Gliomas are the most common human brain tumors and are divided into four stages by WHO classification scheme. Benign gliomas are defined as grades I (Pilocytic astrocytomas) and II (Astrocytomas), whereas grade III (Anaplastic Astrocytomas, AA) and grade IV (Glioblastoma Multiforme, GBM) are malignant. Although both grades III and IV are malignant, the prognoses for these tumors are quite different. The 2-year survival rate for grade III gliomas is 50%, and grade IV is < 20 %. Mechanisms of tumorigenesis are not exactly elucidated in brain tumor cells. The thesis is to study the role of p27 kip1 in human malignant brain tumors. The experimental methods include ribonuclease protection assay (RPA), western blotting, immunohistochemical staining and immunocytochemical staining. mRNAs of p130, p107, Rb, p53 and p27 kip1 in normal brain tissues and brain tumors were overexpressed in most case. The p27kip1 mRNA were expressed in all astrocytomas and GBM, and mRNA quantity of p27kip1 were more in brain tumors than in normal brain tissues. PI3K/Akt pathway regulates several cellular functions such as cell survival and cell proliferation. Active Akt can phosphorylate p27kip1 that may contribute cell cycle from G1 phase to S phase. Skp2 identifies phospho-p27kip1 and promotes p27kip1 degradation. We found p27kip1 overexpression and Akt activation in astrocytomas and GBM. The expression of p-Akt were found in 20 %, 87 % and 71 % in normal brain tissues, astrocytomas and GBM, respectively. Expression of p27kip1 and p-Akt has shown significant correlation in GBM (P = 0.0236). Overexpression of p27kip1 mRNA in brain tumors may be consequence of p-Akt and Skp2.

O Celecoxib, antiinflamatório não esteroidal, inibidor seletivo da COX-2, tem se mostrado um importante agente anticarcinogênico, mas o seu papel no carcinoma epidermóide de boca (CEB) não é totalmente compreendido. Sabe-se que diversas alterações genéticas estão associadas à patogênese do CEB, a neoplasia maligna mais comum de cabeça e pescoço. Algumas dessas alterações comprometem proteínas pertencentes à via de sinalização do Akt, envolvida em diferentes fenômenos celulares. É sabido que Akt pode ativar o fator de transcrição NF-kB, o qual tem importante participação na fisiologia normal e no câncer. A proteína COX-2, descrita inicialmente em processos inflamatórios, está associada com a oncogênese e recentemente tem sido associada com a via de sinalização do Akt e com o NF-kB. Portanto, o objetivo deste estudo foi analisar o efeito do Celecoxib sobre linhagens celulares de carcinoma epidermóide de boca e verificar a localização intracelular e a expressão das proteínas pAkt, NF-kB e COX-2 em linhagens celulares de carcinoma epiermóide de boca após o tratamento com o Celecoxib.Através da técnica de imunofluorescência, foram analisados os padrões de expressão das proteínas pAkt, NFkB e COX-2 em quatro linhagens celulares de carcinoma epidermóide bucal submetidas ao tratamento com Celecoxib, cuja a dose e o tempo foram obtidos a partir de ensaios de viabilidade celular. Também se realizou ensaio de apoptose celular. Como controle utilizou-se células não tratadas com o medicamento. O Celecoxib na dose de 30 M por 24 horas causa apoptose.Na técnica de western blot, somente a linhagem SCC15 apresentou uma diminuição significativa para a COX-2. Entretanto, para p-Akt e NF-kB nenhuma alteração na expressão foi observada entre os grupos controle e tratado.Na imunofluorescência, houve alteração no padrão de expressão das proteínas pAkt, NF-kB e COX-2, quando se comparou os grupos contrele e tratado. Portanto, o Celecoxib pode ser um eficaz agente terapêutico, uma vez que demonstrou grande eficácia na inibição da proliferação celular de linhagens celulares de CEB. / Celecoxib, nonsteroidal anti-inflammatory COX-2 selective inhibitor, has proven to be an important anticancer agent. However its role in oral squamous cell carcinoma (OSCC) is not entirely understood. This is the most common malignancy of head and