O Glioblastoma Multiforme (GBM), tumor maligno do sistema nervoso central, é a forma mais agressiva e maligna entre os astrocitomas. Com tratamento padrão, o tempo médio de sobrevivência é de 10-12 meses. Uma vez que a família do paciente e os cuidadores familiares adoecem juntos, a ajuda psicológica às famílias é considerada essencial. O presente trabalho teve como objetivo estudar a qualidade de vida de cuidadores não-profissionais de pacientes portadores de GBM por meio de um programa psicoeducativo, visando promover condições para o uso produtivo de orientação e informação. A pesquisa envolveu um Grupo Experimental (GE) composto por vinte participantes e um Grupo Controle (GC) composto por dez participantes. Utilizou-se uma Entrevista Psicológica Semi-Dirigida e o instrumento de avaliação de qualidade de vida WHOQOL-bref. O instrumento é composto por duas questões gerais e 24 questões divididas em quatro domínios: físico, psicológico, relações sociais e meio ambiente. Posteriormente os participantes do GE foram incluídos em um programa psicoeducativo, individualmente, composto por quatro sessões temáticas com 45 minutos de duração cada realizadas uma vez ao mês. Os instrumentos foram reaplicados, ao final, para comparar os resultados da qualidade de vida, após a intervenção psicológica. O GC não participou do Programa Psicoeducativo, somente da aplicação dos instrumentos. O discurso do familiar é pautado pelo sentido de que a vida sofreu intensas mudanças. Os pacientes com GBM adquirem um alto grau de dependência dos cuidadores, que demonstram muitas vezes atitude de superproteção que pode resultar em infantilização do paciente. Em relação ao impacto psicossocial, ficou evidente a sobrecarga vivenciada, embora a maioria relate que se sente bem exercendo essa tarefa. Após a participação no Programa Psicoeducativo, todos os participantes apresentaram melhora na qualidade de vida, principalmente no domínio psicológico. Todos os domínios obtiveram diferenças estatisticamente significativas. Tiveram suas dúvidas esclarecidas quanto à doença, tratamento, bem como se sentiram acolhidos e relataram que, após a participação no programa, conseguiram encontrar uma forma de estabelecer comunicação com o paciente. Já no GC, houve uma piora da qualidade de vida em todos os Domínios. O atendimento psicoeducativo se mostrou benéfico, de forma a orientar e informar, além de minimizar o estresse desencadeado pela doença e permitir uma melhora no bem-estar e na qualidade de vida do cuidador familiar. Este vivencia o sentimento de perda iminente, desgaste físico e emocional, e muitas vezes, acaba por esquecer e ignorar seus próprios problemas partilhando os mesmos medos e angústias que o ente querido / Glioblastoma Multiforme (GBM), a malignant tumor of the central nervous system, is the most aggressive and malignant among astrocytomas. Median survival time with standard treatment is 10-12 months. Patient and family are involved together in the illness. Therefore, psychological support to family members is critical. The objective of this study was to investigate the quality of life of non-professional caregivers of patients with GBM by means of a psychoeducational program oriented to provide guidance and information. This research study involved an Experimental Group (EG) of twenty participants and a Control Group (CG) made up of ten participants. A semi-structured psychological interview and the WHOQOL-bref assessment tool were used. The WHOQOL-bref is comprised of two general questions and the remaining questions are distributed in sections that evaluate four domains: physical, psychological, social and environmental. The participants in the EG were then included in an individual psychoeducational program consisting of four monthly thematic sessions of 45 minutes each. At the end of the study, the tools were used again in order to compare quality of life before and after the psychological intervention. The CG was submitted only to the tools, not to the Psychoeducational Program. The basis of the family discourse is the dramatic change in their lives. Patients with GBM become highly dependent on their caregivers, who often show an attitude of overprotection that leads to childish behavior of the patient. In terms of psychosocial impact, caregivers consider these tasks very rewarding, despite the clear burden experienced. After attending the Psychoeducational Program, all participants managed to improve their quality of life, particularly in the psychological domain. All domains showed significant statistical differences. The participants obtained answers to their questions about the disease and the treatment. They felt reassured after the program and managed to find a way to connect with the patient. As to the CG, the quality of life got worse in all domains. The psychoeducational program proved to be beneficial, providing guidance and information, minimizing the stress triggered by the disease, and improving the wellness and quality of life of the family caregiver. These caregivers live with the sense of imminent loss, physical and emotional stress, and often end up by forgetting and ignoring their own problems, sharing the same fears and distress of their beloved ones

O glioblastoma multiforme (GBM) é um dos tumores mais letais e a radioterapia permanece como um dos principais tratamentos. Novas estratégias são necessárias para coibir a resistência ao tratamento, como o silenciamento de fatores de transcrição (FTs). Nossa hipótese é a de que FTs associados a listas de genes diferencialmente expressos, os quais foram selecionados para linhagens de GBM irradiadas, ou comparando amostras de GBM à amostras de tecido cerebral, possam fornecer alvos moleculares que aumentariam a morte das células tumorais, quando silenciados. Foram analisadas a proliferação, morte e ciclo celular, além da formação e diferenciação de neuroesferas, utilizando, em quase todas as etapas, a citometria de fluxo. Os FTs HEB e E2F1, cujas funções principais estão relacionadas à neurogênese e proliferação celular, foram selecionados a partir das análises in silico de GBM irradiados ou não, ou de GBMs comparados a amostras de cérebro normal, respectivamente. Esses FTs encontram-se expressos em linhagens U87, astrócitos primários e neuroesferas provenientes das mesmas, analisadas por Western blot. O silenciamento de HEB e E2F1 na linhagem U87, de forma geral, reduziu a proliferação, induziu morte celular e diminuiu a porcentagem de células em G0/ G1, em pelo menos um dos tempos analisados (24, 48 e 72h) em relação ao grupo transfectado com a sequência scrambled. O silenciamento de HEB e E2F1 reduziu o número de neuroesferas quando comparadas às células transfectadas com a sequência scrambled. Possivelmente, a capacidade anti-proliferativa do silenciamento dos FTs HEB e E2F1 observada no cultivo em monocamada da