Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Neurociências, Florianópolis, 2016. / Made available in DSpace on 2016-05-31T04:09:54Z (GMT). No. of bitstreams: 1 339613.pdf: 3611153 bytes, checksum: 3f8c794d29b943c788477e63cfdb3f2e (MD5) Previous issue date: 2016 / Gliomas, tumores originados de células gliais (astrócitos, oligodendrócito ou epêndima), são os tumores cerebrais primários mais comuns. Lectinas são proteínas de reconhecimento e ligação específicos a carboidratos. O objetivo do presente trabalho foi analisar o possível efeito citotóxico das lectinas ConA, ConBr, ConM, CoxyL, ConGF, DSL (nativa e recombinante), DwL, DvioL, DmrL, DLL, DrfL e CTL, que são extraidas de plantas leguminosas, sobre células C6 de glioma de rato, um glioma com características astrocitárias. Após 24h de incubação com as respectivas lectinas e nas concentrações de 10, 30, 50 e 100µg/mL, foi observado que apenas a lectina CTL não provocou redução da viabilidade, avaliada através do teste de redução do MTT. Baseado na potência de ação citotóxica observada nesse screening as lectinas ConA, ConBr, DvioL e DSL recombinante foram escolhidas para realizar o mesmo teste do MTT, porém também nos tempos 1, 3, 6 e 12 horas, sendo então selecionadas as lectinas ConA e Dviol para investigação das possíveis vias de morte celular envolvidas. Dessa forma, através de ensaios de microscopia de fluorescência, citometria de fluxo e microscopia eletrônica conclui-se que o principal mecanismo responsável pelo efeito citotóxico de ConA e DvioL é a indução de morte celular autofágica, efeito obtido a partir das concentrações de 30µg/mL. Ao realizar ensaios com as lectinas desnaturadas, concluiu-se que o efeito biológico depende da estrutura terciária da lectina, porém, mais estudos sobre a importância do sítio de reconhecimento a carboidratos devem ser realizados, uma vez que os ensaios de bloqueio do sítio de ligação ao açúcar não mostraram claramente uma ação bloqueadora do efeito citotóxico de ConA e DvioL. Os resultados também mostraram que DvioL possui maior potência em relação a ConA na indução de morte autofágica. Este trabalho sugere DvioL com uma molécula com potencial para fututros estudos de terapia anti-tumoral.<br> / Abstract : Gliomas, tumors originating from glial cells (astrocytes, oligodendrocytes or ependymal) are the most common primary brain tumors. Lectins are proteins of specific recognition and binding to carbohydrates. The aim of this study was to analyze the possible cytotoxic effect of the lectins ConA, ConBr, ConM, CoxyL, ConGF, DSL (native and recombinant), DWL, DvioL, DmrL, DLL, DrfL and CTL, which are extracted from leguminous plants, on C6 rat glioma cells, which have astrocytes features. After 24 hours of incubation with the respective lectins and at concentrations of 10, 30, 50 and 100µg/mL, it was observed that only the CTL lectin did not cause reduction in viability as measured by MTT test. Based on the potency of cytotoxic activity observed in this screening, ConA, ConBr, DvioL and DSL recombinant lectins were chosen for the same MTT test after 1, 3, 6 and 12 hours incubation, and then ConA and Dviol lectins were selected for further investigation concerning the possible cell death pathways involved. Thus, by fluorescence microscopy, flow cytometry and electron microscopy assays, it was concluded that the main mechanism responsible for the cytotoxic effect of ConA and DvioL is the induction of autophagic cell death, the effect obtained from the concentration of 30µg/mL. When performing tests with denatured lectins, it was concluded that the biological effect depends on the tertiary structure of the lectin. However, further studies regarding the role of the carbohydrate recognition domain (CRD) deserve to be conducted, since the CRD blocking protocol did not completely abrogated the cytotoxic action of ConA and DvioL. The results also showed that DvioL have greater potency compared to the ConA to induce autophagic death. This study suggests DvioL as a potential molecule to undertake future studies for anti-tumor therapy.

