O glioblastoma multiforme (GBM) é uma neoplasia do sistema nervoso central (SNC), caracterizada por uma elevada capacidade proliferativa e migratória. O desenvolvimento do tumor provoca uma remodelação da matriz extracelular (MEC) que facilita a migração tumoral. Eicosanóides são moléculas lipídicas importantes na carcinogênese e a sua síntese está correlacionada com o grau de desenvolvimento do tumor. As prostaglandinas são eicosanóides envolvidas na estimulação da angiogênese, na adesão celular e proliferação celular. Este estudo tem por objetivo avaliar in vitro o efeito da PGE2 na expressão moléculas da MEC e das moléculas de adesão envolvidas na migração, em células U87MG e U251MG. As células U251MG e U87MG foram tratadas com PGE2 (10µM) e Ibuprofeno (25µM), por um período 48hs. As proteínas da MEC foram analisadas por RT-qPCR após o tratamento. Foram realizadas reações de imunohistoquímica para as moléculas da MEC. As alterações foram encontradas na expressão de laminina, fibronectina, colágeno tipo IV e as integrinas αv , α3 e α5 para células U87MG . Observamos imunomarcação nas linhas celulares para colágeno tipo IV, laminina e fibronectina. Concluímos que o tratamento com IBU e PGE2, afeta a expressão gênica de moléculas de MEC. / Glioblastoma Multiforme (GBM) is a neoplasm of the central nervous system (CNS), characterized by a high proliferative and migratory capacity. Tumor development leads to extracellular matrix (ECM) remodeling and facilitating the migration of these cells. Eicosanoids are important lipid molecules in carcinogenesis, and their synthesis often correlates with the degree of tumor development. Prostaglandins are eicosanoids involved in the stimulation of angiogenesis, cell adhesion and cell proliferation. This study is aimed to evaluate the expression of several ECM molecules involved in migration after altering the concentration of prostaglandins, using human glioma cell lines as an in vitro model. The cell lines U87MG and U251MG were treated with PGE2 (10µM) and Ibuprofen (25µM), for a predetermined period of 48hs. Proteins involved in extracellular matrix were analyzed by RT-qPCR after treatment in vitro. Immunohistochemical reactions were also performed for the ECM molecules. Changes were found in the expression of laminin, fibronectin, type IV collagen and αv, α3 and α5 integrins in cells U87MG. We observed immunostaining in cell lines to type IV collagen, laminin and fibronectin. In conclusion, Ibuprofen and PGE2, affects gene expression of ECM molecules.

Submitted by PPG Biologia Celular e Molecular (bcm@pucrs.br) on 2017-10-11T12:59:22Z No. of bitstreams: 1 PEDRO_VARGAS_DIS.pdf: 3468332 bytes, checksum: 563022a7fa710bed810c652069a0777c (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-10-11T20:09:27Z (GMT) No. of bitstreams: 1 PEDRO_VARGAS_DIS.pdf: 3468332 bytes, checksum: 563022a7fa710bed810c652069a0777c (MD5) / Made available in DSpace on 2017-10-11T20:19:08Z (GMT). No. of bitstreams: 1 PEDRO_VARGAS_DIS.pdf: 3468332 bytes, checksum: 563022a7fa710bed810c652069a0777c (MD5) Previous issue date: 2017-03-10 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Glioblastoma multiform (GBM) is considered the most aggressive tumors of central nervous system (CNS), and the most lethal among primary tumors presenting low survival prognosis, resistance to radiotherapy and chemotherapy. P2Y12 is considered a chemoreceptor