O Sarcoma de Ewing (ES) é um raro tumor de ossos e tecidos moles com uma característica translocação cromossomal, a fusão EWS/FLI-1, que atua sobre diversos processos oncogênicos. O desenvolvimento da resistência à quimioterapia é comum no tumor e continua como uma das principais causas na falha do tratamento. O objetivo desse estudo foi avaliar a expressão de genes após a indução de resistência em linhagens celulares de ES. Foi selecionado um conjunto de genes (CCAR1, TUBA1A, POLDIP2, SMARCA4 e SMARCB1) a partir da mineração da literatura em resistência tumoral para duas drogas utilizadas na terapia de ES, doxorrubicina e vincristina. Descrevemos a expressão de cada gene selecionado antes e após as linhagens SK-ES-1 serem submetidas a um protocolo de indução de resistência para ambos os fármacos, que obteve êxito ao induzir as células à resistência. A expressão relativa dos níveis de mRNA foi avaliada e foi encontrada em maior expressão para os genes SMARCA4, SMARCB1 e POLDIP2, e em menor expressão para os genes TUBA1A e CCAR1, quando comparadas às linhagens de controle não-resistentes de cada quimioterápico. Os resultados sugerem o envolvimento de mecanismos de reparo de dano ao DNA, remodelamento de cromatina via SWI/SNF, atividade de microtúbulos e atividade spliceossomal nos processos de resistência quimioterápica em ES. / Ewing Sarcoma (ES) is a rare bone and soft tissue tumor with a characteristic chromosomal translocation, the fusion protein EWS/FLI-1, that drives several oncogenic processes. The development of resistance to chemotherapy is common and remains as the main cause of treatment failure. The goal of this study was to evaluate the expression of selected genes in ES cell lines after induction of resistance. A set of genes (CCAR1, TUBA1A, POLDIP2, SMARCA4 and SMARCB1) was data mined from tumoral resistance literature for two drugs used in ES therapy, doxorubicin and vincristine. We describe the expression of each selected gene before and after SK-ES-1 cell lines were exposed to a drug resistance inducing protocol for doxorubicin and vincristine. Cell lines were successfully induced to be resistant to doxorubicin and vincristine. The relative mRNA expression levels were upregulated for genes SMARCA4, SMARCB1 and POLDIP2 and downregulated for genes TUBA1A and CCAR1, when comparing resistant and non-resistant ES cell lines for each drug. The results suggest involvement of repair pathways, SWI/SNF chromatin remodeling, microtubule and spliceosomal activity processes in drug resistance mechanisms in ES.

O Sarcoma de Ewing (ES) é um raro tumor de ossos e tecidos moles com uma característica translocação cromossomal, a fusão EWS/FLI-1, que atua sobre diversos processos oncogênicos. O desenvolvimento da resistência à quimioterapia é comum no tumor e continua como uma das principais causas na falha do tratamento. O objetivo desse estudo foi avaliar a expressão de genes após a indução de resistência em linhagens celulares de ES. Foi selecionado um conjunto de genes (CCAR1, TUBA1A, POLDIP2, SMARCA4 e SMARCB1) a partir da mineração da literatura em resistência tumoral para duas drogas utilizadas na terapia de ES, doxorrubicina e vincristina. Descrevemos a expressão de cada gene selecionado antes e após as linhagens SK-ES-1 serem submetidas a um protocolo de indução de resistência para ambos os fármacos, que obteve êxito ao induzir as células à resistência. A expressão relativa dos níveis de mRNA foi avaliada e foi encontrada em maior expressão para os genes SMARCA4, SMARCB1 e POLDIP2, e em menor expressão para os genes TUBA1A e CCAR1, quando comparadas às linhagens de controle não-resistentes de cada quimioterápico. Os resultados sugerem o envolvimento de mecanismos de reparo de dano ao DNA, remodelamento de cromatina via SWI/SNF, atividade de microtúbulos e atividade spliceossomal nos processos de resistência quimioterápica em ES. / Ewing Sarcoma (ES) is a rare bone and soft tissue tumor with a characteristic chromosomal translocation, the fusion protein EWS/FLI-1, that drives several oncogenic processes. The development of resistance to chemotherapy is common and remains as the main cause of treatment failure. The goal of this study was to evaluate the expression of selected genes in ES cell lines after induction of resistance. A set of genes (CCAR1, TUBA1A, POLDIP2, SMARCA4 and SMARCB1) was data mined from tumoral resistance literature for two drugs used in ES therapy, doxorubicin and vincristine. We describe the expression of each selected gene before and after SK-ES-1 cell lines were exposed to a drug resistance inducing protocol for doxorubicin and vincristine. Cell lines were successfully induced to be resistant to doxorubicin and vincristine. The relative mRNA expression levels were upregulated for genes SMARCA4, SMARCB1 and POLDIP2 and downregulated for genes TUBA1A and CCAR1, when comparing resistant and non-resistant ES cell lines for each drug. The results suggest involvement of repair pathways, SWI/SNF chromatin remodeling, microtubule and spliceosomal activity processes in drug resistance mechanisms in ES.