neck regionand it is known that various genetic alterations are associated with its pathogenesis. Some of these changes affect proteins belonging to the Akt signaling pathway, involved in different cellular processes. It is known that Akt can activate the transcription factor NF-kB, which has important role in normal physiology and cancer.The COX-2 proteinwas firstly described in the inflammatory processes, is associated with oncogenesis and has recently been related with the Akt signaling pathwayand with NF-kB. Therefore, the aim of this study was to analyze the effect of Celecoxib on squamous cell carcinoma cell lines and to determine pAkt, NF-kB and COX-2 intracellular localization and levels of expressionin this cell lines after treated with Celecoxib. By immunofluorescence, we analyzed the pAkt, NF-kB and COX-2 expression patterns in four oral squamous cell carcinoma cell lines treated with Celecoxib, which the dose and time were obtained from cell viability assays. Cellular apoptosis assay was also performed. As control the cells were not treated with this drug. Celecoxibcauses apoptosisin the dose of 30 M for 24 hours. In the western blottechnique, only the SCC15 cell line shows a significant decrease for COX-2. However, for p-Akt and NF-kB no change in expression was observed between control and treated groups. On immunofluorescence, there were changes in the pAkt, NFkB and COX-2protein expression pattern when the control group was compared with treated group. Therefore, the Celecoxib can be an effective therapeutic agent, since it has shown great efficacy in thecelular proliferation inhibition of the OSCC cell lines.

O Celecoxib, antiinflamatório não esteroidal, inibidor seletivo da COX-2, tem se mostrado um importante agente anticarcinogênico, mas o seu papel no carcinoma epidermóide de boca (CEB) não é totalmente compreendido. Sabe-se que diversas alterações genéticas estão associadas à patogênese do CEB, a neoplasia maligna mais comum de cabeça e pescoço. Algumas dessas alterações comprometem proteínas pertencentes à via de sinalização do Akt, envolvida em diferentes fenômenos celulares. É sabido que Akt pode ativar o fator de transcrição NF-kB, o qual tem importante participação na fisiologia normal e no câncer. A proteína COX-2, descrita inicialmente em processos inflamatórios, está associada com a oncogênese e recentemente tem sido associada com a via de sinalização do Akt e com o NF-kB. Portanto, o objetivo deste estudo foi analisar o efeito do Celecoxib sobre linhagens celulares de carcinoma epidermóide de boca e verificar a localização intracelular e a expressão das proteínas pAkt, NF-kB e COX-2 em linhagens celulares de carcinoma epiermóide de boca após o tratamento com o Celecoxib.Através da técnica de imunofluorescência, foram analisados os padrões de expressão das proteínas pAkt, NFkB e COX-2 em quatro linhagens celulares de carcinoma epidermóide bucal submetidas ao tratamento com Celecoxib, cuja a dose e o tempo foram obtidos a partir de ensaios de viabilidade celular. Também se realizou ensaio de apoptose celular. Como controle utilizou-se células não tratadas com o medicamento. O Celecoxib na dose de 30 M por 24 horas causa apoptose.Na técnica de western blot, somente a linhagem SCC15 apresentou uma diminuição significativa para a COX-2. Entretanto, para p-Akt e NF-kB nenhuma alteração na expressão foi observada entre os grupos controle e tratado.Na imunofluorescência, houve alteração no padrão de expressão das proteínas pAkt, NF-kB e COX-2, quando se comparou os grupos contrele e tratado. Portanto, o Celecoxib pode ser um eficaz agente terapêutico, uma vez que demonstrou grande eficácia na inibição da proliferação celular de linhagens celulares de CEB. / Celecoxib, nonsteroidal anti-inflammatory COX-2 selective inhibitor, has proven to be an important anticancer agent. However its role in oral squamous cell carcinoma (OSCC) is not entirely understood. This is the most common malignancy of head and neck regionand it is known