Thesis (Ph.D.)--University of Florida, 2005. / Typescript. Title from title page of source document. Document formatted into pages; contains 95 pages. Includes Vita. Includes bibliographical references.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / AvaliaÃÃo dos efeitos da administraÃÃo diÃria, intragÃstrica (ig) e subcutÃnea (sc) prolongada do produto hidrossolÃvel da prÃpolis verde, extraÃda em L-lisina e do aminoÃcido L-lisina ig sobre a angiogÃnese de carcinomas de bexiga e a carcinogÃnese de bexiga induzida pelo BBN (N-butil-N{4-hidroxibutil}nitrosamina) em ratas Wistar. O total de 125 ratas foi distribuÃdo inicialmente em 14 grupos: I, IIA, IIB, III, IV, V, VI, VII, VIII, IX, X, XI, XII e XIII. Os grupos de I a X receberam BBN por 14 semanas em Ãgua de beber. O grupo I foi tratado com prÃpolis ig 150 mg/kg/peso, por 44 s, iniciado 30 dias antes do inÃcio do BBN. Os grupos IIA, III e VIII foram tratados com prÃpolis (150 mg/kg/peso), por 40s, vias sc e ig iniciada simultaneamente com o BBN. Na 32Âs, os animais dos grupos IV, V, VI, VII e IX, apÃs ultrassonografia, foram estratificados e realocados em 4 grupos K, L, M e N, de forma que cada grupo recebesse o mesmo nÃmero de animais sem lesÃo vesical ou com imagem tumoral pequena, mÃdia e grande. As ratas dos grupos L, M e N, foram tratadas ig com: L-lisina (300mg/kg/peso), celecoxibe (30 mg/kg/peso) e prÃpolis (300 mg/kg/peso) respectivamente, da 32 a 40Âs. Os grupos controles positivos (que receberam apenas BBN) IIB e K foram tratados com Ãgua, via sc e oral, respectivamente, por 40 s. Os grupos controles negativos XI, XII e XIII receberam nesta sequÃncia, prÃpolis (150mg/kg/peso), L-lisina (150 mg/kg/peso) e Ãgua ig por 40 semanas (s). Os grupos III e VIII foram reunidos em um Ãnico grupo, o grupo III. Os animais foram sacrificados ao final da 40Âs. A carcinogÃnese foi estudada em todos os grupos. A angiogÃnese foi avaliada nos grupos K, L, M, N III e X, pela quantificaÃÃo da densidade microvascular em hot spots, atravÃs de imunomarcaÃÃo (CD-31), utilizando-se um programa de computador. O Ãndice de carcinogÃnese (21,00) e a incidÃncia de carcinomas (20%) no grupo I, tratado com prÃpolis 30 dias antes do inÃcio do BBN, foram menores (P<005) que os do grupo controle K (39,67% e 66,67%, respectivamente). A densidade microvascular de hot spot em carcinomas de bexigas foi menor (P<0,05) tanto em ratos tratados com prÃpolis por 40 semanas (grupo III), quanto em tratados com prÃpolis da 32 a 40Âs (grupo N), quando comparada com a densidade dos carcinomas de animais que receberam apenas carcinÃgeno (grupo K). A multiplicidade de carcinomas (P= 0, 00267), o Ãndice de carcinogÃnese (P<0,05) a densidade microvascular (P<0,05) no grupo X (tratado com L-lisina) foram maiores que os do grupo controle K. Somando-se a isso, a incidÃncia de carcinomas no grupo X foi de 100% e apresentando tumores mais invasivos (p= 0, 0039) que os verificados no grupo K. O grupo que recebeu apenas L-lisina nÃo apresentou lesÃes vesicais. Isso significa que a L-lisina por si sà nÃo gera carcinomas. NÃo hà descriÃÃo de efeito promotor da L - lisina na carcinogÃnese de bexiga na literatura pesquisada. A incidÃncia de 100% de carcinomas no grupo tratado com L- lisina pode fazer desta modelo para estudo da carcinogÃnese de bexiga. Conclui-se que o produto de prÃpolis verde administrado diariamente na dose de 150 mg/kg/peso ig, diminui a incidÃncia de carcinomas quando iniciada 30 dias antes do BBN e inibe a angiogÃnese de carcinomas de bexiga na dose de 150 mg/kg/peso quando iniciada simultaneamente ou na dose de 300 mg/kg/peso apÃs a 31 semana do inÃcio de BBN. / The present study evaluated the effects of prolonged, daily intragastric (ig) or subcutaneous (sc) administration of water-soluble derivative of green propolis extracted in L-lysine and of ig administration of L-lysine upon Wistar rat bladder carcinomas angiogenesis and bladder carcinogenesis induced with BBN (N-butyl-N-[4-hydroxybutyl] nitrosamine). At baseline 125 rats were distributed in 14 groups (I, IIA, IIB, III, IV, V, VI, VII, VIII, IX, X, XI, XII and XIII). Groups I through X received BBN in drinking water for 14 weeks. Group I was treated with propolis ig (150 mg/kg body weight) during 44 weeks beginning 30 days before challenge with BBN. Groups IIA, III and VIII were treated for 40 weeks with propolis (150 mg/kg) sc and ig initiated concurrently with BBN. By the 32nd week, the animals in Groups IV, V, VI, VII