RESUMEN

We investigated the antiproliferative effect of phenylacetate covalently linked to dextran derivatives (DMCBPA conjugates) on human breast cancer MCF-7 cells. We show that free sodium phenylacetate (NaPA) inhibits the cell growth (IC50 = 14 mM), while an important inhibitory effect is observed for DMCBPA conjugates. The IC50 dose of these conjugates is as low as 1.0 mg/ml, corresponding to 1.3 mM of phenylacetate. The precursors, dextran substituted with methylcarboxylate and benzylamide groups, did not affect the growth of MCF-7 tumor cells. We have observed that MCF-7 cell growth inhibition depends on amount of phenylacetate linked to the conjugate. The data indicated that an optimum antiproliferative effect is more significant when the amount of phenylacetate groups present on the dextran backbone is high. Analysis of doubling time by growth kinetics study shows that conjugates have more time-sustained effect than free NaPA. It is noteworthy that the inhibitory effect is observed at non-toxic concentration. Theses conjugates could be considered as acceptable derivatives to prevent tumor progression.

Asunto(s)

Dextranos/química , Fenilacetatos/química , Antimetabolitos Antineoplásicos/farmacología , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Humanos , Concentración 50 Inhibidora , Cinética , Modelos Químicos , Fenilacetatos/farmacología

RESUMEN

BACKGROUND AND OBJECTIVES: This article presents a new approach for removing the Factor VIII inhibitors (anti-FVIII) in haemophiliac patients by immunoadsorption using an affinity matrix. MATERIALS AND METHODS: Ten blood samples from haemophiliac patients with anti-FVIII were assayed for antibodies, total immunoglobulins, procoagulant proteins and complement C3 protein after circulation over one or two columns filled with the polymers under investigation. RESULTS: These new synthetic sorbents are able to remove in vitro 90% of anti-FVIII from haemophiliac plasma with inhibitors (up to 540 Bethesda Units/ml). Neither coagulation factor adsorption nor effects on complement system activation were observed. CONCLUSIONS: The data presented clearly show that these polymers allow a rapid and efficient reduction of inhibitor titre. In view of the parameters studied, these polymers fulfil the requirements for use in a blood purification process to decrease high inhibitor titres without losing essential proteins.

Asunto(s)

Autoanticuerpos/aislamiento & purificación , Factor VIII/inmunología , Hemofilia A/terapia , Poliestirenos , Autoanticuerpos/sangre , Factores de Coagulación Sanguínea/análisis , Cromatografía de Afinidad , Complemento C3/análisis , Manejo de la Enfermedad , Hemofilia A/inmunología , Humanos , Inmunoglobulinas/sangre , Técnicas de Inmunoadsorción , Resinas Sintéticas/normas

RESUMEN

We have studied the cytostatic effects of sodium phenylacetate (NaPA) in association with several substituted dextrans on human tumor melanoma 1205LU cells. We show that NaPA alone inhibits the growth of these cells (IC50 = 3.9 mM) while a weak inhibitory effect appears at a concentration of 37 microM (10 microg/ml) for a dextran methyl carboxylate benzylamide (LS17-DMCB). The precursors of LS17-DMCB [T40 Dextran and carboxymethyl dextran (LS17-DMC)] did not affect the growth of 1205LU cells. To potentiate the inhibitory activity of NaPA at low concentrations (below 5.6 mM), we have tested NaPA and LS17-DMCB in physical mixture (association) or linked together covalently (this conjugate is termed 'LS17-NaPaC'). We have observed an increase of the 1205LU cell growth inhibition effect with NaPA in association (IC50 1.8 mM). For a concentration of 5 mM of NaPA (free in the case of association or linked in the case of conjugate), the association with dextran derivative exhibits a 4.6-fold higher efficacy than with NaPA alone (9 versus 41% surviving fraction), while the conjugate is 1.3-fold smaller (52% growth inhibition). By performing isobologram analysis of the IC50 data, we have shown a synergistic effect for a particular molar ratio of NaPA and LS17-DMCB (NaPA:LS17-DMCB = 0.35).

