A number of clinical studies have demonstrated that norethisterone (NET), a potent synthetic progestin, restores postmenopausal bone loss, although its mode of action on bone cells is not fully understood, while the effect of naturally occurring progesterone in bone has remained controversial. A recent report claims that the potent effects of NET on osteoblastic cell proliferation and differentiation, mimicking the action of estrogens, are mediated by non-phenolic NET derivatives. To determine whether osteoblasts possess the enzymes required to bioconvert a progesterone receptor (PR) agonist into A-ring reduced metabolites with affinity to bind estrogen receptor (ER), we studied the in vitro metabolism of [³H]-labeled NET in cultured neonatal rat osteoblasts and the interaction of its metabolic conversion products with cytosolic –osteoblast ER, employing a competition analysis. Results indicated that NET was extensively bioconverted (36.4%) to 5α-reduced metabolites, including 5α-dihydro NET, 3α,5α-tetrahydro NET (3α,5α-NET) and 3β,5α-tetrahydro NET (3β,5α-NET), demonstrating the activities of 5α-steroid reductase and two enzymes of the aldo-keto reductases family. Expression of Srd5a1 in neonatal osteoblast was well demonstrated, whereas Srd5a2 expression was not detected. The most striking finding was that 3β,5α-NET and 3α,5α-NET were efficient competitors of [³H]-estradiol for osteoblast ER binding sites, exhibiting affinities similar to that of estradiol. The results support the concept that the interplay of 5α-steroid reductase and aldo-keto reductases in osteoblastic cells, acting as an intracrine modulator system is capable to bioconvert a PR agonist into ER agonists, offering an explanation of the molecular mechanisms NET uses to enhance osteoblastic cell activities.

Breast cancer is a sex steroid hormone-dependent malignant neoplasia. The role of oestradiol in this malignancy has been well documented; however, the involvement of androgens has remained controversial. To determine the role of non-phenolic androgen metabolites in human breast cancer, we studied the metabolism of [¹⁴C] testosterone and [¹⁴C] androstenedione in oestrogen-dependent MCF-7 cells and non-oestrogen-dependent MDA-MB 231 cells, at different substrate concentrations (1–10 μM) and time periods (30 min–48 h). Cultured non-oestrogen-dependent HeLa and yeast cells served as controls. Metabolites were identified and quantified by reverse isotope dilution. A distinctive pattern of androgen metabolism was identified in MCF-7 cells, being the 5α-androstane-3α,17β-diol (3α,5α-diol) and its 3β epimer (3β,5α-diol), the major conversion products of testosterone (48.3%), with 5α-dihydrotestosterone as intermediary. The formation of 3α,5α-diol and 3β,5α-diol (diols) was substrate concentration- and time-dependent, and abolished by finasteride. In contrast, very little of any diol formation was observed in MDA-MB 231, HeLa and yeast cell incubations. Additional enzyme gene expression studies revealed an overexpression of 5α-steroid reductase type-1 in MCF-7 cells, as compared with MDA-MB 231 cells. The oestrogen-like activities of diols were assessed in HeLa cells co-transfected with expression vectors for α or β subtypes of the human oestrogen receptor (hER) genes and for an oestrogen-responsive reporter gene. The results show that 3β, 5α-diol and to a lesser extent 3α,5α-diol bind with high relative affinity to hERα and hERβ.

Both diols induced hER-mediated reporter gene transactivation in a dose–response manner, similar to that induced by oestradiol, though with lower potency, an effect that was abolished by ICI-182 780. Furthermore, 3β,5α-diol and to lesser extent 3α,5α-diol induced MCF-7 cell proliferation. The overall results demonstrated that MCF-7 cells exhibit enhanced expression and activity of androgen-metabolising enzymes, leading to rapid and large diol formation, and provide evidence that these androgen metabolites exert a potent oestrogen-agonistic effect, at genomic level, in oestrogen-dependent breast cancer cells. The data suggest that diols may act as in situ intracrine factors in breast cancer and that its formation can be pharmacologically inhibited.