and IX were ultrasound-scanned, stratified and reallocated into 4 groups (K, L, M and N) so that the groups contained the same number of animals with small, medium and large tumors or without lesions. Groups L, M and N were treated with L-lysine ig (300mg/kg), celecoxib (30 mg/kg) and propolis (300 mg/kg), respectively, from the 32nd to the 40th week. Groups IIB and K (positive control groups receiving BBN only) were treated with water sc and orally, respectively, during 40 weeks. Groups XI, XII and XIII (negative control groups) received propolis (150mg/kg), L-lysine (150 mg/kg) and water ig, respectively, for 40 weeks. Groups III and VIII were merged into a single group (Group III). The animals were euthanized after 40 weeks. Carcinogenesis was studied in all groups. Angiogenesis was evaluated for Groups K, L, M, N, III and X using quantifying microvascular density of hot spots in histological sections stained with the marker CD-31 and analyzed with specific software. The carcinogenesis index (21.00) and the carcinoma incidence (20%) in Group I (treated with propolis 30 days prior to challenge with BBN) were smaller (p<0.05) than for Group K (control; 39.67 and 66.67%, respectively). The microvascular density of bladder carcinoma hot spots was smaller (p<0.05) for rats treated with propolis for 40 weeks (Group III) and rats treated with propolis from the 32nd to the 40th week (Group N) than for rats receiving BBN only (Group K). Carcinoma multiplicity (p=0.00267), carcinogenesis index (p<0.05) and microvascular density (p<0.05) were greater for Group X (treated with L-lysine) than for group K. In addition, the carcinoma incidence in Group X was 100%, and tumors were more invasive (p=0.0039) than in Group K. The group receiving only L-lysine presented no vesical lesions, indicating that L-lysine in itself does not generate carcinomas. Our review of the literature identified no reports of L-lysine acting as a promoter of bladder carcinogenesis. However, the 100% carcinoma incidence observed in the group treated with L-lysine suggests the amino acid may serve as a model for the study of carcinogenesis. In conclusion, daily ig administration of water-soluble derivative of green propolis at 150 mg/kg body weight reduced the incidence of carcinoma when initiated 30 days prior to challenge with BBN, and inhibited angiogenesis in bladder carcinoma at 150 mg/kg when initiated concurrently with BBN or when administered at 300 mg/kg after 31a week of the initiate BBN.

Orientador: Valéria Helena Alves Cagnon Quitete / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T14:16:58Z (GMT). No. of bitstreams: 1 Montico_Fabio_D.pdf: 8683742 bytes, checksum: b24bef7642e19dd250ce77f07cca65c4 (MD5) Previous issue date: 2014 / Resumo: A senescência está associada a modificações hormonais na próstata, resultando em microambiente favorável ao desenvolvimento de lesões neoplásicas. A angiogênese é fundamental para o crescimento tumoral e, na próstata, está sujeita à regulação por andrógenos, sendo sua estimulação um importante evento no estroma reativo associado ao câncer. Assim, a inibição da angiogênese representa terapia promissora no tratamento de neoplasias da próstata. O objetivo deste estudo foi caracterizar aspectos do estroma prostático na senescência e frente a terapias antiangiogênicas e de ablação hormonal, comparando-os com a reação estromal associada a lesões glandulares no modelo TRAMP. Camundongos FVB machos senis (52 semanas) foram submetidos a tratamentos antiangiogênicos com SU5416 (6 mg/kg; i.p.) e/ou TNP-470 (15 mg/kg; s.c.). O bloqueio hormonal foi obtido com finasterida (20 mg/kg; s.c.), isoladamente ou associada a ambos os inibidores. Após 21 dias de tratamento, amostras da próstata dorsolateral foram coletadas para análises morfológicas, imunohistoquímicas e de Western Blotting. A senescência levou à ocorrência de inflamação e de lesões proliferativas na próstata, bem como à maior frequência de células positivas para CD34/VIM e CD34/?SMA e ao aumento de MMP-9, IGFR-1, VEGF, HIF-1?, FGF-2, CD31, VIM e ?SMA, caracterizando semelhança com o microambiente prostático de camundongos TRAMP. Por outro lado, a endostatina e o TGF-? apresentaram-se elevados somente na senescência, mas não ao longo da progressão tumoral no modelo TRAMP. O tratamento com inibidores angiogênicos levou à recuperação e/ou interrupção das alterações glandulares associadas à senescência, mas efeitos diferenciais foram