With the critical role of aberrantly active Signal Transducer and Activator of Transcription (Stat) 3 protein in many human cancers, selective small-molecule inhibitors targeting the dimerization event which is required for stat3 activation, would be valuable as therapeutic agents. And the inhibitors will be useful chemical probes to clarify the complex biological functions of Stat3. By computational and structural analyses of the interaction between Stat3 and the lead dimerization disruptor, S3I-201, we have designed a diverse set of analogs. One of the most active analogs, S3I-201.1066 is derived to contain a cyclo-hexyl benzyl moiety on the amide nitrogen, which increases the binding to the Stat3 SH2 domain. Evidence is presented from in vitro biochemical and biophysical studies that S3I-201.1066 directly interacts with Stat3 or the SH2 domain, with an affinity (K[subscript D]) of 2.74 [micrometer], and disrupts the binding of Stat3 to the cognate pTyr-peptide, GpYLPQTV-NH2, with an IC₅₀ of 23 [micrometer]. Moreover, S3I-201.1066 selectively blocks the association of Stat3 with the epidermal growth factor receptor (EGFR), and inhibits Stat3 tyrosine phosphorylation and nuclear translocation in EGF-stimulated mouse fibroblasts. In cancer cells that harbor aberrant Stat3 activity, S3I-201.1066 inhibits constitutive Stat3 DNA-binding and transcriptional activities. By contrast, S3I-201.1066 has no effect on Src activation or the EGFR-mediated activation of the Erk1/2MAPK pathway. S3I-201.1066 selectively suppresses the viability, survival, and malignant transformation of the human breast and pancreatic cancer lines and the v-Src-transformed mouse fibroblasts harboring persistently active Stat3. Treatment with S3I-201.1066 on malignant cells harboring aberrantly active Stat3 down regulated the expression of c-Myc, Bcl-xL, Survivin, matrix metalloproteinase 9, and VEGF, which are known Stat3-regulated genes important in diverse tumor processes. The in vivo administration of S3I-201.1066 induced significant anti-tumor response in mouse models of human breast cancer, which correlates with the inhibition of constitutively active Stat3 and the suppression of known Stat3-regulated genes. Further computer-aided lead optimization derives higher-affinity (K[subscript D], 504 nM), orally bioavailable Stat3 SH2 domain-binding ligand, BP-1-102 as a structural analog of S3I-201.1066. The most significant modification is the pentafluorobenzene sulfonamide component of BP-1-102, which permits accessibility of a third sub-pocket of the Stat3 SH2 domain surface. BP-1-102-mediated inhibition of aberrantly-active Stat3 in human pancreatic cancer, Panc-1, breast cancer, MDA-MB-231, and prostate (DU145) cancer cells and in the mouse transformed fibroblasts harboring aberrantly-active Stat3. It also disrupts Stat3-NF[kappa]B cross-talk and suppresses the release of granulocyte colony-stimulating factor, soluble intercellular adhesion molecule-1, macrophage-migration-inhibitory factor/glycosylation-inhibiting factor, interleukin-1 receptor antagonist and the serine protease inhibitor (serpin) protein 1, and the expression of c-Myc, Cyclin D1, Bcl-xL, Survivin, and vascular endothelial growth factor expression in vitro and in vivo. Inhibition of tumor cell-associated constitutively-active Stat3 further suppresses focal adhesion kinase and paxillin induction, enhances E-cadherin expression, and down-regulates Kruüppel-like factor 8 expression. Consequently, BP-1-102 selectively suppresses anchorage-dependent and independent growth, survival, migration and invasion of Stat3-dependent tumor cells in vitro. Intravenous or oral gavage delivery of BP-1-102 furnishes micromolar or microgram levels in tumor tissues and inhibits growth of mouse xenografts of human breast and lung tumors. Computer-aided lead optimization has therefore derived a more suitable small-molecule inhibitor as a drug candidate. Our studies of the Stat3 SH2 protein surface and of the interactions between lead agents and the SH2 domain provided significant data to facilitate the structural optimization. From S2I-201 to S3I-201.1066 and to BP-1-102, we note the substantial gain in potency and efficacy, and the pharmacokinetic improvements. The oral bioavailability of BP-1-102 represents a substantial advancement in the discovery of small-molecule Stat3 inhibitors as novel anticancer agents.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed June 5, 2008). Includes bibliographical references.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 229-253.

