RESUMEN
INTRODUCTION: The 37/67 kDa high-affinity laminin receptor (laminin receptor precursor/laminin receptor, LRP/LR) is a multi-faceted cellular receptor. It plays a vital role in the malignancy of various cancer types where it is seen to contribute to invasion, adhesion, apoptosis evasion and angiogenesis. Furthermore, it has been found to play an important role in facilitating the processes leading to neurotoxicity in Alzheimer's disease (AD). Various therapeutic options targeting this receptor have been patented with the outlook on application for the treatment/prevention of these diseases. AREAS COVERED: The various roles that LRP/LR plays in cancer, AD and infectious diseases caused by viruses and bacteria have been examined in detail and an overview of the current patented therapeutic strategies targeting this receptor is given. EXPERT OPINION: Molecular tools directed against LRP/LR, such as antibodies and small interfering RNA, could prove to be effective in the prevention of metastasis and angiogenesis while inducing apoptosis in cancers. Moreover, these strategies could also be applied to AD where LRP/LR is seen to facilitate the production and internalization of the neurotoxic Aß peptide. This review provides a comprehensive overview of the mechanisms by which LRP/LR is involved in eliciting pathogenic events, while showing how the use of patented approaches targeting this receptor could be used to treat them.
Asunto(s)
Diseño de Fármacos , Terapia Molecular Dirigida , Receptores de Laminina/efectos de los fármacos , Proteínas Ribosómicas/efectos de los fármacos , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/fisiopatología , Animales , Antineoplásicos/farmacología , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Patentes como Asunto , ARN Interferente Pequeño/administración & dosificación , Receptores de Laminina/metabolismo , Proteínas Ribosómicas/metabolismoRESUMEN
PURPOSE: All-trans-retinoic-acid (ATRA), the active derivative of vitamin A, is critical in regulating cell cycle as well as inhibiting tumor growth and angiogenesis. It has been used in the clinical treatment of leukemia. 67kDa laminin receptor (67LR), as one of the receptor of laminin, plays an important role in tumor cells invasion, proliferation and metastasis. Current research indicates that 67LR is highly expressed in glioma and is associated with tumor progression. However, the underlying molecular mechanisms, especially the signaling pathways involved, have not been reported yet. Therefore it is of great importance to clarify its downstream targets. METHODS: The U251 glioma cell line was used in this study. Cell Counting Kit-8 was used in cell proliferation assay. Quantitative real-time PCR (qRT-PCR) was used to determine the transcription level of dual specificity phosphatases (DUSPs). Western blot analysis was used to detect the expression of mitogen activated protein kinases (MAPKs) and phosphorylated MAPKs. RESULTS: 67LR could influence the transcription of DUSPs and expression of MAPKs. ATRA could enhance the expression of 67LR in U251 cells and this enhancement was dose-dependent. ATRA was able to inhibit the growth of U251 cells. CONCLUSIONS: ATRA expressed significant therapeutic effect on glioma cells, and 67LR is not the only factor that can influence the proliferation of U251 cells.
Asunto(s)
Neoplasias Encefálicas/enzimología , Fosfatasas de Especificidad Dual/metabolismo , Glioma/enzimología , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Receptores de Laminina/metabolismo , Proteínas Ribosómicas/metabolismo , Antineoplásicos/farmacología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Fosfatasas de Especificidad Dual/genética , Activación Enzimática , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glioma/genética , Glioma/patología , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Fosforilación , Interferencia de ARN , Receptores de Laminina/efectos de los fármacos , Receptores de Laminina/genética , Proteínas Ribosómicas/efectos de los fármacos , Proteínas Ribosómicas/genética , Transcripción Genética , Transfección , Tretinoina/farmacologíaRESUMEN
Axonal regeneration after injury to the CNS is hampered by myelin-derived inhibitors, such as Nogo-A. Natural products, such as green tea, which are neuroprotective and safe for long-term therapy, would complement ongoing various pharmacological approaches. In this study, using nerve growth factor-differentiated neuronal-like Neuroscreen-1 cells, we show that extremely low concentrations of unfractionated green tea polyphenol mixture (GTPP) and its active ingredient, epigallocatechin-3-gallate (EGCG), prevent both the neurite outgrowth-inhibiting activity and growth cone-collapsing activity of Nogo-66 (C-terminal domain of Nogo-A). Furthermore, a synergistic interaction was observed among GTPP constituents. This preventive effect was dependent on 67-kDa laminin receptor (67LR) to which EGCG binds with high affinity. The antioxidants N-acetylcysteine and cell-permeable catalase abolished this preventive effect of GTPP and EGCG, suggesting the involvement of sublethal levels of H2 O2 in this process. Accordingly, exogenous sublethal concentrations of H2 O2 , added