Regulation of ornithine decarboxylase induction by deguelin, a natural product cancer chemopreventive agent.

Deguelin, a plant-derived rotenoid, mediates potent chemopreventive responses through transcriptional regulation of phorbol ester-induced ornithine decarboxylase (ODC) activity. To explore the mechanism of this effect, the activity of this compound was evaluated with a number of model systems. Using cultured mouse epidermal 308 cells, the steady-state levels of both 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ODC mRNA and c-fos were decreased by treatment with deguelin. ODC activity was also inhibited by bullatacin and various antimitotic agents (podophyllotoxin, vinblastine, and colchicine), but only deguelin and bullatacin were active as inhibitors of ODC levels in a TPA-independent c-Myc-mediated induction system using cultured BALB/c c-MycER cells. These results suggest that antimicrotubule effects, as mediated by rotenone, for example, are not responsible for inhibitory activity facilitated by deguelin. This was confirmed by use of an in vitro model of tubulin polymerization in which deguelin and a variety of other rotenoids were investigated and found to be inactive. As anticipated, however, NADH dehydrogenase was inhibited by these rotenoids. Moreover, inhibition of this enzyme correlated with a rapid depletion of ATP levels and potential to inhibit either TPA- or c-Myc-induced ODC activity. It therefore seems that deguelin-mediated interference with transient requirements for elevated energy can inhibit the induction of ODC activity and thereby yield a cancer chemopreventive response.

Sulfonic acid polymers are potent inhibitors of HIV-1 induced cytopathogenicity and the reverse transcriptases of both HIV-1 and HIV-2.

Four novel sulfonic acid polymers were evaluated for their in vitro HIV-1 and HIV-2 reverse transcriptase (RT) inhibitory activity and found to be equipotent against both RTs. The aromatic polymers demonstrated IC50 values that were approximately 10(3)-fold lower than those observed with the aliphatic polymers. Among the aromatic polymers, poly(4-styrenesulfonic acid) (PSS) (MW 8000; IC50 = 0.02 microgram/ml) was 3-fold more potent than poly(anetholesulfonic acid) (PAS) of approximately the same molecular weight range. The activity of PSS polymers increased in proportion to the size of the polymers and, relative to suramin, activity could be enhanced over 200-fold. These polymers also inhibited the cytopathic effect of HIV-1 at concentrations that were non-toxic to MT-4 cells. The potent RT inhibitory properties of these stable sulfonic acid polymers suggest that structure-activity studies are warranted to yield agents capable of inhibiting multiple stages of the viral process.

Discovery of natural product chemopreventive agents utilizing HL-60 cell differentiation as a model.

Terminal differentiation of human promyelocytic leukemia (HL-60) cells can be induced by a variety of chemical agents and this process can be monitored readily by the generation of morphologically, histochemically, and functionally mature granulocytes and monocytes/macrophages. The availability of this model has heightened interest in the possible therapeutic role of inducers of myeloid differentiation for the treatment of leukemia and other neoplasms. In addition, however, potent cancer chemopreventive agents induce HL-60 cell differentiation at very low dose levels. Thus, as part of our search for natural product chemopreventive agents, extracts derived from nearly 400 plants were tested for their potential to induce HL-60 cell differentiation. As a result, 17 plant extracts were judged to be active (ED50 values < or = 4 micrograms/ml). One of most potent leads was an extract derived from Dirca occidentalis Gray (Thymelaeaceae) (ED50, 0.14 micrograms/ml), and bioassay-guided fractionation led to the identification of genkwanin (I), (+/-)-lariciresinol (II) and sitoindoside II (IV) as active principles, with ED50 values of 18.3, 1.1 and 0.069 microM, respectively. Based on these data, we conclude that the HL-60 cell differentiation system is a valid and useful model for the discovery of natural product cancer chemopreventive or chemotherapeutic agents.

Evaluation of the cytotoxic mechanism mediated by baccatin III, the synthetic precursor of taxol.

Baccatin III, which is used as a precursor for the semisynthesis of taxol, showed cytotoxic activity against a variety of cancer cell lines in culture, with ED50 values ranging from approximately 8 to 50 microM. Although the potency of this response is much lower than that mediated by taxol, it was interesting to note that any significant cytotoxic response could be mediated by this compound. Thus, it was considered of potential value to investigate the mechanism of cytotoxic action. Consistent with an antimitotic mode of action, baccatin III induced cultured cells to accumulate in the G2 + M phases of the cell cycle. However, unlike taxol, which potentiates the polymerization of tubulin, baccatin III mediated an antimitotic response through inhibition of the polymerization reaction, similar to colchicine, podophyllotoxin, or vinblastine. Accordingly, baccatin III was unable to reduce the extent of Ca(2+)-induced depolymerization, a hallmark of the biological response mediated by taxol. To further explore the mode of antimitotic activity facilitated by baccatin III, competitive interactions with the colchicine, podophyllotoxin, and vinblastine binding sites of tubulin were investigated. Baccatin III displaced the binding of radiolabeled colchicine or radiolabeled podophyllotoxin, but did not displaced the binding of radiolabeled vinblastine. Greater affinity with the colchicine binding site was observed and the kinetics of inhibition were shown to be mixed. The side chain of taxol, which differentiates the molecule from baccatin III and is known to be of requisite importance for the unique activity mediated by taxol, is not by itself active in any of these processes. Thus, the baccatin III nucleus of taxol may lead to an interaction with tubulin through traditional binding sites. Facilitated by this interaction, the intact molecule of taxol may thereby be permitted to potentiate tubulin polymerization and block cells in the mitotic phase of the cell cycle.

