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1
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Li W, Fu Y, Sun J, Gong H, Yan R, Wang Y. Construction and in vitro evaluation of pH-sensitive nanoparticles to reverse drug resistance of breast cancer stem cells. Discov Oncol 2024; 15:21. [PMID: 38285118 PMCID: PMC10825093 DOI: 10.1007/s12672-024-00873-w] [Citation(s) in RCA: 5] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/19/2023] [Accepted: 01/24/2024] [Indexed: 01/30/2024] Open
Abstract
Breast cancer is a major threat to safety and health of women. The breast cancer stem cells (BCSCs) have multi-drug resistance to chemotherapy drugs, which leads to chemotherapy failure. We proposed a strategy of delivery of tumor-killing drugs and a resistance reversal agent, to enhance inhibition of BCSCs. Here, schisandrin B (SchB)/AP NPs are constructed using acid-grafted-poly (β-amino ester) (ATRA-g-PBAE, AP) grafted polymer nanoparticle encapsulated SchB, with pH-sensitive release function. This drug delivery system has good pharmacological properties and can increase the SchB release with the decrease of pH. The NPs showed cytotoxic effects in reversing ATRA resistance to BCSCs. Lysosomal escape was achieved when the nanoparticles were taken up by BCSCs. In addition, we found that NPs may reverse MDR by inhibiting the expression of P-glycoprotein (P-gp) and affecting the energy supply of drug efflux. This study provides a nanodelivery therapy strategy that reverses BCSCs multidrug resistance (MDR) and demonstrates that it did so by interfering with cancer cell energy metabolism. Therefore, the co-delivery strategy of ATRA and SchB provides a new option for the treatment of breast cancer.
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Affiliation(s)
- Weinan Li
- School of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin, China.
- Key Laboratory of Basic and Application Research of Beiyao, Heilongjiang University of Chinese Medicine), Ministry of Education, Harbin, China.
| | - Yuhan Fu
- School of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin, China
- Key Laboratory of Basic and Application Research of Beiyao, Heilongjiang University of Chinese Medicine), Ministry of Education, Harbin, China
| | - Jialin Sun
- Postdoctoral Research Station, Heilongjiang University of Chinese Medicine, Harbin, China
- Biological Science and Technology Department, Heilongjiang Vocational College for Nationalities, Harbin, China
| | - Hexin Gong
- School of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin, China
- Key Laboratory of Basic and Application Research of Beiyao, Heilongjiang University of Chinese Medicine), Ministry of Education, Harbin, China
| | - Ru Yan
- School of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin, China
- Key Laboratory of Basic and Application Research of Beiyao, Heilongjiang University of Chinese Medicine), Ministry of Education, Harbin, China
| | - Yanhong Wang
- School of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin, China.
- Key Laboratory of Basic and Application Research of Beiyao, Heilongjiang University of Chinese Medicine), Ministry of Education, Harbin, China.
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2
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Candida Efflux ATPases and Antiporters in Clinical Drug Resistance. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2016; 892:351-376. [PMID: 26721282 DOI: 10.1007/978-3-319-25304-6_15] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
An enhanced expression of genes encoding ATP binding cassette (ABC) and major facilitator superfamily (MFS) transport proteins are known to contribute to the development of tolerance to antifungals in pathogenic yeasts. For example, the azole resistant (AR) clinical isolates of the opportunistic human fungal pathogen Candida albicans show an overexpression of CDR1 and/or CaMDR1 belonging to ABC and MFS, superfamilies, respectively. The reduced accumulation (due to rapid efflux) of drugs in AR isolates confirms the role of efflux pump proteins in the development of drug tolerance. Considering the importance of major multidrug transporters, the focus of recent research has been to understand the structure and function of these proteins which could help to design inhibitors/modulators of these pump proteins. This chapter focuses on some aspects of the structure and function of yeast transporter proteins particularly in relation to MDR in Candida.
