The Hedgehog (Hh) signaling pathway plays a critical role in modulating various developmental processes that requires fine tuning of the Hh signal, such that dysregulation can lead to cellular events involved in cancer. To elucidate the factors responsible for aberrant Hh activation and subsequent tumorigenesis, I investigated three distinct modulators of Hh signaling: (1) p53 tumour suppressor (2) primary cilia (3) PTHLH. During chondrocyte development, abnormal Hh signalling can result in benign cartilage tumours, called Enchondroma. As precursor lesions, enchondroma may progress to malignant neoplasia, collectively known as chondrosarcoma. Although the molecular events involved in this progression are poorly understood, inactivation of the p53 tumour suppressor has been identified in approximately one-third of chondrosarcoma. Using an enchondroma mouse model, I showed that p53 deficiency can cause chondrosarcoma to develop. The combined inhibitory effects of Hh and p53 pathways on the pro-apoptotic factor, IGFBP-3, suppressed apoptosis and was demonstrated to play a critical role in the progression to chondrosarcoma. The primary cilium is an organelle that serves as a signaling centre for the Hh pathway to allow for greater control of the signal output. Loss of primary cilia results in abnormal Hh signaling that is associated with cancer and various developmental defects. I observed a depletion of primary cilia in both human Chondrosarcoma and Enchondroma tumours compared to normal cartilage. Analysis of cilia-deficient mice revealed that defective ciliogenesis alone could lead to the formation of benign cartilage tumours. Furthermore, loss of primary cilia potentiated the effect of Hh signaling activation, revealing a novel role in cartilage tumorigenesis. Parathyroid-like hormone (PTHLH) is an essential inhibitor of the Hh pathway during chondrocyte development, however its function as a regulator of Hh in other tissue types are largely unknown. Through activation of PKA, PTHLH suppresses the activation of Gli transcription factors; downstream effectors of the Hh pathway. Using irradiated Ptch+/- mice that exhibit a high incidence of skin and brain tumours, I demonstrated that treatment with PTHLH agonist, PTH (1-34), results in inhibition of the Hh pathway, increased survival and a reduction in tumour incidence and size. Thus, PTH (1-34) may have therapeutic potential for Hhrelated cancers, especially given its known clinical safety in treating osteoporosis.

The Hedgehog (Hh) signaling pathway plays a critical role in modulating various developmental processes that requires fine tuning of the Hh signal, such that dysregulation can lead to cellular events involved in cancer. To elucidate the factors responsible for aberrant Hh activation and subsequent tumorigenesis, I investigated three distinct modulators of Hh signaling: (1) p53 tumour suppressor (2) primary cilia (3) PTHLH. During chondrocyte development, abnormal Hh signalling can result in benign cartilage tumours, called Enchondroma. As precursor lesions, enchondroma may progress to malignant neoplasia, collectively known as chondrosarcoma. Although the molecular events involved in this progression are poorly understood, inactivation of the p53 tumour suppressor has been identified in approximately one-third of chondrosarcoma. Using an enchondroma mouse model, I showed that p53 deficiency can cause chondrosarcoma to develop. The combined inhibitory effects of Hh and p53 pathways on the pro-apoptotic factor, IGFBP-3, suppressed apoptosis and was demonstrated to play a critical role in the progression to chondrosarcoma. The primary cilium is an organelle that serves as a signaling centre for the Hh pathway to allow for greater control of the signal output. Loss of primary cilia results in abnormal Hh signaling that is associated with cancer and various developmental defects. I observed a depletion of primary cilia in both human Chondrosarcoma and Enchondroma tumours compared to normal cartilage. Analysis of cilia-deficient mice revealed that defective ciliogenesis alone could lead to the formation of benign cartilage tumours. Furthermore, loss of primary cilia potentiated the effect of Hh signaling activation, revealing a novel role in cartilage tumorigenesis. Parathyroid-like hormone (PTHLH) is an essential inhibitor of the Hh pathway during chondrocyte development, however its function as a regulator of Hh in other tissue types are largely unknown. Through activation of PKA, PTHLH suppresses the activation of Gli transcription factors; downstream effectors of the Hh pathway. Using irradiated Ptch+/- mice that exhibit a high incidence of skin and brain tumours, I demonstrated that treatment with PTHLH agonist, PTH (1-34), results in inhibition of the Hh pathway, increased survival and a reduction in tumour incidence and size. Thus, PTH (1-34) may have therapeutic potential for Hhrelated cancers, especially given its known clinical safety in treating osteoporosis.