that various genetic alterations are associated with its pathogenesis. Some of these changes affect proteins belonging to the Akt signaling pathway, involved in different cellular processes. It is known that Akt can activate the transcription factor NF-kB, which has important role in normal physiology and cancer.The COX-2 proteinwas firstly described in the inflammatory processes, is associated with oncogenesis and has recently been related with the Akt signaling pathwayand with NF-kB. Therefore, the aim of this study was to analyze the effect of Celecoxib on squamous cell carcinoma cell lines and to determine pAkt, NF-kB and COX-2 intracellular localization and levels of expressionin this cell lines after treated with Celecoxib. By immunofluorescence, we analyzed the pAkt, NF-kB and COX-2 expression patterns in four oral squamous cell carcinoma cell lines treated with Celecoxib, which the dose and time were obtained from cell viability assays. Cellular apoptosis assay was also performed. As control the cells were not treated with this drug. Celecoxibcauses apoptosisin the dose of 30 M for 24 hours. In the western blottechnique, only the SCC15 cell line shows a significant decrease for COX-2. However, for p-Akt and NF-kB no change in expression was observed between control and treated groups. On immunofluorescence, there were changes in the pAkt, NFkB and COX-2protein expression pattern when the control group was compared with treated group. Therefore, the Celecoxib can be an effective therapeutic agent, since it has shown great efficacy in thecelular proliferation inhibition of the OSCC cell lines.

Les stratégies actuelles de lutte contre le cancer consistent à développer de nouveaux traitements à base de molécules d’origine naturelle susceptibles de déclencher l’apoptose de cellules malignes et d’inhiber les principales voies de survie cellulaire. Le premier objectif de ces travaux porte sur les effets anti-prolifératifs et apoptotiques de l’extrait éthanolique de la plante Berberis libanotica sur les lignées érythroleucémiques humaines HEL, K562 et K562 (COX-2+) et sur son effet sur l’expression de la COX-2 dans ces lignées. Nos résultats montrent que l’extrait induit l’apoptose dans les lignées étudiées et ceci par activation de la caspase-3, le clivage de la PARP-1 et la fragmentation de l’ADN. De même, il induit la diminution de l’expression de COX-2. Nous avons démontré que les voies survie de NF-ĸB et p-AKT sont inhibées. Le deuxième objectif de ces travaux consistent à identifier la molécule présente dans cet extrait et qui est capable de déclencher ces effets anticancéreux. Nous avons démontré que la berbérine est la molécule majoritaire dans cet extrait et possède des effets apoptotiques et des effets inhibiteurs des voies de survie, ce qui est similaire aux effets de l’extrait brut.Mots clés :érythroleucémie, apoptose, berberis libanotica, berbérine, COX-2, NF-kB, p-AKT. / The first aim of this study focuses on the apoptotic effect of Berberis Libanotica on human erythroleukemia cell lines HEL, K562, and K562(COX-2+) and it is effect on the expression of COX-2. In light of the reported chemopreventive and chemosensitive effects of natural products on various tumor cells and animal models, we postulated that our Bl extract may mediate their effects through apoptosis induction with suppression of cell survival pathways. We showed that this extract induces apoptosis in eryrhtroleukemia cells by activation of the late markers of caspase-3 activation, PARP cleavage and DNA fragmentation. In the other hand, we showed that Bl extract reduced significantly expression of COX-2 by a dose-dependent manner. In regard to our results, it is clear that the simultaneous inhibition of Akt and NF-κB signalling can significantly contribute to the anticancer effects of Bl extract in human erythroleukemia cells.The second objective of this report is to elucidate wich molecule present in our extract can exert this effects. We found that berberine, the majoritory compound, can induce an apoptotic effect and inhibits the survival pathway of NF-ĸB and p-AKT similarly to the extract.Key words: erythroleukemia, apoptosis, Berberis libanotica ,berberine, COX-2, NF-ĸB, p-AKT

