David J. Kroll

Catalytic inhibition of human DNA topoisomerase IIα by hypericin, a naphthodianthrone from St. John’s wort (Hypericum perforatum)

St. Johns wort (Hypericum perforatum) is the most widely used herbal medicine for the treatment of depression. However, concerns have arisen about the potential of its interaction with other drugs due to the induction of cytochrome P450 isozymes 1A2 and 3A4 by the components hypericin and hyperforin, respectively. Structurally similar natural products are often employed as antitumor agents due to their action as inhibitors of DNA topoisomerases, nuclear enzymes that modify DNA during cellular proliferation. Preliminary findings that hypericin inhibited the DNA relaxation activity of topoisomerase IIalpha (topo II; EC 5.99.1.3) led us to investigate the mechanism of enzyme inhibition. Rather than stabilizing the enzyme in covalent complexes with DNA (cleavage complexes), hypericin inhibited the enzyme prior to DNA cleavage. In vitro assays indicate that hypericin is a potent antagonist of cleavage complex stabilization by the chemotherapeutics etoposide and amsacrine. This antagonism appears to be due to the ability of hypericin to intercalate or distort DNA structure, thereby precluding topo II binding and/or DNA cleavage. Supporting its non-DNA damaging, catalytic inhibition of topo II, hypericin was shown to be equitoxic to both wild-type and amsacrine-resistant HL-60 leukemia cell lines. Moreover, hypericin was incapable of stimulating DNA damage-responsive gene promoters that are activated by etoposide. As with the in vitro topo II assay, antagonism of DNA damage stimulated by 30 microM etoposide was evident in leukemia cells pretreated with 5 microM hypericin. Since many cancer patients experience clinical depression and concomitantly self-medicate with herbal remedies, extracts of St. Johns wort should be investigated further for their potential to antagonize topo II-directed chemotherapy regimens.

Etoposide Induces Transition of 14-3-3 from the Cytoplasma into the Nucleus where it Binds to Topoisomerase II

We have shown previously, that 14-3-3epsilon binds to topoisomerase alpha. The interaction is specific for the epsilon form of the 14-3-3 isoproteins and phosphorylation of the topoisomerase alpha protein enhances the interaction. Etoposide induced DNA damage is completely inhibited in a concentration dependent manner by the binding of 14-3-3epsilon to topoisomerase II alpha. These experiments have been performed in vitro and questions emerged, under which patho-physiological conditions this interaction between topoisomerase alpha, which is physiologically localized in the nucleus, and 14-3-3epsilon, physiologically localized in the cytoplasm, might occur. In this paper, we show that etoposide treatment induces a transition of 14-3-3epsilon from the cytoplasm into the nucleus where it binds to topoisomerase II alpha. These experiments indicate that the inhibition of etoposide induced DNA damage by the binding of 14-3-3epsilon to topoisomerase II alpha might well be a clinically relevant factor for cellular resistance against topoisomerase II inhibitors.