Lors d’une radiothérapie conventionnelle (Rayons X) ou non-conventionnelle (Hadronthérapie), l’impact des irradiations sur les tissus sains pose des questions essentielles de radiobiologie, ces tissus sains se trouvant sur le trajet du faisceau lors du traitement d’une tumeur. Parmi ces questions, quel est l’impact de l’effet Bystander radio-induit ? Ce mécanisme fait intervenir des signaux de stress encore mal identifiés, émis par une ou plusieurs cellules irradiées vers les cellules non irradiées adjacentes ou très proches, pouvant produire des effets biologiques proches de ceux obtenus dans la zone irradiée.Afin d’étudier ce phénomène, nous avons utilisé différentes techniques de biologie moléculaire dont : des tests de clonogénicité permettant d’étudier la survie des cellules après un traitement, des électrophorèses bidimensionnelles permettant l’analyse du protéome cellulaire, des analyses de milieux conditionnés permettant d’identifier les signaux Bystander émis par les cellules irradiées et des expériences de protéo-génomique visant à étudier des cellules en mélange. En parallèle de ces techniques d’étude, nous avons développé un modèle de culture de cellules en 3D via l’utilisation de « pellets » (agrégats cellulaires).Nos résultats ont montré une diminution de la survie des cellules bystander après transfert de milieu provenant de cellules irradiées, l'implication potentiel de certaines cytokines dans la signalisation bystander ainsi que plusieurs protéines candidates pouvant expliquer en partie la réponse bystander. / In conventional radiotherapy (X-ray) or unconventional radiotherapy (Hadrontherapy), the impact of irradiation on healthy tissue raises essential questions of radiobiology, these healthy tissues being in the path of the beam during the treatment of a patient. tumor. Among these questions, what is the impact of the radio-induced Bystander effect? This mechanism involves stress signals still poorly identified, emitted by one or more irradiated cells to adjacent or very close non-irradiated cells, which can produce biological effects close to those obtained in the irradiated zone.To study this phenomenon, we used various molecular biology techniques including: clonogenic assay to study the survival fraction of cells after treatment, two-dimensional electrophoresis allowing the analysis of the cellular proteome, conditioned medium analysis for identify Bystander signals emitted by irradiated cells and proteomembrane experiments aimed at studying cells in a mix. In parallel with these study techniques, we have developed a 3D cell culture model via the use of "pellets" (cell aggregates).Our results showed a decrease in the survival of bystander cells after transfer of medium from irradiated cells, the potential involvement of some cytokines in bystander signaling as well as several candidate proteins that may partly explain the bystander response.

Introduction: NG2/CSPG4 is a unique transmembrane chondroitin sulphate proteoglycan molecule expressed as a core protein and a chondroitin sulphate proteoglycan (CSPG) up to 400kD. NG2/CSPG4 mediates the communication between the extracellular and intracellular compartments through interactions with collagen VI, growth factors and the actin cytoskeleton. NG2/CSPG4 affects cell migration, spreading, apoptosis and proliferation processes. NG2/CSPG4 has been shown to be expressed in developing and adult cartilage where less is known of its function. I tested the hypothesis: NG2/CSPG4 is an important regulator of chondrocytes function and has the potential to be a therapeutic target for treatment of diseases of cartilage such as osteoarthritis and chondrosarcoma. To do this, I had the following aims: 1) investigate whether different types of chondrocytes show variation in the form or distribution of NG2/CSPG4 expression and 2) through a knockdown approach develop a model to study the functional roles of NG2/CSPG4 in human chondrocytes. Materials and Methods: JJ012, a chondrosarcoma cell line, chondrocytes derived from human articular cartilage and C20/A4 an immortalised chondrocyte cell line were used. NG2/CSPG4 expression was investigated by RT-PCR western blotting, flow-cytometry and immunocytochemistry. NG2/CSPG4 interaction with Golgi complex and endoplasmic reticulum (ER) was assessed by double immunofluorescence. Biochemical interactions were assessed by immunoprecipitation and mass spectroscopy. For NG2/CSPG knockdown, a viral transduction method was carried out using 5 different constructs. Different functional roles of NG2/CSPG4 were investigated. The role of NG2/CSPG4 in gene regulation was studied by shRNA knockdown of NG2/CSPG4 in JJ012 cells and RTPCR. Results: NG2/CSPG4 mRNA was detectable in all cells tested. Western blotting showed