RESUMO
Cellular and molecular mechanisms involved in the resistance to cytotoxic heavy metals remain largely to be characterized in mammalian cells. To this end, we have analyzed a metal-resistant variant of the human lung cancer GLC4 cell line that we have selected by a step-wise procedure in potassium antimony tartrate. Antimony-selected cells, termed GLC4/Sb30 cells, poorly accumulated antimony through an enhanced cellular efflux of metal, thus suggesting up-regulation of a membrane export system in these cells. Indeed, GLC4/Sb30 cells were found to display a functional overexpression of the multidrug resistance-associated protein MRP1, a drug export pump, as demonstrated by Western blotting, reverse transcriptase-polymerase chain reaction and calcein accumulation assays. Moreover, MK571, a potent inhibitor of MRP1 activity, was found to markedly down-modulate resistance of GLC4/Sb30 cells to antimony and to decrease cellular export of the metal. Taken together, our data support the conclusion that overexpression of functional MRP1 likely represents one major mechanism by which human cells can escape the cytotoxic effects of heavy metals.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Antimônio/farmacologia , Resistência a Múltiplos Medicamentos , Metais Pesados/farmacologia , Regulação para Cima/efeitos dos fármacos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antimônio/metabolismo , Antineoplásicos/farmacologia , Arsenitos/farmacologia , Western Blotting , Cloreto de Cádmio/farmacologia , Resistencia a Medicamentos Antineoplásicos , Fluoresceínas/metabolismo , Amplificação de Genes/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Neoplasias Pulmonares , Meglumina/farmacologia , Metalotioneína/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Propionatos/farmacologia , Quinolinas/farmacologia , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas , Sulfato de Zinco/farmacologiaRESUMO
We compared the incorporation of arachidonic acid (AA) and eicosapentaneoic acid (EPA) into phospholipids of non-transformed (NT-) and spontaneously-transformed (T-) rat liver epithelial cells (RLEC), and their consequences on DNA-synthesis. In NT-cells, both radioactive fatty acids were preferentially incorporated into phosphatidylcholine (PC). In T-cells, in contrast, AA was predominantly incorporated into phosphatidylethanolamine (PE), whereas EPA remained preferentially incorporated into PC. After pulse labelling, we observed in both cell types a progressive decrease in AA- and EPA-labelled PC associated with an increase in AA- and EPA-labelled PE. Preincubation of NT-cells with increasing concentrations of AA or EPA (0.1 microM to 20 microM) resulted in a concentration-dependent DNA-synthesis stimulation with a stronger effect of AA compared with EPA. In T-cells, the same treatment had no effect on DNA-synthesis.
Assuntos
Ácido Araquidônico/farmacologia , DNA/efeitos dos fármacos , Ácido Eicosapentaenoico/farmacologia , Fígado/efeitos dos fármacos , Fosfolipídeos/metabolismo , Animais , Ácido Araquidônico/metabolismo , Linhagem Celular Transformada , DNA/biossíntese , Ácido Eicosapentaenoico/metabolismo , Epitélio , Fígado/citologia , Fígado/metabolismo , RatosRESUMO
The multidrug resistance-associated protein (MRP) is a drug efflux membrane pump conferring multidrug resistance on tumor cells. In order to look for compounds that can lead to reversal of such a resistance, the antituberculosis compound rifampicin, belonging to the chemical class of rifamycins, was examined for its effect on MRP activity in human multidrug resistant lung cancer GLC4/ADR cells. Rifampicin was shown to increase accumulation of the MRP substrate calcein in GLC4/ADR cells in a dose-dependent manner by inhibiting its MRP-mediated efflux from the cells; it also enhanced intracellular retention of another substrate of MRP such as the anticancer drug vincristine in the resistant cells. By contrast, the antituberculosis drug did not alter cellular levels of accumulation of either calcein or vincristine in parental drug-sensitive GLC4 cells. Other rifamycins such as rifamycin B and rifamycin SV were also demonstrated to increase intracellular accumulation of calcein in GLC4/ADR cells. These results therefore indicate that rifamycins, including rifampicin, probably constitute a new chemical class of modulators down-regulating MRP-mediated drug transport.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Neoplasias Pulmonares/metabolismo , Rifampina/farmacologia , Células Tumorais Cultivadas/metabolismo , Antibióticos Antituberculose/farmacologia , Relação Dose-Resposta a Droga , Resistência a Múltiplos Medicamentos , Fluoresceínas/metabolismo , Humanos , Modelos Químicos , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Rifamicinas/farmacologiaRESUMO
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.
Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , Antineoplásicos/farmacologia , Arsenicais/farmacologia , Óxidos/farmacologia , Transportadores de Cassetes de Ligação de ATP/fisiologia , Trióxido de Arsênio , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Células Tumorais CultivadasRESUMO
Multidrug resistance-associated protein (MRP) and P-glycoprotein are drug efflux pumps conferring multidrug resistance to tumor cells and sharing numerous substrates. In order to determine a flow cytometric assay allowing to analyse MRP activity in cancerous cells in a sensitive and specific manner, cellular accumulation and efflux of the anionic fluorescent dye carboxy-2',7'-dichlorofluorescein (CDF) were studied by flow cytometry using mainly MRP-overexpressing lung GLC4/Sb30 cells and parental GLC4 cells. GLC4/Sb30 cells were found to display reduced accumulation and enhanced efflux of the dye when compared to their parental counterparts. Probenecid, a well known blocker of MRP, strongly enhanced CDF accumulation in GLC4/Sb30 cells through inhibiting efflux of the dye; it also increased CDF levels in GLC4 cells, although to a lesser extent, which may likely be linked to the low, but detectable, expression of MRP in these cells. Comparison of CDF retention with that of calcein demonstrated that the former dye was the most efficiently effluxed by GLC4/Sb30 cells. In contrast to MRP overexpression, that of P-glycoprotein was not found to alter cellular CDF labelling whereas it strongly impaired calcein staining. These results indicate that CDF is a substrate for MRP, but not for P-gp, which may likely be useful for sensitive and specific flow cytometric determination of MRP activity in clinical samples.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Transportadores de Cassetes de Ligação de ATP/fisiologia , Corantes , Resistência a Múltiplos Medicamentos , Fluoresceínas , Proteínas de Neoplasias/fisiologia , Ânions , Citometria de Fluxo , Humanos , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Sensibilidade e Especificidade , Células Tumorais CultivadasRESUMO
The canine model of ventricular tachycardias (VT) induced by programmed stimulation is used routinely in several laboratories to test antiarrhythmic drugs. The aim of the present study was to determine the rate of success and reproducibility of this model. We analyzed a group of 58 dogs that underwent a 2-hr occlusion and were submitted to programmed electrical stimulation at least 4 days after the surgery. Only 29 dogs (50%) were inducible and included in the study, as 22 dogs died following myocardial infarction, and seven dogs were never inducible. Out of 130 trials, 92 (70%) performed on inducible dogs were positive with 11% of nonsustained ventricular tachycardias, 63% of sustained monomorphic ventricular tachycardias, and 26% of ventricular fibrillation. Inducibility decreased over time in a subgroup of 19 dogs that was submitted to four trials during the first month after the infarction (68% of inducible dogs versus 46% in trials 1 and 4, respectively). Ventricular effective refractory period decreased significantly from 146 +/- 7 msec at trial 1 to 114 +/- 6 msec at trial 4, and the severity of the induced ventricular tachycardias increased. This variability should be considered when planning studies on antiarrhythmic drugs in this model.
