Modulation of P-glycoprotein-mediated multidrug resistance by acceleration of passive drug permeation across the plasma membrane

The drug concentration inside multidrug-resistant cells is the outcome of competition between the active export of drugs by drug efflux pumps, such as P-glycoprotein (Pgp), and the passive permeation of drugs across the plasma membrane. Thus, reversal of multidrug resistance (MDR) can occur either by inhibition of the efflux pumps or by acceleration of the drug permeation. Among the hundreds of established modulators of Pgp-mediated MDR, there are numerous surface-active agents potentially capable of accelerating drug transbilayer movement. The aim of the present study was to determine whether these agents modulate MDR by interfering with the active efflux of drugs or by allowing for accelerated passive permeation across the plasma membrane. Whereas Pluronic P85, Tween-20, Triton X-100 and Cremophor EL modulated MDR by inhibition of Pgp-mediated efflux, with no appreciable effect on transbilayer movement of drugs, the anesthetics chloroform, benzyl alcohol, diethyl ether and propofol modulated MDR by accelerating transbilayer movement of drugs, with no concomitant inhibition of Pgp-mediated efflux. At higher concentrations than those required for modulation, the anesthetics accelerated the passive permeation to such an extent that it was not possible to estimate Pgp activity. The capacity of the surface-active agents to accelerate passive drug transbilayer movement was not correlated with their fluidizing characteristics, measured as fluorescence anisotropy of 1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene. This compound is located among the headgroups of the phospholipids and does not reflect the fluidity in the lipid core of the membranes where the limiting step of drug permeation, namely drug flip-flop, occurs.     

Radiopharmaceuticals for assessment of multidrug resistance P-glycoprotein-mediated drug transport activity

Multidrug resistance (MDR) mediated by overexpression of MDR1 P-glycoprotein (Pgp) is one of the best characterized transporter-mediated barriers to successful chemotherapy in cancer patients. Thus, noninvasive interrogation of Pgp-mediated transport activity in vivo would be beneficial in guiding therapeutic choices. Both small organic medicinals as well as metal complexes characterized as transport substrates for Pgp are amenable to incorporation of PET or SPECT radionuclides and may enable noninvasive imaging of Pgp in cancer patients. Toward this objective, clinically approved agents, exemplified by (99m)Tc-Sestamibi and (99m)Tetrofosmin, have already shown promise for the functional evaluation of Pgp-mediated transport activity in human tumors in vivo. In addition, selected agents from an upcoming class of substituted Schiff-base gallium(III) complexes containing an N(4)O(2) donor core in their organic scaffold and capable of generating both SPECT and PET radiopharmaceuticals have also been shown to be promising for noninvasive assessment of Pgp activity in vitro and in vivo.     

Analogues of tetramethylrosamine as transport molecules for and inhibitors of P-glycoprotein-mediated multidrug resistance

Tetramethylrosamine and its thio- and seleno- analogues (TMR-O, TMR-S, and TMR-Se, respectively) were examined for their ability to be transported by Pgp into chemo-resistant CR1R12 cells. Verapamil (7 x 10(-6)M) enhanced the uptake of TMR-O and TMR-S into CR1R12 cells compared to those cultures not previously exposed to verapamil. The uptake of TMR-O and TMR-S in CR1R12 cells in the presence of 7 x 10(-6)M verapamil was equivalent to its uptake in the chemo-sensitive parent cell line AUXB1 in the absence or presence of verapamil. None of the TMR analogues were effective alone as photosensitizers of CR1R12 cells. However, when either TMR-S or TMR-Se was added to CR1R12 cells after 7 x 10(-6)M verapamil exposure for 2h, irradiation of cultures with 5.0J cm(-2) of 350-750 nm light caused significant phototoxicity. TMR-O showed no significant phototoxicity in the presence of verapamil. Chemo-sensitive AUXB1 cells are equally susceptible to phototoxicity using TMR-Se with or without previous exposure to verapamil. The Pgp modulators verapamil and CsA increased the uptake of CAM into CR1R12. Exposure of CR1R12 cells to TMR-S or TMR-Se for 2h in the dark resulted in no significant change in the intracellular accumulation of CAM. However, 1h of light exposure after incubation of cells with TMR-S or TMR-Se resulted in an up to 2-fold increase in CAM uptake.     

