RESUMEN
Osteoarthritis is a chronic degenerative joint disease affecting millions of people worldwide, with no disease-modifying drugs currently available to treat the disease. Tissue inhibitor of metalloproteinases 3 (TIMP-3) is a potential therapeutic target in osteoarthritis because of its ability to inhibit the catabolic metalloproteinases that drive joint damage by degrading the cartilage extracellular matrix. We previously found that suramin inhibits cartilage degradation through its ability to block endocytosis and intracellular degradation of TIMP-3 by low-density lipoprotein receptor-related protein 1 (LRP1), and analysis of commercially available suramin analogues indicated the importance of the 1,3,5-trisulfonic acid substitutions on the terminal naphthalene rings for this activity. Here we describe synthesis and structure-activity relationship analysis of additional suramin analogues using ex vivo models of TIMP-3 trafficking and cartilage degradation. This showed that 1,3,6-trisulfonic acid substitution of the terminal naphthalene rings was also effective, and that the protective activity of suramin analogues depended on the presence of a rigid phenyl-containing central region, with para/para substitution of these phenyl rings being most favourable. Truncated analogues lost protective activity. The physicochemical characteristics of suramin and its analogues indicate that approaches such as intra-articular injection would be required to develop them for therapeutic use.
Asunto(s)
Osteoartritis , Inhibidor Tisular de Metaloproteinasa-3 , Humanos , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Inhibidor Tisular de Metaloproteinasa-3/farmacología , Inhibidor Tisular de Metaloproteinasa-3/uso terapéutico , Suramina/farmacología , Suramina/metabolismo , Suramina/uso terapéutico , Cartílago/metabolismo , Osteoartritis/tratamiento farmacológico , Osteoartritis/metabolismo , Metaloproteasas/metabolismo , Metaloproteasas/farmacología , Metaloproteasas/uso terapéuticoRESUMEN
TIMP-3 is one of four tissue inhibitors of matrix metalloproteinases, the endogenous inhibitors of the matrix metalloproteinase enzymes. These enzymes have an important role in metastasis, in the invasion of cancer cells through the basement membrane and extracellular matrix. TIMP-1, -2 and -4 both promote and inhibit tumour development, in a context-dependent manner, however TIMP-3 is consistently anti-tumourigenic. TIMP-3 is also the only insoluble member of the family, being either bound to the extracellular matrix or the low density lipoprotein-related protein-1, through which it can be endocytosed. Levels of TIMP-3 have also been shown to be regulated by micro RNAs and promoter hypermethylation, resulting in frequent silencing in many tumour types, to the extent that its expression has been suggested as a prognostic marker in some tumours, being associated with lower levels of metastasis, or better response to treatment. TIMP-3 has been shown to have anti-metastatic effects, both through inhibition of matrix metalloproteinases and ADAM family members and downregulation of angiogenesis. This occurs via interactions with receptors including VEGF, via modulation of signaling pathways and due to protease inhibition. TIMP-3 has also been shown to reduce tumour growth rate, most often by inducing apoptosis by stabilisation of death receptors. A number of successful mechanisms of delivery of TIMP-3 to tumour or inflammatory sites have been investigated in vitro or in animal studies. It may therefore be worthwhile further exploring the use of TIMP-3 as a potential anti-metastatic or anti-tumorigenic therapy for many tumour types.
