P-Glycoprotein in cell cultures: a combined approach to study expression, localisation, and functionality in the confocal microscope

Hämmerle, Sibylle P.; Rothen‐Rutishauser, Barbara; Krämer, Stefanie-Dorothea; Günthert, Maja; Wunderli‐Allenspach, Heidi

doi:10.1016/s0928-0987(00)00142-1

Cited by 40 publications

(31 citation statements)

References 35 publications

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“…In these cells, the uropods are the most probable contact sites with either other cells or extracellular matrix components. 21,22 Our results and previous reports on epithelial cells [48][49][50] strongly suggest that P-gp needs to be polarized to fully exert its function. Consistent with this hypothesis, we showed that treatment with ERM antisense oligonucleotides recruited the sensibility of a human lymphoblastoid cell line, CEM-VBL100 cells, to the toxic effect of drugs, consistent with the inhibition of the P-gp-mediated pump, the intracellular retention of the drugs, and the unpolarized redistribution of P-gp on the cell surface.…”

Section: Discussionsupporting

confidence: 72%

P-glycoprotein–actin association through ERM family proteins: a role in P-glycoprotein function in human cells of lymphoid origin

Luciani

Molinari

Lozupone

et al. 2002

Blood

135

140

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P-glycoprotein is a 170-kd glycosylated transmembrane protein, expressed in a variety of human cells and belonging to the adenosine triphosphate-binding cassette transporter family, whose membrane expression is functionally associated with the multidrug resistance phenotype. However, the mechanisms underlying the regulation of P-glycoprotein functions remain unclear. On the basis of some evidence suggesting P-glycoprotein-actin cytoskeleton interaction, this study investigated the association of Pglycoprotein with ezrin, radixin, and moesin, a class of proteins that cross-link actin filaments with plasma membrane in a human cell line of lymphoid origin and that have been shown to link other ionpump-related proteins. To this purpose, a multidrug-resistant variant of CCRF-CEM cells (CEM-VBL100) was used as a model to investigate the following: (1) the cellular localizations of P-glycoprotein and ezrin, radixin, and moesin and their molecular associations; and (2) the effects of ezrin, radixin, and moesin antisense oligonucleotides on multidrug resistance and P-glycoprotein function. The results showed that: (1) P-glycoprotein colocalized and coimmunoprecipitated with ezrin, radixin, and moesin; and (2) treatment with antisense oligonucleotides for ezrin, radixin, and moesin restored drug susceptibility consistently with inhibition of both drug efflux and actin-P-glycoprotein association and induction of cellular redistribution of P-glycoprotein. These data suggest that P-glycoprotein association with the actin cytoskeleton through ezrin, radixin, and moesin is key in conferring to human lymphoid cells a multidrug resistance phenotype. Strategies aimed at inhibiting P-glycoprotein-actin association may be helpful in increasing the efficiency of both antitumor and antiviral therapies. (Blood. 2002;99:641-648)

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Section: Discussionsupporting

confidence: 72%

P-glycoprotein–actin association through ERM family proteins: a role in P-glycoprotein function in human cells of lymphoid origin

Luciani

Molinari

Lozupone

et al. 2002

Blood

135

140

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“…MDR1-MDCK and SKVLB cells were preincubated for 30 min with assay buffer (10 mM Hepes, 0.4 mM K 2 HPO 4 , 25 mM NaHCO 3 , 3.0 mM KCl, 1.2 mM MgSO 4 , 1.4 mM CaCl 2 , 122 mM NaCl, 10 mM glucose) (28,29). After the preincubation period, the assay buffer was removed and replaced with 2.5 ml of assay buffer containing 100 g/ml of rhodamine 123 (rho123) (Sigma).…”

Section: Methodsmentioning

confidence: 99%

Inhibition of Multidrug Resistance by AdamantylGb3, a Globotriaosylceramide Analog

