A funcionalização de superfície nas nanocápsulas contendo doxorrubicina com o peptídeo RGD é uma estratégia promissora devido a ligação preferencial na integrina αvβ3 expressa em células tumorais. Este estudo objetivou o desenvolvimento, caracterização e estudos biológicos de nanocápsulas multiparedes com doxorrubicina e funcionalizadas com RGD. Para isso, na primeira etapa do trabalho foi realizada a síntese do peptídeo RGD. Os produtos obtidos foram caracterizados por análises de infravermelho e RMN de 1H. Na segunda etapa foram desenvolvidas formulações de nanocápsulas com doxorrubicina ou cloridrato de doxorrubicina, e, nanocápsulas multiparedes revestidas com quitosana, íons zinco, RGD ou fenilalanina. Essas suspensões foram caracterizadas através da determinação do pH, diâmetro de partícula por diferentes técnicas, potencial zeta, eficiência de encapsulação e eficiência de associação do RGD na superfície da nanopartícula. Na terceira etapa, foram realizados ensaios de viabilidade celular por MTT após 24 e 72h com as formulações desenvolvidas em células de câncer de mama (MCF7) e glioblastoma humano (U87MG). As formulações apresentaram diferentes valores de citotoxicidade e, utilizando o Gráfico de Pareto foi possível determinar os fatores que exercem maior influencia. Em células MCF7 foi a concentração de fármaco e tempo de tratamento e, nas células U87MG além desses fatores, a funcionalização mostrou-se determinante. Além disso, foi avaliada a captação das nanocápsulas funcionalizadas com RGD e fenilalanina após 24h nas células tumorais e células de queratinócitos humanos (HaCat), com diferentes níveis de expressão da integrina αvβ3. O estudo mostrou menores valores de captação nas células HaCat (sem expressão de integrina αvβ3) para as duas formulações testadas. Finalmente as nanocápsulas funcionalizadas com RGD apresentaram maior captação em células U87MG com maior expressão da integrina αvβ3. / The surface functionalization in nanocapsules containing doxorubicin with RGD peptide is a promising strategy due to preferential binding in the αvβ3 integrin expressed on tumor cells. This study aimed the development, characterization, and biological studies of multiwall nanocapsules containing doxorubicin and functionalized with RGD. For this reason, in the first stage of this study the synthesis of RGD peptide was performed and the products characterized by infrared analysis and 1H NMR. Besides, nanocapsules formulations were developed containing doxorubicin or doxorubicin hydrochloride, and multiwall nanocapsules coated with chitosan, zinc ions, RGD or phenylalanine. These suspensions were characterized by pH determination, particle diameter by different techniques, zeta potential, encapsulation efficiency, and association efficiency of RGD on the surface of the nanoparticle. Additionally, it was performed cell viability assays by MTT after 24 and 72 hours with formulations developed in breast cancer (MCF7) and human glioblastoma cells (U87MG). Formulations showed different cytotoxicity values. The Pareto chart was possible to determine factors that have more influence. In MCF7 cells was drug concentration and treatment time, and U87MG cells, besides these factors, the functionalization was decisive. Furthermore, it was performed the cellular uptake of nanocapsules functionalized with RGD or phenylalanine after 24 hours in tumor cells and human keratinocyte cells (HaCaT), with different levels of expression αvβ3 integrin. The study showed less uptake in HaCaT cells (without expression αvβ3 integrin) for the two formulations applied, and the nanocapsules functionalized with RGD showed more uptake in U87MG cells, with higher expression of integrin αvβ3.

