P-glycoprotein Transport Activity Research Articles

Characterization of a novel 99mTc-carbonyl complex as a functional probe of MDR1 P-glycoprotein transport activity.

Multidrug resistance (MDR) mediated by overexpression of MDR1 P-glycoprotein (Pgp) is one of the best characterized barriers to chemotherapy in cancer patients. Furthermore, the protective function of Pgp-mediated efflux of xenobiotics in various organs has a profound effect on the bioavailability of drugs in general. Thus, there is an expanding requirement to noninvasively interrogate Pgp transport activity in vivo. We herein report the Pgp recognition properties of a novel 99mTc(I)-tricarbonyl complex, [99mTc(CO)3(MIBI)3]+ (Tc-CO-MIBI). Tc-CO-MIBI showed 60-fold higher accumulation in drug-sensitive KB 3-1 cells compared to colchicine-selected drug-resistant KB 8-5 cells. In KB 8-5 cells, tracer enhancement was observed with the potent MDR modulator LY335979 (EC50 = 62 nM). Similar behavior was observed using drug-sensitive MCF-7 breast adenocarcinoma cells and MCF-7/MDR1 stable transfectants, confirming that Tc-CO-MIBI is specifically excluded by overexpression of MDR1 Pgp. By comparison, net accumulation in control H69 lung tumor cells was 9-fold higher than in MDR-associated protein (MRP1)-expressing H69AR cells, indicating only modest transport by MRP1. Biodistribution analysis following tail vein injection of Tc-CO-MIBI showed delayed liver clearance as well as enhanced brain uptake and retention in mdr1a/1b(-/-) gene deleted mice versus wild-type mice, directly demonstrating that Tc-CO-MIBI is a functional probe of Pgp transport activity in vivo.

Characterization of a Novel 99mTc-Carbonyl Complex as a Functional Probe of MDR1 P-Glycoprotein Transport Activity

Multidrug resistance (MDR) mediated by overexpression of MDR1 P-glycoprotein (Pgp) is one of the best characterized barriers to chemotherapy in cancer patients. Furthermore, the protective function of Pgp-mediated efflux of xenobiotics in various organs has a profound effect on the bioavailability of drugs in general. Thus, there is an expanding requirement to noninvasively interrogate Pgp transport activity in vivo. We herein report the Pgp recognition properties of a novel 99mTc(I)-tricarbonyl complex, [99mTc(CO)3(MIBI)3]+ (Tc-CO-MIBI). Tc-CO-MIBI showed 60-fold higher accumulation in drug-sensitive KB 3-1 cells compared to colchicine-selected drug-resistant KB 8-5 cells. In KB 8-5 cells, tracer enhancement was observed with the potent MDR modulator LY335979 (EC50 = 62 nM). Similar behavior was observed using drug-sensitive MCF-7 breast adenocarcinoma cells and MCF-7/MDR1 stable transfectants, confirming that Tc-CO-MIBI is specifically excluded by overexpression of MDR1 Pgp. By comparison, net accumulation in control H69 lung tumor cells was 9-fold higher than in MDR-associated protein (MRP1)-expressing H69AR cells, indicating only modest transport by MRP1. Biodistribution analysis following tail vein injection of Tc-CO-MIBI showed delayed liver clearance as well as enhanced brain uptake and retention in mdr1a/1b(-/-) gene deleted mice versus wild-type mice, directly demonstrating that Tc-CO-MIBI is a functional probe of Pgp transport activity in vivo.

Effect of chronic renal failure on the expression and function of rat intestinal P-glycoprotein in drug excretion.

