verlukast and Lung-Neoplasms

Non-small cell lung cancers (NSCLCs) frequently exhibit resistance to therapeutic drugs, which seriously hampers their treatment. Here, we set out to assess the roles of the multidrug resistance protein 1 (MRP1) and P-glycoprotein (P-gp) in the doxorubicin (DOX) resistance of NSCLC cells, as well as the putative therapeutic efficacy of MRP1 and P-gp blockers on DOX-treated NSCLC cells.. The impact of DOX on cell survival, DOX efflux and MRP1 and P-gp expression was assessed in 5 different NSCLC-derived cell lines (parental CH27, A549, H1299, H460, and DOX resistant CH27) in the absence or presence of MK571 (MRP1 inhibitor) or Verapamil (P-gp inhibitor), under both normoxic and hypoxic conditions.. We found that in response to DOX treatment, NSCLC cells that express high levels of MRP1 and P-gp (such as CH27) showed a better DOX efflux and a higher DOX resistance. MK571 and Verapamil were found to abolish DOX resistance and to act as chemosensitizers for DOX therapy in all cell lines tested. We also found that hypoxia could inhibit MRP1 and P-gp expression in a HIF-1α-dependent manner, abolish DOX resistance and boost the chemosensitizer effect of MK571 and Verapamil on DOX treatment of all the NSCLC cells tested, except the DOX-resistant CH27 cells.

Topics: Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Hypoxia; Cell Line, Tumor; Doxorubicin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Flow Cytometry; Humans; Lung Neoplasms; Multidrug Resistance-Associated Proteins; Propionates; Quinolines; Verapamil

Integration and miniaturization are main advantages of microchip-based systems. Vertical integration of the multiple operations within a multiple-layer chip is expected to satisfy the urgent demand for high-throughput and large-scale applications. This study aimed at establishing a double-layer chip to integrate the operations including the cell culture, the identification of the protein and the detection of the cell viability onto a platform systematically and supplied with flow fresh medium continuously via a syringe pump to mimic the microenvironment in vivo. With this device, human non-small cell lung cancer cell line (SPCA-1) was cultured well; the expression and the activity of multidrug resistance-associated protein (MRP1) were detected by immunofluorescence assay for the cells pretreated with or without MK-571, a known inhibitor of MRP1; apoptosis percentages were assayed for the cells after being treated by the anticancer drug etoposide (VP-16). The results demonstrated that the function of the MRP1 was inhibited by MK-571, and the percentage of apoptotic for the cells pretreated with MK-571 was higher than that of the control (38.2±2.5% versus 12.3±0.85%, p<0.005). All these indicated that the new device could provide a suitable condition for cell culture and functional analysis in biomedical research, and MK-571 is an effective inhibitor of MRP1 associated with the viability of SPCA-1 cell line treated by VP-16.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Survival; Doxorubicin; Drug Resistance, Neoplasm; Enzyme Inhibitors; Equipment Design; Etoposide; Humans; Lung Neoplasms; Microfluidic Analytical Techniques; Microscopy, Fluorescence; Multidrug Resistance-Associated Proteins; Propionates; Quinolines; Rhodamine 123

Defense against toxic endo- and xenobiotics is a major concern of all living species and ABC transporters play a vital role in this defense system. Multidrug resistance associated proteins 1 (MRP1) is a cellular detoxifying factor supposed to transport a wide range of compounds across cell membranes either as GSH conjugates or as co-transport accompanying glutathione transposition. The cellular localization of MRP1 is a determining factor whether the transport function can take place. In this study we have undertaken experiments on the transport activity of MRP1 in cultured human lung tumor cells in order to check whether MRP1 is expressed as a functionally active protein. For this purpose we have adapted a quantitative fluorescence imaging assay to conditions where a small number of attached cells should be repeatedly measured by a non-destructive method. In cultured A549, H358 and H322 cells MRP1 is located in the cell membrane as observed by immunocytochemistry. Efflux of 5,6-carboxy-2'-7'-dichloro-fluorescein (CDF) from lung cells was sensitive toward the MRP1 inhibitor MK571 while verapamil had no effect. On the other hand, efflux of Rhodamin 123, a Pgp-glycoprotein substrate, from lung cells reacted to inhibition by verapamil, while MK571 had no effect. Modulation of glutathion content of lung cells by N-acetyl cystein and buthionine sulfoximine shifted CDF efflux toward higher or lower rates, respectively. These experiments confirm that MRP1 function can be followed in the attached cells in vitro under non-toxic concentrations of the substrates without the need to harvest and destroy the cells.

