Five traditional Chinese medicines were screened for their antiangiogenic activities through zebrafish angiogenic assay. Two of them, Tripterygium wilfordii and Rheum palmatum showed potential in the primary screening. T. wilfordii was selected in further study. / In the further investigation of antiangiogenic activity of triptolide on mammal systems, triptolide showed potent activity in human umbilical vein endothelial cells (HUVECs) assays including proliferation, migration and tube formation assay. It inhibited HUVEC proliferation with IC50 as low as 34 nM. It also showed more potency in HUVEC migration and tube formation assay at as low concentration as nanomolar level than SU5416, a putative VEGFR-2 inhibitor currently in clinic trials. RT-PCR and western blotting analysis showed that the underlying mechanism of triptolide correlated with down-regulation of VEGFR-2 and Tie2 expression and production. Tie2 inhibition appeared to be a later event as compared with VEGFR-2. Tie2 overexpression in HUVEC could attenuate the inhibitory effect of triptolide on HUVEC proliferation. Tie2 knockdown mimicked the inhibition activity of triptolide in tube formation assay. These phenomemon revealed that Tie2 signaling pathway plays a crucial role in triptolide-mediated angiogenesis inhibition. In in vivo Matrigel Plug assay, triptolide showed inhibition effect at as low as 100 nM. / T. wilfordii is an immune-suppressive, anti-inflammatory herb used in China for centuries. Through bioassay-guided purification, three antiangiogenic terpenoids were isolated from the ethyl acetate fraction, namely, celastrol, cangoronine and triptolide. Among them, triptolide manifested the most potent antiangiogenic activities against vessel formation. As low as 0.31microM, triptolide inhibited 20% of vessel formation, and the inhibition reached a plateau of 50% at 1.2 microM. Celatrol reduced vessel formation by more than 30% at 0.62microM, but killed 50% of the embryos at higher concentrations. Cangoronine was much weaker, inhibiting vessel formation by 20% at 2.5microM. These three components all showed stronger antiangiogenic activities than 2-methoxyestradiol, a putative compound currently under clinical trials as an antiangiogenic agent for cancer treatment, as the latter inhibited angiogenesis in zebrafish embryos by 34% at 10microM. The loss of vessel formation in the embryos treated with triptolide was further confirmed using endogenous alkaline phosphatase staining. Semi-quantitative RT-PCR analysis revealed that triptolide dose- and time-dependently reduced the mRNA expression of angiopoietin (angpt2) and tie2 in zebrafish, indicating the involvement of angpt2/tie2 signaling pathway in the antiangiogenic action of triptolide. / This research revealed that zebrafish model is a promising antiangiogenic model for both the screening of antiangiogenic agents from Chinese herbal medicine and the subsequent discovery for the drug targets. Triptolide, an anti-inflammatory component from T. wilfordii, is a potent angiogenic inhibitor through targeting VEGFR-2 and Tie2 pathways in mammal models whereas targeting ang2-tie2 pathway in zebrafish model. The anti-tumor action of triptolide was demonstrated to be partly through inhibition of tumor angiogenesis. Moreover, the potent antiangiogenic action exerted by triptolide at nanomolar dosage level gives proof that it is a promising lead compound for the development of antiangiogenic drug for cancer treatment. (Abstract shortened by UMI.) / He, Mingfang. / Adviser: Paul Pui-Hey Bot. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0247. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 84-106). / 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 Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