In chronic renal failure, the renal excretion of certain drugs is dramatically reduced. To determine whether other routes of drug elimination, such as secretion through the intestinal barrier by intestinal P-glycoprotein can be altered, we compared P-glycoprotein activity, P-glycoprotein protein content, and P-glycoprotein mRNA levels in intestine of control and chronic renal failure rats. Chronic renal failure was surgically induced in rats by partial (7/8) nephrectomy. After 5 weeks, intestinal transport of rhodamine 123, a P-glycoprotein substrate, was carried out using an in vitro model of everted gut sacs. P-glycoprotein protein content was quantified by enzyme-linked immunosorbent assay and P-glycoprotein mRNA expression was evaluated by semi-quantitative reverse transcriptase polymerase chain reaction. A decrease of intestinal rhodamine 123 transport was observed in chronic renal failure rats, pointing to an inhibition of P-glycoprotein activity. Transport was inhibited in both sham-operated rats and rats with chronic renal failure by verapamil and cyclosporin A, but relative inhibition vs baseline was less marked in chronic renal failure than in sham-operated rats. In contrast, no significant differences in levels of P-glycoprotein protein or mRNA were observed between the two groups. Intestinal secretion of rhodamine 123 is mainly mediated by P-glycoprotein. It was reduced in rats with chronic renal failure, reflecting reduced intestinal drug elimination via a decrease in P-glycoprotein transport activity rather than via protein underexpression.

A Continuous Fluorescence Assay for the Study of P-Glycoprotein-Mediated Drug Efflux Using Inside-Out Membrane Vesicles

A fluorimetric procedure for assaying the transport activity of P-glycoprotein (P-gp) using a membrane vesicle model has been developed. In this assay methylene blue is incorporated into inside-out vesicles prepared from human acute lymphoblastic leukemic cells resistant to 100 ng · ml−1vinblastine (VBL100) and their sensitive controls. The fluorescence of a fluorescent derivative of colchicine (fluorescein–colchicine) is quenched as the probe is transported across the vesicle membrane. The fluorescein–colchicine transport was found to be dependent on the presence of P-glycoprotein, required ATP, and was inhibited by vanadate and the reversal agent, verapamil, in a dose-dependent manner. Furthermore, the transport was competed against by the P-gp substrates, vinblastine and methotrexate. The transport of fluorescein–colchicine by P-gp was found to be cooperative (n= 1.23). The assay is rapid, requires small amounts of sample, and removes the need for the radioactive procedures used in the past. The assay should find use in characterizing the transport kinetics of P-gp, for examining and optimizing combinations of chemotherapeutics, and for examining the effects of reversal agents and substrates which potentially compete for transport with the fluorescent substrate probe. Other possible applications include examining P-gp-mediated transport properties of purified P-gp in reconstituted systems.

The human multidrug resistance protein MRP1 translocates sphingolipid analogs across the plasma membrane.

Recently, we have provided evidence that the ABC-transporter MDR1 P-glycoprotein translocates analogs of various lipid classes across the apical plasma membrane of polarized LLC-PK1 cells transfected with MDR1 cDNA. Here, we show that expression of the basolateral ABC-transporter MRP1 (the multidrug resistance protein) induced lipid transport to the exoplasmic leaflet of the basolateral plasma membrane of LLC-PK1 cells at 15 degreesC. C6-NBD-glucosylceramide synthesized on the cytosolic side of the Golgi complex, but not C6-NBD-sphingomyelin synthesized in the Golgi lumen, became accessible to depletion by BSA in the basal culture medium. This suggests the absence of vesicular traffic and direct translocation of C6-NBD-glucosylceramide by MRP1 across the basolateral membrane. In line with this, transport of the lipid to the exoplasmic leaflet depended on the intracellular glutathione concentration and was inhibited by the MRP1-inhibitors sulfinpyrazone and indomethacin, but not by the MDR1 P-glycoprotein inhibitor PSC 833. In contrast to the broad substrate specificity of the MDR1 P-glycoprotein, MRP1 selectively transported C6-NBD-glucosylceramide and C6-NBD-sphingomyelin, the latter only when it was released from the Golgi lumen by brefeldin A. This shows the specific nature of the lipid translocation. We conclude that the transport activity of MDR1 P-glycoprotein and MRP1 must be taken into account in studies on the transport of lipids to the cell surface.

The drug transporter P-glycoprotein limits oral absorption and brain entry of HIV-1 protease inhibitors.