<p>The aims of the studies described in this thesis were to investigate the pharmacokinetics of and cellular resistance to chemotherapy as causes of treatment failure in childhood acute lymphoblastic leukemia (ALL).</p><p>Leukemic cells from 370 children with newly diagnosed ALL were tested by the Fluorometric Microculture Cytotoxicity Assay to measure their resistance to each of ten standard cytotoxic drugs. In the high-risk group, increased in vitro resistance to each of the drugs dexamethasone, etoposide and doxorubicin was associated with a worse clinical outcome. Combining the results for these drugs yielded a drug resistance score, showing a relative risk of relapse in the most resistant group that was 9.8 times higher than in the most sensitive group. In the standard-risk and intermediate-risk groups, final evaluation must await longer follow-up.</p><p>The new cytotoxic agent CHS 828 was equally active in vitro in samples from children with acute myeloblastic leukemia (AML) and ALL, with 50% cell kill at concentrations achievable in vivo. In AML samples CHS 828 also displayed high frequencies of synergistic interactions with four standard drugs. The well-known differences in clinical outcome between Down´s syndrome (DS) and non-DS children with acute leukemia may partly be explained by our finding of differences in drug resistance at the cellular level.</p><p>Pharmacokinetic studies were performed at the start of induction treatment of ALL. Doxorubicin was assayed by reversed-phase liquid chromatography with fluorometric detection, and vincristine by high performance liquid chromatography with electrochemical detection. Plasma doxorubicin concentrations were measured in 107 children after 23 h of a 24-h infusion. The median steady-state concentration in children 4-6 years old, a group known to have a favorable outcome of treatment, was about 50% higher than in those 1-2 and >6 years old Vincristine pharmacokinetics was evaluated in 98 children. There was no correlation between age and total body clearance or any other pharmacokinetic parameters.</p><p>In vitro testing of cellular drug resistance might be useful in predicting the outcome in high-risk ALL. The further exploration of CHS 828 in childhood leukemia seems warranted. There is no pharmacokinetic rationale for the common practice of administering relatively lower doses of vincristine to adolescents than to younger children.</p>

The aims of the studies described in this thesis were to investigate the pharmacokinetics of and cellular resistance to chemotherapy as causes of treatment failure in childhood acute lymphoblastic leukemia (ALL). Leukemic cells from 370 children with newly diagnosed ALL were tested by the Fluorometric Microculture Cytotoxicity Assay to measure their resistance to each of ten standard cytotoxic drugs. In the high-risk group, increased in vitro resistance to each of the drugs dexamethasone, etoposide and doxorubicin was associated with a worse clinical outcome. Combining the results for these drugs yielded a drug resistance score, showing a relative risk of relapse in the most resistant group that was 9.8 times higher than in the most sensitive group. In the standard-risk and intermediate-risk groups, final evaluation must await longer follow-up. The new cytotoxic agent CHS 828 was equally active in vitro in samples from children with acute myeloblastic leukemia (AML) and ALL, with 50% cell kill at concentrations achievable in vivo. In AML samples CHS 828 also displayed high frequencies of synergistic interactions with four standard drugs. The well-known differences in clinical outcome between Down´s syndrome (DS) and non-DS children with acute leukemia may partly be explained by our finding of differences in drug resistance at the cellular level. Pharmacokinetic studies were performed at the start of induction treatment of ALL. Doxorubicin was assayed by reversed-phase liquid chromatography with fluorometric detection, and vincristine by high performance liquid chromatography with electrochemical detection. Plasma doxorubicin concentrations were measured in 107 children after 23 h of a 24-h infusion. The median steady-state concentration in children 4-6 years old, a group known to have a favorable outcome of treatment, was about 50% higher than in those 1-2 and &gt;6 years old Vincristine pharmacokinetics was evaluated in 98 children. There was no correlation between age and total body clearance or any other pharmacokinetic parameters. In vitro testing of cellular drug resistance might be useful in predicting the outcome in high-risk ALL. The further exploration of CHS 828 in childhood leukemia seems warranted. There is no pharmacokinetic rationale for the common practice of administering relatively lower doses of vincristine to adolescents than to younger children.