Reversal of P-glycoprotein-mediated multidrug resistance by doxorubicin and quinine co-loaded liposomes in tumor cells

Multidrug resistance (MDR) is a major obstacle to successful clinical cancer chemotherapy. Currently, there is still unsatisfactory demand for innovative strategies as well as effective and safe reversing agent to overcome MDR. In this study, we developed a novel nanoformulation, in which doxorubicin hydrochloride (DOX) and quinine hydrochloride (QN) were simultaneously loaded into liposomes by a pH-gradient method for overcoming MDR and enhancing cytotoxicity in a doxorubicin-resistant human breast cancer cell line (MCF-7/ADR). The various factors were investigated to optimize the formulation and manufacturing conditions of DOX and QN co-loaded liposomes (DQLs). The DQL showed uniform size distribution and high encapsulation efficiency (over 90%) for both the drugs. Furthermore, DQLs significantly displayed high intracellular accumulation and potential of MDR reversal capability in MCF-7/ADR cells through the cooperation of DOX with QN, in which QN played the role as a MDR reversing agent. The IC₅₀ of DQL0.5:1 with the DOX/QN/SPC weight ratio of 0.5:1:50 was 1.80?±?0.03?μg/mL, which was 14.23 times lower than that of free DOX in MCF-7/ADR cells. And the apoptotic percentage induced by DQL0.5:1 was also increased to 62.2%. These findings suggest that DQLs have great potential for effective treatment of MDR cancer.     

Reversal of P-glycoprotein-mediated multidrug resistance by cholesterol derived from low density lipoprotein in a vinblastine-resistant human lymphoblastic leukemia cell line.

P-glycoprotein (P-gp) is believed to be one of the most common causes of multidrug resistance (MDR) in chemotherapy. Studies have shown that the biosynthesis of cholesterol and cholesterol esters interfere with the function of P-gp. Since low density lipoprotein (LDL) carries a large amount of cholesterol, we investigated the effect of cholesterol derived from LDL on a line of human lymphoblastic leukemia MDR cells, CEM/VLB. Our results demonstrated that, in addition to increased cytotoxicity, the uptake of vinblastine in CEM/VLB cells increased, and LDL subsequently increased the intracellular vinblastine concentrations retained by CEM/VLB cells. The cholesterol levels in the membrane of the MDR cells were restored, while LDL significantly decreased the P-gp-associated ATPase activity. Current studies have shown that LDL leads to the resensitization of CEM/VLB cells to cytotoxic agents, likely through the restoration of cholesterol and reduction of P-gp-associated ATPase in the cell membrane.     

Nilotinib counteracts P-glycoprotein-mediated multidrug resistance and synergizes the antitumoral effect of doxorubicin in soft tissue sarcomas

The therapeutic effect of doxorubicin (DXR) in the treatment of soft tissue sarcomas (STS) is limited by its toxicity and the development of multidrug resistance (MDR), the latter mainly induced by high expression of efflux pumps (e.g., P-glycoprotein [P-gp]). Therefore, the search for alternative therapies, which sensitize these tumors to chemotherapy while maintaining a low toxicity profile, is a rational approach. We assessed efficacy and molecular mechanisms involved in the antiproliferative effects of the tyrosine kinase inhibitors, nilotinib and imatinib, as single agents or in combination with DXR, in human synovial sarcoma SW982 and leiomyosarcoma SK-UT-1 cells. As single compound nilotinib (1-10 μM) was more potent than imatinib inhibiting the growth of SK-UT-1 and SW982 cells by 33.5-59.6%, respectively. Importantly, only nilotinib synergized the antitumoral effect of DXR (0.05-0.5 μM) by at least 2-fold, which clearly surpassed the mere sum of effects according to isobolographic analysis. Moreover, nilotinib in combination with DXR had a sustained effect on cell number (-70.3±5.8%) even 12 days after withdrawal of drugs compared to DXR alone. On the molecular level, only nilotinib fully blocked FBS-induced ERK1 and p38 MAPK activation, hence, reducing basal and DXR-induced up-regulation of P-gp levels. Moreover, efflux activity of the MDR-related proteins P-gp and MRP-1 was inhibited, altogether resulting in intracellular DXR retention. In high-risk STS tumors 53.8% and 15.4% were positive for P-gp and MRP-1 expression, respectively, with high incidence of P-gp in synovial sarcoma (72.7%). In summary, nilotinib exhibits antiproliferative effects on cellular models of STS and sensitizes them to DXR by reverting DXR-induced P-gp-mediated MDR and inhibiting MRP-1 activity, leading to a synergistic effect with potential for clinical treatment.     

