leukotriene-c4 and Carcinoma--Small-Cell

Increased active transport of LTC(4) observed frequently in multidrug-resistant cancer cells have been attributed to ABC-transporter proteins particularly, MRP1. We have demonstrated recently that a novel non-ABC transporter, RLIP76 (RALBP1) can also mediate ATP-dependent transport of GSH-conjugates (GS-E) as well as doxorubicin (DOX). We demonstrate RLIP76 reconstituted in artificial liposomes can catalyze ATP-dependent transport of LTC(4), which can be modulated by PKC-alpha. The ATPase activity of E. coli expressed homogenous RLIP76 was stimulated in a saturable fashion by LTC(4) with half maximal stimulation at 130 nM. Proteoliposomes reconstituted with RLIP76 catalyzed temperature and osmolar sensitive ATP-dependent transport of LTC(4) with K(m) values of 5.1 mM and 210 nM for ATP and LTC(4), respectively. V(max) for transport was found to be 3.2 nmol/min/mg. Colchicine inhibited LTC(4) transport to 50% at 5.8 microM. PKC-alpha catalyzed phosphorylation of RLIP76 and increased its transport activity by 2-3-fold. Membrane vesicles prepared from the small (SCLC) and non-small (NSCLC) lung cancer cell lines as well as HL-60 (leukemia) and U937 (lymphoma) cell lines exhibited ATP-dependent transport of LTC(4), which was inhibited by anti-RLIP76 antibodies. The rate of transport of LTC(4) in SCLC (H69, H378) was half of that observed in NSCLC cell lines but after transfection with RLIP76, the transport rate of LTC(4) in H69 became comparable to that in NSCLC cell lines. Anti-RLIP76 antibodies inhibited LTC(4) transport by 67-81% in all 8 cell lines examined, whereas N-19 anti-MRP1 antibodies inhibited transport of LTC(4) by only 11-26%. These results suggest that RLIP76 is the major LTC(4) transporter in cancer cells and that its transport activity is regulated by PKC-alpha-mediated phosphorylation.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; ATP-Binding Cassette Transporters; Biological Transport; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Cell Line, Tumor; Colchicine; Drug Resistance, Neoplasm; Electrophoresis, Polyacrylamide Gel; GTPase-Activating Proteins; HL-60 Cells; Humans; Leukotriene C4; Mass Spectrometry; Phosphorylation; Protein Kinase C; U937 Cells

The 190-kDa multidrug resistance protein (MRP) has recently been associated with the transport of cysteinyl leukotrienes and several glutathione (GSH) S-conjugates. In the present study, we have examined the transport of leukotriene C4 (LTC4) in membrane vesicles from MRP-transfected HeLa cells (T14), as well as drug-selected H69AR lung cancer cells which express high levels of MRP. V(max) and K(m) values for LTC4 transport by membrane vesicles from T14 cells were 529 +/- 176 pmol mg(-1) min(-1) and 105 +/- 31 nM, respectively. At 50 nM LTC4, the K(m) (ATP) was 70 micron. Transport in T14 vesicles was osmotically-sensitive and was supported by various nucleoside triphosphates but not by non- or slowly-hydrolyzable ATP analogs. LTC4 transport rates in membrane vesicles derived from H69AR cells and their parental and revertant variants were consistent with their relative levels of MRP expression. A 190-kDa protein in T14 membrane vesicles was photolabeled by [3H]LTC4 and immunoprecipitation with MRP-specific monoclonal antibodies (mAbs) confirmed that this protein was MRP. LTC4 transport was inhibited by an MRP-specific mAb (QCRL-3) directed against an intracellular conformational epitope of MRP, but not by a mAb (QCRL-1) which recognizes a linear epitope. Photolabeling with [3H]LTC4 was also inhibitable by mAb QCRL-3 but not mAb QCRL-1. GSH did not inhibit LTC4 transport. However, the ability of alkylated GSH derivatives to inhibit transport increased markedly with the length of the alkyl group. S-Decylglutathione was a potent competitive inhibitor of [3H]LTC4 transport (K(i(app)) 116 nM), suggesting that the two compounds bind to the same, or closely related, site(s) on MRP. Chemotherapeutic agents including colchicine, doxorubicin, and daunorubicin were poor inhibitors of [3H]LTC4 transport. Taxol, VP-16, vincristine, and vinblastine were also poor inhibitors of LTC4 transport but inhibition by these compounds was enhanced by GSH. Uptake of [3H]vincristine into T14 membrane vesicles in the absence of GSH was low and not dependent on ATP. However, in the presence of GSH, ATP-dependent vincristine transport was observed. Levels of transport increased with concentrations of GSH up to 5 mM. The identification of an MRP-specific mAb that inhibits LTC4 transport and prevents photolabeling of MRP by LTC4, provides conclusive evidence of the ability of MRP to transport cysteinyl leukotrienes. Our studies also demonstrate that MRP is capable of mediating ATP-depe

