MDR1 P-glycoprotein Expression Research Articles

Raltegravir does not revert efflux activity of MDR1-P-glycoprotein in human MDR cells

BackgroundRaltegravir (Isentress®)(RALT) has demonstrated excellent efficacy in both treatment-experienced and naïve patients with HIV-1 infection, and is the first strand transfer integrase inhibitor to be approved for use in HIV infected adults worldwide. Since the in vivo efficacy of this class of antiviral drugs depends on their access to intracellular sites where HIV-1 replicates, we analyzed the biological effects induced by RALT on human MDR cell systems expressing multidrug transporter MDR1-P-glycoprotein (MDR1-Pgp).MethodsOur study about RALT was performed by using a set of consolidated methodologies suitable for evaluating the MDR1-Pgp substrate nature of chemical and biological agents, namely: i) assay of drug efflux function; ii) analysis of MDR reversing capability by using cell proliferation assays; iii) monoclonal antibody UIC2 (mAb) shift test, as a sensitive assay to analyze conformational transition associated with MDR1-Pgp function; and iv) induction of MDR1-Pgp expression in MDR cell variant subjected to RALT exposure.ResultsFunctional assays demonstrated that the presence of RALT does not remarkably interfere with the efflux mechanism of CEM-VBL100 and HL60 MDR cells. Accordingly, cell proliferation assays clearly indicated that RALT does not revert MDR phenotype in human MDR1-Pgp expressing cells. Furthermore, exposure of CEM-VBL10 cells to RALT does not induce MDR1-Pgp functional conformation intercepted by monoclonal antibody (mAb) UIC2 binding; nor does exposure to RALT increase the expression of this drug transporter in MDR1-Pgp expressing cells.ConclusionsNo evidence of RALT interaction with human MDR1-Pgp was observed in the in vitro MDR cell systems used in the present investigation, this incorporating all sets of studies recommended by the FDA guidelines. Taken in aggregate, these data suggest that RALT may express its curative potential in all sites were HIV-1 penetrates, including the MDR1-Pgp protected blood/tissue barrier. Moreover RALT, evading MDR1-Pgp drug efflux function, would not interfere with pharmacokinetic profiles of co-administered MDR1-Pgp substrate antiretroviral drugs.

ATP-binding cassette transporters in primary central nervous system lymphoma: Decreased expression of MDR1 P-glycoprotein and breast cancer resistance protein in tumor capillary endothelial cells

Most tumors in the central nervous system are drug resistant partly because of the presence of the blood-brain barrier (BBB) between circulating blood and tumor tissues. Primary central nervous system lymphoma (PCNSL) is one of the exceptions, as it is highly sensitive to high-dose methotrexate (MTX)-based chemotherapy. The aim of this study was to evaluate the BBB function of tumor capillary endothelial cells in PCNSL. Expression of three major drug efflux transporters that belong to the ATP-binding cassette (ABC) superfamily, P-glycoprotein encoded by the human multidrug resistance gene (MDR1 Pgp; ABCB1), breast cancer resistance protein (BCRP; ABCG2), and multidrug resistance-associated protein 1 (MRP1; ABCC1), was evaluated. Immunohistochemistry was performed in capillary endothelial cells of 30 tumor areas from 22 PCNSL cases and compared with that of 30 gliomas. The microenvironment around tumor capillaries was assessed by examining the distribution of astrocytes and by counting the number of macrophages and T-cells, the principal cytokine producers. In PCNSL, expression of MDR1 Pgp and BCRP in tumor capillary endothelial cells was decreased in 63 and 93% of tumor areas examined, respectively, and these reduction levels differed significantly from those of gliomas (P<0.05). When the PCNSLs were further segregated by way of infiltration of tumor cells into three patterns (dense, perivascular and sparse), decreased MDR1 Pgp and BCRP in tumor capillary endothelial cells were much more prominent in dense and perivascular patterns. In all tumors and non-tumor areas of the brain, MRP1 was undetected on capillary endothelial cells. Assessment of the microenvironment around the tumor capillaries suggested that dissociation from astrocytes and infiltration of macrophages and T-cells was involved in the down-regulation of MDR1 Pgp and BCRP in capillary endothelial cells. In conclusion, we report that expression of the major ABC transporters of the BBB, MDR1 Pgp and BCRP, decreases in tumor capillary endothelial cells of PCNSL. Thus, decreased expression may permit delivery of chemotherapeutic agents to the tumor tissues.