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Prasad R, Goffeau A. Yeast ATP-Binding Cassette Transporters Conferring Multidrug Resistance. Annu Rev Microbiol 2012; 66:39-63. [DOI: 10.1146/annurev-micro-092611-150111] [Citation(s) in RCA: 164] [Impact Index Per Article: 12.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Affiliation(s)
- Rajendra Prasad
- School of Life Sciences, Jawaharlal Nehru University, New Delhi, 110067 India;
| | - Andre Goffeau
- Institut des Sciences de la Vie, Université Catholique de Louvain, Louvain-la-Neuve, 1349 Belgium;
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Baro N, Pooput C, Roepe PD. Analysis of chloroquine resistance transporter (CRT) isoforms and orthologues in S. cerevisiae yeast. Biochemistry 2011; 50:6701-10. [PMID: 21744797 PMCID: PMC3155940 DOI: 10.1021/bi200922g] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Previous work from our laboratory optimized MeOH-inducible expression of the P. falciparum malarial parasite transporter PfCRT in P. pastoris yeast. These strains are useful for many experiments but do not allow for inducible protein expression under ambient growth conditions. We have therefore optimized galactose-inducible expression of PfCRT in S. cerevisiae yeast. We find that expression of PfCRT confers CQ hypersensitivity to growing yeast and that this is due to plasma membrane localization of the transporter. We use quantitative analyses of growth rates to compare hypersensitivity for yeast expressing various PfCRT isoforms. We also report successful high level inducible expression of the P. vivax orthologue, PvCRT, and compare CQ hypersensitivity for PvCRT vs PfCRT expressing yeast. We test the hypothesis that hypersensitivity is due to increased transport of CQ into yeast expressing the transporters via direct (3)H-CQ transport experiments and analyze the effect that membrane potential has on transport. The data suggest important new tools for rapid functional screening of PfCRT and PvCRT isoforms and provide further evidence for a model wherein membrane potential promotes charged CQ transport by PfCRT. Data also support our previous conclusion that wild type PfCRT is capable of CQ transport and provide a basis for understanding the lack of correspondence between PvCRT mutations and resistance to CQ in the important malarial parasite P. vivax.
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Affiliation(s)
- Nick Baro
- Department of Chemistry, Department of Biochemistry and Molecular Biology, and Center for Infectious Diseases, Georgetown University, 37 and O Streets, NW Washington, D.C. 20057
| | - Chaya Pooput
- Department of Chemistry, Department of Biochemistry and Molecular Biology, and Center for Infectious Diseases, Georgetown University, 37 and O Streets, NW Washington, D.C. 20057
| | - Paul D. Roepe
- Department of Chemistry, Department of Biochemistry and Molecular Biology, and Center for Infectious Diseases, Georgetown University, 37 and O Streets, NW Washington, D.C. 20057
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Patel JJ, Thacker D, Tan JC, Pleeter P, Checkley L, Gonzales JM, Deng B, Roepe PD, Cooper RA, Ferdig MT. Chloroquine susceptibility and reversibility in a Plasmodium falciparum genetic cross. Mol Microbiol 2010; 78:770-87. [PMID: 20807203 DOI: 10.1111/j.1365-2958.2010.07366.x] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
Mutations in the Plasmodium falciparum chloroquine (CQ) resistance transporter (PfCRT) are major determinants of verapamil (VP)-reversible CQ resistance (CQR). In the presence of mutant PfCRT, additional genes contribute to the wide range of CQ susceptibilities observed. It is not known if these genes influence mechanisms of chemosensitization by CQR reversal agents. Using quantitative trait locus (QTL) mapping of progeny clones from the HB3 × Dd2 cross, we show that the P. falciparum multidrug resistance gene 1 (pfmdr1) interacts with the South-East Asia-derived mutant pfcrt haplotype to modulate CQR levels. A novel chromosome 7 locus is predicted to contribute with the pfcrt and pfmdr1 loci to influence CQR levels. Chemoreversal via a wide range of chemical structures operates through a direct pfcrt-based mechanism. Direct inhibition of parasite growth by these reversal agents is influenced by pfcrt mutations and additional loci. Direct labelling of purified recombinant PfMDR1 protein with a highly specific photoaffinity CQ analogue, and lack of competition for photolabelling by VP, supports our QTL predictions. We find no evidence that pfmdr1 copy number affects CQ response in the progeny; however, inheritance patterns indicate that an allele-specific interaction between pfmdr1 and pfcrt is part of the complex genetic background of CQR.
