P Krishnan1, K F Bastow. 1. Division of Medicinal Chemistry and Natural Products, School of Pharmacy, CB #7360, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Abstract
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 the in vitro mechanisms [17] and primarily involved the use of cultured KB (nasopharyngeal tumor cells) cells and the etoposide-resistant sub-line KB-7d. RESULTS: The KB-7d cells exhibited collateral sensitivity to alpha-lapachone; this supports the possibility of catalytic inhibition of topoisomerase 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 breaks became irreversible at treatment times greater than 1 h. Studies showed that DNA breaks in KB-7d cells were not caused by endonucleases. Use of antioxidants abolished the appearance of cellular DNA breaks; this suggests involvement of the oxidation-reduction cycle of pyranonaphthoquinones in topoisomerase II inhibition; however, irreversible DNA breaks were not a result of drug-induced oxidative stress. CONCLUSIONS: On the basis of the findings, it is proposed that the compounds, on longer incubation with cells, induce abortive dissociation of topoisomerase II from the DNA, leading to an irreversible accumulation of high molecular weight DNA fragments. In addition to establishing topoisomerase II as an intracellular target of alpha-lapachone, the results suggest that both compounds can be classified as neither typical poisons nor as typical catalytic inhibitors of the enzyme. In summary, both compounds are members of a new inhibitor class, and alpha-lapachone, in particular, can be considered a potential lead for the development of drugs to treat multidrug-resistant cell lines with lower expression of topoisomerase II.
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 the in vitro mechanisms [17] and primarily involved the use of cultured KB (nasopharyngeal tumor cells) cells and the etoposide-resistant sub-line KB-7d. RESULTS: The KB-7d cells exhibited collateral sensitivity to alpha-lapachone; this supports the possibility of catalytic inhibition of topoisomerase 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 breaks became irreversible at treatment times greater than 1 h. Studies showed that DNA breaks in KB-7d cells were not caused by endonucleases. Use of antioxidants abolished the appearance of cellular DNA breaks; this suggests involvement of the oxidation-reduction cycle of pyranonaphthoquinones in topoisomerase II inhibition; however, irreversible DNA breaks were not a result of drug-induced oxidative stress. CONCLUSIONS: On the basis of the findings, it is proposed that the compounds, on longer incubation with cells, induce abortive dissociation of topoisomerase II from the DNA, leading to an irreversible accumulation of high molecular weight DNA fragments. In addition to establishing topoisomerase II as an intracellular target of alpha-lapachone, the results suggest that both compounds can be classified as neither typical poisons nor as typical catalytic inhibitors of the enzyme. In summary, both compounds are members of a new inhibitor class, and alpha-lapachone, in particular, can be considered a potential lead for the development of drugs to treat multidrug-resistant cell lines with lower expression of topoisomerase II.
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