Topics: Acetylcysteine; ATP-Binding Cassette Transporters; Biological Assay; Biological Transport; Buthionine Sulfoximine; Cell Culture Techniques; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fluoresceins; Fluorescent Dyes; Fluorometry; Glutathione; Humans; Immunohistochemistry; Lung Neoplasms; Multidrug Resistance-Associated Proteins; Propionates; Quinolines; Sensitivity and Specificity; Verapamil

The present study examined how the multidrug resistance protein (MRP1), which is an ATP-dependent anionic conjugate transporter, also mediates the transport of reduced glutathione (GSH) and the co-transport of the cationic drug, daunorubicin, with GSH in living GLC4/Adr cells. To obtain information on the affinity of GSH for the multidrug resistance protein in GLC4/Adr cells, we investigated the GSH concentration dependence of the ATP-dependent GSH efflux. The intracellular GSH concentration was modulated by preincubation of the cells with 25 microM buthionine sulfoximine, an inhibitor of GSH synthetase, for 0-24 h. The transport of GSH was related to the intracellular GSH concentration up to approximately 5 mM and then plateaued. Fitting of the obtained data according to the Michaelis-Menten equation revealed a Km of 3.4+/-1.4 mM and a Vmax of 1.5+/-0.2x10(-18) mol/cell/s. The ATP-dependent transport of GSH was inhibited by 3-([[3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl]-[(3-dimethylamino-3-oxopropyl)-thio]-methyl]thio)propanoic acid (MK571), with 50% inhibition being obtained with 1.4 microM MK571. We investigated the GSH concentration dependence of the MRP1-mediated ATP-dependent transport of daunorubicin under conditions where the transport of daunorubicin became saturated. The daunorubicin transport was related to the intracellular GSH concentration up to approximately 5 mM and then plateaued. We were therefore in the situation where GSH acted as an activator: its presence was necessary for the binding and transport of daunorubicin by MRP1. However, GSH was also transported by the multidrug resistance protein. The concentration of GSH that gave half the maximal rate of daunorubicin efflux was 2.1+/-0.8 mM, very similar to the Km value obtained for GSH. In conclusion, the rate of daunorubicin efflux, under conditions where the transport of daunorubicin became saturated, and the rate of GSH efflux determined at any intracellular concentration of GSH were very similar, yielding a 1:1 stoichiometry with respect to GSH and daunorubicin transport. These results support a model in which daunorubicin is co-transported with GSH.

Topics: ATP-Binding Cassette Transporters; Biological Transport; Carcinoma, Small Cell; Cyclic P-Oxides; Daunorubicin; Dose-Response Relationship, Drug; Extracellular Space; Gene Expression Regulation, Neoplastic; Glutathione; Humans; K562 Cells; Kinetics; Lung Neoplasms; Multidrug Resistance-Associated Proteins; Nicotinic Acids; Propionates; Quinolines; Time Factors; Tumor Cells, Cultured; Vinblastine

