Enhanced formation of non-phenolic androgen metabolites with intrinsic oestrogen-like gene transactivation potency in human breast cancer cells: a distinctive metabolic pattern

Abstract

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 nonphenolic androgen metabolites in human breast cancer, we studied the metabolism of [ C] testosterone and [14 C] androstenedione in oestrogen-dependent MCF-7 cells and non-oestrogen-dependent MDA-MB 231 cells, at different substrate concentrations (1-10 mM) 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 5a-androstane-3a,17b-diol (3a,5a-diol) and its 3b epimer (3b,5a-diol), the major conversion products of testosterone (48$3%), with 5a-dihydrotestosterone as intermediary. The formation of 3a,5a-diol and 3b,5a-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 5a-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 a or b subtypes of the human oestrogen receptor (hER) genes and for an oestrogen-responsive reporter gene. The results show that 3b, 5a-diol and to a lesser extent 3a,5a-diol bind with high relative affinity to hERa and hERb.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, 3b,5a-diol and to lesser extent 3a,5a-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.

Dimethandrolone (7α,11β-dimethyl-19-nortestosterone) and 11β-methyl-19-nortestosterone are not converted to aromatic A-ring products in the presence of recombinant human aromatase

“…It is not known whether this is due to weak activity intrinsic to the androgens or to their conversion to metabolites with estrogenic activity in T47Dco cells. Both synthetic 19-norandrogen derivatives [19,20] and non-phenolic metabolites formed in human breast cancer cells from androgenic substrates [21] have been shown to exhibit estrogenic activity. These compounds have reduced and hydroxylated A-rings; for example, T is metabolized to 5α-androstane-3α, 17β-diol and its 3β-enantiomer in MCF-7 cells [21].…”

“…Both synthetic 19-norandrogen derivatives [19,20] and non-phenolic metabolites formed in human breast cancer cells from androgenic substrates [21] have been shown to exhibit estrogenic activity. These compounds have reduced and hydroxylated A-rings; for example, T is metabolized to 5α-androstane-3α, 17β-diol and its 3β-enantiomer in MCF-7 cells [21]. The basis for transactivation of 3XERE-LUC by selected androgens in T47Dco cells is being investigated further.…”

“…Thus, in this study we examined the possible conversion of selected androgens to aromatic A-ring derivatives with estrogenic activity using recombinant human aromatase, a preparation devoid of other contaminating enzymatic activities such as would be found in intact cells or isolated microsomes. Another potential route for obtaining compounds with estrogenic activity from androgens or 19-norsteroids is through formation of non-phenolic androgen metabolites by 5α-reduction followed by hydroxylation of the A-ring [19][20][21].DMA and 11β-MNT are not likely to be converted to estrogens by this route either as they are not substrates for 5α-reductase [22]. The most decisive method to determine the metabolism of DMA and 11β-MNT in vivo would be to isolate and characterize all possible metabolites.…”

The anti-estrogenic activity of indole-3-carbinol in neonatal rat osteoblasts is associated with the estrogen receptor antagonist 2-hydroxyestradiol

“…Nonradioactive steroid carriers were detected on the chromatograms using p-anisaldehydesulfuric/acetic acid reagent. Radioactivity was determined using a Packard Tri-Carb liquid scintillation spectrometer (model 1900 TR), as previously described by our group [30]. The rate at which the metabolic conversion products of [ 3 H]-E 1 were formed was expressed as picomoles per milligram of protein/h.…”

Neonatal rat osteoblasts bioconvert testosterone to non-phenolic metabolites with estrogen-like effects on bone cell proliferation and differentiation

“…The biological importance of 3 α ,5 α -and 3 β ,5 α -diols has been derived from studies demonstrating that the administration of 3 β ,5 α -diol induced precocious puberty in immature female rats [ 27 ] and promoted the synthesis of estrogen-dependent progesterone receptors in the mouse uterus [ 28 ]. In addition, both diols have shown estrogen-like gene transactivation potency through α and β ER subtypes [ 29 ]. Although these data indicate estrogenic properties of diols on a variety of reproductive functions, their precise role in bone cells has not yet been elucidated.…”