Asunto(s)

Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Dextranos/farmacología , Melanoma/patología , Fenilacetatos/farmacología , Neoplasias Cutáneas/patología , Animales , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Dextranos/metabolismo , Sinergismo Farmacológico , Humanos , Ratones , Ratones Desnudos , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patología

RESUMEN

Sodium phenylacetate (NaPa) and carboxymethyl benzylamide dextran derivative (CMDB(LS4)) are able to inhibit growth of breast tumour cells. In this study, we explored whether the combination of NaPa and CMDB(LS4)may enhance their respective inhibitory effects on the MCF-7ras cell growth in vitro and in vivo. NaPa inhibited MCF-7ras cell proliferation by reducing the DNA replication concomitantly with a recruitment of cells in G0/G1 phase and by inducing apoptosis in a dose- and time-dependent manner. The addition of CMDB(LS4)potentiated the NaPa antiproliferative effect in the manner dependent on the ratio of CMDB(LS4)and NaPa concentrations. In nude mice, CMDB(LS4)(150 mg kg(-1)) or NaPa (40 mg kg(-1)) administrated twice a week, for 7 weeks inhibited MCF-7ras xenograft growth by 40% and 60%, respectively. The treatment by both, CMDB(LS4)and NaPa, decreased tumour growth by 83% without any toxicity. To better understand the mechanism of NaPa and CMDB(LS4)action we assessed their effect on mitogenic activity of MCF-7ras conditioned medium (CM) on BALBC/3T3 fibroblasts. CMDB(LS4)added to the CM, inhibited its mitogenic activity whereas NaPa had an anti-mitogenic effect when CM was prepared from MCF-7ras cells pretreated with NaPa. Thus, the antiproliferative effects of NaPa and CMDB(LS4)involve 2 different mechanisms explaining, at least in part, the possible synergism between them. Overall, this study points to the potential use of a combination of dextran derivatives with NaPa to inhibit the breast tumour growth.

Asunto(s)

Antimetabolitos Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , División Celular/efectos de los fármacos , Dextranos/farmacología , Fenilacetatos/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Células 3T3 , Animales , Neoplasias de la Mama/patología , Ciclo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Medios de Cultivo Condicionados , Combinación de Medicamentos , Femenino , Fibroblastos/metabolismo , Sustancias de Crecimiento/biosíntesis , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Células Tumorales Cultivadas/metabolismo

RESUMEN

Dextranmethylcarboxylate benzylamide sulfate (DMCBSu), a functionalized dextran, exhibits anticoagulant properties. Its synthesis involves three steps: a carboxymethylation with monochloroacetic acid in alkaline water-iso-propanol, a benzylamidification of some of the methylcarboxylate groups with benzylamine in the presence of a water soluble carbodiimide and a partial sulfation of the remaining hydroxyl groups with SO3-pyridine in dimethylformamide. This procedure yields reproducibly DMCBSu with degrees of substitution in methylcarboxylate (MC), benzylamide (B) and sulfate (Su) groups, respectively, up to 1.61, 0.35 and 1.5, each obtained in one step. For a degree of substitution of methylcarboxylate ca. 1, the presence of sulfate groups is absolutely necessary to confer anticoagulant activities to the samples. In addition, the anticoagulant ability is higher for derivatives bearing benzylamide groups. The anticoagulant ability of DMCBSu increases with the degree of sulfation, reaching 20% of heparin activity for a degree of substitution of Su groups about 1.3.

Asunto(s)

Anticoagulantes/farmacología , Compuestos de Bencilo/farmacología , Dextranos/farmacología , Anticoagulantes/síntesis química , Compuestos de Bencilo/síntesis química , Cromatografía/métodos , Dextranos/síntesis química , Heparina/farmacología , Humanos , Iones , Espectroscopía Infrarroja por Transformada de Fourier

RESUMEN

Carboxymethyl Benzylamide Dextran (CMDB7) displayed an in vitro growth inhibitory activity on breast tumor cells. CMDB7 is able to disrupt the interaction of angiogenic growth factors (FGF2, TGF beta and PDGF) with their membrane receptors. This compound blocks the angiogenesis of MDA-MB435 carcinoma xenografted in mammary fat pad and their lung metastases in nude mice. In this work, we studied the uptake of CMDB7 labeled with 99mTc in cultured human breast cancer MCF-7 cell line and the highly tumorigenic MCF-7ras cell line (Ha-ras-transfected MCF-7 cells) and the in vivo distribution in MCF-7ras tumor-bearing mice. The 99mTc-CMDB7 are stable and the intracellular concentration is time-dependent and reaches a plateau at 180 minutes. 99mTc CMDB7 uptake is much higher in MCF-7ras cells than MCF-7 cells. Since CMDB7 is internalized and could also inhibit cell proliferation by acting at nuclear sites, we investigated the MCF-7ras nuclear localization after cell fractionation. Cell fractionation revealed a cytoplasmic and nuclear internalization of CMDB7. The tumor uptakes of 99mTc-CMDB7 were 0.34%, 0.72% and 0.62% of the administrated doses per gram of tumor tissue at 1 hour, 3 hour and 5 hours respectively after their injection. The blood clearance of 99mTc CMDB7 was very rapid and the liver, spleen and kidney uptakes were very weak. These results confirm the absence of toxicity of CMDB7 and the usefulness of CMDB7 in cancer therapy by targeting breast tumors.