Rosa

Ackerley

Wang

et al. 2008

Journal of Biological Chemistry

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Multidrug resistance (MDR) via the ABC drug transporter (ABCB1), P-glycoprotein (P-gp/MDR1) overexpression, is a major obstacle in cancer chemotherapy. Many inhibitors reverse MDR but, like cyclosporin A (CsA), have significant toxicities. MDR1 is also a translocase that flips glucosylceramide inside the Golgi to enhance neutral glycosphingolipid (GSL) synthesis. We observed partial MDR1/globotriaosylceramide (Gb 3 ) cell surface co-localization, and GSL removal depleted cell surface MDR1. MDR1 may therefore interact with GSLs. AdamantylGb 3 , a water-soluble Gb 3 mimic, but not other GSL analogs, reversed MDR1-MDCK cell drug resistance. Cell surface MDR1 was up-regulated 1 h after treatment with CsA or adaGb 3 , but at 72 h, cell surface expression was lost. Intracellular MDR1 accumulated throughout, suggesting long term defects in plasma membrane MDR1 trafficking. AdaGb 3 or CsA rapidly reduced rhodamine 123 cellular efflux. MDR1 also mediates gastrointestinal epithelial drug efflux, restricting oral bioavailability. Vinblastine apical-to-basal transport in polarized human intestinal C2BBe1 cells was significantly increased when adaGb 3 was added to both sides, or to the apical side only, comparable with verapamil, a standard MDR1 inhibitor. Disulfide cross-linking of mutant MDR1s showed no binding of adaGb 3 to the MDR1 verapamil/cyclosporin-binding site between surface proximal helices of transmembrane segments (TM) 6 and TM7, but rather to an adjacent site nearer the center of TM6 and the TM7 extracellular face, i.e. close to the bilayer leaflet interface. Verotoxin-mediated Gb 3 endocytosis also upregulated total MDR1 and inhibited drug efflux. Thus, a functional interplay between membrane Gb 3 and MDR1 provides a more physiologically based approach to MDR1 regulation to increase the bioavailability of chemotherapeutic drugs. Multidrug resistance (MDR)3 is one of the major obstacles for successful chemotherapy in patients with cancer. The MDR phenotype has been associated with overexpression of P-glycoprotein in cancer cells (1). MDR1 is a 170-kDa plasma membrane glycoprotein that uses ATP to pump hydrophobic molecules out of the cell (2, 3). Its efflux function decreases drug concentration in tumor cells that results in chemotherapy failure. MDR1 is expressed in relatively high levels in the apical membranes of epithelial cells of intestine, kidney, liver, and blood-brain/testes barriers (4). Thus, the presence of MDR1 within the endothelial cells' epithelium can affect the bioavailability and the oral availability of therapeutic drugs (5).Various attempts have been made to reverse this resistance using MDR1 inhibitors that interact with MDR1 and block drug efflux. First generation modulators such as calcium channel blockers, calmodulin inhibitors, and cyclosporin were developed for pharmacological uses other than reversal of MDR and were relatively nonspecific and weak inhibitors that were also substrates of MDR1, and their deleterious toxicities with their use at required concentrations to inhibit MDR1 ...

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“…The human colon carcinoma cell line (Caco-2) was obtained from American Type Culture Collection (Rockville, MD, USA). MDCK cell clones strain II [17] (MDCKII) and its human MDR1 cDNA transfected strain [18] (MDR1-MDCKII) were gifts from Professor Borst P of the Netherlands Cancer Institute (NKI [19] . The inserts were washed twice with warm transport buffer (HBSS containing 1% DMSO [v/v] and 1 mmol/L ascorbic acid [to prevent oxidation of quercetin]).…”

Section: Methodsmentioning

confidence: 99%

Intra-herb pharmacokinetics interaction between quercetin and isorhamentin

Lan

Tian

et al. 2008

Acta Pharmacol Sin

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Aim: Quercetin and isorhamnetin are common constituents of some herb extracts, such as extracts of gingko leaves and total flavones of Hippophae rhamnoides L. The intra-herb pharmacokinetics interactions between isorhamnetin and quercetin were investigated in the present study. Methods: Human MDR1 cDNA transfected MDCKII cells were used to validate whether isorhamnein interacted with P-gp. Caco-2 transport assays and a randomized, 3-way crossover pharmacokinetics study in rats were used to investigate the pharmacokinetics interactions. HPLC was used to determine cell transport samples. The total plasma concentrations of quercetinand isorhamnetin were determined by liquid chromatography tandem mass spectrometry (LC-MS/MS) by treatment with β-glucuronidase and sulfatase. Results: The permeability ratio (absorptive permeability/secretive permeability) of isorhamnetin across human MDR1 cDNA transfected MDCKII cells, Caco-2 cells and wild-type MDCKII cells are 0.25±0.02, 0.74±0.05, and 1.41±0.06, respectively. This result proved the role of P-gp in the cell efflux of isorhamnetin. While co-transporting with each other across Caco-2 cells monolayer, the permeability ratio of isorhamnetin and quercetin increased by 4.3 and 2.2 times. After coadministration with each other to rats,the C max , AUC 0-72 h , and AUC 0-∞ of both isorhamnetin and quercetin significantly increased compared with single administration. Conclusion:The above results proved intra-herb pharmacokinetics interaction between quercetin and isorhamentin. P-gp might play an important role, whereas other drug efflux pumps, such as multi-drug resistance associate protein 2 and breast cancer resistance protein, might be involved. Accordingly, besides the drug-herb interactions, intra-herb interaction might be brought into view with the wide use of herbal-based remedies.

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P-Glycoprotein in cell cultures: a combined approach to study expression, localisation, and functionality in the confocal microscope

Cited by 40 publications

References 35 publications

P-glycoprotein–actin association through ERM family proteins: a role in P-glycoprotein function in human cells of lymphoid origin

P-glycoprotein–actin association through ERM family proteins: a role in P-glycoprotein function in human cells of lymphoid origin

Inhibition of Multidrug Resistance by AdamantylGb3, a Globotriaosylceramide Analog

Intra-herb pharmacokinetics interaction between quercetin and isorhamentin

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