registrados para as drogas. Enquanto o SU5416 atuou principalmente sobre a remodelação tecidual prostática, reduzindo os níveis de MMP-9, VIM e ?SMA bem como a frequência de células positivas para CD34/VIM e CD34/?SMA, o TNP-470 influenciou sobretudo o IGFR-1, o VEGF e o HIF-1?, promovendo inibição de maior amplitude sobre esses fatores. A associação destes inibidores levou à combinação de tais efeitos diferenciais. A finasterida, isoladamente ou associada aos agentes antiangiogênicos, resultou em diminuição dos níveis de MMP-9, IGFR-1, VEGF, HIF-1? e FGF-2 em relação aos controles, embora tenha demonstrado tendência em regular positivamente a expressão de fatores pró-angiogênicos. Todavia, ao contrário da inibição da angiogênese, a ablação hormonal não resultou em diminuição do TGF-?. Assim, concluiu-se que a senescência gerou um microambiente de estroma reativo similar ao do modelo TRAMP, com estímulo aos à proliferação celular, remodelação tecidual, angiogênese e transição endotélio-mesenquimal, certamente propiciando condições favoráveis para o desenvolvimento de lesões prostáticas pré-malignas e malignas. Entretanto, a ausência de adenocarcinoma pouco diferenciado e a menor distribuição das lesões neoplásicas em relação a camundongos TRAMP sugeriram um possível papel protetor da endostatina sobre a próstata na senescência. A terapia antiangiogênica foi eficaz em promover efeitos antitumorais e a inibição da neovascularização, sobretudo frente à combinação dos inibidores, sugerindo que a ação diferencial desses agentes nos processos tumorigênicos resulta em espectro de ação mais amplo para o tratamento. Por fim, ressalta-se que os efeitos benéficos do tratamento com finasterida merecem especial atenção, considerando o potencial desta droga em promover o estroma reativo facilitador do desenvolvimento de desordens glandulares / Abstract: Senescence is associated with hormonal changes in the prostate, leading to a permissive microenvironment for the development of neoplastic lesions. Prostatic angiogenesis is an androgen-regulated process which is fundamental for tumor growth and represents an important event in cancer-associated reactive stroma. Thus, angiogenesis inhibition emerges as a promising therapy in the treatment of prostate neoplasms. The aim herewith was to characterize prostatic stroma during senescence and following antiangiogenic and hormonal ablation therapies, comparing the findings with the reactive stroma phenotype associated to TRAMP model glandular lesions. Elderly male FVB mice (52 week-old) were submitted to antiangiogenic treatments with SU5416 (6 mg/kg; i.p.) and/or TNP-470 (15 mg/kg; s.c.). Hormonal blockage was achieved with finasteride administration (20mg/kg; s.c.), either alone or combined to both inhibitors. After 21 days of treatment, dorsolateral prostate samples were collected for morphological, immunohistochemical and Western Blotting analysis. Senescence led to the occurrence of inflammatory foci and proliferative lesions in the prostate, apart from increased frequency of CD34/VIM and CD34/?SMA positive cells and raised levels of MMP-9, IGFR-1, VEGF, HIF-1?, FGF-2, CD31, VIM and ?SMA, resembling TRAMP mice prostatic microenvironment. On the other hand, endostatin and TGF-? showed higher expression in senescence but not during tumor progression in the TRAMP model. Antiangiogenic treatment resulted in recovery and/or interruption of the senescence-associated glandular changes, showing differential effects for each drug. SU5416 acted mainly on prostatic tissue remodeling, reducing MMP-9, VIM and ?SMA levels as well as the frequency of CD34/VIM and CD34/?SMA positive cells, whereas TNP-470 influenced specially IGFR-1, VEGF and HIF-1?, promoting greater inhibition over these factors. The association between the inhibitors led to synergistic differential effects. Despite presenting a trend to regulate positively pro-angiogenic factor expression, finasteride resulted in decreased MMP-9, IGFR-1, VEGF, HIF-1? and FGF-2 levels relatively to controls, either in isolation or combined to antiangiogenic agents. However, unlike angiogenesis inhibition, hormonal ablation did not lead to decreased TGF-? expression. Thus, it was concluded that senescence created a reactive stroma microenvironment which resembles that verified in the TRAMP model and is characterized by stimulated cell proliferation, tissue remodeling, angiogenesis and endothelial-to-mesenchymal transition. This scenario