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Affiliation(s)
- Jigar J Patel
- The Eck Institute for Global Health, Department of Biological Sciences, University of Notre Dame, 205 Galvin Life Sciences, Notre Dame, IN 46556, USA
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6
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Bottova I, Sauder U, Olivieri V, Hehl AB, Sonda S. The P-glycoprotein inhibitor GF120918 modulates Ca2+-dependent processes and lipid metabolism in Toxoplasma gondii. PLoS One 2010; 5:e10062. [PMID: 20386707 PMCID: PMC2851653 DOI: 10.1371/journal.pone.0010062] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2009] [Accepted: 03/15/2010] [Indexed: 11/18/2022] Open
Abstract
Up-regulation of the membrane-bound efflux pump P-glycoprotein (P-gp) is associated with the phenomenon of multidrug-resistance in pathogenic organisms, including protozoan parasites. In addition, P-gp plays a role in normal physiological processes, however our understanding of these P-gp functions remains limited. In this study we investigated the effects of the P-gp inhibitor GF120918 in Toxoplasma gondii, a model apicomplexan parasite and an important human pathogen. We found that GF120918 treatment severely inhibited parasite invasion and replication. Further analyses of the molecular mechanisms involved revealed that the P-gp inhibitor modulated parasite motility, microneme secretion and egress from the host cell, all cellular processes known to depend on Ca2+ signaling in the parasite. In support of a potential role of P-gp in Ca2+-mediated processes, immunoelectron and fluorescence microscopy showed that T. gondii P-gp was localized in acidocalcisomes, the major Ca2+ storage in the parasite, at the plasma membrane, and in the intravacuolar tubular network. In addition, metabolic labeling of extracellular parasites revealed that inhibition or down-regulation of T. gondii P-gp resulted in aberrant lipid synthesis. These results suggest a crucial role of T. gondii P-gp in essential processes of the parasite biology and further validate the potential of P-gp activity as a target for drug development.
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Affiliation(s)
- Iveta Bottova
- Institute of Parasitology, University of Zurich, Zurich, Switzerland
| | | | | | - Adrian B. Hehl
- Institute of Parasitology, University of Zurich, Zurich, Switzerland
| | - Sabrina Sonda
- Institute of Parasitology, University of Zurich, Zurich, Switzerland
- * E-mail:
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7
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Schulz TA, Choi MG, Raychaudhuri S, Mears JA, Ghirlando R, Hinshaw JE, Prinz WA. Lipid-regulated sterol transfer between closely apposed membranes by oxysterol-binding protein homologues. ACTA ACUST UNITED AC 2010; 187:889-903. [PMID: 20008566 PMCID: PMC2806323 DOI: 10.1083/jcb.200905007] [Citation(s) in RCA: 180] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
The ORP lipid-binding domain can contact two membranes simultaneously to facilitate sterol extraction or delivery at one membrane in response to the lipid composition of the other. Sterols are transferred between cellular membranes by vesicular and poorly understood nonvesicular pathways. Oxysterol-binding protein–related proteins (ORPs) have been implicated in sterol sensing and nonvesicular transport. In this study, we show that yeast ORPs use a novel mechanism that allows regulated sterol transfer between closely apposed membranes, such as organelle contact sites. We find that the core lipid-binding domain found in all ORPs can simultaneously bind two membranes. Using Osh4p/Kes1p as a representative ORP, we show that ORPs have at least two membrane-binding surfaces; one near the mouth of the sterol-binding pocket and a distal site that can bind a second membrane. The distal site is required for the protein to function in cells and, remarkably, regulates the rate at which Osh4p extracts and delivers sterols in a phosphoinositide-dependent manner. Together, these findings suggest a new model of how ORPs could sense and regulate the lipid composition of adjacent membranes.