Asunto(s)

Neoplasias de la Mama/metabolismo , Carcinoma/metabolismo , Dextranos/farmacocinética , Neoplasias Mamarias Experimentales/metabolismo , Animales , Transporte Biológico , Línea Celular Transformada , Núcleo Celular/metabolismo , Femenino , Genes ras , Humanos , Ratones , Ratones Desnudos , Tecnecio , Distribución Tisular , Células Tumorales Cultivadas

RESUMEN

A capillary electrophoresis assay of sulfoesterase activity was developed that overcomes the main drawbacks encountered with the usual methods for sulfate determination in complex biological medium. Conditions are described allowing direct measurement of inorganic sulfate that is enzymatically produced in the reaction mixture. The main features of this method are electrokinetic sample introduction, which allows selective extraction of sulfate from the matrix into the separation capillary, counter-electroosmotic flow migration mode, indirect absorbance detection and use of an internal standard for quantitative performances. Likewise, perfect linearity was obtained for concentrations of sulfate up to 40 ppm. The limits of detection and quantification were 0.2 and 0.6 ppm, respectively. The run-to-run and day-to-day precision are 1 and 4.5%, respectively, for sulfate concentrations varying from 35 ppm down to 1 ppm. The accuracy was established for the synthetic p-nitrocatechol sulfate substrate by comparison with the classical spectrophotometric assay. The method was applied to the kinetic monitoring of the activity of a sulfoesterase extracted from the marine mollusc Pecten maximus on fucoidan, a bioactive sulfated fucose-based polysaccharide derived from brown algae. For the first time, a sulfoesterase activity was shown to be effective on such sulfated polysaccharides.

Asunto(s)

Electroforesis Capilar/métodos , Sulfatos/análisis , Sulfotransferasas/metabolismo , Animales , Electrólitos , Moluscos/enzimología , Polisacáridos/metabolismo , Reproducibilidad de los Resultados , Especificidad por Sustrato

RESUMEN

Some liposomal formulations are now in clinical use. New applications in biology and medicine using targeted liposomes remain an intensive research area. In this context, liposomes constituted of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cholesterol (70/10/20 mol %) were prepared by detergent dialysis and coated with dextran (Dx) or functionalized dextran (FDx), both hydrophobized by a cholesterol anchor which penetrates the lipid bilayer during the vesicle formation. The coating of liposomes with these polysaccharides was performed because chemically modified dextran but not native Dx interacted with vascular cells. The liposome uptake by human endothelial cells was followed using uncoated and coated liposomes radiolabeled with a neutral lipid (3H-cholesterol) and a polar phospholipid (14C-PC). The results indicated for both radiolabels a preferential uptake by endothelial cells of FDx-coated liposomes compared to uncoated or Dx-coated liposomes. Addition to the culture medium of calcium up to 10 mM further enhanced the level and rate of incorporation of FDx-coated liposomes, whereas interaction of endothelial cells with uncoated liposomes or liposomes coated with Dx was poorly affected. Liposome membranes were then labeled with N-(lissamine rhodamine B sulfonyl)diacyl-PE and liposome uptake by endothelial cells was observed by fluorescence microscopy. The punctate intracellular fluorescence of cells incubated at 37 degrees C with fluorolabeled liposomes is indicative of the liposome localization within the endocytotic pathway of the cells. Altogether, these data demonstrate that coating of liposomes with FDx enable specific interactions with human endothelial cells in culture. Consequently, these liposomes coated with bioactive polymers represent an attractive approach as materials for use as drug delivery vehicles targeting vascular cells.