Asunto(s)
Carcinogénesis/patología , Metástasis de la Neoplasia/patología , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Apoptosis/genética , Membrana Basal/metabolismo , Carcinogénesis/genética , Metilación de ADN , Modelos Animales de Enfermedad , Regulación hacia Abajo , Epigénesis Genética , Matriz Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/metabolismo , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Invasividad Neoplásica/prevención & control , Metástasis de la Neoplasia/tratamiento farmacológico , Metástasis de la Neoplasia/genética , Pronóstico , Regiones Promotoras Genéticas , Inhibidor Tisular de Metaloproteinasa-3/genética , Inhibidor Tisular de Metaloproteinasa-3/uso terapéutico , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/uso terapéuticoRESUMEN
Tissue Inhibitor of Metalloproteinase 3 (TIMP3) is a secreted protein that has a great utility to inhibit elevated metalloproteinase (MMP) activity in injured tissues including infarcted cardiac tissue, inflamed vessels, and joint cartilages. An imbalance between TIMP3 and active MMP levels in the local tissue area may cause worsening of disease progression. To counter balance elevated MMP levels, exogenous administration of TIMP3 appeared to be beneficial in preclinical studies. However, the current form of WT-TIMP3 molecule has a limitation to be a therapeutic candidate due to low production yield, short plasma half-life, injection site retention, and difficulty in delivery, etc. We have engineered TIMP3 molecules by adding extra glycosylation sites or fusing with albumin, Fc, and antibody to improve pharmacokinetic properties. In general, the C-terminal fusion of TIMP3 improved expression and production in mammalian cells and extended half-lives dramatically 5-20 folds. Of note, a site-specific glycosylation at K22S/F34N resulted in a higher level of expression and better cardiac function compared to other fusion proteins in the context of left ventricle ejection fraction (LVEF) changes in a rat myocardial infarction model. It appeared that cardiac efficacy depends on a high ECM binding affinity, in which K22S/F34N and N-TIMP3 showed a higher binding to the ECM compared to other engineered molecules. In conclusion, we found that the ECM binding and sustained residence of injected TIMP3 molecules are important for cardiac tissue localization and inhibition of adverse remodeling activity.
Asunto(s)
Proteína ADAM17/antagonistas & inhibidores , Metaloproteinasas de la Matriz/metabolismo , Infarto del Miocardio/tratamiento farmacológico , Proteínas Recombinantes de Fusión/farmacología , Inhibidor Tisular de Metaloproteinasa-3/farmacología , Función Ventricular Izquierda/efectos de los fármacos , Proteína ADAM17/metabolismo , Animales , Línea Celular , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Fibroblastos , Glicosilación , Humanos , Infusiones Intravenosas , Inyecciones Intralesiones , Masculino , Mutación , Infarto del Miocardio/etiología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/uso terapéutico , Inhibidor Tisular de Metaloproteinasa-3/química , Inhibidor Tisular de Metaloproteinasa-3/genética , Inhibidor Tisular de Metaloproteinasa-3/uso terapéutico , Resultado del TratamientoRESUMEN
An imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) contributes to the left ventricle (LV) remodeling that occurs after myocardial infarction (MI). However, translation of these observations into a clinically relevant, therapeutic strategy remains to be established. The present study investigated targeted TIMP augmentation through regional injection of a degradable hyaluronic acid hydrogel containing recombinant TIMP-3 (rTIMP-3) in a large animal model. MI was induced in pigs by coronary ligation. Animals were then randomized to receive targeted hydrogel/rTIMP-3, hydrogel alone, or saline injection and followed for 14 days. Instrumented pigs with no MI induction served as referent controls. Multimodal imaging (fluoroscopy/echocardiography/magnetic resonance imaging) revealed that LV ejection fraction was improved, LV dilation was reduced, and MI expansion was attenuated in the animals treated with rTIMP-3 compared to all other controls. A marked reduction in proinflammatory cytokines and increased smooth muscle actin content indicative of myofibroblast proliferation occurred in the MI region with hydrogel/rTIMP-3 injections. These results provide the first proof of concept that regional sustained delivery of an MMP inhibitor can effectively interrupt adverse post-MI remodeling.