4-Hydroxynonenal (4HNE) is the most prevalent toxic lipid peroxidation product formed during oxidative stress. It exerts its cytotoxicity mainly by the modification of intracellular proteins. The detection of 4HNE-modified proteins in several degenerative disorders suggests a role for 4HNE in the onset of these diseases. Efficient protection mechanisms are required to prevent the intracellular accumulation of 4HNE. The toxicity of 4HNE was tested with the small cell lung cancer cell lines GLC(4) and the multidrug-resistance-protein (MRP1)-overexpressing counterpart GLC(4)/Adr. In the presence of the MRP1 inhibitor MK571 or the GSH-depleting agent buthionine sulphoximine, both cell lines became more sensitive and showed decreased survival. Transport experiments were performed with the (3)H-labelled glutathione S-conjugate of 4HNE ([(3)H]GS-4HNE) with membrane vesicles from GLC(4)-derived cell lines with different expression levels of MRP1. [(3)H]GS-4HNE was taken up in an ATP-dependent manner and the transport rate was dependent on the amount of MRP1. The MRP1 inhibitor MK571 decreased [(3)H]GS-4HNE uptake. MRP1-specific [(3)H]GS-4HNE transport was demonstrated with membrane vesicles from High Five insect cells overexpressing recombinant MRP1. Kinetic experiments showed an apparent K(m) of 1.6+/-0.21 microM (mean+/-S.D.) for MRP1-mediated [(3)H]GS-4HNE transport. In conclusion, MRP1 has a role in the protection against 4HNE toxicity and GS-4HNE is a novel MRP1 substrate. MRP1, together with GSH, is hypothesized to have a role in the defence against oxidative stress.

Topics: Adenosine Triphosphate; Aldehydes; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP-Binding Cassette Transporters; Biological Transport; Buthionine Sulfoximine; Carcinoma, Small Cell; Cell Line; Cell Survival; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glutathione; Humans; Immunoblotting; Insecta; Kinetics; Leukotriene Antagonists; Lipid Peroxidation; Lung Neoplasms; Multidrug Resistance-Associated Proteins; Oxidative Stress; Propionates; Quinolines; Recombinant Proteins; Time Factors; Tumor Cells, Cultured

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.

Topics: Antimony; Antineoplastic Agents; Arsenites; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP-Binding Cassette Transporters; Blotting, Western; Cadmium Chloride; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Fluoresceins; Gene Amplification; Humans; Inhibitory Concentration 50; Lung Neoplasms; Meglumine; Metallothionein; Metals, Heavy; Multidrug Resistance-Associated Proteins; Propionates; Quinolines; RNA, Messenger; Tumor Cells, Cultured; Up-Regulation; Zinc Sulfate

Multidrug resistance-associated protein (MRP) is a transport system that is involved in the elimination of xenobiotics and biologically active endogenous substrates. Recently, the presence of MRP has been demonstrated in cultured brain capillary endothelial cells (BCECs). The time-dependent, functional expression of MRP in porcine BCECs was investigated to assess the value of this cell culture model for drug transport at the blood-brain barrier. Western blot analysis was used to investigate MRP expression in freshly isolated porcine BCECs and compared to MRP expression at days 8 and 10 in culture. Subcellular localization of MRP was investigated by immunocytochemistry with an MRP-specific monoclonal antibody, MRPr1. Functional activity of MRP was assessed by efflux studies with the fluorescent MRP substrate glutathione-methylfluorescein (GS-MF). No significant MRP expression was detected in freshly isolated endothelial cells. However, MRP expression is up-regulated in cell culture in a time-dependent manner. Immunostaining revealed predominantly perinuclear and, to a lesser degree, plasma membrane localization of MRP. At 10 degrees C GS-MF efflux was significantly decreased, indicating the involvement of an energy-dependent transport system. Efflux of GS-MF was apparently inhibited by MK571, a specific inhibitor for MRP. Porcine BCECs demonstrate up-regulation of functional MRP expression during culture, as observed in human tissue, and therefore might serve as a useful in vitro system for studying MRP-mediated blood-brain barrier transport.