Assuntos
Modelos Animais de Doenças , Estimulação Elétrica , Taquicardia Ventricular/fisiopatologia , Animais , Antiarrítmicos/farmacologia , Cães , Eletrofisiologia , Feminino , Masculino , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/fisiopatologia , Reprodutibilidade dos Testes , Taquicardia Ventricular/tratamento farmacológico , Fatores de TempoRESUMO
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.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Transportadores de Cassetes de Ligação de ATP/biossíntese , Proteínas de Transporte/biossíntese , Hormônios/fisiologia , Proteínas de Membrana Transportadoras , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Xenobióticos/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/biossíntese , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Proteínas de Transporte de Ânions , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/fisiologia , Humanos , Insulina/fisiologia , Proteína 2 Associada à Farmacorresistência Múltipla , Regulação para Cima/fisiologiaRESUMO
The human multidrug-resistance protein (MRP1), known to mediate cellular efflux of a wide range of xenobiotics, including anticancer drugs, has also been shown to transport antimony, thereby conferring resistance to this heavy metal. The aim of the present study was to investigate whether other cytotoxic metals could be handled by MRPI using MRP1-overexpressing lung tumor GLC4/Sb30 cells. Such cells were found to be 3.4-, 12.7- and 16.3-fold more resistant than parental GLC4 cells to mercuric ion, arsenite and arsenate, respectively, whereas they remained sensitive to other cytotoxic metals tested such as copper, chromium, cobalt or aluminium. MK571, a potent inhibitor of MRP1 activity, almost totally reversed resistance of GLC4/Sb30 cells to mercuric ions and arsenic while it did not significantly alter sensitivity of GLC4 cells to metals. Arsenate-treated GLC4/Sb30 cells were found to poorly accumulate arsenic through increased MK571-inhibitable efflux of the metal. Arsenate, however, failed to alter MRP1-mediated transport of known MRP1 substrates such as calcein and vincristine. In conclusion, these findings demonstrated that MRP1 likely handled some, but not all, cytotoxic metals such as arsenic and mercuric ions in addition to antimony, therefore resulting in reduced toxicity of these compounds towards MRP1-overexpressing cells.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Metais/toxicidade , Transportadores de Cassetes de Ligação de ATP/análise , Antimônio/metabolismo , Antimônio/toxicidade , Arsênio/metabolismo , Arsênio/toxicidade , Resistência a Múltiplos Medicamentos , Humanos , Neoplasias Pulmonares/patologia , Mercúrio/metabolismo , Mercúrio/toxicidade , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Células Tumorais CultivadasRESUMO
We investigated the substitution of 15(S)-hydroxyeicosatetraenoic acid (15(S)HETE) in phospholipid signaling pathways and its consequences on the growth of non-transformed (NT-) and spontaneously transformed (T-) rat liver epithelia cells (RLEC). 15(S)HETE was selectively incorporated into the sn-2 position of phosphatidylinositol (PI) and at a higher rate into T-RLEC. RLEC rapidly mobilized the resulting 15(S)HETE-containing PI (15(S)HETE-PI) and produced 1-acyl,2-[1(S)HETE]-glycerol. Although total diacylglycerol levels were similar in both cell types, the ratio 1-acyl,2-[15(S)HETE]-glycerol / 15(S)HETE-PI was higher in NT-RLEC, suggesting a lower mobilization of 15(S)HETE-PI in T-RLEC. Using rat brain protein kinase C, 1-stearoyl,2-[15(S)HETE]-glycerol was as potent an in vitro protein kinase C activator as 1-stearoyl,2-arachidonoyl-glycerol. Finally, selective substitution of 15(S)HETE in PI altered DNA synthesis in T-RLEC: whereas low concentrations of 15(S)HETE (1 nM and 10 nM) in these cells were mitogenic, higher concentrations resulted in a 30% inhibition of DNA synthesis.