U87, possam atuar na capacidade de formação de neuroesferas e, consequentemente, podem ter um papel na manutenção das células tronco do GBM. O silenciamento não alterou a radiorresistência da U87 cultivada em monocamada, com exceção dos efeitos do silenciamento de E2F1 em 24 h, em que houve radioproteção. A irradiação não reduziu o número de neuroesferas silenciadas para HEB em comparação ao grupo não irradiado, mas reduziu o número de células presentes nas neuroesferas, indicando uma possível atuação de HEB na resposta à irradiação em neuroesferas, fato este nunca antes descrito. O silenciamento de E2F1 não interferiu na resposta das neuroesferas à radiação. A expressão de CD133 avaliada oito dias após a dissociação das células silenciadas para E2F1 e HEB, cultivadas em meio de diferenciação, foram superiores ao do grupo scrambled, indicando uma possível diminuição na diferenciação celular. O silenciamento dos dois FTs não atuou na seleção positiva de CD133+ após a irradiação, como observado no grupo das neuroesferas transfectadas com a sequência scrambled e irradiadas, comparado às não irradiadas. Assim, E2F1 e HEB mostraram-se alvos interessantes no sentido de reduzir a proliferação, tanto em células U87 cultivadas em monocamada quanto em neuroesferas. / Glioblastoma multiforme (GBM) is one of the most lethal tumors, and radiation therapy remains one of the main treatments. New strategies are needed to suppress typical GBM treatment resistance and transcription factors (TFs) silencing seems to be a promising strategy. Our hypothesis is that TFs associated with lists of differentially expressed genes which were selected for irradiated compared to shamirradiated GBM cell lines, or GBM samples compared to brain tissue samples, could provide molecular targets that are supposed to increase tumor cell death when they are silenced. We analyzed proliferation, cell death and cell cycle progression, besides the formation and differentiation of neurospheres, using several analyses by flow cytometry. The TFs HEB and E2F1, whose primary functions are related to neurogenesis and cell proliferation, were selected from in silico analysis of GBM irradiated or sham-irradiated GBMs and GBM samples compared with normal brain samples, respectively. These TFs were found expressed in U87 GBM cell line, and primary astrocytes, as well as in neurospheres derivated from both, as analyzed by Western blot. Silencing of E2F1 and HEB in U87 cells, reduced proliferation, induced cell death and decreased the percentage of cells at G0/G1 (24, 48 or 72h) compared to the scrambled sequence transfected group. HEB and E2F1 silencing reduced the number of neurospheres when compared to cells transfected with scrambled sequence. Possibly, the anti-proliferative ability of silencing of HEB and E2F1 TFs observed in monolayer culture of U87, may act in neurospheres forming capacity and therefore may play a role in the maintenance of GBM stem cells. In our experiments, gene silencing did not alter the radio-resistance of U87 grown in monolayer. Irradiation did not reduce the number of neurospheres silenced for HEB compared to non-irradiated group, but reduced the number of cells present in neurospheres, indicating a possible role of HEB in response to ionizing irradiation in neurospheres, a fact that was not described yet. The silencing of E2F1 in neurospheres did not affect the response to irradiation. The expression of CD133, as assessed at eight days after the dissociation of cells silenced for E2F1 and HEB (cultured in differentiation culture media), was superior compared with the scrambled group, indicating a possible decrease in cell differentiation. The silencing of both TFs did not influence the positive selection of CD133 after irradiation, as observed in the group of neurospheres transfected with scrambled sequence, and irradiated compared to nonirradiated. Thus, E2F1 and HEB proved to be interesting targets for decreasing proliferation in both U87 cells grown as monolayer or neurospheres.

Glioblastoma (GBM) is the most common and malignant of the glial tumors. These tumors may develop from lower-grade astrocytomas (diffuse astrocytoma; grade II or anaplastic astrocytoma; grade III) through a progressive pathway, but, more frequently, they manifest de novo without any evidence of a pre-malignant lesion. The treatment of GBM includes surgery, radiotherapy, and chemotherapy with temozolomide. Despite improvements in treatment protocols, the median survival of GBM patients remains very low at 12-15 months. The cause of glioma (either development or progression) can be genetic and epigenetic modification driven changes. In contrast to genetic modifications, where DNA sequence is changed, epigenetic modifications are those gene expression regulatory mechanisms which do not involve the change in the DNA sequence. It includes DNA methylation, chromatin modifications and miRNA mediated changes in gene expression. Aberrant DNA methylation is one of the common molecular lesions occurring in the cancer cell. The 5th position of cytosine (CpG) is the most preferred site of DNA methylation in mammalian cells. The methylated cytosines are prone to undergo oxidative deamination, and get mutated to thymine in DNA. Consequently, this led to decrease in CpG abundance in the genome. In normal conditions, promoters of majority of the genes escape methylation, because of which CpG of these regions remain same. This phenomenon led to the restriction of CpGs in the promoter regions of most of the genes. These CpG rich regions of the promoters are known as CpG islands, and the methylation status of these islands have a major role in regulating gene expression. The cancer genome is shown to undergo genome-wide hypomethylation whereas CpG islands undergo hypermethylation compared to normal tissue, resulting in net loss of total methylation, as the CpGs from non-island areas far exceed in number compared to the CpGs from islands. The most studied change of DNA methylation in neoplasms is the silencing of the tumor suppressor genes by CpG island promoter hypermethylation. Apart from few studies, the role of DNA methylation in glioma development and progression is poorly known. With this background, we have focused our study on DNA methylation changes in GBM. To identify GBM specific DNA methylation alterations, we have performed the genome wide methylation profile of 44 GBM and 8 normal samples using Infinium methylation array. Beta value, which is a measure of methylation, was calculated for all the CpG probes. Beta value ranges between 0-1 (from no methylation to complete methylation). We sought to understand the clinical importance, with particular importance to patient survival, of the DNA methylation pattern observed. We also undertook steps to understand the contribution of the differential DNA methylation and the