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Hepatocellular carcinoma (HCC) is one of deadliest cancers worldwide and ranking the third among all cancer-related mortalities. Current effective therapeutic approaches for HCC include surgical resection and trans-arterial embolization (TAE). Chemotherapy remains largely ineffective, and most popular used agents are epirubicin, doxorubicin, cisplatin and 5-FU. Besides, these chemotherapic drugs had potential serious side-effects such as low blood count, hair loss, vomiting, and they rarely present good anti-HCC effect in clinical practice. Our previous studies found that epirubicin injection attenuated the tumor burden of orthotopic Novikoff hepatoma, but caused serious side effects to hosts including reduction in spleen weight, white count, and body weight and high GOT level. Therefore, we aimed to evaluate possible alternative treatment such as COX-2 inhibitor for HCC. Celecoxib is a highly selective COX-2 inhibitor and less toxic than the traditional non-selective NSAIDs. Celecoxib showed relatively low cytotoxicity in Novikoff N1-S1 hepatoma cells and Clone 9 normal hepatocytes with an IC50 of up to 100 microM. Expression analysis revealed that COX-2 expression is very low in N1-S1 cells at protein and mRNA levels. Thus, N1-S1 is a kind of hepatoma cell line with low COX-II level. Interestingly, celecoxib upregulated PTEN expression and decreased AKT phosphorylation in vitro by COX-2 independent pathway, and then oral administration of celecoxib (30 mg/kg) for 7 days showed tendency of tumor suppression of Novikoff hepatoma in rats revealed by ultrasound and computed tomography (CT) scan. Histological analysis revealed that CD31-positive neo-vascularization¡BKi-67-positive cell-proliferation and FOXP3-positive regulatory T cells were found to reduce in celecoxib-treated rats, and then TUNEL-positive apoptotic cells were found to increase in celecoxib-treated rats. Besides, celecoxib-treated rats exhibited no significant side effect. Therefore, oral celecoxib may be a suitable chose of adjuvant therapy in combination with epirubicin or other chemotherapeutic agents for the treatment of HCC.

Background: Since constrictive pericarditis is most often idiopathic and the pathophysiology remains largely unknown, both the diagnosis and the treatment can be challenging. However, by definition, inflammatory processes are central to this disease process. Amniotic membrane patches have been shown to possess anti-inflammatory properties and are believed to be immune privileged. Due to these properties, amniotic membrane patches were applied intraoperatively in a complicated patient presenting with constrictive pericarditis. Case presentation: A patient with a history of multiple cardiac surgeries presented with marked fatigue, worsening dyspnea and sinus tachycardia. He was found to have constrictive physiology during cardiac catheterization, with cardiac MRI demonstrating hepatic vein dilatation, atrial enlargement and ventricular narrowing. After amniotic membrane patch treatment and pericardiectomy, post-operative cardiac MRI failed to demonstrate any appreciable pericardial effusion or inflammation, with no increased T2 signal that would suggest edema. Conclusions: Given the positive results seen in this complex patient, we suggest continued research into the beneficial properties of amniotic membrane patches in cardiac surgery.

