Modulation of P-glycoprotein mediated drug accumulation in multidrug resistant CCRF VCR-1000 cells by chemosensitisers.

P-glycoprotein (PGP) mediated transport of cytostatic drugs out of resistant cancer cells is a major cause of experimental and probably also of clinical multidrug resistance, which often leads to treatment failure during chemotherapy. The broad substrate specificity of PGP strongly restricts effective chemotherapy and diminishes the patients' prognosis. Inhibition of PGP's pumping function by chemosensitisers is one way to restore cellular responsiveness to otherwise ineffective cytostatics. Clinical trials with several chemosensitisers are under way. To date, it is not clear whether a certain chemosensitiser potentiates the action of different cytostatic drugs, transported by PGP equally well, or whether the chemosensitising potency is dependent on the cytostatic drugs used. Therefore, we compared the effects of five potent chemosensitisers on cellular accumulation using [3H]daunomycin, [3H]vincristine and rhodamine-123 as substrates for PGP. The acridonecarboxamide derivative GF 120918 was the most potent compound and a half-maximal effect was seen at concentrations ranging from 5 nM for rhodamine-123 accumulation to 14 and 19 nM for [3H]vincristine or [3H]daunomycin accumulation, respectively. The new chemosensitiser B9203-016 was slightly less effective than GF 120918 in all three test systems. Dexniguldipine was of intermediate potency with half-maximal effects at concentrations between 62 and 194 nM. The cyclic undecapeptide SDZ PSC 833 showed somewhat lower potency ranging from 151 to 331 nM. Cyclosporin A was less potent than SDZ PSC 833. Furthermore, enhancement of drug accumulation produced by each chemosensitiser was similar, regardless of which PGP substrate was measured, that is, the rank order of potency to increase accumulation was the same in each of the assays used. Our data point to similar, if not identical, mechanisms of drug transport by PGP and inhibition of drug transport by chemosensitisers at least for the substrates rhodamine-123, vincristine and daunomycin.

Interaction of cytostatics and chemosensitizers with the dexniguldipine binding site on P-glycoprotein.

The interaction of cytostatics and chemosensitizers with the dexniguldipine binding site of P-glycoprotein was investigated in photoaffinity labeling experiments. A tritiated azidoderivative of the chemosensitizer dexniguldipine with dihydropyridine structure, [3H]B9209-005, was used to irreversibly label P-glycoprotein. The apparent affinity of cytostatics and chemosensitizers to this binding site was estimated from labeling experiments in the presence of increasing concentrations of compounds. From the cytostatics tested, the vinca alkaloids and taxol showed the highest affinity, anthracyclins possessed moderate affinity while methotrexate, ara C and camptothecin, cytostatics not involved in P-glycoprotein-mediated multidrug resistance, were almost inactive. The chemosensitizers GF 120918, cyclosporin A and SDZ PSC-833 inhibited photoincorporation with the highest potency. Steep dose-inhibition curves were obtained with the cyclic peptides and S9788, indicating that these compounds may bind allosterically to a separate binding site. Compounds with dihydropyridine structure with or without chemosensitizing potency were also tested and some structure-activity relationships could be derived from the data. Our data show that inhibition of photoaffinity labeling by [3H]B9209-005 is a valuable and reliable system for measuring the interaction with and potency of chemosensitizing compounds at P-glycoprotein. Furthermore, data obtained in this test system are well suited to investigate structure-activity relationships for chemosensitizers at P-glycoprotein. In addition cytostatics underlying P-glycoprotein-mediated multidrug resistance can be identified.

B9209-005, an azido derivative of the chemosensitizer dexniguldipine-HCl, photolabels P-glycoprotein.

