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1.
Br J Cancer ; 105(11): 1697-707, 2011 Nov 22.
Article in English | MEDLINE | ID: mdl-22027709

ABSTRACT

BACKGROUND: ANG1005 consists of three molecules of paclitaxel conjugated via ester bonds to the 19-amino-acid peptide Angiopep-2. The new chemical agent has been shown to cross the blood-brain barrier (BBB) by receptor-mediated transcytosis via low-density lipoprotein receptor-related protein 1 (LRP1). The experiments here examined the role of LRP1 in the subsequent endocytosis of drug into cancer cells. METHODS: Localisation of ANG1005 and Angiopep-2 was examined by immunohistochemistry and in-vivo near-infrared fluorescence imaging in mice carrying orthotopic glioma tumours. Transport of ANG1005 and Angiopep-2 was examined in U87 glioblastoma cell lines. RESULTS: Systemically administered ANG1005 and Cy5.5Angiopep-2 localised to orthotopic glioma tumours in mice. The glioma transplants correlated with high expression levels of LRP1. Decreasing LRP1 activity, by RNA silencing or LRP1 competitors, decreased uptake of ANG1005 and Angiopep-2 into U87 glioblastoma cells. Conversely, LRP1 expression and endocytosis rates for ANG1005 and Angiopep-2 increased in U87 cells under conditions that mimicked the microenvironment near aggressive tumours, that is, hypoxic and acidic conditions. CONCLUSION: ANG1005 might be a particularly effective chemotherapeutic agent for the wide array of known LRP1-expressing brain and non-brain cancers, in particular those with an aggressive phenotype.


Subject(s)
Brain Neoplasms/metabolism , Glioma/metabolism , Paclitaxel/pharmacokinetics , Receptors, LDL/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Biological Transport , Blood-Brain Barrier/metabolism , Brain Neoplasms/drug therapy , Brain Neoplasms/pathology , Cell Line, Tumor , Endocytosis , Glioma/drug therapy , Glioma/pathology , Hep G2 Cells , Humans , Low Density Lipoprotein Receptor-Related Protein-1 , Male , Mice , Mice, Nude , Paclitaxel/pharmacology , Peptides/pharmacokinetics , Peptides/pharmacology , Phenotype , RNA Interference , Receptors, LDL/genetics , Tumor Microenvironment , Tumor Suppressor Proteins/genetics
2.
Br J Pharmacol ; 155(2): 185-97, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18574456

ABSTRACT

BACKGROUND AND PURPOSE: Paclitaxel is highly efficacious in the treatment of breast, head and neck, non-small cell lung cancers and ovarian carcinoma. For malignant gliomas, paclitaxel is prevented from reaching its target by the presence of the efflux pump P-glycoprotein (P-gp) at the blood-brain barrier. We investigated the utilization of a new drug delivery system to increase brain delivery of paclitaxel. EXPERIMENTAL APPROACH: Paclitaxel molecules were conjugated to a brain peptide vector, Angiopep-2, to provide a paclitaxel-Angiopep-2 conjugate named ANG1005. We determined the brain uptake capacity, intracellular effects and antitumour properties of ANG1005 in vitro against human tumour cell lines and in vivo in human xenografts. We then determined ANG1005 activity on brain tumours with intracerebral human tumour models in nude mice. KEY RESULTS: We show by in situ brain perfusion that ANG1005 enters the brain to a greater extent than paclitaxel and bypasses the P-gp. ANG1005 has an antineoplastic potency similar to that of paclitaxel against human cancer cell lines. We also demonstrate that ANG1005 caused a more potent inhibition of human tumour xenografts than paclitaxel. Finally, ANG1005 administration led to a significant increase in the survival of mice with intracerebral implantation of U87 MG glioblastoma cells or NCI-H460 lung carcinoma cells. CONCLUSIONS AND IMPLICATIONS: These results demonstrate the antitumour potential of a new drug, ANG1005, and establish that conjugation of anticancer agents with the Angiopep-2 peptide vector could increase their efficacy in the treatment of brain cancer.


