Interaction of doxorubicin with ATP: quantification of complexes and effect on its diffusion into DNA-loaded liposomes--implication for ATP-driven transport studies.
Doxorubicin, a drug largely used in chemotherapy, is transported by P-glycoprotein, a protein involved in the multidrug-resistance phenotype. Taking advantage of the doxorubicin fluorescence quenching upon interaction with DNA, a sensitive assay of this active transport can be carried out: quantitative in vitro studies could be achieved with DNA-loaded proteoliposomes, after correction for the doxorubicin passive diffusion through phospholipids. In this paper, we describe experimental conditions that will be relevant to P-glycoprotein studies. Efficient DNA entrapment in preformed liposomes was obtained using the freeze/thawing procedure, and the doxorubicin passive diffusion was quantified in the presence of ATP/Mg2+, the second substrate of P-glycoprotein. The doxorubicin diffusion rate decreases in the presence of ATP, indicating an interaction between doxorubicin and ATP that will hinder any measurement of ATP-driven transport. The interaction between doxorubicin and ATP was studied by fluorescence quenching, octanol/buffer partition coefficient, and diffusion rate into DNA-loaded liposomes. The results give evidence for complex interactions. However, under our experimental conditions, these interactions are only slightly modified in the presence of Mg2+. Since this cation is essential for P-glycoprotein activity, it can be concluded that in these conditions the accurate evaluation of P-glycoprotein-catalyzed doxorubicin transport will be obtained from the Mg2+-sensitive transport into DNA-loaded proteoliposomes.
Chambon
M H
MH
CNRS, Pessac, 33600, France.
Viratelle
O M
OM
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry*; D001692:Biological Transport; D004247:DNA / Q000737:chemistry*; D004058:Diffusion; D004317:Doxorubicin / Q000737:chemistry*; D005453:Fluorescence; D008081:Liposomes
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D008081:Liposomes; D004317:Doxorubicin; D000255:Adenosine Triphosphate; D004247:DNA