for adenosine diphosphate (ADP). Its expression is documented in some cancer types, such as the C6 lineage of rat glioma, renal carcinoma and colon carcinoma. However, its role in the development of tumour progression and the resistance mechanism in chemotherapy are not well elucidated. Currently, it is well established that normal platelet function is an important factor for the progression of tumors, with thrombocytopenic rats demonstrating a significant reduction of metastases. Clopidogrel bisulfate is a drug, which belongs to the class of antiplatelet agents, being a P2Y12 receptor antagonist whose endogenous ligand is ADP. The aim of the present study was to determine the effects of tumor cells exposed to the treatment with clopidogrel. The C6 rat cell line was sensitive to the drug tested (150 ?M) in the viability and cell counts (69.63 ? 8.70) and (53.05 ? 20.06). Both cell lines showed a significant reduction in the number of colonies after 10 days of treatment with clopidogrel. The U251-MG strain demonstrated a significant increase in the G1 phase and a significant reduction in the S phase in the cell cycle (20.32 ? 3.05) and (19.45 ? 2.35) in the concentration of 500 ?M. Other results demonstrate important antiproliferative activity in both tumor lines, suggesting an important participation of the P2Y12 receptor in this process. / O glioblastoma multiforma (GBM) ? considerado o mais agressivo dos tumores do sistema nervoso central (SNC) e o mais letal entre os tumores prim?rios apresentando baixo progn?stico de sobrevida, resist?ncia ? radioterapia e a quimioterapia. O P2Y12 ? considerado um quimiorreceptor para a adenosina difosfato (ADP). Sua express?o ? documentada em algumas linhagens de c?ncer, como a linhagem C6 de glioma de rato, carcinoma renal e carcinoma de c?lon. Contudo, seu papel no desenvolvimento da sinaliza??o e resposta ? quimioterapia n?o est? bem elucidado. Atualmente, est? bem estabelecido que o funcionamento normal das plaquetas ? um importante fator para a progress?o de tumores, sendo que ratos com trombocitopenia demonstraram uma redu??o significativa de met?stases. O bissulfato de clopidogrel ? um f?rmaco pertencente ? classe dos antiagregantes plaquet?rios, sendo um antagonista do receptor P2Y12, cujo ligante end?geno ? o ADP. O presente trabalho teve como objetivo averiguar quais os efeitos ocasionados ?s c?lulas de gliomas quando expostas ao clopidogrel. Para isto, foram utilizadas duas linhagens de gliomas, U251-MG de glioma humano e C6 de ratos, e o tratamento com clopidogrel (150, 300, e 500 ?M), foi avaliado nos par?metros de viabilidade celular, contagem de c?lulas, RT-PCR e citometria de fluxo. A linhagem de rato C6 mostrou-se sens?vel ao f?rmaco testado (150 ?M) com redu??o da viabilidade e contagem celular (69.63?8,70) e (53,05 ? 20.06), respectivamente. Ambas as linhagens tapresentaram uma redu??o significativa no n?mero de col?nias ap?s 10 dias tratamento com clopidogrel. A linhagem U251-MG demonstrou um aumento significativo na fase G1 e uma redu??o significativa na fase S e do ciclo celular (20,32 ? 3.05) e (19.45? 2.35 ) na concentra??o de 500 ?M, por?m nos ensaios de viabilidade e contagem n?o houve diferen?a significativa. Nossos resultados demonstram atividade antiproliferativa importante em ambas as linhagens tumorais, sugerindo uma participa??o importante do receptor P2Y12 neste processo.