Subject(s)
Antineoplastic Agents, Phytogenic/administration & dosage , Brain Neoplasms/drug therapy , Drug Carriers/metabolism , Drug Delivery Systems , Paclitaxel/administration & dosage , Animals , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/therapeutic use , Brain Neoplasms/prevention & control , Disease Models, Animal , Female , Humans , Male , Mice , Mice, Nude , Neoplasm Transplantation , Paclitaxel/chemistry , Paclitaxel/therapeutic use , Peptides/chemistry , Tumor Cells, Cultured/transplantation , Xenograft Model Antitumor Assays
3.
Biochim Biophys Acta ; 1763(10): 1024-30, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16979249

ABSTRACT

Tissue-type plasminogen activator (tPA) and its substrate plasminogen (Plg) are key components in the fibrinolytic system. We have recently demonstrated, that truncated human recombinant soluble melanotransferrin (sMTf) could stimulate the activation of Plg by urokinase plasminogen activator and inhibit angiogenesis. Since various angiogenesis inhibitors were shown to stimulate tPA-mediated plasminogen activation, we examined the effects of sMTf on tPA-dependent fibrinolysis. This study demonstrated that sMTf enhanced tPA-activation of Plg by 6-fold. sMTf also increased the release of [125I]-fibrin fragments by tPA-activated plasmin. Moreover, we observed that the interaction of sMTf with Plg provoked a change in the fibrin clot structure by cleaving the fibrin alpha and beta chains. Overall, the present study shows that sMTf modulates tPA-dependent fibrinolysis by modifying the clot structure. These results also suggest that sMTf properties could involve enhanced dissolution of the provisional extracellular fibrin matrix.


Subject(s)
Extracellular Matrix/metabolism , Fibrin/metabolism , Fibrinolysis , Neoplasm Proteins/pharmacology , Tissue Plasminogen Activator/pharmacology , Antigens, Neoplasm , Cells, Cultured , Clot Retraction , Dose-Response Relationship, Drug , Drug Synergism , Fibrinolysin/metabolism , Humans , Melanoma-Specific Antigens , Plasminogen/metabolism , Recombinant Proteins/pharmacology
5.
Int J Cancer ; 93(1): 62-6, 2001 Jul 01.
Article in English | MEDLINE | ID: mdl-11391622

ABSTRACT

Multidrug resistance (MDR) is associated with the expression of P-glycoprotein (P-gp), an ATP-dependent transporter which expels anti-cancer drugs from cells. In the present study, MDR1 P-gp was immunodetected by Western blot analysis in 60 human brain tumors, including meningiomas, schwannomas, low-grade gliomas (astrocytomas, pilocytic astrocytomas) and high-grade gliomas (anaplastic astrocytomas, glioblastomas and anaplastic oligodendrogliomas). Most samples from primary tumors expressed P-gp at the same levels as normal brain tissue except for schwannomas, in which levels were reduced by 65%, and meningiomas, in which levels were more than 10-fold higher in 7 of 10 samples. P-gp levels were 70% and 95% lower in brain metastases from melanomas and lung adenocarcinomas, respectively, than in normal brain tissue. These results indicate that the majority of primary brain tumors express MDR1 P-gp and that its high expression levels in meningiomas may be a marker for this type of brain tumor.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/analysis , Brain Chemistry , Brain Neoplasms/chemistry , Astrocytoma/chemistry , Brain Neoplasms/secondary , Glioblastoma/chemistry , Humans , Lung Neoplasms/pathology , Melanoma/pathology , Meningioma/genetics , Neurilemmoma/genetics , Oligodendroglioma/chemistry , Reference Values
6.
Biochem Biophys Res Commun ; 281(3): 827-34, 2001 Mar 02.
Article in English | MEDLINE | ID: mdl-11237734