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers in the world and Taiwan. The major factors involved in the molecular pathogenesis for the development of HCC had been explored in recent years. An extensive array of growth factors and their receptors had been identified and may act as positive and negative modulators in different stages of hepatocarcinogenesis. Current therapeutic approaches for HCC include surgical resection (include liver transplantation), trans-arterial embolization (TAE), alcohol injection, etc. However, the effect is limited due to most of the HCC patients present with advanced stages of the disease. Therefore, this underscores the need for the development of novel therapeutic strategies. It is pivotal to set up an orthotopic hepatoma model for the development of novel intervention strategies for HCC. Under the guidance of ultrasound, we are able to create hepatoma in the liver lobe of Sprague-Dawley (SD) rats by injection of Novikoff (N1-S1) hepatoma cells. In addition, sonographic technique was employed for the monitoring of tumor growth in this animal model in the following subprojects. The continuous, non-invasive measurement of orthotopic hepatoma development will be a valuable tool for the evaluation of effects of drugs for treatment of HCC. In Chapter 1, the study employed a relatively non-invasive approach to establish an orthotopic HCC model in immune-competent rats. This was done by ultrasound-guided implantation of cancer cells and the model was used to evaluate the therapeutic efficacy of short-term and low-dose epirubicin chemotherapy. Ultrasound-guided implantation of Novikoff hepatoma cells led to the formation of orthotopic HCC in 60.4% of the SD rats. Moreover, tumor sizes measured by ultrasound significantly correlated with those measured by calipers after sacrificing the animals (P < 0.00001). The rate of tumor induction by ultrasound-guided implantation was comparable to that of laparotomy (55/91, 60.4% vs. 39/52, 75%) and no significant difference in sizes of tumor was noted between the two groups. Moreover, there was a significant correlation in tumor size measurement by ultrasound and computerized tomography. In tumor-bearing rats, short-term and low-dose epirubicin chemotherapy caused a significant reduction in tumor growth, and was found to be associated with enhanced apoptosis and attenuated proliferation as well as a decrease in microvessel density in tumors. In chapter 2, we investigated the chemopreventive effects of celecoxib in the growth of orthotopic rat HCC and the possible signal pathways involved. The status of COX-2 expression in rat Novikoff HCC was consistent with that in human HCC. Both Western blot and PCR tests had proved that N1-S1 was a HCC model presenting with low COX-2 enzymes in tumor cells. Then, low doses of celecoxib was shown to effectively inhibited the proliferation and increased the apoptosis of N1-S1 cells in vitro, which were also safe to the normal hepatocytes. Moreover, chemoprevention by celecoxib inhibiting the HCC tumor growth was shown in rat orthotropic HCC model. Tumor incidence was not affected by the celecoxib prevention, but, tumor weight was found significantly suppressed by the drug. Possible mechanisms of chemoprevention by celecoxib seen in the animal model were thought to be related to the anti-angiogenic, anti-proliferative and anti-hCSC characters of the drug. In chapter 3, we tried to test the combined inhibitory effects of low doses of celecoxib and epirubicin on the growth of HCC. Combined low doses of epirubicin and celecoxib was effective in inhibiting the hepatic cancer stem cells, tumor angiogenesis, tumor cell proliferation, as well as promoting cancer apoptosis. These are compatible with the effects of the individual drugs on HCC growth shown in the previous two chapters. In general, combination therapy expressed more effectiveness in tumor suppression and less bone marrow suppression than the individual drugs used alone. Taken together, ultrasound-guided implantation of Novikoff hepatoma cells is an effective means of establishing orthotopic HCC in SD rats, which is suitable and convenient for therapeutic trial of anti-HCC treatment. In the current study, we had proved the efficacies of low doses of two drugs, epirubicin and celecoxib, acting individually, as well as the combined effects of them in treating HCC in this model.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24132号 / 医博第4872号 / 新制||医||1059(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 平井 豊博, 教授 湊谷 謙司, 教授 羽賀 博典 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

There is mounting evidence to support combined therapy with radiation (RT) and antiangiogenic agents (AA) for the treatment of brain tumors. However, the therapeutic benefit of this combined treatment hinges on the specific dose, schedule, and duration of each treatment. Early biomarkers that reflect tumor physiological responses provide key information that could guide these aspects of treatment. Pre-clinical tumor models are invaluable tools for identifying potential biomarkers, their optimal timing for measurement and their ability to guide therapy in clinical translation. This thesis demonstrates the feasibility and potential of serial MRI to guide the design, delivery and measure of early response to combined AA and RT in a murine intracranial glioma model. We identified promising biomarker changes reflecting early treatment response that may ultimately facilitate individualized spatio-temporal delivery of radiotherapy (RT) and anti-angiogenic agents (AA) for brain tumors.

There is mounting evidence to support combined therapy with radiation (RT) and antiangiogenic agents (AA) for the treatment of brain tumors. However, the therapeutic benefit of this combined treatment hinges on the specific dose, schedule, and duration of each treatment. Early biomarkers that reflect tumor physiological responses provide key information that could guide these aspects of treatment. Pre-clinical tumor models are invaluable tools for identifying potential biomarkers, their optimal timing for measurement and their ability to guide therapy in clinical translation. This thesis demonstrates the feasibility and potential of serial MRI to guide the design, delivery and measure of early response to combined AA and RT in a murine intracranial glioma model. We identified promising biomarker changes reflecting early treatment response that may ultimately facilitate individualized spatio-temporal delivery of radiotherapy (RT) and anti-angiogenic agents (AA) for brain tumors.