Assuntos
Ácidos Hidroxieicosatetraenoicos/química , Fígado/efeitos dos fármacos , Fosfatidilinositóis/fisiologia , Animais , Encéfalo/enzimologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Transformada , Ativação Enzimática , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Fígado/citologia , Fosfatidilinositóis/química , Proteína Quinase C/metabolismo , RatosRESUMO
Bradykinin receptor stimulation results in G-protein-coupled phospholipase activation, initiating protein kinase C (PKC) stimulation and cytosolic free Ca2+ concentration ([Ca2+]i) rises as signalling pathways. Using Rb+ as a tracer for K+, we have studied the mechanisms involved in bradykinin-stimulated Rb+ influx in NIH-3T3 fibroblasts. The furosemide-sensitive Na+/K+/Cl- cotransport and the ouabain-sensitive Na+/K(+)-ATPase were both involved in Rb+ influx under resting conditions with a ratio Na+/K+/Cl- cotransport/Na+/K(+)-ATPase (r) = 0.73. Bradykinin stimulated Rb+ influx (+82.6%) through both systems without changing their ratio (r = 0.72). PKC stimulation by a 15-min-treatment with phorbol 12-myristate 13-acetate (PMA) (2x10(-7) M) increased Rb+ influx in resting cells by 75.7% without affecting r (0.75). PKC inhibition by H-7, and PKC down-regulation by 24-h PMA (10(-6) M) treatment decreased the bradykinin-induced stimulation of Rb+ influx (+31% and +14.9% above control, respectively). Both down-regulation and inhibition of PKC dramatically reduced the furosemide-sensitive Na+/K+/Cl- cotransport, as r fell to 0.239 and 0.032 in bradykinin-stimulated cells after H-7 and 24-h PMA treatments, respectively. BAPTA/AM pretreatment (10(-4) M, 60 min), which complexed with [Ca2+]i, not only prevented the bradykinin-induced [Ca2+]i raise, but also partially inhibited bradykinin-induced Rb+ influx stimulation (+39% above control), without modifying r (0.76). We conclude that stimulation of PKC is a major pathway involved in bradykinin stimulation of Rb+ influx in NIH-3T3 fibroblasts, and that rises in [Ca2+]i participate in bradykinin signalling, possibly through PKC activation. Our data also suggest that active PKC is required for basal and bradykinin-stimulated Na+/K+/Cl- cotransport activity in these cells.
Assuntos
Bradicinina/farmacologia , Proteínas de Transporte/efeitos dos fármacos , Rubídio/farmacologia , ATPase Trocadora de Sódio-Potássio/efeitos dos fármacos , Células 3T3 , Animais , Proteínas de Transporte/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Furosemida/farmacologia , Transporte de Íons , Camundongos , Proteína Quinase C/metabolismo , Simportadores de Cloreto de Sódio-Potássio , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
An attempt was made to determine the relationship between the characteristics of electrical activity of the hypertrophied myocardium of rats at the cellular level and at the level of the whole heart after a one-month left ventricular pressure overload. Such an animal model has already been demonstrated to be highly resistant to epinephrine-induced arrhythmias. Since severe ventricular arrhythmias often occur in patients with cardiac hypertrophy, ventricular vulnerability might depend on some electrophysiological characteristics of the heart related to the stage of hypertrophy. Using the "floating" microelectrode technique, the computed characteristics of cardiac transmembrane action potentials (AP) of the left and right epicardium cells were compared in situ to computed characteristics of the electrocardiograms in anaesthetized control rats (group C) and in rats with left ventricular hypertrophy (group H) induced by a one-month suprarenal constriction of the abdominal aorta. The aortic pressure overload caused a significant (p<0.001) and marked increase in AP duration of left ventricular cells: APD 30 and APD 80 were 29+/-3 ms and 89+/-6 ms, respectively, in group H and 14+/-1 ms and 53+/-2 ms in group C. The same modifications were observed in right ventricular cells when right hypertrophy was present. Simultaneous electrocardiograms exhibited a significant (p<0.01) prolongation of P-R, Q-S and T duration and T wave flattening in group H (63+/-2 ms, 32+/-3 ms, 109+/-5 ms and 0.25+/-0.03 mV as compared with 53+/-1 ms, 20+/-1 ms, 88+/-2 ms and 0.40+/-0.04 mV in group C). After a one-month aortic overload in rats, both left and right ventricles are hypertrophied and have the same electrophysiological characteristics: in this model, at this stage of hypertrophy, some factors favouring ventricular arrhythmias are missing. The corresponding flattening of the T wave in the ECG might be of clinical relevance.
