Celastraceae sesquiterpenes as a new class of modulators that bind specifically to human P-glycoprotein and reverse cellular multidrug resistance.
Overexpression of ABCB1 (MDR1) P-glycoprotein, a multidrug efflux pump, is one mechanism by which tumor cells may develop multidrug resistance (MDR), preventing the successful chemotherapeutic treatment of cancer. Sesquiterpenes from Celastraceae family are natural compounds shown previously to reverse MDR in several human cancer cell lines and Leishmania strains. However, their molecular mechanism of reversion has not been characterized. In the present work, we have studied the ability of 28 dihydro-beta-agarofuran sesquiterpenes to reverse the P-glycoprotein-dependent MDR phenotype and elucidated their molecular mechanism of action. Cytotoxicity assays using human MDR1-transfected NIH-3T3 cells allowed us to select the most potent sesquiterpenes reversing the in vitro resistance to daunomycin and vinblastine. Flow cytometry experiments showed that the above active compounds specifically inhibited drug transport activity of P-glycoprotein in a saturable, concentration-dependent manner (K(i) down to 0.24 +/- 0.01 micromol/L) but not that of ABCC1 (multidrug resistance protein 1; MRP1), ABCC2 (MRP2), and ABCG2 (breast cancer resistance protein; BCRP) transporters. Moreover, sesquiterpenes inhibited at submicromolar concentrations the P-glycoprotein-mediated transport of [(3)H]colchicine and tetramethylrosamine in plasma membrane from CH(R)B30 cells and P-glycoprotein-enriched proteoliposomes, supporting that P-glycoprotein is their molecular target. Photoaffinity labeling in plasma membrane and fluorescence spectroscopy experiments with purified protein suggested that sesquiterpenes interact with transmembrane domains of P-glycoprotein. Finally, sesquiterpenes modulated P-glycoprotein ATPase-activity in a biphasic, concentration-dependent manner: they stimulated at very low concentrations but inhibited ATPase activity as noncompetitive inhibitors at higher concentrations. Sesquiterpenes from Celastraceae are promising P-glycoprotein modulators with potential applications in cancer chemotherapy because of their MDR reversal potency and specificity for P-glycoprotein.
Instituto de Parasitología y Biomedicina López-Neyra, Consejo Superior de Investigaciones Científicas, Granada, Spain.
Francisco
F
Muñoz-Martínez
Peihua
P
Lu
Fernando
F
Cortés-Selva
José María
JM
Pérez-Victoria
Ignacio A
IA
Jiménez
Angel G
AG
Ravelo
Frances J
FJ
Sharom
Francisco
F
Gamarro
Santiago
S
Castanys
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000378:metabolism; D001562:Benzimidazoles / Q000378:metabolism; D001667:Binding, Competitive; D029753:Celastraceae / Q000737:chemistry*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003630:Daunorubicin / Q000493:pharmacokinetics / Q000494:pharmacology; D004095:Dihydropyridines / Q000378:metabolism; D018432:Drug Resistance, Multiple / Q000187:drug effects*; D019008:Drug Resistance, Neoplasm; D005452:Fluoresceins / Q000493:pharmacokinetics; D006801:Humans; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D051379:Mice; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D041681:NIH 3T3 Cells; D019920:Photoaffinity Labels / Q000378:metabolism; D011485:Protein Binding; D012717:Sesquiterpenes / Q000378:metabolism* / Q000494:pharmacology*; D013379:Substrate Specificity; D014747:Vinblastine / Q000493:pharmacokinetics / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate; D001386:Azides; D001562:Benzimidazoles; D003630:Daunorubicin; D004095:Dihydropyridines; D005452:Fluoresceins; D008081:Liposomes; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D019920:Photoaffinity Labels; D012717:Sesquiterpenes; D014747:Vinblastine; C000715087:ABCC2 protein, human; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; C041130:azidopine; C017807:bisbenzimide ethoxide trihydrochloride; C007740:fluorexon; C421109:multidrug resistance-associated protein 1