Orientadores: Fábio Rogério, Roger Chammas / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T17:12:57Z (GMT). No. of bitstreams: 1 Furuzawa_KarinaMie_M.pdf: 6831585 bytes, checksum: 1169d18e1d04229e3df523b6e1bea8d3 (MD5) Previous issue date: 2011 / Resumo: Gliomas são as mais frequentes neoplasias primárias do sistema nervoso central e compõem um grupo heterogêneo, apresentando diferentes graus de malignidade. Glioblastomas (OMS grau IV) representam aproximadamente 50% dos tumores de origem glial e estão associados ao pior prognóstico, com sobrevida média de menos de um ano após o diagnóstico. O gliossarcoma, uma variante do glioblastoma, apresenta componente mesenquimal, além do componente glial. A linhagem celular de glioma utilizada no presente trabalho, NG97ht, é híbrida humano-murina e foi estabelecida a partir de células neoplásicas implantadas no dorso de camundongos nudes. Os tumores que se desenvolveram, por sua vez, são derivados de astrocitoma anaplásico (OMS grau III) ressecado de um paciente masculino de 66 anos. Realizou-se a caracterização morfológica e imunofenotípica de um modelo ortotópico de astrocitoma de alto grau e a análise do padrão de expressão de galectina-3 em tumores derivados da inoculação de células NG97ht. Através de cirurgia estereotáxica, foram inoculadas 5 x 105 células NG97ht no córtex cerebral de camundongos nude (imunodeprimidos) e BALB/c (imunocompetentes) com oito semanas de idade. Foi realizada ressecção dos tumores que se desenvolveram duas ou quatro semanas pós-implante. Análise morfológica e cálculo do índice mitótico foram realizados em cortes dos tumores incluídos em parafina, assim como coloração para detecção de fibras reticulínicas e reações imunoistoquímicas para GFAP, S-100, vimentina, PCNA, galectina-3, CD34, CD56/NCAM, 1A4, desmina, AE1AE3 e sinaptofisina. Além disso, foi realizada semiquantificação da expressão de galectina-3 através de Western Blotting. As características morfológicas e imunofenotípicas da neoplasia indicam um glioma de alto grau, particularmente o gliossarcoma. Os tumores apresentaram alta celularidade, presença de focos necróticos e exibem predominantemente células fusiformes com alto índice mitótico. A expressão de GFAP apresentou alta variabilidade, muitas vezes presente em agrupamentos de células neoplásicas. Foi observada imunopositividade citoplasmática para CD56 e 1A4, indicando diferenciação divergente neuroectodérmica e mesenquimal. Além disso, foi detectada rica e fina rede de fibras reticulínicas intercelulares. A localização da galectina-3 foi nuclear e/ou citoplasmática e sua expressão foi intensa, principalmente nos limites da lesão com o tecido normal adjacente. Não foi detectada diferença significativa na expressão de galectina-3 entre tumores obtidos duas e quatro semanas pós-implante. Em camundongos BALB/c não foi observado crescimento tumoral. Em conclusão, o implante ortotópico de células NG97ht representa uma interessante alternativa para investigações in vivo de gliossarcoma. Considerando-se a falta de estudos sobre o papel da galectina-3 em gliossarcomas e seu possível envolvimento com migração celular, angiogênese e quimiorresistência, a galectina-3 pode ser um potencial alvo para novas estratégias terapêuticas / Abstract: Gliomas are the most frequent primary brain tumors and correspond to various histopathological types and malignancy grades. Glioblastomas (WHO grade IV) account for approximately 50% of glial tumors and are associated with the worst prognoses, with median survival rate of less than 1 year. Gliosarcoma is a rare variant of glioblastoma which displays distinct glial and mesenchymal components. NG97ht is a malignant murine and human hybrid cell line established from a human anaplastic astrocytomaderived tumor engrafted into nude mice. The aims of the present work were morphological and immunophenotypical characterization of an in vivo orthotopic glioma model and analysis of galectin-3 expression pattern in NG97ht cell line-derived tumors. 