associated gene expression changes in GBM development. This work has been divided into three parts: Part I –Identification of GBM specific methylome and development of a DNA methylation prognostic signature for GBM To identify the differentially methylated genes in GBM, we compared the methylation levels of 27,578 CpGs between GBM and normal control samples using statistical methods. We then compared the list of differentially methylated genes with the expression data generated by The Caner Genome Atlas (TCGA) to find out genes whose expression oppositely correlates with the DNA methylation status. This resulted in the identification of 62 genes hypermethylated and down regulated, while 65 genes hypomethylated and up regulated. We believe that this set of differentially methylated genes may play important role in glioma development. Next, to identify GBM specific DNA methylation survival signature, we correlated the survival data of 44 GBM patients with beta values of all the 27,578 probes. Using Cox regression method, we identified a set of 9 genes, whose methylation predicted the survival in GBM patients. A risk score was then calculated using methylation values and regression co-efficient of each of the genes. The methylation risk score was found to be an independent predictor of survival in a multivariate analysis in TCGA data set and the Bent et al data set (independent validation sets). Using methylation risk score, we were able to divide the patients into low and high risk groups with significant difference in survival. To discover the biology behind the difference in the survival of low and high risk groups, we performed network analysis, using differentially expressed genes between low and high risk patients, which revealed an activated NFkB pathway association with poor prognosis. The inhibition of NFkB pathway sensitized the glioma cells for chemotherapeutic drugs only in NFkB activated cell lines, suggesting a pivotal role for NFkB pathway imparting chemoresistance in poor surviving group. Part II -NPTX2, a methylation silenced gene, inhibits NFkB through a p53-PTEN-PI3K-AKT signaling pathway To understand the mechanism behind the prediction of survival by methylation of 9 genes, we took NPTX2 as a candidate gene for further investigation. NPTX2, a risky methylated gene, is highly methylated in high risk group with poor survival, which suggests that it may have a growth inhibitory activity in GBM. Bisulphite sequencing confirmed the hypermethylation status of NPTX2 promoter in GBM samples and glioma cell lines compared to normal brain tissue. As expected, NPTX2 transcript level was significantly down regulated in GBMs and glioma cell lines compared to normal samples, and could be re-expressed upon methylation inhibitor treatment in glioma cells. Exogenous over expression of NPTX2 inhibited proliferation, colony formation and sensitized glioma cells to chemotherapeutic drugs. Moreover, NPTX2 also inhibited soft agar colony formation in vitro, which confirms its growth inhibitory function in GBM. As NPTX2 was methylated and silenced in the high risk group, which has high activation of NFkB pathway, we then checked if NPTX2 could inhibit NFkB activity. Indeed, we observed that NPTX2 overexpression inhibited expression from NFkB dependent luciferase reporter, sequence-specific DNA-binding of NFkB, nuclear translocation of NFkB sub unit (p65) and it also significantly repressed key NFkB target genes. We also show that NPTX2 mediated inhibition of NFkB could be abrogated by co-expression of constitutively active forms of PI3 kinase, AKT and IKKα, suggesting an involvement of PI3K-AKT-IKKα axis in NPTX2 mediated NFkB inhibition. Further, we found that NPTX2 repressed NFkB activity by inhibiting AKT through an ATM-p53-PTEN-PI3K dependent pathway. Thus, these results explain the need for hypermethylation and down regulation of NPTX2 in high risk GBM wherein the NFkB pathway is activated. Part III -Methylation silencing of ULK2, an autophagy gene, is important for astrocyte transformation and cell growth Among the differentially methylated genes (see part I), ULK2 was one of the most hypermethylated and down regulated genes. ULK2 is a known initiator protein in autophagy pathway, which is a type II cell death mechanism. There are many contradictory reports with respect to the role of autophagy in GBM development. For example, it has been shown that autophagy has a tumor suppressor activity and is essential for temozolomide mediated cell toxicity in GBM cells, whereas others studies implicate its involvement in tumor growth and progression. Hence, we carried out experiments to understand the role of ULK2 in GBM development. Using bisulphite sequencing, we validated ULK2 promoter hypermethylation status in GBM and glioma cell lines. In good correlation, ULK2 was found to be down regulated in GBMs and glioma cell lines, which was reexpressed by methylase inhibitor treatment in glioma cell lines. The over expression of ULK2 was found to inhibit colony formation, proliferation and soft agar colony formation of glioma cells. As expected, ULK2 overexpressing cells showed higher autophagy, compared to control cells. Interestingly, we also found increased apoptosis in ULK2 overexpressing cells. The cell death caused by ULK2 overexpression was compromised when cells were treated with 3-MA (an autophagy inhibitor) or Z-VAD-FMK (a pan caspase inhibitor). However, ULK2 failed to inhibit cell growth in autophagy deficient cells (ATG5-/-), thereby suggesting the importance of autophagy in ULK2 induced cell death. Further, ULK2 overexpression, increased catalase degradation and Reactive Oxygen Species (ROS) generation, which suggests that increase in ROS may play a role in ULK2 dependent cell death. In good correlation, N-Acetyl Cysteine, a ROS inhibitor, treatment rescued the cells from ULK2 mediated cell death, confirming the role of ROS in ULK2 induced cell death. Kinase deficient ULK2 overexpression failed to induce cell growth inhibition, autophagy and apoptosis, suggesting kinase activity of ULK2 is important for ULK2 function. Co-transfection of ULK2 inhibited Ras mediated transformation of immortalized normal human astrocytes. Taken together, we have identified and validated ULK2 as one of the DNA methylation silenced genes in GBM. ULK2 was found to be growth inhibitory in GBM cells by increasing autophagy dependent apoptosis. ULK2 inhibited Ras mediated transformation, suggesting essentiality of DNA methylation mediated ULK2 down regulation in GBM. In conclusion, the present work sheds light on the importance of methylation of genes in GBM progression. As observed, two of the genes, NPTX2 and ULK2 play as critical growth inhibitors in GBM. Also, we have identified a robust, independent and a highly sensitive 9 gene methylation signature, for GBM patient’s survival prediction.

