Luteolin, a naturally occurring flavonoid, is abundant in our daily dietary intake. It exhibits a wide spectrum of pharmacological properties, but little is known about its biochemical targets other than the fact that it induces topoisomerase II-mediated apoptosis. In the present study, we show that luteolin completely inhibits the catalytic activity of eukaryotic DNA topoisomerase I at a concentration of 40μM, with an IC50 of 5μM. Preincubation of enzyme with luteolin before adding a DNA substrate increases the inhibition of the catalytic activity (IC50 = 0.66μM). Treatment of DNA with luteolin before addition of topoisomerase I reduces this inhibitory effect. Subsequent fluorescence tests show that luteolin not only interacts directly with the enzyme but also with the substrate DNA, and intercalates at a very high concentration (>250μM) without binding to the minor groove. Direct interaction between luteolin and DNA does not affect the assembly of the enzyme–DNA complex, as evident from the electrophoretic mobility-shift assays. Here we show that the inhibition of topoisomerase I by luteolin is due to the stabilization of topoisomerase-I DNA-cleavable complexes. Hence, luteolin is similar to camptothecin, a class I inhibitor, with respect to its ability to form the topoisomerase I-mediated ‘cleavable complex'. But, unlike camptothecin, luteolin interacts with both free enzyme and substrate DNA. The inhibitory effect of luteolin is translated into concanavalin A-stimulated mouse splenocytes, with the compound inducing SDS–K+-precipitable DNA–topoisomerase complexes. This is the first report on luteolin as an inhibitor of the catalytic activity of topoisomerase I, and our results further support its therapeutic potential as a lead anti-cancer compound that poisons topoisomerases.
Abbreviations used: CT, calf thymus; DTT, dithiothreitol; EMSA, electrophoretic mobility-shift assay; m-AMSA, 4′-(acridinylaminol)-N-(methanesulphonyl)-m-anisidine.
Present address: Room no. 421, Institute of Molecular Biology, Academica Sinica, Nonkang, Taipei 11529, Taiwan, Republic of China.