Η ηπατική καρκινογένεση είναι μια πολυσταδιακή διαδικασία που οδηγεί προοδευτικά στον κακοήθη μετασχηματισμό του ηπατικού κυττάρου, μέσω ποικίλων μοριακών μηχανισμών. Πρόσφατα αναδεικνύεται ολοένα και σε μεγαλύτερο βαθμό, η τεράστια σημασία που έχει για την ανάπτυξη και εξέλιξη του καρκίνου το μικροπεριβάλλον του όγκου, η αλληλεπίδραση δηλαδή των καρκινικών κυττάρων με την εξωκυττάρια ουσία. Ιδιαίτερο ενδιαφέρον παρουσιάζει η αλληλεπίδραση των καρκινικών κυττάρων με την εξωκυττάρια ουσία μέσω των ιντεγκρινών, η οποία φαίνεται ότι εμπλέκεται σε όλα τα στάδια καρκινογένεσης. Σημαντικό μόριο στην προαναφερθείσα διαδικασία αποτελεί η ILK (Integrin-Linked Kinase), μία κινάση σερίνης-θρεονίνης που παρεμβάλλεται στα σηματοδοτικά μονοπάτια που ξεκινούν από ιντεγκρίνες, αυξητικούς παράγοντες και συμμετέχει στη ρύθμιση κομβικών για το καρκινικό κύτταρο λειτουργιών όπως έλεγχο του κυτταρικού κύκλου, απώλεια των δομών συνοχής του κυττάρου, αναστολή της απόπτωσης και ενεργοποίηση της αγγειογένεσης. Διαπιστώθηκε πως το σηματοδοτικό μονοπάτι ILK/p-Akt διαδραματίζει κομβικό ρόλο τόσο στη βιολογία της κίρρωσης όσο και του ηπατοκυτταρικού καρκίνου και πιθανόν αποτελεί μοριακό σύνδεσμο μεταξύ των δύο καταστάσεων, επιβεβαιώνοντας τη διατυπωμένη θεωρία περί άξονα χρόνιας φλεγμονής-ίνωσης (κίρρωσης)-καρκίνου. Επίσης στην παθογένεια της κίρρωσης και του καρκίνου ήπατος, φαίνεται πως ρόλο έχει το φαινόμενο της επιθηλιακής προς μεσεγχυματική μετατροπή (EMT-epithelial to mesenchymal transition). Αυτό αποδεικνύεται από την απώλεια έκφρασης της E-καντχερίνης και τη μεταφορά της β-κατενίνης στον πυρήνα που οδηγούν σε απώλεια των κυτταρικών συνδέσεων από τη μεμβράνη καθώς και από την υπερέκφραση των μορίων ILK και p-Akt. Τέλος η υπερέκφραση survivin και κυκλίνης-D1 στην κίρρωση και στο ηπακυτταρικό καρκίνωμα καταδεικνύουν τη σημασία της αντιαποπτωτικής δραστηριότητας και του αυξημένου κυτταρικού πολλαπλασιασμού στις συγκεκριμένες νοσολογικές οντότητες. / The hepatic carcinogenesis is a many phased process that leads progressively to the malignant transformation of hepatic cell via various molecular mechanisms. Recently, the enormous importance of the tumor microenvironment for the growth and development of cancer, i.e. the interaction between cancer cells and the extacellular matrix, has been proven. Particular interest is presented in the interaction of tumor cells with the extracellular matrix via integrins, which appears to be involved in all the stages of carcinogenesis. An important molecule in the aforementioned process is the ILK (Integrin-Linked Kinase), a serine-threonine kinase that is implicated in the signal transduction pathways that begin from integrines, growth factors and participate in the regulation of nodal for the tumor cell functions, such as control of cell cycle, loss of cell adhesion structures, suppresion of apoptosis and activation of angiogenesis. It has been realized that the signal transduction pathway ILK /p-Akt plays a nodal role so much in the biology of cirrhosis as well as in hepatocellular cancer and probably constitutes a molecular connection between the two conditions, confirming the formulated theory of axis of chronic inflamation-chirrosis-cancer. Also in the pathogenicity of cirrhosis and liver cancer, it appears that the epithelial to mesenchymal transition (EMT) phenomenon plays a role. This is proven by the loss of expression of E-cadherin and the transport of b-catenin in the nucleus that leads to loss of cellular connections from the membrane as well as from the overexpression of molecules ILK and p-Akt. Finally, the overexpression of survivin and cyclin-D1 in cirrhosis and hepatocellular carcinoma show the importance of antiapoptotic activity and increased cellular proliferation in the particular disease entities.

