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International Journal of Cancer Jun 1998Efflux of chemotherapy drugs by P-glycoprotein (P-gp) at the plasma membrane is thought to be a major cause of cancer multidrug resistance. In this report, we show by...
Efflux of chemotherapy drugs by P-glycoprotein (P-gp) at the plasma membrane is thought to be a major cause of cancer multidrug resistance. In this report, we show by flow cytometry that P-gp also concentrates large amounts of 2 different drugs, Hoechst 33342 and daunorubicin, within a cytoplasmic compartment of multidrug resistant CHRC5 cells. A quantitative assay of Hoechst 33342 revealed that cytoplasmic sequestration by P-gp in CHRC5 cells accounted for about half of the amount of Hoechst 33342 accumulated by the drug-sensitive parental Aux BI cells. Daunorubicin sequestered in the cytoplasm of CHRC5 cells could be released by inhibiting P-gp function with cyclosporin A, resulting in cell death. A likely site of drug sequestration is P-gp-containing cytoplasmic vesicles, in which the P-gp is oriented so that drugs are transported and concentrated in the interior of the vesicles. P-gp was detected in the membranes of cytoplasmic vesicles of CHRC5 cells by confocal immunofluorescence microscopy and immunoelectron microscopy with anti-P-gp monoclonal antibodies (MAbs). Vesicular localization of daunorubicin was observed by epifluorescence microscopy. The origin and nature of the P-gp-containing vesicles are unknown, but they do not correspond to endocytic vesicles. Our results directly demonstrate that chemosensitizer-induced release of drugs sequestered in cytoplasmic vesicles by P-gp can be used to overcome multidrug resistance.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Benzimidazoles; Cell Line; Cyclosporine; Daunorubicin; Drug Resistance
PubMed: 9626353
DOI: 10.1002/(sici)1097-0215(19980610)76:6<857::aid-ijc15>3.0.co;2-# -
Current Drug Metabolism May 2009Gender differences in drug concentrations, drug response and toxicity have been attributed to various distinct yet interrelated physiological and molecular mechanisms.... (Review)
Review
Gender differences in drug concentrations, drug response and toxicity have been attributed to various distinct yet interrelated physiological and molecular mechanisms. Drug transporters and metabolising enzymes play an important role in the xenobiotic cascade and are important regulators of drug disposition at the molecular level. The proposal of a dynamic interplay between drug metabolism and efflux has positioned drug transporters as important mediators of gender disparity in respect to drug disposition and therapeutic response. In examining the effects of gender on drug disposition and response we will specifically direct our focus on the role of the predominant drug transporter, P-glycoprotein. This review focuses on the role of the P-glycoprotein as a molecular mediator of gender differences in both drug exposure and response. Differences in transporter expression and function will be discussed together with the molecular basis for the observed difference in drug exposure between the sexes. Gender differences affecting transporter expression and function at the effect compartment and the effect of this on drug response will also be discussed.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Animals; Cytochrome P-450 CYP3A; Gene Expression Regulation; Humans; Pharmacokinetics; Sex Characteristics; Tissue Distribution; Treatment Outcome
PubMed: 19519340
DOI: 10.2174/138920009788498996 -
Current Drug Metabolism Aug 2003Since the generation of the multi-drug resistance 1 (mdr1) gene knockout (KO) mice in the early 90's, these animals have been instrumental to our understanding of the... (Review)
Review
Since the generation of the multi-drug resistance 1 (mdr1) gene knockout (KO) mice in the early 90's, these animals have been instrumental to our understanding of the physiological roles of mdr1 gene product P-glycoprotein. Located in crucial organs such as brain, intestine, liver, and kidney, P-glycoprotein-mediated transport has been shown to affect both the pharmacokinetics and pharmacodynamics of endogenous compounds and xenobiotics. It appears that P-glycoprotein may not be essential for the maintenance of normal body function as suggested by the similarity in life span and serum chemistry values of mdr1 gene KO mice compared to their genetically competent littermates. However, numerous studies have demonstrated that P-glycoprotein limits the brain penetration of many drug substrates. The reduced central nervous system (CNS) access of these compounds has been linked to decreased pharmacological or toxicological effects. In contrast to the critical role that P-glycoprotein plays in the brain, the extent of P-glycoprotein involvement in oral absorption and hepatobiliary or renal excretion of xenobiotics appears more variable. In addition to the mdr1 gene KO model, in vitro cell lines that over-express P-glycoprotein, and clinical trials using P-glycoprotein modulators have allowed for the comparison of in vitro-in vivo and species related difference in P-glycoprotein activity. For the most part, studies have shown reasonable in vitro-in vivo correlations, modest species-related differences, and comparable human-mouse in vivo P-glycoprotein effects on systemic drug disposition. Therefore, the mdr1 gene KO mouse model, when used appropriately, may allow for prediction of CNS drug access and certain drug-drug interaction.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Animals; Drug Design; Drug Resistance, Multiple; Humans; Mice; Mice, Knockout; Pharmaceutical Preparations; Pharmacokinetics; Pharmacology; Species Specificity
PubMed: 12871045
DOI: 10.2174/1389200033489415 -
Journal of Bioenergetics and... Feb 1995Cells expressing elevated levels of the membrane phosphoprotein P-glycoprotein exhibit a multidrug resistance phenotype. Studies involving protein kinase activators and... (Comparative Study)
Comparative Study Review