Design,synthesis and biological evaluation of novel tetrahydroisoquinoline derivatives as P-glycoprotein-mediated multidrug resistance inhibitors

Multidrug resistance (MDR) is one of the main obstacles of clinical chemotherapy. A great deal of research shows that the occurrence of drug resistance in various malignant tumors is closely related to the expression of P-glycoprotein (P-gp) on the surface of the cell membrane. In this paper, based on the structure-activity relationship of phenylethyl tetrahydroisoquinoline, we choose tariquidar as the lead compound for the design and synthesis of 17 novel tetrahydroisoquinoline P-gp inhibitors. Additionally, in vitro and in vivo cytotoxicity assays and reversed MDR activity assays were evaluated. Among them, compound 3 had a good reversal of MDR activity and the reversal mechanism study of it was carried out. All of these results demonstrated that compound 3 was considered to be a promising P-gp-mediated MDR reversal candidate.     

Design,synthesis and evaluation of novel triazole core based P-glycoprotein-mediated multidrug resistance reversal agents

A novel series of triazol-N-ethyl-tetrahydroisoquinoline based compounds were designed and synthesized via click chemistry. Most of the synthesized compounds showed P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) reversal activities. Among them, compound 7 with little cytotoxicity towards GES-1 cells (IC₅₀ >80 μM) and K562/A02 cells (IC₅₀ >80 μM) exhibited more potency than verapamil (VRP) on increasing anticancer drug accumulation in K562/A02 cells. Moreover, compound 7 could significantly reverse MDR in a dose-dependent manner and also persist longer chemo-sensitizing effect than VRP with reversibility. Further mechanism studies revealed that compound 7 in reversing MDR revealed that it could remarkably increase the intracellular accumulation of both rhodamine-123 (Rh123) and adriamycin (ADM) in K562/A02 cells as well as inhibit their efflux from the cells. These results suggested that compound 7 showed more potency than the classical P-gp inhibitor VRP under the same conditions, which may be a promising P-gp-mediated MDR modulator for further development.     

Discovery of aromatic amides with triazole-core as potent reversal agents against P-glycoprotein-mediated multidrug resistance

P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) is a major impediment for clinical cancer therapy. 19 novel aromatic amides with triazole-core as MDR reversal agents were designed and synthesized via click chemistry to reverse MDR. Among them, compound 42 was identified as the most promising candidate with high potency (EC₅₀ = 78.1 ± 5.4 nM), low cytotoxity (SI > 1282) and persistent duration in reversing doxorubicin (DOX) resistance in K562/A02 cells. 42 also enhanced the potency of other P-gp associated cytotoxic agents with different structures. In further study, remarkably increased intracellular accumulation of Rh123 and DOX in K562/A02 cells was achieved by compound 42, while CYP3A4 activity had no change by compound 42. These results indicate that compound 42 as a relatively safe modulator of P-gp-mediated MDR has good potential for further development.     

Reversal of P-glycoprotein-mediated multidrug resistance by 5,6,7,3',4'-pentamethoxyflavone (Sinensetin)

Vif, one of the six accessory genes expressed by HIV-1, is essential for the productive infection of natural target cells. Previously we suggested that Vif acts as a regulator of the viral protease (PR): It prevents the autoprocessing of Gag and Gag-Pol precursors until virus assembly, and it may control the PR activity in the preintegration complex at the early stage of infection. It was demonstrated before that Vif, and specifically the 98 amino acid stretch residing at the N'-terminal part of Vif (N'-Vif), inhibits both the autoprocessing of truncated Gag-Pol polyproteins in bacterial cells and the hydrolysis of synthetic peptides by PR in cell-free systems. Linear synthetic peptides derived from N'-Vif specifically inhibit and bind HIV-1 PR in vitro, and arrest virus production in tissue culture. Peptide mapping of N'-Vif revealed that Vif88-98 is the most potent PR inhibitor. Here we report that this peptide inhibits both HIV-1 and HIV-2, but not ASLV proteases in vitro. Vif88-98 retains its inhibitory effect against drug-resistant HIV-1 PR variants, isolated from patients undergoing long-term treatment with anti-PR drugs. Variants of HIV protease bearing the mutation G48V are resistant to inhibition by this Vif-derived peptide, as shown by in vitro assays. In agreement with the in vitro experiments, Vif88-98 has no effect on the production of infectious particles in cells infected with a G48V mutated virus.     