expression of only a 270kD core protein in JJ012 and C20A4 cells. Using two different anti NG2/CSPG4 antibodies human OA chondrocytes were seen to express multiple molecular weight forms differentially recognised with and without chondroitinase ABC pre-treatment. Expression of NG2/CSPG4 in JJ012 cells was predominantly membrane associated whilst in OA chondrocytes and C20A4 cells, additional, predominant punctuate cytoplasmic distribution was evident. In OA chondrocytes NG2/CSPG4 co-localised with the Golgi complex and ER. Immunoprecipitation and mass spectrometry data demonstrated associations between NG2/CSPG4 and both collagen VI and thrombopoietin in OA chondrocytes. A model of NG2/CSPG4 gene knockdown was achieved in JJ012 chondrosarcoma cell line, known as B3. B3 cells spread more and migrate less than JJ012 cells; with a significant difference observed in migration (after 10hours: the closed area was 81.4% for JJ012 and 54.6% for B3). There was no difference in cell adhesion to collagen I, II, VI and fibronectin. EGTA inhibited cell adhesion to fibronectin in dose dependent manner with no significant difference observed between both JJ012 and B3 cells. EDTA reduced adhesion of B3 cells but not JJ012 to fibronectin. A significant difference in cell proliferation was detected with no change in apoptosis. Following NG2/CSPG4 knockdown in JJ012 cells there was no difference in expression of aggrecan, collagen II and SOX-9. In contrast, B3 cells showed a decreased expression of MPP3 and ADAMTS-4, a complete loss of ADAMTS-5 and increased expression of MMP13. Conclusions: I have identified altered expression and multiple forms of NG2/CSPG4 in different types of chondrocytes and shown association of this molecule with type VI collagen and thrombopoietin. Creation of a chondrocyte cell line that has stable knockdown of NG2/CSPG4 allowed further investigation of NG2/CSPG function in chondrocytes. NG2/CSPG4 knockdown reduced the cellular migration and proliferation and increased the chondrocyte spreading. The adhesion mechanism in JJ012 appears to be calcium dependent. Loss of NG2/CSPG4 induced changes in expression of aggrecanases and MMPs. Altered expression or associations of NG2/CSPG4 with extracellular ligands or intracellular signalling cascades may be important in the pathogenesis of OA by regulating proteolytic activity or apoptosis related pathways. NG2/CSPG4 is a potential therapeutic target in degenerative and neoplastic diseases of cartilage.

Ce travail de thèse s’intègre dans la stratégie développée et brevetée par l’UMR 990 INSERM/UdA, qui consiste à exploiter l’affinité d’un vecteur ammonium quaternaire chargé positivement pour les protéoglycanes, afin d’acheminer des agents de diagnostic et/ou de thérapie, pour la prise en charge du chondrosarcome. Du fait de son abondante matrice extracellulaire de nature chondrogénique, de sa faible vascularisation et de son environnement hypoxique, le chondrosarcome est une tumeur chimio- et radio-résistante. Le seul traitement efficace à ce jour reste la chirurgie qui possède un taux de survie à 10 ans de seulement 21% en cas de rechute et dans les formes les plus graves. Dans ce contexte, une prodrogue cytotoxique activable en hypoxie dérivée du cyclophosphamide, de type nitroimidazole (8-QA) et possédant un ammonium quaternaire, a été développée et évaluée in vitro et in vivo, comparativement à son équivalent non « quaternisé » (8) et à un équivalent « quaternisé » mais non activable en hypoxie (10-QA). In vitro, les études par résonance plasmonique de surface (SPR) ont montré que les deux prodrogues « quaternisées », 8-QA et 10-QA possédaient une affinité plus importante pour l’agrécane que la prodrogue 8 ne possédant pas le vecteur ammonium quaternaire. Par la suite, nous avons développé un modèle de culture en 3 dimensions (sphéroïdes) des cellules de chondrosarcome humain HEMC-SS qui reproduit le microenvironnement hypoxique et riche en protéoglycanes retrouvé dans les tumeurs du cartilage in vivo. Les molécules activables en hypoxie 8-QA et 8 ont une activité cytotoxique supérieure en situation d’hypoxie qu’en normoxie, avec une résistance aux traitements augmentée pour le modèle de sphéroïde comparativement aux cellules cultivées en 2-D. In vivo, sur un modèle de xénogreffe de cellules de chondrosarcome humain HEMC-SS implantées sur des souris SCID, modèle préalablement caractérisé en terme d’hypoxie (imagerie photoacoustique, échographie de contraste, Doppler) et de protéoglycanes, la molécule 8-QA entraine (i) une inhibition significative de la croissance tumorale de 62,1%, alors que la molécule 8 et la molécule 10-QA n’ont aucune activité in vivo ; (ii) une diminution des effets indésirables, améliorant ainsi l’index thérapeutique. La caractérisation