Two simple bioassays continue to be effective for the detection and fractionation monitoring of plant antitumor agents. These are 1) the inhibition of crown gall tumors, induced by plasmid transfer and expression from Agrobacterium tumefaciens, on discs of potato (Solanum tuberosum) tubers and 2) lethality to the larvae of brine shrimp, Artemia salina. In a recent publication (Anderson et. al., 1991), these two bioassays were evaluated for their accuracies at detecting a series of known in vivo active antitumor agents which were supplied (blind) by the National Cancer Institute. In every case, the potato disc assay correctly differentiated between the in vivo actives and inactives (p = 0.008), and the brine shrimp assay (p = 0.033) proved to be superior or equally as accurate, at predicting in vivo activity, as cytotoxicities in a series of human solid tumor cell lines (p = 0.033 - 0.334). Thus, these two simple bioassays are presented as convenient and statistically reliable bench-top supplements, to more expensive and elaborate methods, for the screening, detection, and isolation of plant-derived antitumor agents. Since the initiation of this R01 grant in 1984, approximately one hundred and fifty chemically diverse in vitro and in vivo active plant antitumor agents have been isolated and characterized in our laboratory using this approach. For example, bullatalicin, a novel nonadjacent bis- tetrahydrofuran Annonaceous acetogenin, has been isolated (Hui et al., 1989) and (at Upjohn) showed activity quite comparable to that of cisplatin against human A2780 ovarian tumor xenografts in athymic mice. In addition to a host of active compounds from other known and new phytochemical classes, we have, to date, isolated and characterized twenty-five of these potent Annonaceous acetogenins; and this work has led to the recent determination of their mode of action as very powerful inhibitors of mitochondrial electron transport systems, a bioenergetic site which has been long proposed (Folkers, 1974), but little explored, for antitumor drug development. Piceatannol, a tetrahydroxystilbene, was the first in vivo 3PS active that we isolated using these methods (Ferrigni et al., 1984), and its mechanism of action is now proven as the effective inhibition of several protein tyrosine kinases (Geahlen and McLaughlin, 1989). Our long-term objectives are two-fold: 1) to continue to discover new, potentially useful, plant antitumor leads and 2) to demonstrate, convincingly, that these simple, higher-animal sparing, bench-top bioassays are truly effective in expediting the search for new antitumor leads. Both of these objectives are being met. Our research productivity remains high, and an increasing number of laboratories, worldwide, are quoting our methods in their publications.
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