ABSTRACT

Endothelial cells (EC) were isolated from brain, lung, and renal cortex using magnetic microbeads cross-linked to an antibody directed against the platelet-endothelial cell adhesion molecule-1 (PECAM-1). Levels of endothelial nitric oxide synthase (eNOS) and PECAM-1 were measured by Western blots and both were enriched in the positively selected EC fractions. The multidrug resistance P-glycoprotein (P-gp) was strongly enriched (59-fold) in the EC fraction from brain and was absent in the negative fraction, in which the glial fibrillary acidic protein (GFAP), an astrocyte marker, was present. Lower P-gp levels were detected in EC from renal cortex and lung. Reverse transcription-polymerase chain reaction analysis showed that the mdr1a gene was preferentially expressed in EC fraction from the brain. The mdr1b gene was found in EC from renal cortex whereas both mdr1 genes were detected in EC from lung. Our results indicate that EC can be isolated using microbeads and that the isoform of P-gp found in brain is mostly mdr1a, associated with EC.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Brain/metabolism , Endothelium/metabolism , Kidney/metabolism , Lung/metabolism , Protein Isoforms/metabolism , Animals , Brain/cytology , Endothelium/cytology , Immunomagnetic Separation , Kidney/cytology , Lung/cytology , Platelet Endothelial Cell Adhesion Molecule-1/immunology , Rats , Reverse Transcriptase Polymerase Chain Reaction
7.
Cancer Metastasis Rev ; 20(1-2): 13-25, 2001.
Article in English | MEDLINE | ID: mdl-11831641

ABSTRACT

Malignant brain tumors and brain metastases present a formidable clinical challenge against which no significant advances have been made over the last decade. Multidrug resistance (MDR) is one of the main factors in the failure of chemotherapy against central nervous system tumors. The MDR1 gene encoding P-glycoprotein (P-gp), a drug efflux pump which plays a significant role in modulating MDR in a wide variety of human cancers, is highly expressed in the blood-brain barrier (BBB). The BBB controls central nervous system exposure to many endogenous and exogenous substances. The exact molecular mechanisms by which the BBB is involved in the resistance of brain tumors to chemotherapy remain to be identified. The purpose of this review is to summarize reports demonstrating that P-gp, one of the most phenotypically important markers of the BBB, is present in primary brain tumors and thus plays a crucial role in their clinical resistance to chemotherapy.


Subject(s)
Antineoplastic Agents/therapeutic use , Blood-Brain Barrier , Brain Neoplasms/drug therapy , Drug Resistance, Multiple , Drug Resistance, Neoplasm , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Brain Neoplasms/metabolism , Humans , Multidrug Resistance-Associated Proteins/metabolism
8.
Kidney Int ; 57(4): 1590-8, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10760094

ABSTRACT

BACKGROUND: Administration of the immunosuppressive agent cyclosporine A (CsA) is associated with nephrotoxicity. The main target for CsA, cyclophilin A (CypA), was found in high levels in epithelial cells of renal proximal tubules. In the present study, CypA was immunodetected and characterized following CsA treatment in subcellular fractions of renal cortex. METHOD: The renal content and distribution of CypA was evaluated in untreated rats and in rats treated with a subcutaneous injection of CsA (10 mg. kg-1. day-1) for 10 days. RESULTS: In untreated rats, membrane-bound CypA represents 0.25% of total brush border membrane (BBM) proteins, similar to the proportion found in the soluble fraction. High ionic strength treatment was unable to extract CypA from BBMs, whereas alkaline treatment (Na2CO2, pH 11) and detergent 3 - [(3 - cholamidopropyl) - dimethyl - ammonio] - 1 - propanesulfate (CHAPS) released it from BBMs. These results indicate that CypA is associated with renal BBMs, and that hydrophobic interactions are involved in this association. The CypA distribution was strongly modified in both BBMs and the soluble fraction after CsA treatment, but its affinity for CsA estimated by photoaffinity labeling was unaffected. The CypA expression level decreased by 45% in BBMs, while it increased by 33% in the soluble fraction, compared with control rats. CypA remained associated with the membranes following in vitro incubation of renal BBMs with CsA. However, incubation of CypA with one of its substrates released CypA from renal BBMs. CONCLUSIONS: These experiments suggest that renal BBMs contain a significant amount of CypA and chronic exposure to CsA, and acute exposure to one of CypA substrates may modify its subcellular distribution.


Subject(s)
Cyclosporine/pharmacology , Immunosuppressive Agents/pharmacology , Kidney/metabolism , Peptidylprolyl Isomerase/metabolism , Animals , Immunologic Techniques , Kidney Cortex/metabolism , Male , Microvilli/metabolism , Rats , Rats, Sprague-Dawley , Subcellular Fractions/metabolism , Tissue Distribution/drug effects
9.
Biochim Biophys Acta ; 1478(1): 51-60, 2000 Mar 16.
Article in English | MEDLINE | ID: mdl-10719174