de l’activité antitumorale sur les tumeurs prélevées a mis en évidence une diminution de l’activité mitotique, une augmentation de l’apoptose (p53S15) et une diminution de la prolifération (PCNA) pour les échantillons traités avec la molécule 8-QA. De plus les études d’immunohistochimie ont montré que cette molécule entraine une augmentation des dommages à l’ADN (marquage γH2Ax) retrouvés majoritairement dans les régions hypoxiques (marquage au pimonidazole). La dernière partie de ce travail de thèse a consisté à optimiser, chez des souris NUDE, de nouveaux modèles in vivo de chondrosarcome humain exprimant à la fois un environnement hypoxique et une matrice extracellulaire riche en protéoglycanes pour affiner la caractérisation de l’activité anticancéreuse de la molécule 8-QA. Pour cela nous avons développé un modèle de xénogreffe de sphéroïdes HEMC-SS implantés en situation sous cutanée et un modèle orthotopique de cellules de chondrosarcome humain HEMC-SS implantées en position paratibiale. Après caractérisation, les premiers résultats d’efficacité antitumorale sur ces deux modèles ont confirmé une activité prometteuse de la molécule 8-QA. En conclusion, ces travaux de thèse auront permis de démontrer que l’exploitation des caractéristiques phénotypiques du chondrosarcome par une approche bi-spécifique est une stratégie prometteuse pour la prise en charge thérapeutique de cette pathologie, certes rare mais redoutable. Il a également contribué à montrer l’importance de l’interaction des cellules tumorales avec leur environnement pour la réponse aux traitements de chimiothérapie. / This work takes place in the strategy developed and patented by our lab UMR 990 INSERM/UdA, that exploits the affinity of positively charged quaternary ammonium to proteoglycans, in order to address diagnostic and/or therapeutic agents for chondrosarcoma management. Due to its abundant chondrogenic extracellular matrix, its poor vascularization and its hypoxic microenvironment, chondrosarcoma is chemo- and radio-resistant. The only effective treatment remains surgical resection that could be at the origin of a 10-years survival rate of 21% in case of local relapse of high-grade tumors. In this context, a quaternary ammonium-conjugated hypoxia-activable prodrug, 8-QA, has been developed and studied in vitro and in vivo respectively to its non-quaternary ammonium conjugated derivative, 8, and to a quaternary ammonium-conjugated non-hypoxia activable 10-QA. In vitro, surface plasmon resonance (SPR) studies evidenced that the two quaternary ammonium-conjugated derivatives, 8-QA and 10-QA, highlighted a higher affinity for aggrecan than non-quaternary ammonium conjugated prodrug 8. We have then developed a 3-D culture model (spheroid) of human chondrosarcoma HEMC-SS cell line that was demonstrated to reproduce the hypoxia and proteoglycan-rich microenvironment similar to in vivo cartilaginous neoplasms. Hypoxia-activated prodrugs, 8 and 8-QA, exhibited a higher cytotoxic activity in hypoxia as compared to normoxia, associated to an increased resistance for 3-D culture respectively to 2-D cells. In vivo, on human HEMC-SS xenograft model implanted on SCID mice and previously characterized in term of hypoxia (photoacoustic and contrast echographic imaging, Doppler), 8-QA derivative induced (i) a significant inhibition of tumor growth of 62,1% while 8 and 10- QA derivatives did not exhibit any antitumor activity; (ii) a decrease of side effects, both improving the therapeutic index. Mechanistic analysis on tumors sampled evidenced a decrease of mitotic activity and proliferation (PCNA), an increase of apoptosis (p53S15) for 8-QA treated tissues. Interestingly, immunohistochemistry studies highlighted that 8-QA caused DNA damages (evidenced by γH2Ax marker) which are mainly localized in hypoxic areas (pimonidazole staining). Finally, we optimized, on NUDE mice, new in vivo human chondrosarcoma models highly expressing simultaneously a hypoxic and proteoglycan-rich microenvironment in order to refine the characterization of 8-QA antitumor activity. For this, we developed a xenograft model of HEMC-SS spheroid implanted subcutaneously and an orthotopic model of HEMC- SS cells inoculated in paratibial location. After characterization, first results of antitumor efficacy on these two models confirmed the promising activity of 8-QA derivative. To conclude, this work demonstrates the proof of concept that exploiting the phenotypic features of chondrosarcoma by a bi-selective approach, is a promising innovative strategy for the therapeutic management of this pathology, relatively rare but redoubtable. This work also demonstrates the role of tumor cell interactions with their environment for tumor growth and response to chemotherapy treatments.

