2-Methoxymethylestradiol: a new 2-methoxy estrogen analog that exhibits antiproliferative activity and alters tubulin dynamics.

An estradiol metabolite, 2-methoxyestradiol (2-MeOE(2)), has shown antiproliferative effects in both hormone-dependent and hormone-independent breast cancer cells. Previously, a series of 2-hydroxyalkyl estradiol analogs had been synthesized in our laboratories as potential probes for comparison of estrogen receptor (ER)-mediated versus non-ER-mediated effects in breast cancer cells. A methoxy derivative of 2-hydroxymethyl estradiol was prepared for biological evaluation and comparison with 2-MeOE(2). Estrogenic activity of the synthetic analogs was evaluated in two ways, one by examining affinity of the analogs for the estrogen receptor in MCF-7 cells and the other by examining the ability of the analogs to induce estrogen-responsive gene expression. The analog, 2-methoxymethyl estradiol (2-MeOMeE(2)), demonstrated weak affinity for the estrogen receptor (0.9% of estradiol) and weak ability to stimulate estrogen-induced expression of the pS2 gene (0.02% of estradiol). Antitumor activity was evaluated both in vitro and in vivo. The steroidal nucleus seems to be an attractive target for developing novel tubulin polymerization inhibitors. Additionally, such steroidal compounds may have low toxicity compared to the natural products known to interact with tubulin. Interestingly, 2-MeOMeE(2) inhibited tubulin polymerization in vitro at concentrations of 1 and 3 microM and was more effective than 2-MeOE(2). In cells, 2-MeOMeE(2) was effective in suppressing growth and inducing cytotoxicity in MCF-7 and MDA-MB-231 breast cancer cells. The cytotoxic effects of 2-MeOMeE(2) are associated with alterations in tubulin dynamics, with the frequent appearance of misaligned chromosomes, a significant mitotic delay, and the formation of multinucleated cells. In comparison, 2-MeOE(2) was more effective than 2-MeOMeE(2) in producing cytotoxicity and altering tubulin dynamics in intact cells. Assessment of in vivo antitumor activity was performed in athymic mice containing human breast tumor xenografts. Nude mice bearing MDA-MB-435 tumor xenografts were treated i.p. with 50 mg/kg per day of 2-MeOMeE(2) or vehicle control for 45 days. Treatment with 2-MeOMeE(2) resulted in an approximate 50% reduction in mean tumor volume at treatment day 45 when compared to control animals and had no effect on animal weight. Thus, 2-MeOMeE(2) is an estrogen analog with minimal estrogenic properties that demonstrates antiproliferative effects both in vitro and in the human xenograft animal model of human breast cancer.

Effects of phytoestrogens and synthetic combinatorial libraries on aromatase, estrogen biosynthesis, and metabolism.

Approximately 60% of breast cancer patients have hormone-dependent breast cancer containing estrogen receptors and requiring estrogen for tumor growth. The extent of estrogen biosynthesis and metabolism in the breast cancer tissue microenvironment influences breast-tumor development and growth, and endogenous and exogenous agents may alter the levels of hormonally active estrogens and their metabolites. Isoflavonoid phytoestrogens such as genistein exhibit numerous biochemical activities; however, their effects on estrogen biosynthesis and metabolism in breast cancer cells have not been fully examined. MCF-7 cells (hormone-dependent) and MBA-MB-231 cells (hormone-independent) were treated with genistein (100 nM) for five days and then incubated with radiolabeled estradiol (100 nM, 2.5 microCi) for 0 to 48 h. Media were extracted with ethyl acetate, and the organic residues analyzed by reverse-phase HPLC with a radioactivity flow detector. The major metabolite formed in all cases is estrone, although differences were observed between the cell lines and the various drug treatments. The formation of estrone in untreated MCF-7 cells (approximately 9.3% of radioactivity at 24 h) is relatively limited, in contrast to untreated MDA-MB-231 cells (approximately 32.0% of radioactivity at 24 h). Treatment of MCF-7 cells with 100 nM genistein increased the conversion of estradiol to estrone up to 19.5% in 24 h. The effect of genistein on estrone formation in MDA-MB-231 cells resulted in 37.7% of the radioactivity being estrone. Thus, genistein treatment of breast cancer cells resulted in increased 17-betahydroxysteroid dehydrogenase activity and elevated formation of estrone. Increased levels of oxidative 17-betahydroxysteroid dehydrogenase activity (Type II) were confirmed by Western blots. Therefore, exposure of breast cancer cells to genistein results in elevated conversion of estradiol to estrogenically weaker or inactive metabolites. The regulation of breast-tissue aromatase by exogenous agents such as drugs and environmental agents is being investigated. The benzopyranone-ring system is a molecular scaffold of considerable interest, and this scaffold is found in flavonoid natural products that have weak aromatase inhibitory activity. Medicinal chemistry efforts focus on diversifying the benzopyranone scaffold and utilizing combinatorial chemistry approaches to construct small benzopyranone libraries as potential aro- matase inhibitors. Several compounds in the initial libraries have demonstrated moderate aromatase inhibitory activity in screening assays.

Molecular pharmacology of aromatase and its regulation by endogenous and exogenous agents.

Aromatase (estrogen synthase) is the cytochrome P450 enzyme complex that converts C19 androgens to C18 estrogens. Aromatase activity has been demonstrated in breast tissue in vitro, and expression of aromatase is highest in or near breast tumor sites. Thus, local regulation of aromatase by both endogenous factors as well as exogenous medicinal agents will influence the levels of estrogen available for breast cancer growth. The prostaglandin E2 (PGE2) increases intracellular cAMP levels and stimulates estrogen biosynthesis, and our recent studies have shown a strong linear association between CYP19 expression and the sum of COX-1 and COX-2 expression in breast cancer specimens. PGE2 can bind to four receptor subtypes, EP1-EP4, which are coupled to different intracellular signaling pathways. In primary human breast stromal cell cultures, aromatase activity was significantly induced by PGE2, dexamethasone, and agonists for the EP1 and EP2 receptor subtypes. An EP1 antagonist, SC-19220, inhibited the induction of enzyme activity by PGE2 or 17-phenyltrinor-PGE2, an EP1 agonist. Sulprostone, an EP3 agonist, did not alter aromatase activity levels. Investigations are also underway on the regulation of aromatase by exogenous medicinal agents. Selective steroidal and nonsteroidal agents are effective in inhibiting breast tissue aromatase. The benzopyranone ring system is a molecular scaffold of considerable interest, and this scaffold is found in certain flavonoid natural products that have weak aromatase inhibitory activity. Our novel synthetic route for benzopyranones utilizes readily available salicylic acids and terminal alkynes as starting materials. The synthesis of flavones with diversity on the benzopyranone moiety and at the C-2 position occurs with good to excellent yields using these reaction conditions, resulting in an initial benzopyranone library of thirty compounds exhibiting enhanced and differential aromatase inhibition. Current medicinal chemistry efforts focus on diversifying the benzopyranone scaffold and utilizing combinatorial chemistry approaches to construct small benzopyranone libraries as potential aromatase inhibitors.