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1.
Toxicol Lett ; 206(3): 289-99, 2011 Oct 30.
Article in English | MEDLINE | ID: mdl-21872649

ABSTRACT

1-Nitropyrene (1-NP) is a nitro-polycyclic aromatic hydrocarbon (nitro-PAH) present in diesel exhaust and bound to particular matter in urban air. We show that 1-NP and the referent PAH benzo(a)pyrene (BP) induce apoptosis and a lipid accumulation dependent on cytochrome P450 1A1-metabolites in mouse hepatoma cells, whereas 1-amino-pyrene had no effect. The caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-fmk), inhibits 1-NP-induced apoptosis, but failed to alter 1-NP-triggered lipid accumulation determined by Nile red staining. We further show that cholesterol and fatty acid contents are modified after nitro-PAH exposure and that 1-NP-induced cholesterol level is partially involved in related apoptosis. In parallel, the activity of the stearoyl-CoA desaturase 1 (SCD1), determined by fatty acid analysis, and its expression are reduced by 1-NP. The role of SCD1 in 1-NP-induced apoptosis is demonstrated in cells down-expressing SCD1, in which an increased apoptosis is observed, whereas the SCD1 overexpression elicits the opposite effects. In contrast, changes in SCD1 gene expression have no effect on the induced lipid accumulation. Moreover, 1-NP increases the activity of the AMP-dependent protein kinase (AMPK) leading to a caspase-independent apoptosis. Overall, our study demonstrates that the 1-NP-induced apoptosis is caspase- and AMPK-dependent, and is associated to a decrease of SCD1 expression which results in an alteration of lipid homeostasis.


Subject(s)
Apoptosis/drug effects , Lipid Metabolism/drug effects , Pyrenes/toxicity , AMP-Activated Protein Kinases/physiology , Animals , Benzo(a)pyrene/toxicity , Caspases/physiology , Cell Line, Tumor , Cholesterol/metabolism , Liver Neoplasms, Experimental/metabolism , Liver Neoplasms, Experimental/pathology , Mice , Stearoyl-CoA Desaturase/physiology
3.
J Pharmacol Exp Ther ; 314(2): 693-702, 2005 Aug.
Article in English | MEDLINE | ID: mdl-15860575

ABSTRACT

Polycyclic aromatic hydrocarbons (PAHs) are major carcinogenic environmental contaminants known to exert bone marrow toxicity and to induce leukemias, suggesting that these chemicals target hematopoietic stem cells. To investigate this hypothesis, we studied the effects of PAHs on cell proliferation and differentiation in human hematopoietic CD34+ cell cultures. Benzo(a)pyrene (BP), a prototypical PAH, was shown to markedly impair CD34+ cell expansion and to inhibit CD34+ cell differentiation into various hematological cell lineages, including erythroid, granulomacrophagic, and megakaryocytic lineages. This was associated with the induction of a caspase- and mitochondrion-related apoptosis process. CD34+ progenitor cells were found to exhibit functional expression of the aryl hydrocarbon receptor (AhR), and the use of the pure AhR antagonist 3'-methoxy-4'-nitroflavone partially counteracted the deleterious effects of BP in CD34+ cell cultures, underlining the involvement of AhR in BP toxicity. Additional events such as CYP1A1/1B1-dependent PAH metabolism and adduct formation were also required since 1) 2,3,7,8-tetrachlorodibenzo-p-dioxin, a very potent ligand of the AhR that is poorly metabolized and therefore does not generate reactive metabolites in contrast to PAHs, failed to affect CD34+ cell expansion; 2) the CYP1A1/1B1 inhibitor alpha-naphthoflavone blocked both BP adduct formation and BP toxicity; and 3) benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide, a highly reactive BP metabolite, exerted a marked toxicity toward CD34+ cell cultures. Overall, these data indicate that human hematopoietic CD34+ cells can bioactivate chemical carcinogens such as PAHs and, in this way, constitute targets for such carcinogenic environmental contaminants.