as a bolus dose (5 µM) or more effectively through a steady-state generation (1-2 µM), mimicked GTPP in counteracting the action of Nogo-66. Exogenous H2 O2 mediated this action by bypassing the requirement of 67LR. Taken together, these results show for the first time that GTPP and EGCG, acting through 67LR and elevating intracellular sublethal levels of H2 O2 , inhibit the antineuritogenic action of Nogo-A. Currently, several agents are being evaluated for overcoming axonal growth inhibitors to promote functional recovery after stroke and spinal cord injury. Epigallocatechin-3-gallate (EGCG), present in green tea polyphenol mixture (GTPP), prevents antineuritogenic activity of Nogo-A, a myelin-derived axonal growth inhibitor. The preventive action of EGCG involves the cell-surface-associated 67-kDa laminin receptor and H2 O2 . GTPP may complement ongoing efforts to treat neuronal injuries.>
Asunto(s)
Peróxido de Hidrógeno/farmacología , Proteínas de la Mielina/antagonistas & inhibidores , Proteínas de la Mielina/farmacología , Neuritas/efectos de los fármacos , Oxidantes/farmacología , Polifenoles/farmacología , Receptores de Laminina/efectos de los fármacos , Té/química , Animales , Células Cultivadas , Conos de Crecimiento/efectos de los fármacos , Ratones , Proteínas Nogo , Polifenoles/química , Seudópodos/efectos de los fármacosRESUMEN
Since the administration of synthetic medicines is associated with drug resistance and undesired side effects, utilization of natural compounds could be an alternative and complementary modality to inhibit or prevent the development of tumors. Epigallocatechin 3-O-gallate (EGCG, 1), the major flavan component of green tea, and genistein (2), a soy isoflavonoid, are known to have chemopreventive and chemotherapeutic effects against cancer. This study demonstrated that both flavonoids inhibit cell proliferation, an effect enhanced under serum-free conditions. Compound 1, but not 2, induced downregulation of ErbB1 and ErbB2 in mammary and epidermoid carcinoma cells, and its inhibitory effect on cell viability was mediated by the 67 kDa laminin receptor (67LR). While 1 was superior in inducing cell death, 2 was more efficient in arresting the tumor cells in the G2/M phase. Furthermore, number and brightness analysis revealed that 1 decreased the homoclustering of a lipid raft marker, glycosylphosphatidylinositol-anchored GFP, and it also reduced the co-localization between lipid rafts and 67LR. The main conclusion made is that the primary target of 1 may be the lipid raft component of the plasma membrane followed by secondary changes in the expression of ErbB proteins. Compound 2, on the other hand, must have other unidentified targets.
Asunto(s)
Anticarcinógenos/química , Anticarcinógenos/farmacología , Catequina/análogos & derivados , Flavonoides/farmacología , Genisteína/farmacología , Receptores de Laminina/efectos de los fármacos , Proteínas Ribosómicas/efectos de los fármacos , Té/química , Algoritmos , Anticarcinógenos/aislamiento & purificación , Apoptosis/efectos de los fármacos , Carcinoma de Células Escamosas/metabolismo , Catequina/química , Catequina/aislamiento & purificación , Catequina/farmacología , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Flavonoides/química , Flavonoides/aislamiento & purificación , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Puntos de Control de la Fase G2 del Ciclo Celular/fisiología , Genes erbB-1/genética , Genes erbB-1/fisiología , Genes erbB-2/genética , Genes erbB-2/fisiología , Genisteína/química , Genisteína/aislamiento & purificación , Humanos , Estructura Molecular , Polifenoles/farmacología , Receptores de Laminina/genética , Receptores de Laminina/metabolismo , Receptores de Laminina/fisiología , Glycine max/químicaRESUMEN
IMPORTANCE OF THE FIELD: The 37/67 kDa laminin receptor precursor/laminin receptor (LRP/LR) represents a multifunctional protein located on the cell surface, in the cytosol and the nucleus. The receptor acts as a mediator for cell adhesion, cell proliferation and cell differentiation. It is a key player in invasion and adhesion, major functions of several important metastatic cancer types. The receptor hampers apoptosis thereby favoring cancer progression. LRP/LR plays a major role as a cell surface receptor in prion disorders and may be of considerable importance for other neurodegenerative diseases such as Alzheimer's disease. A series of viruses including Sindbis virus, Dengue virus and Adeno-associated virus use LRP/LR as attachment receptors. Bacteria and Candida albicans use the receptor for pathogenesis. AREAS COVERED IN THIS REVIEW: Background and patented biological approaches for therapeutic modulation of LRP/LR in neurodegenerative diseases, cancer, viral disorders, bacterial and yeast infections. WHAT THE READER WILL GAIN: A comprehensive review of the role of LRP/LR in infectious and non-infectious diseases and an insightful assessment of published or patented biological approaches for the therapeutic modulation of LRP/LR. TAKE HOME MESSAGE: Molecular tools such as antibodies directed against LRP/LR have the potential to act as promising alternative therapeutics for the treatment of various diseases.