Une nouvelle stratégie oncologique, basée sur l’intégration de la radiothérapie nanovectorisée et l’administration loco-régionale, a été évaluée pour le traitement et l’imagerie du glioblastome, le type le plus commun des tumeurs cérébrales primaires. Les dendrimères Gallic Acid-Triethylène Glycol (GATG) sont des nanovecteurs de choix pour délivrer simultanément l’agent thérapeutique (le radioisotope 188Re par son rayonnement béta a été retenu) et l’agent diagnostique (le gadolinium est un agent paramagnétique utilisé en Imagerie par Résonance Magnétique (IRM)). Leur évaluation a été réalisée par administration locorégionale par stéréotaxie sur un modèle de rat F 98. Les données pharmaco-cinétiques ont été également obtenues après injection intraveineuse permettant d’apprécier les propriétés des différents dendrimères synthétisés. Leur apport en terme de confinement au site d’injection représente un avantage majeur de ce nouveau type de radiopharmaceutiques. / A new oncologic strategy, based on the integration of nanovectorized radiotherapy and locoregional delivery, was evaluated for the treatment and imaging of glioblastomas, the most common and lethal type of primary brain tumors. Gallic acidtriethylene glycol (GATG) dendrimers were the nanovectors of choice to deliver the radiotherapeutic 188Re and paramagnetic nuclei Gd3+, with a minimally invasive stereotactic injection, directly depositing the radiotherapeutic dose to the tumor site in a F98 rat glioma model. Intravenous injection was used to further investigate the pharmacokinetics, throughout body distribution and clearance profiles of these dendrimers. Molecular weight and architecture had an important role on the in vivo behavior of the dendrimers. Their use as nanovectors prevented the fast brain clearance of the radionuclide alone, and prolonged the confinement of the internal radiation at the tumor site.

Orientador: Helder Tedeschi / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T19:44:14Z (GMT). No. of bitstreams: 1 Valadares_MarceloGomesCordeiro_M.pdf: 1238842 bytes, checksum: f4f19f2521d318218d181dd5e357d746 (MD5) Previous issue date: 2015 / Resumo: Introdução: O tratamento para gliomas de alto grau consiste em cirurgia seguida de radioterapia e quimioterapia. Mesmo com o melhor tratamento disponível a sobrevida em geral não atinge 2 anos para glioblastoma multiforme e 2 a 5 anos para outros gliomas anaplásicos. Estudos realizados em países em desenvolvimento sugerem que as limitações financeiras dos sistemas públicos de saúde destes países podem dificultar o acesso da população aos recursos necessários, podendo inclusive influenciar negativamente a sobrevida. Neste trabalho analisamos uma série de pacientes portadores de gliomas de alto grau tratados em um hospital público de uma grande região metropolitana brasileira. Comparamos o tratamento oferecido com o preconizado na literatura e verificamos se eventuais limitações nas diversas etapas do tratamento multidisciplinar afetam a qualidade do serviço prestado. Método: Analisamos retrospectivamente prontuários de pacientes portadores de gliomas de alto grau tratados no Hospital de Clínicas da Unicamp entre 2007 e 2013. Resultados: Foram incluídos 135 pacientes. A idade média foi de 57 anos e 2 meses e haviam 2 homens para cada mulher. A unidade de emergência foi a porta de entrada para 89% dos pacientes. No momento do diagnóstico 90.3% já apresentavam sintomas há mais de 2 semanas e 14,8% estavam sintomáticos há mais de 12 semanas. Quimioterapia e radioterapia foram prescritas para 56% dos pacientes. Radioterapia apenas foi o tratamento de escolha para 25% e outros 19% foram encaminhados para cuidados paliativos. Tromboembolismo venoso foi diagnosticado em 26%, infecções relacionadas ao procedimento cirúrgico em 16%. Após a cirurgia a mediana de tempo para início dos tratamentos subsequentes foi: 6.71 semanas para a primeira consulta com o radioterapêuta, 6.28 semanas para a primeira consulta com o oncologista e 12.57 semanas para iniciar a quimioterapia e radioterapia. Progressão tumoral com necessidade de nova abordagem cirúrgica foi observada em 27 pacientes no grupo de terapia tardia (>6 semanas) e em nenhum paciente no grupo de terapia precoce (?6 semanas) (p=0.002). Não foi observada correlação estatística entre a ocorrência de eventos tromboembólicos (p=0.09) ou infecciosos (p=0.057) e a demora para o início do tratamento adjuvante. A mediana de sobrevida de 100 pacientes portadores de glioblastoma multiforme submetidos a todas as formas de tratamento foi de 7.8 meses. Conclusão: Embora o tratamento cirúrgico seguido de radioterapia e quimioterapia esteja disponível no Hospital de Clínicas da Unicamp, apenas 56% dos pacientes são submetidos a todo o tratamento preconizado e apenas 12% iniciam a quimioterapia e radioterapia em menos de 6 semanas após a cirurgia. Estes números são inferiores aos observados em países desenvolvidos, mas compatíveis com outros estudos de países em desenvolvimento. A mesma comparação é feita em relação à sobrevida global dos pacientes diagnosticados com glioblastoma multiforme. Os dados apresentados reforçam a tese de que o tratamento de portadores de gliomas de alto grau é influenciado negativamente pelas limitações presentes na saúde pública de países em desenvolvimento / Abstract: Introduction: The general management of high-grade gliomas is based on surgery plus radiotherapy and chemotherapy. However, in developing countries, the lack of financial