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1.
Nat Prod Commun ; 5(12): 1931-4, 2010 Dec.
Article in English | MEDLINE | ID: mdl-21299123

ABSTRACT

Chromatographic separation of the ethyl acetate extract of roots of Polyalthia cerasoides has led to the isolation of the new compound, 6,8-dihydroxy-7-methoxy-1-methyl-azafluorenone. This compound exhibited potent cytotoxic activities with IC50 values in the range of 2.64-3.58 microg x mL(-1) for A549, GLC4 and GLC4/Adr cells, but was not recognized by ABCC1/MRP1 protein. The compound also showed very strong inhibition of M. tuberculosis using a broth microdilution method, with an MIC value of 0.78 microg x mL(-1), which was equal to that of ofloxacin, one of the four antibiotic drugs used as a positive control.


Subject(s)
Anti-Bacterial Agents/isolation & purification , Antineoplastic Agents, Phytogenic/isolation & purification , Fluorenes/isolation & purification , Polyalthia/chemistry , Anti-Bacterial Agents/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Cell Line, Tumor , Fluorenes/pharmacology , Humans , Mycobacterium tuberculosis/drug effects , Plant Roots/chemistry
2.
Biophys J ; 85(3): 2006-14, 2003 Sep.
Article in English | MEDLINE | ID: mdl-12944313

ABSTRACT

Characterization of rhodamine 123 as functional assay for MDR has been primarily focused on P-glycoprotein-mediated MDR. Several studies have suggested that Rh123 is also a substrate for MRP1. However, no quantitative studies of the MRP1-mediated efflux of rhodamines have, up to now, been performed. Measurement of the kinetic characteristics of substrate transport is a powerful approach to enhancing our understanding of their function and mechanism. In the present study, we have used a continuous fluorescence assay with four rhodamine dyes (rhodamine 6G, tetramethylrosamine, tetramethylrhodamine ethyl ester, and tetramethylrhodamine methyl ester) to quantify drug transport by MRP1 in living GLC4/ADR cells. The formation of a substrate concentration gradient was observed. MRP1-mediated transport of rhodamine was glutathione-dependent. The kinetics parameter, k(a) = V(M)/k(m), was very similar for the four rhodamine analogs but approximately 10-fold less than the values of the same parameter determined previously for the MRP1-mediated efflux of anthracycline. The findings presented here are the first to show quantitative information about the kinetics parameters for MRP1-mediated efflux of rhodamine dyes.


Subject(s)
Multidrug Resistance-Associated Proteins/chemistry , Rhodamines/chemistry , Biophysical Phenomena , Biophysics , Cell Line, Tumor , Chlorine/chemistry , Daunorubicin/pharmacology , Dose-Response Relationship, Drug , Fluorescent Dyes/pharmacology , Glutathione/chemistry , Glutathione/metabolism , Humans , Kinetics , Membrane Potentials , Models, Chemical , Models, Theoretical , Multidrug Resistance-Associated Proteins/physiology , Phenotype , Potassium/chemistry , Rhodamine 123/pharmacology , Time Factors
3.
J Biochem Biophys Methods ; 57(1): 1-16, 2003 Jul 31.
Article in English | MEDLINE | ID: mdl-12834959

ABSTRACT

In order to get more insight into the energetic state of multidrug-resistance (MDR) cell compared with its corresponding sensitive cell, a noninvasive fluorescence method for determining and monitoring the mitochondrial membrane potential (DeltaPsi(m)), using rhodamine B and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was established. Rhodamine B distributes across biological membranes in response to the electrical transmembrane potential. P-glycoprotein- and MRP1-protein-mediated efflux do not create a concentration gradient, leading the cell-rhodamine B system to reach a steady state, where the ratio of cytosolic to extracellular rhodamine B was equal to 1. The mitochondrial matrix rhodamine B concentration was precisely determined as a decrease of rhodamine B fluorescence in the presence of formazan, a rhodamine B fluorescence quencher, which locally accumulates in the matrix of mitochondria. The kinetics of decrease in rhodamine B fluorescence (V(i)) can be used to estimate DeltaPsi(m) using the Nernst equation: DeltaPsi(m)=-61.54 log V(i)-258.46. The DeltaPsi(m) values determined were -160+/-4 mV for K562 cell, -146+/-6 mV for K562/adr cell, -161+/-10 mV for GLC4 cell and -168+/-2 mV for GLC4/adr cell. An increase or a decrease in DeltaPsi(m) consequently followed an increase or a decrease in the cellular ATP contents. An increase ATP content in the two MDR cell lines can protect cells from cytotoxicity induced by pirarubicin.