Asunto(s)
Anticuerpos/administración & dosificación , Sistemas de Liberación de Medicamentos , Receptores de Laminina/efectos de los fármacos , Animales , Adhesión Celular , Diferenciación Celular , Proliferación Celular , Humanos , Patentes como Asunto , Receptores de Laminina/metabolismoRESUMEN
Insulin and (-)-epigallocatechin gallate (EGCG) have been reported to regulate fat cell mitogenesis and adipogenesis, respectively. This study investigated the pathways involved in EGCG modulation of insulin-stimulated mitogenesis in 3T3-L1 preadipocytes. EGCG inhibited insulin stimulation of preadipocyte proliferation in a dose- and time-dependent manner. EGCG also suppressed insulin-stimulated phosphorylation of the insulin receptor-beta, insulin receptor (IR) substrates 1 and 2 (IRS1 and IRS2), and mitogen-activated protein kinase pathway proteins, RAF1, MEK1/2, and ERK1/2, but not JNK. Furthermore, EGCG inhibited the association of IR with the IRS1 and IRS2 proteins, but not with the IRS4 protein. These data suggest that EGCG selectively affects particular types of IRS and MAPK family members. Generally, EGCG was more effective than epicatechin, epicatechin gallate, and epigallocatechin in modulating insulin-stimulated mitogenic signaling. We identified the EGCG receptor [also known as the 67-kDa laminin receptor (67LR)] in fat cells and found that its expression was sensitive to growth phase, tissue type, and differentiation state. Pretreatment of preadipocytes with 67LR antiserum prevented the effects of EGCG on insulin-stimulated phosphorylation of IRS2, RAF1, and ERK1/2 and insulin-stimulated preadipocyte proliferation (cell number and bromodeoxyuridine incorporation). Moreover, EGCG tended to increase insulin-stimulated associations between the 67LR and IR, IRS1, IRS2, and IRS4 proteins. These data suggest that EGCG mediates anti-insulin signaling in preadipocyte mitogenesis via the 67LR pathway.
Asunto(s)
Adipocitos/efectos de los fármacos , Fármacos Antiobesidad/farmacología , Camellia sinensis , Catequina/análogos & derivados , Proliferación Celular/efectos de los fármacos , Insulina/metabolismo , Receptores de Laminina/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Células 3T3-L1 , Adipocitos/metabolismo , Animales , Fármacos Antiobesidad/aislamiento & purificación , Camellia sinensis/química , Catequina/aislamiento & purificación , Catequina/farmacología , Relación Dosis-Respuesta a Droga , Sueros Inmunes , Proteínas Sustrato del Receptor de Insulina/metabolismo , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/metabolismo , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-raf/metabolismo , Receptores de Laminina/inmunología , Receptores de Laminina/metabolismo , Factores de TiempoRESUMEN
As large amounts of porcine cartilage are discarded as waste in daily life, it is necessary to find new uses for them. We extracted polysaccharide from cartilage and performed in vitro and in vivo experiments in cancer cells. A mouse breast-cancer pulmonary metastasis model was set up, and we tried to determine the mechanism of the inhibition of metastasis by cartilage PS (polysaccharide). Effects on tumour size and the progression of metastasis indicated that cartilage PS can obviously inhibit metastasis in breast-cancer cells. The levels of LNR1 (laminin receptor 1), alphavbeta3 integrin and MMP-9 (matrix metalloproteinase-9) in mice treated or not with cartilage PS showed significant differences. Cartilage PS inhibited the growth of MCF-7 human breast adenocarcinoma cells, but had little effect on normal cells. Cartilage PS can inhibit the activity of the MMP-2 and the MMP-9 by decreasing the levels of LNR1 and alphavbeta3 integrin to inhibit metastasis further. In summary, we conclude that cartilage PS can act as a specific anti-metastatic agent in breast-cancer cells.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Cartílago/química , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/secundario , Metástasis de la Neoplasia/tratamiento farmacológico , Polisacáridos/farmacología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Integrina alfaVbeta3/efectos de los fármacos , Integrina alfaVbeta3/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz , Ratones , Trasplante de Neoplasias , Polisacáridos/aislamiento & purificación , Receptores de Laminina/efectos de los fármacos , Receptores de Laminina/metabolismo , Proteínas Ribosómicas , PorcinosRESUMEN
The epidemiological studies and recent data have provided convinced evidence that green tea and its major constituent epigallocatechin gallate (EGCG) might have the potential to lower the risk of cancers in humans. Metal ions, such as zinc and cadmium, which are necessary to our health, are important factors inducing many diseases including prostate cancer in the condition of absence or excess. EGCG can satisfactorily exhibit complex chemistry with metal ions because of multiple hydroxyl states, which in turn changes their bioactivities and metabolism pathways. This paper presents the results of an investigation of the cytotoxicity of EGCG against PC-3 prostate cancer cells in the presence and absence of Cd2+ in vitro. The results showed that both EGCG and Cd2+ suppressed viability and clonegenecity of PC-3 cells, and the suppression effect was enhanced when EGCG added with Cd2+. Although Cd2+ up-regulated the 67 kDa laminin receptor (67LR), which is a migration-associated protein, the cell migration ability was not significantly increased after each treatment. We also found that EGCG and Cd2+ directly interacted with mitochondrial, and the mixture of EGCG and Cd2+ (EGCG+Cd2+) significantly caused loss of the mitochondrial membrane potential, decrease of the ATP content and activation of caspase-9 compared with EGCG treated alone. Taken together, these findings suggest that Cd2+ enhanced the cytotoxicity of EGCG to PC-3 cells by up-regulating the 67LR and the mitochondria-mediated apoptosis pathway.