Prognostic significance of MDR-1 P-glycoprotein expression in breast cancer

Objective To investigate the expression of MDR-1 P-glycoprotein(MDR-1 Pgp) in breast cancer and analyze its correlation to the biological behavior and prognosis of the disease. Methods The expression of MDR-1 Pgp was examined in 75 cases of breast cancer patients by using three different monoclonal antibodies(JSB1, C219 and C494) with S-P immunohistochemisty. These patients were followed up for 5 years, and the correlation between MDR-1 Pgp expression, survival rate and lymph metastasis was analyzed. Results Positive detection of MDR-1 Pgp by JSB1, C219 and C494 in 75 cases of breast cancer was 86.7%, 48% and 85.3%, respectively. MDR-1 Pgp expression was not related to ages of patients ( P > 0.05). JSB1-detected expression of MDR-1 Pgp was related to lymph node metastasis( P < 0.05); while C219 and C494 were not( P > 0.05). The patients with MDR-1 Pgp expression positively detected by either two of the three antibodies, had five-year survival rate that was significantly higher than those positively detected by all the three antibodies( P < 0.05). Conclusion Three antibodies should be used simultaneously to detect MDR-1 Pgp expression in breast cancer. Positive MDR-1 Pgp expression in breast cancer detected by all the three antibodies may represent a poor prognosis; while positive MDR-1 Pgp detection by JSB1 and C494 is associated with lymph metastasis.

Influence of Different Chemicals on MDR-1 P-Glycoprotein Expression and Activity in the HK-2 Proximal Tubular Cell Line

P-glycoprotein (Pgp), the MDR-encoded membrane transporter, is physiologically expressed in normal tissues with excretory functions, including kidney proximal tubules. In a preliminary report we have shown that HK-2, an immortalized cell line from normal human proximal tubule, expresses a functional Pgp and may be considered a valuable model for in vitro investigations on the Pgp role(s) in human renal pathophysiology. The present investigation was designed to further characterize the properties of HK-2 Pgp by exploring its responsiveness to a variety of exogenous or endogenous modulators. HK-2 cells were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 supplemented with 5% FCS in the absence or in the presence of modulators. Pgp mRNA expression was studied by RT-PCR and the amount of Pgp was determined by Western blotting. Pgp activity was assessed by intracellular rhodamine-123 (R-123) accumulation. RT-PCR showed that HK-2 cells express MDR-1, but not MDR-3. Both MDR-1 Pgp and MDR-1 mRNA were significantly increased in cells cultured in the presence of cyclosporin A (CsA), 1,25(OH) 2D 3, platelet activating factor, dexamethasone (Dex), or aldosterone. Verapamil (Vp), cimetidine, and trimethoprim did not affect HK-2 Pgp expression. Conversely, 2-acetylaminofluorene strongly downregulated Pgp expression. Vp, CsA, 1,25(OH) 2D 3 and Dex significantly increased R-123 intracellular retention, indicating the inhibition of Pgp-mediated transport. Drug-pretreated, Pgp-overexpressing cells showed increased Pgp activity and were less susceptible to toxic concentrations of CsA. MDR-1 Pgp in HK-2 appears to be responsive to many compounds, including classical Pgp inhibitors and putative physiological substrates, but some of its pharmacological properties are different from those described in other experimental, in particular nonhuman, cell models.