Currently available HIV-1 protease inhibitors are potent agents in the therapy of HIV-1 infection. However, limited oral absorption and variable tissue distribution, both of which are largely unexplained, complicate their use. We tested the hypothesis that P-glycoprotein is an important transporter for these agents. We studied the vectorial transport characteristics of indinavir, nelfinavir, and saquinavir in vitro using the model P-glycoprotein expressing cell lines L-MDR1 and Caco-2 cells, and in vivo after intravenous and oral administration of these agents to mice with a disrupted mdr1a gene. All three compounds were found to be transported by P-glycoprotein in vitro. After oral administration, plasma concentrations were elevated 2-5-fold in mdr1a (-/-) mice and with intravenous administration, brain concentrations were elevated 7-36-fold. These data demonstrate that P-glycoprotein limits the oral bioavailability and penetration of these agents into the brain. This raises the possibility that higher HIV-1 protease inhibitor concentrations may be obtained by targeted pharmacologic inhibition of P-glycoprotein transport activity.

Characterization of phosphine complexes of technetium(III) as transport substrates of the multidrug resistance P-glycoprotein and functional markers of P-glycoprotein at the blood-brain barrier.

The multidrug resistance (MDR1) P-glycoprotein functions as a broad specificity efflux transporter of structurally diverse natural product and xenobiotic compounds. P-glycoprotein also is an important component of the functional blood-brain barrier. To enable further studies of function and modulation of MDR1 P-glycoprotein in vitro and in vivo, two novel phosphine technetium(III) complexes were designed and characterized: trans-[2,2'-(1, 2-ethanediyldiimino)bis(1, 5-methoxy-5-methyl-4-oxo-hexenyl)]bis[methylbis(3-methoxy-1- propyl)ph osphine]Tc(III) (Tc-Q58) and trans-[5,5'-(1,2-ethanediyl diimino)bis(2-ethoxy-2-methyl-3-oxo-4-pentenyl)]bis[dimethyl(3- methox y-1-propyl)phosphine)]Tc(III) (Tc-Q63). In human drug-sensitive KB 3-1 cells and multidrug-resistant KB 8-5 and 8-5-11 derivative cell lines, expressing nonimmunodetectable, low, and high levels of MDR1 P-glycoprotein, respectively, accumulation of Tc-Q58 and Tc-Q63 was inverse to expression of the transporter. Differences between drug-sensitive and multidrug-resistant cells, while detectable at picomolar concentrations of each radiopharmaceutical, were independent of tracer concentration. Ratios of tracer accumulation in KB 3-1 and 8-5 cells were 62.3 and 48.1 for Tc-Q58 and Tc-Q63, respectively. Cell contents of Tc-Q58 and Tc-Q63 were enhanced up to 60-fold in MDR cells by known modulators of MDR1 P-glycoprotein, while drugs not in the multidrug-resistant phenotype had no effect on their accumulation. In KB 8-5 cells, potency of modulators was GF120918 >> cyclosporin A > verapamil. Accumulation of Tc-Q58 and Tc-Q63 in Sf9 insect cells infected with a recombinant baculovirus containing human MDR1 P-glycoprotein was reduced in a GF120918-reversible manner (EC50 </= 70 nM) compared with cells infected with a wild-type baculovirus. By contrast, cell contents of Tc-Q58 or Tc-Q63 in Sf9 cells expressing the homologous MDR3 P-glycoprotein did not differ from wild-type virus. Demonstrating molecular targeting of these complexes in vivo, distribution and retention of Tc-Q58 in brain tissue of FVB mice treated with a saturating dose of GF120918 and mice deficient in the mdr1a gene [mdr1a (-/-)] were enhanced 180% and 520% over control, respectively. Exploiting the gamma-emission spectrum of 99mTc, increased uptake of Tc-Q58 in brain tissue of mdr1a (-/-) mice was readily detected noninvasively by scintigraphic imaging. Thus, both Tc-Q58 and Tc-Q63 are demonstrated to be substrates for transport by MDR1 P-glycoprotein, broadening the specificity of this transporter to include phosphine-containing metal complexes. As shown with Tc-Q58, these Q complexes can be used to detect transport activity and modulation of MDR1 P-glycoprotein in vitro and to directly monitor the functional status of P-glycoprotein at the blood-brain barrier in vivo.