Cells expressing elevated levels of the membrane phosphoprotein P-glycoprotein exhibit a multidrug resistance phenotype. Studies involving protein kinase activators and inhibitors have implied that covalent modification of P-glycoprotein by phosphorylation may modulate its biological activity as a multidrug transporter. Most of these reagents, however, have additional mechanisms of action and may alter drug accumulation within multidrug resistant cells independent of, or in addition to, their effects on the state of phosphorylation of P-glycoprotein. The protein kinase(s) responsible for P-glycoprotein phosphorylation has(ve) not been unambiguously identified, although several possible candidates have been suggested. Recent biochemical analyses demonstrate that the major sites of phosphorylation are clustered within the linker region that connects the two homologous halves of P-glycoprotein. Mutational analyses have been initiated to confirm this finding. Preliminary data obtained from phosphorylation- and dephosphorylation-defective mutants suggest that phosphorylation of P-glycoprotein is not essential to confer multidrug resistance.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Amino Acid Sequence; Animals; Cell Membrane; DNA Mutational Analysis; Drug Resistance, Multiple; Humans; Models, Structural; Molecular Sequence Data; Phosphorylation; Protein Kinases; Protein Structure, Secondary; Recombinant Proteins; Sequence Homology, Amino Acid
PubMed: 7629052
DOI: 10.1007/BF02110331 -
GMHC Treatment Issues : the Gay Men's... Jan 2002
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; HIV; HIV Infections; Humans
PubMed: 11871250
DOI: No ID Found -
Die Pharmazie Feb 2002The expression and function of P-glycoprotein (P-gp), an ATP-dependent efflux pump, were examined in rats pretreated with dexamethasone (DEX), an inducer of P-gp, and in... (Review)
Review
The expression and function of P-glycoprotein (P-gp), an ATP-dependent efflux pump, were examined in rats pretreated with dexamethasone (DEX), an inducer of P-gp, and in rats with glycerol-induced acute renal failure (ARF) and with CCl4-induced acute hepatic failure (AHF). DEX pretreatment increased the P-gp level and its functional activity in the intestine. In contrast, in ARF and AHF rats, the in vivo P-gp function was systemically suppressed, even though the level of P-gp remained unchanged or rather increased. In Caco-2 cells, the plasma collected from diseased rats exhibited a greater inhibitory effect on P-gp function than did plasma from control rats. A higher-plasma level of corticosterone, an endogenous P-gp substrate/inhibitor, was observed in the disease rats. These findings indicate that the actual in vivo function of P-gp cannot be predicted merely from the expression level of P-gp, and suggest that some endogenous P-gp-related compounds such as corticosterone participate in the regulation of in vivo P-gp function in diseased states.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Acute Kidney Injury; Animals; Disease; Heart Failure; Rats
PubMed: 11878184
DOI: No ID Found -
Current Topics in Medicinal Chemistry 2010Design of inhibitors of P-glycoprotein still represents a challenging task for medicinal chemists. The polyspecificity of the transporter combined with the limited... (Review)
Review
Design of inhibitors of P-glycoprotein still represents a challenging task for medicinal chemists. The polyspecificity of the transporter combined with the limited structural information renders rational drug design approaches rather ineffective. Within this article we will exemplify how recent insights into structure and mechanism of P-glycoprotein may aid in design of potent inhibitors.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Antineoplastic Agents; Binding Sites; Biological Transport; Drug Design; Humans; Molecular Structure
PubMed: 20645918
DOI: 10.2174/156802610792928004 -
Pharmacology & Therapeutics Feb 2007This year marks the 30th anniversary of the discovery of the multidrug resistance (MDR) ATP-binding cassette (ABC) transporter P-glycoprotein (P-gp). Since then a... (Review)
Review
This year marks the 30th anniversary of the discovery of the multidrug resistance (MDR) ATP-binding cassette (ABC) transporter P-glycoprotein (P-gp). Since then a considerable research effort has attempted to provide a greater understanding of the biological enigma of "multidrug" efflux. Moreover, the growing correlation between P-gp expression and a negative prognosis or poor outcome for chemotherapy has sparked significant interest in the generation of inhibitors. How close are we to overcoming the unwanted actions of P-gp in resistant cancer following 30 years of research? The initial inhibitors were pre-existing clinically used compounds and exploited the broad specificity of P-gp. Unfortunately, the concentrations required to inhibit P-gp meant that these compounds generated considerable toxicity. Pharmacological investigations progressed to rational design using the 1st generation compounds as a template structure. Inherent toxicity of the drugs was reduced; however, pharmacokinetic interactions with the anticancer drugs were unsustainable. Generation of the most recent of inhibitors employed combinatorial chemistry to produce a handful of potent and selective P-gp inhibitors. Some of these drugs have progressed to clinical trials with poor results or in some cases, undisclosed progress. There remains a clear need for the generation of P-gp inhibitors and this review describes the potential for a structure-based design to facilitate this undertaking. In particular, the plethora of functional data can provide important regions on the protein that could conceivably be exploited as inhibitor targets.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Animals; Drug Design; Drug Resistance, Neoplasm; Humans
PubMed: 17208306
DOI: 10.1016/j.pharmthera.2006.10.003 -
Molecular Vision Nov 2002The retinal pigment epithelium (RPE) is a transporting epithelial monolayer that controls hydration and composition of the subretinal space. P-glycoprotein is an...