Influence of Different Chemicals on MDR-1 P-Glycoprotein Expression and Activity in the HK-2 Proximal Tubular Cell Line

P-glycoprotein (Pgp), the MDR-encoded membrane transporter, is physiologically expressed in normal tissues with excretory functions, including kidney proximal tubules. In a preliminary report we have shown that HK-2, an immortalized cell line from normal human proximal tubule, expresses a functional Pgp and may be considered a valuable model for in vitro investigations on the Pgp role(s) in human renal pathophysiology. The present investigation was designed to further characterize the properties of HK-2 Pgp by exploring its responsiveness to a variety of exogenous or endogenous modulators. HK-2 cells were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 supplemented with 5% FCS in the absence or in the presence of modulators. Pgp mRNA expression was studied by RT-PCR and the amount of Pgp was determined by Western blotting. Pgp activity was assessed by intracellular rhodamine-123 (R-123) accumulation. RT-PCR showed that HK-2 cells express MDR-1, but not MDR-3. Both MDR-1 Pgp and MDR-1 mRNA were significantly increased in cells cultured in the presence of cyclosporin A (CsA), 1,25(OH)2D3, platelet activating factor, dexamethasone (Dex), or aldosterone. Verapamil (Vp), cimetidine, and trimethoprim did not affect HK-2 Pgp expression. Conversely, 2-acetylaminofluorene strongly downregulated Pgp expression. Vp, CsA, 1,25(OH)2D3 and Dex significantly increased R-123 intracellular retention, indicating the inhibition of Pgp-mediated transport. Drug-pretreated, Pgp-overexpressing cells showed increased Pgp activity and were less susceptible to toxic concentrations of CsA. MDR-1 Pgp in HK-2 appears to be responsive to many compounds, including classical Pgp inhibitors and putative physiological substrates, but some of its pharmacological properties are different from those described in other experimental, in particular nonhuman, cell models.

Increased MDR1 P-glycoprotein expression in methotrexate resistance: Comment on the article by Yudoh et al

Arthritis & RheumatismVolume 43, Issue 7 p. 1661-1662 LetterFree to Read Increased MDR1 P-glycoprotein expression in methotrexate resistance: Comment on the article by Yudoh et al Proton Rahman MD, Proton Rahman MD Memorial University of Newfoundland, St. John's, Newfoundland, CanadaSearch for more papers by this authorDonna Hefferton, Donna Hefferton Memorial University of Newfoundland, St. John's, Newfoundland, CanadaSearch for more papers by this authorDesmond Robb MD, Desmond Robb MD Memorial University of Newfoundland, St. John's, Newfoundland, CanadaSearch for more papers by this author Proton Rahman MD, Proton Rahman MD Memorial University of Newfoundland, St. John's, Newfoundland, CanadaSearch for more papers by this authorDonna Hefferton, Donna Hefferton Memorial University of Newfoundland, St. John's, Newfoundland, CanadaSearch for more papers by this authorDesmond Robb MD, Desmond Robb MD Memorial University of Newfoundland, St. John's, Newfoundland, CanadaSearch for more papers by this author First published: 26 March 2001 https://doi.org/10.1002/1529-0131(200007)43:7<1657::AID-ANR39>3.0.CO;2-ZCitations: 3AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume43, Issue7July 2000Pages 1661-1662 RelatedInformation

Increased MDR1 P-glycoprotein expression in methotrexate resistance: Comment on the article by Yudoh et al

Increased MDR1 P-glycoprotein expression in methotrexate resistance: Comment on the article by Yudoh et al

Novel gallium(III) complexes transported by MDR1 P-glycoprotein: potential PET imaging agents for probing P-glycoprotein-mediated transport activity in vivo