Glioblastoma multiforme é um câncer do sistema nervoso central agressivo e invasivo. Apesar dos avanços terapêuticos com cirurgia, radiação e quimioterapia, o tempo de sobrevida média permanece reduzido. Os polifenóis têm sido relatados por apresentarem várias atividades biológicas, incluindo atividade anticâncer. Nesse estudo, foi avaliada a potencial atividade antiglioma do extrato de Achyrocline satureioides (marcela) e seus principais flavonoides em modelos de glioblastoma in vitro e in vivo. O extrato de A. satureioides (100 μg/mL) assim como os flavonoides isolados (quercetina, 3-O-metilquercetina, luteolina e achyrobichalcona; em 10 μM ou doses menores) reduziram a proliferação de três linhagens celulares de glioma (C6, U87 e U251). A combinação do extrato de A. satureioides com temozolomida (TMZ) demonstrou elevada atividade antiproliferativa comparada com o quimioterápico sozinho em uma menor concentração (50 μg/mL). Cabe ressaltar que nenhuma toxicidade foi observada nas células sadias do cérebro (astrócitos primários, neurônios primários e culturas organotípicas de fatias do hipocampo) na concentração que afetou as células de gliomas. Entretanto, a administração do extrato de A. satureioides no modelo tumoral em ratos não apresentou o mesmo efeito conforme observado in vitro. Além disso, ele foi capaz de bloquear o efeito do TMZ. Esses resultados geram preocupação uma vez que extratos de plantas poderiam afetar a atividade de fármacos anticâncer, especialmente considerando que o chá de marcela é amplamente consumido pela população da América do Sul e indicado para alívios de sintomas como dores de cabeça e náuseas. Portanto, mais estudos in vivo usando os flavonoides isolados são necessários para investigar a potencial atividade antiglioma observada in vitro. / Glioblastoma multiforme is an aggressive and invasive central nervous system cancer. Despite the therapeutic improvements with surgery, radiation, and chemotherapy, the median survival times remain dismal. Polyphenols have been reported to have numerous biological activities, including anticancer activity. In this study, the potential antiglioma activity of Achyrocline satureioides (marcela) extract and its main flavonoids was evaluated both in vitro and in vivo in glioblastoma models. A. satureioides extract (100 μg/mL) as well as the isolated flavonoids (quercetin, 3-O-methyl-quercetin, luteolin and achyrobichalcone; at 10 μM or lower doses) reduced proliferation of three glioma cell lines (C6, U87 and U251). Combination of A. satureioides extract with temozolomide (TMZ) showed enhanced antiproliferative activity compared to the chemotherapeutic alone at a lower concentration (50 μg/mL). Importantly, no toxicity was observed in healthy cells of the brain (primary astrocytes, primary neurons, and organotypic hippocampal slice cultures) at the concentration that affected glioma cells. However, administration of A. satureioides extract did not show the same effect in a rat tumor model as observed in vitro. In addition, it blocked the effect of TMZ. These results raise concern since plant extracts could affect the activity of anticancer drugs, especially considering that marcela tea is widely consumed by the South American population and indicated for relief of symptoms such as headaches and nausea. Therefore, further in vivo studies using the isolated flavonoids are required to investigate their potential antiglioma activity observed in vitro.

Glioblastoma is the most common adult malignant primary brain tumor with one of the worst prognosis. With a survival of 10 to 12 months, glioblastoma remains one of the most challenging disease to treat. The standard treatment method involves maximal possible resection of the tumor followed by radiation and chemotherapy. However, the short half-life of most chemotherapeutic drugs, high systemic toxicity and inability to cross the blood brain barrier inhibits effective delivery of the chemotherapeutics to the tumor. An ideal drug delivery system can reach the tumor site with high efficiency and continuously release the drug at the tumor site for an extended period. Adult stem cells including neural stem cells (NSC) and mesenchymal stem cells (MSC) have inherent tumor trophic properties allowing for site-specific delivery of chemotherapeutics. They can also be genetically engineered to secrete the chemotherapeutic drug continuously making them ideal candidates for cell-based delivery system for treating glioblastoma. MSC have been isolated from a wide range of sources including bone marrow, umbilical cord, adipose tissue, liver, multiple dental tissues and induced pluripotent stem cells. MSC are also easily amenable to viral modification allowing for easy manipulation to produce chemotherapeutic drugs. Additionally, more than 350 clinical trials using MSC have successfully established the safety of using MSC for cell-based therapies. Collectively these factors have led to the widespread use of MSC in cancer therapy. MSC have been successfully transduced to produce chemotherapeutic drugs to treat glioma, melanoma, lung cancer, ovarian cancer and breast cancer. Despite the multitudes of advantages that cell therapy provides they are limited in three main domains (1) Low cell retention and survival at the site of the tumor (2) In ability to co-deliver multiple therapeutics and (3) In ability to deliver drugs other than peptide based drugs. This thesis details the work to engineer mesenchymal stem cells to tackle these three issues and develop a system that can increase the efficacy of glioblastoma treatment. To increase the cellular retention and survival we engineered MSC to form multicellular spheroids and cell sheets. To co-delivery multiple therapeutics we engineered MSC to form MSC/DNA-templated nanoparticle hybrid cluster to co-deliver drugs for cancer therapy. The system showed superior performance due to the increased retention of the cells and nanoparticle at the tumor site. Finally, to deliver drugs other peptide based we engineered graphene oxide cellular patches for mesenchymal stem cells. Graphene oxide can carry diverse therapeutics and can kill the cancer cells without affecting the cellular viability of MSC.