Subject(s)
Antigens, CD34/metabolism , Carcinogens/toxicity , Hematopoietic Stem Cells/drug effects , Polycyclic Aromatic Hydrocarbons/toxicity , Apoptosis/drug effects , Benzo(a)pyrene/metabolism , Biotransformation/drug effects , Blotting, Western , Carcinogens/metabolism , Caspase 3 , Caspase 9 , Caspases/metabolism , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Colony-Forming Units Assay , Environmental Pollutants/toxicity , Flow Cytometry , Hematopoietic Stem Cells/metabolism , Humans , Membrane Potentials/drug effects , Mitochondria/drug effects , Patch-Clamp Techniques , Polycyclic Aromatic Hydrocarbons/metabolism , RNA/biosynthesis , RNA/genetics , RNA/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction
4.
Anticancer Drugs ; 16(3): 255-61, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15711177

ABSTRACT

ATP-binding cassette (ABC) transporters [P-glycoprotein and multidrug resistance (MDR)-associated proteins (MRPs)] confer MDR to tumor cells. In this work, we investigated doxorubicin resistance in three thyroid carcinoma cell lines. The effects of sodium butyrate (NaB) on doxorubicin-induced cytotoxicity and on transcription of three MDR genes were also studied. Thyroid cell lines established from anaplastic (8505C) and two poorly differentiated follicular (FTC 238 and FTC 133) cancers were cultured for 24 or 48 h in the presence of NaB (0, 0.25, 0.5 and 1 mM) alone or combined with increased doses of doxorubicin. Cytotoxicity was assessed using the MTT test. MDR1, MRP1 and MRP2 mRNA expression was studied by RT-PCR. After a 24- or 48-h incubation, doxorubicin alone induced cytotoxicity in the three cell lines. NaB significantly (p<0.0001) increased the doxorubicin-induced cytotoxicity. MRP1 transcripts were expressed in the three non-treated cell lines. MDR1 and MRP2 mRNAs were both present in 8505C, but absent in FTC 133 or FTC 238 cell lines, respectively. Treatment with NaB for 24 or 48 h induced no change in MRP1 and MRP2 levels, but increased MDR1 expression in 8505C and FTC 238 cell lines comparably to alkaline phosphatase activity. In conclusion, MRP1 and sometimes MDR1 and MRP2 are expressed in the tested cell lines. NaB potentiates doxorubicin-induced cytotoxicity independently of the ABC transporters. The combination of doxorubicin and NaB might have clinical implications for thyroid cancer therapy.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Antineoplastic Agents/therapeutic use , Butyric Acid/pharmacology , Doxorubicin/therapeutic use , Drug Resistance, Multiple/drug effects , Thyroid Neoplasms/drug therapy , ATP-Binding Cassette Transporters/drug effects , Drug Interactions , Drug Resistance, Multiple/genetics , Humans , Thyroid Neoplasms/metabolism , Tumor Cells, Cultured
5.
J Neurochem ; 88(1): 23-31, 2004 Jan.
Article in English | MEDLINE | ID: mdl-14675146

ABSTRACT

The blood-brain barrier (BBB) plays an important role in controlling the passage of molecules from blood to brain extracellular fluid. The multidrug efflux pump P-glycoprotein (P-gp) is highly expressed in the luminal membrane of brain endothelium and contributes to the formation of a functional barrier to lipid-soluble drugs such as anticancer agents. The mdr1a P-gp-encoding gene is exclusively expressed in the rodent BBB. Primary cultures of rat brain endothelial cells and GP8.3 cells showed a dramatic decrease in mdr1a mRNA level and some expression of mdr1b mRNA. GPNT cells, derived from GP8.3 cells after transfection with a puromycin resistance gene, were chronically treated with 5 microg/mL puromycin, a P-gp substrate. Compared with rat brain endothelial cells and GP8.3 cells, GPNT cells exhibited a very high level of expression of mdr1a mRNA together with a moderate level of mdr1b mRNA expression. Accordingly, P-gp expression and activity were strongly increased. When GP8.3 and puromycin-starved GPNT cells were treated with puromycin, mdr1a expression was selectively increased. High expression of mdr1a mRNA in GPNT cells may thus be related to the chronic treatment with puromycin. We conclude that GPNT cells may be used as a valuable rat in vitro model for studying the regulation of mdr1a expression at the BBB level.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B/genetics , ATP-Binding Cassette Transporters/genetics , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Puromycin/pharmacology , ATP Binding Cassette Transporter, Subfamily B/metabolism , ATP-Binding Cassette Transporters/metabolism , Animals , Antimetabolites, Antineoplastic/pharmacology , Antineoplastic Agents, Phytogenic/pharmacokinetics , Blood-Brain Barrier/drug effects , Blood-Brain Barrier/metabolism , Brain/blood supply , Capillaries/cytology , Capillaries/metabolism , Cell Line , Dose-Response Relationship, Drug , Endothelium, Vascular/cytology , Gene Expression/drug effects , Protein Synthesis Inhibitors/pharmacology , RNA, Messenger/metabolism , Rats , Vincristine/pharmacokinetics , ATP-Binding Cassette Sub-Family B Member 4
6.
Ann Biol Clin (Paris) ; 60(6): 663-72, 2002.
Article in French | MEDLINE | ID: mdl-12446230