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Vincristine is a chemotherapeutic agent used to treat various types of cancers with effective clinical outcomes. However, its effects include a number of disorders related to balance, such as ataxia, tremors and peripheral neuropathy. These damages are likely associated with poisoning by the drug in the central and peripheral nervous systems. There is evidence that guarana (Paullinia cupana) may have neuroprotective effects on the central nervous system. The aim of this study was to evaluate the effects of vincristine and guarana on cell viability and oxidative metabolism in vitro cell cultures of mice brain and cerebellum. The cells were incubated for 24 and 72 hours in a specific medium (DMEM) and treated with vincristine at a concentration 0.009 mM for 24 hours and 0.0007 mM for 72 hours and at concentrations of 10, 30, 100 and 300μg/m Lof guarana extract. Incubation was followed by MTT and Pico Green ® tests and evaluation of catalase, superoxide dismutase and thiol activity (cell viability), as well as TBARS, protein carbonyls and dichlorodihydrofluorescein (oxidative metabolism). The results indicated that vincristine may cause cytotoxic effects in mice cerebellum and brain and that guarana may reverse this cytotoxicity in concentrations 100 e 300μg/mL. / A vincristina é um quimioterápico utilizado no tratamento de vários tipos de neoplasias com grandes resultados clínicos, porém, seus efeitos incluem uma série de alterações do equilíbrio, como ataxia, tremores e neuropatia periférica. Estes danos estão provavelmente associados à intoxicação provocada pelo fármaco no sistema nervoso central e periférico. Há indícios de que o guaraná (Paullinia cupana) possui componentes capazes de produzir neuroproteção central. O objetivo deste estudo foi avaliar os efeitos da vincristina e do guaraná sobre a viabilidade celular e o metabolismo oxidativo no cérebro e no cerebelo de camundongos em cultura celular in vitro. As células foram incubadas por 24 e 72 horas em meio específico (DMEM) e tratadas com vincristina na concentração 0,009 μM para 24 horas e 0,0007 μM para 72 horas e com as concentrações de 10, 30, 100 e 300μg/mL de extrato de guaraná. Após, realizou-se os testes MTT, Pico Green®, atividade das enzimas catalase e superóxido dismutase e tióis (viabilidade celular); TBARS, diclorodihidrofloresceína e carbonilação de proteínas (metabolismo oxidativo). Os resultados indicaram que a vincristina pode causar efeitos citotóxicos em cerebelo e cérebro de camundongos e que o guaraná pode reverter essa citotoxicidade, principalmente nas concentrações de 100 e 300 μg/mL.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The Vincristine is a chemotherapy used for various types of cancers, and is one of the major side effects the impairment of motor function and balance, as well as the oxidative and inflammatory unbalance. Therefore, the present study aimed to evaluate the effect of physical exercise on the balance and in the oxidative metabolism of rats treated with anti-inflammatory Vincristine. For this, rats weighing between 270-300 g were divided into two groups: Trained Group (n = 20, 6 weeks of swimming, 1:00/day, 5 days/week, with 5% overload body weight) and Sedentary Group (n = 20, placebo swimming in a tank with water to 5 cm deep, with no extra charge). After 6 weeks the two groups (n = 20 of each) were treated with a single dose of weekly 0, 5 mg/kg of vincristine sulfate, for two weeks, totaling a cumulative dose of 1 mg/kg intraperitoneal. The rats used in control groups (n = 10 n = 10 trained and sedentary) received the same dose of saline (NaCl). For analysis