Sobrevida após radioterapia em pacientes com glioma de alto grau Introdução: Gliomas de alto grau são os principais tumores primários do sistema nervoso central em adultos, cujo prognóstico permanece invariavelmente ruim, mesmo com a terapia atual: cirurgia e quimioirradiação. Na radioterapia utiliza-se de técnicas conformacionais (3DRT) e de intensidade modulada do feixe (IMRT). Objetivos: Avaliar a sobrevida dos pacientes tratados na instituição e os fatores que influenciam os resultados. Métodos: Análise retrospectiva dos tratamentos terapêuticos de pacientes diagnosticados com gliomas de alto grau entre 2009 e 2014 e tratados com radioterapia no Serviço de Radioterapia do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (HCRP-USP). Resultados: A sobrevida mediana obtida foi 16,63 meses, 29 pacientes (61,7%) foram à óbito e os demais encontravam-se em seguimento até o momento da análise. IMRT correspondeu à modalidade de tratamento em 68,1% dos casos, a duração média da radioterapia foi de 56 dias, com intervalo médio (demora) entre cirurgia e o início da radioterapia de 61,7 dias (27 a 123 dias). Observamos que idade, ressecção macroscópica total, tipo histológico e utilização de temozolomida adjuvante influenciaram a taxa de sobrevida (p<0,05). A sobrevida global estimada é de 18 meses (estimativa Kaplan Meyer). Nossa série avaliou os pacientes tratados e os dados corroboram os já relatados na literatura, respaldando o protocolo de tratamento institucional. / Introduction: High-grade gliomas are the main primary tumors of the central nervous system in adults, it`s prognosis remains invariably bad, even with current therapy: chemoradiation after surgery. Radiotherapy is currently employed as conformational techniques (3DRT) or intensity modulated beam (IMRT) with or without temozolomide. Objectives: To evaluate survival of patients treated with radiation and factors influencing results. Methods: Retrospective analysis of patients diagnosed with high-grade gliomas between 2009 and 2014 and treated with radiotherapy at the University Hospital of the Faculty of Medicine of Ribeirão Preto, University of São Paulo (HCRP-USP). Results: Median survival was 16.63 months, 29 patients (61.7%) died and the others were in follow-up at the time of analysis. IMRT was used in 68.1% of cases, the mean duration of radiotherapy obtained of 56 days, with a mean interval (delay) between the surgery/diagnosis and radiotherapy was 61.7 days (27-123 days). We found that age, total gross resection, histological type and use of adjuvant temozolomide influenced the survival rate (p <0.05). The estimated overall survival was 18 months (Kaplan Meyer estimator). Our data was similar to those reported in the literature, supporting the institutional treatment protocol.

Sobrevida após radioterapia em pacientes com glioma de alto grau Introdução: Gliomas de alto grau são os principais tumores primários do sistema nervoso central em adultos, cujo prognóstico permanece invariavelmente ruim, mesmo com a terapia atual: cirurgia e quimioirradiação. Na radioterapia utiliza-se de técnicas conformacionais (3DRT) e de intensidade modulada do feixe (IMRT). Objetivos: Avaliar a sobrevida dos pacientes tratados na instituição e os fatores que influenciam os resultados. Métodos: Análise retrospectiva dos tratamentos terapêuticos de pacientes diagnosticados com gliomas de alto grau entre 2009 e 2014 e tratados com radioterapia no Serviço de Radioterapia do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (HCRP-USP). Resultados: A sobrevida mediana obtida foi 16,63 meses, 29 pacientes (61,7%) foram à óbito e os demais encontravam-se em seguimento até o momento da análise. IMRT correspondeu à modalidade de tratamento em 68,1% dos casos, a duração média da radioterapia foi de 56 dias, com intervalo médio (demora) entre cirurgia e o início da radioterapia de 61,7 dias (27 a 123 dias). Observamos que idade, ressecção macroscópica total, tipo histológico e utilização de temozolomida adjuvante influenciaram a taxa de sobrevida (p<0,05). A sobrevida global estimada é de 18 meses (estimativa Kaplan Meyer). Nossa série avaliou os pacientes tratados e os dados corroboram os já relatados na literatura, respaldando o protocolo de tratamento institucional. / Introduction: High-grade gliomas are the main primary tumors of the central nervous system in adults, it`s prognosis remains invariably bad, even with current therapy: chemoradiation after surgery. Radiotherapy is currently employed as conformational techniques (3DRT) or intensity modulated beam (IMRT) with or without temozolomide. Objectives: To evaluate survival of patients treated with radiation and factors influencing results. Methods: Retrospective analysis of patients diagnosed with high-grade gliomas between 2009 and 2014 and treated with radiotherapy at the University Hospital of the Faculty of Medicine of Ribeirão Preto, University of São Paulo (HCRP-USP). Results: Median survival was 16.63 months, 29 patients (61.7%) died and the others were in follow-up at the time of analysis. IMRT was used in 68.1% of cases, the mean duration of radiotherapy obtained of 56 days, with a mean interval (delay) between the surgery/diagnosis and radiotherapy was 61.7 days (27-123 days). We found that age, total gross resection, histological type and use of adjuvant temozolomide influenced the survival rate (p <0.05). The estimated overall survival was 18 months (Kaplan Meyer estimator). Our data was similar to those reported in the literature, supporting the institutional treatment protocol.