Topics: Adenosine Triphosphate; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biological Transport; Carcinoma, Small Cell; Cations, Divalent; Cell Line; Cell Membrane; Glutathione; HeLa Cells; Humans; Kinetics; Leukotriene B4; Leukotriene C4; Lung Neoplasms; Membrane Proteins; Osmolar Concentration; Recombinant Proteins; Sucrose; Transfection; Tumor Cells, Cultured; Vinblastine; Vincristine

In addition to its ability to confer resistance to a range of natural product type chemotherapeutic agents, multidrug resistance protein (MRP) has been shown to transport the cysteinyl leukotriene, LTC4, and several other glutathione (GSH) S-conjugates. We now demonstrate that its range of potential physiological substrates also includes cholestatic glucuronidated steroids. ATP dependent, osmotically sensitive transport of the naturally occurring conjugated estrogen, 17 beta-estradiol 17-(beta-D-glucuronide) (E(2)17 beta G), was readily demonstrable in plasma membrane vesicles from populations of MRP-transfected HeLa cells (Vmax 1.4 nmol mg-1 min-1, K(m) 2.5 micron). The involvement of MRP was confirmed by demonstrating that transport was completely inhibited by a monoclonal antibody specific for an intracellular conformational epitope of the protein. MRP-mediated transport of LTC4, was competitively inhibited by E(2)17 beta G (K(i(app)) 22 micron), despite the lack of structural similarity between these two substrates. Competitive inhibition of [3H]E(2)17 beta G transport was also observed with a number of other cholestatic conjugated steroids. All of these compounds prevented photolabeling of MRP with [3H]LTC4, demonstrating that the cholestatic steroid and leukotriene conjugates compete either for the same or possibly overlapping sites on the protein. Consistent with the presence of overlapping but non-identical sites, studies using chemotherapeutic drugs to inhibit MRP-mediated E(2)17 beta G transport indicated that daunorubicin had the highest relative potency of the drugs tested, whereas it was the least potent inhibitor of LTC4 transport. Non-cholestatic steroids glucuronidated at the 3 position of the steroid nucleus, such as 17 beta-estradiol 3-(beta-D-glucuronide), did not compete for transport of E(2)17 beta G by MRP, nor did they inhibit photolabeling of the protein with [3H]LTC4. These data identify MRP as a potential transporter of cholestatic conjugated estrogens and demonstrate site-specific requirements for glucuronidation of the steroid nucleus.

Topics: Adenosine Triphosphate; Animals; Antibodies, Monoclonal; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Bile Acids and Salts; Binding, Competitive; Biological Transport; Carcinoma, Small Cell; Cell Line; Cell Membrane; Epitopes; Estradiol; Glucuronates; HeLa Cells; Humans; Kinetics; Leukotriene C4; Lung Neoplasms; Mice; Protein Conformation; Recombinant Proteins; Transfection; Tumor Cells, Cultured

The ATP-dependent glutathione S-conjugate export pump (GS-X pump) has been suggested to play a role in the mechanism of cisplatin resistance. The purpose of this study was to determine the relationship between intracellular glutathione (GSH) levels and GS-X pump activity and whether GS-X pump overexpression results in cisplatin resistance. We transfected the human gamma-glutamylcysteine synthetase (gamma-GCS) gene into a human small-cell lung cancer cell line, SBC-3, producing SBC-3/GCS. The intracellular GSH content of SBC-3/GCS was twice that of the parental line, its GS-X pump activity was significantly enhanced and cellular cisplatin accumulation decreased. SBC-3/GCS showed higher resistance (relative resistance value of 7.4) to cisplatin than the parental line SBC-3. These data indicate that gamma-GCS gene overexpression induces cellular cisplatin resistance associated with increases in both the GSH content and GS-X pump activity, resulting in reduced cisplatin accumulation. In conclusion, GS-X pump expression is related to cellular GSH metabolism and involved in cisplatin resistance.

Topics: Adenosine Triphosphate; Base Sequence; Carcinoma, Small Cell; Carrier Proteins; Cell Division; Cell Line; Cell Membrane; Cisplatin; Drug Resistance, Neoplasm; Gene Expression; Gene Library; Glutamate-Cysteine Ligase; Glutathione; Humans; Kinetics; Leukotriene C4; Lung Neoplasms; Membrane Transport Proteins; Molecular Sequence Data; Recombinant Proteins; Thymus Gland; Transfection; Tumor Cells, Cultured