ABSTRACT

We have investigated the effects of different biologically active components from natural products, including green tea polyphenols (GTP), resveratrol, genistein and organosulfur compounds from garlic, on matrix metalloproteinase (MMP)-2, MMP-9 and MMP-12 activities. GTP caused the strongest inhibition of the three enzymes, as measured by fluorescence assays using gelatin or elastin as substrates. The inhibition of MMP-2 and MMP-9 caused by GTP was confirmed by gelatin zymography and was observed for MMPs associated with both various rat tissues and human brain tumors (glioblastoma and pituitary tumors). The activities of MMPs were also measured in the presence of various catechins isolated from green tea including (-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate(ECG), (-)-epigallocatechin (EGC), (-)-epicatechin (EC) and (+)-catechin (C). The most potent inhibitors of these activities, as measured by fluorescence and by gelatin or casein zymography, were EGCG and ECG. GTP and the different catechins had no effect on pancreatic elastase, suggesting that the effects of these molecules on MMP activities are specific. Furthermore, in vitro activation of proMMP-2 secreted from the glioblastomas cell line U-87 by the lectin concanavalin A was completely inhibited by GTP and specifically by EGCG. These results indicate that catechins from green tea inhibit MMP activities and proMMP-2 activation.


Subject(s)
Catechin/analogs & derivatives , Matrix Metalloproteinase Inhibitors , Tea , Animals , Catechin/chemistry , Catechin/pharmacology , Cell Line , Concanavalin A , Cricetinae , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Enzyme Precursors/antagonists & inhibitors , Gelatinases/antagonists & inhibitors , Guanosine Triphosphate/pharmacology , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Metalloendopeptidases/antagonists & inhibitors , Mice , Molecular Structure , Swine
10.
FEBS Lett ; 466(2-3): 219-24, 2000 Jan 28.
Article in English | MEDLINE | ID: mdl-10682831

ABSTRACT

A significant proportion of P-glycoprotein (P-gp) and caveolin was co-localized in caveolae isolated from resistant (CH(R)C5) cells overexpressing P-gp and from drug-sensitive Chinese hamster ovary cells (AuxB1). The proportion of P-gp and caveolin associated with caveolar microdomains was higher in CH(R)C5 cells grown in the presence of P-gp substrates (cyclosporin A or colchicine) than in untreated CH(R)C5 cells. Coimmunoprecipitation of P-gp and caveolin from CH(R)C5 lysates suggests that there is a physical interaction between them. Furthermore, co-localization of P-gp and caveolin was found in caveolae from brain capillaries, indicating that this association also takes place in vivo.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Brain/blood supply , Capillaries/metabolism , Amino Acid Sequence , Animals , CHO Cells , Cricetinae , Humans , Membrane Proteins/metabolism , Precipitin Tests , Tumor Cells, Cultured
11.
Am J Physiol ; 277(6): F832-40, 1999 12.
Article in English | MEDLINE | ID: mdl-10600929

ABSTRACT

The expression of two members of the ATP-binding cassette family of transport proteins, P-glycoprotein (P-gp) and the canalicular multispecific organic anion transporter (cMOAT or Mrp2), was evaluated in renal brush-border membranes (BBM) and various rat tissues after cisplatin treatment. One administration of cisplatin (5 mg/kg) increased P-gp expression by >200-300% in renal BBM and in crude membranes from liver and intestine. The increase in P-gp expression in the kidney was also detected in photolabeling experiments, suggesting the induction of functional P-gp. cMOAT expression was increased by >10-fold in renal BBM after cisplatin administration, although it had no effect on liver cMOAT expression. The increase in the levels of both proteins was maximal at 2 days after cisplatin treatment and lasted for at least 8 days. These results indicate that a single administration of cisplatin induces overexpression of P-gp and cMOAT in specific tissues. This may be of significant relevance to the design of clinical trials using cisplatin as a single chemotherapeutic agent or in combination with other drugs.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Carrier Proteins/genetics , Cisplatin/pharmacology , Gene Expression Regulation/drug effects , Kidney/drug effects , Animals , Anion Transport Proteins , Brain/metabolism , Capillaries/metabolism , Cell Membrane/metabolism , Cerebrovascular Circulation , Kidney/metabolism , Liver/metabolism , Lung/metabolism , Male , Microvilli/metabolism , Organ Specificity , Rats , Rats, Sprague-Dawley
12.
Biochem Cell Biol ; 77(1): 47-58, 1999.
Article in English | MEDLINE | ID: mdl-10426286