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Affiliation(s)
- Timothy A Schulz
- Laboratory of Cell Biochemistry and Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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8
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Lekostaj JK, Natarajan JK, Paguio MF, Wolf C, Roepe PD. Photoaffinity labeling of the Plasmodium falciparum chloroquine resistance transporter with a novel perfluorophenylazido chloroquine. Biochemistry 2008; 47:10394-406. [PMID: 18767816 PMCID: PMC2724749 DOI: 10.1021/bi8010658] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Abstract
Several models describing how amino acid substitutions in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) confer resistance to chloroquine (CQ) and other antimalarial drugs have been proposed. Further progress requires molecular analysis of interactions between purified reconstituted PfCRT protein and these drugs. We have thus designed and synthesized several perfluorophenyl azido (pfpa) CQ analogues for PfCRT photolabeling studies. One particularly useful probe (AzBCQ) places the pfpa group at the terminal aliphatic N of CQ via a flexible four-carbon ester linker and includes a convenient biotin tag. This probe photolabels PfCRT in situ with high specificity. Using reconstituted proteoliposomes harboring partially purified recombinant PfCRT, we analyze AzBCQ photolabeling versus competition with CQ and other drugs to probe the nature of the CQ binding site. We also inspect how pH, the chemoreversal agent verapamil (VPL), and various amino acid mutations in PfCRT that cause CQ resistance (CQR) affect the efficiency of AzBCQ photolabeling. Upon gel isolation of AzBCQ-labeled PfCRT followed by trypsin digestion and mass spectrometry analysis, we are able to define a single AzBCQ covalent attachment site lying within the digestive vacuolar-disposed loop between putative helices 9 and 10 of PfCRT. Taken together, the data provide important new insight into PfCRT function and, along with previous results, allow us to propose a model for a single CQ binding site in the PfCRT protein.
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Affiliation(s)
| | | | | | - Christian Wolf
- Department of Chemistry, University, Washington, D.C. 20057
- Center for Infectious Disease, Georgetown University, Washington, D.C. 20057
| | - Paul D. Roepe
- Program in Tumor Biology, University, Washington, D.C. 20057
- Department of Chemistry, University, Washington, D.C. 20057
- Center for Infectious Disease, Georgetown University, Washington, D.C. 20057
- Department of Biochemistry and Cellular and Molecular Biology, University, Washington, D.C. 20057
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9
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Enhanced susceptibility to antifungal oligopeptides in yeast strains overexpressing ABC multidrug efflux pumps. Antimicrob Agents Chemother 2008; 52:4057-63. [PMID: 18794383 DOI: 10.1128/aac.01648-07] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The susceptibility to several oligopeptide and amino acid antifungals of a Saccharomyces cerevisiae strain carrying multiple deletions in yeast multidrug resistance genes was compared to transformants containing the CDR1, CDR2, or MDR1 genes that encode the major Candida albicans drug efflux pumps. Recombinant yeast strains overexpressing Cdr1p and Cdr2p showed enhanced susceptibilities to all tested oligopeptide antifungals. The enhanced susceptibilities of multidrug-resistant yeast strains to oligopeptide antifungals corresponded to higher rates of oligopeptide uptake. Yeast cells overexpressing Cdr1p or Cdr2p effluxed protons at higher rates than the reference cells lacking these ABC transporters. An increased plasma membrane electrochemical gradient caused by the functional overexpression of Cdr1p or Cdr2p appeared to increase cellular susceptibility to oligopeptide antifungals by stimulating their uptake via oligopeptide permeases.