5 x 105 NG97ht cells were stereotactically inoculated into cerebral cortex of 8-week-old nude or BALB/c mice and tumors were resected after two or four weeks. Morphological analysis and calculation of mitotic index were made in paraffinembedded sections, as well as immunohistochemical reactions for GFAP, CD56, 1A4, AE1AE3 and galectin-3. Semi-quantification of galectin-3 expression was obtained through Western Blotting. The neoplastic features favour a high-grade glioma, particularly gliosarcoma. Tumors were predominantly constituted by spindle cells with high mitotic index and showed elevated cellular density and some necrotic areas. GFAP was expressed by groups of tumoral cells. Positivity for CD56 and 1A4 was cytoplasmic and indicates neuroectodermic and mesenchymal divergent differentiation. Localization of galectin-3 was nuclear and/or cytoplasmic and its expression was intense, mainly at tumor borders with adjacent tissue. There was no significant difference in galectin-3 expression between tumors resected after two or four weeks. Tumor growth was not detected in BALB/c mice. In conclusion, the orthotopic implant of NG97ht cells represents an interesting alternative for gliosarcoma in vivo studies. Considering the lack of investigation about the role of galectin-3 in gliosarcomas and the possible involvement of galectin-3 in cellular migration, angiogenesis and chemoresistance, it should be a potentially good target for new therapeutic strategies / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Hsp90 (heat shock protein 90) est une protéine chaperonne ubiquitaire et conservée impliquée dans le repliement et la réparation de protéines dites « clientes ». Parmi ces protéines, de nombreuses sont impliquées dans des phénomènes oncogéniques, faisant de hsp90 une cible d’intérêt dans le traitement du cancer. Hsp90 est constituée de trois domaines, un domaine N-terminal site de l’hydrolyse de l’ATP, nécessaire à sa fonction ; un domaine intermédiaire où vient se fixer la protéine cliente et un domaine C-terminal impliqué dans la dimérisation, étape indispensable pour le repliement de la protéine cliente. De nombreux inhibiteurs ont été synthétisés en ciblant ces différents domaines. Les inhibiteurs N-terminaux sont efficaces, à l’instar de la Geldanamycine en termes d’activité anti-tumorale mais des effets secondaires ainsi que des résistances au traitement ont limité leur utilisation en pratique clinique. En effet, l’inhibition N-terminale induit une réponse au stress caractérisée par une augmentation de hsp90 et de ses co-chaperonnes, souvent associée à une résistance au traitement et un pronostic défavorable. La novobiocine, un antibiotique coumarinique, est capable d’inhiber le domaine C-terminal de hsp90, sans induire de réponse au stress. Ainsi de nombreux dérivés de cette molécule ont été synthétisés, parmi lesquels on trouve le 6BrCaQ. Cette molécule induit l’apoptose et le blocage dans le cycle cellulaire sur plusieurs lignées cellulaires (dont MCF-7 et MDA-MB-231) et provoque la dégradation de plusieurs protéines clientes impliquées dans le développement tumoral mais sa faible solubilité limite son administration in vivo.Dans cette thèse, une forme liposomale du 6BrCaQ a été développée et étudiée sur des lignées cellulaires de cancer de prostate, de sein et de leucémie aigüe myéloïde in vitro et in vivo sur un modèle orthotopique de cancer du sein (MDA-MB-231 luc-GFP). Le 6BrCaQ liposomal est capable d’ induire de l’apoptose, de bloquer le cycle cellulaire sur différentes lignées cellulaires (PC-3, MDA-MB-231 et MOLM-13) mais également de ralentir la migration cellulaire sur PC-3 (test de comblement de blessure). De plus, le 6BrCaQ liposomal entre en synergie avec la doxorubicine (cellules PC-3) et la daunorubicine (cellules MOLM-13). Au niveau moléculaire, les liposomes de 6BrCaQ modifient l’expression protéique de Hsp90 sans modifier celle d’Hsp70 sur PC-3 alors que les gènes codant pour les Hsp70 semblent être légèrement induits dans MDA-MB-231. Les résultats in vivo ont montré un ralentissement de la croissance tumorale sur un modèle de cancer du sein orthotopique (MDA-MB-231-luc2-GFP) dès 13 jours de traitement pour une dose de 1 mg/kg injectée une fois par semaine. Des analyses histologiques ont révélé une augmentation de la proportion des zones nécrotiques dans le groupe traité par rapport au contrôle et une diminution significative de la prolifération cellulaire (marquage au KI67) intra-tumorale.Par ailleurs, Hsp90 possède