resources may delay the treatment, and probably affect patients¿ outcome. Based on this, the aim of this study is to review the clinical data of high-grade glioma patients treated in a brazilian referral public hospital. Methods: We retrospectively analyzed the clinical data with emphasis on the management options (surgery, radiotherapy and chemotherapy) of high grade glioma patients from 2007 to 2014 at the University of Campinas (Unicamp) hospital. Results: A total of 135 patients (27% of all brain tumors) had the diagnosis of high-grade glioma and were included. The mean age of high-grade glioma patients at the time of diagnosis was 57 years and 2 months. There were 90 men (67%) and 44 women (33%). The outpatient referral system was responsible for only 15 (11%) admissions with 120 (89%) patients being admitted through the emergency unit. Headache was the main complaint and 90.3% of the patients had been symptomatic for over 2 weeks and 14,8% for over 12 weeks. After surgery 56% were treated with chemotherapy and radiotherapy. Radiotherapy was the single therapy for 25% of the cases. Another 19% were referred to supportive care. After surgery patients took a median 6.71 weeks to the first radiotherapy consultation, 6.28 weeks to the first clinical oncology consultation and a median 12.57 weeks to start radiation. Only 12% started radiation in less than 6 weeks. Tumor progression requiring a second surgery was observed in 27 patients in the delayed therapy group (> 6weeks) compared with no patients in the early therapy (< 6 weeks) group (P=.002). Glioblastoma Multiforme patients overall survival was 7.8 months. Conclusion: Although surgery, radiation and temozolamide were available at our institution only 56% of the patients received all these treatments and only 12% started radiation in less than 6 weeks. These numbers fail in comparison to many studies published in developed countries but are similar to others published in developing nations. The same applies to the overall survival calculated for glioblastoma patients. This study supports that financial constrictions in public health care in developing countries have a negative impact on high-grade glioma treatment / Mestrado / Neurologia / Mestre em Ciências Médicas

No description available.

To determine the characteristics, treatments and outcomes of patients with glioblastoma multiforme (GBM) or gliosarcoma (GS) and metastases outside of the central nervous system (CNS).

Background: Highly infiltrative growth and resistance to radiation as well as chemotherapy contribute to a poor prognosis of glioblastoma. To improve the survival of patients with glioblastoma, further research to uncover the complex signaling network is essential. Due to the central role of the signaling adaptor lamellipodin in nervous system development and cell migration, a function of lamellipodin in glioblastoma is conceivable. However, the specific function of lamellipodin in the invasion and radioresistance of glioblastoma is so far entirely unknown. Therefore, the present work investigates the invasion and radioresistance of glioblastoma cells and the underlying signaling mechanism under the influence of lamellipodin. Material and Methods: Expression of lamellipodin was evaluated in human astrocytes and nine glioblastoma cell lines by Western blot analysis. Localization of lamellipodin in glioblastoma cells was analyzed by applying immunofluorescence staining. The effects of lamellipodin silencing by siRNA on the glioblastoma characteristics invasion, survival, and residual DNA double strand breaks (DSB) upon X-ray irradiation, proliferation, apoptosis, and senescence were investigated. Alterations in molecular signaling were analyzed by phosphoproteome analysis upon control and lamellipodin depletion combined with/-out X-ray irradiation in A172 cells. Colony formation assays were performed after single and double knockdown of lamellipodin and the affected proteins to connect latter findings to clonogenic survival. Direct lamellipodin binding partners were explored using mass spectrometry analysis. Validation of protein-protein interaction was determined by immunoprecipitation and proximity ligation assay. Clonogenic survival assay was performed after triple knockdown of lamellipodin, previous proteins identified by phosphoproteome, and the direct interaction partner of lamellipodin. Hierarchical analysis was performed by analyzing the expression and phosphorylation of the determined proteins by immunoblot. Results: Lamellipodin was shown to be expressed and phosphorylated in varying amounts in the glioblastoma cell culture panel, with a preferential localization in membrane protrusions and the cytoplasm. The siRNA-specific depletion of lamellipodin tremendously decreased glioblastoma invasion and proliferation while exerting no impact on apoptosis or senescence. Moreover, seven of the nine studied glioblastoma cell lines were radiosensitized by lamellipodin silencing without affecting the number of residual DNA double strand breaks, while its overexpression improved radiation survival. Mechanistically, the loss of lamellipodin impaired the phosphorylation of nine proteins (EIF2A, EGFR, FOS, MKK6, NFKBIA, PRKAA2, RSK2, SRC, and TAK1), which are mostly implicated in the EGFR-MAPK signaling. The combinational silencing of lamellipodin and the relevant proteins achieved overall radiosensitization in A172 and U343MG cells. Furthermore, mass spectrometric analysis of lamellipodin