ABSTRACT

The interaction between P-glycoprotein (P-gp) from membranes isolated from multidrug-resistant Chinese hamster ovary cells and cyclosporin A (CsA) analogues and its metabolites was characterized. Screening of these latter as chemosensitizers was performed using three different assays: (i) vinblastine uptake, (ii) photoaffinity labeling by [125I]iodoaryl azidoprazosin, and (iii) P-gp ATPase activity. Oxidation of the hydroxyl group at position I of CsA (200-096), CsG (215-834), or CsD (PSC-833) increased their inhibition of P-gp. CsA analogues (208-032, 208-183) modified at position 11 retained their ability to inhibit P-gp while analogues modified at position 2 (CsC and CsD) lost their efficiency. The inhibitions induced by metabolites of CsA were also compared to those obtained with CsG metabolites. From all the molecules tested, PSC-833 and 280-446 peptolide were the strongest inhibitors. Our results indicate that modifications of CsA analogues at position 1 and 2 are critical for their interaction with P-gp and that CsA metabolites retain a portion of the inhibitory activity of the parent drug.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , Cyclosporins/pharmacology , Endosomes/metabolism , Vinblastine/pharmacokinetics , Adenosine Triphosphate/metabolism , Animals , CHO Cells , Cell Membrane/drug effects , Cell Membrane/metabolism , Cells, Cultured , Cricetinae , Cyclosporine/chemistry , Cyclosporine/metabolism , Cyclosporine/pharmacology , Dose-Response Relationship, Drug , Models, Chemical , Photoaffinity Labels , Time Factors , Verapamil/pharmacology
13.
FEBS Lett ; 442(2-3): 208-14, 1999 Jan 15.
Article in English | MEDLINE | ID: mdl-9929003

ABSTRACT

The expression of P-glycoprotein (P-gp) and canalicular multispecific organic anion transporter (cMOAT or Mrp2) was evaluated by Western blotting analysis of rat tissues isolated following daily administration (1 mg kg(-1) day(-1)) of dexamethasone over 4 days. Dexamethasone rapidly increased P-gp expression more than 4.5- and 2-fold in liver and lung, respectively, while it was decreased 40% in kidney. cMOAT expression was increased 2-fold in liver and kidney following dexamethasone treatment. The levels of both proteins returned to control values by 6 days after the conclusion of dexamethasone administration. These results indicate that dexamethasone can modulate P-gp and cMOAT expression in specific rat tissues and may have significant relevance for patients treated with dexamethasone as a single agent or in combination therapy with other drugs.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Carrier Proteins/metabolism , Dexamethasone/pharmacology , Gene Expression/drug effects , ATP Binding Cassette Transporter, Subfamily B, Member 1/immunology , Animals , Anion Transport Proteins , Blotting, Western , Carrier Proteins/immunology , Dexamethasone/administration & dosage , Drug Resistance, Multiple , Kidney/drug effects , Kidney/enzymology , Kidney/metabolism , Liver/drug effects , Liver/enzymology , Liver/metabolism , Lung/drug effects , Lung/enzymology , Lung/metabolism , Male , Membranes/enzymology , Molecular Weight , Protein Isoforms/immunology , Protein Isoforms/metabolism , Rats , Rats, Sprague-Dawley
14.
Biochemistry ; 37(51): 18110-8, 1998 Dec 22.
Article in English | MEDLINE | ID: mdl-9922180