Das Glioblastom ist mit 70 % aller Gliome der häufigste humane Hirntumor mit sehr ungünstiger Prognose. Es konnte gezeigt werden, dass das natürlich vorkommende Dipeptid Carnosin (β-Alanyl-L-histidin) die Proliferation von Glioblastomzellen inhibiert. Diese Wirkung des Carnosins konnte ebenfalls in vivo nachgewiesen werden. Da Carnosin auch einen Einfluss auf den ATP-Haushalt der Glioblastomzellen besitzt, war das Ziel dieser Arbeit einen Wirkungsort von Carnosin zu identifizieren, womit die ATP mindernden und proliferationshemmenden Eigenschaften erklärt werden können. Es wurde untersucht, ob Carnosin den Energiemetabolismus der Glioblastome beeinflusst. Dabei konnte mithilfe zellbiochemischer Methoden gezeigt werden, dass die untersuchten Zelllinien nicht von der Energieversorgung durch die mitochondriale oxidative Phosphorylierung abhängen, da sich weder Hemmung (KCN) noch Entkopplung (DNP) der Elektronentransportkette auf den zellulären ATP-Gehalt auswirkten. Carnosin hingegen verringerte den ATP-Spiegel dieser Zellen. Die Hemmung der Glykolyse durch Oxamat (LDH-Hemmung), bewirkte einen starken Abfall des intrazellulären ATP-Spiegels, worauf Carnosin keinen zusätzlichen Effekt mehr besaß. Carnosin konnte eine Wirkung auf die glykolytische ATP-Synthese zugesprochen werden. Da ein direkter, molekularer Wirkungsort auf diesem Weg nicht identifiziert werden konnte, wurde parallel untersucht, ob sich über Proteomanalysen der Glioblastomzelllinie T98G ein Wirkungsort, bzw. -mechanismus bestimmen lässt. Anhand der Methode der zweidimensionalen Gelelektrophorese (2D-GE) konnten 31 signifikant differenziell exprimierte Proteine detektiert werden, von denen 6 Proteine (VBP-1, OLA-1, TALDO 1, UROD, BAG-2, GRPEL1) über MALDI-TOF-Analysen identifiziert wurden. In Western-Blot-Analysen konnte ein Protein (VBP-1), neben T98G, auch in primären Glioblastomzelllinien als differenziell exprimiert nachgewiesen werden. Anhand der zellbiologischen und proteinbiochemischen Untersuchungen konnte einerseits eine Verbindung des Carnosins zum HIF1α-Signalweg und andererseits zur generellen posttranslationalen Peptidprozessierung hergestellt werden. Der direkte Nachweis eines Einflusses von Carnosin auf HIF1α wurde aber bisher nicht erbracht.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-09-04T17:32:14Z No. of bitstreams: 1 Vanessa Investigacao do efeito cititóxico....pdf: 1637760 bytes, checksum: 53a0a5f0c495bc04a29d54e8f4415870 (MD5) / Made available in DSpace on 2012-09-04T17:32:14Z (GMT). No. of bitstreams: 1 Vanessa Investigacao do efeito cititóxico....pdf: 1637760 bytes, checksum: 53a0a5f0c495bc04a29d54e8f4415870 (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / Investigar a capacidade antitumoral do extrato metanólico de Bixa orellana em células neoplásicas de Glioblastoma multiforme (GL-15) e Glioma murino (C6), sem toxicidadade para as células astrocitárias normais in vitro. Métodos e resultados: Caracterização do extrato por