Asunto(s)

Materiales Biocompatibles , Dextranos , Endotelio Vascular/metabolismo , Liposomas , Materiales Biocompatibles/química , Calcio/farmacología , Línea Celular , Dextranos/química , Portadores de Fármacos , Estabilidad de Medicamentos , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Colorantes Fluorescentes , Humanos , Ensayo de Materiales , Propiedades de Superficie

RESUMEN

The functionalized dextrans termed carboxymethyl benzylamide sulfonate dextran (CMDBS) represent a family encompassing a wide range of polymers. These soluble macromolecular compounds, which are substituted with specific chemical functional groups, are designed to interact with living systems. By analogy with glycosaminoglycan heparin, a natural highly charged anionic polysaccharide that exerts a variety of biological effects, we postulated that CMDBS compounds also possess binding sites capable of specific interactions with biological constituents, depending on the overall composition of the polymer. The synthesis and heparin-like properties of these CMDBS have been extensively investigated. Thus, it appears that dextran derivatives can mimic the action of heparin in regard to its interactions with antithrombin and serine proteases involved in blood coagulation. Other derivatives interact with various components of the immune system or with adhesive proteins such as fibronectin in modulating the proliferation of Staphylococcus aureus. Because they are able to stimulate wound healing in various in vivo models, these polysaccharides may also constitute a family of tissue repair agents because of their protecting and potentiating effects with heparin binding growth factors. Moreover, dextran derivatives in contact with cells such as endothelial cells, smooth muscle cells, or tumoral cells can affect both cell proliferation and metabolism. It appears that these bioactive polymers are also efficient tools to investigate the precise mechanism of action of individual biological activities by contrasting their mode of action to that of heparin. In addition to their numerous biological properties and biospecificity, functionalized dextrans are relatively simple to manufacture and exempt of donor contaminant, which make them attractive in a variety of clinical applications.

Asunto(s)

Materiales Biocompatibles/química , Dextranos/química , Animales , Antiinfecciosos/química , Antiinfecciosos/farmacología , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Anticoagulantes/química , Anticoagulantes/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Antivirales/química , Antivirales/farmacología , Materiales Biocompatibles/farmacología , Secuencia de Carbohidratos , División Celular/efectos de los fármacos , Dextranos/farmacología , Fibrinolíticos/química , Fibrinolíticos/farmacología , Humanos , Técnicas In Vitro , Ensayo de Materiales , Datos de Secuencia Molecular , Estructura Molecular

RESUMEN

CMDB (carboxymethyldextran-benzylamide) are dextrans statistically substituted with carboxymethyl and benzylamide groups which can mimick some of the biological properties of heparin. It has previously been shown that CMDB inhibit autocrine growth of breast tumor cells (Bagheri-Yarmand et al., Biochem. Biophys. Res. Commun. 239: 424-428, 1997) and selectively displace fibroblast growth factor 2 (FGF-2) from its receptor. Here, we used circular dichroism and fluorescence anisotropy measurements to show that the conformation of FGF-2 was significantly altered upon its binding to CMDB and to short CMDB fragments prepared within this study. CMDB and fragments formed a stable 1:1 complex with FGF-2, with affinities being estimated as 20+/-10 nM from fluorescence anisotropy analysis. No such a complex was formed with insulin-like growth factor (IGF-1) or epidermal growth factor (EGF). CMDB competed with the FGF-2 receptor for binding to FGF-2 but did not disturb the binding of IGF-1 and EGF to their receptors. Thus, our results highlight the selectivity of CMDB and their fragments towards FGF-2. Heparin, however, competes with CMDB and their fragments for binding to FGF-2. The carboxymethyl and benzylamide groups of these molecules likely interact directly with a heparin-binding region of FGF-2. The resulting change in conformation disturbs the binding of FGF-2 to its receptor and consecutively its mitogenic activity.

Asunto(s)

Dextranos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Unión Competitiva , Células Cultivadas , Dicroismo Circular , Reactivos de Enlaces Cruzados , Dextranos/química , Dextranos/farmacología , Factor 2 de Crecimiento de Fibroblastos/química , Polarización de Fluorescencia , Humanos , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Estereoisomerismo

