Action of calcium antagonists on multidrug resistant cells

Cano‐Gauci, Danielle F.; Riordan, John R.

doi:10.1016/0006-2952(87)90139-0

Cited by 133 publications

(24 citation statements)

References 23 publications

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“…Colchicine did not inhibit P-gp photolabeling with 125I-NASAV and also did not compete with vinblastine photoaffinity labeling (9), indicating the possible existence of a different drug binding site or different P-gp for this agent that is not affected by verapamil or vinblastine or perhaps the presence of a colchicine acceptor that is not related to P-gp. These results are consistent with the demonstration that verapamil was not effective in altering colchicine accumulation by MDR cells (32). Similarly, the effect of verapamil on reversing Vinca alkaloid resistance was shown to be much greater than its effect on other natural product drugs against which cells express cross-resistance (33).…”

Section: Discussionsupporting

confidence: 80%

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Photoaffinity labeling of the multidrug-resistance-related P-glycoprotein with photoactive analogs of verapamil.

Safa¹

1988

Proc. Natl. Acad. Sci. U.S.A.

184

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Verapamil, a phenylalkylamine calcium channel blocker, has been shown to reverse multidrug resistance in tumor cells, possibly by increasing drug retention through interaction with an outward drug transporter of the resistant cells. In this study two photoactive radioactive analogs of verapamil, N-(p-azido[3,5-31H]benzoyl)aminomethyl verapamil and N-(p-azido[3-'25I]salicyl)aminomethyl verapamil, were synthesized and used to identify the possible biochemical target(s) for verapamil in multidrug-resistant DC-3F/VCRd-5L Chinese hamster lung cells selected for resistance to vincristine. The results show that a specifically labeled 150-to 180-kDa membrane protein in resistant cells was immunoprecipitated with a monoclonal antibody specific for Pglycoprotein. Phenylalkylamine binding specificity was established by competitive blocking ofspecific photolabeling with the nonradioactive photoactive analogs as well as with verapamil.Photoaffinity labeling was also inhibited by 50 jzM concentrations of the calcium channel blockers nimodipine, nifedipine, nicardipine, azidopine, bepridil, and diltiazem and partially by prenylamine. Bay K8644, a calcium channel agonist, also inhibited P-glycoprotein photolabeling. Moreover, P-glycoprotein labeling was inhibited in a dose-dependent manner by vinblastine with half-maximal inhibition at 0.2 jiM compared to that by verapamil at 8 jiM. Photolabeling was also partially inhibited by two of the drugs to which these cells are crossresistant, doxorubicin and actinomycin D, at 100 jM, but not by colchicine. These data provide direct evidence that Pglycoprotein has broad drug recognition capacity and that it serves as a molecular target for calcium channel blocker action in reversing multidrug resistance.Multidrug resistance (MDR) refers to patterns of crossresistance that develop in tumor cells selected by using a single natural-product drug. Exposure to agents such as vinblastine, vincristine, doxorubicin, or colchicine confers resistance to a wide range of compounds with no apparent structural or functional similarities (1, 2) to the selective agent. MDR is frequently characterized by diminished drug accumulation in resistant cells compared to drug-sensitive cells (3,4). This reduced accumulation often correlates with the concomitant over-expression of a 150-to 180-kDa integral membrane glycoprotein, P-glycoprotein (P-gp) or gp150-180, which is produced in MDR cells in proportion to their level of drug resistance (5-7). Experiments with vinblastine photoactive analogs revealed the specific interaction of vinblastine with P-gp in plasma membranes from MDR cells and suggested that this protein may mediate cellular drug accumulation by binding to drugs and regulating their membrane transport (8,9). Based on sequence data, it has been shown that significant homology exists between P-gp and bacterial transport proteins, that P-gp has two nucleotide binding domains (10-12) that bind ATP (13), that the gene coding for this protein generates the MDR phenotype when transfected into dru...

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Section: Discussionsupporting

confidence: 80%

“…Such binding may result in the active efflux of verapamil (18,32) and may account for decreased verapamil accumulation in MDR cells (32). Interestingly, despite this reduced accumulation, MDR cells are more sensitive to verapamil than are parental sensitive cells (37,38).…”

Section: Discussionmentioning

confidence: 99%

Photoaffinity labeling of the multidrug-resistance-related P-glycoprotein with photoactive analogs of verapamil.

Safa¹

1988

Proc. Natl. Acad. Sci. U.S.A.