Les glioblastomes multiformes (GBM) comptent parmi les tumeurs au pronostic le plus sombre. L’extraordinaire capacité invasive des cellules tumorales rend toutes les interventions thérapeutiques actuelles totalement impuissantes. Une sous-population de Cellules Souches de Glioblastome (CSG) hautement invasive est responsable de la récurrence tumorale. Dans le cerveau, les GBM migrent principalement le long des vaisseaux sanguins au sein de l’espace périvasculaire riche en laminine, fibronectine et collagène ainsi qu’en suivant l’alignement des fibres myélinisées du corps calleux. La Matrice Extracellulaire (MEC) de ces régions joue un rôle important dans l’invasion des GBM, mais les mécanismes mis en jeu n’ont pas été complètement dévoilés. De plus, le développement de nouvelles thérapies anti-migratrices ciblant l’interaction des GBM avec la MEC reste encore limité. Dans le but de mimer la composition biochimique et les propriétés mécaniques de la MEC cérébrale et d’étudier leur rôle(s) dans la migration des CSG, nous avons développé un nouveau support de nanofibres (NF) alignées et fonctionnalisées avec de la laminine. Mes travaux de thèse ont montré que les NF génèrent un microenvironnement tridimensionnel (3D) favorisant l’adhésion et la migration des CSG. Cette adhésion est améliorée en comparaison avec les supports planaires (SP) conventionnels (2D) et récapitule mieux les mécanismes d’interaction des CSG avec la MEC au cours de l’invasion dans le modèle murin de tumeurs xénogreffées. Dans ces conditions physiologiques plus convenables générées par les NF, la variation des composantes biochimiques et mécaniques de la MEC affecte la migration des CSG. La présence ou l’absence de laminine régule le mode migratoire et l’orientation de fibres contrôle la direction de migration des CSG. D’un autre coté, l’altération de la glycosylation des protéines de la surface cellulaire module l’interaction des cellules tumorales du cerveau avec la MEC et augmente leur invasion. La deuxième partie de mes travaux de thèse a permis de démontrer que les glycomimétiques phostines « 3.1a » réorganisent le processus de la N-glycosylation des CSG diminuant leur invasivité in vitro et in vivo en inhibant les voies de signalisation de la kinase FAK et du récepteur de TGF-β impliqués dans l’interconnexion cellule-MEC. / Glioblastoma Multiforme (GBM) is a biologically aggressive tumor with an extremely poor prognosis. The highly invasive capacity of a subpopulation of Glioblastoma Initiating Cells (GIC) makes complete surgical resection impossible. GBM dissemination occurs along preexisting brain structures such as the perivascular space rich in laminin, fibronectine and collagen as well as the aligned myelinated fibers of the corpus callosum. The Extracellular Matrix (ECM) of these cerebral regions plays an important role during GBM invasion, but the underlying mechanisms remain largely unknown. Accordingly, the development of new anti-migratory therapies targeting the cell-ECM interactions is lacking. In order to mimic the compositional and physical properties of the cerebral ECM and to investigate their role(s) in GBM invasion, we have set up a new aligned nanofibers (NF)scaffold functionalized with laminin. My work demonstrated that the NFs constitute a tridimensional (3D) microenvironment supporting GIC adhesion and migration. The cell-ECM adhesion is improved on the NF in comparison to the conventional 2D planar surfaces (PS). Furthermore, the mechanisms of GIC interaction with the ECM on the NF are similar to those observed in the human GBM xenograft murine model. In this physiologically more relevant 3D microenvironment reproduced by the NF, the variation of the different biochemical and mechanical components of the ECM affects the migration of GIC. The presence or absence of laminin on the NF regulates the mode of migration and the orientation of the fibers dictates the direction of migration of GIC. On the other hand, the glycosylation that decorates cell surface proteins modulates the interaction of GBM tumor cells with the ECM and its alteration increases their invasion. The second part of my thesis demonstrated that the glycomimetics phostines « 3.1a » remodel the N-glycosylation of GIC and decrease their invasivity in vitro and in vivo via the inhibition of FAK and TGFβ-R signaling pathways known to be implicated in the cell-ECM intercommunication.

