scholarly journals Substrate Specificity of Aglaia loheri Active Isolate towards P-glycoprotein in Multidrug-Resistant Cancer Cells

2016 ◽  
Vol 11 (11) ◽  
pp. 1934578X1601101
Author(s):  
Else Dapat ◽  
Sonia Jacinto ◽  
Thomas Efferth
Keyword(s):  
Substrate Specificity ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Competitive Inhibition ◽  
Competitive Inhibitor ◽  
Multidrug Resistant ◽  
Contributory Factor ◽  
Acetoxymethyl Ester ◽  
P Glycoprotein ◽  
Drug Concentrations

Multidrug resistance (MDR) is a major contributory factor in the failure of chemotherapy. Concrete interpretation of P-glycoprotein (P-gp) substrate specificity, whether a substance is a substrate or an inhibitor, represents an important feature of a compound's pharmaceutical profiling in drug design and development. In this work, the P-gp substrate specificity of Maldi 531.2[M+H]+, a phenol ester from Aglaia loheri Blanco leaves was investigated. This study focuses on the effect of Maldi 531.2[M+H]+ on P-gp ATPase activity, which was examined by measuring the amount of inorganic phosphates (Pi) released as a result of ATP hydrolysis. To test the effects of Maldi 531.2[M+H]+ on MDR activity, an attempt to combine Maldi 531.2[M+H]+ with a potent P-gp substrate such as verapamil was performed. As a result of this combination treatment, two distinct patterns of interaction with P-gp activity were determined by a calcein-acetoxymethyl ester (AM) assay. Depending on the concentratgion, both stimulation and inhibition of MDR activity were observed at certain drug concentrations suggesting biphasic reactions, which can be understood as cooperative stimulation and competitive inhibition, respectively. Verapamil is a strong substrate to P-gp. Substrate specificity of Maldi 531.2[M+H]+ may be less than the substrate specificity of verapamil, but it acts additively together with low concentrations of verapamil in stimulating ATPase activity. On the one hand, verapamil and Maldi 531.2[M+H]+ exerted cooperative stimulation on P-gp. On the other hand, Maldi 531.2[M+H]+ acts as competitive inhibitor at higher concentrations.

scholarly journals Transmembrane segment 1 of human P-glycoprotein contributes to the drug-binding pocket

2006 ◽  
Vol 396 (3) ◽  
pp. 537-545 ◽  
Author(s):  
Tip W. Loo ◽  
M. Claire Bartlett ◽  
David M. Clarke
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Binding Pocket ◽  
Drug Binding ◽  
Covalent Attachment ◽  
Acetoxymethyl Ester ◽  
Glycoprotein P ◽  
P Glycoprotein ◽  
Cysteine Scanning ◽  
Cysteine Scanning Mutagenesis

P-glycoprotein (P-gp; ABCB1) actively transports a broad range of structurally unrelated compounds out of the cell. An important step in the transport cycle is coupling of drug binding with ATP hydrolysis. Drug substrates such as verapamil bind in a common drug-binding pocket at the interface between the TM (transmembrane) domains of P-gp and stimulate ATPase activity. In the present study, we used cysteine-scanning mutagenesis and reaction with an MTS (methanethiosulphonate) thiol-reactive analogue of verapamil (MTS-verapamil) to test whether the first TM segment [TM1 (TM segment 1)] forms part of the drug-binding pocket. One mutant, L65C, showed elevated ATPase activity (10.7-fold higher than an untreated control) after removal of unchanged MTS-verapamil. The elevated ATPase activity was due to covalent attachment of MTS-verapamil to Cys65 because treatment with dithiothreitol returned the ATPase activity to basal levels. Verapamil covalently attached to Cys65 appears to occupy the drug-binding pocket because verapamil protected mutant L65C from modification by MTS-verapamil. The ATPase activity of the MTS-verapamil-modified mutant L65C could not be further stimulated with verapamil, calcein acetoxymethyl ester or demecolcine. The ATPase activity could be inhibited by cyclosporin A but not by trans-(E)-flupentixol. These results suggest that TM1 contributes to the drug-binding pocket.