RESUMEN

We previously showed that carboxymethyl benzylamide dextran (CMDB7) prevents tumor growth and tumor angiogenesis by binding to angiogenic growth factors, thereby preventing them from reaching their receptors on tumor or stromal cells (Bagheri-Yarmand et al. Br. J. Cancer, 78: 111-118, 1998; Bagheri-Yarmand et al. Cell Growth Differ., 9: 497-504, 1998). In this study, CMDB7 inhibited neovessel formation within the fibroblast growth factor 2-enriched matrigel in mice, and its anticancer effect was then tested in a metastatic breast cancer model. Human MDA-MB435 cells were injected into the mammary fat pad of nude mice, and breast tumors developed within 1 week; all of the mice had lung metastases at 12 weeks. CMDB7 treatment (50, 150, or 300 s.c. or 300 i.v. mg/kg/week for 10 weeks) reduced the incidence of lung metastases to 12%. Histological analysis showed markedly less tumor neovascularization in the CMDB7-treated mice. Pulmonary metastasis incidence was strongly dependent on the intratumoral neoangiogenesis in primary tumors.

Asunto(s)

Anticarcinógenos/uso terapéutico , Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/prevención & control , Dextranos/uso terapéutico , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Neovascularización Patológica/prevención & control , Tejido Adiposo , Animales , Colágeno , Modelos Animales de Enfermedad , Combinación de Medicamentos , Femenino , Factor 2 de Crecimiento de Fibroblastos/farmacología , Humanos , Laminina , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Proteoglicanos , Receptores de Estrógenos/metabolismo , Trasplante Heterólogo , Células Tumorales Cultivadas

RESUMEN

Insulin was covalently attached to two terpolymers of N-(2-hydroxypropyl) methacrylamide, N-methacryloyldiglycine and a) R-(-)-1-methyl-2-methacryloylamidoethyl 2-acetamido-2-deoxy-beta-D-glucopyranoside or b) S-(+)-1-methyl-2-methacryloylamidoethyl 2-acetamido-2-deoxy-beta-D-glucopyranoside. The mitogenic effect of both conjugates on vascular smooth muscle cell proliferation was investigated. The results indicated that insulin bound to both carriers with pendant N-acetylglucosaminyl groups possesses hypoglycemic activity but not the mitogenic effect of native insulin. This study shows that for these insulin conjugates, the effect does not depend on the steric configuration of the sugar-containing monomer units incorporated in the terpolymer. A hypothesis is developed that some competition is taking place between N-acetylglucosaminyl groups on the polymeric insulin carrier and the same moieties in the insulin receptor expressed on the surface of smooth muscle cells leading to a lack of mitogenic activity.

Asunto(s)

Insulina/administración & dosificación , Mitógenos/farmacología , Músculo Liso Vascular/efectos de los fármacos , Animales , Aorta/efectos de los fármacos , División Celular/efectos de los fármacos , Hipoglucemiantes/farmacología , Insulina/farmacología , Masculino , Músculo Liso Vascular/citología , Polímeros/administración & dosificación , Ratas , Ratas Sprague-Dawley , Ratas Wistar

RESUMEN

The highly tumorigenic human breast cancer MCF-7ras line (Ha-ras-transfected MCF-7 cell line) loses estrogen dependence and secretes diffusible growth factors that support its own tumor growth in vivo. Our previous studies showed that carboxymethyl benzylamide dextran (CMDB7) inhibits the growth of breast MCF-7 and MCF-7ras cell lines. In this study, we have shown that conditioned medium (CM) from MCF-7 and MCF-7ras cells stimulated the DNA synthesis of BALB/c3T3 fibroblasts and that CMDB7 strongly inhibited these mitogenic effects in a dose-dependent manner. Neutralizing antibodies against platelet-derived growth factor (PDGF) partially inhibited the mitogenic effect of MCF-7ras CM. The flow cytometry analysis of the cell cycle showed that the CM of tumor cells increased the percentage of fibroblasts in S phase and that CMDB7 blocked them in G0/G1 phase. CMDB7 inhibited the mitogenic effect of PDGF-BB and transforming growth factor (TGF) beta1 but not those of epidermal growth factors and insulin-like growth factor on BALB/c3T3 fibroblasts. CMDB7 increased the electrophoretic mobility of radiolabeled PDGF-BB and TGF-beta1, apparently by forming a stable complex with these factors. On intact BALB/c3T3 fibroblasts, binding of iodinated growth factors (125I-TGF-beta1 and 125I-PDGF) to their receptors was completely displaced by CMDB7. In vivo studies demonstrated that s.c. injection of CMDB7 inhibited by 66% the tumor growth of MCF-7ras xenografts in nude mice. These results showed that CMDB7 inhibits the mitogenic effect of growth factors released from MCF-7 and MCF-7ras cells and suppresses tumor growth in the MCF-7ras model.