The basement membrane protein laminin-332 (laminin-5) mediates both stable cell adhesion and rapid cell migration, and thus has the potential to either restrain or promote tumor cell metastasis. The major cellular receptors for laminin-332 are integrin α3β1, which mediates rapid tumor cell migration, and integrin α6β4, which often mediates stable cell attachment. Tetraspanin protein CD151 interacts directly with both α3β1 and α6β4 integrins and with other tetraspanins, thereby promoting α3β1 and α6β4 association with tetraspanin-enriched microdomains on the cell surface. To explore the possibility of selectively modulating tumor cell responses to laminin-332, we re-expressed a series of CD151 mutants in epidermoid carcinoma cells with near total, RNAi- mediated silencing of endogenous CD151. CD151's interactions with its integrin partners or its interactions with other tetraspanins were selectively disrupted by specific mutations in the CD151 large extracellular loop (EC2 domain) or in intracellular CD151 palmitoylation sites, respectively. CD151- integrin association and CD151-tetraspanin association were both important for α3β1 integrin- dependent initial adhesion and rapid migration on laminin-332. Remarkably, however, only CD151-integrin association was required for stable, α6β4 integrin-dependent cell attachment on laminin-332. In gap-filling assays, where CD151-silenced cells moved more rapidly than WT cells, again, only CD151-integrin association was required to restrict movement into the gap, suggesting that both α3β1 and α6β4 integrin must be able to associate with CD151 in order restrict group motility. In addition, we found that a QRD amino acid motif in the CD151 EC2 domain that had been thought to be crucial for CD151-integrin interaction is not essential for CD151-integrin association or for CD151's ability to promote several different integrin functions. These new data suggest potential strategies for selectively modulating migratory cell responses to laminin-332, while leaving stable cell attachment on laminin-332 intact.

Integrins are transmembrane heterodimer protein receptors that mediate adherence to both the intracellular cytoskeleton and extracellular matrix. They play a major role in cellular adhesion and the breadth of their importance in biology is only recently being understood. The ability to detect concentrations of integrins on the cell surface, spatially resolve them, and study the dynamics of their behavior would be a significant advance in this field. Ultimately, the ability to detect dynamic changes of integrins on the surface of a cell maybe possible by developing a combined device such as an atomic force microscope (AFM) and surface enhanced Raman spectroscopy (SERS) system. However, the focus of this research is to first determine if integrins can be detected using SERS. Surface enhanced Raman spectroscopy (SERS) is technique used to detect the presence of analytes at the nanomolar level or below, through detection of inelastically scattered light. This thesis discusses the detection of integrins employing SERS as the detection modality. Integrins have been detected, in solution, using two silver colloids as the enhancing surface. Two silver colloid preparation methods are compared by ease of formulation and degree of enhancement in this thesis. Citrate and hydroxylamine hydrochloride (HA-HCl) reduced silver colloids were prepared through wet chemistry,compared using UV-Vis light spectroscopy, and tested for surface enhancement using adenine (a strong SERS active molecule), and two different integrins, (alpha)V(beta)3 and (alpha)5(beta)1. Results indicated that both colloids demonstrate SERS activity for varying concentrations of adenine as compared to standard non-enhanced Raman, however, only the citrate reduced colloid showed significant enhancement effect for the integrins.

In the Xenopus embryo, cell rearrangements during early development require the dynamic modulation of adhesion. Cells primarily use the integrin family of transmembrane receptors for attachment to and interpretation of the extracellular environment. While acting as adhesion receptors, integrins also have bidirectional signalling properties essential for driving cellular movements. The regulation of integrin activity is thought to stem from cytoplasmic assemblies of constitutively expressed molecules. PINCH (Particularly Interesting New cysteine-histidine rich protein), an adapter protein, is part of an IPP complex that has emerged as a key signalling scaffold indispensable for integrin function in vitro. As such, I tested the hypothesis that PINCH regulates integrin function in the Xenopus embryo. Xenopus PINCH was successfully cloned using RT-PCR. The predicted amino acid sequence of PINCH shares a 98% similarity with mammalian orthologs, and comprises of five highly conserved LIM domains. PINCH mRNA and protein are ubiquitously expressed throughout embryogenesis. In situ hybridization indicates that PINCH mRNA is expressed in the blastocoel roof and the pre-involution mesoderm. The localization and temporal expression of PINCH suggests a role in mediating cell adhesive events during gastrulation. A functional approach was used to examine the role of PINCH during gastrulation. I used site-directed mutagenesis to generate non-functional LIM1 (LIM1mut) and LIM4 (LIM4mut) domains that have been proposed to bind ILK and Grb4 respectively. Over-expression of PINCH leads to a delay in blastopore closures, while the expression of both LIM1mut and LIM4mut relieve this inhibition at lower concentrations. Further analysis indicates that PINCH, LIM1mut, and LIM4mut inhibit FN matrix assembly independent of integrin adhesion. Contradictory to in vitro studies, co-immunoprecipitation analysis indicates that endogenous PINCH does not bind ILK, confirming an integrin-independent role during gastrulation. Furthermore, in the embryo PINCH is found at cell boundaries but does not appear to directly modulate cadherin adhesion. As such this thesis provides evidence that PINCH regulates cell intercalation movements independent of integrin and cadherin receptors and raises the possibility that the LIM4 domain is involved in PINCH regulation of cell adhesion during early development.