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10
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Sawamiphak S, Sophasan S, Endou H, Boonchird C. Functional expression of the rat organic anion transporter 1 (rOAT1) in Saccharomyces cerevisiae. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2005; 1720:44-51. [PMID: 16325760 DOI: 10.1016/j.bbamem.2005.09.021] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/11/2005] [Revised: 08/30/2005] [Accepted: 09/27/2005] [Indexed: 10/25/2022]
Abstract
Organic anion transporter 1 (OAT1) is localized in the basolateral membrane of the proximal tubule in the kidney and plays an essential role in eliminating a wide range of organic anions, preventing their toxic effects on the body. Structural and functional studies of the transporter would be greatly assisted by inexpensive and rapid expression in the yeast Saccharomyces cerevisiae. The gene encoding rat OAT1 (rOAT1) contains many yeast non-preferred codons at the N-terminus and so was modified by fusion of the favored codon sequence of a hemagglutinin (HA) epitope preceding the start codon. The modified gene was cloned into several yeast expression plasmids, both integrative and multicopy, with either ADH1 promoter or GAL1 promoter in order to find a suitable expression system. Compared with the wild type gene, a substantial increase in rOAT1 expression was achieved by modification in the translational initiation region, suggesting that the codon chosen at the N-terminus influenced its expression. The highest inducible expression of rOAT1 was obtained under GAL1 promoter in 2 mu plasmid. A large fraction of rOAT1 was glycosylated in yeast, unaffected by growth temperature. The recombinant yeast expressing rOAT1 showed an increase in the uptake of p-aminohippurate (PAH) and this showed a positive correlation with rOAT1 expression level. Location of rOAT1 predominantly in the yeast plasma membrane confirmed correct processing. The importance of glycosylation for rOAT1 targeting was also shown. To our knowledge, this is the first successful functional expression of rOAT1 in the yeast S. cerevisiae.
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Affiliation(s)
- Suphansa Sawamiphak
- Department of Biotechnology, Faculty of Science, Mahidol University, Rama 6 Road, Phayathai, Rajathevee Bangkok 10400, Thailand
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11
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Peer WA, Bandyopadhyay A, Blakeslee JJ, Makam SN, Chen RJ, Masson PH, Murphy AS. Variation in expression and protein localization of the PIN family of auxin efflux facilitator proteins in flavonoid mutants with altered auxin transport in Arabidopsis thaliana. THE PLANT CELL 2004; 16:1898-911. [PMID: 15208397 PMCID: PMC514169 DOI: 10.1105/tpc.021501] [Citation(s) in RCA: 269] [Impact Index Per Article: 12.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/17/2023]
Abstract
Aglycone flavonols are thought to modulate auxin transport in Arabidopsis thaliana via an as yet undefined mechanism. Biochemical studies suggest that flavonoids interact with regulatory proteins rather than directly with the PIN auxin efflux facilitator proteins. Auxin transport is enhanced in the absence of flavonoids (transparent testa4 [tt4]) and reduced in the presence of excess flavonols (tt7 and tt3). Steady state PIN mRNA levels in roots inversely correlate with auxin movement in tt mutants. PIN gene transcription and protein localization in flavonoid-deficient mutants appear to be modulated by developmental cues and are auxin responsive. Modulation of PIN gene expression and protein distribution by localized auxin accumulations occurs in the wild type as well. Flavonoids inhibit auxin transport primarily at the shoot apex and root tip and appear to modulate vesicular cycling of PIN1 at the root tip. In some auxin-accumulating tissues, flavonoid increases and changes in flavonoid speciation are subsequent to auxin accumulation.
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Affiliation(s)
- Wendy Ann Peer
- Department of Horticulture, Purdue University, West Lafayette, Indiana 47907-2010, USA.
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12
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Dragomir A, Roomans GM. Increased chloride efflux in colchicine-resistant airway epithelial cell lines. Biochem Pharmacol 2004; 68:253-61. [PMID: 15193997 DOI: 10.1016/j.bcp.2004.03.037] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2003] [Accepted: 03/15/2004] [Indexed: 11/30/2022]
Abstract
Colchicine has been proposed as a treatment to alleviate chronic lung inflammation in cystic fibrosis patients and clinical trials are ongoing. Our aim was to investigate whether chronic exposure of cystic fibrosis cells to colchicine can affect their ability to transport chloride in response to cAMP. Colchicine-resistant cells were selected by growing in medium containing nanomolar concentrations of the drug. While microtubuli were affected by acute exposure to colchicine, they appeared normal in colchicine-resistant cells. Colchicine-resistant clones had higher expression of multidrug resistance proteins compared to untreated cells. Cystic fibrosis transmembrane conductance regulator (CFTR) labelling by immunocytochemistry showed no significant changes. The intracellular chloride concentration and basal chloride efflux of the cystic fibrosis treated cells increased significantly compared with untreated cells, while for the cAMP-stimulated Cl-efflux there was no significant change. The results suggest that colchicine promotes chloride efflux via alternative chloride channels. Since this is an accepted strategy for pharmacological treatment of cystic fibrosis, the results strengthen the notion that colchicine would be beneficial to these patients.
