The WHO classification of brain tumors is based on histological features and the aggressiveness of the tumor is classified from grade I to IV, where grade IV is the most aggressive. Today, the correlation between prognosis and tumor grade is the most important component in tumor classification. High grade gliomas, glioblastomas, are associated with poor prognosis and a median survival of 14 months including all available treatments. Low grade meningiomas, usually benign grade I tumors, are in most cases cured by surgical resection. However despite their benign appearance grade I meningiomas can, without any histopathological signs, in some cases develop bone invasive growth and become lethal. Thus, it is necessary to improve conventional treatment modalities, develop new treatment strategies and improve the knowledge regarding the basic pathophysiology in the classification and treatment of brain tumors. In this thesis, both proteomics and metabolomics have been applied in the search for biomarkers or biomarker patterns in two different types of brain tumors, gliomas and meningiomas. Proteomic studies were carried out mainly by surface enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS). In one of the studies, isobaric tags for relative and absolute quantitation (iTRAQ) labeling in combination with high-performance liquid chromatography (HPLC) was used for protein detection and identification. For metabolomics, gas-chromatography time-of-flight mass spectrometry (GC-TOF-MS) has been the main platform used throughout this work for generation of robust global metabolite profiles in tissue, blood and cell cultures. To deal with the complexity of the generated data, and to be able to extract relevant biomarker patters or latent biomarkers, for interpretation, prediction and prognosis, bioinformatic strategies based on chemometrics were applied throughout the studies of the thesis. In summary, we detected differentiating protein profiles between invasive and non-invasive meningiomas, in both fibrous and meningothelial tumors. Furthermore, in a different study we discovered treatment induce protein pattern changes in a rat glioma model treated with an angiogenesis inhibitor. We identified a cluster of proteins linked to angiogenesis. One of those proteins, HSP90, was found elevated in relation to treatment in tumors, following ELISA validation. An interesting observation in a separate study was that it was possible to detect metabolite pattern changes in the serum metabolome, as an effect of treatment with radiotherapy, and that these pattern changes differed between different patients, highlighting a possibility for monitoring individual treatment response.  In the fourth study of this work, we investigated tissue and serum from glioma patients that revealed differences in the metabolome between glioblastoma and oligodendroglioma, as well as between oligodendroglioma grade II and grade III. In addition, we discovered metabolite patterns associated to survival in both glioblastoma and oligodendroglioma. In our final work, we identified metabolite pattern differences between cell lines from a subgroup of glioblastomas lacking argininosuccinate synthetase (ASS1) expression, (ASS1 negative glioblastomas), making them auxotrophic for arginine, a metabolite required for tumor growth and proliferation, as compared to glioblastomas with normal ASS1 expression (ASS1 positive). From the identified metabolite pattern differences we could verify the hypothesized alterations in the arginine biosynthetic pathway. We also identified additional interesting metabolites that may provide clues for future diagnostics and treatments. Finally, we were able to verify the specific treatment effect of ASS1 negative cells by means of arginine deprivation on a metabolic level.

