Analysis of the Promoter-Specific Estrogenic Potency of the Phytoestrogens Genistein, Daidzein and Coumestrol

• Abstract
• Materials and Methods
• Acknowledgments
• References

Abstract

Estrogens modulate the transcription of sensitive genes either via binding of the activated ER to responsive elements in their promoter region or via binding of the activated ER to transcription factors like NFκB. In this study we have analyzed the effects of the phytoestrogens daidzein, coumestrol and genistein in promoter-specific reporter gene systems. The dose-dependent ability to stimulate an ERE-bearing reporter in MVLN breast cancer cells was compared to the dose-dependent ability to repress the IL-1β-stimulated reporter in U2OS osteosarcoma cells. Coumestrol, daidzein and genistein stimulate the expression of the ERE-dependent reporter in MVLN cells and repress the activity of the IL-6 promoter in U2OS cells in a dose-dependent manner. Interestingly, the relative potency of all phytoestrogens to repress the activity of the IL-6 promoter in U2OS cells was much higher than their potency to stimulate the ERE-dependent reporter in MVLN cells. We assume that the demonstrated promoter-specific potency therefore could be an important mechanism to explain a tissue-specific action of some of these compounds.

A large number of plant-derived estrogens has been identified so far and the term phytoestrogens (PE) is used for those compounds with the ability to perform estrogenic effects in vertebrates. PEs have achieved distinct attention for a number of reasons. Their impact on human health and the potential risks or benefits of consumption [1] are still debatable. It was assumed that PEs do not uniformly mimic estrogen’s activity [2]. An answer to the question if these substances may exert potentially organ-selective actions would be of great pharmacological relevance.

Estrogens have been demonstrated to regulate gene activity in a promoter-specific manner. On the one hand they can modulate the transcription of sensitive genes via binding to estrogen-responsive elements in the genes promoter region [3]. In this way of action ER subtypes and specific cofactors are involved [4]. This is an important mechanistic explanation for the principle of tissue-selective action. An alternative way of activity has been shown by binding of the activated ER to transcription factors such as, for example, NFκB, modulating the activity of a variety of specific genes [5]. One of theses genes is the cytokine and growth factor IL-6, an essential modulator of bone metabolism in mammals [6].

In this study, we have addressed the question of whether distinct phytoestrogens possess differences in their promoter-specific potency. Therefore, we have analyzed the effects of the phytoestrogens daidzein, coumestrol and genistein in promoter-specific reporter gene systems representing the mentioned molecular mechanisms of estrogen promoter interaction. These three PEs were selected because of their comparably high exposure via dietary intake (soy products) as well as the usage of soy and red clover extracts in treatment of menopausal complaints. Consequently, these PEs are much better characterized than others [7], [8]. The dose-dependent ability to stimulate an ERE-bearing vitellogenin-2 promoter/luciferase reporter construct in MVLN breast cancer cells [9], [10] was compared to the dose-dependent ability to repress the IL-1β-stimulated activity of an IL-6-promotor/luciferase reporter construct [11] in U2OS osteosarcoma cells. Coumestrol, daidzein and genistein stimulate the expression of the ERE-dependent reporter in MVLN cells and repress the activity of the IL-6 promoter in U2Os cells in a dose-dependent manner. Interestingly, all phytoestrogens very potently repress the activity of the IL-6 promoter in U2OS cells (Fig. [2]). The potency of daidzein (EC₅₀ : 8 × 10 ^{- 10} M) in this system was higher than the potency of the reference substance raloxifen and only 16-fold lower than E2. In contrast, the potency of the phytoestrogens to stimulate the ERE-dependent reporter in MVLN cells was rather low (Fig. [1]). In this system the potency of daidzein, for example, was 60,000-fold lower than that of E2.

Our results implicate a differentiated promoter-specific potency of diverse phytoestrogens. Obviously the ability of the phytoestrogens to inhibit the IL-1β-stimulated expression of the IL-6 gene was much higher than their ability to stimulate the activity of an ERE-controlled promoter (Table [1]). We assume that the demonstrated promoter-specific potency could be an important mechanism enabling us to explain a tissue-specific action of some of these compounds.

Materials and Methods

17β-Estradiol, 4-hydroxytamoxifen, daidzein, coumestrol and genistein were obtained from Sigma (Deissenhofen, Germany). Raloxifen was provided by Schering AG (Berlin, Germany).

MVLN cells [9], [10], a bioluminescent MCF-7-derived cell line, were used to study the modulation of estrogenic activity. The MVLN cells were cultured as formerly described [9], [10]. For testing of the estrogenicity of the naringenins the MVLN cells, a bioluminescent MCF-7-derived cell line to study the modulation of estrogenic activity, was used. MVLN cells were stably transfected with a vitellogenin-2 promoter/luciferase reporter construct which assayed for reporter enzyme activity of firefly luciferase. The luciferase activity represented a direct measurement for the bioactivity of the tested compounds.