immunoprecipitates demonstrated that the lamellipodin interactome alters in response to X ray irradiation conditions. RICTOR was confirmed as a direct linker of lamellipodin to the EGFR-MAPK signaling by immunoprecipitation and proximity ligation assay. In addition, triple depletion of lamellipodin, RICTOR, and EGFR resulted in similar degrees of radiosensitization as reported for lamellipodin knockdown, highlighting their superimposable role in glioblastoma radiation response. In line, lamellipodin silencing increased EGFR expression and phosphorylation, while lamellipodin phosphorylation was decreased upon EGFR deficiency. Conclusion: The results uncover the crucial function of lamellipodin for invasion, proliferation, and radiosensitivity of glioblastoma cells. Based on molecular analyses, lamellipodin was discovered as a determinant of EGFR signaling by interacting with RICTOR. Based on these data, the complexity of the signaling networks conducting radiation survival is broadened by adding the adaptor protein lamellipodin. / Hintergrund: Ein stark infiltrierendes Wachstum und Resistenzen gegenüber Bestrahlung und Chemotherapie tragen zu einer schlechten Prognose des Glioblastoms bei. Um die Therapie des Glioblastoms zu verbessern, ist die weitere Erforschung des komplexen Signalnetzwerkes notwendig. Aufgrund der essentiellen Rolle des Signaladaptors Lamellipodin für die Entwicklung des Nervensystems und der Migration von Zellen ist eine Funktion von Lamellipodin im Glioblastom vorstellbar. Die spezifische Funktion von Lamellipodin in der Invasion und Strahlenresistenz des Glioblastoms ist allerdings bisher völlig unbekannt. Die vorliegende Arbeit untersucht daher die Invasion und Strahlenresistenz von Glioblastomzellen sowie den zugrundliegenden molekularen Mechanismus im Hinblick auf Lamellipodin. Material und Methoden: Die Expression von Lamellipodin wurde in humanen Astrozyten und neun Glioblastom-Zelllinien mittels Western Blot Analyse untersucht. Die Lokalisierung von Lamellipodin in Glioblastomzellen wurde mit Immunfluoreszenzfärbung analysiert. Die Auswirkungen eines Lamellipodin-Knockdown mittels siRNA auf die Glioblastom-Eigenschaften Invasion, Überleben und residuale DNA-Doppelstrangbrüche (DSB) bei Röntgenbestrahlung, Proliferation, Apoptose und Seneszenz wurden untersucht. Veränderungen in molekularen Signalwegen wurden durch Phosphoproteomanalyse nach Kontroll- und Lamellipodin-Knockdown in Kombination mit und ohne Röntgenbestrahlung in A172 Zellen analysiert. Koloniebildungsassays wurden nach einfachem und doppeltem Knockdown von Lamellipodin und den betroffenen Proteinen durchgeführt, um letztere Ergebnisse mit dem klonogenen Überleben in Verbindung zu bringen. Direkte Lamellipodin-Bindungspartner wurden mittels massenspektrometrischer Analyse untersucht. Die Validierung der Protein-Protein-Interaktion wurde durch Immunpräzipitation und Proximity Ligation Assay bestimmt. Ein klonogenes Überlebensassay wurde nach dreifachem Knockdown von Lamellipodin, durch das Phosphoproteom identifizierten Proteinen und dem direkten Interaktionspartner von Lamellipodin durchgeführt. Die hierarchische Analyse erfolgte durch Analyse der Expression und Phosphorylierung der ermittelten Proteine mittels Immunblot. Ergebnisse: Die Expression und Phosphorylierung von Lamellipodin war unterschiedlich in den Glioblastomzelllinien. Die siRNA-vermittelte Deletion von Lamellipodin verringerte die Invasion und Proliferation von Glioblastomzellen enorm, während die Reduktion von Lamellipodin keine Auswirkungen auf Apoptose oder Seneszenz hatte. Darüber hinaus wurden sieben der neun untersuchten Glioblastomzelllinien durch das Ausschalten von Lamellipodin radiosensibilisiert, ohne dass sich dies auf die Anzahl der residualen DNA-Doppelstrangbrüche auswirkte, während die Überexpression von Lamellipodin das Überleben nach Bestrahlung verbesserte. Mechanistisch beeinträchtigte der Verlust von Lamellipodin die Phosphorylierung von neun Proteinen (EIF2A, EGFR, FOS, MKK6, NFKBIA, PRKAA2, RSK2, SRC und TAK1), die hauptsächlich an der EGFR-MAPK-Signalübertragung beteiligt sind. Die kombinierte Ausschaltung von Lamellipodin und den relevanten Proteinen führte zu einer allgemeinen Radiosensibilisierung in den Zelllinien A172 und U343MG. Darüber hinaus zeigte die massenspektrometrische Analyse von Lamellipodin-Immunpräzipitaten, dass sich das Lamellipodin-Interaktom als Reaktion auf Röntgenbestrahlung verändert. RICTOR wurde durch Immunpräzipitation und Proximity Ligation Assay als direktes Bindeglied von Lamellipodin zum EGFR-MAPK-Signalweg identifiziert. Darüber hinaus führte die dreifache Deletion von Lamellipodin, RICTOR und EGFR zu einem ähnlichen Grad an Radiosensibilisierung wie der Knockdown von Lamellipodin, was ihre gemeinsame Rolle bei der Strahlenreaktion von Glioblastomen unterstreicht. Darüber hinaus erhöhte die Ausschaltung von Lamellipodin die EGFR-Expression und -Phosphorylierung, während die Lamellipodin-Phosphorylierung bei EGFR-Mangel verringert wurde. Schlussfolgerung: Die Ergebnisse decken die entscheidende Funktion von Lamellipodin für die Invasion, Proliferation und Radiosensitivität von Glioblastomzellen auf. Basierend auf molekularen Analysen wurde Lamellipodin als eine Determinante der EGFR-Signalübertragung durch Interaktion mit RICTOR identifiziert. Auf der Grundlage dieser Daten wird die Komplexität des Signalnetzwerks, welches das Überleben durch Strahlung reguliert, durch das Adaptorprotein Lamellipodin erweitert.