No description available.

O metabolismo do triptofano (Trp) se dá pela via das quinureninas (QUIN), pela via serotoninérgica (SER) e pela via das aminas traço. A primeira gera QUIN e uma variedade de outros metabólitos secundários. Quando conduzida pela enzima indolamina 2,3 dioxigenase (IDO) contribui para os fenômenos de tolerância e imune escape de células tumorais; e quando conduzida pela triptofano 2,3 dioxigenase (TDO) no fígado, participa na síntese da niacina e NAD. A via SER leva à formação do neurotransmissor serotonina (SER), que pode gerar o hormônio melatonina (MEL), respectivamente e outros metabólitos biologicamente ativos. Outra via menos estudada, a via das aminas traço, produz produtos neuroativos. Dada a abrangência e importância das rotas metabólicas do Trp, nós desenvolvemos e validamos uma metodologia bioanalítica robusta, seletiva e sensível por cromatografia líquida de alta eficiência (HPLC), acoplado espectrometria de massas (MS) para a determinação simultânea do Trp e seus 15 metabólitos. Para tanto, escolhemos para a avaliação das três vias, linhagens de glioma humano. A escolha por este tipo celular deveu-se ao grande interesse de estudos de metabolismo de Trp em células tumorais, no qual células de glioma tem sido modelo. Nos ensaios com as células de glioma acompanhamos os efeitos de um indutor e inibidores da primeira etapa de metabolização do Trp pela via das quinureninas, ou seja, IFN-&#947; (indutor da IDO), 1-metiltriptofano (1-MT; inibidor competitivo da IDO) e 680C91 (inibidor seletivo da TDO). Pudemos observar o impacto que a indução ou a inibição do primeiro passo teve sobre os metabólitos subsequentes e as diferenças no metabolismo das duas linhagens estudadas, A172 e T98G. A linhagem T98G só tem atividade de IDO, enquanto que a A172 tem tanto atividade IDO quanto TDO. A indução por IFN-&#947; mostrou que essa citocina não só atua na formação da via QUIN, mas possui um impacto modesto nas demais rotas. Observamos também que a inibição do 1-MT mostrou seu impacto nos metabólitos invdividualmente, do que a simples relação Trp-QUIN. Contudo, nosso resultados nos permitiu mostrar pela primeira vez a descrição completa dessas vias, em especial nessas linhagens celulares, podendo supor estratégias terapêuticas nessas rotas que estão relacionadas a progressão ou não tumoral. / The tryptophan metabolism (Trp) takes place by means of kynurenine (QUIN), by the serotonin pathway (SER) and by the pathway of trace amines synthesis. The first generates QUIN and a variety of other secondary metabolites. When driven by the enzyme indoleamine 2,3 dioxygenase (IDO) contributes to the phenomena of tolerance and immune escape of tumor cells; and when conducted by tryptophan 2,3 -dioxygenase (TDO) in the liver, participates in the niacin synthesis NAD. The SER pathway leads to the serotonin neurotransmitter (SER) formation, which can generate the hormone melatonin (MEL), respectively and other biologically active metabolites. Another less studied amines trace synthesis pathway produces neuroactive products. Given the scope and importance of Trp metabolic pathways,we developed and validated a robust, sensitive and selective bioanalytical method by high performance liquid chromatography (HPLC) coupled mass spectrometry (MS) for simultaneous determination of TRP and its 16 metabolites. Therefore, we chose to evaluate the three routes, glioma cell lines. The initial choice of this type of cell was due to the great interest in Trp metabolism studies in tumor cells, which glioma cells has been a model. In assays with glioma cells, we followed the effects of an inductor and inhibitors of the first stage of Trp metabolism, via the kynurenine pathway, or IFN -&#947; (IDO inducer) 1- methyltryptophane (1- MT; competitive IDO inhibitor) and 680C91 (selective TDO inhibitor). We could observe the first step induction or inhibition impact had over the further metabolites and the metabolism differences between the two studied strains, A172 and T98G. The T98G glioma cell has only IDO activity, while the A172 has both IDO and TDO activity as well. The IFN-&#947; indution showed that this cytokine not only acts in the formation of QUIN route, but has a modest impact on the others routes. Inhibition of IDO showed that the competitive inhibitor has activity in itself than a simple Trp-QUIN relationship. However, our results allow us to show the first time the complete description of these pathways, in particular, in these cell lines that can assume therapeutic strategies in these routes that are related or not with tumor progression.