Le cancer de la vessie représente un vrai problème de santé publique, avec une surveillance et suivi clinique à long terme en raison de l’importance des fréquences de récidives. La chimiothérapie reste souvent inefficace. L’objectif de cette thèse est donc la recherche de marqueurs sérologiques et moléculaires à valeur pronostique dans le cancer de la vessie qui peuvent servir à prédire la maladie. D’abord, nous avons étudié trois facteurs de transcriptions des lymphocytes T activées qui sont T-bet, GATA-3 et Bcl-6. Nous avons montré une surexpression de T-bet chez les malades à stade invasif et de haut grade, cependant, la surexpression de GATA-3 et Bcl-6 a été corrélée au stade superficiel et de bas grade. La survie a été corrélée avec le groupe des malades sans histoires de récidive ou progression et avec la surexpression de Bcl-6 et GATA-3. Cependant les malades qui expriment fortement T-bet répondent mieux au BCG. Ensuite, nous avons visé la détection de FasL et TRAIL solubles dans le sérum des malades atteints du cancer vésicale. Nous avons montré une surexpression de sFasL et sTRAIL chez les malades à stade superficiel et de bas grade. Le rôle anti-tumoral de ces cytokines a été confirmé sur deux lignées du cancer de la vessie montrant que le traitement avec le sérum riche en sFasL ou en sTRAIL diminue la viabilité cellulaire in vitro. A la fin de cette thèse, nous avons testé l’activation p-Akt dans la tumeur vésicale. Nous avons montré une surexpression de p-Akt au sein des tumeurs comparées au tissu sain adjacent, et au sein des malades à stade invasif et de haut grade. Akt semble être un marqueur de progression tumorale dans le cancer de la vessie. / Bladder cancer is the first most common urogenital cancer in men in Tunisia, with a high recurrence rate. Patients with muscle-invasive disease develop metastasis. The need for expensive continuous surveillance. In this thesis we try to search some candidate biomarkers. Their use for cancer staging and personalization of therapy at the time of diagnosis in order to identify a better treatment could improve patient care. The aim of this first part of our study was to investigate the clinical significance of three immune cell-related transcription factors, T-bet, GATA-3 and Bcl-6 in Tunisian patients with bladder cancer. We found that T-bet level was significantly higher in invasive carcinoma with high- grade. However, T-bet is predictive of response to BCG. On the contrary, the expression of GATA-3 and Bcl-6 was significantly higher in non-invasive carcinoma with low grade. We furthermore studied the effect of activation of soluble FasL and TRAIL molecule in bladder cancer. We demonstrate that the mean serum level of sFasL was higher in patients than in normal donors. sFasL was only higher than in sera of healthy donors where patients had superficial stage and low- and medium-grade cancer. sTRAIL was significantly lower in sera from patients with invasive and high-grade bladder carcinoma than in controls. Finally, we demonstrate that p-Akt levels in patients with invasive carcinoma and high-grade bladder cancer were significantly elevated compared to patients with non-invasive and low grade bladder cancer. Altogether, our results suggest that Akt activation can provide useful prognostic information.