Asunto(s)
Anticarcinógenos/farmacología , Apoptosis/efectos de los fármacos , Cadmio/farmacología , Catequina/análogos & derivados , Regulación hacia Arriba/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Caspasa 9/efectos de los fármacos , Caspasa 9/metabolismo , Catequina/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Receptores de Laminina/efectos de los fármacos , Receptores de Laminina/metabolismo , Té/químicaRESUMEN
PCK3145 is a synthetic peptide corresponding to amino acids 31-45 of prostate secretory protein 94 which can reduce experimental skeletal metastases and prostate tumor growth. These anti-metastatic and anti-tumoral effects of PCK3145 are partially explained by the in-vivo and in-vitro decrease in matrix metalloproteinase (MMP)-9 extracellular levels through as yet unidentified molecular mechanisms of action. Gelatin zymography and immunoblots were used to monitor the levels of secreted MMP-9 from HT-1080 cells. Flow cytometry was used to monitor HT-1080 cell surface binding of FITC-labeled PCK3145 and biotin-labeled laminin. PCK3145-coated cell culture dishes were used to monitor cell adhesion. HT-1080 cell lysates were used for immunoblotting of HuR, extracellular signal-regulated protein kinase (ERK) and phospho-ERK. Total RNA was isolated and RT-PCR used to monitor HuR gene expression. We found that PCK3145 bound to the HT-1080 cell surface and that this binding rapidly triggered ERK phosphorylation that, ultimately, led to a reduction of secreted MMP-9. Laminin inhibited both cell surface binding and ERK phosphorylation by PCK3145. Overexpression of the 67-kDa laminin receptor led to an increased binding of the cells to PCK3145. HuR, a protein that can bind to and stabilize MMP-9 mRNA, was found to be downregulated by PCK3145. The mitogen-activated protein kinase/ERK (MEK) inhibitor PD98059 as well as native laminin and SIKVAV laminin-derived peptide prevented that downregulation. Our data suggest that PCK3145 rapidly triggers intracellular signaling through cell surface laminin receptors. This leads to decreased HuR expression and subsequent destabilization of MMP-9 transcripts. This is the first molecular evidence demonstrating the intracellular signaling and anti-metastatic mechanism of action of PCK3145 that leads to the inhibition of MMP-9 secretion.
Asunto(s)
Inhibidores de la Metaloproteinasa de la Matriz , Fragmentos de Péptidos/farmacología , Proteínas de Secreción Prostática/farmacología , Receptores de Laminina/metabolismo , Transducción de Señal/efectos de los fármacos , Antígenos de Superficie/metabolismo , Neoplasias Óseas/enzimología , Neoplasias Óseas/prevención & control , Neoplasias Óseas/secundario , Línea Celular Tumoral , Proteínas ELAV , Proteína 1 Similar a ELAV , Flavonoides/farmacología , Humanos , Laminina/farmacología , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Oligopéptidos/farmacología , Fragmentos de Péptidos/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patología , Proteínas de Secreción Prostática/química , Proteínas de Secreción Prostática/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas de Unión al ARN/metabolismo , Receptores de Laminina/efectos de los fármacosRESUMEN
Transforming growth factor-beta1 (TGF-beta1) has the ability to induce epithelial cell migration while stopping proliferation. In this study, we show that, concomitant to promoting migration of normal human keratinocytes in vitro, TGF-beta1 induced a marked decrease in their adhesion capacity to processed alpha3-containing laminin 5-coated surfaces. Indeed, the expression levels of alpha3 and alpha6 integrin subunit mRNA and protein, as well as the cell surface alpha3beta1 and alpha6beta4 integrins, were down-regulated. Recent studies showed that keratinocytes over express and deposit laminin 5 during migration and we have shown that laminin 5 found in the matrix of TGF-beta1 induced migrating keratinocytes is present in its unprocessed form [Décline and Rousselle, 2001: J. Cell Sci. 114:811-823]. We show here that TGF-beta1 treatment of the cells promoted a significant increase in their adhesion to the alpha3 chain carboxy-terminal LG4/5 subdomain and that this interaction is likely to be mediated by a heparan sulfate proteoglycan type of receptor. Our results indicate that alpha6beta4 and alpha3beta1 integrin interactions with laminin 5 are diminished during migration while a specific interaction occurs between an additional cellular receptor and the alpha3 LG4/5 module present on unprocessed laminin 5.