O tratamento dos gliomas malignos cerebrais é um dos grandes desafios da medicina atualmente, pois, apesar do grande avanço no conhecimento destes tumores, o prognóstico de vida dos portadores desta doença é muito ruim. Foram estudados retrospectivamente 61 pacientes com diagnóstico de glioblastoma multiforme ou astrocitoma anaplásico, no período de 1998 a 2002, com o objetivo de avaliar o uso do Paclitaxel como potencializador do tratamento radioterápico destes tumores. Todos os pacientes foram tratados inicialmente com cirurgia para retirada ampla do volume tumoral (mínimo de 80%) seguido de tratamento com radioterapia fracionada e reforço com radiocirurgia estereotáxica. Em caso de crescimento tumoral, após o tratamento inicial dos pacientes com KPS > 70, novo tratamento cirúrgico e nova radiocirurgia foram indicados. Destes 61 pacientes, 32 receberam tratamento com Paclitaxel, na dose de 100mg/m2 e 29 pacientes não receberam nenhum tipo de quimioterápico. Os grupos foram comparados, em relação ao tipo histológico, faixa etária, sexo e localização tumoral, não havendo diferenças estatisticamente significantes entre os mesmos. Os pacientes de ambos os grupos tiveram acompanhamento laboratorial antes, durante e após o tratamento com paclitaxel e foram acompanhados até o óbito causado pela doença. Foram excluídos do estudo os portadores de tumor que foram a óbito por outras causas. A análise dos resultados mostrou que não houve diferenças estatísticas em relação à sobrevida média do grupo tratado com Paclitaxel e o grupo sem o tratamento (p=1,000). Da mesma forma, a comparação entre os pacientes com glioblastomas (p=0,8933) e com astrocitomas anaplásicos (p=0,5920) de ambos os grupos não mostrou diferença estatística em relação à sobrevida. O número de craniotomias( p=0,5268) e o número de radiocirurgias (p=0,3666) foram semelhantes estatisticamente. Os estudos laboratoriais realizados durante o tratamento no grupo que recebeu o paclitaxel, não mostraram alterações que levassem à suspensão do tratamento. A análise dos resultados deste estudo permitiu concluir que o uso do paclitaxel, concomitante ao tratamento radioterápico dos gliomas malignos cerebrais, não mostrou nenhum ganho adicional na sobrevida dos pacientes portadores destes tumores e, pela análise da necessidade de novo tratamento durante o curso da doença, não potencializou os efeitos da radioterapia. Palavras-chave: Paclitaxel, gliomas malignos, radioterapia / Nowadays, the treatment of the malignant cerebral gliomas is one of the greatest challenges for neurosurgons. Despite of the advance regarding these tumors? knowledge expectance of life for these patients is very bad. The main purpose of this study was to evaluate the use of paclitaxel to enhance the radiotherapy treatment in those tumors. Sixty-one patients with diagnosis for glioblastoma multiforme or anaplastic astrocytoma in the period of 1998 to 2002 were, retrospectively, studied. All patients were initially treated with surgery in order to remove a wide portion of the tumor?s volume (minimum of 80%). Then, the patients were treated with fractionated radiotherapy and reinforcement with stereotactic radiosurgery. If there was an increase in the tumor after the initial treatment, the patients with a KPS higher than 70 had new treatment with surgery as well as with radiosurgery. Among the 61 patients, 32 were treated with a 100 mg/m2 dose of paclitaxel, and 29 of then did not have any kind of chemotherapy treatment. Comparisons bettwen both regardhg to the histological type, age, gender and location of the tumor showed no differences . Patients of both groups had a laboratory follow-up before, during and after the treatment with paclitaxel. All of them were followed until their death, which was caused by the disease. Patients that died from other diseases were not included in the study. The analysis of the results indicated that there were no statistics differences regarding the mean survival time between the groups treated or nor treated with paclitaxel ( p=1,000). Likewise, a comparison between the glioblastomas (p=0,8933) and the anaplastic astrocytomas (p=0,5920) of both groups did not indicate any statistic difference regarding to the survival time. The was no statistic difference between the number of craniotomies (p=0,5268) as well as between the number of radiosurgeries. (p=0,3666). The laboratory studies held during the treatment of the group that received the paclitaxel did not show any changes which could lead to cease the treatment. Hence the results led to the conclusion that the treatment of malignant cerebral gliomas with paclitaxel and radiotherapy treatment at the same time did not give any additional gain in the patients?survival. Regarding to the demand for new treatment throughout the disease, there was no enhance of the radiotherapy effects with the Paclitaxel. Key words: Paclitaxel, malignant gliomas, radiotherapy.