Subject(s)
Adenosine Triphosphate/metabolism , Doxorubicin/analogs & derivatives , Fluorescent Dyes , Intracellular Membranes/metabolism , Mitochondria/metabolism , Rhodamines , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Adenosine Triphosphate/analysis , Cell Line, Tumor , Doxorubicin/pharmacology , Doxorubicin/toxicity , Drug Resistance, Multiple , Flow Cytometry , Formazans , Humans , Intracellular Membranes/chemistry , Intracellular Membranes/drug effects , Membrane Potentials , Mitochondria/chemistry , Mitochondria/drug effects , Multidrug Resistance-Associated Proteins/metabolism , Spectrometry, Fluorescence , Tetrazolium Salts
4.
Chem Pharm Bull (Tokyo) ; 51(6): 728-30, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12808255

ABSTRACT

5-Aryl-3-(4-hydroxyphenyl)-1,3,4-oxadiazole-2(3H)-thiones 3 were prepared by cyclocondensation of 1-(4-hydroxyphenyl)-2-aroylhydrazines with thiophosgene. All compounds exhibited antiproliferation activity in K562, IC(50) ranging from 24 to 94 micro M comparable efficacy with apigenin and genistein and showed more potent antiproliferation of K562/adr cells, highly expressing P-glycoprotein. Compounds 3g, 3e and 3a inhibited the function of P-glycoprotein with the alpha(0.5) equal to 10+/-3 micro M, 21+/-5 micro M and 34+/-7 micro M, respectively.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , Antineoplastic Agents/chemical synthesis , Oxazoles/chemical synthesis , Thiones/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Survival/drug effects , Humans , Inhibitory Concentration 50 , K562 Cells , Magnetic Resonance Spectroscopy , Molecular Structure , Oxazoles/chemistry , Oxazoles/pharmacology , Thiones/chemistry , Thiones/pharmacology
5.
Biochem Pharmacol ; 65(6): 969-77, 2003 Mar 15.
Article in English | MEDLINE | ID: mdl-12623128

ABSTRACT

Multidrug resistance (MDR) in tumour cells is often caused by the overexpression of two transporters the P-glycoprotein (P-gp) and the multidrug resistance-associated protein (MRP1) which actively pump out multiple chemically unrelated substrates across the plasma membrane. A clear distinction in the mechanism of translocation of substrates by MRP1 or P-gp is indicated by the finding that, in most of cases, the MRP1-mediated transport of substrates is inhibited by depletion of intracellular glutathione (GSH), which has no effect on their P-gp-mediated transport. The aim of the present study was to quantitatively characterise the transport of anionic compounds dihydrofluorescein and fluorescein (FLU). We took advantage of the intrinsic fluorescence of FLU and performed a flow cytometric analysis of dye accumulation in the wild-type drug sensitive GLC4 that do not express MRP1 and its MDR subline which display high level of MRP1. The measurements were made in real time using intact cells. The kinetics parameters, k(a)=V(M)/K(m), which is a measure of the efficiency of the transporter-mediated efflux of a substrate, was very similar for the two FLU analogues. They were highly comparable with values for k(a) of other negatively charged substrates, such as GSH and calcein. The active efflux of both FLU derivatives was inhibited by GSH depletion.


Subject(s)
Fluorescein/metabolism , Fluoresceins/metabolism , Membrane Transport Proteins , Multidrug Resistance-Associated Proteins/metabolism , Biological Transport , Cyclic P-Oxides/pharmacology , Flow Cytometry , Humans , Kinetics , Leukotriene Antagonists/pharmacology , Multidrug Resistance-Associated Protein 2 , Nicotinic Acids/pharmacology , Propionates/pharmacology , Quinolines/pharmacology , Tumor Cells, Cultured
6.
Eur J Biochem ; 270(3): 476-85, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12542697

ABSTRACT

Multidrug resistance (MDR) in tumour cells is often caused by the overexpression of the plasma drug transporter P-glycoprotein (P-gp). This protein is an active efflux pump for chemotherapeutic drugs, natural products and hydrophobic peptides. Despite the advances of recent years, we still have an unclear view of the molecular mechanism by which P-gp transports such a wide diversity of compounds across the membrane. Measurement of the kinetic characteristics of substrate transport is a powerful approach to enhancing our understanding of their function and mechanism. The aim of the present study was to further characterize the transport of several rhodamine analogues, either positively charged or zwitterionic. We took advantage of the intrinsic fluorescence of rhodamines and performed a flow-cytometric analysis of dye accumulation in the wild-type drug sensitive K562 that do not express P-gp and its MDR subline that display high levels of MDR. The measurements were made in real time using intact cells. The kinetic parameter, ka = VM/km, which is a measure of the efficiency of the P-gp-mediated efflux of a substrate was similar for almost all the rhodamine analogues tested. In addition these values were compared with those determined previously for the P-gp-mediated efflux of anthracycline. Our conclusion is that the compounds of these two classes of molecules, anthracyclines and rhodamines, are substrates of P-gp and that their pumping rates at limiting low substrate concentration are similar. The findings presented here are the first to show quantitative information about the kinetic parameters for P-gp-mediated efflux of rhodamine analogues in intact cells.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/physiology , Fluorescent Dyes/metabolism , Rhodamines/metabolism , Antineoplastic Agents/pharmacology , Biological Transport , Doxorubicin/pharmacology , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Flow Cytometry , Humans , K562 Cells/metabolism
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