Asunto(s)
Adhesión Celular/efectos de los fármacos , Comunicación Celular/fisiología , Movimiento Celular/fisiología , Queratinocitos/fisiología , Laminina/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Comunicación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Citoesqueleto/efectos de los fármacos , Regulación hacia Abajo , Matriz Extracelular/metabolismo , Humanos , Integrina alfa3beta1/biosíntesis , Integrina alfa3beta1/efectos de los fármacos , Integrina alfa3beta1/genética , Integrina alfa6beta4/biosíntesis , Integrina alfa6beta4/efectos de los fármacos , Integrina alfa6beta4/genética , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Masculino , Fenotipo , Receptores de Laminina/efectos de los fármacos , Receptores de Laminina/metabolismo , Factor de Crecimiento Transformador beta1 , Cicatrización de HeridasRESUMEN
Elastin is a major component of the extracellular matrix. Elastin peptides derived from its degradation are present in human sera. Elastin peptides induce on fibroblasts, phagocytic cells, lymphocytes, smooth muscle cells and endothelial cells, a variety of biological effects mediated by the elastin-laminin receptor which has been demonstrated to be present on the membrane of these cells. The transduction pathway of the ELR receptor involves the activation of phospholipase C (PLC) by a pertussis toxin sensitive G-protein. PLC induces the production of inositol trisphosphate (IP3) leading to the increase of the intracellular free calcium on one hand, and of diacylglycerol (DAG) which stimulates the translocation to the membrane of PKC leading to the phosphorylation of members of the MAPK family, such as p42/p44 MAPK. Considering the multiple biological effects of ELR the elucidation of the complexity of the signaling pathways will help to better modulate it, mainly in pathological situations such as atherosclerosis.
Asunto(s)
Elastina/fisiología , Receptores de Superficie Celular/fisiología , Receptores de Laminina/fisiología , Animales , Señalización del Calcio/efectos de los fármacos , Quimiotaxis/efectos de los fármacos , Proteínas de Unión al GTP/efectos de los fármacos , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Queratinocitos/efectos de los fármacos , Linfocitos/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , N-Formilmetionina Leucil-Fenilalanina/farmacología , Proteínas de Neoplasias/fisiología , Péptidos/farmacología , Toxina del Pertussis , Fagocitos/efectos de los fármacos , Fosforilación , Procesamiento Proteico-Postraduccional , Ratas , Receptores de Superficie Celular/efectos de los fármacos , Receptores de Laminina/efectos de los fármacos , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismo , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
We have examined the mechanism of signaling by the 67 kDa YIGSR binding protein of laminin and its properties in neuroblastoma cells. Ligand displacement analysis showed that the interaction with the C(YIGSR)3-NH2 peptide amide is of intermediate affinity (1.5 x 10[-7] M). Cross-linking experiments with sulfo-MBS detected an additional protein with a molecular mass of 116 kDa that binds the YIGSR sequence. Incubation of neuroblastoma cells with C(YIGSR)3-NH2 peptide amide or antibody directed against the 67 kDa laminin binding protein induces tyrosine phosphorylation of proteins with a molecular mass ranging from 115 to 130 kDa and another heterogeneous protein group of 32 kDa.
