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 activity correlate with significantly higher expression of 20α-HSO, 3α-HSO2 and 3α-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 5α-pregnanes and decreased production of anti-cancer 20α – and 3α-4-pregnenes.
Highlights
Recent observations indicate that human tumorous breast tissue metabolizes progesterone differently than nontumorous breast tissue
The results provide the first demonstration of the expression of messenger RNA for 5αR type 1 (5αR1; SRD5A1), 5αR type 2 (5αR2; SRD5A2), 20α-HSO (AKR1C1), 3α-hydroxysteroid oxidoreductase (3α-HSO) type 2 (3α-HSO2; AKR1C3), 3α-HSO type 3 (3α-HSO3; AKR1C2) and 3β-HSO in breast cell lines
The findings provide the first evidence that the conversion of progesterone to the cancer promoting 5α-pregnanes is significantly higher in the human tumorigenic breast cell lines, MCF-7 (ER+/PR+), MDA-MB-231 (ER-/PR-) and T47D (ER+/PR+), than in the nontumorigenic cell line, MCF-10A (ER-/PR-)
Summary
Recent observations indicate that human tumorous breast tissue metabolizes progesterone differently than nontumorous breast tissue. 5α-reduced metabolites (5αpregnanes, shown to stimulate cell proliferation and detachment) are produced at a significantly higher rate in tumorous tissue, indicating increased 5α-reductase (5αR) activity. The activities of 3α-hydroxysteroid oxidoreductase (3α-HSO) and 20α-HSO enzymes appeared to be higher in normal tissues. The conversion to 5α-pregnanes, requiring the action of 5α-reductase (5αR), is significantly higher in tumorous than in nontumorous human breast tissue [1]. Exposure of human breast cell lines (MCF-7, MCF-10A, and ZR-75-1) to 5α-pregnanes results in changes associated with neoplasia, including increased proliferation and decreased attachment [1], depolymerization of F-actin [2] and decreases in adhesion plaque-associated vinculin [2]. Specific but separate, high-affinity receptors for the 5α-pregnane, 5α-pregnane-3,20-dione and the 4-pregnene, 3α-hyroxy-4-pregnen-20-one, have been identified in the plasma membrane fractions of breast cancer cells [3]
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