Topics: Animals; ATP-Binding Cassette Transporters; Blotting, Western; Brain Chemistry; Bronchodilator Agents; Capillaries; Carcinoma, Small Cell; Endothelium, Vascular; Humans; Immunohistochemistry; In Vitro Techniques; Lung Neoplasms; Multidrug Resistance-Associated Proteins; Propionates; Quinolines; Subcellular Fractions; Swine; Tumor Cells, Cultured; Up-Regulation

MRP is a member of the ABC trafficking proteins thought to mediate the transport of glutathione S-conjugates and amphiphilic natural products. However, unlike P-glycoprotein, the biochemical mechanism by which MRP mediates the resistance to cytotoxic drugs is not clear. In this report, we describe the interactions of a quinoline-based drug, N-{4-[1-hydroxy-2-(dibutylamino)ethyl] quinolin-8-yl}-4-azidosalicylamide (IAAQ), with MRP. Our results demonstrate the ability of IAAQ to photoaffinity label a 190 kDa protein in resistant Small Cell Lung Cancer cells (H69/AR) but not in the parental H69 cells. The photoaffinity labeling of the 190 kDa protein with IAAQ was both saturable and specific. The identity of the 190 kDa protein, as MRP, was confirmed by immunoprecipitation with the monoclonal antibody, QCRL-1. Furthermore, a molar excess of LTC4, MK 571 or vinblastine inhibited the photoaffinity labeling of MRP with IAAQ in intact cells and plasma membranes. Cell growth and drug transport studies showed H69/AR cells to be less sensitive to and to accumulate less IAAQ than the parental H69 cells. In addition, MK 571 and doxorubicin increased the sensitivity to and the accumulation of IAAQ in H69/AR cells. Together, the results of this study show for the first time the direct binding of unaltered cytotoxic drug to MRP. Moreover, given the structural similarities between IAAQ and MK 571, we suggest that MK 571 modulates MRP-mediated resistance by direct binding to MRP.

Topics: Affinity Labels; Antibodies, Monoclonal; ATP-Binding Cassette Transporters; Binding Sites; Carcinoma, Small Cell; Cell Division; Doxorubicin; Drug Resistance, Multiple; Humans; Kinetics; Lung Neoplasms; Multidrug Resistance-Associated Proteins; Propionates; Quinolines; Salicylates; Tumor Cells, Cultured; Vinblastine

The multidrug resistant cell lines HL60/AR and GLC4/ADR show high overexpression of the gene encoding the multidrug resistance associated protein MRP compared to their drug sensitive parental counterparts. This and the virtual absence of mdr1/P-glycoprotein gene expression was proven by a complementary DNA polymerase chain reaction (cDNA-PCR) approach. Applying a 72-hour tetrazolium based colorimetric MTT-assay we demonstrate on both MDR sublines a dose-dependent modulation of drug resistances by the leukotriene LTD4 receptor antagonist MK571. A complete reversal of vincristine resistances was achieved at final MK571 concentrations of 30 microM (HL60/AR) or 50 microM (GLC4/ADR) which by itself did not disturb cellular proliferation. The drug resistance of a mdr1/P-gp overexpressing multidrug-resistant HL60 subline, in contrast, was not significantly affected by MK571. Similar effects were seen using the glutathione (GSH) synthesis inhibitor buthionine sulfoximine (BSO). Our results point to a relationship between MRP and a conjugate transporter and identify MK571 as a new tool structure for developing modulators specific for a MRP associated multidrug resistance.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP-Binding Cassette Transporters; Base Sequence; Carcinoma, Small Cell; Cell Line; Cell Survival; Clone Cells; Daunorubicin; DNA Primers; Dose-Response Relationship, Drug; Drug Resistance, Multiple; Gene Expression; Humans; Leukemia, Promyelocytic, Acute; Leukotriene Antagonists; Lung Neoplasms; Membrane Proteins; Molecular Sequence Data; Multidrug Resistance-Associated Proteins; Polymerase Chain Reaction; Propionates; Quinolines; Receptors, Immunologic; Receptors, Leukotriene; Tumor Cells, Cultured; Vincristine