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.

The key role of estrogens on osteoblastic cell function is well documented; however, the role of progesterone (P) and synthetic progestins remains controversial. While several reports indicate that P has no significant effects on bone cells, a number of clinical studies have shown that 19-norprogestins restore postmenopausal bone loss. The mechanisms by which 19-norprogestins induce estrogen-like effects on bone cells are not fully understood. To assess whether the actions of 19-norprogestins on osteoblasts are mediated by their non-phenolic metabolites, we studied the effects of norethisterone (NET), levonorgestrel (LNG), and two of their A-ring reduced derivatives upon cell proliferation and differentiation in neonatal rat osteoblasts. Osteoblast function was assessed by determining cell DNA, cell-associated osteocalcin and calcium content, alkaline phosphatase activity, and mineral deposition. P failed to induce changes on osteoblasts, while NET and LNG exerted a number of actions. The most striking finding was that the 3β,5α- and 3α,5α-tetrahydro derivatives of NET and LNG induced osteoblast proliferation and differentiation with higher potency than those exerted by their parent compounds, mimicking the effects of estradiol. Interestingly, osteoblast differentiation and mineral deposition induced by NET and LNG were abolished by finasteride, a 5α-reductases inhibitor, while the potent effect on osteoblast proliferation induced by progestin derivatives was abolished by a steroidal antiestrogen. Results demonstrate that A-ring reduced derivatives of NET and LNG exhibit intrinsic estrogen-like potency on rat osteoblasts, offering a plausible explanation for the mechanism of action of 19-norprogestins in bone restoration in postmenopausal women and providing new insights for hormone replacement therapy research.

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.