Submitted by Suzana Dias (suzana.dias@famerp.br) on 2018-10-19T20:34:14Z No. of bitstreams: 1 CarlosHenriqueViésidoNascimentoFilho_tese.pdf: 4165435 bytes, checksum: e1126fdbe7f8fe5e5640b7a27f4c69e2 (MD5) / Made available in DSpace on 2018-10-19T20:34:14Z (GMT). No. of bitstreams: 1 CarlosHenriqueViésidoNascimentoFilho_tese.pdf: 4165435 bytes, checksum: e1126fdbe7f8fe5e5640b7a27f4c69e2 (MD5) Previous issue date: 2018-03-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancies worldwide. Antiangiogenic drugs are a conventional treatment, although it has been shown to stimulate hypoxic areas inside the tumor, and hypoxia has been associated with metastasis as well as accumulation of cancer stem cell (CSC) in breast cancer. Objective: To study the effects of hypoxia on HNSCC behavior and accumulation of cancer stem cell. Material and methods: Culture of HN6, HN12 and HN13 tumor cells under hypoxic conditions was carried out for 6hrs, 12hrs, and 24hrs under an O2 concentration of 1%. The content of cancer stem cell was determined using Aldefluor assay according to the manufacturer's (STEMCELL Technologies) to detect ALDH1A1Bright cells in combination with APC mouse anti-human CD44. Tumor invasion under hypoxic conditions was carried out using Millicell Cell Culture Inserts (Millipore, Billerica, MA, USA). Control tumor cells were maintained in DMEM supplemented with 10% FBS and 1% antibiotics at normoxia levels (~21% of O2), while hypoxia group was maintained at 1% of O2. We used a PTEN inhibitor (BPV) concentrations of 1nM, 5nM, and 10nM. Results: PTEN expression is downregulated under hypoxic conditions. Hypoxia-induced PTEN downregulation triggered an epithelial-mesenchyme transition (EMT) phenotype and accumulation of CSC. Plus, we showed antagonistic expression of PTEN and HIF-1α when targeted disruption of PTEN (shPTEN HN13 cell line) strongly suggesting that loss of PTEN activates the PI3K/mTOR pathway in association with HIF-1αupregulated. Validation of our results in xenograft animal models showed colocalization of ALDH1A1Bright cells and hypoxic areas along with the loss of nuclear localization of PTEN. Conclusion: Thus, we show that hypoxia-driven downregulation of PTEN plays a crucial role in tumor aggressiveness and that the potential use of CSC-targeted therapies should be considered during the administration of anti-angiogenic drugs to minimize tumor resistance. / O câncer de cabeça e pescoço (CCP) é a sexta neoplasia mais comum no mundo. Os fármacos antiangiogênicos constituem um tratamento efetivo na redução da massa tumoral, porém estimulam áreas de hipóxia no interior do tumor. O aumento de áreas em hipóxia tem sido associado às metástases e ao acúmulo de células-tronco tumorais (CTT) no câncer de mama. Objetivos: Com base nestas evidências, o presente estudo avaliou os efeitos da hipóxia no comportamento de células de CCP e verificou o acúmulo de CTT. Material e métodos: As linhagens celulares HN6, HN12 e HN13 foram cultivadas em condições de hipóxia por 6, 12 e 24 horas, sob uma concentração de O2 menor que 2%. As CTT foram identificadas por meio da determinação de elevados níveis do conteúdo enzimático aldehyde dehydrogenases (ALDH1A1Bright), utilizando o Kit Aldefluor, em combinação com elevados níveis de CD44, uma glicoproteína de superfície celular, conjugado com o fluoróforo allophycocyanin (APC). O ensaio de invasão tumoral sob condições de hipóxia foi realizado utilizando Millicell Cell Culture Inserts. Como controle foram utilizadas células tumorais mantidas em meio DMEM suplementado com 10% de SFB e 1% de antibiótico, em níveis de normóxia (~21% de O2). O composto bisperoxovanadium (BPV), um inibidor de tirosino fosfatase, foi utilizado nas concentrações de 1nM, 5nM e 10 nM para reduzir os níveis celulares de PTEN. Complementar ao tratamento com BVP foi realizado o silenciamento gênico utilizando segmentação direcionada de PTEN (linhagem celular shPTEN HN13 e como controle pGIPZ). Resultados: Os resultados apontam uma menor expressão do PTEN durante condições de hipóxia. Demonstrou-se a expressão antagonista de PTEN e HIF-1α por meio do silenciamento gênico com a segmentação direcionada, sugerindo, fortemente, que a perda de PTEN ativa a via PI3K/mTOR em associação com a alta expressão de HIF-1α. Além da regulação negativa do PTEN pela hipóxia, foi identificado o desencadeamento do processo epitélio-mesênquimal (EMT) em combinação com o acúmulo de CTT. A validação dos resultados em modelos animais de xenoenxerto evidenciou a co-localização de células ALDH1A1 positivas em áreas de hipóxia juntamente com a perda da expressão nuclear de PTEN. Conclusão: Este trabalho mostrou que a regulação negativa do PTEN durante períodos de hipóxia desempenha um papel fundamental no acúmulo de CTT e aumento da agressividade tumoral. Os resultados indicam que o tratamento para CCP utilizando agentes antiangiogênicos deve ser pareado com terapias voltadas à destruição de CTT, minimizando, assim, a aquisição de um fenótipo de resistência tumoral.