Η παρούσα διπλωματική εργασία αποτελεί μία βιβλιογραφική μελέτη επάνω στην υποκλασματοποιημένη ακτινοθεραπεία του γλοιοβλαστώματος. Το πολύμορφο γλοιοβλάστωμα είναι ο πιο επιθετικός τύπος πρωτοπαθούς εγκεφαλικού όγκου με δυσμενείς προγνωστικούς δείκτες. Η θεραπεία του γλοιοβλαστώματος έχει προβληματίσει καθώς τα αποτελέσματα από τις διάφορες θεραπευτικές προσεγγίσεις είναι απογοητευτικά. Στο πρώτο μέρος της εργασίας έχουν δοθεί στοιχεία για την παθοφυσιολογία της νόσου και έχει γίνει ανάλυση της επίδρασης της ακτινοβολίας πρώτα στην οργανική ύλη και στην συνέχεια στο γλοιοβλάστωμα. Αμέσως μετά, εξετάστηκε, ο ρόλος της ακτινοθεραπείας κάνοντας εκτενή αναφορά στις βασικές της αρχές και στις τεχνολογικές εξελίξεις των ακτινοθεραπευτικών τεχνικών σε σχέση με το γλοιοβλάστωμα. Έγινε αναφορά σε κάθε μια χωριστά και παρουσιάζονται τα πλεονεκτήματα και τα μειονεκτήματα όλων των τεχνικών στο γλοιοβλάστωμα. Ακολούθως, η εργασία αυτή εστίασε την προσοχή της στην υποκλασματοποίηση της ακτινοθεραπείας, περιγράφοντας τον ορισμό της και παρουσιάζοντας τις διαστάσεις της. Στο τέλος του πρώτου μέρους αφιερώθηκε ένα μικρό κομμάτι στις γονιδιακές θεραπείες, καθώς δεν είναι λίγοι οι ερευνητές που αναζητούν ευθύνες σε συγκεκριμένα γονίδια και ελπίζουν να ανοίξει ο δρόμος σε νέες γονιδιακές θεραπευτικές προσεγγίσεις στο γλοιοβλάστωμα. Στο δεύτερο μέρος της εργασίας έγινε αναλυτική αναφορά στις μελέτες που έχουν γίνει σχετικά με την υποκλασματοποιημένη ακτινοθεραπεία του γλοιοβλαστώματος και παρατέθηκαν τα σημαντικότερα συμπεράσματα ολόκληρης της βιβλιογραφικής ανασκόπησης στο θέμα. Ένας συγκεντρωτικός πίνακας με επιλεγμένα στοιχεία όλων των μελετών ολοκλήρωσε τη διπλωματική εργασία αυτή. / The present dissertation is a literature study on the hypofractionated radiotherapy of glioblastoma. Glioblastoma multiform is the most aggressive type of primary brain tumour with unfavourable prognostic indicators. Treatment of glioblastoma has puzzled since the results of the different therapeutic approaches are disappointing. In the first part, information was given concerning the pathophysiology of the disease and an analysis have been made on the effect of radiation in the first organic material and then in glioblastoma. Then, the role of radiotherapy was examined, with extensive reference to basic principles and technological developments in radiotherapeutic techniques compared to glioblastoma. A reference was made to each single technique and then all advantages and disadvantages on glioblastoma were presented. Thereafter, the dissertation focuses its attention on subfractionation of radiotherapy, describing its definition and showing its dimensions. At the end of the first part a small chapter was devoted in gene therapies, as there are a few researchers looking into specific genes and hope to open the road to new gene therapeutic approaches in glioblastomas. The second part was a detailed report to studies that have been made on hypofractionated radiotherapy of glioblastoma and the most important conclusions of the literature review on the subject have been cited. Finally, a summary table of selected data of all studies concludes this dissertation.

Glioblastoma is the commonest form of brain neoplasm and among the most malignant forms of cancer. The identification of a subpopulation of self-renewing and multipotent cancer stem cells within glioblastoma has revealed a novel cellular target for the treatment of this disease. The role of developmental cell signaling pathways in these cell populations remains poorly understood. Herein, we examine the role of the Notch signaling pathway in glioblastoma stem cells. In this thesis we have demonstrated that the canonical Notch pathway is active in glioblastoma stem cells and functions to inhibit neuronal lineage commitment in a subset of patient derived glioblastoma stem cells in vitro. Gamma secretase (γ-secretase) small molecule inhibitors or dominant-negative co-activators inhibit glioblastoma stem cell proliferation and induce neuronal lineage commitment in a fashion that synergizes with Wingless pathway activation via GSK-3β blockade. Our data suggest that subsets of patient samples show a Notch gene expression profile that predicts their abilities to undergo neuronal lineage differentiation in response to γ-secretase small molecule inhibitors. Additionally, the data suggests that Notch may perturb the relative fractions of cells undergoing symmetric division, in favour of asymmetric division, limiting clonal expansion from single cells. These data may have important implications for treating human glioblastoma, and suggest that in addition to inhibition of proliferation, influencing lineage choice of the tumor stem cells may be a mechanism by which these tumors may be pharmacologically inhibited.