Background: Multidrug resistance (MDR) mediated by expression of MDR1 P-glycoprotein (Pgp) represents one of the best characterized barriers to chemotherapy in cancer patients. Positron emission tomography (PET) agents for analysis of Pgp-mediated drug transport activity in vivo would enable noninvasive assessment of chemotherapeutic regimens and MDR gene therapy. Results: Candidate Schiff-base phenolic gallium(III) complexes were synthesized from their heptadentate precursors and gallium(III)acetylacetonate. Crystal structures demonstrated a hexacoordinated central gallium with overall trans-pseudo-octahedral geometry. Radiolabeled 67Ga-complexes were obtained in high purity and screened in drug-sensitive (Pgp −) and MDR (Pgp +) tumor cells. Compared with control, lead compound 6 demonstrated antagonist-reversible 55-fold lower accumulation in Pgp-expressing MDR cells. Furthermore, compared with wild-type control, quantitative pharmacokinetic analysis showed markedly increased penetration and retention of 6 in brain and liver tissues of mdr1a/b (−/−) gene disrupted mice, correctly mapping Pgp-mediated transport activity at the capillary blood–brain barrier and hepatocellular biliary cannalicular surface in vivo. Conclusions: These results indicate that gallium(III) complex 6 is recognized by MDR1 Pgp as an avid transport substrate, thereby providing a useful scaffold to generate 68Ga radiopharmaceuticals for molecular imaging of Pgp transport activity in tumors and tissues in vivo using PET.

MDR1-Pgp 170 expression in human bronchus.

MDR1 P-glycoprotein (Pgp 170), a member of the adenosine triphosphate (ATP) binding cassette transporters, acts as an efflux pump for various hydrophobic agents, particularly for xenobiotics such as benzo(a)pyrene. It has also been shown to regulate cell-volume activated chloride channels. Pgp 170 could, therefore, be of particular importance in cellular mechanisms of defence in the airways and in the control of mucus layer composition. For these reasons, we evaluated the precise localization of Pgp 170 in human adult airways. Fresh non-neoplastic bronchial specimens were collected from 33 patients (26 smokers, four exsmokers and three nonsmokers) who underwent surgery for lung carcinoma. The presence of MDR1 messenger ribonucleic acid (mRNA) was demonstrated by reverse transcriptase chain reaction (RT-PCR) in bronchial epithelial cells collected by gentle scraping from either smokers, exsmokers or nonsmokers. Immunodetection of Pgp 170 using a panel of monoclonal antibodies (MRK16, JSB1, C219, C494) was performed either on cryostat or paraffin-embedded sections of histologically normal bronchial tissue. Pgp 170 was constantly detected with intense labelling at the apical surface of ciliated epithelial cells from the surface epithelium or ciliated collecting ducts, and on apical and lateral surfaces of serous cells from bronchial glands. No staining of mucus-secreting cells was observed. Pgp 170 was also demonstrated at the luminal surface of endothelial cells of bronchial capillaries. In conclusion, the expression of MDR1 P-glycoprotein in bronchial structures, particularly at the epithelial apical surface, suggests important roles for this transmembrane protein in human airways.

Involvement of MDR1 P-glycoprotein in multifactorial resistance to methotrexate.

Cellular resistance to methotrexate (MTX) is believed to be unaffected by expression of MDR1 P-glycoprotein (Pgp), a pleiotropic efflux pump acting on different hydrophobic compounds that enter cells by passive diffusion. A series of human leukemic CCRF-CEM sublines, isolated by multi-step selection for very high resistance to MTX, exhibit multiple mechanisms of MTX resistance, including decreased carrier-mediated uptake of MTX and DHFR gene amplification. These sublines show cross-resistance to drugs of the multi-drug resistance (MDR) family, which is correlated with relative resistance to MTX. The MTX-selected sublines show increased expression and function of the MDR1 gene, based on the measurement of MDR1 mRNA, Pgp and rhodamine 123 accumulation. Sequence analysis of the MDR1 cDNA from MTX-selected CCRF-CEM cells revealed no mutations in the protein coding region. MTX resistance in these cell lines is partially reversible by a Pgp-specific monoclonal antibody (MAb) UIC2 and a monovalent FaB fragment of UIC2. Our results indicate that Pgp can contribute to multifactorial resistance to MTX.