Characterization of functional assays of multidrug resistance P-glycoprotein transport activity.

P-glycoprotein-mediated multidrug resistance has emerged as one of the most attractive targets to improve anticancer therapy. The P-glycoprotein functions as an energy-dependent, membrane transport pump capable of decreasing the intracellular concentration of a broad range of chemotherapeutic agents. Pharmaceuticals which inhibit P-glycoprotein transport activity are currently being evaluated in clinical trials. Characterization of P-glycoprotein functional activity is critical in determining if these multidrug resistance reversal agents improve therapeutic responses of tumors expressing P-glycoprotein. In this report, we directly compare and characterize assays using rhodamine 123, dimethyloxadicarbocyanine iodide (DiOC2), [3H]daunorubicin and hexakis(2-methoxyisobutyl isonitrile)technetium(I) ([(99m)Tc]Sestamibi) as P-glycoprotein transport probes to quantitate functional activity. The accumulation of certain substrates is concentration dependent and the parameters which determine probe accumulation are impacted by the level of P-glycoprotein expression. In addition, higher concentrations of reversal agents are required to inhibit multidrug resistance in cell lines expressing higher levels of P-glycoprotein. Furthermore, the concentration of reversal agents required to inhibit completely P-glycoprotein transport activity is higher than generally recognized. Thus, the level of P-glycoprotein expression may confound intersample comparisons unless sensitive probes are used in combination with saturating concentrations of potent reversal agents. These results highlight the importance of carefully characterizing assay systems under uniform conditions to quantitate P-glycoprotein function.

Taxuspines A ∼ C, new taxoids from Japanese yew Taxus cuspidata inhibiting drug transport activity of P-glycoprotein in multidrug-resistant cells

Three new taxoids, taxuspines A ∼ C ( 1 ∼ 3), possessing different skeletons from usual taxane diterpenoids consisting of a 6/8/6-membered ring system have been isolated together with known taxoids ( 4 ∼ 12) from stems of the Japanese yew Taxus cuspidata Sieb. et Zucc. and the structures elucidated on the basis of spectroscopic data. Most of the taxoids increased cellular accumulation of vincristine in multidrug-resistant tumor cells, while taxol ( 12) did not show such an activity. The taxoids ( 1 ∼ 11) showed no or weak cytotoxicity and among them, 2 and 6 reduced appreciably CaCI 2-induced depolymerization of microtubules. These results suggest that some taxoids may be useful for overcoming multidrug resistance in tumor cells.

Identification of the major phosphorylation domain of murine mdr1b P-glycoprotein. Analysis of the protein kinase A and protein kinase C phosphorylation sites.

P-glycoprotein is phosphorylated in cells, and it has been suggested that phosphorylation may regulate the drug transport activity of P-glycoprotein. Domain mapping, utilizing a combination of cyanogen bromide digestion and immunoblot analysis, was used to reveal the major phosphorylation sites in murine mdr1b P-glycoprotein. After labeling of J7.V1-1 cells with [32P]Pi, or labeling membranes with [gamma-32P]ATP and either protein kinase A or protein kinase C, it was found that the majority of the label was contained within a single cyanogen bromide fragment (amino acid 627-682) that encompassed the majority of the linker region. The in vitro protein kinase C phosphorylation sites within this fragment were analyzed by a combination of fast atom bombardment mass spectrometry (FABMS) and two-dimensional phosphopeptide mapping. FABMS analysis of a protein kinase C-phosphorylated synthetic peptide, corresponding to a segment of the linker region of P-glycoprotein, identified serine 669 as the single site of phosphorylation. Comparison of two-dimensional tryptic phosphopeptide maps prepared from synthetic peptide and P-glycoprotein, both of which were phosphorylated in vitro with protein kinase C, revealed that serine 669 was also the major phosphorylation site in the intact glycoprotein. The in vitro protein kinase A phosphorylation site was identified as serine 681 by site-directed mutagenesis. Inspection of the gene organization and the deduced amino acid sequence of mdr1b P-glycoprotein revealed that the linker region, although shorter than the R domain (55 versus 241 amino acids), fits the operational definition of the R domain of cystic fibrosis conductance regulator. Like the R domain, the linker region is encoded by a single exon, is highly charged with alternating acidic and basic side chains, and contains several protein kinase A/protein kinase C consensus phosphorylation sites. Since the R domain is believed to be involved in the regulation of cystic fibrosis conductance regulator function by phosphorylation, it is possible that the linker region plays a similar regulatory role in P-glycoprotein function.

