Selected Novel Flavones Inhibit the DNA Binding or the DNA Religation Step of Eukaryotic Topoisomerase I

Abstract

Topoisomerases are involved in many aspects of DNA metabolism such as replication and transcription reactions. Camptothecins, which stabilize the covalent intermediate of topoisomerase I and DNA are effective, though toxic, drugs for cancer therapy. In this study, a new class of topoisomerase I inhibitors was identified, and their mode of action was characterized using recombinant human topoisomerase I preparations and human HL-60 leukemic cells. Quercetin and the related natural flavones, acacetin, apigenin, kaempferol, and morin, inhibit topoisomerase I-catalyzed DNA religation. In contrast to camptothecin, these compounds do not act directly on the catalytic intermediate and also do not interfere with DNA cleavage. However, formation of a ternary complex with topoisomerase I and DNA during the cleavage reaction inhibits the following DNA religation step. 3,3,4,7-Tetrahydroxy-substituted flavones stabilize the covalent topoisomerase I-DNA intermediate most efficiently. Enhanced formation of covalent topoisomerase I-DNA complexes was also demonstrated in human HL-60 cells. In contrast, synthetic 3,5-dibromo-4-hydroxy-3-methylflavones bind selectively to topoisomerase I in its non-DNA-bound form and block the following DNA binding step. As a consequence, these synthetic flavonoids are capable of counteracting topoisomerase I-directed effects of camptothecin. Inhibition of DNA binding is obtained by voluminous hydrophobic substituents in 6-position of the flavone structure. Our data show that selective inhibitors of both half-reactions of topoisomerase I can be derived from the flavone structure.

Betulinic Acid, a Catalytic Inhibitor of Topoisomerase I, Inhibits Reactive Oxygen Species–Mediated Apoptotic Topoisomerase I–DNA Cleavable Complex Formation in Prostate Cancer Cells but Does Not Affect the Process of Cell Death

“…DU145 cells were cultured in six-well plates with or without appropriate drugs. Nuclear extracts were prepared as described (12,19). Briefly, cells were suspended in hypotonic buffer [10 mmol/L Tris-HCl (pH 7.5), 0.1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L benzamidine hydrochloride, and 5 mmol/L DTT] and homogenized.…”

Apoptosis is induced in leishmanial cells by a novel protein kinase inhibitor withaferin A and is facilitated by apoptotic topoisomerase I–DNA complex

“…Nuclear fraction was isolated as described. 31 Briefly, cells were suspended in hypotonic buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.1 mM EGTA, 1 mM PMSF, 1 mM benzamidine hydrochloride and 5 mM DTT) and homogenized. The homogenate was centrifuged for 10 min at 10 000 Â g. The pellets were washed and were the source of nuclear fraction.…”

MAPK signaling in neurodegeneration: influences of flavonoids and of nitric oxide

“…Since flavonoids have been reported to bind to benzodiazepine binding sites of GABA-A and adenosine receptors [52,132], they might also exert an effect on the mPTP and, therefore, modulate cytochrome c release. In addition flavonoids have been found to bind to ATP-binding sites of proteins [43] such as the mitochondrial ATPase [53], calcium plasma membrane ATPase [10] protein kinase A [164], PKC [117] and topoisomerase [17] all of which must be considered in intracellular responses to oxidative insults. Thus, flavonoids might be able to modulate mitochondrial functions by binding to ATP binding sits on the ANT, ATPases or others.…”