Asunto(s)
Hidrogel de Polietilenoglicol-Dimetacrilato/química , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/metabolismo , Inhibidor Tisular de Metaloproteinasa-3/administración & dosificación , Inhibidor Tisular de Metaloproteinasa-3/uso terapéutico , Remodelación Ventricular/fisiología , Animales , Modelos Animales de Enfermedad , Hidrogel de Polietilenoglicol-Dimetacrilato/administración & dosificación , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Remodelación Ventricular/efectos de los fármacosRESUMEN
BACKGROUND: Tumor necrosis factor (TNF)-alpha and its receptors play a critical role in the inflammatory cascade after hepatic ischemia/reperfusion injury. TNF-alpha converting enzyme (TACE) or disintegrin and metalloproteinase (ADAM)-17 is a metalloproteinase disintegrin that specifically cleaves precursor TNF-alpha to its mature form and is involved in the ectodomain shedding of TNF receptors. The regulation of TACE is poorly understood and its role in liver injury and/or regeneration is unknown. METHODS: Male Wistar rats were subjected to 10 or 30 min of partial warm hepatic ischemia followed by 3 to 24 hr of reperfusion. Serum and/or hepatic TACE, TNF-alpha, TNF receptor 1 (TNFR1), and interleukin (IL)-6 levels were assessed by enzyme-linked immunosorbent assay, real-time reverse-transcriptase polymerase chain reaction, and/or Western blot. RESULTS: Low levels of TACE were detected in normal liver tissue. Both 10 and 30 min warm ischemia resulted in a rise in TACE expression which peaked six hr after reperfusion. TNF-alpha, TNFR1, and IL-6 levels were up-regulated in a pattern similar to TACE messenger RNA (mRNA) levels. Moreover, selective inhibition of TACE activity by specific inhibitor tissue inhibitor of metalloproteinase (TIMP)-3 at dosages of 100 or 1000 ng/kg body weight showed significant decrease of circulating TNF-alpha and serum alanine transferase (ALT) levels and histological improvement of hepatic ischemia/reperfusion injuries. CONCLUSIONS: TACE expression and its activity, as measured by increases in TNF-alpha, TNFR1, and IL-6 levels, are increased following hepatic ischemia/reperfusion injury, implying that TACE plays an important role in hepatic ischemia/reperfusion injury. Amelioration of hepatic ischemia/reperfusion injury after inhibition of TACE activity by TIMP-3 suggests that TACE inhibition may play an important role in preventing liver ischemia/reperfusion injury warranting further experimental and clinical study.
Asunto(s)
Proteínas ADAM/antagonistas & inhibidores , Hígado/enzimología , Daño por Reperfusión/prevención & control , Inhibidor Tisular de Metaloproteinasa-3/farmacología , Proteína ADAM17 , Animales , Humanos , Interleucina-6/análisis , Interleucina-6/metabolismo , Hígado/efectos de los fármacos , Hígado/patología , Masculino , Ratas , Ratas Wistar , Receptores Tipo I de Factores de Necrosis Tumoral/análisis , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Daño por Reperfusión/enzimología , Daño por Reperfusión/patología , Inhibidor Tisular de Metaloproteinasa-3/uso terapéutico , Factor de Necrosis Tumoral alfa/análisis , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
We have studied the effect of a newly identified tumor suppressor tissue inhibitor of metalloproteinases- 3 (TIMP-3) on the growth of human melanoma and squamous-cell carcinoma (SCC). Adenoviral delivery of the TIMP-3 gene to human melanoma (A2058) and SCC (UT-SCC-7) cells ex vivo inhibited tumorigenesis after subcutaneous (s.c.) injection of the infected cells into SCID/SCID mice. Three daily consecutive intratumoral injections of 1.4x10(9) plaque-forming units (pfu) of TIMP-3 adenovirus (RAdTIMP-3) inhibited the growth of preestablished melanoma and SCC xenografts in SCID/SCID mice, whereas growth of control virus-injected tumors was not affected. The antitumor effect of RAdTIMP-3 was obtained with in vivo adenoviral transduction efficiency of 8-10%, and it was more potent than that of adenovirally delivered p53. Adenovirusmediated expression of TIMP-3 potently reduced gelatinolytic activity, increased the number of apoptotic cells, and inhibited vascularization of melanomas. Escalation of the adenoviral dose to three rounds of three daily consecutive injections with 1.4x10(9) pfu of RAdTIMP-3 every 6 days entirely inhibited growth of injected melanomas for 32 days. Mixing RAdTIMP-3-infected A2058 cells with uninfected cells in 1:1 ratio in culture resulted in death of all cells in 96 hours. Adenovirally delivered TIMP-3 was also expressed by A2058 cells in soluble form into the culture medium, where it exerted a cytotoxic effect on uninfected A2058 cell cultures after relocating to the cell layer. These results identify TIMP-3 as a novel type of secreted tumor suppressor, which has antiinvasive, antiangiogenic, and proapoptotic effects in vivo, and which displays a potent bystander effect validating further exploration of its applicability in human cancer gene therapy.