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Affiliation(s)
- Anca Dragomir
- Department of Medical Cell Biology, University of Uppsala, Box 571, 751 23 Uppsala, Sweden.
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Chen B, Jin F, Lu P, Lu XL, Wang PP, Liu YP, Yao F, Wang SB. Effect of mitogen-activated protein kinase signal transduction pathway on multidrug resistance induced by vincristine in gastric cancer cell line MGC803. World J Gastroenterol 2004; 10:795-9. [PMID: 15040019 PMCID: PMC4727016 DOI: 10.3748/wjg.v10.i6.795] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: To investigate the correlation between mitogen-activated protein kinase (MAPK) signal transduction pathway and multidrug resistance (MDR) in MGC803 cells.
METHODS: Western blot was used to analyze the expression of MDR associated gene in transient vincristine (VCR) induced MGC803 cells, which were treated with or without the specific inhibitor of MAPK, PD098059. Morphologic analysis of the cells treated by VCR with or without PD098059 was determined by Wright-Giemsa staining. The cell cycle analysis was performed by using flow cytometric assay and the drug sensitivity of MGC803 cells which were exposed to VCR with or without PD098059 was tested by using MTT assay.
RESULTS: Transient exposure to VCR induced P-gp but not MRP1 or GST-π expression in MGC803 cells and the expression of P-gp was inhibited by PD098059. Apoptotic bodies were found in the cells treated with VCR or VCR+PD098059. FCM results indicated that more MGC803 cells showed apoptotic phenotype when treated by VCR and PD098059 (rate: 31.23%) than treated by VCR only (rate: 18.42%) (P < 0.05). The IC50 (284 ± 13.2 μg/L) of MGC803 cells pretreated with VCR was 2.24-fold as that of negative control group (127 ± 17.6 μg/L) and 1.48-fold as that of the group treated with PD098059 (191 ± 27.9 μg/L).
CONCLUSION: This study shows that the expression of P-gp can be induced by transient exposure to VCR and this induction can be prevented by PD098059, which can block the activity of MAPK. MAPK signal transduction pathway may play some roles in modulating MDR1 expression in gastric cancer.
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Affiliation(s)
- Bo Chen
- Department of Surgical Oncology, the First Affiliated Hospital, China Medical University, Shenyang 110001, Liaoning Province, China.
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Zhang H, Howard EM, Roepe PD. Analysis of the antimalarial drug resistance protein Pfcrt expressed in yeast. J Biol Chem 2002; 277:49767-75. [PMID: 12351620 DOI: 10.1074/jbc.m204005200] [Citation(s) in RCA: 58] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Mutations in the novel membrane protein Pfcrt were recently found to be essential for chloroquine resistance (CQR) in Plasmodium falciparum, the parasite responsible for most lethal human malaria (Fidock, D. A., Nomura, T., Talley, A. K., Cooper, R. A., Dzekunov, S. M., Ferdig, M. T., Ursos, L. M., Sidhu, A. B., Naude, B., Deitsch, K. W., Su, X. Z., Wootton, J. C., Roepe, P. D., and Wellems, T. E. (2000) Mol. Cell 6, 861-871). Pfcrt is localized to the digestive vacuolar membrane of the intraerythrocytic parasite and may function as a transporter. Study of this putative transport function would be greatly assisted by overexpression in yeast followed by characterization of membrane vesicles. Unfortunately, the very high AT content of malarial genes precludes efficient heterologous expression. Thus, we back-translated Pfcrt to design idealized genes with preferred yeast codons, no long poly(A) sequences, and minimal stem-loop structure. We synthesized a designed gene with a two-step PCR method, fused this to N- and C-terminal sequences to aid membrane insertion and purification, and now report efficient expression of wild type and mutant Pfcrt proteins in the plasma membrane of Saccharomyces cerevisiae and Pichia pastoris yeast. To our knowledge, this is the first successful expression of a full-length malarial parasite integral membrane protein in yeast. Purified membranes and inside-out plasma membrane vesicle preparations were used to analyze wild type versus CQR-conferring mutant Pfcrt function, which may include effects on H(+) transport (Dzekunov, S., Ursos, L. M. B., and Roepe, P. D. (2000) Mol. Biochem. Parasitol. 110, 107-124), and to perfect a rapid purification of biotinylated Pfcrt. These data expand on the role of Pfcrt in conferring CQR and define a productive route for analysis of important P. falciparum transport proteins and membrane associated vaccine candidates.