ABSTRACT

Diagnosis of haematological malignancies is based on multiparametric analysis such as morphology, phenotype and genotype studies. Some entities are only defined by one of these approach. Flow-cytometry (FCM) is useful to determined the normal counterpart of the tumoral process and its differentiation status within the involved lineage. Furthermore, FCM is able to detect clonality in B or T proliferations and criteria for malignancies such as abnormal phenotype. Finally it also specifies prognosis criterias. Among the different haematological malignancies, acute lymphoblastic leukaemia (ALL) can be diagnosed using FCM, whereas acute myeloblastic leukaemia diagnosis is only confirmed by this methodology, which could moreover determine prognosis factors. A scoring system (EGIL) determine the normal counterpart of tumoral cells using a panel of different markers. Immunophenotyping is also useful in chronic lymphoproliferative disorders, such as chronic lymphocytic leukaemia (CLL) by using a similar scoring system (so-called Matutes scoring). Since FCM is able to detect simultaneously numerous cell markers it could be more accurate than immunohistochemistry for the diagnosis of follicular lymphoma, mantle cell lymphoma or hairy cell leukaemia. Finally, during treatment follow-up, minimal residual disease characterised by the detection of rare specific events, may be examined using FCM, in some situations.


Subject(s)
Flow Cytometry/methods , Hematologic Neoplasms/diagnosis , Antibodies, Monoclonal , Biomarkers, Tumor/analysis , Genotype , Hematologic Neoplasms/genetics , Hematologic Neoplasms/pathology , Humans
7.
Cell Biol Toxicol ; 18(4): 221-33, 2002.
Article in English | MEDLINE | ID: mdl-12206135

ABSTRACT

The multidrug resistance-associated protein 2 (MRP2) is an ATP-binding cassette transporter involved in biliary, renal, and intestinal secretion of numerous organic anions, including endogenous compounds such as bilirubin and exogenous compounds such as drugs and toxic chemicals. Its expression can be modulated in various physiopathological situations, notably being markedly decreased during liver cholestasis and upregulated in some cancerous tissues. In addition, MRP2 levels are altered in hepatocytes in response to hormones such as glucocorticoids and to structurally unrelated drugs such as rifampicin, phenobarbital, ritonavir, and cisplatin. The chemical carcinogen 2-acetylaminofluorene and chemopreventive agents such as oltipraz and sulforaphane also markedly increased MRP2 expression in liver parenchymal cells. Interestingly, most of the chemical inducers of MRP2 act on drug-metabolizing enzymes, indicating a coordinated regulation of these detoxifying proteins; cellular mechanisms involved are, at least partly, common and may be related to nuclear hormone receptors such as the pregnane X receptor. Owing to the major role played by MRP2 in elimination of drugs and endogenous compounds, modulation of its expression may lead to adverse effects or to changes in drug pharmacokinetics.


Subject(s)
Gene Expression Regulation , Membrane Transport Proteins , Multidrug Resistance-Associated Proteins/biosynthesis , Animals , Carcinogens/pharmacology , Cell Membrane/metabolism , Cholestasis/metabolism , Glucocorticoids/pharmacology , Hormones/metabolism , Humans , Liver/cytology , Liver/drug effects , Multidrug Resistance-Associated Protein 2 , Multidrug Resistance-Associated Proteins/genetics , Pregnane X Receptor , RNA, Messenger/metabolism , Rats , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Steroid/metabolism , Transcription, Genetic , Xenobiotics/pharmacology
8.
Br J Haematol ; 116(4): 834-8, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11886388

ABSTRACT

Multidrug resistance protein (MRP) activity was investigated in 44 newly diagnosed acute myeloid leukaemia (AML) patients using a functional assay based on efflux of carboxy-2',7'-dichlorofluorescein, an anionic dye handled by both MRP1 and MRP2. Elevated MRP transport was detected in 29% of cases, but was not significantly correlated with sex, age, white blood cell count at diagnosis or karyotype. In contrast, it was associated with secondary AML (P = 0.002), CD34 positivity (P = 0.041) and P-glycoprotein activity (P = 0.01). There was a lower rate of complete remission in MRP-positive patients versus MRP-negative patients (23% versus 81%; P = 0.001); overall survival was also better for MRP-negative patients (P = 0.004). These data indicate a probable role for MRP activity in the clinical outcome of AML.