scholarly journals Effects of steroids and verapamil on P-glycoprotein ATPase activity: progesterone, desoxycorticosterone, corticosterone and verapamil are mutually non-exclusive modulators

1996 ◽  
Vol 317 (2) ◽  
pp. 515-522 ◽  
Author(s):  
Stéphane ORLOWSKI ◽  
Lluis M. MIR ◽  
Jean BELEHRADEK ◽  
Manuel GARRIGOS
Keyword(s):  
Atpase Activity ◽  
Binding Sites ◽  
Drug Transport ◽  
Competitive Inhibition ◽  
Membrane Vesicles ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Glycoprotein P ◽  
P Glycoprotein ◽  
Synergistic Modulation

P-glycoprotein (P-gp) is a membranous ATPase responsible for the multidrug resistance (MDR) phenotype. Using membrane vesicles prepared from the highly resistant cell line DC-3F/ADX we studied the influence on P-gp ATPase activity of four progesterone derivatives which specifically bind to P-gp and reverse MDR. Progesterone and desoxycorticosterone stimulate P-gp ATPase activity with, respectively, apparent concentrations giving half-maximal activation of 20–25 μM and 40–50 μM, and activation factors of 2.3 (at 100 μM progesterone) and 1.8 (at 170 μM desoxycorticosterone). Hydrocortisone above 100 μM stimulates P-gp ATPase activity while corticosterone has no apparent stimulating effect. Our data are consistent with the location of the binding sites for the progesterone derivatives on the P-gp membranous domain. The effects of these steroids on verapamil-stimulated P-gp ATPase activity support a non-competitive mechanism, i.e. the binding sites for verapamil and steroids are mutually non-exclusive for P-gp ATPase modulation. A similar non-competitive inhibition of progesterone-stimulated P-gp ATPase activity by desoxycorticosterone or by corticosterone leads to the conclusion that these steroids, although sharing related structures, have distinct modulating sites on P-gp. As expected from their mutually non-exclusive interactions on P-gp, progesterone and verapamil when mixed induce a synergistic modulation of P-gp ATPase activity. Since drug transport by P-gp is believed to be coupled to its ATPase activity, a corresponding synergistic effect of these two modulators for the inhibition of P-gp-mediated drug resistance can be expected.

Wilforine resensitizes multidrug resistant cancer cells via competitive inhibition of P-glycoprotein

Phytomedicine ◽  
2020 ◽  
Vol 71 ◽  
pp. 153239
Author(s):  
Ying-Tzu Chang ◽  
Yu-Chao Lin ◽  
Lijuan Sun ◽  
Wei-Chieh Liao ◽  
Charles C.N. Wang ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Competitive Inhibition ◽  
Multidrug Resistant ◽  
P Glycoprotein

scholarly journals Anlotinib Reverses Multidrug Resistance (MDR) in Osteosarcoma by Inhibiting P-Glycoprotein (PGP1) Function In Vitro and In Vivo

2022 ◽  
Vol 12 ◽  
Author(s):  
Gangyang Wang ◽  
Lingling Cao ◽  
Yafei Jiang ◽  
Tao Zhang ◽  
Hongsheng Wang ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Mouse Model ◽  
Atpase Activity ◽  
Multidrug Resistant ◽  
Chemotherapeutic Agents ◽  
Protein Expression Level ◽  
Nude Mouse Model ◽  
P Glycoprotein