Effects of multidrug resistance (MDR1) P-glycoprotein expression levels and coordination metal on the cytotoxic potency of multidentate (N4O2) (ethylenediamine)bis[propyl(R-benzylimino)]metal(III) cations.

Enhanced mitochondrial transmembrane potentials in tumor cells have been proposed to confer tumor-selective-targeting properties to modestly lipophilic monocationic compounds. To explore the potential cytotoxic activity of lipophilic cationic metallopharmaceuticals containing a highly flexible hexadentate N4O2 Schiff-base phenolic ligand, we first synthesized precursors H3Mabi (1) and H3DMabi (2) by condensation of an appropriate linear tetraamine with substituted salicylaldehydes. The desired N4O2 ligands, (ethylenediamine)-N,N'-bis[propyl[(2-hydroxy-3-methoxybenzyl)imino]] and (ethylenediamine)-N,N'-bis[propyl[2-hydroxy-4,6-dimethoxybenzyl)-imino]] (R-ENBPI), were obtained by cleavage of the imidazolidine ring, and their corresponding monocationic complexes were produced by reaction with appropriate hydrated salts or acetylacetonates of Al(III), Fe(III), Ga(III), and In(III). All complexes were stable to neutral hydrolysis. In human epidermal carcinoma KB-3-1 cells, cytotoxic potencies of racemic mixtures of these complexes were in the low micromolar range and, for a given ligand, depended on the identity of the coordinating central metal. The active 4,6-dimethoxy-ENBPI complexes were more potent than their 3-methoxy analogs, while the free ligands and metal(III) ions showed little or no cytotoxic activity. Furthermore, in colchicine-selected KB-8-5 multidrug resistant (MDR) cells, modest cellular expression of human MDR1 P-glycoprotein conferred protection from the cytotoxic activities of Al(III), Fe(III), and Ga(III) R-ENBPI complexes indicating that these complexes were recognized as transport substrates by the P-glycoprotein efflux transporter. However, the cytotoxic activities of the corresponding In(III) complexes, while among the lowest in potencies, were also not altered by expression of MDR1 P-glycoprotein. Thus, for the Group III elements, human cells were capable of distinguishing R-ENBPI complexes formed of the same ligands with different metals. Furthermore, selected R-ENBPI metal(III) complexes may be useful as novel anticancer metallopharmaceuticals.

Non-P-glycoprotein mediated mechanism for multidrug resistance precedes P-glycoprotein expression during in vitro selection for doxorubicin resistance in a human lung cancer cell line : Baas F, Jongsma APM, Broxterman HJ et al. Division of Molecular Biology, The Netherlands Cancer Institute, Plasmanlaan 121, 1066 CX Amsterdam. Cancer Res 1990;50:5392–5398

Two different mechanisms that contribute to multidrug resistance (MDR) were found in derivatives of the human squamous lung cancer cell line SW-1573. The parental cell line has a low amount of mdr1 P-glycoprotein mRNA. In three independent selections for doxorubicin resistance, MDR variants arose in which mdr1 P-glycoprotein mRNA and protein was not detectable. Selection on higher doxorubicin concentrations gave rise to variants containing high levels of mdr1 mRNA, due to transcriptional activation of the mdr1 gene. Upon continued selection for higher levels of doxorubicin resistance, the mdr1 gene became amplified, resulting in an additional increase in the level of mdr1 mRNA. The cross-resistance pattern of the sublines that lack mdr1 P-glycoprotein expression is different from that seen in the mdr1 overexpressing cells. Both types of MDR cell lines are resistant to doxorubicin, daunorubicin, etoposide, colchicine, gramicidin D, and vincristine. However, in the non-P-glycoprotein-mediated MDR cell lines, resistance levels are lower and a preferential resistance for etoposide is seen.