CONICET | Buscador de Institutos y Recursos Humanos

ABSTRACTBackground: Overexpression of P-glycoprotein (P-gp), linked to multidrug resistance (MDR), is one of the underlying obstacles to the success of chemotherapy. Purpose: To identify agents with P-gp inhibitory properties in plants of Argentina and study the mechanism of action of the active principle isolated from the most active species. Methods: Doxorubicin accumulation assay by flow cytometry was employed to determine inhibition of P-gp-mediated efflux by extracts, fractions and compounds in the P-gp overexpressing MDR leukemia cell, Lucena 1. The effect of the active molecule was further confirmed by rhodamine 123 accumulation and resistance reversal assays. Molecular modelling was performed to investigate the mode of binding of the ligand to P-gp. MTT assays using peripheral blood mononuclear cells and hemolysis assays were carried out to preliminarily explore the toxicity of the active principle. Results: The screening of the ethanol extracts from 80 plants demonstrated that the most effective extract was that from Ligaria cuneifolia. Its bioassay-guided isolation yielded the non-cytotoxic pentacyclic triterpene betulin as active agent. This efficiently inhibited P-gp mediated efflux, as demonstrated by the enhancement of the intracellular accumulation of doxorubicin and rhodamine 123 from 1.56 µM in Lucena 1. Betulin was also able to render Lucena 1 sensitive to Dox from 0.39 µM. The docking studies revealed that betulin tightly binds to a key region of the TMDs, with a binding mode overlapping one main site of doxorubicin and, more interestingly, emulating the same contacts as tariquidar, as revealed by the residue energetic analysis from molecular dynamics simulations.Conclusion: These findings provide important evidence that betulin may be a safe and promising entity to be further investigated to develop agents able to overcome P-gp-mediated MDR.