Introdução: O Câncer de próstata (CaP) é o mais comum do homem brasileiro. É importante a identificação de alterações moleculares que possam prever o seu desenvolvimento e potencial biológico. Polimorfismos de nucleotídeo único (SNP) são alterações da seqüência do DNA onde somente uma base é trocada com uma freqüência superior a 1% na população, que podem levar a modificações estruturais e funcionais na proteína, ou afetar a sua quantidade, e podem constituir marcadores de predisposição e prognóstico de neoplasias. Metaloproteinases (MMP) são proteínas da família de enzimas proteolíticas, que degradam a matriz extracelular, e SNP na sua estrutura têm sido associados ao comportamento de tumores. Objetivos: Avaliar a freqüencia de SNP nos genes das MMP1, 2, 7 e 9, em pacientes com CaP e grupo controle, relacionando com suscetibilidade para o desenvolvimento da doença e previsão de seu potencial biológico. Material e Métodos: A amostra é constituída por tecido não tumoral de 100 indivíduos com CaP, e 100 amostras controle representadas por soro de indivíduos saudáveis, sem câncer de próstata. O DNA foi obtido utilizando protocolos convencionais de extração. Para genotipagem foi utilizada técnica de identificação de base única com uso de sondas marcadas com fluoróforos (Taqman®) pela técnica de reação em cadeia da polimerase em tempo real. As freqüências alélicas foram calculadas e a comparação entre os grupos foi feita utilizando-se o teste de qui-quadrado com valor de significância de 0,05. Resultados: Nos genes das MMP1 a freqüência do genótipo homozigoto polimórfico esteve mais presente no grupo controle que no CaP (p>0,001). No gene da MMP9 o alelo polimórfico esteve mais presente em pacientes com CaP (p>0,001), e em tumores com escore de Gleason6 (p=0,003). No gene da MMP2 de acordo com estadiamento patológico o alelo polimórfico foi mais freqüente em tumores pT3 (p=0,026) e Gleason maior ou igual a 7(p=0,042). Conclusão: Nossos resultados sugerem que o polimorfismo no gene da MMP1 está associado a um caráter de proteção aos indivíduos quanto ao desenvolvimento do CaP. O polimorfismo no gene da MMP9 está associado a um aumento no risco de desenvolvimento desta neoplasia, e quando analisamos as associações com os fatores prognósticos encontramos uma correlação com tumores de melhor prognóstico. Por outro lado o polimorfismo do gene da MMP2 se associa a tumores não órgãoconfinados / Introduction: Prostate cancer (PCa) is the most frequent tumor in males in Brazil. Research has been directed for the identification of molecular markers that can predict the PCa predisposition and prognosis. Single nucleotide polymorphisms (SNPs) are genome variations, present in a frequency of 1% or more. The matrix metalloproteinases (MMPs) are a family of enzymes responsible for the degradation of extracellular matrix. SNPs have been demonstrated in the promoter region of these genes and have been associated with development and progression of some cancers. Objective: To investigate the correlation between polymorphisms of MMP1, 2, 7, 9 with susceptibility and classical prognostic parameters in PCa. Patients and methods: The sample is constituted by normal tissue of 100 patients with PCa, and 100 healthy men as controls (serum). DNA genomic was extracted from paraffin blocks and serum using conventional protocols. The DNA sequence containing the polymorphic sites was amplified by Real-Time polymerase chain reaction, using fluorescent probes (Taqman®). The allelic frequency was calculated and the comparison between the groups was made using the qui-square test with value of significance of 0.05. Results: The polymorphic homozygote genotype of the MMP1 was more frequent in the control group than in the PCa (p<0.001). The polymorphic allele of MMP9 was more frequent in the PCa group (p<0.001), and in tumors Gleason6 (p=0,003). The polymorphic allele of MMP2 was more frequent in tumors of higher stage (pT3) (p=0.026) and higher Gleason Score (7) (p=0.042). Conclusion: We have shown that MMP1 polymorphism is more frequent in the control group, than in patients with PCa, it may be associated to protection for the development of PCa. The MMP9 polymorphism was related to higher risk for development of this neoplasia, but associated with lower Gleason score. MMP2 polymorphism was associated with non organconfined disease.