Synthesis of betulinic acid derivatives with activity against human melanoma.

Betulinic acid has been modified at C-3, C-20, and C-28 positions and the toxicity of the derivatives has been evaluated against cultured human melanoma (MEL-2) and human epidermoid carcinoma of the mouth (KB) cell lines. This preliminary investigation demonstrates that simple modifications of the parent structure of betulinic acid can produce potentially important derivatives, which may be developed as antitumor drugs.

Specific inhibition of human immunodeficiency virus type 1 reverse transcriptase mediated by soulattrolide, a coumarin isolated from the latex of calophyllum teysmannii.

Soulattrolide (1), a coumarin isolated from Calophyllum teysmannii latex, was found to be a potent inhibitor of HIV-1 reverse transcriptase (RT) with an IC50 of 0.34 microM. Inhibition was remarkably specific, with no appreciable activity being observed toward HIV-2 RT, AMV RT, RNA polymerase, or DNA polymerases alpha or beta.

A mixed micellar formulation suitable for the parenteral administration of taxol.

Taxol is a promising antitumor agent with poor water solubility. Intravenous administration of a current taxol formulation in a non-aqueous vehicle containing Cremophor EL may cause allergic reactions and precipitation upon aqueous dilution. In this study a novel approach to formulate taxol in aqueous medium for i.v. delivery is described. The drug is solubilized in bile salt (BS)/phospholipid (PC) mixed micelles. The solubilization potential of the mixed micelles increased as the total lipid concentration and the molar ratio of PC/BS increased. Precipitation of the drug upon dilution was avoided by the spontaneous formation of drug-loaded liposomes from mixed micelles. The formulation can be stored in a freeze-dried form as mixed micelles to achieve optimum stability, and liposomes can be prepared by simple dilution just before administration. As judged by a panel of cultured cell lines, the cytotoxic activity of taxol was retained when formulated as a mixed-micellar solution. Further, for the same solubilization potential, the mixed-micellar vehicle appeared to be less toxic than the standard nonaqueous vehicle of taxol containing Cremophor EL.

Cytotoxic constituents of the bark of Plumeria rubra collected in Indonesia.

By bioactivity-directed fractionation, six cytotoxic constituents have been characterized from the bark of Plumeria rubra collected in Indonesia. Three iridoids, fulvoplumierin [1], allamcin [2], and allamandin [3], as well as 2,5-dimethoxy-p-benzoquinone [4], were found to be active constituents of the P. rubra petroleum-ether- and CHCl3-soluble extracts. Cytotoxic compounds isolated from the H2O-soluble extract of the bark were the iridoid plumericin [5], and the lignan liriodendrin [6]. Each of these substances was found to demonstrate general cytotoxic activity when evaluated with a panel of cell lines composed of murine lymphocytic leukemia (P-388) and a number of human cancer cell-types (breast, colon, fibrosarcoma, lung, melanoma, KB). Five additional iridoids, 15-demethylplumieride [7], plumieride [8], alpha-allamcidin [9], beta-allamcidin [10], and 13-O-trans-p-coumaroylplumieride [11], were obtained as inactive constituents. Compound 7 was found to be a novel natural product, and its structure was determined by spectroscopic methods and by conversion to plumieride [8]. The configuration of the C-4 stereocenter was unambiguously assigned for compounds 9 and 10, and certain nmr reassignments have been provided for compound 1.

1H- and 13C-nmr assignments of phyllanthin and hypophyllanthin: lignans that enhance cytotoxic responses with cultured multidrug-resistant cells.

Complete 1H-nmr data and unambiguous assignments of the 13C-nmr spectra of phyllanthin [1] and hypophyllanthin [2] were obtained through extensive nmr studies, including homonuclear COSY, homonuclear decoupling, APT, HETCOR, nOe difference, selective INEPT, and COLOC experiments. The absolute configuration of hypophyllanthin [2] was determined by cd. Neither of these lignans demonstrated significant cytotoxic activity when evaluated with a battery of cultured mammalian cells, but both were found to enhance the cytotoxic response mediated by vinblastine with multidrug-resistant KB cells. In addition, 1 was found to displace the binding of vinblastine with membrane vesicles derived from this cell line, suggesting an interaction with the P-glycoprotein.