Integrins are transmembrane heterodimer protein receptors that mediate adherence to both the intracellular cytoskeleton and extracellular matrix. They play a major role in cellular adhesion and the breadth of their importance in biology is only recently being understood. The ability to detect concentrations of integrins on the cell surface, spatially resolve them, and study the dynamics of their behavior would be a significant advance in this field. Ultimately, the ability to detect dynamic changes of integrins on the surface of a cell maybe possible by developing a combined device such as an atomic force microscope (AFM) and surface enhanced Raman spectroscopy (SERS) system. However, the focus of this research is to first determine if integrins can be detected using SERS. Surface enhanced Raman spectroscopy (SERS) is technique used to detect the presence of analytes at the nanomolar level or below, through detection of inelastically scattered light. This thesis discusses the detection of integrins employing SERS as the detection modality. Integrins have been detected, in solution, using two silver colloids as the enhancing surface. Two silver colloid preparation methods are compared by ease of formulation and degree of enhancement in this thesis. Citrate and hydroxylamine hydrochloride (HA-HCl) reduced silver colloids were prepared through wet chemistry,compared using UV-Vis light spectroscopy, and tested for surface enhancement using adenine (a strong SERS active molecule), and two different integrins, (alpha)V(beta)3 and (alpha)5(beta)1. Results indicated that both colloids demonstrate SERS activity for varying concentrations of adenine as compared to standard non-enhanced Raman, however, only the citrate reduced colloid showed significant enhancement effect for the integrins.

The objective of the presented research is to examine the relationship between the cellular microenvironment and biochemical response of metastatic cells. Clinically recognized as a trait of cancer progression, the cellular microenvironment can have variable and distinct mechanical properties that are processed via cellular mechanosensing, resulting in a cellular biochemical response. A range of studies investigating the interactions between the cellular micromechanical environment and the cell's molecular response during disease progression have been made, yet remain absent of quantitative characterization of many of these coordinated responses. The fundamental inquiry that drives the following research attempts to elucidate how a cell perceives the physical microenvironment and converts that signal to a biochemical response. With the goal of providing insight to such responses, the presented research seeks to elucidate the following questions: (1) What are the integrated effects of ECM stiffness, ECM architecture, and breast cancer cell metastatic potential on cell migration? (2) How does endogenous tissue transglutaminase (tTG) cross-linking of the ECM scaffold effect ECM mechanical properties? (3) How does the architecture and stiffness of the extracellular matrix (ECM) effect the systems-level cellular migration and signaling response? (4) What are the integrated effects of ECM architecture and the targeted knockdown of integrin [beta]1 and MT1-MMP on cellular metastatic potential? The presented research utilizes an interdisciplinary approach, integrating experimental mechanics, biochemical analysis, cellular biology techniques, covalent chemistry, and various microscopy techniques, to investigate these events. In short, cancerous cells are cultured atop or within synthetic collagen type I ECMs of varying mechanical stiffness and structure. These cells are subsequently analyzed by molecular analysis and immunoassays, including quantitative PCR, Western blotting, and gelatin zymography, to acquire measures of the cellular response to perturbations of micromechanical environment. Time-lapse microscopy experiments and subsequent image analyses enable observations of cellular migratory potential through synthetic ECMs. Results indicate that cooperative synergy between ECM properties, cell-matrix adhesion, and pericellular proteolysis drive cell migratory potential of highly invasive tumorigenic cell populations. Collectively, these findings contribute to the cancer biology and mechanobiology fields by systematically extending current insights of matrix mechanics, cellular signaling, and cellular migratory potential in cancer. / text