Le travail présenté dans ce manuscrit s’inscrit dans une stratégie de vectorisation d’agents diagnostiques et thérapeutiques, vers les protéoglycanes du chondrosarcome, via une structure chimique de reconnaissance : un ammonium quaternaire. Nous nous sommes intéressés aux deux aspects de la prise en charge du chondrosarcome : l’aspect diagnostique par imagerie scintigraphique et l’aspect thérapeutique. Pour l’imagerie, nous avons validé l’intérêt du radiotraceur 99m Tc-NTP 15-5 dans le diagnostic précoce, le staging et le suivi de la réponse au traitement dans un modèle de chondrosarcome de rat. Ce dernier démontre une accumulation spécifique et rapide au niveau du tissu tumoral, et ce, dès les stades précoces de la pathologie. Actuellement en phase de transfert vers la clinique pour la pathologie de l’arthrose, nos résultats ont permis de proposer une extension de l’application au chondrosarcome. Pour la thérapie, nous avons confirmé, in vitro, que la greffe du vecteur ammonium quaternaire, sur un agent alkylant, le melphalan, ne modifiait pas son activité cytotoxique. In vivo, les résultats obtenus avec le dérivé ammonium quaternaire du melphalan, ont mis en évidence un ralentissement net de la croissance tumorale associée à une diminution considérable du syndrome myélodysplasique associé au traitement. Ces résultats prouvent tout l’intérêt de la vectorisation pour l’amélioration de l’index thérapeutique et nous ont permis de valider la preuve de concept pour la vectorisation d’agent cytotoxique. Une autre approche de thérapie a consisté à greffer le vecteur sur des dérivés du cyclophosphamide clivables en hypoxie. Parmi les composés synthétisés, l’un d’entre eux a montré, in vitro, un différentiel de cytotoxicité hypoxie/normoxie intéressant. Son évaluation in vivo dans le modèle de xénogreffe de chondrosarcome HEMC-SS a démontré une inhibition importante de la croissance tumorale. En nous intéressant à l’environnement hypoxique de la tumeur, nous offrons ainsi une approche encore plus spécifique de la thérapie. L’ensemble de ces travaux offrent un véritable espoir pour le management du chondrosarcome, pathologie certes rare mais néanmoins redoutable. / Our strategy consists in using the quaternary ammonium function, that exhibits a high affinity for proteoglycans of extracellular matrix, as a selective carrier to chondrosarcoma of (i) radioisotopes for a targeted in vivo imaging or (i) cytotoxics for a targeted therapeutic approach. For diagnosis application, a radiotracer radiolabeled with 99m Tc ( 99m Tc-NTP 15-5) was designed and tested in vivo in an orthotopic Swarm chondrosarcoma model. For therapeutic application, a quaternary ammonium derivative of melphalan, Melphalan-AQ, was synthesized and assessed in vitro (cytotoxic activity, cell cycle distribution) and in vivo in the orthotopic Swarm chondrosarcome model (antitumor efficacy, toxicity). Since chondrosarcoma is characterized by hypoxia, research was focused on the vectorization of hypoxia-activated prodrugs. For diagnosis, 99m Tc-NTP 15-5 radiotracer highly accumulated in tumoral tissue at very early stage of pathology while no palpable nor measurable tumor could be assessed. For therapy, chondrosarcoma bearing rats treated with Melphalan-AQ showed a significant tumor growth inhibition with associated heamatological side effects being significantly reduced as compared to Melphalan. Among all phosphoramidate-AQ derivatives synthetised, one was selected on the basis of cytotoxic activity improvement under hypoxia respectively to normoxia. In vivo, this compound demonstrated a significant tumoral growth inhibition in the hypoxic HEMC-SS chondrosarcoma model. This work underlines the potential of the proteoglycans vectorization strategy developed in our lab, for a targeted management of chondrosarcoma including: (i) the first and only radiopharmaceutical able to provide in vivo a specific diagnosis and staging of the tumoral pathology of cartilage and (ii) a promising targeted antineoplastic approach exploiting both the chondrogenic and hypoxic features of chondrosarcoma.