certainly provided favorable conditions for the development of pre-malignant and malignant prostatic lesions. However, the absence of poor-differentiated adenocarcinoma and the lesser distribution of neoplastic lesions in relation to TRAMP mice suggested a possible protective role of endostatin on the prostate during aging. Antiangiogenic therapy was efficient in promoting antitumor effects and neovascularization inhibition, especially following the combination of inhibitors, suggesting that the differential action of these agents on tumorigenic processes results in a broader spectrum of effects for the treatment. Finally, it is noteworthy that beneficial effects from finasteride treatment must be looked carefully, considering the capacity of this drug to promote a reactive stroma microenvironment favorable to the development of glandular disorders / Doutorado / Anatomia / Doutor em Biologia Celular e Estrutural

Abstract Cancer progression is a complex multi-step process. Two critical steps in tumor growth and invasion are the proteolytic processing of the extracellular matrix environment and the angiogenic switch enabling blood supply into the tumor. Matrix metalloproteases (MMPs) are a group of proteolytic enzymes involved in physiological and pathological extracellular matrix processing. Trypsinogen, a serine protease, is one of the first proteolytic enzymes characterized. The amount of one of its isoforms, tumor associated trypsinogen-2 (TAT-2) correlates with the malignant phenotype of several forms of cancers. Both of these protease groups are critically dependent on their activation from latent proforms to fully active enzymes. We found that the overproduction of TAT-2 in malignant oral squamous cell carcinoma cell line was associated with elevated proMMP-9 (but not proMMP-2) activation, as well as enhanced cancer cell intravasation in an in vivo model. This indicates that TAT-2 and MMP-9 activation play a role in the invasive growth of oral carcinomas. Proteases are involved in angiogenesis, the formation of new blood vessels, in several ways. One mechanism is the release of cryptic anti-angiogenic molecules from larger extracellular matrix components. Endostatin is one such cryptic endogenous inhibitor of angiogenesis. Certain MMPs were able to cleave endostatin from its parent molecule, collagen XVIII. The endostatin fragments generated by MMP-3, -7, -9, -13 and -20 inhibited angiogenesis in a similar fashion as the native endostatin. The regulation between MMPs and endostatin was shown to be reciprocal, as endostatin was able to block the activation and activities of MMP-2, -9 and -13. The inhibition of these tumor-associated MMPs explains at least in part the anti-tumor activity of endostatin. Endostatin not only affects endothelial cell growth as is usually thought, but it also inhibits the migration of oral carcinoma cells. In addition, cell density and proper concentration were proven to be critical for the activity of endostatin. Arresten is another endogenous inhibitor of angiogenesis and tumor growth derived from type IV collagen. We confirmed that arresten binds to integrin α1β1 on endothelial cell surface. We found that this binding is functionally significant for the anti-angiogenic properties of arresten, as tumors planted to integrin α1 knockout mice or endothelial cells derived from those mice did not respond to arresten treatment.

No / Anti-vascular effects of the novel Vinca alkaloid, vinflunine have been investigated in the MAC 15A transplantable murine colon adenocarcinoma model and compared with those induced by the most recently identified clinically useful third generation Vinca. Administration of the maximum tolerated dose of either vinflunine (50 mg kg-1) or vinorelbine (8 mg kg-1) resulted in significant tumour growth delay with subsequent histological analysis revealing substantial haemorrhagic necrosis. This suggested possible anti-vascular effects and these were confirmed by Hoechst 33342 perfusion studies. Vinflunine, currently undergoing Phase I trials in Europe, was found to be at least as effective as the clinically active vincristine and vinorelbine in this model and, remarkably, produced anti-vascular effects at doses much lower than the maximum tolerated dose. Although vinflunine caused apoptosis in HUVEC monolayer cultures this event did not occur within the first 8 hours of exposure whereas vascular shutdown in vivo was observed within the first 4 hours.