Subject(s)
Leukemia, Myeloid/blood , Multidrug Resistance-Associated Proteins/analysis , ATP Binding Cassette Transporter, Subfamily B/analysis , Acute Disease , Adolescent , Adult , Aged , Antigens, CD34/analysis , Biomarkers/analysis , Female , Humans , Leukemia, Myeloid/drug therapy , Leukemia, Myeloid/mortality , Male , Middle Aged , Remission Induction , Survival Rate , Treatment Outcome
9.
Ann Pharm Fr ; 60(6): 380-5, 2002 Nov.
Article in French | MEDLINE | ID: mdl-12514503

ABSTRACT

Membrane transport proteins play a major role in hepato-biliary secretion of xenobiotics. Some of them, especially OATPs and OCT1, are present at the vascular pole of hepatocytes and mediate uptake of xenobiotics into parenchymal liver cells from blood whereas others, such as P-glycoprotein and MRP2, are ABC transporters present at the canalicular domain of hepatocytes and responsible for the transmembrane passage into bile of drugs or their metabolites. Many endogenous or exogenous factors, including drug metabolizing enzyme inducers, alter expression of hepatic transporters whose activity can moreover be inhibited by various structurally-unrelated compounds. Such changes of expression and/or activity of membrane transport proteins may contribute to some drug interactions.


Subject(s)
Carrier Proteins/metabolism , Liver/metabolism , Membrane Proteins/metabolism , Pharmaceutical Preparations/metabolism , Animals , Bile/metabolism , Humans , Membranes/metabolism
10.
Hum Immunol ; 62(10): 1073-80, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11600213

ABSTRACT

P-glycoprotein (P-gp), an ATP-binding cassette (ABC) drug efflux pump, has been recently shown to play an important role in the physiology of Langherans cells, a subtype of dendritic cells (DC) found in the skin. The present study was designed to investigate expression and activity of P-gp and of multidrug resistance-associated protein (MRP), another ABC efflux pump sharing numerous substrates with P-gp, in human monocyte-derived DC. Immunolabeling experiments and dye efflux assays indicated that such cells displayed elevated levels of MRP activity and expression when compared to those present in parental monocytes. Generation of DC from monocytes in the presence of the MRP inhibitor indomethacin did not, however, alter the capacity of DC to stimulate allogeneic T cells proliferation in mixed lymphocyte reaction. In addition, indomethacin did not inhibit the up-regulation of the CD1a, a marker occurring during the differentiation of monocytes into DC. In contrast to that of MRP, functional expression of P-gp was not detected in monocyte-derived DC. Such antigen presenting cells that constitute a promising tool for antitumor vaccinal therapy therefore display differential expression of the efflux pumps P-gp and MRP.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis , Dendritic Cells/metabolism , Drug Resistance, Multiple/immunology , Monocytes/metabolism , Biological Transport/drug effects , Biological Transport/immunology , Cell Differentiation/drug effects , Cell Differentiation/immunology , Cells, Cultured , Coculture Techniques , Dendritic Cells/cytology , Dendritic Cells/drug effects , Dendritic Cells/immunology , Fluoresceins/metabolism , Fluorescent Antibody Technique, Indirect , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , HL-60 Cells , Humans , Immunophenotyping , Interleukin-4/pharmacology , K562 Cells , Lymphocyte Culture Test, Mixed , Monocytes/cytology , Monocytes/drug effects , Monocytes/immunology , Probenecid/pharmacology
11.
Toxicology ; 167(1): 37-46, 2001 Oct 05.
Article in English | MEDLINE | ID: mdl-11557128

ABSTRACT

Biliary elimination of endogenous compounds and xenobiotics usually requires carrier-mediated systems allowing movement across the canalicular membrane of hepatocytes. The major systems implicated belong to the ATP binding cassette transporter family: P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2), principally mediate the passage into the bile of cationic and anionic compounds, respectively, whereas the bile salt export pump (BSEP) handles biliary acids and also some anticancer drugs. Expression of these canalicular proteins can be altered in response to various hormones and structurally unrelated xenobiotics. Indeed, glucocorticoids up-regulate expression of both MRP2 and BSEP in rat hepatocytes, whereas insulin induces P-gp. P-gp expression is also up-regulated by numerous chemical carcinogens, such as polycyclic aromatic hydrocarbons and 2-acetylaminofluorene and by some anticancer drugs, such as anthracyclins. 2-Acetylaminofluorene also induces MRP2; in addition, expression of this transporter in liver cells is increased in response to various drugs, such as the barbiturate phenobarbital, the chemopreventive agent, oltipraz and the anticancer drug, cisplatin. Most of the chemical inducers acting on canalicular transporter levels are well-known to up-regulate some hepatic drug metabolizing enzymes, suggesting a coordinate regulation of liver detoxifying proteins in response to these compounds.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis , ATP-Binding Cassette Transporters/biosynthesis , Carrier Proteins/biosynthesis , Hormones/physiology , Membrane Transport Proteins , Multidrug Resistance-Associated Proteins , Xenobiotics/pharmacology , ATP Binding Cassette Transporter, Subfamily B/biosynthesis , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 11 , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Animals , Anion Transport Proteins , Carrier Proteins/genetics , Carrier Proteins/metabolism , Gene Expression Regulation/drug effects , Glucocorticoids/physiology , Humans , Insulin/physiology , Multidrug Resistance-Associated Protein 2 , Up-Regulation/physiology
12.
J Pharmacol Exp Ther ; 298(1): 234-9, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11408547