Overexpression of the multidrug resistance (MDR)-related protein P-glycoprotein (PGP1), which actively extrudes chemotherapeutic agents from cells and significantly decreases the efficacy of chemotherapy, is viewed as a major obstacle in osteosarcoma chemotherapy. Anlotinib, a novel tyrosine kinase inhibitor (TKI), has good anti-tumor effects in a variety of solid tumors. However, there are few studies on the mechanism of anlotinib reversing chemotherapy resistance in osteosarcoma. In this study, cellular assays were performed in vitro and in vivo to evaluate the MDR reversal effects of anlotinib on multidrug-resistant osteosarcoma cell lines. Drug efflux and intracellular drug accumulation were measured by flow cytometry. The vanadate-sensitive ATPase activity of PGP1 was measured in the presence of a range of anlotinib concentrations. The protein expression level of ABCB1 was detected by Western blotting and immunofluorescence analysis. Our results showed that anlotinib significantly increased the sensitivity of KHOSR2 and U2OSR2 cells (which overexpress PGP1) to chemotherapeutic agents in vitro and in a KHOSR2 xenograft nude mouse model in vivo. Mechanistically, anlotinib increases the intracellular accumulation of PGP1 substrates by inhibiting the efflux function of PGP1 in multidrug-resistant cell lines. Furthermore, anlotinib stimulated the ATPase activity of PGP1 but affected neither the protein expression level nor the localization of PGP1. In animal studies, anlotinib in combination with doxorubicin (DOX) significantly decreased the tumor growth rate and the tumor size in the KHOSR2 xenograft nude mouse model. Overall, our findings suggest that anlotinib may be useful for circumventing MDR to other conventional antineoplastic drugs.

Inhibition of P-glycoprotein by cyclosporin A analogues and metabolites

1999 ◽  
Vol 77 (1) ◽  
pp. 47-58 ◽  
Author(s):  
Michel Demeule ◽  
Alain Laplante ◽  
Arash Sepehr-Araé ◽  
Édith Beaulieu ◽  
Diana Averill-Bates ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Cyclosporin A ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Multidrug Resistant ◽  
Hydroxyl Group ◽  
Glycoprotein P ◽  
Ovary Cells ◽  
Psc 833 ◽  
P Glycoprotein

The interaction between P-glycoprotein (P-gp) from membranes isolated from multidrug-resistant Chinese hamster ovary cells and cyclosporin A (CsA) analogues and its metabolites was characterized. Screening of these latter as chemosensitizers was performed using three different assays: (i) vinblastine uptake, (ii) photoaffinity labeling by [125I]iodoaryl azidoprazosin, and (iii) P-gp ATPase activity. Oxidation of the hydroxyl group at position 1 of CsA (200-096), CsG (215-834), or CsD (PSC-833) increased their inhibition of P-gp. CsA analogues (208-032, 208-183) modified at position 11 retained their ability to inhibit P-gp while analogues modified at position 2 (CsC and CsD) lost their efficiency. The inhibitions induced by metabolites of CsA were also compared to those obtained with CsG metabolites. From all the molecules tested, PSC-833 and 280-446 peptolide were the strongest inhibitors. Our results indicate that modifications of CsA analogues at position 1 and 2 are critical for their interaction with P-gp and that CsA metabolites retain a portion of the inhibitory activity of the parent drug.Key words: P-glycoprotein, cyclosporin A, vinblastine uptake, photolabeling, ATPase activity.

scholarly journals Multidrug-resistant cancer cells contain two populations of P-glycoprotein with differently stimulated P-gp ATPase activities: evidence from atomic force microscopy and biochemical analysis

2005 ◽  
Vol 388 (2) ◽  
pp. 563-571 ◽  
Author(s):  
Stéphane BARAKAT ◽  
Landry GAYET ◽  
Guila DAYAN ◽  
Stéphane LABIALLE ◽  
Adina LAZAR ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Atpase Activity ◽  
Cancer Cells ◽  
Multidrug Resistant ◽  
Cholesterol Depletion ◽  
Force Microscopy ◽  
Atomic Force ◽  
P Glycoprotein ◽  
Optimal Value