Glioblastoma multiforme (GBM) is the most lethal primary central nervous system tumor, with median survival after diagnosis of less than 12 months because dissemination into the brain parenchyma limits the long-term effectiveness of surgical resection, and because GBM cells are resistant to radiation and chemotherapy. This sad dismal prognosis for patients with GBM emphasizes the need for greater understand of the fundamental biology of the disease. Invasion is one of the major causes of treatment failure and death from glioma, because disseminated tumor cells provide the seeds for tumor recurrence. Inflammation is increasingly recognized as an important component of invasion. In the brain, inflammation can occur by activation of microglia, the resident macrophages of the brain, or by tumor-associated blood macrophages. Therefore, we hypothesize that activity of the innate immune system in the brain can influence tumor progression by secreting cytokines such as Tumor Necrosis Factor alpha (TNF-α). In this study, we show that patient-derived glioma spheres undergo morphological changes in response to TNF‑α that are associated with changes in migration behavior in vitro. These morphological changes include appearance of tumor islands in site different from where the primary tumor cells were seeded. We further showed that TNF‑α treated cells significantly increased expression of cell adhesion molecules such as CD44 and VCAM-1. Furthermore, we demonstrate increased cell density also caused increased in expression of cell adhesion molecules. The extent to which these are recapitulated in vivo will be investigated.

Astrocytic tumors are the most common malignancies of the central nervous system, accounting for more than 60% of all primary brain tumors. The prognosis for high grade astrocytoma patients is dismal and there is no curative treatment, today. A molecular hallmark of astrocytic tumors is dysregulated receptor tyrosine kinase signaling, especially of the epidermal growth factor receptor (EGFR, ErbB1). The aim of the present thesis was to identify endogenous human proteins that downregulate the function of the ErbB1 receptor. We identified a human gene family, the leucine-rich repeats and immunoglobulin-like domains family (LRIG), consisting of LRIG1, LRIG2 and LRIG3, which might fulfill this criterion. Two candidates were identified, LRIG1 and LRIG2, which genes were localized to regions frequently deleted in human cancers, chromosome bands 3p14 and 1p13, respectively. LRIG1 and LRIG2 mRNA were expressed in all tissues analyzed, with high expression in brain and other organs. The LRIG mRNA were predicted to encode integral membrane proteins. Antibodies against LRIG1 and LRIG2 were developed and the protein expression was analyzed. LRIG1 was found to have an apparent molecular weight of 143 kDa and was expressed in most tissues with high expression in glandular tissues of the breast and prostate, and the peptic cells of the stomach. LRIG2 was slightly smaller and had an apparent molecular weight of 132 kDa. The LRIG proteins were localized to the cell surface and to perinuclear structures, where LRIG1 co-localized with the trans-Golgi network and early endosomes. LRIG1 was found to restrict growth factor signaling by enhancing receptor ubiquitylation and degradation. We showed that LRIG1 interacted with all four members of the ErbB family and induced their downregulation. The interaction with ErbB1 involved both the LRR-domains and the Ig-like domains of LRIG1. LRIG1 enhanced receptor degradation by recruiting the E3 ubiquitin ligase c-Cbl to the LRIG1-ErbB1 complex. LRIG1, LRIG2, and LRIG3 were expressed in glioma cell lines and tumor tissues. The LRIG expression was analyzed in 404 astrocytic tumor samples. We found that perinuclear LRIG protein expression correlated with increased survival of patients with astrocytic tumors. Especially perinuclear LRIG3 showed strong correlations with patient survival and tumor cell proliferation index. In summary, this thesis contains the discovery and characterization of human LRIG1 and LRIG2. LRIG1 was found to interact with ErbB receptors and downregulate their function. In a clinical material, expression of LRIG proteins correlated with survival in patients with astrocytic tumors.