ABSTRACT

Metals, such as arsenic or cadmium, have recently been demonstrated to interact with metabolic pathways, including phase I and phase II enzymes and the phase III efflux pump P-glycoprotein. In the present study, we investigated the effects of heavy metals and metalloids on the expression of the multidrug resistance-associated protein 2 (MRP2), a major hepatic transporter. Treatment of primary rat hepatocytes by sodium arsenite [As(III)], sodium arsenate and potassium antimony tartrate, but not cadmium chloride, was shown to markedly increase MRP2 mRNA and protein levels; As(III)-mediated induction was dose- and time-dependent and paralleled a strong increase in MRP2 amounts as assessed by Western blotting. As(III) was also demonstrated to markedly up-regulate MRP2 gene expression in primary human hepatocytes. MRP2 mRNA induction occurring in As(III)-treated rat hepatocytes was fully blocked by actinomycin D, indicating that it required active gene transcription. It was associated with an activation of the c-Jun N-terminal kinase pathway and with a reduction of cellular glutathione levels. Quercetin, a flavonoid compound known to block As(III)-related induction of P-glycoprotein, was also found to prevent up-regulation of MRP2 gene expression in rat hepatocytes exposed to As(III). Such an effect was unlikely to be due to alteration of JNK pathway since quercetin failed to abolish As(III)-induced JNK phosphorylation. It may rather be linked to the increase of cellular glutathione levels by quercetin, thus limiting the depleting effects of As(III) on glutathione amounts. Finally, these results confirm that some metals strongly regulate expression of detoxifying proteins, including biliary drug transporters.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B/drug effects , Arsenites/pharmacology , Hepatocytes/drug effects , Membrane Transport Proteins , Multidrug Resistance-Associated Proteins , RNA, Messenger/drug effects , Sodium Compounds/pharmacology , ATP Binding Cassette Transporter, Subfamily B/metabolism , Animals , Antimony Potassium Tartrate/pharmacology , Arsenates/pharmacology , Cadmium Chloride/pharmacology , Cells, Cultured , Enzyme Inhibitors/pharmacology , Hepatocytes/metabolism , JNK Mitogen-Activated Protein Kinases , Male , Mitogen-Activated Protein Kinases/drug effects , Mitogen-Activated Protein Kinases/metabolism , Multidrug Resistance-Associated Protein 2 , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Schistosomicides/pharmacology , Transcription, Genetic/drug effects , Transcription, Genetic/physiology
13.
Biochem Pharmacol ; 61(11): 1387-91, 2001 Jun 01.
Article in English | MEDLINE | ID: mdl-11331074

ABSTRACT

The human multidrug-resistance protein (MRP1) confers resistance to some heavy metals such as arsenic and antimony, mainly through mediating an increased cellular efflux of metal. However, it was recently suggested that arsenic, used under its trioxide derivative form as anticancer drug, is not handled by MRP1. The aim of the present study was to test this hypothesis in MRP1-overexpressing human lung tumor GLC4/Sb30 cells. Using the cytotoxicity MTT assay, GLC4/Sb30 cells were found to be 10.8-fold more resistant to arsenic trioxide (As2O3) than parental GLC4 cells. MK571, a potent inhibitor of MRP1 activity, almost totally reversed resistance of GLC4/Sb30 cells, but did not alter the sensitivity of GLC4 cells. Moreover, As2O3-loaded GLC4/Sb30 cells poorly accumulated arsenic through an increased MK571-sensitive efflux of metal. Finally, depletion of cellular glutathione levels in buthionine sulfoximine-treated GLC4/Sb30 cells was found to result in increased accumulation and reduced efflux of arsenic in cells exposed to As2O3, outlining the glutathione-dependence of MRP1-mediated transport of the metal. These results indicate that MRP1 overexpression in human tumor cells can confer resistance to As2O3, which may limit the clinical use of this anticancer drug for treatment of MRP1-positive tumors.