également des isoformes et des analogues localisés dans des organites intracellulaires, parmi lesquelles, TRAP-1, localisée au niveau de la mitochondrie, impliquée dans la rgulation du métabolisme mitochondrial et qui pourrait jouer un rôle dans la progression tumorale et les métastases. Le déqualinium (DQ) est capable de cibler la mitochondrie. Dans une seconde partie du travail, des liposomes encapsulant le DQ ont été formulés dans le but de vectoriser le 6BrCaQ vers la mitochondrie. Toutefois, face à la difficulté d’encapsuler le DQ dans des liposomes, une étude de physico-chimie sur l’interaction DQ/liposomes a été mise en place pour comprendre comment le DQ agit sur les bicouches phospholipidiques. Cette étude a révélé que, malgré une capacité de ciblage mitochondrial des liposomes, le DQ était difficile à encapsuler dans les milieux salins et n’était pas inerte sur les bicouches lipidiques ce qui limite son utilisation pour la formulation de liposomes ciblant la mitochondrie. / Hsp90 (Heat shock protein 90) is an ubiquitous and well-conserved chaperone protein involved in the folding and the repair of « client » proteins. Among these proteins, several are involved in oncogenic phenomena making hsp90 an interesting target for cancer therapy. Hsp90 consists of three domains ; a N-terminal domain as the ATP hydrolysis site ; a middle domain where the client proteins binds and a C-terminal domain involved in the dimerization, a necessary step to refold the client protein. Several inhibitors were synthesized to target these different domains. N-terminal inhibitors such as Geldanamycin ; were shown to be very efficient but side effects and resistance to the treatment limited their clinical use. Indeed, N-terminal inhibition induces a stress response characterized by an increase of hsp90 and its co-chaperones which is often associated with resistance to the treatment and poor prognosis. Novobiocin, a coumarin antibiotic, is capable of inhibiting the C-terminal domain of hsp90, without inducing a stress response. Several derivatives of this molecule have been synthesized, including 6BrCaQ. The latter was effective in terms of apoptosis induction and cell cycle blockade on several cell lines (MCF-7, MDA-MB-231) and induced pro-tumoral client protein degradation but its low solubility limits its in vivo administration. In this thesis, a liposomal formulation of 6BrCaQ has been developed and studied on in prostate cancer cell lines and acute myelogenous leukemia in vitro and in vivo in an orthotopic model of breast cancer (MDA-MB-231 luc-GFP). Liposomal 6BrCaQ showed ability to induce apoptosis, to block the cell cycle on several cell lines (PC-3 and MDA-MB-231) and slow down migration of PC-3 cells (wound healing assay). Liposomal 6BrCaQ is able to synergize with doxorubicine or daunorubicine in PC-3 cells and in MOLM-13 cells, respectively. Moreover, protein and RNA expression profiles show that in PC-3 cells liposomal 6BrCaQ downregulates Hsp90 protein and in MDA-MB-231 cells slightly upregulates Hsp70 gene expression. Results obtained during in vivo experiments on the breast orthotopic model revealed a slow downslowdown of tumor growth after 13 days for a dose of 1 mg/kg injected weekly. Histological analysis revealed necrosis in treated groups and aassociated with a decreased cell proliferation (ki67 staining).Hsp90 also has isoforms and analogues localized in intracellular organelles, including, TRAP-1, localized in the mitochondrion and probably implicated in malignant progression through its role in the regulation of the mitochondrial metabolism. Dequalinium (DQ) demonstrated ability to target the mitochondrion. In a second part of the work, liposomes encapsulating DQ have been formulated in order to target 6BrCaQ to mitochondria. However, faced with the difficulty of encapsulating the DQ in liposomes, a physical chemistry study on the interaction DQ / liposomes was established to understand how DQ acts on phospholipid bilayers. Though a mitochondrial targeting capacity, this study revealed DQ was difficult to encapsulate in liposomes in saline medium and not completely inert on lipid bilayers limiting its use to target liposomes to mitochondria.

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