O metabolismo do triptofano (Trp) se dá pela via das quinureninas (QUIN), pela via serotoninérgica (SER) e pela via das aminas traço. A primeira gera QUIN e uma variedade de outros metabólitos secundários. Quando conduzida pela enzima indolamina 2,3 dioxigenase (IDO) contribui para os fenômenos de tolerância e imune escape de células tumorais; e quando conduzida pela triptofano 2,3 dioxigenase (TDO) no fígado, participa na síntese da niacina e NAD. A via SER leva à formação do neurotransmissor serotonina (SER), que pode gerar o hormônio melatonina (MEL), respectivamente e outros metabólitos biologicamente ativos. Outra via menos estudada, a via das aminas traço, produz produtos neuroativos. Dada a abrangência e importância das rotas metabólicas do Trp, nós desenvolvemos e validamos uma metodologia bioanalítica robusta, seletiva e sensível por cromatografia líquida de alta eficiência (HPLC), acoplado espectrometria de massas (MS) para a determinação simultânea do Trp e seus 15 metabólitos. Para tanto, escolhemos para a avaliação das três vias, linhagens de glioma humano. A escolha por este tipo celular deveu-se ao grande interesse de estudos de metabolismo de Trp em células tumorais, no qual células de glioma tem sido modelo. Nos ensaios com as células de glioma acompanhamos os efeitos de um indutor e inibidores da primeira etapa de metabolização do Trp pela via das quinureninas, ou seja, IFN-&#947; (indutor da IDO), 1-metiltriptofano (1-MT; inibidor competitivo da IDO) e 680C91 (inibidor seletivo da TDO). Pudemos observar o impacto que a indução ou a inibição do primeiro passo teve sobre os metabólitos subsequentes e as diferenças no metabolismo das duas linhagens estudadas, A172 e T98G. A linhagem T98G só tem atividade de IDO, enquanto que a A172 tem tanto atividade IDO quanto TDO. A indução por IFN-&#947; mostrou que essa citocina não só atua na formação da via QUIN, mas possui um impacto modesto nas demais rotas. Observamos também que a inibição do 1-MT mostrou seu impacto nos metabólitos invdividualmente, do que a simples relação Trp-QUIN. Contudo, nosso resultados nos permitiu mostrar pela primeira vez a descrição completa dessas vias, em especial nessas linhagens celulares, podendo supor estratégias terapêuticas nessas rotas que estão relacionadas a progressão ou não tumoral. / The tryptophan metabolism (Trp) takes place by means of kynurenine (QUIN), by the serotonin pathway (SER) and by the pathway of trace amines synthesis. The first generates QUIN and a variety of other secondary metabolites. When driven by the enzyme indoleamine 2,3 dioxygenase (IDO) contributes to the phenomena of tolerance and immune escape of tumor cells; and when conducted by tryptophan 2,3 -dioxygenase (TDO) in the liver, participates in the niacin synthesis NAD. The SER pathway leads to the serotonin neurotransmitter (SER) formation, which can generate the hormone melatonin (MEL), respectively and other biologically active metabolites. Another less studied amines trace synthesis pathway produces neuroactive products. Given the scope and importance of Trp metabolic pathways,we developed and validated a robust, sensitive and selective bioanalytical method by high performance liquid chromatography (HPLC) coupled mass spectrometry (MS) for simultaneous determination of TRP and its 16 metabolites. Therefore, we chose to evaluate the three routes, glioma cell lines. The initial choice of this type of cell was due to the great interest in Trp metabolism studies in tumor cells, which glioma cells has been a model. In assays with glioma cells, we followed the effects of an inductor and inhibitors of the first stage of Trp metabolism, via the kynurenine pathway, or IFN -&#947; (IDO inducer) 1- methyltryptophane (1- MT; competitive IDO inhibitor) and 680C91 (selective TDO inhibitor). We could observe the first step induction or inhibition impact had over the further metabolites and the metabolism differences between the two studied strains, A172 and T98G. The T98G glioma cell has only IDO activity, while the A172 has both IDO and TDO activity as well. The IFN-&#947; indution showed that this cytokine not only acts in the formation of QUIN route, but has a modest impact on the others routes. Inhibition of IDO showed that the competitive inhibitor has activity in itself than a simple Trp-QUIN relationship. However, our results allow us to show the first time the complete description of these pathways, in particular, in these cell lines that can assume therapeutic strategies in these routes that are related or not with tumor progression.