Asunto(s)

Anticoagulantes/metabolismo , Neoplasias de la Mama/metabolismo , Dextranos/farmacología , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Células 3T3/efectos de los fármacos , Animales , Anticuerpos/farmacología , Becaplermina , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Relación Dosis-Respuesta a Droga , Factor de Crecimiento Epidérmico/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Femenino , Humanos , Factor I del Crecimiento Similar a la Insulina/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/farmacología , Neoplasias Mamarias Experimentales/prevención & control , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Factor de Crecimiento Derivado de Plaquetas/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/inmunología , Unión Proteica , Proteínas Proto-Oncogénicas c-sis , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta/efectos de los fármacos , Células Tumorales Cultivadas

RESUMEN

Our previous studies showed that carboxymethyl benzylamide dextran (CMDB7) blocks basic fibroblast growth factor (FGF-2)-dependent cell proliferation of a human breast epithelial line (HBL100), suggesting its potential role as a potent antiangiogenic substance. The derived cell line (HH9), which was transformed with the hst/FGF4 gene, has been shown to be highly proliferative in vitro and to induce angiogenic tumours in nude mice. We show here that CMDB7 inhibits the mitogenic activities of the conditioned media from HBL 100 and HH9 cells in a dose-dependent manner. When HH9 cells were injected s.c. into nude mice, CMDB7 treatment (300 mg kg(-1) week(-1)) suppressed the tumour take and the tumour growth by about 50% and 80% respectively. Immunohistochemical analysis showed a highly significant decrease, by more than threefold, in the endothelial density of viable tumour regions, together with a significant increase in the necrosis area. This antiangiogenic activity of CMDB7 was further demonstrated by direct inhibition of calf pulmonary artery (CPAE) and human umbilical vein (HUVEC) endothelial cell proliferation and migration in vitro. In addition, we showed that CMDB7 inhibits specifically the mitogenic effects of the growth factors that bind to heparin such as FGF-2, FGF-4, platelet-derived growth factor (PDGF-BB) and transforming growth factor (TGF-beta1), but not those of epidermal growth factor (EGF) and insulin-like growth factor (IGF-1). These results demonstrate that CMDB7 inhibits FGF-2/FGF-4-dependent tumour growth and angiogenesis, most likely by disrupting the autocrine and paracrine effects of growth factors released from the tumour cells.

Asunto(s)

Dextranos/farmacología , Factor 2 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Neovascularización Patológica/prevención & control , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Células 3T3 , Animales , Neoplasias de la Mama/patología , División Celular/efectos de los fármacos , Línea Celular Transformada/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Femenino , Factor 4 de Crecimiento de Fibroblastos , Fibroblastos/efectos de los fármacos , Humanos , Ratones , Ratones Desnudos , Neovascularización Patológica/inducido químicamente , Trasplante Heterólogo , Células Tumorales Cultivadas/efectos de los fármacos

RESUMEN

Porous silica exhibits excellent mechanical properties for use as a stationary phase for high-performance liquid chromatography. However, negative surface charges make it unusable in its native state. For this reason, silica beads are coated with dextran polymers carrying a calculated amount of diethylaminoethyl groups. Both the minimization of non-specific interactions and the hydrophilic character of such supports allow their functionalization with biospecific ligands and finally their use in high-performance affinity chromatography of biological products. The use of these modified supports in high-performance affinity chromatography requires a better understanding of various characteristics of stationary phases. For this purpose, several techniques were utilized, in particular, size-exclusion chromatography and adsorption of radiolabelled albumin. These methods provided complementary information on the structure of these supports. Coated silica-based supports were functionalized with sialic acid by means of different coupling agents. The affinity of these supports for insulin was determined by the establishment of adsorption isotherms and by high-performance affinity chromatography, to evidence the relationships between structural characteristics of the supports and their separation properties. The study of interactions between these supports and insulin allowed us to show the importance of the coupling method on the performances of supports in affinity chromatography.