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Affiliation(s)
- Hanbang Zhang
- Department of Chemistry, Lombardi Cancer Center, Georgetown University, 37th and O Streets, Washington, D. C. 20057-1227, USA
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15
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Krishnamurthy S, Dixit BL, Gupta CM, Milewski S, Prasad R. ABC transporters Cdr1p, Cdr2p and Cdr3p of a human pathogen Candida albicans are general phospholipid translocators. Yeast 2002; 19:303-18. [PMID: 11870854 DOI: 10.1002/yea.818] [Citation(s) in RCA: 86] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022] Open
Abstract
We have used fluorescent 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-tagged phospholipid analogues, NBD-PE (phosphatidylethanolamine), NBD-PC (phosphatidylcholine) and NBD-PS (phosphatidylserine), to demonstrate that Cdr1p and its other homologues, Cdr2p and Cdr3p, belonging to the ATP-binding cassette (ABC) superfamily behave as general phospholipid translocators. Interestingly, CDR1 and CDR2, whose overexpression leads to azole resistance in C. albicans, elicit in-to-out transbilayer phospholipid movement, while CDR3, which is not involved in drug resistance, carries out-to-in translocation of phospholipids between the two monolayers of plasma membrane. Cdr1p, Cdr2p and Cdr3p could be further distinguished on the basis of their sensitivities to different inhibitors. For example, the in-to-out activity associated with Cdr1p and Cdr2p is energy-dependent and sensitive to sulphydryl blocking agents such as N-ethylmaleimide (NEM) and cytoskeleton disrupting agent cytochalasin E, while Cdr3p-associated out-to-in activity is energy-dependent but insensitive to NEM and cytochalasin E. We found that certain drugs, such as fluconazole, cycloheximide and miconazole, to which Cdr1p confers resistance could also affect in-to-out transbilayer movement of NBD-PE, while the same drugs had no effect on Cdr3p-mediated out-to-in translocation of NBD-PE. The ineffectiveness of these drugs to affect Cdr3p mediated out-to-in phospholipid translocation further confirms the inherent difference in the directionality of phospholipid translocation between these pumps. Notwithstanding the role of some of the Cdrps in drug resistance, this study clearly demonstrates that these ABC transporters of C. albicans are phospholipid translocators and this function could represent one of the physiological functions of such large family of proteins.
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Luker GD, Flagg TP, Sha Q, Luker KE, Pica CM, Nichols CG, Piwnica-Worms D. MDR1 P-glycoprotein reduces influx of substrates without affecting membrane potential. J Biol Chem 2001; 276:49053-60. [PMID: 11598111 DOI: 10.1074/jbc.m105192200] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
MDR1 (multidrug resistance) P-glycoprotein (Pgp; ABCB1) decreases intracellular concentrations of structurally diverse drugs. Although Pgp is generally thought to be an efflux transporter, the mechanism of action remains elusive. To determine whether Pgp confers drug resistance through changes in transmembrane potential (E(m)) or ion conductance, we studied electrical currents and drug transport in Pgp-negative MCF-7 cells and MCF-7/MDR1 stable transfectants that were established and maintained without chemotherapeutic drugs. Although E(m) and total membrane conductance did not differ between MCF-7 and MCF-7/MDR1 cells, Pgp reduced unidirectional influx and steady-state cellular content of Tc-Sestamibi, a substrate for MDR1 Pgp, without affecting unidirectional efflux of substrate from cells. Depolarization of membrane potentials with various concentrations of extracellular K(+) in the presence of valinomycin did not inhibit the ability of Pgp to reduce intracellular concentration of Tc-Sestamibi, strongly suggesting that the drug transport activity of MDR1 Pgp is independent of changes in E(m) or total ion conductance. Tetraphenyl borate, a lipophilic anion, enhanced unidirectional influx of Tc-Sestamibi to a greater extent in MCF-7/MDR1 cells than in control cells, suggesting that Pgp may, directly or indirectly, increase the positive dipole potential within the plasma membrane bilayer. Overall, these data demonstrate that changes in E(m) or macroscopic conductance are not coupled with function of Pgp in multidrug resistance. The dominant effect of MDR1 Pgp in this system is reduction of drug influx, possibly through an increase in intramembranous dipole potential.