Methotrexate (MTX) is an antimetabolite anticancer agent that is used in treatment of multiple cancers, such as acute lymphoblastic leukaemia and osteosarcoma. A lack of selective tumour toxicity is one of the major problems associated with MTX chemotherapy, especially when given at high doses, as in high dose MTX (HDMTX) therapy. MTX causes various toxicity problems including life-threatening nephrotoxicity, haematological toxicity and neurotoxicity. Overcoming this toxicity is of great importance and has been attempted in various ways, not least via the design of prodrugs. The concept of tumour protease, and specifically matrix metalloproteinase (MMP), activated prodrugs was the focus of the work described in this thesis. This concept relies upon attachment of an MMP-sensitive peptide sequence to a specific site in a drug structure, so as to inactive it. The activity of the parent drug is restored once it is activated by the MMPs in the tumour microenvironment. In this work, different MMP-sensitive peptide sequences linked to MTX were synthesised, resulting in 63 MTX prodrugs. The MMP-mediated activation of these conjugates in tumour tissues (specifically HT1080 homogenates) ex vivo was assessed and the results were compared to the activation of these conjugates in various normal tissues specifically liver, kidney and lung. Specific criteria were established for the selection of promising conjugates for more detailed study. From 7 promising compounds, compound 75 was identified as the lead prodrug, demonstrating selective MMP activation, as indicated by inhibition of its activation by broad spectrum MMP inhibitor ilomastat. The pharmacokinetics of compound 75 was studied in tumour (HT1080) xenograft-bearing mice and the results were compared to those obtained from administration of equimolar doses of conventional MTX. Compound 75 led to enhanced tumour concentrations of MTX, with reduced exposure to normal tissues in vivo compared to conventional MTX therapy. Furthermore, the efficacy of equimolar doses of compound 75 and directly dosed MTX in reduction of HT1080 volume were compared. Superior antitumour activity was observed with compound 75 compared to MTX treatment. Compound 75 is the first example of an MMP-activated prodrug to be reported with enhanced therapeutic index, as evidenced by a full in vivo pharmacokinetic analysis and normal tissue metabolism data. The data presented in thesis support the concept of MMP-activated prodrug development, and form a strong foundation upon which to develop a clinicallyuseful MTX prodrug, with the potential to enhance efficacy and reduce toxicity to the patient.

Improvements in medical care over recent decades have increased the number of premature and low birth weight infants that survive hypoxic-ischemic (H-I) insults. Because there is a rising incidence in diseases associated with these events, it is critical to develop effective therapies to treat the various resulting neuropathies. Extracellular matrix constitutes the majority of brain parenchyma. Lecticans and matrix-degrading proteases including ADAMTSs (a disintegrin and metalloproteinase with thrombospondin repeats) and matrix metalloproteinases (MMPs) exert effects on cell viability and may be associated with either protective or destructive processes after H-I. Both ADAMTSs (Cross et al. 2006; Tian et al. 2007) and MMPs (del Zoppo et al. 2007; Gu et al. 2005; Rosenberg et al. 2001) have been associated with pathological states in brain, yet the relative contributions of lecticans, ADAMTSs and MMPs to inflammation and cell death remain unknown. In the present study, the first series of experiments were conducted to characterize cellular damage and neuroinflammation in the postnatal day 7 rat after exposure to H-I, and to determine if cell death and inflammation were associated with alterations in lectican expression. Data showed that reduced brevican expression occurred 4 days after H-I in lesioned hippocampus. Additionally, reduced versican expression in white matter was concomitant with pre-OL cell death at this endpoint. In contrast, both lecticans were elevated at later endpoints (14, 21 days) that were associated with increased neuroinflammation and cavitary infarction. These data suggest that lectican loss is associated with cell death at the early endpoint, whereas increased lectican deposition over time likely leads to glial scar formation and a reduced capacity for neuroplasticity. Two subsequent series of experiments were conducted to determine the relative contributions of matrix-degrading proteases to injury, and whether proteolytic activity was associated with neuroinflammatory events. The first objective was to determine whether treatment with AG3340, a selective inhibitor of gelatin-degrading MMPs, or the anti-inflammatory compound minocycline, could provide neuroprotection when administered at a delayed time point after insult, and to compare the efficacy of AG3340 with that of the well-known anti-inflammatory compound minocycline. Data showed that both compounds effectively dampened the recruitment of microglia/macrophages to the lesion site when administered 24 hrs after H-I. These effects were associated with reduced neurodegeneration, indicating that these compounds neuroprotect at a clinically relevant time point. The final series of experiments tested whether these compounds could neuroprotect in an ex vivo model of oxygen glucose deprivation (OGD) that lacks peripheral immune cell involvement, thus providing insight into the relative contributions of resident microglia and gelatinase activity to the inflammatory sequelae. Results showed that both compounds blocked the OGD-induced increase in gelatinase activity and were neuroprotective in the absence of peripheral immune cells. Taken together, these data indicate that resident microglia contribute to H-I injury through gelatinase activation. Thus, the present study demonstrates that gelatin-degrading MMPs are important targets to consider when developing therapies to combat neonatal H-I injury.