Glioblastoma is the commonest form of brain neoplasm and among the most malignant forms of cancer. The identification of a subpopulation of self-renewing and multipotent cancer stem cells within glioblastoma has revealed a novel cellular target for the treatment of this disease. The role of developmental cell signaling pathways in these cell populations remains poorly understood. Herein, we examine the role of the Notch signaling pathway in glioblastoma stem cells. In this thesis we have demonstrated that the canonical Notch pathway is active in glioblastoma stem cells and functions to inhibit neuronal lineage commitment in a subset of patient derived glioblastoma stem cells in vitro. Gamma secretase (γ-secretase) small molecule inhibitors or dominant-negative co-activators inhibit glioblastoma stem cell proliferation and induce neuronal lineage commitment in a fashion that synergizes with Wingless pathway activation via GSK-3β blockade. Our data suggest that subsets of patient samples show a Notch gene expression profile that predicts their abilities to undergo neuronal lineage differentiation in response to γ-secretase small molecule inhibitors. Additionally, the data suggests that Notch may perturb the relative fractions of cells undergoing symmetric division, in favour of asymmetric division, limiting clonal expansion from single cells. These data may have important implications for treating human glioblastoma, and suggest that in addition to inhibition of proliferation, influencing lineage choice of the tumor stem cells may be a mechanism by which these tumors may be pharmacologically inhibited.

Proliferating tissues, including embryonic and tumour tissues, preferentially employ aerobic glycolysis to support cell growth. This reliance on glycolysis even in the presence of oxygen, referred to as the “Warburg Effect”, may confer a proliferative, survival and invasive advantage and be exploited therapeutically. In this thesis, we demonstrate that the glycolytic enzyme Hexokinase 2 (HK2) is crucial for the “Warburg Effect” in human Glioblastoma Multiforme (GBM), the most common and therapeutically resistant malignant brain tumour. In contrast to normal brain and low-grade gliomas, GBMs exhibited a marked increase in HK2 expression, but not HK1, particularly in perinecrotic, hypoxic regions and its expression predicted poor overall survival of GBM patients. Stable loss of HK2 in GBM cells restored oxidative phosphorylation (OXPHOS)-mediated glucose metabolism, with increased oxygen consumption and decreased lactic acid production, an effect not seen with loss of glycolytic enzymes HK1 or PKM2. Furthermore, HK2 depletion resulted in decreased proliferation in vitro and in vivo and increased sensitivity to apoptotic inducers such as radiation and chemotherapy, both common adjuvant therapies of GBMs. Intracranial xenografts of GBM cells with reduced HK2 demonstrated significantly increased survival with decreased proliferation and angiogenesis yet enhanced invasiveness. In contrast, exogenous HK2 expression in GBM cells promoted proliferation, therapeutic resistance and intracranial growth. This was dependent partly on the PI3K/AKT dependent translocation of HK2 to the mitochondrial membrane. Stable loss of glycolytic enzymes HK2, HK1 and PKM2 reduced GBM proliferation but differentially altered the PI3K/AKT/mTOR and AMPK signaling pathways, the extent to which may influence whether a cell preferentially undergoes autophagy or apoptosis as the primary mode of cell death. Collectively, targeting enzymes employed by the tumour to modulate its energy metabolism, such as HK2 in GBMs, may favourably alter its therapeutic sensitivity to radiation and both classical and novel chemotherapeutic agents.

No description available.

No description available.