Chondrosarcomas are malignant cartilage tumors that do not respond to traditional chemotherapy or radiation. The 5-year survival rate of histologic grade III chondrosarcoma is less than 30%. To achieve a greater understanding of chondrosarcoma tumorigenesis, a model for human chondrosarcoma has been established in a rat system. The model, known as the Swarm rat chondrosarcoma (SRC), resembles human chondrosarcoma and provides a system to study tumor growth and progression. Here we examined the influence of the tumor microenvironment and the impact of genome-wide hypomethylation on the behavior of SRC tumors, two factors known to contribute fundamentally to the development and progression of solid tumors. Previous studies with SRC revealed that tumor microenvironment can significantly influence chondrosarcoma malignancy, but the underlying biologic mechanisms have not been defined. To address this issue we carried out epigenetic and gene expression studies on the SRC tumors that were initiated at different transplantation sites. The epigenetic analysis revealed that microenvironmental changes could promote global DNA hypomethylation in SRC cells. Subsequent gene expression analyses revealed that the transplantation site had a significant impact on the gene expression profiles of SRC tumors. These SRC tumors had unique gene expression profiles, and we were able to identify genes that were differentially expressed between SRC tumors originating from different transplantation sites. Functional analyses of two differentially expressed genes, thymosin-beta-4 and c-fos, provided insight into the role that these genes may play in the development and progression of chondrosarcoma.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010.

Orientadores: Eliane Maria Ingrid Amstalden, Mauricio Etchebehere / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T18:23:37Z (GMT). No. of bitstreams: 1 Cintra_FranciscoFontes_D.pdf: 8878005 bytes, checksum: 10099149390ddadf73aa7dcc9cbabe33 (MD5) Previous issue date: 2012 / Resumo: Introdução: Os condrossarcomas são tumores cartilaginosos com manifestações clínicas e morfológicas diversas. A identificação do comportamento biológico em processos neoplásicos é essencial para definir a conduta terapêutica e a estimativa prognóstica. Distinguir condrossarcoma de baixo grau de encondroma é difícil. A angiogênese tem sido relacionada à taxa de crescimento tumoral e prognóstico, e a expressão de marcadores, como a COX-2, com o comportamento biológico. A análise da angiogênese induzida pelo tumor e da imuno-expressão da COX-2 poderia auxiliar na determinação do prognóstico do paciente, bem como na distinção entre encondroma e condrossarcoma de baixo grau. Objetivos: estudar o valor da angiogênese, do uso de marcadores de estimativa de índole biológica maligna e padrão arquitetural dos vasos (padrão vascular), por meio da técnica de imuno-histoquímica, no diagnóstico diferencial do encondroma e condrossarcoma de baixo grau, assim como na estimativa do prognóstico dos pacientes com estes tumores. Métodos: 21 encondromas e 58 condrossarcomas convencionais foram selecionados para este estudo, sendo separados em 3 grupos: I- 21 encondromas; II- 31 condrossarcomas grau 1; III- 27 condrossarcomas graus 2 e 3. Os espécimes foram estudados quanto às suas características histopatológicas, marcadores de angiogênese (VEGF, CD34 e CD105) e expressão da COX-2. As informações clínicas foram resgatadas dos prontuários. Os tumores foram avaliados de forma cega e simultânea, por dois observadores, para a escolha de cinco áreas com a maior concentração de células/vasos imuno-marcados ("hot-spots"), definidas em consenso, em aumento de X400. A estimativa da expressão do VEGF e da COX-2 foi mensurada por meio da contagem das células positivas e negativas nas áreas de maior expressão ("hot spots") e determinada a proporção média. A vascularização foi aferida com o marcador CD34, utilizando-se do método de Chalkley. A análise da neoangiogênese foi medida por meio do anticorpo anti-CD105. A avaliação do padrão vascular dos tumores foi feita segundo o método de Kalinski. Os resultados foram tabulados e tratados