Os gliomas representam mais de 70% dos tumores cerebrais primários. Os glioblastomas multiformes são gliomas malignos caracterizados por baixa incidência, mas altas taxas de mortalidade. Apesar da responsividade inicial ao tratamento padrão realizado com o quimioterápico alquilante temozolomida (TMZ), poucos foram os avanços para o prognóstico dos pacientes nos últimos 10 anos. Isso deve-se ao fato desses tumores serem raramente passíveis de ressecção cirúrgica e apresentarem alta taxa de recorrência. Além disso, a eficácia de seu tratamento encontra barreiras como efeitos colaterais indesejáveis e resistência quimioterápica. Nesse cenário, a descoberta de novas substâncias que possam atuar com efeito aditivo ou sinérgico e aumentem a sensibilização de células tumorais ao tratamento, torna-se uma estratégia terapêutica no campo da oncologia. O ditelureto de difenila (DTDF) é um composto orgânico contendo telúrio que apresenta interessantes efeitos biológicos in vitro, como antioxidante, quimioprotetivo, citotóxico e antitumoral. Sendo assim, o objetivo deste estudo foi avaliar os efeitos do DTDF e do quimioterápico TMZ, em regimes isolados e em associação. Para tal foram investigados seus efeitos citotóxicos, após exposição aguda e crônica, em culturas celulares de glioma não-resistente (M059J) e resistente à TMZ (GBM). No ensaio de viabilidade celular a TMZ apresentou citotoxicidade para as linhagens celulares testadas, com valor de IC50 maior na linhagem resistente do que quando comparado à linhagem não-resistente. Este dado foi confirmado pelo teste de duplicação cumulativa de população e, também, pela coloração com laranja de acridina, após o tratamento de 120 h, por observar-se um aumento na frequência de células positivas para a formação de organelas vesiculares ácidas em ambas as linhagens, sendo predominantemente em células GBM. Além disso, foi observado que o tratamento associando DTDF e a TMZ apresentou uma maior citotoxicidade quando comparado aos tratamentos isolados, após 120 h de tratamento. Portanto, o DTDF sensibilizou as células ao tratamento com TMZ. Essa sensibilização ocorreu em níveis aproximados para ambas as linhagens, sendo os efeitos do DTDF independentes do perfil de resistência à TMZ. Em conjunto, os dados desse trabalho sugerem o uso do DTDF em associação à TMZ como uma estratégia para reduzir as doses de TMZ empregadas na clínica e diminuir efeitos colaterais aos pacientes em tratamento de glioma / Gliomas represent more than 70% of primary brain tumors. Malignant gliomas are characterized by low incidence, but high mortality rates. Despite the initial responsiveness to the standard treatment with the chemotherapeutic alkylating temozolomide (TMZ), few advances have been made in the prognosis of patients in the last 10 years. This is due to the fact that these tumors are rarely amenable to surgical resection and have a high rate of recurrence. Moreover, the effectiveness of this treatment encounters barriers such as undesirable side effects and chemotherapeutic resistance. In this scenario, the discovery of new substances that may act with additive or synergistic effect and increase the sensitization of tumor cells to the treatment becomes a therapeutic strategy in the field of oncology. Diphenyl ditelluride (DPDT) is a derivative of tellurium used in various reactions of organic synthesis and has interesting in vitro biological effects, as antioxidant, chemoprotective, cytotoxic and antitumor agent. Therefore this work aimed to evaluate the cytotoxic effects of this organotellurium compound and the chemotherapeutic, TMZ, in isolated and in association regimens, after acute and chronic exposure, of non-resistant (M059J) and TMZ- resistant (GBM) glioma cells. Through