tem como objetivo comparar TAE com polivinil álcool (PVA) e TAE com microesferas (ME) na SPE, assim como quanto à resposta tumoral e aos índices de sobrevida. Foi realizada uma coorte histórica de 80 pacientes, 48 tratados com PVA e 32 com ME por meio de embolização superseletiva. A SPE foi graduada na escala do Southwest Oncology Group Criteria. Resposta tumoral foi estudada por meio do mRECIST e a sobrevida pelo método de Kaplan-Meier. Houve diferença estatisticamente significativa na SPE, que esteve presente em 56.2% dos casos embolizados com ME e em 29.1% dos pacientes embolizados com PVA (p=0.02). Não houve diferenças significativas em relação à resposta local (p=0.369) e à sobrevida (p=0.679). Os índices de sobrevida encontrados em 12, 18, 24, 36 e 48 meses foram de 97.9, 88.8, 78.9, 53.4 e 21.4% no grupo PVA e de 100, 92.9, 76.6, 58.8 e 58% no grupo ME, respectivamente. (p= 0.734). O presente estudo concluiu que TAE com ME promoveu mais sintomas relacionados à SPE em comparação à TAE com PVA, sem benefício significativo na resposta tumoral ou na sobrevida. / Transarterial chemoembolization (TACE) and transarterial embolization (TAE) have improved survival rates of patients with unresectable hepatocellular carcinoma (HCC), though the optimal TACE/TAE embolic agent has not yet been identified. Post embolization syndrome (PES) is a common condition after TACE or TAE. It has been not sufficiently described in CHC studies, and there is no study focused on the comparison of PES with two different embolic agents. We designed a study to compare PVA and ME-based TAE on PES, local response and survival rates. Eight HCC patients submitted to TAE in a single center were retrospectively studied. TAE was performed by superselective catheterization followed by embolization with PVA or ME. PES was analyzed according to the Southwest Oncology Group Toxicity Criteria. Tumor response was evaluated using mRECIST criteria. Survival rates were based on Kaplan-Meier curves. Forty-eight patients were PVA-treated, and 32 ME-treated. There were no differences between the groups at the baseline, regarding age, sex, BCLC stage, Child-Pugh score, and tumoral features. ME group had significant more PES (p=0.02) and a trend to prolonged hospital length of stay (p=0.05) than PVA group, although there were no differences on survival (p=0.679) and tumoral response (p=0.369). Medians for survival time were 38.2 months (ME), and 34.3 months (PVA). Survival rates at 12, 18, 24, 36 and 48 months were 97.9, 88.8, 78.9, 53.4 and 21.4% in PVA-TAE, and 100, 92.9, 76.6, 58.8 and 58%, respectively, in ME-TAE group (p= 0.734). ME-TAE leads to more PES than PVA-TAE with no benefit on survival rates or local response.