Η ΒΥΠ διαθέτει αντίτυπο της διατριβής σε έντυπη μορφή στο βιβλιοστάσιο διδακτορικών διατριβών που βρίσκεται στο ισόγειο του κτιρίου της. / Τα κακοήθη γλοιώματα συνιστούν τον πιο κοινό τύπο καρκίνου του κεντρικού νευρικού συστήματος στους ενήλικες και χαρακτηρίζονται από υψηλό βαθμό αγγειογένεσης και διήθησης στον παρακείμενο φυσιολογικό ιστό. Αν και οι θεραπευτικές προσεγγίσεις που χρησιμοποιούνται περιλαμβάνουν χειρουργική επέμβαση, ακτινοθεραπεία και χημειοθεραπεία, ποσοστό μεγαλύτερο του 90% των ασθενών εμφανίζουν υποτροπή της νόσου και η συνολική επιβίωση είναι εξαιρετικά χαμηλή. Τα παραπάνω γεγονότα εντείνουν την ανάγκη για την εφαρμογή πιο αποτελεσματικών θεραπευτικών σχημάτων, καθώς και την ανακάλυψη νέων μορίων-στόχων για τη θεραπεία του συγκεκριμένου τύπου καρκίνου. Η HARP είναι ένας εκκρινόμενος αυξητικός παράγοντας με μοριακό βάρος 18 kDa που έχει ισχυρή συγγένεια με την ηπαρίνη. Η HARP εκφράζεται κυρίως κατά την εμβρυϊκή ανάπτυξη και απομονώθηκε για πρώτη φορά ως πρωτεΐνη που προάγει την επέκταση των νευριτών στον αναπτυσσόμενο εγκέφαλο. Στην HARP έχει αποδοθεί ένας σημαντικός αριθμός βιολογικών δράσεων, όπως η συμμετοχή της στον πολλαπλασιασμό, στη μετανάστευση και στη διαφοροποίηση των κυττάρων, στο σχηματισμό οστών, στη χονδρογένεση, στη σπερματογένεση, στην ογκογένεση και στην αγγειογένεση. Η HARP εκφράζεται σε διάφορες καρκινικές σειρές από μηνιγγίωμα, νευροβλάστωμα, αστροκυττάρωμα, μελάνωμα, μικροκυτταρικό τύπο καρκίνου του πνεύμονα και γλοιοβλάστωμα. Κλινικές μελέτες έχουν αναφέρει αυξημένα επίπεδα της HARP στον ορό αίματος ασθενών με καρκίνο του κόλον του εντέρου, με καρκίνο του παγκρέατος και με καρκίνο του μαστού. Στην παρούσα μελέτη, προσπαθήσαμε να διερευνήσουμε το ρόλο της HARP στην ανάπτυξη όγκου και στην αγγειογένεση που προκαλούν τα κύτταρα C6, μετασχηματίζοντας τα κύτταρα αυτά με την αντινοηματική αλληλουχία για το cDNA της HARP και καταστέλλοντας την έκφρασή της στα κύτταρα C6. Μείωση της ενδογενούς έκφρασης της πρωτεΐνης της HARP στα κύτταρα C6 (κύτταρα AS-C6) προκάλεσε στατιστικά σημαντική αύξηση στον πολλαπλασιασμό, στην ανάπτυξη των κυττάρων σε τρισδιάστατο περιβάλλον και στη μετανάστευση. Όταν κύτταρα AS-C6 εμβολιάστηκαν σε χοριοαλλαντοϊκή μεμβράνη εμβρύου όρνιθας, οδήγησαν σε σημαντική αύξηση της καρκινικής αγγειογένεσης, σε σύγκριση με τις μεμβράνες που είχαν εμβολιαστεί με μη μετασχηματισμένα κύτταρα C6 ή με κύτταρα C6 που είχαν μετασχηματιστεί μόνο με το πλασμίδιο-φορέα (κύτταρα ΡC-C6). Με παρόμοιο τρόπο, θρεπτικό μέσο καλλιέργειας των κυττάρων AS-C6 προκάλεσε στατιστικά σημαντική αύξηση στον πολλαπλασιασμό, στη μετανάστευση και στο σχηματισμό ψευδαγγείων των ενδοθηλιακών κυττάρων in vitro σε σύγκριση με την επίδραση του θρεπτικού μέσου καλλιέργειας των κυττάρων C6 και ΡC-C6. Η έκφραση της HARP φαίνεται να είναι απαραίτητη για την ανασταλτική δράση αυξημένων ενδοκυτταρικών επιπέδων cAMP στον πολλαπλασιασμό και τη μετανάστευση των κυττάρων C6 και αύξηση των ενδοκυτταρικών επιπέδων cAMP οδήγησε σε αύξηση της έκφρασης και έκκρισης HARP, πιθανά μέσω ενεργοποίησης του μεταγραφικού παράγοντα AP-1. Τέλος, παρατηρήθηκε σημαντική αύξηση των επιπέδων του mRNA και της πρωτεΐνης του αυξητικού παράγοντα των ενδοθηλιακών κυττάρων (vascular endothelial growth factor, VEGF) σε καλλιέργειες των κυττάρων AS-C6 σε σχέση με τα κύτταρα C6 και PC-C6. H HARP βρέθηκε να συν-ανοσοκατακρημνίζεται