Novobiocin-induced accumulation of etoposide (VP-16) in WEHI-3B D+ leukemia cells.
A previous report from this laboratory demonstrated that novobiocin produced supra-additive cytotoxicity when combined with etoposide (VP-16) or teniposide (VM-26) in WEHI-3B D+ and A549 cells. The increase in cytotoxicity was accompanied by an increase in the formation of drug-stabilized protein-DNA covalent complexes. We now report that novobiocin increased the amount of VP-16-induced covalent complexes between the 170 kDa form of topoisomerase II and DNA in WEHI-3B D+ cells, as measured by the band-depletion immunoblotting assay, while it did not affect the extractable topoisomerase II activity, measured by the unknotting of P4 phage DNA and by a DNA cleavage assay. Novobiocin progressively increased the steady-state concentration of intracellular VP-16. Removal of novobiocin resulted in a rapid return of VP-16 to levels comparable to those seen with VP-16 alone. The increased accumulation of VP-16 was accounted for by an increase in the exchangeable fraction only. The novobiocin-mediated increase in the steady-state concentration of VP-16 occurred whether novobiocin was added simultaneously with VP-16 or was added after a steady-state level of VP-16 had been achieved. Novobiocin did not affect the initial rate of uptake of VP-16; however, it inhibited the efflux of the epipodophyllotoxin. In fact, when cells were loaded with the same level of VP-16 in the presence or absence of novobiocin, the efflux curves in the presence or absence of novobiocin were significantly different. We conclude that the inhibition of VP-16 efflux by novobiocin is responsible for the increase in VP-16 accumulation, leading to increased formation of VP-16-stabilized topoisomerase-II-DNA covalent complexes and increased cytotoxicity.
Lorico
A
A
Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06510.
Rappa
G
G
Sartorelli
A C
AC
D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D004250:DNA Topoisomerases, Type II / Q000502:physiology; D004347:Drug Interactions; D005047:Etoposide / Q000493:pharmacokinetics* / Q000627:therapeutic use; D007942:Leukemia, Experimental / Q000188:drug therapy / Q000201:enzymology / Q000378:metabolism*; D051379:Mice; D009675:Novobiocin / Q000494:pharmacology*; D059005:Topoisomerase II Inhibitors; D014407:Tumor Cells, Cultured
D059005:Topoisomerase II Inhibitors; D009675:Novobiocin; D005047:Etoposide; D004250:DNA Topoisomerases, Type II