of motor function and balance were performed after chemotherapy applications, on 1, 7, 8,15 days: Rotarod tests (balance), Grip Test (muscle strength), the Open field (exploration) and the hot plate (peripheral sensitivity). At the end of the assessment (day 15) cerebellum samples were taken for analysis of oxidative stress (DCFH, SOD and CAT) and blood samples for analysis of Picogreen and CBC. In behavioral assessment, there was a decrease in body balance in rats exposed to the VCR and, also, a significant protection of physical exercise. There was no significant decrease in muscle strength and exploitation in the open field of rats exposed to VCR, but there was no effect per se of physical exercise, which increased muscle strength and the distance and the operating time standing in the open field in mice. There was significant change in peripheral sensitivity in mice treated and prior exercise was able to normalize this damage. In biochemical analysis we observed a significant protective effect of exercise in trained/VCR rats, once ROS levels were reduced (DCFH analysis) and antioxidant system enzymes of the cerebellum SOD and CAT modulated positively. In the analysis of observed Picogreen exercise protection against DNA damage in the trained/VCR group. The CBC trained /VCR rats they kept the parameters analyzed within the reference values when compared to sedentary/VCR. Therefore, the exercise proved to be a great ally in protecting the body and balance the immune system front of chemotherapy toxicity Vincristine. / A vincristina é um quimioterápico usado para diversos tipos de cânceres, e tem como um dos principais efeitos colaterais o comprometimento da função motora e equilíbrio, bem como o desbalanço oxidativo e inflamatório. Portanto, o presente estudo teve como objetivo avaliar o efeito do exercício físico no equilibrio e no metabolismo oxidativo-inflamatório de ratos tratadas com Vincristina. Para isso, ratos com peso entre 270-300g foram divididos em dois grupos: Grupo treinado (n=20, 6 semanas de natação, 1h/dia, 5 dias/semana, com sobrecarga de 5% do peso corporal) e Grupo sedentário (n=20, natação placebo em um tanque com água a 5cm de profundidade, sem carga extra). Após as 6 semanas os dois grupos (n=20 de cada) foram tratados com dose única semanal de 0,5mg/kg de sulfato de vincristina, durante duas semanas, totalizando uma dose acumulada de 1 mg/kg, por via intraperitoneal. Os ratos utilizados nos grupos controle (n=10 treinados e n=10 sedentários) receberam a mesma dose de solução salina (NaCl). Para análise da função motora e equilíbrio foram realizados, após as aplicações de quimioterapia, nos dias 1, 7, 8 e 15 os testes Rotarod (equilíbrio), Grip-Test (força muscular), Campo aberto (exploração) e o Placa quente (sensibilidade periférica). Ao final das avaliações (dia 15) foram retiradas as amostras de cerebelo para análise de estresse oxidativo (DCFH, SOD e CAT) e amostras de sangue para análise de Picogreen e hemograma. Na avaliação comportamental, observou-se uma diminuição do equilíbrio corporal nos ratos expostos à VCR e, também, uma proteção significativa do exercício físico. Não houve diminuição significativa da força muscular e da exploração no campo aberto dos ratos expostos à VCR, mas houve efeito per se do exercício físico, que aumentou a força muscular e a distância percorrida e o tempo de exploração em pé no Campo aberto nos ratos treinados. Houve alteração significativa na sensibilidade periférica nos ratos tratados e o exercício físico prévio foi capaz de normalizar este dano. Nas análises bioquímicas observamos um efeito protetor significativo do exercício físico nos ratos treinados/VCR, uma vez que os níveis de ROS foram diminuídos (análise de DCFH) e as enzimas do sistema antioxidante do cerebelo SOD e CAT modularam positivamente. Na análise de Picogreen, observamos uma proteção do exercício físico contra o dano de DNA no grupo treinado/VCR. No hemograma os ratos treinados/VCR, mantiveram os parâmetros analisados dentro dos valores de referência quando comparados aos sedentários/VCR. Sendo assim, o exercício físico mostrou-se um grande aliado na proteção do equilíbrio corporal e do sistema imunológico frente à toxicidade do quimioterápico Vincristina.