με τον VEGF σε θρεπτικό μέσο καλλιέργειας των κυττάρων C6 και pC-C6, το οποίο υποδηλώνει μια άμεση αλληλεπίδραση μεταξύ των δύο αυτών αυξητικών παραγόντων. Συνοψίζοντας, τα αποτελέσματα της παρούσας εργασίας προτείνουν ότι ο αυξητικός παράγοντας HARP έχει αρνητικό ρόλο στη ρύθμιση της ανάπτυξη όγκου και της αγγειογένεση που επάγουν τα κύτταρα C6, πιθανά ρυθμίζοντας την έκφραση ή/και τη διαθεσιμότητα άλλων αυξητικών παραγόντων. / Malignant gliomas, the most common type of brain tumors in adults, present a remarkable degree of neovascularisation and invasiveness into surrounding tissues. Although combinations of surgery, radiotherapy and chemotherapy are used, more than 90% of the patients experience local recurrence and their survival is extremely low. These facts stress the need for more effective therapeutic strategies and new targets for tumour therapy. Heparin affin regulatory peptide (HARP), also known as pleiotrophin or heparin-binding growth-associated molecule, is an 18-kDa secreted growth factor that has high affinity for heparin. HARP is mainly expressed during embryonic development, in early postnatal rat and bovine brain and was first isolated as a major neurite outgrowth-promoting protein of developing brain. Since then, a number of biological activities have been well established for HARP, such as its role in cellular proliferation, migration and differentiation and its involvement in bone formation, chondrogenesis, spermatogenesis, tumor growth and angiogenesis. HARP is expressed in various cancer cell lines, derived from meningiomas, neuroblastomas, astrocytomas, melanomas, small cell lung cancer (SCLC) cell lines and glioblastomas. Clinical studies have shown elevated serum levels and tumor expression of HARP in patients with colon, stomach, pancreatic and breast cancer. Ιn the present study, we tried to elucidate the role of HARP in the growth and angiogenicity of C6 glioma cells by using an antisense strategy for inhibition of HARP expression in rat C6 glioma cells. Decrease of the expression of endogenous HARP protein in C6 cells (AS-C6 cells) significantly increased proliferation rate and anchorage-independent growth of cells. Implantation of AS-C6 cells onto chicken embryo chorioallantoic membranes resulted in a significant increase of tumor-induced angiogenesis, compared with those induced by non transfected or C6 cells transfected with the plasmid alone (PC-C6 cells). In the same line, conditioned medium from AS-C6 cells significantly increased endothelial cell proliferation, migration and tube formation in vitro compared with the effect of C6 or PC-C6 cells. HARP expression is required for cAMP-induced inhibition of C6 cell proliferation and migration and cAMP increased the expression and secretion of HARP by C6 cells, possibly through activation of Activator Protein 1. Finally, a significant increase in vascular endothelial growth factor (VEGF) mRNA and protein levels were observed in cultures of AS-C6 cells compared with C6 or PC-C6 cells. HARP was co-immunoprecipitated with VEGF from the conditioned medium of C6 and PC-C6 cells, which indicates a direct interaction between the two factors. Collectively, these data suggest that HARP negatively regulates tumor growth and angiogenesis induced by C6, possibly through regulation of the expression or/and activation of other growth factors.

