JOURNAL OF MEDICINAL FOOD Volume 2, Numbers 3-4, 1999 Mary Ann Liebert, Inc. Application of Topoisomerase Assays in the Evaluation of Natural Products as Antitumor Agents ANDREAS CONSTANTINOU and GEORGE SALTI ABSTRACT Initially, DNA topoisomerase (topo) inhibitors found clinical applications as antibiotics and cancer chemotherapeutic agents. Recently, we demonstrated that plant flavonoids that inhibit mammalian topo I or topo II might be useful as cancer chemopreventive agents (Constanti- nou et al., 1995b). Phytochemicals can inhibit DNA topoisomerases in different ways; de- pending on the mode and the type of enzyme, these can be classified as topo I poisons, topo II poisons, topo I antagonists, or topo II antagonists. Correctly classifying topo inhibitors is critical because it provides an important lead as to whether the plant agent can be useful in chemoprevention or in chemotherapy. We outline below a strategy that was designed to iden- tify and classify topo I and II inhibitors. SOME OF THE MOST POTENT ANTITUMOR AGENTS currently being used in cancer chemother- apy or being evaluated as chemopreventive agents inhibit DNA topoisomerase (topo) I or II. In eukaryotes, topos are involved in the processes of DNA replication, transcription, and recombination, and they play key roles in cell proliferation and differentiation (Heck and Earnshaw, 1986; Constantinou et al., 1989; Kiguchi et al., 1990). Transient DNA strand breakage is an integral component of both topo I and topo II normal enzymatic activities. In the presence of certain inhibitors known as topo I or topo II poisons, a reaction intermediate be- tween the enzyme and DNA (known as the "cleavable complex") becomes stabilized, giv- ing rise to DNA breaks (Liu, 1989; Robinson and Osheroff, 1991; Osheroff, 1989). Cancer cells characterized by increased topo II activity are especially susceptible to the DNA-damag- ing and cytotoxic effects of topo II poisons (Constantinou et al, 1990; Shin et al, 1990; Tan- abe, et al., 1991). Paradoxically, not only topo II poisons but also poisons of topo I (whose lev- els are generally not higher in cancer cells) have found applications in cancer chemotherapy. Antitumor drugs such as teniposide (VM-26), etoposide (VP-16), ellipticine, doxorubicin, and amsacrine are mechanistically topo II poisons. Drugs such as irinotecan and topotecan (both camptothecin derivatives) are mechanistically topo I poisons. A different class of topo inhibitors includes agents that do not stabilize the covalent en- zyme-DNA complex but rather hinder its for- mation. These inhibitors have been named cat- alytic inhibitors or topo antagonists, because they oppose the DNA cleavage reaction of the target enzyme (Shin et al., 1990). Novobiocin, merbarone, aclarubicin, fostriecin, bis(2,6-diox- opiperazine) derivatives, and gossypol are some representative topo II catalytic inhibitors exhibiting antitumor effects (Creighton et al., 1969; Witiak et al., 1978; Pedersen-Bjergaard et al, 1984; Rao et al., 1985; Adlakha et al., 1989; Tanabe et al., 1991). Catalytic inhibitors often Department of Surgical Oncology, College of Medicine, University of Illinois at Chicago, Chicago, IL. 167