Submitted by Suzana Dias (suzana.dias@famerp.br) on 2018-11-09T18:31:25Z No. of bitstreams: 1 JessicaHelenaPires_dissert.pdf: 3617808 bytes, checksum: 1df14ad1ee40275580d520f969311d14 (MD5) / Made available in DSpace on 2018-11-09T18:31:25Z (GMT). No. of bitstreams: 1 JessicaHelenaPires_dissert.pdf: 3617808 bytes, checksum: 1df14ad1ee40275580d520f969311d14 (MD5) Previous issue date: 2018-03-06 / Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP / Breast cancer represents the second type of tumor that has the highest mortality rates, being the most common among women. The causes of these high mortality rates are related to high proliferation and metastasis, and for tumor progression, the growth of new blood vessels, angiogenesis is required. This event can be stimulated by several factors, such as insulin-like growth factor 1 receptor (IGF-1R), hypoxia-inducible factor 1-alpha (HIF-1α) and vascular endothelial growth factor (VEGF). Several molecules are involved in the control of angiogenesis such as melatonin and microRNAs (miRNAs). The miRNAs can induce the gene silencing of genes related to angiogenesis by pairing with certain specific messenger RNA (mRNA), resulting in the degradation of this molecule. Melatonin (N-acetyl-5-methoxytryptamine), the main hormone produced and secreted by the pineal gland, has several physiological functions and a proven antiangiogenesis action. This hormone can regulate miRNAs and genes related to this process. Objective: To evaluate the ability of melatonin to modulate miR-152-3p and its targets in triple-negative breast cancer cells. Material and Method: After the determination of the melatonin concentration to be used, the differential expression of the miRNAs in the MDA-MB-468 strain after the melatonin treatment was evaluated using the plate RT² Profiler™ PCR Array Human Breast Cancer containing 84 miRNAs related to breast cancer. An in-silico analysis was performed to select a miRNA involved in angiogenesis and its potential target genes. Overexpression of miR-152-3p was performed on MDA-MB-468 and MDA-MB-231 cells by transient transfection and after relative quantification of their expression and their target genes IGF-1R, VEGF and HIF-1α was evaluated by real-time PCR. Quantification of the protein expression of the genes was verified by immunocytochemistry. Results: The cell viability assay in the MDA-MB-468 cell line demonstrated that cells treated with 1 mM melatonin had the lowest viability (p <0.05). Analysis of the miRNAs by PCR Array in the MDA-MB-468 cell line showed six positively regulated miRNAs and seven negatively regulated miRNAs after treatment with melatonin. Evaluation of gene expression demonstrated that miR-152-3p overexpression was influenced by melatonin, leading to increased expression of the genes IGF-1R, HIF-1α and VEGF in MDA-MB-468 cells. In the MDA-MB-231 cell line, melatonin did not influence the expression of miR-152-3p and decreased the expression of the target genes. Finally, immunocytochemistry revealed that melatonin and overexpression of miR-152-3p were able to decrease the protein expression of IGF-1R, HIF-1α and VEGF in the MDA-MB-468 and MDA-MB-231 cells. Conclusions: Melatonin was able to modulate the expression of miR-152-3p and its target genes involved in angiogenesis in triple-negative breast cancer. Therefore, this study confirms the action of melatonin on the important cellular event of angiogenesis, a determinant process for the progression of the disease and also indicates it as a potential therapeutic protocol for triple-negative breast cancer. / O câncer de mama representa o segundo tipo tumoral que possui os maiores índices de mortalidade, sendo o mais comum entre as mulheres. As causas desses altos índices de mortalidade têm relação com a alta proliferação e formação de metástases, e para a progressão tumoral, é necessário o crescimento de novos vasos sanguíneos, a angiogênese. Este evento pode ser estimulado por diversos fatores, como o receptor tipo 1 do fator de crescimento semelhante à insulina (IGF-1R), o fator induzido por hipóxia 1 alfa (HIF-1α) e o fator de crescimento endotelial vascular (VEGF). Os miRNAs podem induzir o silenciamento de genes relacionados com a angiogênese pelo pareamento com determinado RNA mensageiro (RNAm) específico, resultando na degradação desta molécula. A melatonina (N-acetil-5-metoxitriptamina), principal hormônio produzido e secretado pela glândula pineal, possui diversas funções fisiológicas e comprovada ação antitumoral, inclusive ação antiangiogênica. Esse hormônio pode regular miRNAs e genes relacionados a esse processo. Objetivo: Avaliar a capacidade da melatonina em modular o miR-152-3p e seus alvos, em células de câncer de mama triplo-negativo. Material e Método: Após a definição