Novel mechanism of cellular DNA topoisomerase II inhibition by the pyranonaphthoquinone derivatives alpha-lapachone and beta-lapachone

Purpose: The mechanisms of intracellular topoisomerase II inhibition by the pyranonaphthoquinone derivatives alpha -lapachone and beta -lapachone were studied. Methods: Cell-based mechanistic studies were designed based on th e in vitro mechanisms [17] and primarily involved the use of cultured KB (n asopharyngeal tumor cells) cells and the etoposide-resistant sub-line KB-7d . Results: The KB-7d cells exhibited collateral sensitivity to alpha -lapac hone; this supports the possibility of catalytic inhibition of topoisomeras e II in the cells. Interestingly, both compounds induced an increase (two- to threefold) in reversible double-stranded DNA breaks in cell lines with a reduced expression of topoisomerase II. However, these drug-induced DNA br eaks became irreversible at treatment times greater than 1 h. Studies showe d that DNA breaks in KB-7d cells were not caused by endonucleases. Use of a ntioxidants abolished the appearance of cellular DNA breaks; this suggests involvement of the oxidation-reduction cycle of pyranonaphthoquinones in to poisomerase II inhibition; however, irreversible DNA breaks were not a resu lt of drug-induced oxidative stress. Conclusions: On the basis of the findi ngs, it is proposed that the compounds, on longer incubation with cells, in duce abortive dissociation of topoisomerase II from the DNA, leading to an irreversible accumulation of high molecular weight DNA fragments. In additi on to establishing topoisomerase II as an intracellular target of alpha -la pachone, the results suggest that both compounds can be classified as neith er typical poisons nor as typical catalytic inhibitors of the enzyme. In su mmary, both compounds are members of a new inhibitor class, and alpha -lapa chone, in particular, can be considered a potential lead for the developmen t of drugs to treat multidrug-resistant cell lines with lower expression of topoisomerase II.