ABSTRACT

The binding site of cyclosporin A to P-glycoprotein was characterized by using a multidrug-resistant Chinese hamster ovary cell line. P-glycoprotein photolabeled with diazirine-cyclosporin A analogue was purified by a two-step process involving continuous elution electrophoresis followed by wheat germ agglutinin-agarose precipitation. The cyclosporin A covalently bound to P-glycoprotein and to subsequent proteolytic fragments was detected by Western blot analysis using a monoclonal antibody against cyclosporin A. Proteolytic digestion of purified P-glycoprotein by V8 generated a major fragment of 15 kDa photolabeled by cyclosporin A, while proteolysis of P-glycoprotein photolabeled by [125I]-iodoaryl azidoprazosin generated a major fragment of 7 kDa. Limited proteolysis of cyclosporin A-photolabeled P-glycoprotein with trypsin indicated that the major binding site for cyclosporin A was in the C-terminal half of the protein. This cyclosporin A binding site was further characterized with chemical agents (N-chlorosuccinimide, cyanogen bromide, and 2-nitro-5-thiocyanobenzoate). These three chemical agents established a proteolytic profile of P-glycoprotein for fragments photolabeled with cyclosporin A and for fragments that contained the C494 and C219 epitopes. The smallest fragments generated by these chemical agents include the transmembrane domains (TMs) 10, 11, and 12 of P-glycoprotein. When the fragments generated by these chemical agents are aligned, the region that binds cyclosporin A is reduced to the 953-1007 residues. These combined results suggest that the major binding site of cyclosporin A occurs between the end of TM 11 and the end of TM 12.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Cyclosporine/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/isolation & purification , Animals , Azirines/metabolism , Binding Sites , CHO Cells , Cricetinae , Cyclosporins/metabolism , Electrophoresis, Polyacrylamide Gel , Membrane Proteins/isolation & purification , Membrane Proteins/metabolism , Peptide Fragments/metabolism , Photoaffinity Labels/metabolism , Serine Endopeptidases/metabolism , Succinimides/metabolism , Sulfhydryl Reagents/metabolism , Thiocyanates/metabolism , Ultraviolet Rays
15.
Biochem J ; 326 ( Pt 2): 539-44, 1997 Sep 01.
Article in English | MEDLINE | ID: mdl-9291129

ABSTRACT

Luminal membranes of the vascular endothelium were isolated from brain, heart and lungs by modification of their density. The presence of P-glycoprotein (P-gp) was detected by Western blotting in luminal membranes from the endothelium of the three tissues. Strong enrichment in brain capillary luminal membranes, compared with brain capillaries (17-fold) and whole membranes (400-500-fold), indicates that P-gp is mainly located on the luminal side of the brain endothelium. Western blotting was also performed with antibodies directed against GLUT1, glial fibrillary acidic protein, adaptin, IP3R-3, integrins alphav and collagen IV as controls to determine whether the preparations were contaminated by other membranes. Strong enrichment of GLUT1 in brain capillary luminal membranes (9.9-fold) showed that the preparation consisted mainly of endothelial cell plasma membranes. Poor enrichment of glial fibrillary acidic protein (1.4-fold) and adaptin (2.4-fold) and a decreased level of IP3R-3, integrins alphav and collagen IV excludes the possibility of major contamination by astrocytes or internal and anti-luminal membranes. High levels of P-gp in the luminal membranes of brain capillary endothelial cells suggests that it may play an important role in limiting the access of anti-cancer drugs to the brain.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis , Brain/blood supply , Endothelium, Vascular/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/chemistry , Affinity Labels , Animals , Azides/metabolism , Blotting, Western , Brain/metabolism , Capillaries/chemistry , Capillaries/metabolism , Cell Membrane/chemistry , Cell Membrane/metabolism , Cerebral Cortex/blood supply , Endothelium, Vascular/chemistry , Glycosylation , Iodine Radioisotopes , Male , Prazosin/analogs & derivatives , Prazosin/metabolism , Rats , Rats, Sprague-Dawley
16.
J Biol Chem ; 272(10): 6647-52, 1997 Mar 07.
Article in English | MEDLINE | ID: mdl-9045695

ABSTRACT

The interaction between P-glycoprotein (140-180 kDa) from the multidrug-resistant Chinese hamster ovary cell line CHRC5 and cyclosporin A was characterized using three different photoactivable cyclosporin A analogs. Two monoclonal antibodies, which are able to discriminate between two major domains of cyclosporin A (the cyclophilin and calcineurin binding domains), were used to detect the photolabeled proteins. A protein of 155 kDa corresponding to P-glycoprotein was much more strongly photolabeled in membranes of CHRC5 cells than in membranes of their drug-sensitive parent cell line AuxB1. The antitumor drug vinblastine and the reversal agents verapamil and cyclosporin A inhibited the photolabeling, and the nonimmunosuppressive derivative PSC-833 caused a stronger inhibition than cyclosporin A. P-glycoprotein photolabeled with cyclosporin A analogs was only detected with the monoclonal antibody that recognizes cyclosporin A and its metabolites, indicating that the calcineurin binding domain recognized specifically by the other antibody is not exposed. These results suggest that the portion of cyclosporin A that binds to calcineurin plays a role in the interaction of cyclosporin A with P-glycoprotein.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/chemistry , Cyclosporine/chemistry , Animals , Antibodies, Monoclonal , Azirines/chemistry , Binding Sites , Binding, Competitive , Blotting, Western , CHO Cells , Cricetinae , Photochemistry
17.
Anal Biochem ; 230(2): 239-47, 1995 Sep 20.
Article in English | MEDLINE | ID: mdl-7503413