Asunto(s)
Laminina/metabolismo , Precursores de Proteínas , Receptores de Laminina/metabolismo , Tirosina/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos/metabolismo , Anticuerpos/farmacología , Sitios de Unión , Reactivos de Enlaces Cruzados , Humanos , Ratones , Datos de Secuencia Molecular , Neuroblastoma/metabolismo , Fragmentos de Péptidos/farmacología , Fosfatidilinositol Diacilglicerol-Liasa , Fosforilación/efectos de los fármacos , Proteínas/química , Proteínas/metabolismo , Ratas , Receptores de Laminina/efectos de los fármacos , Receptores de Laminina/inmunología , Transducción de Señal , Células Tumorales Cultivadas , Fosfolipasas de Tipo C/farmacologíaRESUMEN
Gliomas are characterized by their extensive invasion into the brain parenchyma. Recently it has been shown that normal brain cells can produce laminin, fibronectin and collagen type IV when confronted by invading glioma cells. Laminin stimulates cell migration of several human glioma cell lines in vitro. This migration can be inhibited by adding blocking monoclonal antibodies (MAbs) against the most expressed integrin subunits, alpha3 and beta1. Previous studies have shown that glioma cell migration, invasion and growth are stimulated by epidermal growth factor (EGF). However, MAb directed against the EGF receptor (EGFR) did only partly inhibit the invasive process in vitro. Since laminin has regional peptide homology with EGF (EGF-like repeats), the present work was aimed at studying how two human glioma cell lines exposed to antibodies to the EGFR, reacted to laminin stimulated migration. Furthermore, we wanted to study which role the EGFR and the laminin receptor integrin subunits alpha3 and beta1 play during glioma cell invasion. EGFR expression of two glioma cell lines, AN1/lacZ and U-251/lacZ was studied by flow cytometry and immunofluorescence microscopy. A cell migration assay was used to study effects of MAbs against EGFR on migration from laminin-stimulated tumor spheroids. Tumor cell invasion was evaluated by using an in vitro co-culture model, where normal fetal brain cell aggregates were confronted with multicellular tumor spheroids. The results show that both cell lines expressed EGFR, AN1/lacZ 4-fold more than U-251/lacZ. MAb against EGFR inhibited the laminin-stimulated migration only from AN1/lacZ spheroids. MAbs against alpha3 and beta1 integrin subunits inhibited glioma cell invasion in vitro. The present work indicates possible connections between laminin-stimulated cell migration and the EGFR expression on glioma cells. These elements contribute to the characteristic features of glioma cells and may be an important part of the complex relationships between growth factors, integrins and extracellular matrix during glioma cell invasion.
Asunto(s)
Movimiento Celular/fisiología , Factor de Crecimiento Epidérmico/metabolismo , Glioma/patología , Receptores de Laminina/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Antígenos CD/efectos de los fármacos , Antígenos CD/metabolismo , División Celular , Movimiento Celular/efectos de los fármacos , Receptores ErbB/efectos de los fármacos , Receptores ErbB/inmunología , Receptores ErbB/metabolismo , Citometría de Flujo , Glioma/tratamiento farmacológico , Glioma/metabolismo , Humanos , Integrina alfa3 , Integrina beta1/efectos de los fármacos , Integrina beta1/metabolismo , Integrinas/efectos de los fármacos , Integrinas/metabolismo , Microscopía Fluorescente , Invasividad Neoplásica , Ratas , Receptores de Laminina/efectos de los fármacos , Esferoides Celulares , Células Tumorales CultivadasRESUMEN
Altered glomerular epithelial cell attachment to the glomerular basement membrane is an important pathogenetic factor in increased glomerular permeability to proteins. We have previously presented evidence that antibodies reactive with integrin matrix receptors on glomerular epithelial cells inhibit adhesion of these cells and may be involved in the production of proteinuria in vivo. Therefore, we utilized intact glomeruli in an in vitro system to directly assess the effect of anti-beta 1-integrin antibody on glomerular permeability. Permeability to albumin (Palb) was calculated from the volume response of glomeruli to a transcapillary oncotic gradient. Anti-beta 1-integrin increased Palb in a dose- and time-dependent manner. Palb was increased to 0.70 +/- 0.05 whereas normal rabbit IgG had no effect (0.10 +/- 0.04). F(ab')2 fragments of antibody increased Palb to a similar degree whereas Fab fragments had no effect (0.10 +/- 0.06). Cross-linking of Fab fragments, however, with a second antibody restored their ability to increase Palb (0.60 +/- 0.09), demonstrating the importance of integrin cross-linking in producing the observed effect. Intact, F(ab')2 and Fab fragments of anti-beta 1 antibody all inhibited adhesion of glomerular epithelial cells to fibronectin, laminin, and types I and IV collagen, although the degree of inhibition by Fab fragments was significantly less on collagens. No cytotoxic effects were observed with anti-beta 1 antibody or its fragments. These results suggest that antibodies to integrin matrix receptors on glomerular cells alter cell interactions with the glomerular basement membrane and lead to increased glomerular permeability to proteins via a process that is initiated by integrin cross-linking rather than through simple interference with cell adhesion per se.
