RESUMO
We provide proof-of-concept evidence for a new class of therapeutics that target growth factor:extracellular matrix (GF:ECM) interactions for the management of breast cancer. Insulin-like growth factor-I (IGF-I) forms multiprotein complexes with IGF-binding proteins (IGFBP) and the ECM protein vitronectin (VN), and stimulates the survival, migration and invasion of breast cancer cells. For the first time we provide physical evidence for IGFBP-3:VN interactions in breast cancer patient tissues; these interactions were predominantly localized to tumor cell clusters and in stroma surrounding tumor cells. We show that disruption of IGF-I:IGFBP:VN complexes with L(27)-IGF-II inhibits IGF-I:IGFBP:VN-stimulated breast cancer cell migration and proliferation in two- and three-dimensional assay systems. Peptide arrays screened to identify regions critical for the IGFBP-3/-5:VN and IGF-II:VN interactions demonstrated IGFBP-3/-5 and IGF-II binds VN through the hemopexin-2 domain, and VN binds IGFBP-3 at residues not involved in the binding of IGF-I to IGFBP-3. IGFBP-interacting VN peptides identified from these peptide arrays disrupted the IGF-I:IGFBP:VN complex, impeded the growth of primary tumor-like spheroids and, more importantly, inhibited the invasion of metastatic breast cancer cells in 3D assay systems. These studies provide first-in-field evidence for the utility of small peptides in antagonizing GF:ECM-mediated biologic functions and present data demonstrating the potential of these peptide antagonists as novel therapeutics. Mol Cancer Ther; 15(7); 1602-13. ©2016 AACR.
Assuntos
Neoplasias da Mama/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Somatomedinas/metabolismo , Vitronectina/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Imuno-Histoquímica , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/química , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fator de Crescimento Insulin-Like I/química , Ligantes , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Somatomedinas/química , Vitronectina/químicaRESUMO
The majority of the population experience successful wound-healing outcomes; however, 1-3% of those aged over 65 years experience delayed wound healing and wound perpetuation. These hard-to-heal wounds contain degraded and dysfunctional extracellular matrix (ECM); yet, the integrity of this structure is critical in the processes of normal wound healing. Here, we evaluated a novel synthetic matrix protein for its ability to act as an acellular scaffold that could replace dysfunctional ECM. In this regard, the synthetic protein was subjected to adsorption and diffusion assays using collagen and human dermal tissues; evaluated for its ability to influence keratinocyte and fibroblast attachment, migration and proliferation and assessed for its ability to influence in vivo wound healing in a porcine model. Critically, these experiments demonstrate that the matrix protein adsorbed to collagen and human dermal tissue but did not diffuse through human dermal tissue within a 24-hour observation period, and facilitated cell attachment, migration and proliferation. In a porcine wound-healing model, significantly smaller wound areas were observed in the test group compared with the control group following the third treatment. These data provide evidence that the synthetic matrix protein has the ability to function as an acellular scaffold for wound-healing purposes.
Assuntos
Alicerces Teciduais , Vitronectina/uso terapêutico , Ferimentos Penetrantes/terapia , Animais , Técnicas de Cultura de Células , Derme/metabolismo , Modelos Animais de Doenças , Feminino , Fibroblastos/efeitos dos fármacos , Humanos , Queratinócitos/efeitos dos fármacos , Suínos , Vitronectina/farmacocinética , CicatrizaçãoRESUMO
Chronic wounds are an important health problem because they are difficult to heal and treatment is often complicated, lengthy and expensive. For a majority of sufferers the most common outcomes are long-term immobility, infection and prolonged hospitalisation. There is therefore an urgent need for effective therapeutics that will enhance ulcer healing and patient quality of life, and will reduce healthcare costs. Studies in our laboratory have revealed elevated levels of purine catabolites in wound fluid from patients with venous leg ulcers. In particular, we have discovered that uric acid is elevated in wound fluid, with higher concentrations correlating with increased wound severity. We have also revealed a corresponding depletion in uric acid precursors, including adenosine. Further, we have revealed that xanthine oxidoreductase, the enzyme that catalyses the production of uric acid, is present at elevated levels in wound fluid. Taken together, these findings provide evidence that xanthine oxidoreductase may have a function in the formation or persistence of chronic wounds. Here we describe the potential function of xanthine oxidoreductase and uric acid accumulation in the wound site, and the effect of xanthine oxidoreductase in potentiating the inflammatory response.