Synthesis and biological activity of 4-alkoxy chalcones: potential hydrophobic modulators of P-glycoprotein-mediated multidrug resistance

A series of 4-alkoxy-2′,4′,6′-trihydroxychalcones have been synthesized and evaluated for their ability to inhibit P-glycoprotein-mediated multidrug resistance (MDR) by direct binding to a purified protein domain containing an ATP-binding site and a modulator-interacting region. The introduction of hydrophobic alkoxy goups at position 4 led to much more active compounds as compared to the parent chalcone. The binding affinity increased as a function of the chain length, up to the octyloxy derivative for which a K_D of 20 nM was obtained.     

Design,synthesis and biological evaluation of LBM-A5 derivatives as potent P-glycoprotein-mediated multidrug resistance inhibitors

A novel series of P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) inhibitors with triazol-N-phenethyl-tetrahydroisoquinoline or triazol-N-ethyl-tetrahydroisoquinoline scaffold were designed and synthesized via click chemistry. Most of the synthesized compounds showed higher reversal activity than verapamil (VRP). Among them, the most potent compound 4 showed a comparable activity with the known potent P-gp inhibitor WK-X-34 with lower cytotoxicity toward K562 cells (IC₅₀ >100 μM). Compared with VRP, compound 4 exhibited more potency in increasing drug accumulation in K562/A02 MDR cells. Moreover, compound 4 could significantly reverse MDR in a dose-dependent manner and also persist longer chemo-sensitizing effect than VRP with reversibility. Further mechanism studies revealed that compound 4 could remarkably increase the intracellular accumulation of Adriamycin (ADM) in K562/A02 cells as well as inhibit rhodamine-123 (Rh123) efflux from the cells. These results suggested that compound 4 may represent a promising candidate for developing P-gp-mediated MDR inhibitors.     

The membrane lipid environment modulates drug interactions with the P-glycoprotein multidrug transporter.

The P-glycoprotein multidrug transporter functions as an ATP-driven efflux pump for a large number of structurally unrelated hydrophobic compounds. Substrates are believed to gain access to the transporter after partitioning into the membrane, rather than from the extracellular aqueous phase. The binding of drug substrates to P-glycoprotein may thus be modulated by the properties of the lipid bilayer. The interactions with P-glycoprotein of two drugs (vinblastine and daunorubicin) and a chemosensitizer (verapamil) were characterized by quenching of purified fluorescently labeled protein in the presence of various phospholipids. Biphasic quench curves were observed for vinblastine and verapamil, suggesting that more than one molecule of these compounds may bind to the transporter simultaneously. All three drugs bound to P-glycoprotein with substantially higher affinity in egg phosphatidylcholine (PC), compared to brain phosphatidylserine (PS) and egg phosphatidylethanolamine (PE). The nature of the lipid acyl chains also modulated binding, with affinity decreasing in the order egg PC > dimyristoyl-PC (DMPC) > dipalmitoyl-PC (DPPC). Following reconstitution of the transporter into DMPC, all three compounds bound to P-glycoprotein with 2-4-fold higher affinity in gel phase lipid relative to liquid-crystalline phase lipid. The P-glycoprotein ATPase stimulation/inhibition profiles for the drugs were also altered in different lipids, in a manner consistent with the observed changes in binding affinity. The ability of the drugs to partition into bilayers of phosphatidylcholines was determined. All of the drugs partitioned much better into egg PC relative to DMPC and DPPC. The binding affinity increased (i.e., the value of Kd decreased) as the drug-lipid partition coefficient increased, supporting the proposal that the effective concentration of the drug substrate in the membrane is important for interaction with the transporter. These results provide support for the vacuum cleaner model of P-glycoprotein action.     

Relationship between multidrug resistance,hypersensitivity to resistance modifiers and cell volume in Chinese hamster ovary cells.

We have previously shown that very high levels of hypersensitivity to several resistance modifiers are correlated with increasing multidrug resistance in a series of Chinese hamster ovary cell lines. We have now selected a new member of the series which is an exception to this correlation in that although it is almost twice as multidrug resistant as the cell line from which it was derived, it shows much less hypersensitivity to resistance modifiers. Level of resistance modifier hypersensitivity correlated with the level of reduction of verapamil accumulation in these cells, and with the density of P-glycoprotein, but since the selection of this cell line has involved a doubling of cell volume, it was not correlated with total amount of P-glycoprotein.     