Asunto(s)
Integrina beta1/inmunología , Glomérulos Renales/inmunología , Receptores de Laminina/efectos de los fármacos , Albúminas/metabolismo , Animales , Reacciones Antígeno-Anticuerpo/fisiología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/inmunología , Células Cultivadas , Reactivos de Enlaces Cruzados/farmacología , Epitelio/efectos de los fármacos , Epitelio/ultraestructura , Glomérulos Renales/citología , Glomérulos Renales/efectos de los fármacos , Microscopía Electrónica , Permeabilidad/efectos de los fármacos , Proteinuria/etiología , RatasRESUMEN
Biophysical studies were conducted on the action of laminin through membrane receptors of cancer cells. The results showed that variations occurred in the thermodynamic properties of membrane proteins, the mobility of hydrocarbon chains of membrane lipids, and the permeability and transportation pathways of the membrane.
Asunto(s)
Laminina/farmacología , Proteínas de la Membrana/metabolismo , Receptores de Laminina/efectos de los fármacos , Animales , Transporte Biológico Activo , Calcio/metabolismo , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Neoplasias Hepáticas Experimentales/patología , Lípidos de la Membrana/metabolismo , Ratones , Células Tumorales CultivadasRESUMEN
The 67 kDa laminin receptor (67LR), one of several cell surface laminin-binding proteins, is involved in the interactions between cancer cells and laminin during tumor invasion and metastasis. A 37 kDa polypeptide (37LRP), previously identified as the 67LR precursor, is abundantly present in the cytoplasm and has been implicated in polysome formation. To better understand the cellular localization of the 67LR and its precursor, transmission electron microscopic studies of human melanoma A2058 cells were carried out using immunogold labeling and a variety of antibodies: (a) affinity purified antibodies directed against 37LRP cDNA-derived synthetic peptides; (b) anti-67LR monoclonal antibodies raised against intact human small cell lung carcinoma cells; and (c) monoclonal antibodies against the subunits of the integrin laminin receptor, alpha 6 beta 1. Double-labeling immunocyto-chemistry revealed that anti-67LR monoclonal antibodies as well as anti-37 LRP antibodies recognized antigens that were localized in the cytoplasm in electron dense structures. As expected, cell membrane labeling was also observed. Surprisingly, alpha 6 and beta 1 integrin subunits were detected in the same cytoplasmic structures positive for the 67LR and the 37LRP. After addition of soluble laminin to A2058 cells in suspension, the number of labeled cytoplasmic structures increased especially in the vicinity of the plasma membrane, and were exported onto the cell surface. Neither fibronectin nor BSA induced such an effect. The data demonstrate that the 67 LR and the 37 LRP antibodies detect colocalized antigens that are in cytoplasmic structures with alpha 6 beta 1 integrin.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Integrinas/metabolismo , Laminina/farmacología , Receptores de Laminina/metabolismo , Membrana Celular/metabolismo , Citoplasma/metabolismo , Humanos , Integrina alfa6beta1 , Melanoma/metabolismo , Melanoma/secundario , Melanoma/ultraestructura , Microscopía Inmunoelectrónica , Peso Molecular , Invasividad Neoplásica , Receptores de Laminina/química , Receptores de Laminina/efectos de los fármacos , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/ultraestructuraRESUMEN
1. The effect of antiulcer agent, ebrotidine, on the expression of gastric mucosal laminin receptor during ulcer healing was investigated. 2. Rats with acetic acid-induced chronic gastric ulcers were treated twice daily for 14 consecutive days either with ebrotidine at 100 mg/kg or placebo, and then at different stages of treatment used for the isolation and quantitation of gastric mucosal laminin receptor. 3. The binding assays revealed that the ulcer healing was accompanied by an increase in mucosal expression of laminin receptor. A 2.7-fold increase in the receptor expression occurred by 4th day following the development of ulcer and reached a maximum of 8.6-fold increase by the 14th day when the ulcer was essentially healed. 4. Treatment with ebrotidine caused accelerated ulcer healing (7 days), which was accompanied by a remarkable enhancement in the laminin receptor expression. A 2.5-fold increase in the receptor expression over that of controls occurred by the 4th day of ebrotidine treatment, and a 1.7-fold increase was still observed at the 14th day of treatment. 5. The results suggest that ebrotidine, by evoking enhanced mucosal cell laminin receptor expression, promotes reepithelization and, hence, hastens the ulcer healing.