O fator de crescimento endotelial vascular (VEGF) tem sido considerado o principal indutor da angiogênese tumoral por sua ligação a receptores específicos tirosina-quinase presentes nas células endoteliais (VEGFR). Uma importante via reguladora da angiogênese é caracterizada pela ligação do VEGF ao seu receptor VEGFR-2 ativando a via de sinalização de PI3-K, que estimula a expressão da proteína antiapoptótica Bcl-2 elevando a expressão de citocinas pró-angiogênicas CXCL-8 e CXCL-1, o que resulta na proliferação de células endoteliais. Recentemente, foi descoberta uma droga com ação antiangiogênica denominada de PTK787/ZK 222584 (PTK/ZK), que se mostrou um potente inibidor do receptor tirosina-quinase de VEGF, sem aparente efeito citotóxico em células que não expressam esses receptores. Visando a melhor compreensão do mecanismo de ação e da via de atuação antiangiogênica VEGF/VEGFR do PTK/ZK nos carcinomas espinocelulares de cabeça e pescoço, foram desenvolvidos estudos in vitro e in vivo avaliando-se a expressão de Bcl-2, CXCL-8 e CXCL-1. No presente estudo foi utilizado um modelo experimental de angiogênese humana em camundongos imunodeprimidos que receberam co-implantes de células tumorais e endoteliais ou apenas células endoteliais, tratados com administração oral de PTK/ZK por 21 dias. A progressão tumoral nos animais foi avaliada por meio de imagem de bioluminescência. Após o sacrifício dos camundongos, os implantes foram incluídos em parafina sendo obtidos cortes de 3µm para análise da densidade vascular e de 7µm para a técnica de microdissecção a laser visando a avaliação de RNA por meio de RT-PCR e PCR em tempo real nas células malignas e endoteliais. Concomitantemente, experimentos in vitro de co-cultura de células tumorais e endoteliais foram realizados para a análise de expressão de Bcl-2, CXCL-8 e CXCL-1 avaliados por meio de RT-PCR, Western Blot e teste ELISA. Os resultados demonstraram uma redução importante da expressão de Bcl-2, bem como de CXCL-8, em células endoteliais in vitro e in vivo quando tratados com PTK/ZK. Além disso, houve uma redução significativa na densidade vascular nos implantes dos animais tratados com o medicamento. Estes resultados reforçam a ação antiangiogênica do PTK/ZK nas células endoteliais sugerindo uma provável via de sinalização na qual a redução da expressão da proteína antiapoptótica Bcl-2 causou uma diminuição da expressão da molécula pró-angiogênica CXCL-8, o que resultou numa menor densidade vascular e, conseqüentemente, menor crescimento tumoral. Conclui-se, portanto, que o PTK/ZK mostrou ser um medicamento antiangiogênico eficaz que poderá, no futuro, ser utilizado como terapêutica adjuvante nos pacientes portadores de carcinomas espinocelulares de cabeça e pescoço / The Vascular Endothelial Growth Factor (VEGF) has been considered the most important mediator in inducing tumor angiogenesis. It binds to specfic receptors known as Vascular Endothelial Growth Factor Receptor (VEGFR). An important pathway for angiogenesis is VEGF binding to VEGFR-2 and through PI3-K stimulates expression of antiapoptotic protein Bcl-2, inducing expression of proangiogenic cytokines CXCL-8 and CXCL-1, resulting in endothelial cell proliferation. A recently developed antiangiogenic drug is PTK787/ZK 222584 (PTK/ZK), a potent tyrosine kinase VEGFR inhibitor, without aparent cytotoxic effect on cells without expression of these receptors. For the better understanding of this mechanism and the angiogenic VEGF/VEGFR pathway of PTK/ZK in head and neck squamous cell carcinoma, it was developed in vitro and in vivo studies evaluating Bcl-2, CXCL-8 and CXCL-1 expression. A human angiogenesis experimental model was used in immunodeficient mice that received implants of tumor and endothelial cells or endothelial cells only, treated with PTK/ZK orally administered during 21 days. Tumor progression was evaluated by bioluminescence imaging. After mice sacrifice, the implants were paraffin embedded and 3µm sections were obtained for microvessel density analisis and 7µm sections for tumor and endothelial cells RNA retrieving by laser microdissection and analyzed by RT-PCR and Real time PCR. In vitro studies with co-culture of tumor and endothelial cells were analyzed by RT-PCR, Western Blot and ELISA to evaluate Bcl-2, CXCL-8 and CXCL-1 expression. The in vitro and in vivo results showed important downregulation of Bcl-2 as well as CXCL-8 expression in endothelial cells when treated with PTK/ZK. In addition, it was observed a significant reduction of microvessel density in implants of animals treated with the drug. These results suggest that the antiangiogenic mechanism of PTK/ZK on endothelial cells seems to occur by downregulation of antiapoptotic Bcl-2 expression leading to downregulation of pro-angiogenic CXCL-8, resulting in less tumor growth. In conclusion, PTK/ZK seems to be an efficient antiangiogenic drug for future adjuvant therapy of patients with head and neck squamous cell carcinoma.