Assuntos
Aorta/fisiopatologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Potenciais de Ação , Animais , Eletrocardiografia , Masculino , Ratos , Ratos Wistar , Fatores de TempoRESUMO
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.
Assuntos
Proteínas de Transporte/metabolismo , Fígado/metabolismo , Proteínas de Membrana/metabolismo , Preparações Farmacêuticas/metabolismo , Animais , Bile/metabolismo , Humanos , Membranas/metabolismoRESUMO
Cisplatin is one of the most effectively used chemotherapeutic agents for cancer treatment. However, in humans, important cytotoxic side effects are observed including dose-limiting renal damage and profound gastrointestinal symptomatology. The toxic responses to cisplatin in mice are similar to those in human patients. Here, we evaluated whether the acid sphingomyelinase (Asm) mediates at least some of the toxic in vivo effects of cisplatin. To this end, we determined the toxic effects of a single intraperitoneal dose of cisplatin (27 mg/kg) in wild type (Asm(+/+)) and Asm-deficient mice (Asm(-/-)). Tissue injury and apoptosis were determined histologically on hematoxylin-eosin and TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated nick end labeling) stainings 3, 12, 36 and 72 h after treatment. Our results revealed severe toxicity of cisplatin in Asm(+/+) mice with increased numbers of apoptotic cells in the thymus and small intestine. In marked contrast, Asm(-/-) mice were resistant to cisplatin and no apoptosis was observed in these organs after treatment. Moreover, cisplatin treatment primarily triggered apoptosis of endothelial cells in microvessels of intestine and thymus, an effect that was absent in mice lacking Asm. The data thus suggest that at least some toxic effects of cisplatin are mediated by the Asm in vivo resulting in early death of endothelial cells and consecutive organ damage.
Assuntos
Antineoplásicos/efeitos adversos , Cisplatino/efeitos adversos , Citoproteção/genética , Gastroenteropatias/induzido quimicamente , Trato Gastrointestinal/efeitos dos fármacos , Esfingomielina Fosfodiesterase/genética , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Cisplatino/farmacologia , Citoproteção/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Gastroenteropatias/patologia , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/patologia , Genes p53 , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Camundongos Knockout , Esfingomielina Fosfodiesterase/fisiologiaRESUMO
We have previously described benzamide derivatives that inhibited tumor necrosis factor (TNF) production from activated macrophages (Mphi) probably by interacting with a protein kinase C (PKC)-dependent pathway. To investigate their mode of action further, we first tested their effect on isolated PKC in vitro, using the selective inhibitor bisindolylmaleimide (BIM) as a positive control. We found that our representative compound JM34 did not inhibit PKC activity in vitro. We then investigated pathways located downstream of PKC and focused on the Raf1/MEK1,2/Erk1,2 cascade known to be preferentially activated by PKC activators such as phorbol esters. We found that JM34 dose-dependently inhibited Erk2 phosphorylation in Mphi stimulated by phorbol dibutyrate and calcium ionophore (maximal inhibition of 85% at 300 microM). BIM at 3 microM totally abrogated Erk2 phosphorylation. After stimulation with endotoxin or zymosan, Erk2 phosphorylation was only partially inhibited (25-30%) by JM34 or BIM, which confirmed that PKC-independent events were also involved in Erk2 phosphorylation. Because activated Erk2 has been shown to activate phospholipase A2, we tested the effect of JM34 and BIM on the release of arachidonate metabolites from activated Mphi. We found that both products partially inhibited the release of arachidonate metabolites from zymosan-activated Mphi at levels comparable to their inhibition of Erk2 phosphorylation. In contrast, JM34 and BIM markedly differed in their ability to inhibit TNF production. Taken together, our results suggest that JM34 inhibited the PKC-dependent pathway of Erk2 phosphorylation, which may fully account for its inhibitory effect on phospholipase A2 activation. However, the inhibition of TNF release by JM34 probably involved inhibition of an additional pathway, distinct from the Erk1/Erk2 cascade.