Cancer metastasis is a multi-stage process initiated by the cancer cell acquiring the ability to migrate. The protein profile of such a cell undergoes dramatic changes including changes in integrin expression. Integrins play a major role in cell adhesion, motility, differentiation, blood clotting, tissue organization and cell growth as well as cancer cell migration, invasion and metastasis. Integrin a6, which can pair with integrin b4 or b1 is a laminin receptor and is detected in epithelial cells. Earlier studies have reported uPA mediated integrin a6 cleavage in prostate cancer resulting in loss of the ligand binding domain. Site-directed mutagenesis studies have identified the cleavage site to be at R594R595 located in the "stalk" region of the integrin a6. Prostate cancer cells PC3N-a6-RR cells, bearing a R594R595 to A594A595 mutation, engineered to express the uncleavable form of integrin a6 were found to migrate 6.4 folds lesser on Laminin-1 as compared to the PC3N-a6-WT cells which expressed the wild-type integrin a6. This result suggests that integrin a6 cleavage enhances migration. Prostate cancer is known to metastasize to the bone. Injection of the PC3N-a6-WT cells in mouse femurs resulted in increased bone destruction and pain behavior when compared to the femurs injected with PC3N-a6-RR cells indicating that the integrin a6 cleavage could affect and modify the bone microenvironment. An observation that complete conversion of integrin a6 to a6p was not observed in cell lines even in presence of excess uPA suggested a regulatory mechanism. Integrins are known to associate with many proteins including tetraspanins, which are transmembrane proteins, that function as protein adapters. Integrin a6 was found to be refractory to uPA mediated cleavage when complexed with tetraspanin CD151. The amount of integrin a6 available for cleavage increased when CD151 levels were decreased by CD151 siRNA treatment. These results suggest that the integrin a6 available and unavailable for cleavage can be modulated by interaction with CD151 and hence affect the migratory potential of the cell. Collectively these data suggest that integrin a6 cleavage can enhance cell migration, initiate signals to modify the tumor microenvironment and can be regulated by interaction with tetraspanin CD151.

Expression of A6 integrin, a laminin receptor, on tumor cell surfaces is associated with reduced patient survival and increased metastasis in a variety of tumors. In prostate cancer, tumor extra capsular escape occurs in part via laminin coated nerves and vascular dissemination, resulting in clinically significant bone metastases. Our group previously identified a novel form of A6 integrin, called A6p, generated by urokinase (uPA) dependent cleavage of the laminin binding domain from the tumor cell surface. Although functional consequences of cleavage have been characterized, little is known about how this process is regulated.Regulation of uPA mediated cleavage was identified by a laterally interacting protein expressed on the cellular surface. A direct interaction between the urokinase receptor (uPAR) and A6 integrin was characterized. This direct interaction was responsible for the extracellular cleavage of A6. Transient knockout of A3 integrin, a known interacting partner of uPAR, increased uPAR association with A6 integrin and enhanced production of A6p. Analysis of tissue obtained from human prostate tumors confirmed uPAR and A6 integrin expression in invasive disease. Taken together the results demonstrate a novel and dynamic role for uPAR regulation of integrin dependent adhesion through lateral interaction.Using the known conformation sensitivity of integrin function to I determined if engagement of the extracellular domain by antibodies inhibited integrin cleavage and the extravasation step of metastasis. Both endogenous and inducible levels of A6p were inhibited by engaging the extracellular domain of A6 with monoclonal antibody J8H. J8H inhibited tumor cell invasion through Matrigel. A SCID mouse model of extravasation and bone metastasis produced detectable, progressive osteolytic lesions within three weeks of intracardiac injections. Injection of tumor cells, pre-treated with J8H, delayed the appearance of metastases. Validation of the A6 cleavage effect on extravasation was confirmed through a genetic approach using tumor cells transfected with uncleavable A6 integrin. Uncleavable A6 integrin significantly delayed the onset and progression of osseous metastases out to 6 weeks post injection. The results suggest that A6 integrin cleavage permits extravasation of human prostate cancer cells from circulation to bone and can be manipulated to prevent metastasis.

