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

Abstract

Background: Recent observations indicate that human tumorous breast tissue metabolizes progesterone differently than nontumorous breast tissue. Specifically, 5alpha-reduced metabolites (5alpha-pregnanes, shown to stimulate cell proliferation and detachment) are produced at a significantly higher rate in tumorous tissue, indicating increased 5alpha-reductase (5alphaR) activity. Conversely, the activities of 3alpha-hydroxysteroid oxidoreductase (3alpha-HSO) and 20alpha-HSO enzymes appeared to be higher in normal tissues. The elevated conversion to 5alpha-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).

Methods: For 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 5alphaR type1 (SRD5A1), 5alphaR type 2 (SRD5A2), 20alpha-HSO (AKR1C1), 3alpha-HSO type 2 (AKR1C3), 3alpha-HSO type 3 (AKR1C2) and 3beta-HSO (HSD3B1/HSD3B2) in the four cell lines using 18S rRNA as an internal control.

Results: The relative 5alpha-reductase activity, when considered as a ratio of 5alpha-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 5alpha-pregnanes in the tumorigenic cells, respectively, than in the nontumorigenic MCF-10A cells. Conversely, the 20alpha-HSO and 3alpha-HSO activities were significantly higher (p < 0.001) in MCF-10A cells than in the other three cell types. In the MCF-10A cells, 20alpha-HSO activity was 8-14-fold higher and the 3alpha-HSO activity was 2.5-5.4-fold higher than in the other three cell types. The values of 5alphaR:20alpha-HSO ratios were 16.9-32.6-fold greater and the 5alphaR:3alpha-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 5alphaR1 (p < 0.001), and lower expression of 20alpha-HSO (p < 0.001), 3alpha-HSO2 (p < 0.001), 3alpha-HSO3 (p < 0.001) in MCF-7, MDA-MB-231 and T-47D cells than in MCF-10A cells.

Conclusion: The findings provide the first evidence that the 5alphaR activity (leading to the conversion of progesterone to the cancer promoting 5alpha-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 5alphaR activity coincides with significantly greater expression of 5alphaR1. On the other hand, the activities of 20alpha-HSO and 3alpha-HSO are higher in the MCF-10A cells than in MCF-7, MDA-MB-231 and T-47D cells; these differences in activity correlate with significantly higher expression of 20alpha-HSO, 3alpha-HSO2 and 3alpha-HSO3 in MCF-10A cells. Changes in progesterone metabolizing enzyme expression (resulting in enzyme activity changes) may be responsible for stimulating breast cancer by increased production of tumor-promoting 5alpha-pregnanes and decreased production of anti-cancer 20alpha--and 3alpha-4-pregnenes.

Figure 1

Sample autoradiographs of TLCs showing ¹⁴C-labeled metabolites of progesterone in MCF-7 and MCF-10A cells following an 8-hour incubation with [¹⁴C]progesterone (P). The main metabolites identified and quantified for these enzyme activity studies are: (1) 5α-pregnane-3,20-dione, (2) 5α-pregnan-3α-ol-20-one, (3) 5α-pregnan-20α-ol-3-one, (4) 5α-pregnan-3β-ol-20-one, (5) 4-pregnen-3α-ol-20-one (3αHP), (6) 4-pregnen-20α-ol-3-one (20αDHP), (7) 5α-pregnane-3α (β), 20α-diol, (8) 4-pregnene-3α,20α-diol. The extracts were spotted at the origin and the plates were run 2 × in solvent system 1 and 3 × in solvent system 2 in the directions indicated, as described under Methods, and the films were exposed for 8 days. Note greater size/intensity of 5α-pregnane spots (Nos. 1–4, 7) in MCF-7 cells, and greater size/intensity of 4-pregnene spots (No. 5 and 6) in MCF-10A cells.

Figure 6

Progesterone metabolizing enzymes and pathways in MCF-7, MDA-MB-231, T-47D, and MCF-10A cells. Progesterone is converted to 5α-pregnane-3,20-dione (5α-P-3,20-dione) by 5αR, to 4-pregnen-3α-ol-20-one (3αHP) by 3α-HSO, or to 4-pregnen-20α-ol-3-one (20αDHP) by 20α-HSO. Each of the 4-pregnenes can be 5α-reduced by 5αR, in a one-way reaction, to the respective 5α-pregnane. The activities of 3α-HSO and 20α-HSO are reversible and control reductive and oxidative inter-conversions within the 4-pregnenes (inner ring of metabolites) or 5α-pregnanes (outer ring of metabolites). Identification of 5α-pregnan-3β-ol-20-one as a metabolite of all four cell types, indicates the presence of 3β-HSO activity, but at this time a full pathway of 3β-reduction/oxidation is not identified.

Figure 2

Relative 5α-reductase (5αR) activities in MCF-7, MDA-MB-231, T-47D, and MCF-10A cells, calculated (mean ± SEM; n = 3–6) as (a) percent of total metabolites, (b) ratio of 5α-pregnanes:4-pregnenes, and (c) pmoles per hour per 10⁶ cells of 5α-reduced metabolites produced. In (a) the mean levels of 4-pregnenes are significantly different from the mean levels of 5α-pregnanes at p < 0.001. In (b) and (c), *, **, and *** indicate significant differences from MCF-10A cells at p < 0.05, p < 0.01, and p < 0.001, respectively.

Figure 3

Activities (mean ± SEM; n = 3–6) of (a) 20α-HSO, (b) 3α-HSO and (c) 3β-HSO in MCF-7, MDA-MB-231, T-47D, and MCF-10A cells, calculated from the metabolites as pmoles/hour/10⁶ cells. ***, significantly different from MCF-7, MDA-MB-231, and T-47D cells at p < 0.001.

Figure 4

Ratios of (a) 5αR:20α-HSO and (b) 5αR:3α-HSO activities in MCF-7, MDA-MB-231, T-47D, and MCF-10A cells. *, **, and *** indicate significant differences from MCF-10A cells at p < 0.05, p < 0.01, and p < 0.001, respectively.

Figure 5

Representative gels showing the relative expression of (a) 5αR1, (b) 5αR2, (c) 20α-HSO, (d) 3α-HSO type 2, and (e) 3α-HSO type 3 in MCF-7 (lane 2), MDA-MB-231 (lane 3), T-47D (lane 4), and MCF-10A (lane 5) cells. In each case aliquots of cDNA were amplified with gene-specific primers, run on 9% polyacrylamide gels and the bands were quantified as described under Methods. Each right panels shows computer-assisted quantification of data calculated as ratios against 18S rRNA (mean ± SEM) from 6 separate experiments. *** significantly different from MCF-7, MDA-MB-231, and T-47D cells at p < 0.001.