Synthesis and characteristics of allylic 4-pregnene-3,20-diols found in gonadal and breast tissues

Wiebe, John P.; Dave, Vinod; Stothers, J. B.

doi:10.1016/0039-128x(86)90095-4

Cited by 10 publications

(5 citation statements)

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“…[1,2,6,7- 3 H]3α-Hydroxy-4-pregnen-20-one (3αHP) was synthesized from [1,2,6,7- 3 H]progesterone by published procedures [9]. Unlabeled 3αHP, 3βHP, and 4-pregnene-3α (β),20α(β)-diols were prepared as described previously [10,11]. Other unlabeled steroids were purchased from Steraloids Inc (Wilton, N.H.) or Sigma Chemical Co. (Oakville, ON).…”

Section: Methodsmentioning

confidence: 99%

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

Wiebe

Lewis

2003

BMC Cancer

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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...

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Section: Methodsmentioning

confidence: 99%

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

Wiebe

Lewis

2003

BMC Cancer

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“…The reduction of progesterone was performed as described earlier by Wiebe et al, with slight modifications [ 29 ]. Carbonyls at the allylic C-3 and at C-20 were reduced using aluminum isopropoxide in 2-propanol ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The exact structures of the individual isolated derivatives were assigned by comparison of their recorded spectroscopic data with those previously published [ 29 ]. As described, the chemical shifts of the characteristic signals (specially the methyl groups at positions 18, 19 and 21) and the specific splitting patterns allowed unambiguous assignment of the stereochemistry of both hydroxyl groups in each of the four pregn-4-ene-3,20-diols ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The strategies currently available for the analysis of progesterone metabolites include immunoassays [ 23 ], one or two dimension thin-layer chromatography [ 2 , 24 , 25 ], high pressure liquid chromatography (HPLC) [ 26 , 27 ], gas chromatography/mass spectrometry (GC/MS) [ 14 , 28 , 29 ], liquid chromatography/mass spectrometry (LC/MS) [ 30 ] and liquid chromatography—tandem mass spectrometry (LC-MS/MS). The reliability of immunoassays has been questioned due to the high probability of cross-reactivity between different stereoisomers, and it also requires large sample volumes, especially when a steroid is present at low concentrations [ 31 , 32 ].…”

Section: Introductionmentioning

confidence: 99%

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Combined Liquid Chromatography–Tandem Mass Spectrometry Analysis of Progesterone Metabolites

et al. 2015

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Progesterone has a number of important functions throughout the human body. While the roles of progesterone are well known, the possible actions and implications of progesterone metabolites in different tissues remain to be determined. There is a growing body of evidence that these metabolites are not inactive, but can have significant biological effects, as anesthetics, anxiolytics and anticonvulsants. Furthermore, they can facilitate synthesis of myelin components in the peripheral nervous system, have effects on human pregnancy and onset of labour, and have a neuroprotective role. For a better understanding of the functions of progesterone metabolites, improved analytical methods are essential. We have developed a combined liquid chromatography—tandem mass spectrometry (LC-MS/MS) method for detection and quantification of progesterone and 16 progesterone metabolites that has femtomolar sensitivity and good reproducibility in a single chromatographic run. MS/MS analyses were performed in positive mode and under constant electrospray ionization conditions. To increase the sensitivity, all of the transitions were recorded using the Scheduled MRM algorithm. This LC-MS/MS method requires small sample volumes and minimal sample preparation, and there is no need for derivatization. Here, we show the application of this method for evaluation of progesterone metabolism in the HES endometrial cell line. In HES cells, the metabolism of progesterone proceeds mainly to (20S)-20-hydroxy-pregn-4-ene-3-one, (20S)-20-hydroxy-5α-pregnane-3-one and (20S)-5α-pregnane-3α,20-diol. The investigation of possible biological effects of these metabolites on the endometrium is currently undergoing.

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“…Finally, a steroid of progesterone was also reduced using this system in a yield of 98% (entry 7, Table 6) and the configuration of the product was confirmed by comparing its 1 H NMR spectrum (using CDCl 3 as solvent) with that found in the literature. 30 These obvious improvements in activity prove that A-Hp-Br is an efficient catalyst to reduce highly lipophilic (water insoluble) aldehydes and ketones in water.…”

Section: Entrymentioning

confidence: 99%

Highly efficient reduction of carbonyls, azides, and benzyl halides by NaBH₄ in water catalyzed by PANF-immobilized quaternary ammonium salts

Lin

et al. 2016

Green Chem.

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Synthesis and characteristics of allylic 4-pregnene-3,20-diols found in gonadal and breast tissues

Cited by 10 publications

References 20 publications

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

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

Combined Liquid Chromatography–Tandem Mass Spectrometry Analysis of Progesterone Metabolites

Highly efficient reduction of carbonyls, azides, and benzyl halides by NaBH₄ in water catalyzed by PANF-immobilized quaternary ammonium salts

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Synthesis and characteristics of allylic 4-pregnene-3,20-diols found in gonadal and breast tissues

Cited by 10 publications

References 20 publications

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

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

Combined Liquid Chromatography–Tandem Mass Spectrometry Analysis of Progesterone Metabolites

Highly efficient reduction of carbonyls, azides, and benzyl halides by NaBH4 in water catalyzed by PANF-immobilized quaternary ammonium salts

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Highly efficient reduction of carbonyls, azides, and benzyl halides by NaBH₄ in water catalyzed by PANF-immobilized quaternary ammonium salts