the cell viability assay, it was shown that TMZ is cytotoxic for both cell lines tested, showing a higher IC50 value in the resistant line when compared to the other line. This data was confirmed by the cumulative population doubling test. In addition, by the acridine orange staining, it was verified that autophagy might favor the chemoresistance, although not being the main resistance mechanism in the lines tested. It was observed that DPDT clearly has a dose-dependent cytotoxic effect on the M059J and GBM cell lines, in a lower concentration range than that used with TMZ. DPDT sensitized the cells to TMZ treatment as evidenced by the decline in cell viability. It is important to point out that this sensitization occurred in low and approximate IC50 values after both 24 h and 120 h of treatment, being the effects of the DPDT independent of the resistance profile to TMZ. Taken together, data from this work suggest the use of DPDT in association with TMZ as an interesting strategy to reduce the doses of TMZ used in the clinic and to reduce side effects to patients under treatment of glioma.

O tratamento dos gliomas malignos cerebrais é um dos grandes desafios da medicina atualmente, pois, apesar do grande avanço no conhecimento destes tumores, o prognóstico de vida dos portadores desta doença é muito ruim. Foram estudados retrospectivamente 61 pacientes com diagnóstico de glioblastoma multiforme ou astrocitoma anaplásico, no período de 1998 a 2002, com o objetivo de avaliar o uso do Paclitaxel como potencializador do tratamento radioterápico destes tumores. Todos os pacientes foram tratados inicialmente com cirurgia para retirada ampla do volume tumoral (mínimo de 80%) seguido de tratamento com radioterapia fracionada e reforço com radiocirurgia estereotáxica. Em caso de crescimento tumoral, após o tratamento inicial dos pacientes com KPS > 70, novo tratamento cirúrgico e nova radiocirurgia foram indicados. Destes 61 pacientes, 32 receberam tratamento com Paclitaxel, na dose de 100mg/m2 e 29 pacientes não receberam nenhum tipo de quimioterápico. Os grupos foram comparados, em relação ao tipo histológico, faixa etária, sexo e localização tumoral, não havendo diferenças estatisticamente significantes entre os mesmos. Os pacientes de ambos os grupos tiveram acompanhamento laboratorial antes, durante e após o tratamento com paclitaxel e foram acompanhados até o óbito causado pela doença. Foram excluídos do estudo os portadores de tumor que foram a óbito por outras causas. A análise dos resultados mostrou que não houve diferenças estatísticas em relação à sobrevida média do grupo tratado com Paclitaxel e o grupo sem o tratamento (p=1,000). Da mesma forma, a comparação entre os pacientes com glioblastomas (p=0,8933) e com astrocitomas anaplásicos (p=0,5920) de ambos os grupos não mostrou diferença estatística em relação à sobrevida. O número de craniotomias( p=0,5268) e o número de radiocirurgias (p=0,3666) foram semelhantes estatisticamente. Os estudos laboratoriais realizados durante o tratamento no grupo que recebeu o paclitaxel, não mostraram alterações que levassem à suspensão do tratamento. A análise dos resultados deste estudo permitiu concluir que o uso do paclitaxel, concomitante ao tratamento radioterápico dos gliomas malignos cerebrais, não mostrou nenhum ganho adicional na sobrevida dos pacientes portadores destes tumores e, pela análise da necessidade de novo tratamento durante o curso da doença, não potencializou os efeitos da radioterapia. Palavras-chave: Paclitaxel, gliomas malignos, radioterapia / Nowadays, the treatment of the malignant cerebral gliomas is one of the greatest challenges for neurosurgons. Despite of the advance regarding these tumors? knowledge expectance of life for these patients is very bad. The main purpose of this study was to evaluate the use of paclitaxel to enhance the radiotherapy treatment in those tumors. Sixty-one patients with diagnosis for glioblastoma multiforme or anaplastic astrocytoma in the period of 1998 to 2002 were, retrospectively, studied. All patients were initially treated with surgery in order to remove a wide portion of the tumor?s volume (minimum of 80%). Then, the patients were treated with fractionated radiotherapy and reinforcement with stereotactic radiosurgery. If there was an increase in the tumor after the initial treatment, the patients with a KPS higher than 70 had new treatment with surgery as well as with radiosurgery. Among the 61 patients, 32 were treated with a 100 mg/m2 dose of paclitaxel, and 29 of then did not have any kind of chemotherapy treatment. Comparisons bettwen both regardhg to the histological type, age, gender and location of the tumor showed no differences . Patients of both groups had a laboratory follow-up before, during and after the treatment with paclitaxel. All of them were followed until their death, which was caused by the disease. Patients that died from other diseases were not included in the study. The analysis of the results indicated that there were no statistics differences regarding the mean survival time between the groups treated or nor treated with paclitaxel ( p=1,000). Likewise, a comparison between the glioblastomas (p=0,8933) and the anaplastic astrocytomas (p=0,5920) of both groups did not indicate any statistic difference regarding to the survival time. The was no statistic difference between the number of craniotomies (p=0,5268) as well as between the number of radiosurgeries. (p=0,3666). The laboratory studies held during the treatment of the group that received the paclitaxel did not show any changes which could lead to cease the treatment. Hence the results led to the conclusion that the treatment of malignant cerebral gliomas with paclitaxel and radiotherapy treatment at the same time did not give any additional gain in the patients?survival. Regarding to the demand for new treatment throughout the disease, there was no enhance of the radiotherapy effects with the Paclitaxel. Key words: Paclitaxel, malignant gliomas, radiotherapy.

published_or_final_version / Surgery / Master / Master of Philosophy

The identification of the isocitrate dehydrogenase-1 mutation (IDH-1 R132H) in human gliomas has been an important prognostic tool for diagnosing tumor subtype and determining prognosis value in patient care. However; it has not influenced current treatment practices because its role in glioma development is still not fully understood. IDH-1 is a cytoplasmic enzyme that normally functions to mediate the conversion of isocitrate to α-ketogluterate (α-KG). Extensive characterization of the IDH-1 R132H mutation demonstrated that it primarily results in defects associated with (1) decreased efficiency of normal IDH-1 enzymatic activity, (2) elevated production of R-2-hydroxygluterate (R-2HG), and (3) altered genome-wide DNA hypermethylation. Interestingly, the somatic point mutation is exclusively seen in the Proneural glioma subtype (PN), which has phenotypic similarities to oligodendrocyte progenitor cells (OPCs). This suggests that these tumors arise from OPCs or progenitors that acquire an OPC-like phenotype. Thus far, the tumorigenic potential of IDH-1 R132H has not been extensively investigated in current in vivo models of GBM. Beyond its altered enzymatic affinity, the mechanism which facilitates OPC transformation is still not understood. In order to investigate the effects of the IDH-1 R132H mutation during PN gliomagenesis, we built upon previous work by our research group to design a novel in vivo experimental murine glioma system. While IDH-1 R132H mutations have been implicated as the earliest event in PN glioma formation the tumorigenic potential of this metabolic mutation on OPCs of the subcortical white matter has thus far not been tested. In this dissertation I describe my experimental approach and findings for specifically addressing two aims for understanding the relationship of the IDH-1 R132H mutation, OPCs and gliomagenesis: (1) determine whether the expression of the IDH-1 R132H mutant at tumor induction can effect glioma initiation and progression during gliomagenesis in a manner that is phenotypically and molecularly distinct from IDH-1 WT gliomagenesis; and (2) determine how expression of the IDH-1 R132H mutation and 2-HG are affecting OPC lineage development