por métodos estatísticos. Resultados: Foi encontrada associação entre evolução desfavorável e as seguintes variáveis: 1- condrossarcomas de moderado e alto graus; 2- localização em ossos chatos; 3- alta densidade microvascular; 4- padrão arquitetural caracterizado por diminuição do tamanho dos lóbulos com aumento de vasos na sua periferia (subtipo B de Kalinski) ou franca vascularização intra-lobular (subtipo C de Kalinski). Também foi observada associação entre o aumento da angiogênese e condrossarcomas de ossos chatos. Houve correlação entre aumento da densidade microvascular e hiperexpressão da COX-2. A hiperexpressão do VEGF relacionou-se com os padrões vasculares Kalinski B e C. As variáveis analisadas não permitiram diferenciar condrossarcoma de baixo grau de encondroma. Não foi encontrada associação entre o prognóstico e idade, sexo, ou expressão da COX-2, CD105 e VEGF. Conclusão: A avaliação da densidade microvascular e do padrão vascular podem ser uma ferramenta prognóstica adicional no manejo de pacientes com condrossarcoma. A densidade microvascular maior em condrossarcomas de ossos chatos pode estar associada à evolução desfavorável, nesta localização. Nenhuma das variáveis estudadas mostrou-se útil para diferenciar os condrossarcomas de baixo grau dos encondromas / Abstract: Background: Chondrosarcomas are a heterogeneous group of cartilaginous tumors with varied clinical and morphological features. Like any other cancer, the distinction between benign and malignant nature, in cartilaginous bone tumors, is essential for the determination of treatment modality and prognosis evaluation. The distinction between enchondroma and grade 1 chondrosarcoma is very difficult. Angiogenesis has been associated with tumor growth and prognosis, and the expression of COX-2, with the biological nature. Analysis of tumor-induced angiogenesis and immuno-expression of COX-2 could possibly assist in determining patient prognosis as well as distinguish between enchondroma and low grade chondrosarcoma. Objectives: To study the value of immunohistochemical markers of angiogenesis, biological malignancy in the differential diagnosis of cartilaginous tumors and architectural vascular pattern, as well as in estimating prognosis of patients. Methods: 21 enchondromas and 58 conventional chondrosarcomas were chosen and classified in three groups: I- enchondromas (n=21); II- low grade chondrosarcomas (n=31); III- moderate and high grade chondrosarcomas (n=27). All specimens were evaluated for their clinical findings, histopathological markers of angiogenesis (VEGF, CD34 and CD105) and expression of COX-2. The clinical data were retrieved from the medical records. Tumors were evaluated, blindly and simultaneously, by two observers for the analysis of the immunohistochemical preparations. VEGF and COX-2 immunostaining evaluation was determined by the proportion of positive cells in five "hot spots", defined by consensus, in X400 magnification. For CD34 and CD105, five areas were chosen with the highest concentration of micro-vessels ("hot spots"), through the use of the Chalkley graticule for counting. We further evaluated the vascular pattern of the tumor according to Kalinski. The results were tabulated for statistical analysis. Results: Fourteen patients had poor evolution (recurrence, metastasis and death). There were no significant differences regarding the follow-up time between groups. The following factors were statistically associated with poor prognosis: 1- chondrossarcomas of intermediate and high grades; 2- localization in flat bones; 3- increased micro-vascular density (over-expression of CD34); 4- tumor architectural pattern characterized by decrease of the size of the lobules with increased vascularization in their periphery (Kalinski vascular pattern B) and marked intra-lobular vascularization (Kalinski vascular pattern C). A positive association was found between increased angiogenesis and flat bones chondrosarcomas. COX-2 expression was high in tumors with high microvascular density. VEGF over-expression correlated with Kalinski B/C vascular patterns. None of the variables proved to be useful to differentiate