Considerable interest exists about the localization of P-gp (P-glycoprotein) in DRMs (detergent-resistant membranes) of multidrug resistant cancer cells, in particular concerning the potential modulating role of the closely related lipids and proteins on P-gp activity. Our observation of the opposite effect of verapamil on P-gp ATPase activity from DRM and solubilized-membrane fractions of CEM-resistant leukaemia cells, and results from Langmuir experiments on membrane monolayers from resistant CEM cells, strongly suggest that two functional populations of P-gp exist. The first is located in DRM regions: it displays its optimal P-gp ATPase activity, which is almost completely inhibited by orthovanadate and activated by verapamil. The second is located elsewhere in the membrane; it displays a lower P-gp ATPase activity that is less sensitive to orthovanadate and is inhibited by verapamil. A 40% cholesterol depletion of DRM caused the loss of 52% of the P-gp ATPase activity. Cholesterol repletion allowed recovery of the initial P-gp ATPase activity. In contrast, in the solubilized-membrane-containing fractions, cholesterol depletion and repletion had no effect on the P-gp ATPase activity whereas up to 100% saturation with cholesterol induced a 58% increased P-gp ATPase activity, while no significant modification was observed for the DRM-enriched fraction. DRMs were analysed by atomic force microscopy: 40–60% cholesterol depletion was necessary to remove P-gp from DRMs. In conclusion, P-gp in DRMs appears to contain closely surrounding cholesterol that can stimulate P-gp ATPase activity to its optimal value, whereas cholesterol in the second population seems deprived of this function.

scholarly journals Conformational changes of P-glycoprotein by nucleotide binding

1997 ◽  
Vol 328 (3) ◽  
pp. 897-904 ◽  
Author(s):  
Guichun WANG ◽  
Roxana PINCHEIRA ◽  
Mei ZHANG ◽  
Jian-Ting ZHANG
Keyword(s):  
Conformational Changes ◽  
Drug Transport ◽  
Atp Hydrolysis ◽  
Human Cancer ◽  
Limited Proteolysis ◽  
Membrane Vesicles ◽  
Multidrug Resistant ◽  
Conformational States ◽  
Human Cancer Cells ◽  
P Glycoprotein

P-glycoprotein (Pgp) is a membrane protein that transports chemotherapeutic drugs, causing multidrug resistance in human cancer cells. Pgp is a member of the ATP-binding cassette superfamily and functions as a transport ATPase. It has been suggested that the conformation of Pgp changes in the catalytic cycle. In this study, we tested this hypothesis by using limited proteolysis as a tool to detect different conformational states trapped by binding of nucleotide ligands and inhibitors. Pgp has high basal ATPase activity; that is, ATP hydrolysis by Pgp is not rigidly associated with drug transport. This activity provides a convenient method for studying the conformational change of Pgp induced by nucleotide ligands, in the absence of drug substrates which may generate complications due to their own binding. Inside-out membrane vesicles containing human Pgp were isolated from multidrug-resistant SKOV/VLB cells and treated with trypsin in the absence or presence of MgATP, Mg-adenosine 5ʹ-[β,γ-imido]triphosphate (Mg-p[NH]ppA) and MgADP. Changes in the proteolysis profile of Pgp owing to binding of nucleotides were used to indicate the conformational changes in Pgp. We found that generation of tryptic fragments, including the loop linking transmembrane (TM) regions TM8 and TM9 of Pgp, were stimulated by the binding of Mg-p[NH]ppA, MgATP and MgADP, indicating that the Pgp conformation was changed by the binding of these nucleotides. The effects of nucleotides on Pgp conformation are directly associated with the binding and/or hydrolysis of these ligands. Four conformational states of Pgp were stabilized under different conditions with various ligands and inhibitors. We propose that cycling through these four states couples the Pgp-mediated MgATP hydrolysis to drug transport.

Sign in / Sign up

Export Citation Format

Share Document