Orientadores: Laura Sterian Ward, Antônio Hugo José Fróes Marques Campos / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T01:02:11Z (GMT). No. of bitstreams: 1 Rocha_AngelicaGomes_M.pdf: 1644333 bytes, checksum: e3503fb6e2c8209f7a6696df111d6a78 (MD5) Previous issue date: 2014 / Resumo: Marcadores de malignidade, especialmente capazes de distinguir lesões de padrão folicular, que sejam de fácil implantação na rotina do diagnóstico de nódulos tireoidianos, continuam sendo extremamente necessários, dado o crescente aumento de nódulos tireoidianos diagnosticados nos últimos anos. A via HGF/c-Met/STAT3 está relacionada com desenvolvimento e progressão tumoral, sendo que a expressão de c-Met, HGF e de STAT3 foram descritas em grande parte dos carcinomas papilíferos de tireoide (CPT), mas não em tecido tireoidiano normal, sugerindo sua relação com o desenvolvimento e progressão do CPT. Para avaliar a utilidade da expressão proteica de c-Met, HGF, STAT3, e de sua proteína fosforilada (pSTAT3) no diagnóstico e no prognóstico de pacientes com nódulos tireoidianos, analisamos 356 tecidos tireoidianos, sendo 153 carcinomas papilíferos (CPT), dos quais 95 eram clássicos (CPC), 47 carcinomas papilíferos variante folicular (CPVF), e 11 carcinomas papilíferos de células altas (CPCA); 34 carcinomas foliculares (CFT), 34 adenomas foliculares (AF), 124 bócios e 11 tecidos normais. Todos os pacientes foram tratados e acompanhados de acordo com um mesmo protocolo padrão por 1-10 anos (Mo=5 anos). Áreas representativas do tecido foram selecionadas para a construção de uma lâmina de tissue microarray (TMA) que foi submetida à técnica de imunoistoquímica e analisada pelo score de Allred. A expressão citoplasmática de c-Met foi capaz de diferenciar nódulos malignos de benignos (p<0,0001, sensibilidade 86%, especificidade 76%, VPP 77%, VPN 86%); CPT de CF (p=0,0003, sensibilidade 96%, especificidade 31%, VPP 87%, VPN 63%); variante folicular de CPT de CF (p=0,0232 sensibilidade 93%, especificidade 31%, VPP 66%, VPN 77%); assim como variante folicular de CPT de AF (p=0,0003, sensibilidade 93%, especificidade 50%, VPP 68%, VPN 87%). Além disso, a expressão de c-Met se correlacionou com tireoidite (p<0,0001) e multifocalidade (p=0,0028), mas não com presença de cápsula, invasão, tamanho do tumor, estadiamento TNM, e presença de metástase no diagnóstico e na evolução. A expressão nuclear de STAT3 diferenciou os nódulos benignos dos malignos (p<0,0001, sensibilidade 83%, especificidade 74%, VPP 75%, VPN 83%); CF de AF (p=0,0457, sensibilidade 80%, especificidade 52%, VPP 65%, VPN 71%); bócios de CPT variante folicular (p<0,001, sensibilidade 89%, especificidade 65%, VPP 91%, VPN 60%); bócio de CF (p<0,0001, sensibilidade 89%, especificidade 80%, VPP 95%, VPN 60%); bócio de AF (p=0,0005, sensibilidade 89%, especificidade 80%, VPP 95%, VPN 60%). Além disso, a expressão de STAT3 se correlacionou com tireoidite (p=0,0095) e multifocalidade (p<0,0001), mas não com presença de cápsula, invasão, tamanho do tumor, estadiamento TNM, e presença de metástase no diagnóstico e na evolução. A expressão de pSTAT3 e HGF não auxiliou no diagnóstico dos nódulos, e tampouco se correlacionou com características de agressividade dos tumores. Conclui-se que as proteínas c-Met e STAT3 podem ser consideradas marcadores clínicos úteis na rotina de laboratórios, uma vez que foram capazes de diferenciar os nódulos malignos dos benignos, alguns tipos histológicos dos nódulos, além de se correlacionarem com fatores de agressividade dos tumores / Abstract: Malignancy markers, especially the ones that are capable of distinguishing follicular lesions and with easy deployment in the routine diagnosis of thyroid nodules are much needed, given the increasing number of thyroid nodules in recent years. The HGF/c-Met/STAT3 pathway is related to the development and progression of many types of cancers, and c-Met, HGF and STAT3 expression were described in most papillary thyroid carcinomas (PTC), but not in normal thyroid tissue, suggesting it is related with the development and progression of PTC. To evaluate the usefulness of c-Met, HGF, STAT3, and its phosphorylated form (pSTAT3) protein expression in the diagnosis and prognosis of patients with thyroid nodules, we analyzed 356 thyroid tissues, including 153 papillary carcinomas (PTC), 95 classical type, 47 follicular variants of papillary carcinoma, and 11 tall cells carcinomas; 34 follicular carcinomas (FC), 34 follicular adenomas (FA), 124 goiters and 11 normal tissues. All patients were treated and monitored according to the same standard protocol for 1-10 years (Mo = 5 years). Representative