P-glycoprotein is an energy-dependent drug extrusion pump for a variety of anticancer drugs and is involved in the development of multidrug resistance in cancer. Dexniguldipine-HCl is a potent chemosensitizer for P-glycoprotein-mediated multidrug resistance in vitro, and clinical phase I/II trials are underway. To investigate the mechanisms of chemosensitization and to identify the binding sites for dexniguldipine-HCl on target proteins involved in chemosensitization, [3H]B9209-005, an azido derivative of dexniguldipine-HCl, was synthesized and used as a photoaffinity ligand. In two models of multidrug resistance reversal, i.e., sensitization to vincristine and modulation of rhodamine-123 uptake, B9209-005 and dexniguldipine-HCl showed identical biological activities. Photoaffinity labeling experiments with [3H]B9209-005 in cell membranes from multidrug-resistant CCRF ADR-5000 cells, in comparison with labeling experiments with [3H]azidopine (an established photoaffinity ligand for P-glycoprotein), showed that [3H]B9209-005 labeled two proteins, with apparent molecular masses of 170 and 95 kDa. The pharmacological specificity of labeling was demonstrated by inhibition of photoincorporation by several cytostatic drugs transported by P-glycoprotein, as well as by chemosensitizers. Immunoprecipitation of the labeled proteins with the P-glycoprotein-specific monoclonal antibody C 219 and with a site-directed polyclonal antibody to the amino-terminal sequence of P-glycoprotein (amino acids 389-406) identified these proteins as intact P-glycoprotein and the amino-terminal fragment thereof. No specific labeling was obtained in the drug-sensitive parent cell line CCRF-CEM, which is devoid of significant P-glycoprotein expression. Maximal labeling of 17 pmol of the 170-kDa protein/mg of crude membrane protein was obtained. The affinity of [3H]B9209-005 for binding to and photoincorporation into P-glycoprotein was 5-fold greater than that of [3H]azidopine, and photoincorporation of [3H]B9209-005 showed a different photoincorporation pattern, compared with [3H]azidopine, in that the latter compound was incorporated specifically into the carboxyl-terminal 55-kDa fragment of P-glycoprotein. In contrast to [3H]azidopine, no specific labeling of this fragment was obtained with [3H]B9209-005, indicating different binding sites for or different photoincorporation of the two dihydropyridine ligands. Because B9209-005 carries the photoreactive azido group in the dihydropyridine moiety, whereas the azido group of azidopine is located in the side chain, these results suggest that the dihydropyridine moiety of the two compounds probably interacts with the amino-terminal part of P-glycoprotein, whereas the side chains react preferentially with the carboxyl-terminal 55-kDa fragment.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of dexniguldipine-HC1 on rhodamine-123 accumulation in a multidrug-resistant leukaemia cell line: comparison with other chemosensitisers.

In the clinical therapy of cancer, resistance to many cytostatic drugs is a major cause of treatment failure. Among other mechanisms, the expression and pumping activity of P-glycoprotein (PGP) in the membrane of resistant cancer cells is responsible for the reduced uptake of cytostatics. The blockade or inhibition of PGP activity by chemosensitisers seems to be a tenable way to restore sensitivity to antineoplastic drugs and therapeutic efficacy. In the present work the influence of the new chemosensitiser dexniguldipine on rhodamine-123 accumulation in multidrug-resistant leukaemia cells was investigated. Dexniguldipine increases cellular rhodamine-123 accumulation dose-dependently.pEC50 values (-log concentration of drug showing a half maximal effect) in accumulation studies are dependent on pH of the test system and are in the range of 6.5 (pH 7.2) to 7.2 (pH 8.0) for dexniguldipine. In comparison with other chemosensitisers such as SDZ PSC 833, cyclosporin A, verapamil, dipyridamole, quinidine and amiodarone, dexniguldipine is the most potent drug in this test system. In addition to equilibrium measurements of rhodamine-123 accumulation, efflux of rhodamine-123 was analysed in the absence and presence of chemosensitisers. A clear dose-dependency was seen and, moreover, a dramatic decrease in efflux rates was achieved in the presence of chemosensitisers. The described system can be used to investigate PGP-mediated drug transport on a pharmacological and biochemical basis.