Asunto(s)
Adenoviridae/genética , Antineoplásicos/farmacología , Efecto Espectador , Vectores Genéticos , Melanoma/terapia , Inhibidor Tisular de Metaloproteinasa-3/uso terapéutico , Animales , Antineoplásicos/metabolismo , Apoptosis , Carcinoma de Células Escamosas/metabolismo , Terapia Genética , Humanos , Melanoma/metabolismo , Metaloendopeptidasas/antagonistas & inhibidores , Ratones , Ratones SCID , Trasplante de Neoplasias , Neovascularización Patológica/metabolismo , Neovascularización Patológica/prevención & control , Inhibidores de Proteasas/uso terapéutico , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-3/genética , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Transducción Genética , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/metabolismoAsunto(s)
Artritis , Endopeptidasas , Proteínas de la Matriz Extracelular , Metaloendopeptidasas , Proteínas ADAM , Proteína ADAM17 , Agrecanos , Animales , Artritis/tratamiento farmacológico , Artritis/etiología , Endopeptidasas/fisiología , Humanos , Lectinas Tipo C , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/fisiología , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/uso terapéutico , Proteoglicanos/metabolismo , Inhibidor Tisular de Metaloproteinasa-3/farmacología , Inhibidor Tisular de Metaloproteinasa-3/uso terapéuticoRESUMEN
OBJECTIVE: To investigate the anti-cancer effect of tissue inhibitor of metalloproteinase-3 (TIMP-3) and its mechanism of action. METHODS: Recombinant TIMP-3(rTIMP-3) was prepared and used to study its effect on tumor growth and metastases in mice, and its effect on angiogenesis in chicken embryo allantoic membrane (CAM). RESULTS: rTIMP-3 was able to inhibit growth of subcutaneously implanted S180 sarcoma and hepatic carcinoma H22 in a dose-dependent manner. When the rTIMP-3 was given i.p. at a daily dose of 20 mg/kg body weight for 10 days, the growth of S180 sarcoma and H22 carcinoma was inhibited by 63.2% and 73.4%, respectively. The metastases of i.v. injected Lewis lung carcinoma(3LLC) was also suppressed by rTIMP-3, with an inhibition rate of 59.8%. rTIMP-3 was found to significantly inhibit angiogenesis in CAM assay. CONCLUSION: rTIMP-3 has anti-cancer activity, and the mechanism of action might be associated with its inhibition on extracellular matrix degradation and anti-angiogenesis.
Asunto(s)
Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Sarcoma 180/tratamiento farmacológico , Inhibidor Tisular de Metaloproteinasa-3/uso terapéutico , Animales , Carcinoma Pulmonar de Lewis/secundario , Embrión de Pollo , Relación Dosis-Respuesta a Droga , Femenino , Masculino , Ratones , Trasplante de Neoplasias , Neovascularización Patológica/tratamiento farmacológico , Proteínas Recombinantes/uso terapéutico , Células Tumorales CultivadasRESUMEN
Matrix metalloproteinases (MMPs), or matrixins, are a family of zinc endopeptidases that play a key role in both physiological and pathological tissue degradation. Normally, there is a careful balance between cell division, matrix synthesis and matrix degradation, which is under the control of cytokines, growth factors and cell matrix interactions. The MMPs are involved in remodelling during tissue morphogenesis and wound healing. Under pathological conditions, this balance is altered: in arthritis, there is uncontrolled destruction of cartilage; in cancer, increased matrix turnover is thought to promote tumour cell invasion. The demonstration of a functional role of MMPs in arthritis and tumour metastasis raises the possibility of therapeutic intervention using synthetic MMP inhibitors with appropriate selectivity and pharmacokinetics. As the process of drug discovery focuses on structure-based design, efforts to resolve the 3-dimensional structures of the MMP family have intensified. Several novel MMP inhibitors have been identified and are currently being investigated in clinical trials. The structural information that is rapidly accumulating will be useful in refining the available inhibitors to selectively target specific MMP family members. In this review, we focus on the role of MMPs and their inhibitors in tumour invasion, metastasis and angiogenesis, and examine how MMPs may be targeted to prevent cancer progression.