Subject(s)
ATP-Binding Cassette Transporters/biosynthesis , Antineoplastic Agents/pharmacology , Arsenicals/pharmacology , Oxides/pharmacology , ATP-Binding Cassette Transporters/physiology , Arsenic Trioxide , Cell Survival/drug effects , Drug Resistance, Neoplasm/physiology , Drug Screening Assays, Antitumor , Humans , Multidrug Resistance-Associated Proteins , Tumor Cells, Cultured
14.
Biochem Biophys Res Commun ; 282(1): 257-63, 2001 Mar 23.
Article in English | MEDLINE | ID: mdl-11264000

ABSTRACT

Expression of multidrug resistance-associated protein 2 (MRP2), an efflux pump contributing to biliary secretion of xenobiotics, was investigated in primary rat and human hepatocytes exposed to sulforaphane, a naturally-occurring chemopreventive agent. Northern blot indicated that sulforaphane increased MRP2 mRNA levels in primary rat hepatocytes; it also induced expression of drug metabolizing enzymes such as glutathione S-transferase A1/2 isoforms and NAD(P)H:quinone oxidoreductase in a dose-response and time-course manner similar to that observed for the upregulation of MRP2 transcripts. This sulforaphane-related increase of MRP2 mRNAs paralleled increased expression of 190 kD MRP2 protein as assessed by Western blotting; it was fully abolished by the transcription inhibitor actinomycin D. MRP2 induction was associated with increased cellular production of reactive oxygen species (ROS) and addition of dimethyl sulfoxide, that reduced sulforaphane-related formation of ROS, and also decreased MRP2 mRNA levels in sulforaphane-treated primary rat hepatocytes; this suggests that sulforaphane-mediated production of ROS may contribute to MRP2 induction. This link between ROS and MRP2 regulation was further supported by the increase of MRP2 expression occurring in response to t-butylhydroquinone, known to regulate drug metabolizing enzymes through ROS formation. In addition to rat cells, primary human hepatocytes exposed to sulforaphane also displayed induced MRP2 expression evidenced at both mRNA and protein levels. All these observations strongly support the conclusion that the export pump MRP2 can be classified among the detoxifying proteins that are regulated by sulforaphane and that are thought to contribute, at least in part, to its anticarcinogenic properties.


Subject(s)
Hepatocytes/drug effects , Mitochondrial Proteins , Reactive Oxygen Species , Ribosomal Proteins/metabolism , Saccharomyces cerevisiae Proteins , Thiocyanates/pharmacology , Animals , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Hepatocytes/metabolism , Isothiocyanates , Male , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Ribosomal Proteins/genetics , Sulfoxides
15.
Life Sci ; 68(11): 1323-31, 2001 Feb 02.
Article in English | MEDLINE | ID: mdl-11233999

ABSTRACT

Multidrug resistance proteins (MRPs) such as MRP1, MRP2 and MRP3 are membrane efflux pumps involved in multidrug resistance and handling organic anions. In the present study, MRP activity was investigated in normal mature leucocytes and CD34-positive hematopoietic cells from peripheral blood using the flow cytometric carboxy-2',7'-dichlorofluorescein (CF) efflux assay. Basal and similar cellular exports of CF, an anionic fluorescent dye substrate for MRP1 and MRP2 transporters, were evidenced in lymphocytes whatever their subsets (CD3, CD4, CD8, CD20 and CD56 cells), in CD14 monocytes and in CD15 granulocytes whereas higher CF efflux was found in CD34 cells. Such outwardly-directed transports of CF were inhibited by known blockers of MRP function such as probenecid whereas the P-glycoprotein modulator verapamil did not alter the retention of the dye in the blood leukocytes. Peripheral mature blood leukocytes were moreover found to express MRP1 mRNAs and MRP1 protein as assessed by Northern-blot and Western-blot analyses, whereas MRP2 and MRP3 transcripts were not present or only at very low levels. Mature leukocytes therefore display basal constitutive MRP-related transport activity regardless of cell lineage and likely related to MRP1 expression whereas higher MRP-related efflux can be detected in peripheral CD34 hematopoietic cells.