Made available in DSpace on 2016-01-26T18:55:27Z (GMT). No. of bitstreams: 1 DISSETACAO_COMPLETA__16_04_09.pdf: 1755631 bytes, checksum: da3df5aaf8868dbddec8a1a0b3481970 (MD5) Previous issue date: 2009-02-13 / The transmissible venereal tumor (TVT) is a contagious neoplasm of round cells of dogs of very frequent casuistry. The standard treatment consists of chemotherapy, and the most effective treatment is the vincristine sulphate alone, however the resistance emergence to this agent has been taking the association with other drugs. Recent studies demonstrated the antitumoral effect of the avermectins when associated to the vincristine in the treatment of some neoplasms. Therefore, the objective of this work was to compare the effectiveness of the standard protocol with vincristine only and the ivermectin + vincristine association through histopathologycal and cytological analysis of the tumor. The samples analyses reveal a more precocious cure of TVT in the associated protocol. These results suggest that the protocol of the vincristine associated to ivermectin can be an excellent therapeutic alternative for the treatment of TVT in the future. / O tumor venéreo transmissível (TVT) é uma neoplasia de células redondas que acomete cães de casuística muito freqüente. O tratamento padrão consiste no uso de antineoplásicos, sendo de eleição a vincristina como agente único, porém o aparecimento de resistência a este fármaco tem levado a associação com outras drogas. Estudos recentes demonstraram o efeito antitumoral das avermectinas quando associadas à vincristina no tratamento de alguns tipos de neoplasias. Portanto, o objetivo deste trabalho foi comparar a eficácia do protocolo padrão com o uso único de vincristina e o protocolo da associação de vincristina e ivermectina através da análise histopatológica e citológica do tumor. As análises histopatológicas e citológicas revelarem uma cura mais precoce do TVT no protocolo associado. Estes resultados sugerem que o protocolo vincristina associada à ivermectina pode ser no futuro uma excelente alternativa terapêutica para o tratamento do TVT.

Made available in DSpace on 2016-07-18T17:53:05Z (GMT). No. of bitstreams: 1 DISSETACAO_COMPLETA__16_04_09.pdf: 1755631 bytes, checksum: da3df5aaf8868dbddec8a1a0b3481970 (MD5) Previous issue date: 2009-02-13 / The transmissible venereal tumor (TVT) is a contagious neoplasm of round cells of dogs of very frequent casuistry. The standard treatment consists of chemotherapy, and the most effective treatment is the vincristine sulphate alone, however the resistance emergence to this agent has been taking the association with other drugs. Recent studies demonstrated the antitumoral effect of the avermectins when associated to the vincristine in the treatment of some neoplasms. Therefore, the objective of this work was to compare the effectiveness of the standard protocol with vincristine only and the ivermectin + vincristine association through histopathologycal and cytological analysis of the tumor. The samples analyses reveal a more precocious cure of TVT in the associated protocol. These results suggest that the protocol of the vincristine associated to ivermectin can be an excellent therapeutic alternative for the treatment of TVT in the future. / O tumor venéreo transmissível (TVT) é uma neoplasia de células redondas que acomete cães de casuística muito freqüente. O tratamento padrão consiste no uso de antineoplásicos, sendo de eleição a vincristina como agente único, porém o aparecimento de resistência a este fármaco tem levado a associação com outras drogas. Estudos recentes demonstraram o efeito antitumoral das avermectinas quando associadas à vincristina no tratamento de alguns tipos de neoplasias. Portanto, o objetivo deste trabalho foi comparar a eficácia do protocolo padrão com o uso único de vincristina e o protocolo da associação de vincristina e ivermectina através da análise histopatológica e citológica do tumor. As análises histopatológicas e citológicas revelarem uma cura mais precoce do TVT no protocolo associado. Estes resultados sugerem que o protocolo vincristina associada à ivermectina pode ser no futuro uma excelente alternativa terapêutica para o tratamento do TVT.