Reduced mRNA levels for the multidrug-resistance genes in cAMP-dependent protein kinase mutant cell lines.

We have previously shown that in Chinese hamster ovary (CHO) cells, a mutant cell line with a defective regulatory subunit (RI) for the cAMP-dependent protein kinase (Abraham et al: Mol. Cell. Biol., 7:3098-3106, 1987), and a transfectant cell line expressing the same mutant kinase, showed increased sensitivity to a number of drugs that are known to be substrates for the multidrug transporter (P-glycoprotein). In the current study we have investigated the mechanism by which cAMP-dependent protein kinase controls drug resistance. We report here that the sensitivity of the kinase defective CHO cell lines to multiple drugs results from decreased RNA levels for the multidrug-resistance gene. Similar results were obtained with mouse Y1 adrenal cells. Wild-type Y1 cells had high levels of P-glycoprotein due to expression of both the mdr1b and mdr2 genes, whereas the cAMP-dependent protein kinase mutant Kin 8 cells had decreased RNA levels for these genes. A Kin 8 transfectant with restored cAMP-dependent protein kinase activity recovered mdr expression, indicating a cause and effect relationship between the protein kinase mutations and mdr expression. No changes in nuclear run-off assays could be detected, suggesting a non-transcriptional mechanism of regulation. Wild-type Y1 cells are more drug sensitive despite having higher levels of P-glycoprotein than the mutant cells. This paradoxical result may be explained by the higher rate of synthesis of steroids by the wild-type Y1 cells, which appear to be inhibitors of P-glycoprotein transport activity.

Genetic basis of multidrug resistance of tumor cells

Multidrug resistance in animal cells is defined as the simultaneous resistance to a variety of compounds which appear to be structurally and mechanistically unrelated. One type of multidrug resistance is characterized by the decreased accumulation of hydrophobic natural product drugs, a phenotype which is mediated by an ATP-dependent integral membrane multidrug transporter termed P-glycoprotein or P170. The gene coding for P170 is called MDR. The nucleotide-binding domain of P-glycoprotein shares sequence homology with a family of bacterial permease ATP-binding components. In addition, P170 as a whole is structurally very similar to a number of prokaryotic and eukaryotic proteins believed to be involved in transport activities. This review summarizes our current knowledge of the molecular biology and clinical significance of MDR expression and P-glycoprotein transport activity, as well as some theories about the function of this protein in normal cells.

Correlation of protein kinase C translocation, P-glycoprotein phosphorylation and reduced drug accumulation in multidrug resistant human KB cells

Treatment of drug-resistant human KB carcinoma cells (KB-V1) with 0.2 μM phorbol 12-myristate 13-acetate (PMA) resulted in increases of 4-fold in both membrane-associated protein kinase C activity and phosphorylation of P-glycoprotein. The response was essentially complete after 30 min and was relatively stable, since both of these parameters remained elevated above basal levels in cells exposed to PMA for 24 hours. In contrast, long-term PMA treatment of drug-sensitive KB-3 cells caused complete depletion of protein kinase C. The rate of accumulation of [ 3H]vinblastine in KB-V1 cells was 0.8 ± 0.1 pmol/mg/30 min in the absence, and 1.9 ± 0.2 pmol/mg/30 min in the presence, of 20 μM verapamil. Preincubation of cells with PMA resulted in a time-dependent decrease, up to 60% after 24 hours, in both of these values. These results suggest that protein kinase C mediated phosphorylation stimulates the drug transport activity of P-glycoprotein.