Subject(s)
Antigens, CD34/analysis , DNA-Binding Proteins/metabolism , Fungal Proteins/metabolism , Hematopoietic Stem Cells/metabolism , Leukocytes/metabolism , Mitochondrial Proteins , Multidrug Resistance-Associated Proteins , Pyruvate Dehydrogenase Complex , Ribosomal Proteins/metabolism , Saccharomyces cerevisiae Proteins , DNA-Binding Proteins/genetics , Dihydrolipoyllysine-Residue Acetyltransferase , Flow Cytometry , Fluoresceins/metabolism , Fluorescent Dyes/metabolism , Fungal Proteins/genetics , Granulocytes/metabolism , HL-60 Cells , Humans , Leukocytes/chemistry , Lymphocyte Subsets , Lymphocytes/metabolism , Monocytes/metabolism , MutS Homolog 3 Protein , RNA, Messenger/analysis , Ribosomal Proteins/genetics
16.
Br J Pharmacol ; 132(3): 778-84, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11159731

ABSTRACT

1. Glibenclamide, a sulphonylurea widely used for the treatment of non-insulin-dependent diabetes mellitus, has been shown to inhibit the activities of various ATP-binding cassette (ABC) transporters. In the present study, its effects towards multidrug resistance protein 1 (MRP1), an ABC efflux pump conferring multidrug resistance and handling organic anions, were investigated. 2. Intracellular accumulation of calcein, an anionic dye substrate for MRP1, was strongly increased by glibenclamide in a dose-dependent manner in MRP1-overexpressing lung tumour GLC4/Sb30 cells through inhibition of MRP1-related calcein efflux. By contrast, glibenclamide did not alter calcein levels in parental control GLC4 cells. Another sulphonylurea, tolbutamide, was however without effect on calcein accumulation in both GLC4/Sb30 and GLC4 cells. 3. Glibenclamide used at 12.5 microM was, moreover, found to strongly enhance the sensitivity of GLC4/Sb30 cells towards vincristine, an anticancer drug handled by MRP1. 4. Efflux of carboxy-2',7'-dichlorofluorescein, an anionic dye handled by the ABC transporter MRP2 sharing numerous substrates with MRP1 and expressed at high levels in liver, was also strongly inhibited by glibenclamide in isolated rat hepatocytes. 5. In summary, glibenclamide reversed MRP1-mediated drug resistance likely through inhibiting MRP1 activity and blocked organic anion efflux from MRP2-expressing hepatocytes. Such effects associated with the known inhibitory properties of glibenclamide towards various others ABC proteins suggest that this sulphonylurea is a general inhibitor of ABC transporters.


Subject(s)
ATP-Binding Cassette Transporters/antagonists & inhibitors , Glyburide/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , Analysis of Variance , Antineoplastic Agents, Phytogenic/pharmacology , Cell Survival , Drug Interactions , Drug Screening Assays, Antitumor , Humans , Hypoglycemic Agents/pharmacology , Lung Neoplasms/pathology , Multidrug Resistance-Associated Proteins , Tumor Cells, Cultured , Vincristine/pharmacology
17.
Toxicology ; 156(2-3): 109-17, 2001 Jan 02.
Article in English | MEDLINE | ID: mdl-11164613

ABSTRACT

Rat liver epithelial cells resistant to the chemical carcinogen 3MC, termed F258/3MC cells and generated by long-term exposure of parental F258 cells to the PAH, were characterized, especially with respect to expression of multidrug resistance transporters such as P-glycoprotein, MRP1 and MRP2. F258/3MC cells were found to be cross-resistant to other PAHs such as BP and dimethylbenz(a)anthracene but remained sensitive to known substrates of multidrug resistance efflux pumps such as doxorubicin and vincristine. They did not display either decreased cellular PAH accumulation or increased PAH efflux. In addition, P-glycoprotein and MRP2 mRNA levels were not, or only barely detected, in F258/3MC cells and in their parental counterparts whereas these PAH-resistant and sensitive cells showed closed levels of MRP1 mRNAs and activity. Moreover, P-gp- and MRP1-overexpressing cells were shown to display similar accumulation and efflux of BP than those found in P-gp- and MRP1-negative control cells. These data therefore suggest that multidrug resistance transporters do not contribute to PAH resistance in PAH-selected liver cells.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Carcinogens/toxicity , Hepatocytes/metabolism , Membrane Transport Proteins , Polycyclic Aromatic Hydrocarbons/toxicity , 9,10-Dimethyl-1,2-benzanthracene/toxicity , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Animals , Benzopyrenes/toxicity , Cells, Cultured , Cytochrome P-450 CYP1A1/biosynthesis , Cytochrome P-450 CYP1A1/genetics , DNA Adducts/analysis , DNA Adducts/drug effects , Doxorubicin/pharmacology , Drug Resistance, Multiple , Hepatocytes/cytology , Hepatocytes/drug effects , Methylcholanthrene/toxicity , Multidrug Resistance-Associated Protein 2 , Multidrug Resistance-Associated Proteins , RNA, Messenger/metabolism , Rats , Up-Regulation , Vincristine/pharmacology
18.
Anticancer Res ; 21(6A): 3915-9, 2001.
Article in English | MEDLINE | ID: mdl-11911269

