As a result of bioassay-guided fractionation, betulinic acid, a pentacyclic triterpene, was identified as a melanoma-specific cytotoxic agent. In follow-up studies conducted with athymic mice carrying human melanomas, tumour growth was completely inhibited without toxicity. As judged by a variety of cellular responses, antitumour activity was mediated by the induction of apoptosis. Betulinic acid is inexpensive and available in abundant supply from common natural sources, notably the bark of white birch trees. The compound is currently undergoing preclinical development for the treatment or prevention of malignant melanoma.

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There is a clear association between excessive exposure to estrogens and the development of cancer in several tissues including breast and endometrium. The risk factors for women developing these cancers are all associated with longer estrogen exposure, as may be facilitated by early menses, late menopause and long-term estrogen replacement therapy. Equilenin (1,3,5(10),6,8-estrapentaen-3-ol-17-one) or its 17-hydroxylated analogs make up 15% of the most widely prescribed estrogen replacement formulation, Premarin, and yet there is very little information on the human metabolism of these estrogens. In this study, we synthesized the catechol metabolite of equilenin, 4-hydroxyequilenin, and examined how aromatization of the B ring affects the formation and reactivity of the o-quinone (3,5-cyclohexadien-1,2-dione). 4-Hydroxyequilenin-o-quinone is much more redox-active and longer-lived than the endogenous catechol estrone-o-quinones, which suggests that the mechanism(s) of toxicity of the former could be quite different. Interestingly, the rate of reduction of the 4-hydroxyequilenin-o-quinone is increased at least 13-fold in the presence of NAD(P)H:quinone oxidoreductase (DT-diaphorase). Once NADH is consumed however, the catechol auto-oxidized rapidly to the o-quinone. NADH consumption was accompanied by dicumarol-sensitive oxygen uptake both with the purified enzyme and with cytosol from human melanoma cells with high levels of DT-diaphorase activity. P450 reductase and rat liver microsomes also catalyzed NADPH consumption and oxygen uptake. 4-Hydroxyestrone-o-quinone was also rapidly reduced by NAD(P)H; however, this o-quinone does not auto-oxidize and once the o-quinone is reduced the reaction terminates. Including oxidative enzymes in the incubation completes the redox couple and 4-hydroxyestrone-o-quinone behaves like 4-hydroxyequilenin-o-quinone. These data suggest that reduction of estrogen-o-quinones may not result in detoxification. Instead this could represent a cytotoxic mechanism involving consumption of reducing equivalents (NADH/NADPH) as well as formation of superoxide and other reactive oxygen species leading to oxidative stress. Finally, we have compared the cytotoxicity of 4-hydroxyequilenin with that of the estrone catechols in human melanoma cells. 4-Hydroxyequilenin is 5-fold more toxic in these cells compared with 4-hydroxyestrone (ED50 = 7.8 versus 38 microM, respectively) suggesting that formation of the longer-lived redox-active 4-hydroxyequilenin-o-quinone was responsible for the cytotoxic differences. These results substantiate the conclusion that the involvement of quinoids in catechol estrogen toxicity depends on a combination of the rate of formation of the o-quinone, the lifetime of the o-quinone, and the electrophilic/redox reactivity of the quinoids.

The risk factors for women developing breast and endometrial cancers are all associated with a lifetime of estrogen exposure. Estrogen replacement therapy in particular has been correlated with a slight increased cancer risk. Previously, we showed that equilenin, a minor component of Premarin (Wyeth-Ayerst), was metabolized to highly cytotoxic quinoids which caused oxidative stress and alkylation of DNA in vitro [Bolton, J. L., Pisha, E., Zhang, F., and Qiu, S. (1998) Chem. Res. Toxicol. 11, 1113-1127]. In this study, we have compared the chemistry of the major catechol metabolite of equilin (4-hydroxyequilin), which is found in several estrogen replacement formulations, to the equilenin catechol (4-hydroxyequilenin). Unlike endogenous catechol estrogens, both equilin and equilenin were primarily converted by rat liver microsomes to 4-hydroxylated rather than 2-hydroxylated o-quinone GSH conjugates. With equilin, a small amount of 2-hydroxyequilin GSH quinoids were detected (4-hydroxyequilin:2-hydroxyequilin ratio of 6:1); however, no peaks corresponding to 2-hydroxyequilenin were observed in incubations with equilenin. These data suggest that unsaturation in the B ring alters the regiochemistry of P450-catalyzed hydroxylation from primarily 2-hydroxylation for endogenous estrogens to 4-hydroxylation for equine estrogens. 4-Hydroxyequilenin-o-quinone reacts with GSH to give two mono-GSH conjugates and one di-adduct. The behavior of 4-hydroxyequilin was found to be more complex than 4-hydroxyequilenin as conjugates resulting from 4-hydroxyequilenin were detected in addition to the 4-hydroxyequilin-GSH adducts. The mechanism of decomposition of 4-hydroxyequilin likely involves isomerization to a quinone methide which readily aromatizes to 4-hydroxyequilenin followed by autoxidation to 4-hydroxyequilenin-o-quinone. Similar results were obtained with 2-hydroxyequilin, although, in contrast to 4-hydroxyequilenin, 2-hydroxyequilenin does not autoxidize and the reaction stops at the catechol. Since 4-hydroxyequilin is converted to 4-hydroxyequilenin and 4-hydroxyequilenin-o-quinone, similar effects were observed for this equine catechol, including consumption of NAD(P)H likely by the 4-hydroxyequilenin-o-quinone, depletion of molecular oxygen by 4-hydroxyequilenin or its semiquinone radical, and alkylation of deoxynucleosides and DNA by 4-hydroxyequilenin quinoids. Finally, preliminary studies conducted with the human breast tumor cell line MCF-7 demonstrated that the cytotoxic effects of the catechol estrogens from estrone, equilin, and 2-hydroxyequilenin were similar, whereas 4-hydroxyequilenin was a much more potent cytotoxin ( approximately 30-fold). These results suggest that the catechol metabolites of equine estrogens have the ability to cause alkylation/redox damage in vivo primarily through formation of 4-hydroxyequilenin quinoids.

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