4-Hydroxyequilenin (4-OHEN) is a major phase I metabolite of the equine estrogens present in Premarin, a widely prescribed hormone replacement formulation. 4OHEN is autoxidized to potent cytotoxic quinoids and forms ROS, which may contribute to the chemical mechanism for estrogen carcinogenesis. 4-OHEN can be metabolized to 4-methoxyequilenin (4-McOEN) by catechol-o-methyl transferase and 4-MeOEN could be theoretically demethylated to 4-OHEN by cytochrome P450. In order to investigate the participation of equine estrogen metabolites in the hormonal carcinogenic mechanism, the estrogenic activities of 4-OHEN and 4-MeOEN were compared to the endogenous catechol estrogen metabolite 4-hydroxyestradiol in an ERα (+) MCF-7 cell line. Our data demonstrated that the nanomolar estrogenic potency of 4-OHEN and 4McOEN was mediated via ER-ERE interaction, leading to estrogen-responsive gene transcription and cell proliferation in breast cancer cells.

In previous studies, oxidation of the catechol metabolite of estrone (4-OHE) and the β-naphthohydroquinone (β-NHQ) metabolite of equilenin gave o-quinones that produced ROS and damaged DNA by adduction and oxidation. The relative importance of contributions from hormonal carcinogenesis versus chemical carcinogenesis pathways remain unknown, as do the relative contributions to chemical carcinogenesis from DNA alkylation versus DNA oxidation. To differentiate between hormonal and chemical carcinogenesis pathways in ER(+) cells, catechol or β-NHQ warheads were conjugated to the selective estrogen receptor modulator (SERM) desmethylarzoxifene (DMA). These quinone prodrugs with ER delivery (QPED) were anticipated to reveal the capacity of o-quinone warheads to damage DNA selectively in ER(+) cells, in the absence of confounding estrogenic activity, and thereby to provide information on potential contributions to the chemical carcinogenesis of 4-OHE and 4-OHEN. In our results, both DMA-QPEDs were antiestrogens with submicromolar potency in mammary and endometrial cell assays. Conjugation of warheads to DMA increased cytotoxicity accompanied by induction of apoptosis and activation of caspase-3/7. Activation of caspase-3/7, induction of apoptosis, and cytotoxicity were all increased significantly in ER(+) cells for the DMA-QPEDs. ROS production was localized in the nucleus for conjugates in ER(+) cells. The novel conjugation of quinone warheads to an ER-targeting SERM gives ER-dependent, and enhanced apoptosis in mammary cancer cells of potential application in cancer therapy.