Angiotensin II signals cardiovascular disease through metalloproteinases including MMP-2, MMP-7 and ADAM-17/TACE. We hypothesized that these metalloproteinases regulate each other at the transcriptional level. Further, MMP-2, being a major gelatinase in cardiac and vascular tissue, could mediate angiotensin II-induced cardiovascular disease. We studied the development of hypertension (by tail cuff plethysmography), cardiac hypertrophy (by M-mode echocardiography and qRT-PCR analysis of hypertrophy marker genes) and fibrosis (by collagen staining and qRT-PCR analysis of fibrosis marker genes) in mice receiving angiotensin II. Angiotensin II induced hypertension, cardiac hypertrophy and fibrosis which correlated with an upregulation of MMP-2. Downregulation of MMP-2 by pharmacological inhibition and RNA interference attenuated hypertension but not cardiac hypertrophy or fibrosis. Downregulation of MMP-7 or ADAM-17/TACE by RNA interference attenuated angiotensin II-induced MMP-2 upregulation as well as hypertension, cardiac hypertrophy and fibrosis. We conclude that MMP-2 selectively mediates angiotensin II-induced hypertension under the transcriptional control of MMP-7 and ADAM-17/TACE.

Development of the secondary palate (SP) is a complex event despite the small area it encompasses. Problems with SP development can lead to a cleft palate, which is one of the most common birth disorders. The matrix metalloproteinases (MMPs) are required for proper SP development, but a functional role for any one of them remains unknown. MMP-25 is a candidate MMP to have a functional role in SP formation as genetic scans of the DNA of human cleft palate patients indicate a common mutation at a region upstream of the Mmp-25 gene. The purpose of this thesis is to investigate gene expression of Mmp-25 in the developing mouse SP, whether it has a functional role in mouse SP development and begin to identify factors potentially upstream of Mmp-25 expression.<p> Mmp-25 mRNA and protein is found at all SP developmental stages in mice with highest expression at embryonic day (E) 13.5 when analyzed by quantitative real-time PCR and western blotting. Immunohistochemistry localizes MMP-25 protein primarily to the plasma membranes of palate shelf epithelial cells with secondary expression in apical mesenchymal cells. Mmp-25 knockdown with siRNA in palatal cultures resulted in a significant decrease in palate shelf fusion and persistence of the medial edge epithelium in vitro. Mmp-25 mRNA and protein levels are significantly decreased in vitro when cultured palate shelves are incubated in growth medium with 5 ìg/ml of a TGFâ3-neutralizing antibody. Mmp-25 gene expression is highest at E12.5 and E13.5, which corresponds to increasing palate shelf growth downward alongside the tongue. Immunohistochemistry localized MMP-25 protein expression predominantly in the epithelium of the palate shelves, but also in areas of the mesenchyme that were immediately adjacent to the epithelium and apical in location. Knockdown of Mmp-25 expression resulted in palate shelf fusion being impaired and significant medial edge epithelium remaining in contacted areas. Bioneutralization of TGFâ3 resulted in a significant decrease in Mmp-25 gene expression. These data suggest a functional role for MMP-25 in mouse SP development by removing extra-cellular matrix barriers to increased palate shelf growth and place its expression downstream of TGF-â3 signaling. This is the first research to present a role for a single MMP in mouse SP development.

