Activity and expression of progesterone metabolizing 5α-reductase, 20α-hydroxysteroid oxidoreductase and 3α(β)-hydroxysteroid oxidoreductases in tumorigenic (MCF-7, MDA-MB-231, T-47D) and nontumorigenic (MCF-10A) human breast cancer cells

Abstract

BackgroundRecent observations indicate that human tumorous breast tissue metabolizes progesterone differently than nontumorous breast tissue. Specifically, 5α-reduced metabolites (5α-pregnanes, shown to stimulate cell proliferation and detachment) are produced at a significantly higher rate in tumorous tissue, indicating increased 5α-reductase (5αR) activity. Conversely, the activities of 3α-hydroxysteroid oxidoreductase (3α-HSO) and 20α-HSO enzymes appeared to be higher in normal tissues. The elevated conversion to 5α-pregnanes occurred regardless of estrogen (ER) or progesterone (PR) receptor levels. To gain insight into these differences, the activities and expression of these progesterone converting enzymes were investigated in a nontumorigenic cell line, MCF-10A (ER- and PR-negative), and the three tumorigenic cell lines, MDA-MB-231 (ER- and PR-negative), MCF-7 and T-47D (ER- and PR-positive).MethodsFor the enzyme activity studies, either whole cells were incubated with [14C]progesterone for 2, 4, 8, and 24 hours, or the microsomal/cytosolic fraction was incubated for 15–60 minutes with [3H]progesterone, and the metabolites were identified and quantified. Semi-quantitative RT-PCR was employed to determine the relative levels of expression of 5αR type1 (SRD5A1), 5αR type 2 (SRD5A2), 20α-HSO (AKR1C1), 3α-HSO type 2 (AKR1C3), 3α-HSO type 3 (AKR1C2) and 3β-HSO (HSD3B1/HSD3B2) in the four cell lines using 18S rRNA as an internal control.ResultsThe relative 5α-reductase activity, when considered as a ratio of 5α-pregnanes/4-pregnenes, was 4.21 (± 0.49) for MCF-7 cells, 6.24 (± 1.14) for MDA-MB-231 cells, 4.62 (± 0.43) for T-47D cells and 0.65 (± 0.07) for MCF-10A cells, constituting approximately 6.5-fold, 9.6-fold and 7.1 fold higher conversion to 5α-pregnanes in the tumorigenic cells, respectively, than in the nontumorigenic MCF-10A cells. Conversely, the 20α-HSO and 3α-HSO activities were significantly higher (p < 0.001) in MCF-10A cells than in the other three cell types. In the MCF-10A cells, 20α-HSO activity was 8-14-fold higher and the 3α-HSO activity was 2.5-5.4-fold higher than in the other three cell types. The values of 5αR:20α-HSO ratios were 16.9 – 32.6-fold greater and the 5αR:3α-HSO ratios were 5.2 – 10.5-fold greater in MCF-7, MDA-MB-231 and T-47D cells than in MCF-10A cells. RT-PCR showed significantly higher expression of 5αR1 (p < 0.001), and lower expression of 20α-HSO (p < 0.001), 3α-HSO2 (p < 0.001), 3α-HSO3 (p < 0.001) in MCF-7, MDA-MB-231 and T-47D cells than in MCF-10A cells.ConclusionThe findings provide the first evidence that the 5αR activity (leading to the conversion of progesterone to the cancer promoting 5α-pregnanes) is significantly higher in the tumorigenic MCF-7, MDA-MB-231 and T-47D breast cell lines than in the nontumorigenic MCF-10A cell line. The higher 5αR activity coincides with significantly greater expression of 5αR1. On the other hand, the activities of 20α-HSO and 3α-HSO are higher in the MCF-10A cells than in MCF-7, MDA-MB-231 and T-47D cells; these differences in act...

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

“…The on-going studies in our laboratories indicate that diols bind with low affinity to the human androgen receptor (hAR) and induce very limited hAR-mediated transactivation, particularly the 3a,5a-diol (A E Lemus, P Damian-Matsumura, R GarciaBecerra, L Gonzalez, D Orolaz, F Larrea & G Perez, Unpublished observations). The finasteride-induced inhibition of DHTand diols formation in MCF-7 cells, as demonstrated in this study, may have additional interest, since various locally formed A-ring-reduced metabolites of progesterone , Wiebe & Muzia 2001, Wiebe & Lewis 2003 are capable of inducing cell proliferation in human breast cancer, through a novel, non-genomic mechanism (Weiler & Wiebe 2000). The overall results demonstrated that finasteride inhibits the formation of oestrogenic steroids (diols) generated downstream of DHT, as suggested recently by Ishikawa et al (2006).…”

“…These results confirm and extend the report of Wiebe et al (2000), who, using radiolabelled progesterone as substrate, demonstrated an enhanced 5a-steroid reductase activity and also an overexpression of the 5a-steroid reductase type-1 gene in MCF-7 cells, as compared with oestrogen-resistant breast cells (MCF-10A). Furthermore, Wiebe & Lewis (2003) Steroid competitor (nM) Figure 7 Binding affinities of 3a,5a-and 3b,5a-androstanediols (diols) to human oestrogen receptors (hER). Cytosol aliquots of HeLa cells transfected with either hERa or hERb gene expression vectors were incubated with 1 nM [ 3 H] oestradiol at 4 8C, for 48 h, in the absence or presence of increasing concentrations of radioinert 3a,5a-diol, 3b,5a-diol and oestradiol (E 2 ).…”

Progesterone metabolites in breast cancer

“…The 4-pregnenes can be further reversibly converted to 4-pregnene-3a(3b),20a-diol. The same metabolic pathways were subsequently demonstrated in four different breast cell lines (Wiebe & Lewis 2003) and had been previously identified in a number of tissues, including gonads, pituitary, and hypothalamus (Wiebe 1997). In addition, in the human breast cell lines, the final major product was 5a-pregnane-3b,6a-diol-20-one, indicating the presence of 6a-hydroxylase, an enzyme that was also present in tissues at minor activity levels.…”

The effects of synthetic 19-norprogestins on osteoblastic cell function are mediated by their non-phenolic reduced metabolites

“…Furthermore, using co-transfected cell expression systems, we have shown that non-phenolic reduced metabolites of 19-norprogestins are able to transactivate estrogen-dependent genes mediated through human ERa, but not through ERb (Larrea et al 2001, García-Becerra et al 2002, behaving as selective ERa modulators, with ligand-receptor structural and functional responses similar to those induced with naturally occurring E 2 . Additional studies on the effects of progestin reduced derivatives on breast tissue are required, particularly since an overexpression of the 5a-steroid reductase type 1 gene has been reported in breast cancer tumors and cells (Suzuki et al 2001, Wiebe & Lewis 2003.…”