Asunto(s)
Antiulcerosos/uso terapéutico , Bencenosulfonatos/uso terapéutico , Mucosa Gástrica/efectos de los fármacos , Receptores de Laminina/efectos de los fármacos , Úlcera Gástrica/tratamiento farmacológico , Tiazoles/uso terapéutico , Cicatrización de Heridas/efectos de los fármacos , Secuencia de Aminoácidos , Animales , División Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Mucosa Gástrica/química , Masculino , Datos de Secuencia Molecular , Ratas , Ratas Sprague-Dawley , Receptores de Laminina/análisis , Úlcera Gástrica/metabolismoRESUMEN
In this paper, we describe the changes of microfilament assembly and 3H-TdR incorporation in mouse ascites liver cancer cells under the action of concanavalin A (ConA) and laminin (LN). We have also studied the variation of 3H-TdR incorporation induced by destroying microfilaments with cytochalasin B (CB) following ConA and LN binding with their membrane receptors. It was found that ConA and LN interactions with their membrane receptors could induce the assembly of microfilaments below the membrane and promote DNA synthesis in these cells, but this effect was inhibited when microfilaments were destroyed by CB treatment. These results suggest that microfilaments might play a role in transferring signals from the membrane to the nucleus.
Asunto(s)
Citoesqueleto de Actina/fisiología , Concanavalina A/farmacología , Laminina/farmacología , Transducción de Señal/efectos de los fármacos , Citoesqueleto de Actina/efectos de los fármacos , Animales , Citocalasina B/farmacología , ADN de Neoplasias/biosíntesis , Neoplasias Hepáticas Experimentales/patología , Ratones , Receptores de Concanavalina A/efectos de los fármacos , Receptores de Laminina/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Cranin is a 120 kDa integral membrane glycoprotein which binds laminin under conditions of physiologic ionic strength in a calcium-dependent manner. Here, binding of cranin to laminin has been characterized using both ligand-blotting assays and laminin affinity bead assays. Binding was specifically inhibited by anti-laminin antibodies against the A chain terminal domain G, but not by several other region-specific antibodies. Dextran sulfate, fucoidin, and sulfatide were potent inhibitors of binding (50% inhibition at 0.03, 0.5, and 1.7 micrograms/ml, respectively); heparin was a weaker inhibitor (50% inhibition approximately 5 micrograms/ml), and mannan and chondroitin sulfate were not inhibitory at 100 micrograms/ml. Binding was not inhibited by lactose or the A chain peptide PA22-2. The mobility of the broad, fuzzy cranin band was shifted after digestion with neuraminidase, N-glycanase, and O-glycanase, though none of these treatments decreased band heterogeneity nor destroyed the ability to bind laminin. Cranin bound to Jacalin lectin, which recognizes the Gal beta 1-3GalNAc linkage expressed in certain classes of mucins. These findings indicate that cranin binds at or near the high affinity sulfatide-binding site previously mapped to the E3 domain of laminin, which is known to exhibit bioactivity for neural cells. In view of the extremely low abundance of cranin in brain membranes (approximately 0.005%), its avid laminin-binding activity is remarkable, and strongly suggests that cranin may play a physiologic role in regulating specific neural cell interactions.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Laminina/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Laminina/metabolismo , Sulfoglicoesfingolípidos/metabolismo , Animales , Química Encefálica/efectos de los fármacos , Embrión de Pollo , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/inmunología , Sulfato de Dextran/farmacología , Distroglicanos , Glicósido Hidrolasas/metabolismo , Glicósido Hidrolasas/farmacología , Hidrólisis , Indicadores y Reactivos , Laminina/química , Laminina/inmunología , Ligandos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/inmunología , Membranas/metabolismo , Oxidación-Reducción , Polisacáridos/farmacología , Unión Proteica/efectos de los fármacos , Receptores de Laminina/química , Receptores de Laminina/efectos de los fármacos , Sulfoglicoesfingolípidos/farmacologíaRESUMEN
The level of 67 kDa laminin receptor (67LR) expression on breast and colon tumor cell surfaces was previously shown to be correlated with the capacity of tumor cells to metastasize. In the present work we investigate the effects of progestins and estrogen on the expression of 67LR in two sublines of the T47D human breast cancer cells: weakly tumorigenic, poorly invasive parental T47D cells and a highly tumorigenic, more invasive T47Dco subclone. Immunoblotting with an affinity purified antibody directed against a synthetic peptide recognizes the 67LR in these cells. 67LR expression in the T47Dco subclone is 5.5-fold higher than in their parental T47D cells. Treatment of T47D cells with 1 nM of the synthetic progestin R5020 results in a 4-fold increase in 67LR protein expression. Estrogen also induced 67LR expression, but only by 1.5-fold. The progestin-stimulated expression of the 67LR correlates with a 4.3-fold increase in attachment of T47D cells to laminin. A monoclonal antibody, mAb 13, directed against beta 1 integrin, completely blocks the attachment of T47D cells to fibronectin, only partially inhibits the attachment of T47D cells to laminin, and appears not to affect the progestin-stimulated laminin attachment of T47D cells. A new antiprogestin, ZK 112.993, significantly inhibits both progestin-stimulated 67LR expression and the increased attachment to laminin. These results suggest a possible role for progestin in mediating one of the multiple events thought to be important in metastasis of steroid receptor positive human breast cancer cells.