da concentração de melatonina a ser utilizada pelo ensaio de viabilidade celular, foi avaliada a expressão diferencial dos miRNAs na linhagem MDA-MB-468, após o tratamento com melatonina, utilizando-se a placa RT² Profiler™ PCR Array Human Breast Cancer que contêm 84 miRNAs relacionados ao câncer de mama. Uma análise in silico foi realizada para seleção de um miRNA envolvido na angiogênese e seus potenciais genes-alvo. A superexpressão do miR-152-3p foi realizada nas células MDA-MB-468 e MDA-MB-231 por transfecção transiente e após, a quantificação relativa de sua expressão e de seus genes-alvo IGF-1R, VEGF e HIF-1α foram avaliadas por PCR em tempo real. A quantificação da expressão proteica dos genes foi verificada por imunocitoquímica. Resultados: O ensaio de viabilidade celular na linhagem MDA-MB-468, demonstrou que as células tratadas com 1 mM de melatonina tiveram os menores valores de viabilidade (p<0,05). A análise dos miRNAs por PCR Array na linhagem MDA-MB-468 apontou seis miRNAs regulados positivamente e sete miRNAs regulados negativamente, após o tratamento com a melatonina. A avaliação da expressão gênica demonstrou que a superexpressão do miR-152-3p foi influenciada pela melatonina, levando ao aumento da expressão dos genes IGF-1R, HIF-1α e VEGF na linhagem MDA-MB-468. Já na linhagem MDA-MB-231 a melatonina não influenciou a expressão do miR-152-3p e diminuiu a expressão dos genes-alvo. Por fim, a imunocitoquímica revelou que a melatonina e a superexpressão do miR-152-3p foram capazes de diminuir a expressão proteica de IGF-1R, HIF-1α e VEGF nas linhagens MDA-MB-468 e MDA-MB-231. Conclusões: A melatonina foi capaz de modular a expressão do miR-152-3p e de seus genes-alvo envolvidos com a angiogênese no câncer de mama triplo-negativo. Portanto, este estudo confirma a ação da melatonina no importante evento celular de angiogênese, processo determinante para a progressão da doença e ainda indicá-la como potencial protocolo terapêutico para o câncer de mama triplo-negativo.

Thesis (Ph. D.)--University of Florida, 2003. / Title from title page of source document. Includes vita. Includes bibliographical references.

Inhibition of angiogenesis is an effective and low toxic therapeutic avenue for the treatment of cancer patients in addition to traditional interventions. Majority of current available angiogenesis inhibitors for cancer therapies are growth factor inhibitors and small molecule tyrosine kinase inhibitors. A number of endogenous proteins and/or proteolytic fragments of extracellular matrix proteins are shown to have the activity of inhibition of angiogenesis by directly targeting endothelial cells. Structural analyses have indicated that a common structure of anti-parallel β-sheet with a highly positively charged surface presents in many of those inhibitors. This common structural feature is critical for the maintenance of their anti-angiogenic function. With this structural information, we have designed and developed a new class of anti-angiogenic proteins by integrating the short anti-parallel β-sheet forming sequences of endogenous anti-angiogenic proteins into a stable host protein, the extracellular domain-1 of cluster of differentiation 2 molecule (CD2D1). 1D 1H NMR spectra analyses indicated that the designed anti-angiogenic protein (ref to as ProAgio) folded as a β-sheet structure similar to that of the parental protein, CD2D1. ProAgio inhibited the growth of human umbilical vein cells (HUVECs) without affecting the growth of epithelial cells, suggesting a specific effect to endothelial cells. ProAgio effectively reduced endothelial tubules formed by the co-culture of HUVECs and PC3 cells on matrix gel in vitro. The designed anti-angiogenic protein was further site-specifically PEGylated in order to improve PK/PD properties and reduce immunogenicity. Examinations with PC3 xenografts showed that both ProAgio and the PEGylated ProAgio dramatically inhibited tumor growth. Immunofluorescence staining analyses of the endothelial marker CD31 indicated dramatic decreases in tumor vessels in lengths and branching points. Histological and immunofluorescence staining analyses of tissue slices of major organs indicated that there were no pathological damages to the tissue structure or disruption of normal vessels associated with the treatment of our designed anti-angiogenic agent. Overall, our studies developed a novel anti-angiogenesis agent that may have great clinical potentials. Our concept of protein design can be extended to the development of other novel protein drugs.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Feb. 9, 2009). Includes bibliographical references.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 155-170.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 190-220.