All the results showed that TCMs can provide a source for discovering anti-angiogenic agents for the treatment of cancer, and all these experiments in the zebrafish and mammalian models further confirmed the value of zebrafish model in anti-angiogenic drug discovery. / Angiogenesis refers to the formation of new blood capillaries from pre-existing ones, and is essential in a series of normal physiological processes such as embryonic development and pathological responses. However, persistent unregulated angiogenesis causes "angiogenic diseases" such as diabetic retinopathy, tumor growth and metastasis, rheumatoid arthritis, and inflammatory diseases. The linkage between angiogenesis, tumor growth and metastasis was first hypothesized by Dr. Judah Folkman in the 1970s, and now this controversial idea is widely accepted and the inhibition of angiogenesis, or anti-angiogenesis, is considered as a promising anticancer therapeutic strategy. Bevacizumab (Avastin RTM by Genentech Inc.), the first approved anti-angiogenic drug by U.S. FDA in 2004, is a humanized monoclonal antibody to inhibit endothelial cell proliferation and angiogenesis for the treatment of metastatic colorectal cancer, non-small cell lung cancer, advanced breast cancer, glioblastoma, metastatic renal cell cancer. / Anti-angiogenic therapy in cancer treatment has led to the development of compounds designed to control a tumor's growth by blocking its ability to develop a blood supply. The development of agents with different mechanisms of action requires powerful preclinical models for the analysis and optimization of the therapy. Some in vitro and in vivo anti-angiogenic assays are already developed, for example, Human Umbilical Vein Endothelial Cell (HUVEC) assay, Chorioallantoic Membrane assay, Matrigel plug assay et al. Zebrafish, as a relatively new model organism, is firmly established as a powerful research platform for many areas of biology and drug discovery, allowing the testing of bioactive compounds in a whole organism and in cells undergoing normal cell-cell and cell-matrix interactions. Many anti- and pro-angiogenic molecules tested in zebrafish demonstrated similar effects to those observed in humans or other mammalian models. Besides providing a powerful platform for drug screening, zebrafish model can also be used for probing biological processes, and generate insights into mechanisms. / Cancer is a generic term for a large group of diseases that can affect any part of the body, which causes a vast medical problem and is a leading cause of death worldwide nowadays. However, for many years the main methods of treating cancer have been surgery, radiotherapy and chemotherapy. Among these treatments, chemotherapy has played a major role in cancer therapy for half a century. Despite improving managements and efforts, it is not surprising that the prognosis has not greatly improved because of the limitations of current therapies, such as toxicity, inherent and acquired resistance, and metastatic spread. This calls for novel cancer therapies and new group of anticancer agents for selectively targeting cancers without or with lower toxicity to normal tissues. / Traditional Chinese medicines (TCMs) have long been recognized as a rich source for discovering drugs, and various TCMs and their components have shown anti-angiogenic properties. In this thesis study, as a continuing pursuit for elucidating the anti-angiogenic properties of TCMs, our attention is focused on those with effects of anti-inflammation, anti-rheumatoid arthritis and anti-cancer. On zebrafish screening model, three of the selected TCMs, Rheum palmatum, Alpinia oxyphylla (seeds), and Kaempferia galanga showed potential anti-angiogenic activity, indicating the existence of potent anti-angiogenic components in these herbs. The ethyl acetate fraction of R. palmatum showed strong inhibition of vessel formation in zebrafish embryos. Further testing of the anthraquinones of this herb showed three of them displayed potent anti-angiogenic activities. The most potent compound---rhein could inhibit HUVEC migration and affect the mRNA expression of vegfa, kdr, angiopoietin1/2 and tie1/2; The n-hexane and ethyl acetate fractions of A. oxyphylla and K. galangal showed anti-angiogenic potentials both in zebrafish and HUVEC assays. The n-hexane and ethyl acetate fractions of A. oxyphylla could both inhibit the proliferation, migration and tube formation processes of HUVEC. And the most potential component, trans-ethyl-p-methoxycinnamate from K. galanga, could inhibit HUVEC migration and tube formation, and reduce all gene expressions involved in angiogenesis process except for vegfa. / He, Zhiheng. / Adviser: Wei Ge. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 87-108). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