espectrofotometria por absorção de luz visível apresentou picos em 286, 363 e 435 nm. Determinou-se os teores de bixina e compostos fenólicos – 0,17 mg/mg e 0,05 mg por equivalência de pirogalol/mg de extrato seco, respectivamente. A citoxicidade foi investigada pelo teste de Brometo de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolium. A Mínima Concentração Citotóxica (MCC) para GL-15 foi 180 e 80 μg/mL para C6, para astrócitos foi 240 μg/mL, após 48 horas de tratamento. O teste de exclusão com azul tripan confirmou a EC50 para GL-15 após 24 horas. A análise morfológica foi realizada por microscopia de contrataste de fase e fluorescência. Comprovou-se a diminuição de células neoplásicas e alterações celulares na MCC em astrócitos. A capacidade de fluorescência foi comprovada em GL-15. A citotoxicidade não depletou GSH. Investigou-se alterações de ciclo celular e morte celular por citometria de fluxo. Alterações de ciclo celular não foram evidenciadas. O tipo de morte celular foi investigado com marcação para anexina V e Iodeto de Propídio comprovou morte por necrose em GL-15 e por apoptose tardia em C6, os astrócitos apresentaram valores pequenos de morte por apoptose tardia e necrose. Conclusão: Os dados indicam um potencial antitumoral das substâncias presentes neste extrato para células neoplásicas sem ser tóxico para células normais. / This work investigated the hypothesis that the methanol extract of Bixa orellana decreases the viability of GL-15 and C6 cells, without being toxic to normal astrocytes in vitro. Methods: The methanol extract of B. orellana seeds was obtained and characterized by UV-Vis spectrophotometry. The cytotoxic effects were assayed in vitro using the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide method. The cell morphology was investigated by phase contrast and fluorescence microscopy. The depletion of reduced glutathione (GSH) was observed by fluorescence microscopy. The effects on cell cycle and the mode of cell death was studied by flow cytometry. Results: The contents of bixin and phenol were 0.17 mg/mg of extract and 0.05 mg of pyrogallol equivalent/mg of extract, respectively. Three peaks were observed at 286, 363, and 435 nm. The extract killed cells in a dose-dependent manner. The minimum cytotoxic concentrations in the two tumoral cells (GL-15 and C6) were respectively: 180 μg/mL and 80 μg/mL, meanwhile in astrocytes it was 240 μg/mL after 48 hours. The trypan blue assay confirmed the cytotoxic effect to GL-15 cells. Morphological degeneration of treated glioma cells was observed. The same was observed with astrocytes, but at a higher concentration. Treated cells became fluorescent, probably due to incorporation of bixin. The treatment neither depleted GSH, nor interfered on the cell cycle. The main kind of cell death was necrosis. Conclusions: Compounds present in B. orellana seeds are potentially cytotoxic to glioma cells, meanwhile primary astrocytes are more resistant.