ABSTRACT

P-Glycoprotein is an integral membrane protein which mediates the energy-dependent efflux of various antitumor agents from multidrug-resistant cancer cells. Surface plasmon resonance was used for the detection of P-glycoprotein after solubilization from drug-resistant and drug-sensitive Chinese hamster ovary cells and for the analysis of its interaction with cyclosporin A, a competitive inhibitor of drug efflux. Detection of P-glycoprotein relied on its binding to the monoclonal antibody C219 which was immobilized on a sensor chip. Binding of Zwittergent 3-14-solubilized P-glycoprotein to the antibody was concentration-dependent and reflected the relative abundance of P-glycoprotein in both cell lines. It was abolished when C219 was omitted or replaced by a rabbit anti-mouse IgG antibody and considerably reduced after precipitation of P-glycoprotein with wheat germ agglutinin. Preincubation of solubilized proteins with cyclosporin A increased the amount of protein bound to the antibody by approximately 30%. These results indicate that surface plasmon resonance is well suited to the detection of P-glycoprotein from biological samples and shows promise as a tool for the study of its interaction with different drugs.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/analysis , Biosensing Techniques , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Animals , Antibodies, Monoclonal/immunology , CHO Cells , Cricetinae , Cyclosporine/metabolism , Drug Resistance, Multiple , Kinetics , Mice , Rabbits , Solubility
18.
Biochim Biophys Acta ; 1233(1): 27-32, 1995 Jan 26.
Article in English | MEDLINE | ID: mdl-7833346

ABSTRACT

P-glycoprotein (P-gp), an active efflux pump of antitumor drugs, is strongly expressed in endothelial cells of the blood brain barrier (BBB). Two proteins (155 and 190 kDa) were detected by Western blot analysis of beef and rat capillaries with the monoclonal antibody (MAb) C219. In order to characterize the nature of these proteins, their profile of solubilization by different detergents was established and compared with that of P-gp from the CHRC5 tumoral cell line. The 155 kDa protein (p155) of capillaries and the P-gp of CHRC5 cells were well solubilized by deoxycholate and Elugent, whereas the 190 kDa kDa protein (p190) was only solubilized by sodium dodecylsulfate (SDS). Both proteins have different patterns of extraction by Triton X-114, p155 partitioning as a membrane protein, while p190 was insoluble. Deglycosylation of capillary proteins resulted in a 27-28 kDa decrease in the apparent molecular weight of p155, similar to that observed for the P-gp of CHRC5 cells, but a decrease of only 7-8 for p190. Only p155 was immunoprecipitated by MAb C219. These results suggest that only p155 is the P-gp in BBB and that MAb C219 cross-reacts with a 190 kDa MDR-unrelated glycosylated protein. Consequently, the use of this antibody, which is frequently used to detect P-gp in tumors, could be a pitfall of immunohistochemistry screening for cancer tissues and lead to false positive in the diagnosis of MDR.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/analysis , Blood-Brain Barrier , Brain Chemistry , Endothelium, Vascular/chemistry , Membrane Proteins/analysis , ATP Binding Cassette Transporter, Subfamily B, Member 1/immunology , Amino Acid Sequence , Animals , Brain/blood supply , Capillaries/chemistry , Cattle , Cricetinae , Cricetulus , Drug Resistance, Multiple , Molecular Sequence Data , Precipitin Tests , Rats , Tumor Cells, Cultured
19.
Biochem Cell Biol ; 72(3-4): 143-51, 1994.
Article in English | MEDLINE | ID: mdl-7818848