Human tumor cell progression and metastasis is partially dependent on the ability of tumor cells to adhere to the proteins of the extracellular matrix and migrate to distant locations. Using a combinatorial screening approach, six novel D-amino acid containing peptides were identified and analyzed for their ability to adhere to human prostate tumor cells, support tumor cell adhesion and inhibit tumor cell adhesion to ECM proteins. Two peptides, RZ-3 (kmviywkag) and HYD1 (kikmviswkg) bound to tumor cell surfaces. A scrambled peptide derivative of HYD1, HYDS (wiksmkivkg) is not active. As immobilized ligands, RZ-3 and HYD1 can support prostate tumor cell adhesion. Prostate tumor cell adhesion to immobilized RZ-3 and HYD1 is integrin dependent. Soluble RZ-3 and HYD1 inhibits tumor cell adhesion to extracellular matrix proteins in a concentration dependent manner. These results indicate that RZ-3 and HYD1 are biologically active D-amino acid containing peptides that can support tumor cell adhesion and can inhibit tumor cell adhesion to immobilized extracellular matrix proteins.Cell migration is dependent on adhesive interactions with the extracelluar matrix. These interactions induce signaling and cytoskeletal responses necessary for migration. HYD1 completely blocks random haptotactic migration and inhibits invasion of prostate carcinoma cells on laminin-5. This effect is adhesion independent and reversible. The inhibition of migration by HYD1 involves a dramatic remodeling of the actin cytoskeleton resulting in increased stress fiber formation and actin colocalization with cortactin at the cell membrane. HYD1 interacts with a6 and a3 integrin subunits and elevates laminin-5 dependent intracellular signals including focal adhesion kinase, mitogen activated protein kinase kinase, and extracellular signal-regulated kinase. The scrambled derivative of HYD1, HYDS, does not interact with the a6 or a3 integrin subunits and is not biologically active. The minimal element for bioactivity of HYD1 was determined using alanine-substituted analogs of HYD1 and N- and C-terminal deletion mutants of HYD1. The minimal element necessary to block cell migration on laminin-5 and activate cell signaling through ERK is xikmviswxx. Taken together, these results indicate that HYD1 is a biologically active integrin-targeting peptide that reversibly inhibits tumor cell migration on laminin-5 and uncouples phosphotyrosine signaling from cytoskeletal dependent migration.

Numerous studies have identified that elevated plasma concentrations of lipoprotein(a) [Lp(a)] are an emerging risk factor for a variety of atherothrombotic disorders. Apolipoprotein(a) [apo(a)], the unique glycoprotein component of Lp(a), consists of tandem repeats of a plasminogen kringle (K) IV-like domain, followed by sequences homologous to the plasminogen KV and protease domains. Apo(a)/Lp(a) has been consistently shown to regulate endothelial function and inhibit plasminogen activation. In the present study, we have demonstrated that apo(a), signaling via integrin alphaVbeta3, is the functional unit in Lp(a) to stimulate in vitro endothelial cell (EC) proliferation and migration, and activate focal adhesion kinase (FAK) and mitogen-activated protein kinases (MAPK) in cultured ECs. Both apo(a) and Lp(a) have also been shown to reduce the levels of active and total transforming growth factor (TGF)-beta in cultured EC medium in an integrin alphaVbeta3–dependent manner. Despite the stimulatory effects of apo(a) on EC proliferation and migration, we have further confirmed an inhibitory effect of apo(a) on EC in vitro angiogenesis using a fibrin gel tube formation assay. We have provided evidence proving apo(a) inhibits angiogenesis through inhibition of plasminogen activation, and this inhibitory effect is dependent on the presence of apo(a) KV domain. Lastly, apo(a) is shown to reduce the protein levels of annexin A2 and S100A10 in ECs, which implies another potential mechanism by which apo(a)/Lp(a) could impair plasminogen activation on cell surface. In summary, we have discovered the first complete outside-in signaling pathway elicited by apo(a)/Lp(a) in ECs and have built up a connection between the ability of apo(a) to inhibit plasminogen activation and its inhibition of angiogenesis. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2009-09-25 18:29:47.106