184

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“…Drug Cytotoxicity Assays-Cytotoxicity assays were based on published methods (Cano-Gauci and Riordan, 1987). Briefly, cells were seeded in 24-well dishes at a density of 4 ϫ 10 3 cells/well in 1 ml of DMEM with 10% fetal calf serum.…”

Section: Cloning Of the Mutated Mdr1 Genes Into A Yeast Expression Vementioning

confidence: 99%

Protein Kinase C-mediated Phosphorylation Does Not Regulate Drug Transport by the Human Multidrug Resistance P-glycoprotein

Goodfellow

Sardini

Ruetz

et al. 1996

Journal of Biological Chemistry

116

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P-glycoprotein (P-gp) is an active transporter that can confer multidrug resistance by pumping cytotoxic drugs out of cells and tumors. P-gp is phosphorylated at several sites in the "linker" region, which separates the two halves of the molecule. To examine the role of phosphorylation in drug transport, we mutated P-gp such that it could no longer be phosphorylated by protein kinase C (PKC). When expressed in yeast, the ability of the mutant proteins to confer drug resistance, or to mediate [ 3 H]vinblastine accumulation in secretory vesicles, was indistinguishable from that of wild type P-gp. A matched pair of mammalian cell lines were generated expressing wild type P-gp and a non-phosphorylatable mutant protein. Mutation of the phosphorylation sites did not alter P-gp expression or its subcellular localization. The transport properties of the mutant and wild type proteins were indistinguishable. Thus, phosphorylation of the linker of P-gp by PKC does not affect the rate of drug transport. In light of these data, the use of agents that alter PKC activity to reverse multidrug resistance in the clinic should be considered with caution.

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“…substances that are bound by P-glycoprotein and inhibit its drug excluding function (3). The medically important substrates of P-glycoprotein comprise anticancer drugs such as Vinca alkaloids and anthracyclines (2)(3)(4) and approved drugs that turned out to be potent modulators such as verapamil (5,6), calcium antagonists (7), and cyclosporins (8).…”

mentioning

confidence: 99%

Localization of the Iodomycin Binding Site in Hamster P-glycoprotein

Demmer¹,

Thole²,

Kubesch³

et al. 1997

Journal of Biological Chemistry

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P-glycoprotein, the overexpression of which is a major cause for the failure of cancer chemotherapy in man, recognizes and transports a broad range of structurally unrelated amphiphilic compounds. This study reports on the localization of the binding site of P-glycoprotein for iodomycin, the Bolton-Hunter derivative of the anthracycline daunomycin. 125 I]iodomycin had been predominantly incorporated into the fragment 230 -312 of isoform I of hamster P-glycoprotein. According to models based on hydropathy plots, the amino acid sequence 230 -312 forms the distal part of transmembrane segment 4, the second cytoplasmic loop, and the proximal part of transmembrane segment 5 in the Nterminal half of P-glycoprotein. The binding site for iodomycin is recognized with high affinity by vinblastine and cyclosporin A. P-glycoprotein, which belongs to the large family of ABC 1 transporters (1), binds and transports a broad range of structurally unrelated compounds (2). Its overexpression may cause the phenomenon of multidrug resistance (MDR) during cancer chemotherapy, whereby the tumor cells become resistant to a variety of antineoplastic agents due to a reduced intracellular accumulation of drugs (2-4). The MDR phenotype can be overcome by modulators, i.e. substances that are bound by P-glycoprotein and inhibit its drug excluding function (3). The medically important substrates of P-glycoprotein comprise anticancer drugs such as Vinca alkaloids and anthracyclines (2-4) and approved drugs that turned out to be potent modulators such as verapamil (5, 6), calcium antagonists (7), and cyclosporins (8).The major issue of how P-glycoprotein can handle so many substrates has been mainly approached by photoaffinity labeling and mutagenesis studies. Mutants that arose spontaneously during drug selection of cells and thereby changed their resistance profile were detected in the first cytoplasmic loop of human MDR1 protein (Gly 185 3 Val) (9) and in transmembrane segment (TM) 6 of hamster P-glycoprotein (Gly 338 3 Ala/Ala 339 3 Pro) (10). Active mutagenesis identified further motifs and positions in the N-and C-terminal half of P-glycoprotein which influence substrate specificity (11, 12). For example, the exchange of amino acids in the first cytoplasmic loop (11), TM5, TM6, TM10, TM12 (13-16), and the cytosolic linker peptide (17) resulted in all in an altered multidrug resistance phenotype, suggesting that during binding and transport the substrate is recognized by multiple residues located either in the cytosolic, membraneous, or ectoplasmic domains.Hydropathy plots deduced from the primary sequence predict that P-glycoprotein consists principally of two symmetrical halves, each of which contain a membrane domain with six membrane-spanning segments and a subsequent cytosolic nucleotide binding fold (18 -20). The topology of P-glycoprotein in vivo, however, may be more variable than predicted, e.g. under certain experimental conditions, TM segments were detected outside the membrane and loops postulated to face the cytosol were foun...

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Action of calcium antagonists on multidrug resistant cells

Cited by 133 publications

References 23 publications

Photoaffinity labeling of the multidrug-resistance-related P-glycoprotein with photoactive analogs of verapamil.

Photoaffinity labeling of the multidrug-resistance-related P-glycoprotein with photoactive analogs of verapamil.

Protein Kinase C-mediated Phosphorylation Does Not Regulate Drug Transport by the Human Multidrug Resistance P-glycoprotein

Localization of the Iodomycin Binding Site in Hamster P-glycoprotein

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