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Benzamidas/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Macrófagos/efeitos dos fármacos , Proteína Quinase C/fisiologia , Animais , Ácido Araquidônico/metabolismo , Diglicerídeos/fisiologia , Relação Dose-Resposta a Droga , Indóis/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Maleimidas/farmacologia , Proteína Quinase 1 Ativada por Mitógeno , Fosforilação , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Expression of multidrug-resistance-associated protein (MRP), a drug efflux pump transporting a wide range of xenobiotics, including anti-cancer drugs and chemical carcinogens, and present at low levels in normal hepatocytes, was investigated in rat hepatoma cells. Northern-blot analysis allowed detection of high levels of MRP mRNA in rat diethylnitrosamine-induced hepatocarcinomas when compared with normal liver. Similarly, elevated expression of MRP transcripts were evidenced in 6 rat hepatoma cell lines of different origins, especially in HTC cells, that, in contrast, failed to express mRNA of the canalicular multispecific organic anion transporter (cMOAT), an efflux pump sharing numerous substrates with MRP. HTC cells were also found by Western blotting to display much higher amounts of MRP than those observed in normal hepatocytes. In contrast, the MRP gene copy number was similar both in hepatoma HTC cells and in hepatocytes, as assessed by Southern blotting. Analysis of MRP-related transport using 3 types of MRP substrates, namely, the fluorescent glutathione-bimane, the anionic dye calcein and the cationic anti-cancer drug vincristine, demonstrated that HTC cells displayed cellular efflux of these 3 compounds, an efflux strongly inhibited by MRP modulators such as indomethacin. These results indicate that MRP is over-expressed and functional in rat hepatoma cells and may therefore be included in the de-toxifying pathways that are altered during hepatocarcinogenesis and are thus thought to contribute to the known multidrug resistance of liver tumors.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Neoplasias Hepáticas Experimentais/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Indometacina/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Probenecid/farmacologia , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Células Tumorais Cultivadas , Verapamil/farmacologia , Vincristina/farmacocinéticaRESUMO
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.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/efeitos dos fármacos , Arsenitos/farmacologia , Hepatócitos/efeitos dos fármacos , Proteínas de Membrana Transportadoras , Proteínas Associadas à Resistência a Múltiplos Medicamentos , RNA Mensageiro/efeitos dos fármacos , Compostos de Sódio/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Tartarato de Antimônio e Potássio/farmacologia , Arseniatos/farmacologia , Cloreto de Cádmio/farmacologia , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Hepatócitos/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno , Masculino , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína 2 Associada à Farmacorresistência Múltipla , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Esquistossomicidas/farmacologia , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologiaRESUMO
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.