tissue areas were selected for the construction of a tissue microarray (TMA) which was subjected to immunohistochemistry and analyzed by the Allred score. The cytoplasmic expression of c-Met was able to differentiate malignant from benign nodules (p <0.0001, sensitivity 86%, specificity 76%, PPV 77%, 86% NPV); PTC from FCT (p = 0.0003, sensitivity 96%, specificity 31%, PPV 87%, 63% NPV); follicular variant of PTC from FCT (p = 0.0232 sensitivity 93%, specificity 31%, PPV 66%, NPV 77%); as well as follicular variant of CPT from FA (p = 0.0003, sensitivity 93%, specificity 50%, PPV 68%, 87% NPV). Furthermore, c-Met expression was correlated to the presence of thyroiditis (p<0.0001) and multifocality (p=0.0028), but not with the presence of capsule, invasion, tumour size, TNM staging, and metastasis at diagnosis or evolution of the disease. The nuclear expression of STAT3 differentiated benign from malignant nodules (p <0.0001, sensitivity 83%, specificity 74%, PPV 75%, NPV 83%); FCT from FA (p = 0.0457, sensitivity 80%, specificity 52%, PPV 65%, NPV 71%); goiter follicular variant of PTC (p <0.001, sensitivity 89%, specificity 65%, PPV 91%, 60% NPV); goiter from FCT (p <0.0001, sensitivity 89%, specificity 80%, PPV 95%, NPV 60%); goiter from FA (p = 0.0005, sensitivity 89%, specificity 80%, PPV 95%, NPV 60%). In addition, STAT3 expression was associated with thyroiditis (p=0.0095) and multifocality (p<0.0001), but not with the presence of capsule, invasion, tumour size, TNM staging, and metastasis at diagnosis or evolution of the disease. The expression of pSTAT3 and HGF did not help the diagnostic of nodules and was not correlated with any tumour characteristic of aggressiveness. We concluded that c-Met and STAT3 could be useful as molecular markers in laboratory routine, helping to differentiate malignant from benign nodules, and some histological types of nodules, and was also associated with some tumour characteristics of aggressiveness / Mestrado / Ensino em Saúde / Mestra em Clínica Médica

Orientadores: Liliana Aparecida Lucci de Angelo Andrade, Fatima Aparecida Böttcher-Luiz / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T02:59:06Z (GMT). No. of bitstreams: 1 Pietro_Luciana_D.pdf: 3492137 bytes, checksum: 723d823e8ddb16925da7aa8f48f22ea1 (MD5) Previous issue date: 2013 / Resumo: A invasão do endométrio pelo trofoblasto extraviloso é fundamental no desenvolvimento do feto e da placenta, processo este controlado por fatores ligados à atividade imunológica e hormonal que, quando alterada, pode resultar em interrupção da gestação e/ou geração das chamadas doenças trofoblásticas gestacionais. Em algumas situações, pode haver evolução para o coriocarcinoma, neoplasia maligna do trofoblasto, em que há evidências da atuação das moléculas ligadas ao processo de fusão celular e inflamação. Porém, os estudos neste tema são incipientes e inconclusivos. Considerando essas informações, o objetivo deste trabalho é estudar de forma comparativa a expressão das citocinas LIF, IL-6 e do ativador de transcrição STAT-3, além da telomerase, em material de aborto, de placenta normal a termo e de coriocarcinoma. Métodos: a expressão destas moléculas foi avaliada pelos métodos: imunoistoquímica (IHQ), imunofluorescência (IF), Western Blotting (WB) e Real-Time PCR (RT-PCR), em amostras de material de aborto, placenta normal a termo e coriocarcinoma (N=12 cada um). Os ensaios de WB e Real-Time PCR empregaram material a fresco de placenta normal a termo e seu cultivo celular e cultura da linhagem BeWo. Resultados: no material de aborto, as reações de IHQs evidenciaram expressão moderada de IL-6 em 58,4% dos casos e intensa de STAT-3 em 33,3%. Na placenta normal, observou-se intensa marcação de IL-6 em 50% e de STAT- 3 em 16,7% dos casos, enquanto que, no coriocarcinoma, houve expressão intensa de IL-6 em 50% e de STAT-3 em 75% dos casos. Por outro lado, as reações para LIF tiveram expressão nula em todos os três grupos. Pelo WB houve expressão proteica de IL-6 apenas no material fresco de placenta normal e ausência de expressão na sua cultura primária e na linhagem BeWo; LIF não foi expresso em todos os grupos estudados. STAT-3 foi detectado no citoplasma em todos os grupos, entretanto, a expressão nuclear da STAT-3 fosforilada (pSTAT-3) não foi observada na IF e nem pelo WB. Na análise gênica pelo RTPCR houve forte expressão de IL-6 e STAT-3 no material fresco de placenta normal e expressão muito fraca na cultura primária de placenta normal e na linhagem BeWo; a expressão de LIF foi muito fraca em todos os grupos. Apenas a linhagem BeWo demonstrou forte expressão gênica da