A proteína APE1 (do inglêsApurinic/Apyrimidinicendonuclease 1/ Redox Factor-1 - APE1/REF-1) é uma enzima multifuncional, cuja expressão encontra-se frequentemente aumentada em gliomas. Além de apresentar atividade no reparo por excisão de base (BER), o gene APE1 também atua como fator de redução, mantendo fatores de transcrição (FTs) em um estado reduzido ativo. A via BER de reparo do DNA tem sido apontada como um possível fator de resistência a terapias baseadas no uso de agentes alquilantes, tais como temozolomida (TMZ). No presente trabalho, utilizou-se a estratégia de inibição da transcrição do gene APE1 pelo método de RNA interferente(siRNA) e tratamento com a droga TMZ nas células de glioblastoma (GBM), T98G (resistente à TMZ) e U87MG (sensível à TMZ), a fim de verificar a influência do silenciamento do gene APE1 sobre as respostas celulares à droga avaliadas por vários ensaios, bem como os efeitos sobre a expressão transcricional dos genes alvos dos FTs regulados por APE1. O silenciamento de APE1 e o tratamento das células T98G com a TMZ foram eficazes no sentido de reduzir a proliferação e a capacidade clonogênica, além de intervir na progressão do ciclo celular com bloqueio na fase S. Tais efeitos foram acompanhados pelo aumento da indução de danos no DNA e da expressão de H2AX fosforilada (H2AX), o que justifica a queda na sobrevivência celular. O mesmo efeito não foi observado nas células U87MG silenciadas para APE1 e tratadas com a TMZ, havendo o predomínio dos efeitos causados pela TMZ, exceto por uma leve indução de danos no DNA e de H2AX. Adicionalmente, nas células T98G silenciadas e tratadas, verificou-se uma moderada indução de apoptose, que foi observada ao longo dos tempos avaliados (1 a 10 dias), com uma leve indução de caspase-3 (5 dias); nessas células, observou-se também a indução (3,8 vezes) de morte celular autofágica (5 dias). Entretanto, nas células U87MG,a indução de apoptose foi baixa e não houve indução de morte por autofagia, sugerindo outros mecanismos de morte envolvidos na eliminação dessas células em resposta ao tratamento com a TMZ. O silenciamento de APE1 causou uma redução acentuada na invasão das células T98G, de forma similar à observada nas células somente tratadas com a TMZ, sendo que a combinação (silenciamento de APE1 e tratamento com a droga) resultou em um efeito aditivo, enquanto que nas células U87MG a combinação foi eficaz no sentido de reduzir a proporção de células invasivas, fato não observado nas condições isoladas. Os genes COX2 e VEGF, alvos dos FT NFB e HIF-1 (regulados por APE1) foram reprimidos nas células T98G enquanto que o gene VEGF foi induzido nas células U87MG, entretanto, tais alterações no padrão de expressão transcricional foram observadas somente em resposta ao tratamento com a TMZ, independentemente do silenciamento de APE1, indicando nenhuma mudança na atividade redox de APE1, possivelmente pela existência de proteínas APE1 remanescentes na célula. Além disso, a expressão proteica de NFBp65(ser563) foi aumentada em ambas as linhagens silenciadas e tratadas com a TMZ, provavelmente devido à inibição da proliferação celular. Em geral, os resultados do presente trabalho demonstraram que a estratégia de inibição do gene APE1 (participante da via BER) mostrou-se potencialmente viável, suportando a contribuição do BER na resistência à TMZ, visto que nas condições testadas, observou-se uma sensibilização das células de GBM, com efeito restrito às células de GBM resistentes (linhagem T98G), sendo pouco eficaz no sentido de sensibilizar as células sensíveis (linhagem U87MG) a esse agente. Assim, há que considerar as características genéticas de cada linhagem de GBM, visto que estas são cruciais para as respostas apresentadas pelas células aos tratamentos empregados. / APE1 (Apurinic/Apyrimidinic endonuclease 1/ Redox Factor-1 - APE1/REF-1) protein is a multifunctional enzyme whose expression is often increased in gliomas. Besides presenting activity in base excision repair (BER), APE1 also acts as a reduction factor, maintaining transcription factors (TFs) in an active reduced state. The BER pathway has been implicated as a possible factor of resistance to therapies based on the use of alkylating agents such as temozolomide (TMZ). In the present study, we have been using a strategy of small interference RNA (siRNA) to down-regulate the APE1 gene under conditions of treatment with TMZ in T98G (resistant to TMZ) and U87MG (sensitive to TMZ), glioblastoma (GBM), in order to determine the effects of APE1 gene silencing on cellular responses to this drug, evaluated by several assays, as well as the effects on the transcriptional expression of target genes of TFs regulated by APE1. APE1 silencing and TMZ treatment was effective to reduce the cell proliferation and clonogenic capacity of T98G cells, in addition to interfering in the cell cycle progression (S-phase arrest). These effects were accompanied by induction of DNA damage and phosphorylation of H2AX (H2AX), which may explain the decrease in cell survival. The same effect was not observed in silenced U87MG and TMZ-treated cells, being observed a predominance of the effects caused by TMZ itself, except for a slight induction of DNA damage and H2AX. Additionally, in silenced T98G and TMZ-treated cells, there was a moderate induction of apoptosis, as observed over time (1 to 10 days), with a slight induction of caspase-3 (on day 5); for those cells, we also observed autophagic induction (3.8 fold) at day 5. However, the induction of apoptosis and autophagy in U87MG cells was very low, suggesting that other mechanisms of cell death might be involved in the elimination of GBM cells under TMZ treatment. APE1 silencing caused a marked reduction on the invasiveness of T98G cells, similarly to that observed in TMZ treated cells, while the combination (APE1 silencing and drug treatment) led to an additive effect. For U87MG, the treatment combination was effective in reducing the proportion of invasive cells, in spite of an absence of any effect produced by each isolated condition tested. Regarding to the expression profile of target genes of TFs regulated by the APE1 redox activity, it was observed that COX2 and VEGF genes, targets of FTs NFB and HIF-1, were down-regulated in T98G while VEGF gene showed induced in U87MG cells; however, such alterations in the transcriptional expression pattern were observed only in response to TMZ treatment, independently of APE1 gene silencing, indicating no change in the APE1 redox activity, possibly due to the presence of APE1 remaining proteins inside cells. In addition, NFBp65(ser563) protein expression was increased in both cell lines (silenced and treated with TMZ), probably due to the reduced cell proliferation rates. In general, the present results show that the strategy of APE1 gene knockdown was potentially viable, supporting the BER contribution of the mechanism of TMZ resistance, since under the conditions tested, there was a sensitization of GBM cells. However, this effect was restricted to the resistant cell line (T98G cells). Thus, it should be considered the genetic characteristics of each GBM cell line, since these are crucial to the cellular responses to the conditions tested in the present work.