Development of the secondary palate (SP) is a complex event despite the small area it encompasses. Problems with SP development can lead to a cleft palate, which is one of the most common birth disorders. The matrix metalloproteinases (MMPs) are required for proper SP development, but a functional role for any one of them remains unknown. MMP-25 is a candidate MMP to have a functional role in SP formation as genetic scans of the DNA of human cleft palate patients indicate a common mutation at a region upstream of the Mmp-25 gene. The purpose of this thesis is to investigate gene expression of Mmp-25 in the developing mouse SP, whether it has a functional role in mouse SP development and begin to identify factors potentially upstream of Mmp-25 expression.<p> Mmp-25 mRNA and protein is found at all SP developmental stages in mice with highest expression at embryonic day (E) 13.5 when analyzed by quantitative real-time PCR and western blotting. Immunohistochemistry localizes MMP-25 protein primarily to the plasma membranes of palate shelf epithelial cells with secondary expression in apical mesenchymal cells. Mmp-25 knockdown with siRNA in palatal cultures resulted in a significant decrease in palate shelf fusion and persistence of the medial edge epithelium in vitro. Mmp-25 mRNA and protein levels are significantly decreased in vitro when cultured palate shelves are incubated in growth medium with 5 ìg/ml of a TGFâ3-neutralizing antibody. Mmp-25 gene expression is highest at E12.5 and E13.5, which corresponds to increasing palate shelf growth downward alongside the tongue. Immunohistochemistry localized MMP-25 protein expression predominantly in the epithelium of the palate shelves, but also in areas of the mesenchyme that were immediately adjacent to the epithelium and apical in location. Knockdown of Mmp-25 expression resulted in palate shelf fusion being impaired and significant medial edge epithelium remaining in contacted areas. Bioneutralization of TGFâ3 resulted in a significant decrease in Mmp-25 gene expression. These data suggest a functional role for MMP-25 in mouse SP development by removing extra-cellular matrix barriers to increased palate shelf growth and place its expression downstream of TGF-â3 signaling. This is the first research to present a role for a single MMP in mouse SP development.

Three protease inhibitor-like proteins (PILPs) identified from Bungarus multicinctus genome are structurally homologous with Kunitz-type proteinase inhibitor. The goal of the present study is to explore whether PILPs exhibit an inhibitory action on matrix metalloproteinase 2 (MMP-2) activity. Unlike PILP-1 and PILP-2, PILP-3 was found to inhibit MMP-2 activity as evidenced by specific substrate assay. Moreover, in vitro migration and invasion assays, and wound-healing assay showed that PILP-3 suppressed the migration and invasion of human neuroblastoma SK-N-SH cells. Pull-down assay and dot blotting-binding assay proved an interaction between PILP-3 and MMP-2. Nevertheless, PILP-3 did not affect either expression or secretion of MMP-2 in SK-N-SH cells. In terms of highly structural similarity between PILP-2 and PILP-3, two chimeric mutants in which amino acids at N-terminus and C-terminus of PILP-3 were substituted by those of PILP-2 were prepared. In contrast to N-terminus chimera, C-terminus mutant of PILP-3 was unable to inhibit MMP-2 activity and showed a reduction in binding with MMP-2. Taken together, our data suggest that PILP-3 may be a useful template for rational designing pharmaceutical agent in inhibiting MMP-2 activity.

Thesis (Ph. D.)--Auburn University, 2008. / Abstract. Vita. Includes bibliographical references (p. 62-77).

Reproduction de : Thèse doctorat : Pharmacie.Biochimie-Biologie moléculaire : Reims : 2007. / Titre provenant de l'écran-titre. Bibliogr. p.186-218.

Reproduction de : Thèse doctorat : Médecine. Biochimie et biologie moléculaire : Reims : 2007. / Titre provenant de l'écran-titre. Bibliogr.