telomerase, contrastando com a completa falta de expressão no material fresco de placenta normal e em sua cultura primária. Conclusão: A intensa expressão IHQ de IL-6 e STAT-3 no coriocarcinoma indica a atuação de ambas na carcinogênese. A expressão proteica de IL-6 no material fresco de placenta normal e sua ausência no material de cultura primária e na linhagem BeWo pode ser ocasionado pelo contato célula-a-célula nas culturas aderentes, inibindo o crescimento celular e, consequentemente, as vias de sinalização. A falta de expressão da pSTAT-3 tanto na IF como por WB demonstra que a via JAK-STAT está sendo desativada. A ausência de expressão de LIF, em todos os métodos estudados, sugere que esta citocina poderia estar sendo inibida por meio de proteínas SOCS3 ou, atuando, de modo indireto, na proliferação celular do coriocarcinoma. O aumento da atividade da telomerase nas células BeWo reforça sua relação com o fenótipo maligno e a aponta como um bom marcador para progressão da doença / Abstract: The invasion of the endometrium by extravillous trophoblast is a fundamental process in the growth of the fetus and placenta. The process is controlled by factors related to the immune and hormonal activity that, when changed, may result in termination of pregnancy and development of so-called gestational trophoblastic diseases. In some cases, changes can result in malignancy, in which some molecules play a role in cell fusion process and inflammation, although studies in this area are inconclusive. Considering this information, the study had the aim of investigating the expression of cytokines LIF, IL-6, STAT- 3 and the function of telomerase to understand their participation in abortion, in normal at term placenta and choriocarcinoma. Methods: The expression of the molecules was assessed by immunohistochemical assay (IHC), immunofluorescence (IF), Western Blotting (WB) and Real-Time PCR (RT - PCR) using fixed material from biopsies of abortions, normal at term placentas and choriocarcinoma along with fresh tissue of normal at term placenta and their primary culture and BeWo cell line. Paraffin embedded material used in IHC and IF assays were obtained from the Department of Pathology files. Tests of WB and Real-Time PCR employed fresh material, obtained from cell cultures of normal at term placenta and the BeWo line. Results: IHC reactions to abortion biopsies showed moderate staining for IL-6 in 58.4% of cases and intense for STAT-3 in 33.3 % of cases. In biopsies of normal placenta, there was intense reaction for IL-6 in 50% of cases, intense for STAT-3 in 16.7%; choriocarcinoma showed intense staining for IL- 6 in 50% of cases and also for STAT-3 in 75% of cases. On the other hand, LIF expression was missing in all three groups. WB analyses showed IL-6 protein in fresh material from normal placentas, but no expression in placenta primary cultures and BeWo line. LIF was absent in all groups. Cytoplasmic STAT-3 was observed in all groups, while the nuclear expression of phosphorylated STAT-3 was absent. On gene analyses a strong expression of IL-6 and STAT- 3 was observed from fresh normal placenta, but very weak expression in primary cultures of normal placenta and BeWo cell line. LIF expression was very weak in all groups. In regard to the gene expression of telomerase, it was strong in the BeWo line which contrasted with its complete lack of expression in fresh normal placenta and its primary culture. Conclusion: The high expression of IL-6 and STAT-3 in biopsies of choriocarcinoma indicates the role of both in tumor progression. Regarding protein expression, the presence of IL-6 in the material from fresh normal placenta, and its absence in primary culture and BeWo line may be caused by the cell-to-cell contact cultures by inhibiting cell growth and thus signaling pathways. However, the lack of expression of phosphorylated STAT-3 whether through IF or WB shows that its JAK-STAT pathway is inhibited. Lack of expression of the LIF suggests that it might be involved indirectly in choriocarcinoma cell proliferation or be inhibited by SOCS3 protein. Moreover, the increased telomerase activity of BeWo cells enhances their relation to the malignant phenotype and indicates a good marker for disease progression / Doutorado / Ciencias Biomedicas / Doutora em Ciências Médicas

Dissertation (Ph.D.)--University of South Florida, 2009. / Includes vita. Includes bibliographical references. Also available online.

Dissertation (Ph.D.)--University of South Florida, 2009. / Title from PDF of title page. Document formatted into pages; contains 149 pages. Includes vita. Includes bibliographical references.