Orientador: Sara Teresinha Olalla Saad / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-13T00:12:07Z (GMT). No. of bitstreams: 1 Bigarella_CarolinaLouzao_D.pdf: 7284027 bytes, checksum: e4e836fe8140fc48012754178445127b (MD5) Previous issue date: 2009 / Resumo: ARHGAP21 é uma proteína pertencente à família RhoGAP e apresenta atividade GAP sobre Cdc42 e RhoA, interage com ARF-GTPases e com a-catenina, modulando a dinâmica da actina associada às membranas do Golgi e a integridade das junções aderentes. Devido ao relato da elevada expressão de ARHGAP21 em tecido nervoso surgiu o objetivo da presente tese que foi avaliar a expressão e a função de ARHGAP21 no tecido neural normal e neoplásico. Nossos resultados mostraram que ARHGAP21 localiza-se no núcleo e na região peri-nuclear de vários tipos celulares, e nos prolongamentos celulares de neurônios primários, e interage com FAK na região peri-nuclear. Os resultados da depleção de ARHGAP21 por moléculas de shRNAi indicaram que ARHGAP21 inibe a migração de células derivadas de glioblastoma multiforme através do controle negativo de ARHGAP21 sobre Cdc42, pela inativação da sinalização FAK?p130CAS, e pelo controle da secreção de metaloprotease-2. Além disso, a expressão da proteína ARHGAP21 correlaciona-se com o grau tumoral de astrocitomas in vivo, indicando a existência de um possível controle negativo de ARHGAP21 sobre a transformação ainda maior destas células, já que sua depleção in vitro resultou em aumento do grau tumoral. Em tecido cerebral normal, evidenciamos elevada expressão de ARHGAP21 em córtex e cerebelo humano. Além disso, o gene ARHGAP21 murino mostrou-se altamente expresso em E17, dia em que termina a migração de precursores neurais e que coincide com a queda de expressão de Mmp-2. Portanto, ARHGAP21 demonstrou, em sistema nervoso normal, controle semelhante sobre a expressão do gene Mmp-2 ao observado em linhagens de glioblastoma. Nossos resultados sugerem que ARHGAP21 pode ser uma poderosa reguladora da migração celular em diferentes tecidos e, assim, ter papel crucial no controle da progressão de diferentes tipos tumorais. / Abstract: : ARHGAP21 is a RhoGAP protein with GAP activity over Cdc42 and RhoA, it also interacts with ARF-GTPases and with a-catenin, controlling actin dynamics on Golgi membranes and the integrity of adherens junctions, respectively. Due to ARHGAP21 high expression levels in nervous tissues has emerged the objective of the present thesis that was evaluate the expression levels and the function of ARHGAP21 in the normal and neoplasic nervous system. Our results evidenced that ARHGAP21 localizes to the nucleus and perinuclear region of several cell types, and on cell protrusions in primary mouse neurons, and interacts with FAK in the perinuclear region. Results of ARHGAP21 depletion by shRNAi molecules evidenced that ARHGAP21 inhibits glioblastoma cell migration through the negative control of Cdc42, the inhibition of FAK?p130CAS signaling, and through the control of metaloprotease 2 secretion. Besides that, ARHGAP21 expression correlates to tumor grade on astrocytomas samples, indicating the existence of a negative control of ARHGAP21 on astrocytoma cellular transformation, since its depletion in vitro resulted in higher malignity of T98G glioblastoma cell line. In normal cerebral tissue, ARHGAP21 murine gene is highly expressed on E17 animals, day in which neuronal precursor's migration finishes and when there is a reduction in mmp-2 expression. Therefore, ARHGAP21 showed in normal nervous system, similar control of mmp-2 gene expression as observed in glioblastoma cell lines. Our results suggest that ARHGAP21 might be a master regulator of cellular migration in different tissues and, like this, it may has a crucial role in the control of tumor progression. / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Fisiopatologia Medica

Yes / Glioblastoma (GBM) has a dismal prognosis. Evidence from preclinical tumor models and human trials indicates the role of GBM initiating cells (GIC) in GBM drug resistance. Here, we propose a new treatment option with tumor enzyme-activatable, combined therapeutic and diagnostic (theranostic) nanoparticles, which caused specific toxicity against GBM tumor cells and GICs. The theranostic cross-linked iron oxide nanoparticles (CLIO) were conjugated to a highly potent vascular disrupting agent (ICT) and secured with a matrix-metalloproteinase (MMP-14) cleavable peptide. Treatment with CLIO-ICT disrupted tumor vasculature of MMP-14 expressing GBM, induced GIC apoptosis and significantly impaired tumor growth. In addition, the iron core of CLIO-ICT enabled in vivo drug tracking with MR imaging. Treatment with CLIO-ICT plus temozolomide achieved tumor remission and significantly increased survival of human GBM bearing mice by more than 2 fold compared to treatment with temozolomide alone. Thus, we present a novel therapeutic strategy with significant impact on survival and great potential for clinical translation. / Heike E Daldrup-Link, NIH, R21CA176519 and R21CA190196; Sanjiv Sam Gambhir, NIH, 1U54CA199075; Jessica Klockow, NCI training grant, T32CA118681, Robert A. Falconer, University of Bradford, UoB-66031