Structure-activity relationships of some taxoids as multidrug resistance modulator

1,7-Deoxy-4-deacetylbaccatin III (12) and its five analogues 6-9, 13, and their oxetane ring opened derivatives 14, 16, and 17, which were synthesized from taxinine, showed significant activity as MDR reversal agent by the assay of the calcein accumulation toward MDR human ovarian cancer 2780AD cells. The most effective compound 12 in this assay is actually efficient for the recovery of cytotoxic activity of paclitaxel (taxol), adriamycin (ADM), and vincristine (VCR) toward MDR 2780AD cells at the same level toward parental 2780 cells. This activity of 12 is very interesting because baccatin III (4) has no such MDR reversal activity but has cytotoxic activity. The essential functional groups inducing such a difference in biological activity between 4 and 12 are 4alpha-acetoxyl for 4 and 4alpha-hydroxyl for 12. In seven compounds possessing MDR reversal activity, compound 12 is the most desirable compound for anti-MDR cancer reversal agent, because it has the highest accumulation ability of anticancer agent in MDR cancer cells and weak cytotoxic activity. Compounds 8 and 13 showed significant cytotoxic activity toward HepG2 and VA-13, respectively, as well as MDR reversal activity. They are expected to become lead compounds for new types of anticancer agent or anti-MDR cancer agent.     

Modulation of P-glycoprotein-mediated drug transport by alterations in lipid fluidity of rat liver canalicular membrane vesicles.

P-glycoprotein (P-gp) is believed to function as an ATP-dependent efflux pump for natural product anti-cancer drugs in multidrug-resistant (MDR) tumor cells and in certain normal tissues. P-gp has been localized to the apical plasma membrane of the bile canaliculus where it has been shown to transport [3H]daunomycin. In this study, we investigated whether alterations in membrane lipid fluidity of canalicular membrane vesicles (CMV) could modulate the P-gp-mediated accumulation of [3H]daunomycin and [3H]vinblastine. Accumulation of both cytotoxic agents was stimulated by ATP, exhibited temperature dependence and osmotic sensitivity, and followed Michaelis-Menten kinetics. Alterations in CMV lipid fluidity were induced by the known fluidizers, 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate (A2C) and benzyl alcohol, and were assessed by fluorescence polarization techniques using the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). Both A2C (2.5-5.0 microM) and benzyl alcohol (10-20 mM) produced a dose-dependent increase in CMV lipid fluidity. Moreover, both fluidizers, at the above doses, significantly inhibited (p < 0.05) the ATP-dependent accumulation of [3H]daunomycin. [3H]Vinblastine accumulation was also inhibited by A2C (p < 0.05). Lower doses of A2C (0.6 microM) and benzyl alcohol (1 mM) failed to influence either lipid fluidity or P-gp-mediated drug accumulation. Kinetic analysis revealed that A2C (5.0 microM) noncompetitively inhibited [3H]daunomycin accumulation and uncompetitively inhibited [3H]vinblastine accumulation with apparent Ki values of approximately 1.5 and approximately 1.2 microM, respectively. Verapamil competitively inhibited P-gp-mediated accumulation of [3H]daunomycin but failed to alter the fluidity of CMV. Taken together, the present results demonstrate that while increases in membrane fluidity of CMV are not necessarily required to inhibit P-gp-mediated drug accumulation, they can inhibit these processes, at least in CMV. Alterations in the physical state of CMV, therefore, appear to be at least one important modulator of P-gp function.     

The multidrug resistance P-glycoprotein modulates cell regulatory volume decrease.

Cell volume is frequently down-regulated by the activation of anion channels. The role of cell swelling-activated chloride channels in cell volume regulation has been studied using the patch-clamp technique and a non-invasive microspectrofluorimetric assay for changes in cell volume. The rate of activation of these chloride channels was shown to limit the rate of regulatory volume decrease (RVD) in response to hyposmotic solutions. Expression of the human MDR1 or mouse mdr1a genes, but not the mouse mdr1b gene, encoding the multidrug resistance P-glycoprotein (P-gp), increased the rate of channel activation and the rate of RVD. In addition, P-gp decreased the magnitude of hyposmotic shock required to activate the channels and to elicit RVD. Tamoxifen selectively inhibited both chloride channel activity and RVD. No effect on potassium channel activity was elicited by expression of P-gp. The data show that, in these cell types, swelling-activated chloride channels have a central role in RVD. Moreover, they clarify the role of P-gp in channel activation and provide direct evidence that P-gp, through its effect on chloride channel activation, enhances the ability of cells to down-regulate their volume.