Proliferating tissues, including embryonic and tumour tissues, preferentially employ aerobic glycolysis to support cell growth. This reliance on glycolysis even in the presence of oxygen, referred to as the “Warburg Effect”, may confer a proliferative, survival and invasive advantage and be exploited therapeutically. In this thesis, we demonstrate that the glycolytic enzyme Hexokinase 2 (HK2) is crucial for the “Warburg Effect” in human Glioblastoma Multiforme (GBM), the most common and therapeutically resistant malignant brain tumour. In contrast to normal brain and low-grade gliomas, GBMs exhibited a marked increase in HK2 expression, but not HK1, particularly in perinecrotic, hypoxic regions and its expression predicted poor overall survival of GBM patients. Stable loss of HK2 in GBM cells restored oxidative phosphorylation (OXPHOS)-mediated glucose metabolism, with increased oxygen consumption and decreased lactic acid production, an effect not seen with loss of glycolytic enzymes HK1 or PKM2. Furthermore, HK2 depletion resulted in decreased proliferation in vitro and in vivo and increased sensitivity to apoptotic inducers such as radiation and chemotherapy, both common adjuvant therapies of GBMs. Intracranial xenografts of GBM cells with reduced HK2 demonstrated significantly increased survival with decreased proliferation and angiogenesis yet enhanced invasiveness. In contrast, exogenous HK2 expression in GBM cells promoted proliferation, therapeutic resistance and intracranial growth. This was dependent partly on the PI3K/AKT dependent translocation of HK2 to the mitochondrial membrane. Stable loss of glycolytic enzymes HK2, HK1 and PKM2 reduced GBM proliferation but differentially altered the PI3K/AKT/mTOR and AMPK signaling pathways, the extent to which may influence whether a cell preferentially undergoes autophagy or apoptosis as the primary mode of cell death. Collectively, targeting enzymes employed by the tumour to modulate its energy metabolism, such as HK2 in GBMs, may favourably alter its therapeutic sensitivity to radiation and both classical and novel chemotherapeutic agents.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from first page of PDF file (viewed on June 10, 2009). Includes bibliographical references.

Ecteinascidina 743 (ET-743) é uma nova droga isolada de um tunicado marinho, a Ecteinascidia turbinata, que está na fase III dos estudos clínicos por sua marcada atividade anticâncer. Apesar de seu mecanismo de ação não estar completamente elucidado, tem sido demonstrado que a ET-743 se liga ao DNA formando adutos covalentes com o N2 da guanina. Além disso, a ET-743 tem sido relatada como potente inibidora da transcrição. No presente estudo, utilizou-se como modelo para a investigação dos efeitos antiproliferativos deste composto a linhagem celular derivada de glioblastoma humano, U-251 MG. Uma vez que o foco principal de atenção nos estudos sobre o mecanismo de ação da ET-743 esteja concentrado em suas interações com o DNA, a autora buscou avaliar outros aspectos de sua atividade antiproliferativa, quais sejam, o seu efeito sobre a distribuição das células no ciclo celular, sobre a atividade de enzimas associadas ao processo de apoptose, bem como sobre o conteúdo celular da proteína Hsp70. Em incubações de 0,5 nM por 48 h, a ET-743 causou um significante acúmulo das células na fase G2M do ciclo celular, o mesmo ocorrendo com doses mais elevadas (1,0 e 1,5) e incubações mais prolongadas (72 h). A ET-743 induziu morte celular dose-dependente e este efeito foi significativamente prevenido pelo inibidor de caspases z-VAD-fmk. Contudo, não foi observado aumento significativo nos níveis de Hsp70 após tratamento com ET-743. Considerando que alta expressão de Hsp70 é um dos principais mecanismos de proteção das células em condições de estresse, incluindo-se o tratamento com drogas citotóxicas, a não elevação de seus níveis na presença da ET-374 pode estar, ao menos em parte, relacionada à citotoxicidade produzida por este agente na linhagem estudada.