Asunto(s)

Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión/métodos , Insulina/química , Ácido N-Acetilneuramínico , Adsorción , Cromatografía en Gel , Humanos , Radioisótopos de Yodo , Marcaje Isotópico , Microesferas , Albúmina Sérica/química , Dióxido de Silicio

RESUMEN

Heparin and heparan sulfates are regulators of cellular events including adhesion, proliferation and migration. In particular, the antiproliferative effect of heparin on smooth muscle cell (SMC) growth is well described. However, its mechanism of action remains unclear. Numerous results suggest an endocytosis mediated by a still unknown heparin receptor on vascular SMCs. In order to identify a putative heparin receptor on SMCs that could be involved in heparin signalling, affinity chromatography supports were developed. In this paper, we describe high-performance liquid affinity chromatography (HPLAC) supports obtained from silica beads coated with dextran polymer substituted by a calculated amount of diethylaminoethyl functions. With a polysaccharide dextran layer, this type of support can be grafted with specific ligands, such as heparin, using conventional coupling methods. In a previous work, we demonstrated, using butanedioldiglycidyl ether, that silica stationary phases coupled to heparin could be used for the fast elution and good peak resolution of heparin-binding proteins. In the present work, an affinity chromatographic fraction of SMC membrane extracts was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and six heparin-binding proteins from dodecyloctaethyleneglycol monoether-solubilized SMCs were observed. Their Mr values were between 40 and 70 kDa, with three major protein bands at 66, 45 and 41 kDa. These results indicate the usefulness of the chromatographic method for purifying heparin binding proteins from SMC membrane.

Asunto(s)

Membrana Celular/química , Cromatografía de Afinidad/métodos , Cromatografía Líquida de Alta Presión/métodos , Músculo Liso Vascular/química , Receptores de Superficie Celular/aislamiento & purificación , Animales , Aorta , Células Cultivadas , Reactivos de Enlaces Cruzados , Detergentes , Dextranos , Heparina/farmacología , Microesferas , Ratas , Ratas Sprague-Dawley , Dióxido de Silicio , Solubilidad

RESUMEN

Glycosaminoglycans (GAGs) such as heparan sulfates are complex carbohydrate polymers. These structural components of the extracellular matrix are essential for the adhesion, migration, and regulation of cellular growth. To understand the physiological role of GAGs and GAG analogues, a practical approach consists of labeling and detecting them in cell extracts, or analyzing binding domains and their distributions into the cells. We propose a convenient and reliable method for preparing and labeling amino-enriched, polysaccharides with the fluorescent derivative 5-[(4,6-dichlorotriazine-2-yl)amino]-fluorescein (DTAF). Radioiodination is then performed on the DTAF moiety. This method was applied to polysaccharides known to inhibit vascular smooth-muscle cell (SMC) proliferation such as functionalized dextrans derived from poly(alpha 1-6 glucose) and fucan, poly(L-fucose 4-sulfate) extracted from brown seaweed. Using autoradiography and confocal microscopy, we observed the fixation and internalization of labeled antiproliferative products in SMCs from rat aorta. These probes can be useful for the understanding of polysaccharide-cell interactions. In addition, the method presented here can be applied to various synthetic or natural biomedical materials.

Asunto(s)

Endocitosis , Músculo Liso Vascular/metabolismo , Polisacáridos/metabolismo , Aminoácidos , Animales , Autorradiografía , División Celular , Fluoresceínas , Colorantes Fluorescentes , Radioisótopos de Yodo , Microscopía Confocal , Músculo Liso Vascular/citología , Ratas , Ratas Sprague-Dawley

RESUMEN

The biochemical and pharmacological properties of bioactive peptides and proteins can be altered by conjugation with polymers. This report describes site-specific attachment of insulin to activated carboxyl groups of carboxymethyl dextran (CMD, MW=51000) through the GlyA1 insulin amino group. On average, three or four insulin molecules were grafted to a CMD linear chain. Coupled insulin molecules were properly folded, and the bioactivity of conjugated insulin in the blood glucose depression assay was 9.6 IU/mg, which was only 2.6 times less than that for native insulin. The cell growth study indicated that the CMD-insulin conjugate was as mitogenic as insulin on vascular smooth muscle cells, whereas the starting CMD polymer was not. The insulin receptor binding constant of the conjugate (3.6 x 10[9] M[-1]) compared well with that of native insulin (7.6 x 10[9] M[-1]), indicating that the CMD chain does not present any major constraints to binding. Plasma clearance of CMD-insulin obeyed a two-compartment pharmacokinetic (PK) model with a CMD-insulin conjugate plasma elimination half-life of 114.1 min, which was significantly longer than that of soluble Zn-insulin (12.4 min). In contrast, pharmacodynamic (PD) profiles (blood glucose lowering effects) after intravenous (iv) administration of the conjugate or insulin in rats were not different. Subcutaneous (sc) administration of the conjugate resulted in a significantly prolonged plasma profile with a noncompartmental PK parameter mean residence time (MRT) of 103.5 min which was significantly longer than that of soluble Zn-insulin (40.5 min). This was reflected in the protracted PD effect of sc administered conjugate with time needed to reach minimum glucose concentration Tnadir of 95.7 min, which was significantly longer than that of insulin (62 min). We conclude that the conjugation of insulin to CMD leads to a bioactive conjugate with a delayed sc PD profile showing prolonged response, resembling intermediate acting insulin preparations.