ABSTRACT

We have previously demonstrated that proton NMR spectra of fatty acid chains in erythroleukemia K562 wild-type cells and their MDR1 counterparts show variations related to the phenotype over-expressing the P-glycoprotein (P-gp). Human lung cancer cells whose multidrug resistance (MDR) counterparts over-express the multidrug resistance-associated protein MRP1 have not yet been studied by NMR. Both P-gp and MRP1 belong to the same ATP-binding cassette transporter superfamily. A comparison of NMR spectra from both these multidrug-resistance phenotypes showed that the results previously obtained on the MDR1 family are not valid for MRP1. Furthermore, flow cytofluorimetry studies with external phosphatidylserine labelling showed that P-gp and MRP1 overexpressions have strong but differentiated effects on cell lipid pools.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis , Drug Resistance, Multiple/physiology , Lipid Metabolism , Multidrug Resistance-Associated Proteins/biosynthesis , Phosphatidylserines/metabolism , Carcinoma, Small Cell/metabolism , Carcinoma, Small Cell/pathology , Drug Resistance, Neoplasm , Humans , K562 Cells/cytology , K562 Cells/metabolism , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Nuclear Magnetic Resonance, Biomolecular/methods , Protons
19.
Biochem Pharmacol ; 60(12): 1967-75, 2000 Dec 15.
Article in English | MEDLINE | ID: mdl-11108814

ABSTRACT

Organic anion secretion by human hepatocytes was characterized using primary liver parenchymal cell cultures and the anionic fluorescent dye carboxy-2',7'-dichlorofluorescein (CF). Probenecid, a well-known common blocker of the membrane transport process for anions, was shown to increase CF accumulation in primary human hepatocytes by inhibiting cellular CF efflux in a dose-dependent manner, thereby establishing the presence of an efflux system for organic anions in cultured hepatocytes. Outwardly directed transport of CF from hepatocytes was found to be temperature-dependent; it was not altered by changes in the ionic composition of the incubation medium used in efflux experiments. In addition to probenecid, various structurally and functionally unrelated xenobiotics such as glibenclamide, rifampicin, vinblastine, MK-571, indomethacin, and cyclosporin A were shown to inhibit secretion of CF by primary human hepatocytes, thus suggesting that organic anion excretion by human liver may be impaired by various drugs. Northern blot and Western blot analyses of the expression of multidrug resistance proteins (MRP), such as MRP1 and MRP2, which are known to mediate cellular outwardly directed transport of organic anions indicated that MRP2 was present at substantial levels in cultured human hepatocytes as well as in their in vivo counterparts, whereas MRP1 expression was only barely detectable. These results therefore suggest that MRP2, unlike MRP1, may contribute to the organic anion efflux system displayed by primary human hepatocytes and inhibited by a wide range of xenobiotics.


Subject(s)
Fluoresceins/metabolism , Hepatocytes/drug effects , Xenobiotics/pharmacology , Anion Transport Proteins , Anions/metabolism , Biological Transport/drug effects , Carrier Proteins/metabolism , Cells, Cultured , Hepatocytes/metabolism , Humans
20.
Toxicology ; 153(1-3): 203-19, 2000 Nov 16.
Article in English | MEDLINE | ID: mdl-11090958

ABSTRACT

Transport across hepatocyte plasma membranes is a key parameter in hepatic clearance and usually occurs through different carrier-mediated systems. Sinusoidal uptake of compounds is thus mediated by distinct transporters, such as Na(+)-dependent or Na(+)-independent anionic transporters and by some cationic transporters. Similarly, several membrane proteins located at the apical pole of hepatocytes have been incriminated in the excretion of compounds into the bile. Indeed, biliary elimination of anionic compounds, including glutathione S-conjugates, is mediated by MRP2, whereas bile salts are excreted by a bile salt export pump (BSEP) and Class I-P-glycoprotein (P-gp) is involved in the secretion of amphiphilic cationic drugs, whereas class II-P-gp is a phospholipid transporter. The expression of hepatic transporters and their activity are regulated in various situations, such as ontogenesis, carcinogenesis, cholestasis, cellular stress and after treatment by hormones and xenobiotics. Moreover, a direct correlation between a defect and the absence of transporter with hepatic disease has been demonstrated for BSEP, MDR3-P-gp and MRP2.


Subject(s)
Bile Acids and Salts/metabolism , Carrier Proteins/metabolism , Hydroxysteroid Dehydrogenases , Liver/metabolism , Membrane Glycoproteins , Animals , Carrier Proteins/biosynthesis , Humans , Liver Neoplasms/metabolism
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