Assuntos
Regulação da Expressão Gênica , Proteínas de Membrana Transportadoras , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , Animais , Carcinógenos/farmacologia , Membrana Celular/metabolismo , Colestase/metabolismo , Glucocorticoides/farmacologia , Hormônios/metabolismo , Humanos , Fígado/citologia , Fígado/efeitos dos fármacos , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Receptor de Pregnano X , RNA Mensageiro/metabolismo , Ratos , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Transcrição Gênica , Xenobióticos/farmacologiaRESUMO
12(S)-Hydroxyeicosatetraenoic acid (12-HETE), the 12-lipoxygenase-derived metabolite of arachidonic acid, was incorporated into membrane phospholipids (PL) in various cells. PL are precursors of diacylglycerol (DAG), a protein kinase C (PKC) activator involved in cell-growth signaling. We studied 12-HETE incorporation into PL in non-transformed (NT-) and spontaneously transformed (T-) rat liver epithelial cells (RLEC), and its consequence on DNA synthesis. NT- and T-RLEC incorporated 12-HETE predominantly into phosphatidylcholine (PC). 12-HETE was incorporated at a greater rate, and with a higher phosphatidylethanolamine (PE)/PC ratio in T-cells. Preincubation of RLEC with 12-HETE at > or = 0.5 microM partially inhibited basal DNA synthesis in NT- and T-RLEC. Preincubation of NT-RLEC with 12-HETE (2.5 microM) also decreased bradykinin-stimulated DNA synthesis. Unstimulated RLEC produced 1-acyl-2-(12-HETE)DAG which was increased in NT-RLEC by bradykinin stimulation. Finally, 1-stearoyl-2-(12-HETE)DAG was as potent an in vitro PKC activator as 1-stearoyl-2-arachidonyl-DAG. These data demonstrate that 12-HETE incorporation into PL resulted in the production of active 12-HETE-containing DAG, together with reduced DNA synthesis.
Assuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Diglicerídeos/biossíntese , Fígado/citologia , Fosfolipídeos/biossíntese , Animais , Divisão Celular/efeitos dos fármacos , DNA/biossíntese , Ativação Enzimática , Células Epiteliais , Epitélio/metabolismo , Humanos , Recém-Nascido , Fígado/metabolismo , Proteína Quinase C/metabolismo , RatosRESUMO
Expression of the canalicular multispecific organic anion transporter (cMOAT), an efflux pump involved in biliary secretion of xenobiotics, was investigated in rat hepatocytes exposed to the chemopreventive agent oltipraz. Northern blotting indicated that this compound increased cMOAT mRNA levels in primary cultured hepatocytes. Such an induction of cMOAT transcripts was demonstrated to be dose-dependent and started as early as 4 h treatment; in addition, western blotting showed increased levels of 190 kDa cMOAT in oltipraz-treated primary rat hepatocytes when compared with their untreated counterparts. In contrast, administration of oltipraz to rats failed to enhance hepatic cMOAT mRNA and protein amounts whereas it was found to induce liver expression of glutathione S-transferase P1, a well-known oltipraz-regulated drug metabolizing enzyme. These data therefore suggest that cMOAT up-regulation occurring in rat hepatocytes in response to oltipraz may be restricted to in vitro situations and is therefore unlikely to be directly involved in the in vivo chemopreventive properties of oltipraz.
Assuntos
Anticarcinógenos/farmacologia , Proteínas de Transporte/genética , Fígado/efeitos dos fármacos , Pirazinas/farmacologia , Animais , Proteínas de Transporte de Ânions , Células Cultivadas , Fígado/metabolismo , Masculino , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tionas , Tiofenos , Regulação para CimaRESUMO
Using Rb+ as a K+ tracer and atomic absorption spectrophotometry for measuring the Rb+ stable isotope, we studied K+ transport systems and their regulation by protein kinase C in nontransformed and spontaneously transformed rat liver epithelial cells. Ouabain-sensitive Na+/K(+)-ATPase and the furosemide-sensitive Na+/K+/Cl- cotransport had comparable activity ratios in both cell types (0.92 and 1 in nontransformed and transformed rat liver epithelial cells, respectively). The protein kinase C activators, dioctanoylglycerol and phorbol myristate acetate, partly inhibited the Na+/K+/Cl- cotransport in both cell types but their effect was stronger in nontransformed cells, suggesting that, in transformed cells, the Na+/K+/Cl- cotransport had partly lost the ability to be inhibited by protein kinase C. In both cell types, phorbol myristate acetate had little and dioctanoylglycerol had no inhibitory effect on Na+/K(+)-ATPase. Furosemide (1 mM) partly inhibited the [3H]thymidine incorporation in both cell types, suggesting an involvement of the Na+/K+/Cl- cotransport in rat liver epithelial cell growth.