Assuntos
Ácido Úrico/metabolismo , Úlcera Varicosa/fisiopatologia , Cicatrização/fisiologia , Xantina Desidrogenase/fisiologia , Alopurinol/uso terapêutico , Doença Crônica , Inibidores Enzimáticos/uso terapêutico , Sequestradores de Radicais Livres/uso terapêutico , Radicais Livres/metabolismo , Humanos , Inflamação/fisiopatologia , Úlcera Varicosa/tratamento farmacológico , Cicatrização/efeitos dos fármacos , Xantina Desidrogenase/antagonistas & inibidoresRESUMO
Numerous studies have reported associations between IGF-I and other extra cellular matrix (ECM) proteins, including fibronectin (FN), integrins, IGF-binding proteins (IGFBPs) and through IGFBPs, with vitronectin (VN). Nevertheless, the precise nature and mechanisms of these interactions are still being characterised. In this paper, we discuss transglutaminases (TGases) as a constituent of the ECM and provide evidence for the first time that IGF-I is a lysine (K)-donor substrate to TGases. When IGF-I was incubated with an alpha-2 plasmin inhibitor-derived Q peptide in the presence of tissue transglutaminase (TG2), an IGF-I:Q peptide cross-linked species was detected using Western immunoblotting and confirmed by mass spectrometry. Similar findings were observed in the presence of Factor XIIIa (FXIIIa) TGase. To identify the precise location of this K-donor TGase site/s on IGF-I, all the three IGF-I K-sites, individually and collectively (K27, K65 and K68), were substituted to arginine (R) using site-directed mutagenesis. Incubation of these KâR IGF-I analogues with Q peptide in the presence of TG2 or FXIIIa resulted in the absence of cross-linking in IGF-I analogues bearing arginine substitution at site 68. This established that K68 within the IGF-I D-domain was the principal K-donor site to TGases. We further annotated the functional significance of these KâR IGF-I analogues on IGF-I mediated actions. IGF-I analogues with KâR substitution within the D-domain at K65 and K68 hindered migration of MCF-7 breast carcinoma cells and correspondingly reduced PI3-K/AKT activation. Therefore, this study also provides first insights into a possible functional role of the previously uncharacterised IGF-I D-domain.
Assuntos
Fator de Crescimento Insulin-Like I/química , Fator de Crescimento Insulin-Like I/metabolismo , Lisina/metabolismo , Transglutaminases/metabolismo , Sequência de Aminoácidos , Movimento Celular/efeitos dos fármacos , Reagentes de Ligações Cruzadas/metabolismo , Ativação Enzimática/efeitos dos fármacos , Fator XIIIa/metabolismo , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Cinética , Células MCF-7 , Espectrometria de Massas , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor IGF Tipo 1/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Especificidade por Substrato/efeitos dos fármacos , Ressonância de Plasmônio de Superfície , Vitronectina/farmacologiaRESUMO
Transglutaminases are confounding enzymes which are known to play key roles in various cellular processes. In this paper, we aim to bring together several pieces of evidence from published research and literature that suggest a potentially vital role for transglutaminases in receptor tyrosine kinases (RTK) signalling. We cite literature that confirms and suggests the formation of integrin:RTK:transglutaminase complexes and explores the occurrence and functionality of these complexes in a large fraction of the RTK family.
