Energy transfer to analyse membrane-integrated mitoxantrone in BCRP-overexpressed cells.
The binding and the diffusion of mitoxantrone (MTX) through the plasma membrane was performed by Förster resonance energy transfer (FRET) from the membrane fluorescent donor (4Di-10ASP) to the co-localized acceptor MTX. The MTX addition to living 4Di-10ASP-tagged cells resulted in the rapid quenching of the probe emission (1s), revealing the MTX binding to the outer leaflet. Then, a slower quenching (about 90s) occurred which corresponded to the MTX flip-flop into the inner leaflet. Changes of MTX integration into the plasma membrane were described in BCRP-overexpressed cells (HCT-116R) treated with (i) the BCRP inhibitor fumitremorgin C (FTC), (ii) cyclosporin A (CSA) and (iii) benzyl alcohol (BA). Treatments with FTC or CSA showed 80% and 40% higher flip-flop of MTX from the outer to the inner leaflet of HCT-116R cells. The addition of BA clearly increased the MTX integration into both outer and inner leaflets. Confocal fluorescence microscopy displayed that FTC, CSA and BA enhanced MTX accumulation in HCT-116R. In conclusion, Fumitremorgin C and agents modulating MTX accumulation resulted in higher MTX integration in the resistant cell membrane and could disrupt the membrane cohesion. This energy transfer method appears well-adapted to describe the drug diffusion through the plasma membrane of living cells.
Breuzard
Gilles
G
Unité MéDIAN CNRS UMR 6142, IFR53, UFR de Pharmacie, 51 rue Cognacq-Jay, 51096 Reims Cedex, France.
El-Khoury
Victoria
V
Millot
Christine
C
Manfait
Michel
M
Millot
Jean-Marc
JM
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D019905:Benzyl Alcohol / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D016572:Cyclosporine / Q000494:pharmacology; D004058:Diffusion; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D015870:Gene Expression; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008942:Mitoxantrone / Q000493:pharmacokinetics*; D009363:Neoplasm Proteins / Q000235:genetics*
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D007211:Indoles; D009363:Neoplasm Proteins; D016572:Cyclosporine; D008942:Mitoxantrone; C010522:tryptoquivaline; D019905:Benzyl Alcohol