O fator de crescimento endotelial vascular (VEGF) tem sido considerado o principal indutor da angiogênese tumoral por sua ligação a receptores específicos tirosina-quinase presentes nas células endoteliais (VEGFR). Uma importante via reguladora da angiogênese é caracterizada pela ligação do VEGF ao seu receptor VEGFR-2 ativando a via de sinalização de PI3-K, que estimula a expressão da proteína antiapoptótica Bcl-2 elevando a expressão de citocinas pró-angiogênicas CXCL-8 e CXCL-1, o que resulta na proliferação de células endoteliais. Recentemente, foi descoberta uma droga com ação antiangiogênica denominada de PTK787/ZK 222584 (PTK/ZK), que se mostrou um potente inibidor do receptor tirosina-quinase de VEGF, sem aparente efeito citotóxico em células que não expressam esses receptores. Visando a melhor compreensão do mecanismo de ação e da via de atuação antiangiogênica VEGF/VEGFR do PTK/ZK nos carcinomas espinocelulares de cabeça e pescoço, foram desenvolvidos estudos in vitro e in vivo avaliando-se a expressão de Bcl-2, CXCL-8 e CXCL-1. No presente estudo foi utilizado um modelo experimental de angiogênese humana em camundongos imunodeprimidos que receberam co-implantes de células tumorais e endoteliais ou apenas células endoteliais, tratados com administração oral de PTK/ZK por 21 dias. A progressão tumoral nos animais foi avaliada por meio de imagem de bioluminescência. Após o sacrifício dos camundongos, os implantes foram incluídos em parafina sendo obtidos cortes de 3µm para análise da densidade vascular e de 7µm para a técnica de microdissecção a laser visando a avaliação de RNA por meio de RT-PCR e PCR em tempo real nas células malignas e endoteliais. Concomitantemente, experimentos in vitro de co-cultura de células tumorais e endoteliais foram realizados para a análise de expressão de Bcl-2, CXCL-8 e CXCL-1 avaliados por meio de RT-PCR, Western Blot e teste ELISA. Os resultados demonstraram uma redução importante da expressão de Bcl-2, bem como de CXCL-8, em células endoteliais in vitro e in vivo quando tratados com PTK/ZK. Além disso, houve uma redução significativa na densidade vascular nos implantes dos animais tratados com o medicamento. Estes resultados reforçam a ação antiangiogênica do PTK/ZK nas células endoteliais sugerindo uma provável via de sinalização na qual a redução da expressão da proteína antiapoptótica Bcl-2 causou uma diminuição da expressão da molécula pró-angiogênica CXCL-8, o que resultou numa menor densidade vascular e, conseqüentemente, menor crescimento tumoral. Conclui-se, portanto, que o PTK/ZK mostrou ser um medicamento antiangiogênico eficaz que poderá, no futuro, ser utilizado como terapêutica adjuvante nos pacientes portadores de carcinomas espinocelulares de cabeça e pescoço / The Vascular Endothelial Growth Factor (VEGF) has been considered the most important mediator in inducing tumor angiogenesis. It binds to specfic receptors known as Vascular Endothelial Growth Factor Receptor (VEGFR). An important pathway for angiogenesis is VEGF binding to VEGFR-2 and through PI3-K stimulates expression of antiapoptotic protein Bcl-2, inducing expression of proangiogenic cytokines CXCL-8 and CXCL-1, resulting in endothelial cell proliferation. A recently developed antiangiogenic drug is PTK787/ZK 222584 (PTK/ZK), a potent tyrosine kinase VEGFR inhibitor, without aparent cytotoxic effect on cells without expression of these receptors. For the better understanding of this mechanism and the angiogenic VEGF/VEGFR pathway of PTK/ZK in head and neck squamous cell carcinoma, it was developed in vitro and in vivo studies evaluating Bcl-2, CXCL-8 and CXCL-1 expression. A human angiogenesis experimental model was used in immunodeficient mice that received implants of tumor and endothelial cells or endothelial cells only, treated with PTK/ZK orally administered during 21 days. Tumor progression was evaluated by bioluminescence imaging. After mice sacrifice, the implants were paraffin embedded and 3µm sections were obtained for microvessel density analisis and 7µm sections for tumor and endothelial cells RNA retrieving by laser microdissection and analyzed by RT-PCR and Real time PCR. In vitro studies with co-culture of tumor and endothelial cells were analyzed by RT-PCR, Western Blot and ELISA to evaluate Bcl-2, CXCL-8 and CXCL-1 expression. The in vitro and in vivo results showed important downregulation of Bcl-2 as well as CXCL-8 expression in endothelial cells when treated with PTK/ZK. In addition, it was observed a significant reduction of microvessel density in implants of animals treated with the drug. These results suggest that the antiangiogenic mechanism of PTK/ZK on endothelial cells seems to occur by downregulation of antiapoptotic Bcl-2 expression leading to downregulation of pro-angiogenic CXCL-8, resulting in less tumor growth. In conclusion, PTK/ZK seems to be an efficient antiangiogenic drug for future adjuvant therapy of patients with head and neck squamous cell carcinoma.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2007. / Härtill 4 uppsatser.