Assuntos
Receptores Proteína Tirosina Quinases/fisiologia , Transglutaminases/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Humanos , Integrinas/fisiologia , Mapas de Interação de Proteínas , Transdução de SinaisRESUMO
Chronic venous leg ulcers are a major health issue and represent an often overlooked area of biomedical research. Nevertheless, it is becoming increasingly evident that new approaches to enhance healing outcomes may arise through better understanding the processes involved in the formation of chronic wounds. We have for the first time shown that the terminal purine catabolite uric acid (UA) is elevated in wound fluid (WF) from chronic venous leg ulcers with relative concentrations correlating with wound chronicity. We have also shown a corresponding depletion in UA precursors, including adenosine, with increased wound severity. Further, we have shown that xanthine oxidase, the only enzyme in humans that catalyses the production of UA in conjunction with a burst of free radicals, is active in chronic WF. Taken together, this provides compelling evidence that xanthine oxidase may play a critical role in the formation of chronic wounds by prolonging the inflammatory process.
Assuntos
Úlcera da Perna/metabolismo , Ácido Úrico/metabolismo , Idoso , Western Blotting , Doença Crônica , Exsudatos e Transudatos/química , Humanos , Masculino , Pessoa de Meia-Idade , Ultrafiltração , Xantina Oxidase/fisiologiaRESUMO
Several different advanced treatments have been used to improve healing in chronic wounds, but none have shown sustained success. The application of topical growth factors (GFs) has displayed some potential, but the varying results, high doses and high costs have limited their widespread adoption. Many treatments have ignored the evidence that wound healing is driven by interactions between extracellular matrix proteins and GFs, not just GFs alone. We report herein that a clinical Good Manufacturing Practice-grade vitronectin:growth factor (cVN:GF) complex is able to stimulate functions relevant to wound repair in vitro, such as enhanced cellular proliferation and migration. Furthermore, we assessed this complex as a topical wound healing agent in a single-arm pilot study using venous leg ulcers, as well as several 'difficult to heal' case studies. The cVN:GF complex was safe and re-epithelialisation was observed in all but 1 of the 30 patients in the pilot study. In addition, the case studies show that this complex may be applied to several ulcer aetiologies, such as venous leg ulcers, diabetic foot ulcers and pressure ulcers. These findings suggest that further evaluation is warranted to determine whether the cVN:GF complex may be an effective topical treatment for chronic wounds.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/administração & dosagem , Úlcera por Pressão/tratamento farmacológico , Úlcera Varicosa/tratamento farmacológico , Vitronectina/administração & dosagem , Cicatrização/efeitos dos fármacos , Administração Tópica , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Doença Crônica , Pé Diabético/tratamento farmacológico , Quimioterapia Combinada , Feminino , Humanos , Projetos Piloto , Úlcera por Pressão/patologia , Resultado do Tratamento , Úlcera Varicosa/patologiaRESUMO
Successful wound repair and normal turnover of the extracellular matrix relies on a balance between matrix metalloproteinases (MMPs) and their natural tissue inhibitor of metalloproteinases (TIMPs). When overexpression of MMPs and abnormally high levels of activation or low expression of TIMPs are encountered, excessive degradation of connective tissue and the formation of chronic ulcers can occur. One strategy to rebalance MMPs and TIMPs is to use inhibitors. We have designed a synthetic pseudopeptide inhibitor with an amine linker group based on a known high-affinity peptidomimetic MMP inhibitor and have demonstrated inhibition of MMP-1, -2, -3, and -9 activity in standard solutions. The inhibitor was also tethered to a polyethylene glycol hydrogel using a facile reaction between the linker unit on the inhibitor and the hydrogel precursors. After tethering, we observed inhibition of the MMPs although there was an increase in the IC50s that was attributed to poor diffusion of the MMPs into the hydrogels, reduced activity of the tethered inhibitor, or incomplete incorporation of the inhibitor into the hydrogels. When the tethered inhibitors were tested against chronic wound fluid, we observed partial inhibition in proteolytic activity suggesting this approach may prove useful in rebalancing MMPs within chronic wounds.