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Affiliation(s)
- G D Luker
- Molecular Imaging Center, Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA
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Milewski S, Mignini F, Prasad R, Borowski E. Unusual susceptibility of a multidrug-resistant yeast strain to peptidic antifungals. Antimicrob Agents Chemother 2001; 45:223-8. [PMID: 11120970 PMCID: PMC90265 DOI: 10.1128/aac.45.1.223-228.2001] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The susceptibility of Saccharomyces cerevisiae JG436 multidrug transporter deletion mutant, Deltapdr5, to several antifungal agents was compared to that of JG436-derived JGCDR1 and JGCaMDR1 transformants, harboring the CDR1 and CaMDR1 genes, encoding the main drug-extruding membrane proteins of Candida albicans. The JGCDR1 and JGCaMDR1 yeasts demonstrated markedly diminished susceptibility to the azole antifungals, terbinafine and cycloheximide, while that to amphotericin B was unchanged. Surprisingly, JGCDR1 but not JGCaMDR1 cells showed enhanced susceptibility to peptidic antifungals, rationally designed compounds containing inhibitors of glucosamine-6-phosphate synthase. It was found that these antifungal oligopeptides, as well as model oligopeptides built of proteinogenic amino acids, were not effluxed from JGCDR1 cells. Moreover, they were taken up by these cells at rates two to three times higher than by JG436. The tested oligopeptides were rapidly cleaved to constitutive amino acids by cytoplasmic peptidases. Studies on the mechanism of the observed phenomenon suggested that an additive proton motive force generated by Cdr1p stimulated uptake of oligopeptides into JGCDR1 cells, thus giving rise to the higher antifungal activity of FMDP [N(3)-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid]-peptides.
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Affiliation(s)
- S Milewski
- Department of Pharmaceutical Technology and Biochemistry, Technical University of Gdańsk, Gdańsk, Poland.
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Ferté J. Analysis of the tangled relationships between P-glycoprotein-mediated multidrug resistance and the lipid phase of the cell membrane. EUROPEAN JOURNAL OF BIOCHEMISTRY 2000; 267:277-94. [PMID: 10632698 DOI: 10.1046/j.1432-1327.2000.01046.x] [Citation(s) in RCA: 136] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
P-glycoprotein (Pgp), the so-called multidrug transporter, is a plasma membrane glycoprotein often involved in the resistance of cancer cells towards multiple anticancer agents in the multidrug-resistant (MDR) phenotype. It has long been recognized that the lipid phase of the plasma membrane plays an important role with respect to multidrug resistance and Pgp because: the compounds involved in the MDR phenotype are hydrophobic and diffuse passively through the membrane; Pgp domains involved in drug binding are located within the putative transmembrane segments; Pgp activity is highly sensitive to its lipid environment; and Pgp may be involved in lipid trafficking and metabolism. Unraveling the different roles played by the membrane lipid phase in MDR is relevant, not only to the evaluation of the precise role of Pgp, but also to the understanding of the mechanism of action and function of Pgp. With this aim, I review the data from different fields (cancer research, medicinal chemistry, membrane biophysics, pharmaceutical research) concerning drug-membrane, as well as Pgp-membrane, interactions. It is emphasized that the lipid phase of the membrane cannot be overlooked while investigating the MDR phenotype. Taking into account these aspects should be useful in the search of ways to obviate MDR and could also be relevant to the study of other multidrug transporters.
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Affiliation(s)
- J Ferté
- Service de Biophysique des Protéines et des Membranes, DSV-DBCM-SBPM, CEA, Centre de Saclay, Gif-sur-Yvette, France.
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