and whether this is facilitating these glial progenitors to acquire a phenotype that resembles cellular transformation. I found that delivery of the IDH-1 R132H mutations to OPCs in the adult murine subcortical white matter was by itself not sufficient to form tumors; however, when expressed in the context of TP53 tumor suppressor deletions and PDGF, tumors formed and expressed both a histological and molecular signature resembling PN human gliomas. These murine gliomas were molecularly and metabolically distinct from IDH-1 wildtype murine gliomas, in that they showed elevated 2-HG production and evidence of a transcriptional and translational signature that was consistent with significantly increased protein synthesis activity. These particular results provided two major innovations in the study of gliomagenesis: (1) the first murine model of PN IDH-1 R132H mutant gliomagenesis initiated from the delivery of genetic alterations to OPCs, and (2) the isolation and stable propagation of tumor cells isolated from these gliomas that retain expression of the metabolic mutations. More importantly, my findings provide the first account of the IDH-1 R132H mutations driving a cumulative increase in global translation. Alternatively, when I directly tested the effects of the IDH-1 R132H mutation in culture OPCs in the absence of additional genetic alterations, I found that the mutation has a role in impairing differentiation by retaining expression of immature lineage markers (PDGFR and NG2) and keeping OPCs highly proliferative when stimulated to differentiate. This is further supported by the inability of OPCs that expressed the IDH-1 R132H mutation to increase mRNA levels of myelin proteins during differentiation. Directly treating OPCs with 2-HG during differentiation recapitulated a cellular phenotype associated with reduced expression of a mature oligodendroctye marker (O1) and the retention of an immature profile (PDGFR+/O1-). However, while expression of IDH-1 R132H mutation had no effect on cell viability, 2-HG had a dose-dependent effect in decreasing cell viability both in conditions of OPC proliferation and differentiation. Analysis of translation activity in OPCs in response to IDH-1 R132H or R-2HG treatment revealed that both conditions had very distinct patterns in the cellular distribution of global protein synthesis. These experiments provide a preliminary account of how the IDH-1 R132H mutation is negatively influencing the ability for OPCs to undergo normal lineage development and informs on impaired cell cycle exit playing a role in facilitating the acquisition of a transformed cellular phenotype. The capacity for OPCs to undergo cellular transformation into fully-formed PN IDH-1 R132H mutant gliomas has not been previously investigated. In this study, I engineered an experimental approach for targeting this progenitor population. I was able to address this gap in current understanding by providing comprehensive analysis and characterization of a murine model of the IDH-1 R132H mutation. By investigating the effects of IDH-1 R132H expression in non-transformed OPCs and PN gliomagenesis, I was able to identify a process of cellular adaption where the presence of the mutation throughout gliomagenesis defines a cellular context that is distinct from the setting of normal OPCs. This process involves global alterations in translation activity. Furthermore, I believe that the mechanism by which this mutation drives tumor formation may be relevant to the mechanisms that are unique to the development and lineage progression of OPCs. Through the use of in vivo murine models, in vitro glial progenitor systems and an integrated molecular approach for identifying metabolic and phenotypic alterations, I was able to provide an account of my graduate research work dedicated to characterizing the effects of IDH-1 R132H in a mouse model of PN glioma and the effect of IDH1R132H on non-neoplastic OPCs as a novel approach to exploring its effects at early stages of gliomagenesis in the process of cellular transformation.

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 82-97) Also available in print.