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Orientadora: Profa. Dra. Maria Cristina Carlan da Silva / Tese (doutorado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2018. / A relação entre a presença do Citomegalovírus Humano (HCMV) e diversos tipos de tumores tem sido alvo de investigação por décadas, e em especial, em Glioblastoma Multiforme (GBM). O GBM é o mais maligno tumor do Sistema Nervoso Central (SNC), com baixa sobrevida mesmo após o tratamento. Nosso grupo e outros demonstraram a presença do HCMV neste tipo de tumor restrita a uma pequena quantidade de células, além disso, vários mecanismos de aumento de malignidade tumoral mediados pelo HCMV já foram demonstrados in vitro e in vivo. As principais drogas utilizadas durante o tratamento quimioterápico, Temozolamida (TMZ) e Carmustina (BCNU), levam a adição de adutos no DNA, criando erros durante a replicação celular. Estudos demonstram que o HCMV pode modular vias de reparo de DNA em fibroblastos, entretanto, o possível efeito durante a quimioterapia não é conhecido. Com isto, neste trabalho avaliou-se a infecção viral de linhagens celulares de GBM bem como a resposta das mesmas ao tratamento com TMZ e BCNU. Demonstrou-se que linhagens de GBM U87MG, A172, U251MG e TP365MG são suscetíveis a infecção por HCMV, mas não igualmente permissíveis as linhagens virais laboratoriais (AD169) e clínica (TB40), provavelmente refletindo a infectividade do vírus nos tumores. Observou-se também uma diminuição na viabilidade celular nas linhagens TP365MG e U251MG infectadas com HCMV, de maneira dependente da multiplicidade de infecção (MOI). Na presença concomitante de drogas e vírus também ocorre a diminuição de viabilidade MOI dependente, especificamente em TP365MG tratada com BCNU. Em suma, os resultados indicam que a permissibilidade do HCMV em células de GBM é determinada por fatores celulares/hospedeiro e não por fatores virais. Por fim, o vírus também não aumenta a resistência ao tratamento por TMZ e BCNU em linhagens U251MG e TP365MG, entretanto, devido a heterogeneidade tumoral mais estudos são necessários em outros tipos celulares, especialmente em células tronco tumorais, levando a um melhor entendimento do possível efeito do vírus durante o tratamento quimioterápico / The relationship between the Human Cytomegalovirus (HCMV) and different types of tumors has been investigated for many decades, especially in Glioblastoma Multiforme (GBM). GBM is the most malignant tumor of the Nervous Central System (SNC), with a low overall survival even after treatment. We and others demonstrated the presence of HCMV in this type of tumor, restricted to a small number of cells, and various studies showed that the virus can increase cell malignancy both in vitro and in vivo. The chemotherapeutic agents Temozolomide (TMZ) and Carmustine (BCNU) used in GBM treatment lead to insertion of adducts in the DNA, creating errors during replication. It is well known that HCMV can modulate DNA repair pathways in fibroblasts, however, the possible effect of the virus during chemotherapy is not known. In this work we analyzed viral infection in GBM cell lines and their response to the TMZ and BCNU. We demonstrated that A172, U251MG and TP365MG GBM cell lines are susceptible to HCMV infection, but not equally permissive to both laboratory (AD169) and clinical strains (TB40), likely reflecting infection in the tumor. Additionally, we show a decrease in cell viability in TP365MG and U251MG infected with HCMV, in a MOI dependent manner. In infected and treated with chemotherapeutic drugs cells, there is also a reduction in cell viability MOI dependent in TP365MG treated with BCNU. Overall our results indicate that HCMV permissiveness in GBM cells is determined by host/cellular rather than viral factors and that the virus does not increase cell resistance to chemotherapeutic drugs in U251MG and TP365MG. Due to the tumor heterogeneity, more studies need to be performed in other tumor cells, especially in cancer stem cells, to lead to a better understanding of the possible effect of the virus during chemotherapy.