Yes / Despite considerable progress with our understanding of glioblastoma multiforme (GBM) and the precise delivery of radiotherapy, the prognosis for GBM patients is still unfavorable with tumor recurrence due to radioresistance being a major concern. We recently developed a cross-linked iron oxide nanoparticle conjugated to azademethylcolchicine (CLIO-ICT) to target and eradicate a subpopulation of quiescent cells, glioblastoma initiating cells (GICs), which could be a reason for radioresistance and tumor relapse. The purpose of our study was to investigate if CLIO-ICT has an additive therapeutic effect to enhance the response of GBMs to ionizing radiation. Methods: NSG™ mice bearing human GBMs and C57BL/6J mice bearing murine GBMs received CLIO-ICT, radiation, or combination treatment. The mice underwent pre- and post-treatment magnetic resonance imaging (MRI) scans, bioluminescence imaging (BLI), and histological analysis. Tumor nanoparticle enhancement, tumor flux, microvessel density, GIC, and apoptosis markers were compared between different groups using a one-way ANOVA and two-tailed Mann-Whitney test. Additional NSG™ mice underwent survival analyses with Kaplan–Meier curves and a log rank (Mantel–Cox) test. Results: At 2 weeks post-treatment, BLI and MRI scans revealed significant reduction in tumor size for CLIO-ICT plus radiation treated tumors compared to monotherapy or vehicle-treated tumors. Combining CLIO-ICT with radiation therapy significantly decreased microvessel density, decreased GICs, increased caspase-3 expression, and prolonged the survival of GBM-bearing mice. CLIO-ICT delivery to GBM could be monitored with MRI. and was not significantly different before and after radiation. There was no significant caspase-3 expression in normal brain at therapeutic doses of CLIO-ICT administered. Conclusion: Our data shows additive anti-tumor effects of CLIO-ICT nanoparticles in combination with radiotherapy. The combination therapy proposed here could potentially be a clinically translatable strategy for treating GBMs.

Glioblastoma is the most malignant primary brain tumor with the average patients surviving only one year after diagnosis, even with aggressive therapy. The formation of numerous micro-tumors dispersed into the brain due to rapid invasion of tumor cells, presents the primary challenge to the surgical removal of tumors and limits the effectiveness of current treatments. This dissertation presents studies aimed at understanding the molecular mechanisms regulating invasion of human glioblastoma cells. Transplantation of human glioblastoma cells in the zebrafish brain showed that the knockdown of calpain 2, a calcium-activated protease, resulted in a three fold decrease in the tumor cell invasion. The result was further verified in the organotypic mouse brain slices where the knockdown cells demonstrated 2-fold decrease in the area of dispersal compared to control cells. Our data show that calpain 2 plays a role in the process of tumor cell angiogenesis. Glioblastoma cells were transplanted into the brain of zebrafish expressing GFP in the blood vessels and we observed that 23% of animals injected with control tumor cells demonstrated angiogenesis. In contrast, only 9% of fish that received calpain 2 knockdown cells showed the formation of new vessels. Consistent to the reports from human glioblastoma patients and rodent models, we did not observe metastasis of transplanted cells outside of the brain in the zebrafish, supporting for the use of zebrafish as an important model for glioblastoma cell invasion studies. These results provide evidence that calpain 2 protease activity is required for the dispersal of glioblastoma cells in the brain microenvironment. To determine the mechanism of calpain 2 regulation of tumor cell invasion, proteolysis of filamin by calpain 2 was studied. Filamin is an important actin cross-linking protein which develops orthogonal actin networks in the periphery of the cell. In this study, we show that the expression of filamin inhibits glioblastoma cell invasion. Hence, knocking down filamin expression by 80% resulted in 220% increase in the invasion of glioblastoma cells through Matrigel extracellular matrix. The regulated proteolysis of filamin is a potential mechanism to facilitate the cyclic turnover of actin orthogonal networks which is required for glioblastoma cell invasion. In this study, we identified a novel mechanism that the PI3 kinase activity regulates the cleavage of filamin by calpain 2 in glioblastoma cells. Binding of a membrane phospholipid phosphatidylinositol (3,4,5) triphosphate [PtdIns (3,4,5)-P₃] to filamin induces its proteolysis by calpain 2 after the amino acid lysine 268, removing the actin binding domain which in-turn abolishes the actin binding ability of filamin. / Graduation date: 2011 / Access restricted to the OSU Community at author's request from Jan. 31, 2011 - Jan. 31, 2012

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