ABSTRACT

Phosphorylation, protein carboxyl methylation, and ADP-ribosylation were assayed in renal basolateral membranes and brush border membranes isolated from rats treated by subcutaneous administration of 5 or 10 mg/(kg.day) of cyclosporin A (CsA) for 10 days to investigate potential alterations in signal transduction in kidney cortex. Protein carboxyl methylation of class II measured in membranes and in cytosolic fraction was not affected by CsA treatment. ADP-ribosylation performed in the presence of pertussis or cholera toxin was also similar in control and treated rats. However, changes in phosphorylation of endogenous substrates were observed in membranes and cytosol isolated from rats treated with 10 mg/(kg.day) of CsA. Phosphorylation was increased for two brush border membrane proteins (56 and 77 kilodaltons (kDa)) by 47 and 24% and for two basolateral membrane proteins (51 and 80 kDa) by 28 and 29%, respectively. In the cytosolic fraction, phosphorylation of two proteins (31 and 65 kDa) was increased by 37% and that of 25- and 43-kDa proteins was reduced by 29%. Protein kinase A, protein kinase C, and tyrosine protein kinase activities were also determined in membranes. Increases in protein kinase C and tyrosine protein kinase activities were observed in basolateral membranes, but not in brush border membranes after cyclosporin A administration. Endogenous substrates for tyrosine kinase were also detected with an antiphosphotyrosine (PY20) monoclonal antibody. Densitometric analysis indicated that the phosphorylation of three proteins of high molecular masses (61, 132, and 183 kDa) was stimulated by CsA in basolateral membranes.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Cyclosporine/pharmacology , Kidney/metabolism , Phosphoproteins/metabolism , Adenosine Diphosphate Ribose/metabolism , Animals , Cell Membrane/metabolism , Cholera Toxin/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology , Kidney/drug effects , Methylation , Microvilli/metabolism , Molecular Weight , Pertussis Toxin , Phosphorylation , Protein Kinase C/metabolism , Protein-Tyrosine Kinases/metabolism , Rats , Rats, Sprague-Dawley , Virulence Factors, Bordetella/metabolism
20.
Am J Physiol ; 260(4 Pt 2): F518-24, 1991 Apr.
Article in English | MEDLINE | ID: mdl-1672793

ABSTRACT

The activity of various enzymes and transport systems was studied in renal brush-border membrane vesicles (BBMV) isolated from rats injected daily with cyclosporin. Alkaline phosphatase (AP) was strongly stimulated: 55 and 113% increases were obtained in BBMV isolated from rats injected with 10 mg cyclosporin/kg for 5 and 10 days. The affinity of the enzyme remained unaltered, but maximal activity (Vmax) showed a strong increase of 2.4-fold between control and treated animals. In addition to the phosphatase activity, phosphate binding to AP also showed a dose-dependent stimulation by cyclosporin treatment: 44 and 70% increases in animals treated for 5 days with 5 and 10 mg cyclosporin/kg. However, the activity of aminopeptidase M was not affected by these treatments, and polyacrylamide gel electrophoresis of BBMV revealed no alterations in the profile of membrane proteins, suggesting the specificity of cyclosporin interaction with alkaline phosphatase. Na(+)-dependent amino acid and D-glucose transport systems remained unaffected by cyclosporin treatment. The Na(+)-independent transport system for lysine and the Na(+)-H+ antiporter activity were also unaltered. In contrast, the initial rate of phosphate uptake decreased by 28% after administration of cyclosporin (10 mg/kg) for 5 days: the Michaelis constant (Km) and Vmax decreased from 137 to 85 microM and from 1.49 to 1.07 pmol.micrograms-1.5 s-1, respectively. "In vitro" studies with membranes isolated from untreated rats were also undertaken by preincubating membranes with cyclosporin. Neither alkaline phosphatase nor the transport systems were affected under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Alkaline Phosphatase/metabolism , Cyclosporins/pharmacology , Kidney/metabolism , Microvilli/metabolism , Phosphates/metabolism , Absorption , Aminopeptidases/metabolism , Animals , Biological Transport/drug effects , CD13 Antigens , Cell Membrane/drug effects , Cell Membrane/metabolism , Electrophoresis, Polyacrylamide Gel , Kidney/drug effects , Kinetics , Membrane Proteins/isolation & purification , Membrane Proteins/metabolism , Phosphates/urine , Rats , Rats, Inbred Strains , Sodium/pharmacology
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