Assuntos
Isoenzimas/antagonistas & inibidores , Inibidores de Metaloproteinases de Matriz , Peptidomiméticos/química , Ativação Enzimática , Humanos , Hidrogéis/química , Modelos Moleculares , Estrutura Molecular , Peptidomiméticos/síntese química , Polietilenoglicóis/química , Conformação ProteicaRESUMO
Numerous studies have reported links between insulin-like growth factors (IGFs) and the extra-cellular matrix protein vitronectin (VN). We ourselves have reported that IGF-I binds to VN via IGF-binding proteins (IGFBPs) to stimulate HaCaT and MCF-7 cell migration. Here, we detail the functional evaluation of IGFBP-1, -2, -3, -4 and -6 in the presence and absence of IGF-I and VN. The data presented here, combined with our prior data on IGFBP-5, suggest that IGFBP-3, -4 and -5 are the most effective at stimulating cell migration in combination with IGF-I and VN. In addition, we demonstrate that different regions within IGFBP-3 and -4 are critical for complex formation. Furthermore, we examine whether multi-protein complexes of IGF-I and IGFBPs associated with fibronectin and collagen IV are also able to enhance functional biological responses.
Assuntos
Movimento Celular/efeitos dos fármacos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Vitronectina/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Colágeno Tipo IV/metabolismo , Fibronectinas/metabolismo , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Domínios e Motivos de Interação entre Proteínas , Vitronectina/metabolismoRESUMO
Chronic ulcers are an important and costly medical issue, imposing considerable pain, reduced mobility and decreased quality of life. The common pathology in these chronic wounds is excessive proteolytic activity, resulting in degradation of key factors critical to the ulcer's ability to heal. Matrix metalloproteinases (MMPs), a large family of zinc-dependent endopeptidases, have been shown to have increased activity in chronic wound fluid (CWF), with many authors suggesting that they need to be inhibited for the ulcer to heal. The studies we report here show that the excessive MMP activity in CWF can be inhibited with the bisphosphonate alendronate, in the form of a sodium salt, a functionalised analogue, and tethered to a poly(2-hydroxy methacrylate) (PHEMA) hydrogel. Furthermore, these functionalised alendronate hydrogels appear to be biologically inert as assessed in a three-dimensional ex vivo human skin equivalent model. Together, these results highlight the potential use of a tethered MMP inhibitor to inhibit protease activity in wound fluid. This approach may improve wound healing as it still allows MMPs to remain active in the upper cellular layers of the ulcer bed where they perform vital roles in wound healing; thus may offer an attractive new device-orientated wound therapy.
Assuntos
Líquidos Corporais/metabolismo , Difosfonatos/administração & dosagem , Portadores de Fármacos/química , Hidrogéis/química , Inibidores de Metaloproteinases de Matriz , Úlcera/tratamento farmacológico , Úlcera/enzimologia , Cicatrização/efeitos dos fármacos , Difosfonatos/química , Ativação Enzimática/efeitos dos fármacos , Humanos , Teste de MateriaisRESUMO
Complexes comprised of IGF-I, IGF-binding proteins and the ECM protein vitronectin (VN) stimulate cell migration and growth and can replace the requirement for serum for the ex vivo expansion of cells, as well as promote wound healing in vivo. Moreover, the activity of the complexes is dependent on co-activation of the IGF-I receptor and VN-binding integrins. In view of this we sought to develop chimeric proteins able to recapitulate the action of the multiprotein complex within a single molecular species. We report here the production of two recombinant chimeric proteins, incorporating domains of VN linked to IGF-I, which mimic the functions of the complex. Further, the activity of the chimeric proteins is dependent on co-activation of the IGF-I- and VN-binding cell surface receptors. Clearly the use of chimeras that mimic the activity of growth factor:ECM complexes, such as these, offer manufacturing advantages that ultimately will facilitate translation to cost-effective therapies.