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Abstract
The work presented in this dissertation further characterized the activity and mechanism of action of four major classes of bioflavonoids (flavones, flavonols, isoflavones, and catechins) against human topoisomerase IIα and β. These enzymes are required to remove knots and tangles from DNA. Results observed in vitro and in cultured human cells demonstrated that several compounds significantly increase the DNA cleavage activity of both enzyme isoforms. These findings suggest that the proposed health-promoting and clastogenic properties of bioflavonoids are mediated, at least in part, through type II topoisomerases.
Genistein, a major soy isoflavone, was the most active compound. The C5–OH mediates genistein binding, and the C4'–OH contributes to the actions of this compound against topoisomerase II. Protein mutagenesis indicated that genistein interacts near the active site of the enzyme. Further studies demonstrated that genistein acts in a non-covalent manner via a mechanism that does not require redox chemistry.
In contrast to genistein, (–)-epigallocatechin gallate (EGCG), the most abundant and bioactive polyphenol in green tea, enhanced topoisomerase IIα- and β-mediated DNA cleavage through a mechanism that requires redox chemistry and covalent adduction to the enzyme. These results suggest that dietary polyphenolic compounds utilize multiple mechanisms to enhance enzyme-mediated DNA cleavage.
Based on structure-activity studies with flavonols and catechins, a set of rules were proposed that predicts the mechanism by which these compounds and related bioflavonoids act against human topoisomerase II. The rules are related to the chemical substituents on the bioflavonoid B and C rings. These results provide insight into the structural features in bioflavonoids that dictate their actions against the enzyme.
Finally, relationships between the persistence of topoisomerase II-mediated cleavage complexes and the toxicity of compounds that target the enzyme were examined. Findings from these studies suggest a correlation between the persistence of drug-induced cleavage complexes mediated by topoisomerase IIα and β and the cytotoxicity of compounds targeted toward the enzyme. These results provide a rationale for preferentially targeting the cytotoxic effects mediated by the a isoform by altering the administration of the anticancer drug, etoposide.
