FIGURE 4.

Apigenin suppresses topoisomerase 1 activity. (A) Apigenin suppresses topoisomerase 1 activity in whole cells. HT-29 cells were treated with apigenin for 48 h at the concentrations indicated. The original supercoiled DNA is shown (‘Sup’) as well as the effect of treating cells with 30 nM SN38 (positive control for Topo1 inhibition). The lowest band is the supercoiled DNA that is successively uncoiled (ladder) by cellular Topo1 activity to yield the final uncoiled structure (topmost band). Maximal uncoiling is present with untreated cells (“0 µM apigenin”) and this is inhibited by increasing concentrations of apigenin or exposure to SN38 (more intense lowest band). Representative of three independent experiments. (B) Representative result showing the gel separation of supercoiled plasmid (“Sup”), the effect of incubation with rhTopo1 alone and the result of co-incubation with apigenin at concentrations shown. As for panel A the lowest band is input supercoiled DNA that shows uncoiling to higher bands in the presence of Topo1, in this case rhTopo1. Increasing concentrations of apigenin added directly to the reaction mixture inhibit Topo1 activity, such that at 1,000 µM apigenin the uncoiling activity of the enzyme is essentially blocked. (C) rhTopo1 activity was measured in the presence of apigenin (•) or luteolin (◆) at the concentrations shown. Representative of three independent experiments.