low-grade chondrosarcoma from enchondroma. No statistical correlation was found between the prognosis and the following variables: age, gender, and expression of COX-2, CD105 and VEGF. Conclusion: Evaluation of microvessel density and vascular pattern can be useful as additional prognostic tools in the management of patients with chondrosarcoma. Higher microvessel density in chondrosarcomas of flat bones may be associated with unfavorable outcome in this location. None of the variables studied in this work proved useful to differentiate low-grade chondrosarcomas from enchondromas / Doutorado / Anatomia Patologica / Doutor em Ciências Médicas

Les cucurbitacines et les parabènes sont des substances d'origine naturelle appartenant à la famille des triterpéniques cycliques et des esters de l'acide-hydroxybenzoïque, respectivement. Ils sont connus pour posséder chez l'homme plusieurs effets pharmacologiques potentiellement intéressants. Le but de ce travail est d'étudier la biotransformation d'une série de trois cucurbitacines et de cinq parabènes dans le foie humain in vitro, essentiellement par glucuronoconjugaison et hydrolyse et caractériser l'effet cytotoxique des cucurbitacines sur une lignée cellulaire du chondrosarcome humain SW 1353. Les résultats ont montré que les cucurbitacines I et D sont plus cytotoxiques que la cytochalasine D vis-à-vis SW 1353 et entraînent l'apoptose des cellules après 12h de traitement à 1 µM alors que la cucurbitacine E présente une cytotoxicité à 10 µM. L'étude in vitro de biotransformation chez l'homme montre que les cucurbitacines sont surtout sulfo- et glucuronoconjuguées (méthodes radiochimiques) par les isoformes de la famille 1A essentiellement. La cucurbitacine E est également activement hydrolysées (Km 22 µM et Vmax 571 nmol/mg /min) en cucurbitacine I dans le foie humain. Par contre, ces substances sont très faiblement hydroxylées. Les acides oléanolique, ursolique et l'érythrodiol, de structure apparentée aux cucurbitacines, sont aussi glucuronoconjuguées au même taux dans les microsomes hépatiques humains mais avec des affinités 100 fois plus élevées. Les parabènes sont rapidement hydrolysés par des estérases dans les microsomes hépatiques humains en acide 4-hydroxybenzoïque, et leur hydrolyse diminue avec la longueur de la chaîne alkyle. Les parabènes, l'acide caféique, le tyrosol et l'hydroxytyrosol, de structure apparentée aux parabènes, sont activement glucuronoconjuguées par les UGTs des familles 1A et 2B. Cependant, l?acide 4-hydroxybenzoïque n'est pas un bon substrat pour les UGTs / Cucurbitacins and parabens are natural compounds belonging to cyclic triterpenoid and alkyl ester of 4-hydroxybenzoic acid, respectively. They are known for their potential valuable pharmacological effects. The aim of this work was to study the biotransformation of a series of three cucurbitacins and six parabens in human liver, in vitro, essentially by glucuronidation and hydrolysis reactions, and by comparision with other natural substances structurally related, and to characterize the cytotoxicity effect of cucurbitacins on a human chondrosarcoma cell line, SW 1353. The results showed that cucurbitacins I and D showed more cytotoxicity than cytochalasin D and led to cell apoptosis after 12h of treatment at 1 ?M. Cucurbitacin E showed its cytotoxic effect at 10 µM. The in vitro study of cucurbitacins biotransformation in man showed that these substances are sulfated, glucuronidated (radiochemical methods). Moreover, cucurbitacin E was actively hydrolyzed (Km 22 µM and Vmax 571 nmol/mg protein/min) leading to cucurbitacin I in man liver. On the other hand, they are weakly hydroxylated. Oleanolic acid, ursolic acid and erythrodiol, chemically related to cucurbitacins, were glucuronidated at the same rate in human liver microsomes, however, with affinities 100 times greater. Parabens were quickly hydrolyzed in human liver microsomes by esterases and hydrolysis decreased with side chain length. Parabens, caffeic acid, tyrosol and hydroxytyrosol were actively glucuronidated by the UGTs of both families 1A and 2B. However, p-hydroxybenzoic acid is not a good substrate for UGTs