PURPOSE
The retinal pigment epithelium (RPE) is a transporting epithelial monolayer that controls hydration and composition of the subretinal space. P-glycoprotein is an ATP-binding cassette transport protein known to transport a wide range of hydrophobic compounds. The expression of P-glycoprotein in barrier epithelial cells suggests that it could serve a normal protective function, possibly clearing potentially harmful substances from sensitive compartments, like the subretinal space. The present study is designed to determine the expression and activity of P-glycoprotein in normal human RPE.
METHODS
RT-PCR and direct sequencing were employed to examine the presence of mdr1 mRNA in cultured human RPE. P-glycoprotein-specific antibodies were employed in Western blotting to identify P-glycoprotein in cultured human RPE and in an established RPE cell line (D407). Anti-P-glycoprotein antibodies were also used to localize the protein in frozen, formaldehyde-fixed sections of native human RPE/choroid by immunohistochemistry. Finally, rhodamine uptake was performed in cultured human RPE monolayers to assess P-glycoprotein activity. The inhibitory antibody 4E3 and reversins 121 and 205 were used to block transport activity.
RESULTS
P-glycoprotein is expressed, and is active, in human RPE tissue not exposed to any known inducers of P-glycoprotein. RT-PCR yielded a 546 bp product that was 100% identical in sequence to published data for the mdr1 isoform of human P-glycoprotein. Western blotting demonstrated expression at the protein level, with specific bands observed at about 220 and 165 kD. In native tissue, P-glycoprotein immunoreactivity was predominantly membrane associated, with localization to both apical and basolateral cell membranes. Finally, P-glycoprotein expressed in human RPE is active. Steady-state rhodamine accumulation was increased in the presence of compounds reported to block P-glycoprotein mediated rhodamine efflux.
CONCLUSIONS
Human RPE, not exposed to inducer treatment, expresses P-glycoprotein with localization to both apical and basal cell surfaces. Basolateral P-glycoprotein could serve a protective function for the neural retina helping to clear unwanted substances from subretinal space. The finding that P-glycoprotein is also on the apical surface suggests possible additional roles for P-glycoprotein in the RPE.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Adult; Blotting, Western; Cells, Cultured; Fluorescent Antibody Technique, Indirect; Humans; Microscopy, Fluorescence; Pigment Epithelium of Eye; RNA, Messenger; Reverse Transcriptase Polymerase Chain Reaction; Rhodamines
PubMed: 12432340
DOI: No ID Found -
International Review of Cytology 1999This review presents a survey of studies of the movement of chemotherapeutic drugs into cells, their extrusion from multidrug-resistant (MDR) cells overexpressing... (Review)
Review
This review presents a survey of studies of the movement of chemotherapeutic drugs into cells, their extrusion from multidrug-resistant (MDR) cells overexpressing P-glycoprotein (Pgp), and the mode of sensitization of MDR cells to anticancer drugs by Pgp modulators. The consistent features of the kinetics from studies of the operation of Pgp in cells were combined in a computer model that enables the simulation of experimental scenarios. MDR-type drugs are hydrophobic and positively charged and as such bind readily to negatively charged phospholipid head groups of the membrane. Transmembrane movement of MDR-type drugs, such as doxorubicin, occurs by a flip-flop mechanism with a lifetime of about 1 min rather than by diffusion down a gradient present in the lipid core. A long residence time of a drug in the membrane leaflet increases the probability that P-glycoprotein will remove it from the cell. In a manner similar to ion-transporting ATPases, such as Na+,K(+)-ATPase, Pgp transports close to one drug molecule per ATP molecule hydrolyzed. Computer simulation of cellular pharmacokinetics, based on partial reactions measured in vitro, show that the efficiency of Pgp, in conferring MDR on cells, depends on the pumping capacity of Pgp and its affinity toward the specific drug, the transmembrane movement rate of the drug, the affinity of the drug toward its pharmacological cellular target, and the affinity of the drug toward intracellular trapping sites. Pgp activities present in MDR cells allow for the efficient removal of drugs, whether directly from the cytoplasm or from the inner leaflet of the plasma membrane. A prerequisite for a successful modulator, capable of overcoming cellular Pgp, is the rapid passive transbilayer movement, allowing it to reenter the cell immediately and thus successfully occupy the Pgp active site(s).
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Animals; Antineoplastic Agents; Cell Membrane Permeability; Computer Simulation; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Humans; Solubility; Water
PubMed: 10331240
DOI: 10.1016/s0074-7696(08)62148-8