A micróglia é essencial para a homeostase do Sistema Nervoso Central (SNC), função neuro-imune inata, e exerce papel importante na neurodegeneração, envelhecimento cerebral e tumorigênese. Gliomas difusos são tumores cerebrais primários caracterizados por crescimento infiltrativo e altas taxas de heterogeneidade, o que torna a doença praticamente incurável. Avanços em análises genéticas caracterizaram alterações moleculares relacionadas ao tempo de sobrevida e à resposta clínica desses pacientes, especialmente em glioblastomas (GBM). No entanto, a tumorigenicidade dos gliomas não é controlada unicamente por suas alterações genéticas. As interações entre as células tumorais, a micróglia residente e os macrófagos/monócitos infiltrados desempenham um papel crucial na modulação do crescimento e agressividade do glioma. Neste estudo, analisamos o fenótipo de ativação da micróglia/macrófagos em gliomas, incluindo astrocitomas e oligodendroglimas de diferentes graus de malignidade, apresentamos o perfil de expressão gênica da população pura de micróglia cortical e do tecido cerebral total correspondente. Usando sequenciamento de DNA de alta performance, classificamos as amostras de GBM em Proneural, Clássico e Mesenquimal. Em seguida, avaliamos os status de ativação da micróglia/macrófagos dessas amostras. Apesar do alto grau de heterogeneidade, pudemos observar níveis mais altos dos marcadores mielóides (IBA1, CD11b and CD68) em tumores astrocíticos comparados a tumores de origem oligodendrocítica e ao tecido não-neoplásico. Marcadores de anti-inflamação, como CD163, foram mais abundantes em astrocitomas, bem como em GBMs do subtipo Mesenquimal e Clássico; enquanto que marcadores de pró-inflamação, como IL1-beta, mostraram uma expressão mais heterogênea entre as amostras. Em seguida, micróglia foi isolada de 25 amostras de córtex parietal provenientes de autópsia de indivíduos cognitivamente preservados e foi feito o RNA-seq. Os resultados foram comparados à micróglia de camundongo e a outras células mielóides. Boa parte dos genes expressos pela micróglia humana foram similares àqueles expressos pela micróglia murina, como CX3CR1, P2YR12 e ITGAM. Porém, foram identificados genes de característica imune, abundantemente expressos na micróglia humana e não identificados na micróglia de camundongos, como TLR, Fcy, receptores do tipo SIGLEC, e fatores de transcrição NLRC5 e CIITA. A comparação dos dados de expressão gênica da micróglia com monócitos e macrófagos identificou novos marcadores que distinguem a micróglia humana de outras células mielóides. Nossos dados sobre a micróglia em gliomas sugerem características de imunossupressão e de pró-crescimento em tumores de pior prognóstico, ligado a um fenótipo específico de ativação das células mielóides. Este é o primeiro estudo a identificar o transcriptoma da micróglia humana pura, demonstrando que ela é claramente diferente da micróglia murina e de outras células mielóides. Esses resultados abrem portas para estudos de populações específicas de células mielóides em gliomas / Microglia are essential for central nervous system (CNS) homeostasis and innate neuroimmune function, and play important roles in neurodegeneration, brain aging and tumorigenesis. Diffuse gliomas are primary brain tumors characterized by infiltrative growth and high heterogeneity, which renders the disease mostly incurable. Advances in genetic analysis have characterized molecular alterations leading to impact on patients\' overall survival and clinical outcome, particularly in glioblastoma (GBM). However, glioma tumorigenicity is not controlled uniquely by its genetic alterations. The crosstalk between tumor cells, resident microglia and infiltrating monocytes/macrophages plays a crucial role in modulating glioma growth and aggressiveness. Here, we assess the activation status of microglia/macrophages in gliomas,including astrocytomas and oligodendrogliomas of different grades of malignancy, and present the gene expression profile of pure cortical human microglia and corresponding unsorted brain tissue. Using high-throughput DNA sequencing, we have classified GBM samples in Proneural, classical and mesenchymal. Next, we evaluated the activation status of microglia/macrophages within these samples. Despite the great heterogeneity, we observed higher levels of myeloid markers (IBA1, CD11b and CD68) in astrocytic tumors compared to oligodendrocytic ones and to non-neoplastic (NN) tissue. Anti-inflammation markers, such as CD163, are also more abundant in astrocytomas, as well as in the mesenchymal and classical GBM subtypes, while pro-inflammation markers, such as IL1-beta, show a more widespread expression throughout samples. Next, microglia were isolated from the parietal cortex of 25 autopsy samples of cognitively preserved humans and RNA sequenced. Overall, genes expressed by human microglia are similar to mouse microglia, such as CX3CR1, P2YR12, and ITGAM. Interestingly, a number of immune genes, not identified as mouse microglia signature genes, were abundantly expressed in human microglia, such as TLR, Fcy and SIGLEC receptors and NLRC5 and CIITA transcription factors. Comparison of microglia to monocyte and macrophage expression data underscored the CNS-specific functions of microglia and new markers were identified that distinguish human microglia from other myeloid cells. Our glioma-related data suggests an immune-suppressive and growth supportive characteristic for tumors with worse clinical outcome, linked to an activation profile of myeloid cells. This data is the first comprehensive pure human microglia gene expression profile; human microglia clearly differ from mouse microglia and other myeloid cells. These results will help further studies focusing on pure myeloid cells populations in glioma