Angiogênese é a formação de novos capilares a partir de vasos pré-existentes, mediada por eventos de sinalização bioquímica que determinam proliferação, migração, diferenciação e morte celular e controlam crescimento e remodelação tecidual. A beta2-glicoproteína I (beta2GPI) é uma proteína plasmática com ação sobre a função vascular e a aterogênese. Monomêros de beta2GPI apresentam efeito anti-inflamatório e anticoagulante; a clivagem enzimática favorece sua dimerização e induz o aparecimento de efeitos opostos. Resultados anteriores mostraram que monômeros e dímeros de beta2GPI têm efeitos diferentes sobre a proliferação e a diferenciação de células endoteliais em culturas bidimensionais utilizadas como modelo de angiogênese. Os monômeros e dímeros de beta2GPI foram obtidos por purificação fracionada e caracterizada por SDS-PAGE e ELISA, como descrito. Neste trabalho, foram utilizadas culturas tridimensionais de células humanas vasculares de cordão umbilical (HUVEC) sobre Matrigel foram utilizadas para identificar efeitos de monômeros e dímeros da beta2GPI sobre a proliferação, migração e formação de estruturas de interação celular in vitro. O monômero de ?2GPI atuou como um fator de diferenciação endotelial dependente da densidade de plaqueamento, induzindo nas culturas tridimensionais de HUVECs a formação de fenótipos alongados, prolongamentos e estruturas de interação célula-célula. A fração dimérica modulou negativamente a proliferação de HUVECs. A membrana corioalantóide (CAM) de embriões de galinha foi empregada para estudar efeitos da beta2GPI sobre a angiogênese. In ovo, o dímero de beta2GPI impediu a angiogênese e induziu a morte embrionária 48h após a exposição, enquanto o monômero permitiu o desenvolvimento do embrião até o 10º dia, apesar de induzir mudanças precoces no desenvolvimento dos vasos da membrana corioalantóide. As estruturas da microvasculatura foram analisadas através de uma abordagem morfológica quantitativa, baseada na classificação de padrões binários locais (LBP). Alvos moleculares de beta2GPI relatados anteriormente foram considerados como fonte dos efeitos observados in vitro e in ovo. Os resultados obtidos suportam dados anteriores sobre a inibição da via de sinalização de anexina-2/Akt pela beta2GPI. Adicionalmente, sugere-se a via de sinalização Notch como um alvo do efeito antiangiogênico de da beta2GPI / Angiogenesis is the formation of new capillaries from pre-existing vessels, mediated by biochemical signaling events that determine proliferation, migration, differentiation and cell death, and control of tissue growth and remodeling. beta2-glycoprotein I (beta2GPI) is a plasma protein active on vascular function and atherogenesis. ?2GPI monomers present anti-inflammatory and anticoagulant effects. Enzymatic cleavage favors beta2GPI dimerization and induces the appearance of opposing effects. Previous results have shown that beta2GPI monomers and dimers induce different effects upon the proliferation and differentiation of endothelial cells in two-dimensional cultures used as an angiogenesis model. beta2GPI monomers and dimers were obtained by fractioned purification and characterized by SDS-PAGE and ELISA, as described. In this work, three-dimensional Human Umbilical Vein Endothelial Cells (HUVEC) cultures on Matrigel were used to investigate the effects of beta2GPI monomers and dimers upon proliferation, migration and in vitro formation of cellular interaction structures. The beta2GPI monomer performed as a density-dependent endothelial differentiation factor, inducing the formation of elongated phenotypes, membrane extensions and cell-cell interaction structures in three-dimensional HUVEC cultures; the dimeric fraction negatively modulated the proliferation and differentiation of HUVECs. The chorioallantoic membrane (CAM) of chicken embryos was employed to study the effects of beta2GPI upon angiogenesis. In ovo, the beta2GPI dimer prevented angiogenesis and induced embryonic death after 48h exposure, while the monomer allowed embryo development up to the 10th day, despite it induced early changes in the development of chorioallantoic membrane vessels. Microvasculature structures were evaluated through a quantitative morphology approach, based on local binary pattern classification. Previously reported molecular beta2GPI targets were then considered as the source of the observed effects in vitro and in ovo. The obtained results support previous data on the inhibition of the annexin-2/Akt signaling pathway by beta2GPI. Additionally, the Notch signaling pathway is suggested as a target of the antiangiogenic effect of beta2GPI