Les microtubules, composants clés du cytosquelette des cellules eucaryotes, sont des polymères de tubuline très dynamiques et impliqués dans une grande variété de processus cellulaires. Leur rôle essentiel dans le cycle cellulaire a fait d’eux une cible validée en thérapie anticancéreuse. Malgré l’efficacité clinique des agents ciblant les microtubules (ACM), les effets secondaires compliquent l’utilisation. Nous avons cherché à identifier des vulnérabilités génétiques qui peuvent être exploitées pour diminuer la dose requise tout en maintenant l'efficacité, et donc réduire les effets secondaires. En collaboration avec le laboratoire Tyers à l’IRIC, nous avons réalisé un criblage génétique basé sur la létalité synthétique avec des agents antiprolifératifs, dont les ACMs. Nous avons sélectionné les gènes dont l’extinction sensibilisait les cellules aux ACMs. J’ai confirmé que l’invalidation de chacun des gènes GNA13, SEPHS1, DLGAP5 et des gènes QRICH1, DLGAP5 sensibilisaient les cellules NALM6 au docétaxel et la vincristine respectivement. En revanche, aucune invalidation de ces gènes n'a augmenté la sensibilité au docétaxel dans les cellules U2OS. En plus de son effet avec le docétaxel, le gène GNA13 s’est distingué être une cible particulièrement intéressante. En effet, la perte complète de GNA13 augmente considérablement la fréquence et la gravité d’erreurs de ségrégation des chromosomes dans les cellules U2OS. Cette augmentation n’a pas été rectifiée à la suite d’un traitement avec la molécule UMK57, connue pour réduire le taux d’erreurs de ségrégation des chromosomes. De manière intéressante, la perte complète de GNA13 augmente également la fréquence des erreurs de ségrégation des chromosomes dans les cellules RPE1, cellules non-cancéreuses et stables au niveau chromosomique. Cela suggère que la perte complète de GNA13 ne nécessite pas de transformation ni d'instabilité chromosomique, comme conditions préalables pour exacerber l'instabilité chromosomique. L’ensemble de ces résultats ouvre une nouvelle voie de stratégies thérapeutiques anticancéreuses, à savoir, le traitement des cancers présentant une mutation des gènes QRICH1, DLGAP5, GNA13, et SEPHS1 avec de faibles doses d’ACMs. En particulier, GNA13 est fréquemment muté dans certains lymphomes. De plus, les résultats obtenus démontrent que la perte complète de GNA13 aggrave l’instabilité chromosomique et par conséquent, pourrait être impliquée dans la cancérogenèse. / Microtubules, key components of the eukaryotic cytoskeleton, are highly dynamic polymers of tubulin implicated in a wide variety of cellular processes. Their essential roles in the cell cycle have made them a valid target in cancer therapy. Despite the clinical efficacy of microtubule targeting agents (MTA), their use is hampered by side effects. We sought to identify genetic vulnerabilities that can be exploited to decrease the required dose while maintaining efficacy, and therefore reduce side effects. In collaboration with the Tyers laboratory at IRIC, we carried out a genetic screen based on synthetic lethality with antiproliferative agents, including MTAs. We have selected genes whose knockout sensitized cells to MTAs. I have confirmed that the knockout of GNA13, SEPHS1, DLGAP5, and QRICH1, DLGAP5, sensitize NALM6 cells to docetaxel and vincristine respectively. However, no knockout of these genes increased the sensitivity to docetaxel in U2OS cells. In addition to its effect with docetaxel, GNA13 stood out as being a particularly exciting target. GNA13 knockout increased the frequency and severity of chromosome segregation errors in U2OS cells. This increase was not corrected following treatment with UMK57, a molecule known to reduce the rate of chromosome segregation errors. Interestingly, the GNA13 knockout also increased the frequency of chromosome segregation errors in non-cancerous and chromosomally stable RPE1 cells. This suggests that GNA13 does not require transformation nor chromosomal instability as prerequisites for exacerbating chromosomal instability. Overall, these results open up a new avenue of anticancer therapeutic strategies, namely, the treatment of cancers presenting mutations in QRICH1, DLGAP5, GNA13, and SEPHS1 with lower doses of MTAs. In particular, GNA13 is frequently mutated in certain lymphomas. In addition, the results obtained demonstrate that GNA13 knockout exacerbates chromosomal instability and, therefore, could be involved in carcinogenesis.

No description available.