Asunto(s)

Dextranos/química , Insulina/química , Insulina/farmacología , Animales , Aorta Torácica , Glucemia/metabolismo , División Celular/efectos de los fármacos , Células Cultivadas , Dextranos/metabolismo , Semivida , Insulina/metabolismo , Tasa de Depuración Metabólica , Músculo Liso Vascular/citología , Pliegue de Proteína , Ratas , Ratas Sprague-Dawley , Receptor de Insulina/metabolismo , Espectrofotometría Ultravioleta , Relación Estructura-Actividad

RESUMEN

Silica-based packing materials induce non-specific interactions with proteins in aqueous media because of the nature of their surface, mainly silanol groups. Therefore, the silica surface has to be modified in order to be used as stationary phase for the High Performance Size-Exclusion Chromatography (HPSEC) of proteins. For this purpose, porous silica beads were coated with hydrophilic polymer gels (dextrans of different molecular weights) carrying a calculated amount of diethyl-aminoethyl groups (DEAE). Actually, as shown by HPSEC, these dextran modified supports minimize non-specific adsorption for proteins and pullulans in aqueous solution. Then, in order to change the pore size in response to temperature, temperature responsive polymer of poly(N-isopropylacrylamide) (PIPAAm) was introduced into the surface of dextran-DEAE on porous silica beads. The structure of these supports before and after modification was alternately studied by Scanning Electronic Microscopy (SEM) and Scanning Force Microscopy (SFM). An adsorption of radiolabelled albumin was performed to complete our study. Silica modifications by dextran-DEAE and PIPAAm improve the neutrality of the support and minimize the non-specific interactions between the solid support and proteins in solution. At low temperature, the support having PIPAAm exhibits a high resolution domain in HPSEC and finally permits a better resolution of proteins and pullulans. At higher temperature, hydrophobic properties of PIPAAm produce interactions with some proteins and trigger off a slight delay of their elution time.

Asunto(s)

Resinas Acrílicas , Cromatografía en Gel/instrumentación , Cromatografía en Gel/métodos , Dióxido de Silicio , Temperatura , Resinas Acrílicas/síntesis química , Resinas Acrílicas/química , Adsorción , Humanos , Microscopía de Fuerza Atómica , Microscopía Electrónica de Rastreo , Concentración Osmolar , Albúmina Sérica Radioyodada/metabolismo , Dióxido de Silicio/síntesis química , Dióxido de Silicio/química , Propiedades de Superficie

RESUMEN

Modified polystyrene resins containing sulphonate groups and tyrosyl sulphamide or tyrosyl methyl ester sulphamide groups have been investigated with respect to their potential for selective binding of anti-Factor VIII inhibitory antibodies from plasma. Adsorption of total immunoglobulin G and of a monoclonal antibody to Factor VIII was measured following addition of the radioiodinated proteins to normal plasma, plasma depleted of Factor VIII by adsorption on a resin coupled to anti-Factor VIII antibody, and haemophiliac plasma containing various levels of inhibitory anti-Factor VIII antibody. Depletion of anti-Factor VIII antibody from the haemophiliac plasmas by incubation with the resins was also measured by Bethesda assay. The modified resins and their corresponding unmodified "controls' showed similar binding of total immunoglobulin G. However, only resins containing either sulphonate or a combination of sulphonate and tyrosyl sulphamide groups showed evidence of selective adsorption of anti-Factor VIII antibody from plasma.

Asunto(s)

Anticuerpos/sangre , Anticuerpos/química , Factor VIII/antagonistas & inhibidores , Factor VIII/inmunología , Hemofilia A/sangre , Inmunoglobulina G/química , Poliestirenos/química , Adsorción , Factor VIII/análisis , Hemofilia A/inmunología , Humanos , Inmunoglobulina G/sangre , Radioisótopos de Yodo