ER-negative U2OS cells were cultured in D-MEM without phenol red, 5 % FCS, 1 % glutamine and 2 % penicillin-streptomycin at 37 °C and 7.5 % CO₂. To test the ability of the analyzed compounds to repress the activity of the IL-1-stimulated IL-6 promoter, U2OS cells were double transfected with pHEGO (ERα) expression vector [12] and a pIL-6-LUC reporter gene construct [11] by the use of LipofectinReagent (Gibco BRL). After transfixion, the cells were incubated overnight with the respective test substances in the in the presence of 1.8 nM IL-1β. After cell lysis the activity of the reporter enzyme luciferase was determined by the use of the LucLite Kit (Packard Bio Science). Detection of luminescence was performed in a LumiCount plate luminometer (Packard Bio Science).

Hormonal treatments were performed using serial dilutions in 3 - 5 replications for each experiment with the test compounds in ethanol. Dilutions ranged from 1 × 10 ^{- 11} - 10 ^{- 5} M. Positive and negative controls included ethanolic dilutions of estradiol (10 ^{- 12} - 1 × 10 ^{- 8} M), Raloxifen (10 ^{- 11} - 1 × 10 ^{- 7} M) and vehicle controls were used. The standard deviation is illustrated in the bar diagrams by error bars. Using MVLN cells, the estrogenicity of the compounds were tested in a dilution range from 10 ^{- 8} - 10 ^{- 6} M. In U2OS cells as reference 10 ^{- 8} M estradiol was used as positive control while (the vehicle) ethanol was used as negative control. To test for the involvement of the ER-α, the estrogenic effects were antagonized by the antiestrogen 4-hydroxytamoxifen.

Acknowledgments

This paper was supported by the Deutsche Forschungsgemeinschaft grants Vo410/6-3 and Di 716/9-1.

References

• 1 Knight D C, Eden J A. A review of the clinical effects of phytoestrogens.  Obstet Gynecol. 1996;  87 897-904
  PubMedSearch in Google Scholar
• 2 Krishnan V, Heath H, Bryant H U. Mechanism of action of estrogens and selective estrogen receptor modulators.  Vitam Horm. 2000;  60 123-47
  PubMedSearch in Google Scholar
• 3 O’Lone R, Frith M C, Karlsson E K, Hansen U. Genomic targets of nuclear estrogen receptors.  Mol Endocrinol. 2004;  18 1859-75
  CrossrefPubMedSearch in Google Scholar
• 4 McDonnell D P. The molecular determinants of estrogen receptor pharmacology.  Maturitas. 2004;  48 (Suppl 1) 7-12
  CrossrefPubMedSearch in Google Scholar
• 5 McKay L I, Cidlowski J A. Molecular control of immune/inflammatory responses: interactions between nuclear factor-kappa B and steroid receptor-signaling pathways.  Endocr Rev. 1999;  20 435-59
  CrossrefPubMedSearch in Google Scholar
• 6 Kurebayashi S, Miyashita Y, Hirose T, Kasayama S, Akira S, Kishimoto T. Characterization of mechanisms of interleukin-6 gene repression by estrogen receptor.  J Steroid Biochem Mol Biol. 1997;  60 11-7
  CrossrefPubMedSearch in Google Scholar
• 7 Cos P, De Bruyne T, Apers S, Vanden Berghe D, Pieters L, Vlietinck A J. Phytoestrogens: recent developments.  Planta Med. 2003;  69 589-99
  Thieme ConnectPubMedSearch in Google Scholar
• 8 Krebs E E, Ensrud K E, MacDonald R, Wilt T J. Phytoestrogens for treatment of menopausal symptoms: a systematic review.  Obstet Gynecol. 2004;  104 824-36
  CrossrefPubMedSearch in Google Scholar
• 9 Pons M, Gagne D, Nicolas J C, Mehtali M. A new cellular model of response to estrogens: a bioluminescent test to characterize (anti) estrogen molecules.  Biotechniques. 1990;  9 450-9
  PubMedSearch in Google Scholar
• 10 Demirpence E, Duchesne M J, Badia E, Gagne D, Pons M. MVLN cells: a bioluminescent MCE-7-derived cell line to study the modulation of estrogenic activity.  Steroid Biochem Mol Biol. 1993;  46 355-64
  PubMedSearch in Google Scholar
• 11 Pottratz S T, Bellido T, Mocharla H, Crabb D, Manolagas S C. 17beta-Estradiol inhibits expression of human interleukin-6 promoter-reporter constructs by a receptor-dependent mechanism.  J Clin Invest. 1994;  93 944-50
  PubMedSearch in Google Scholar
• 12 Tora L, White J, Brou C, Tasset D, Webster N, Scheer E. et al . The human estrogen receptor has two independent nonacidic transcriptional activation functions.  Cell. 1989;  59 477-87
  CrossrefPubMedSearch in Google Scholar

Dr. Oliver Zierau

Institut für Zoologie

Technische Universität Dresden

Zellescher Weg 20

01217 Dresden

Phone: +49-351-4633-7841

Fax: +49-351-4633-1923

Email: Oliver.Zierau@mailbox.tu-dresden.de

References

• 1 Knight D C, Eden J A. A review of the clinical effects of phytoestrogens.  Obstet Gynecol. 1996;  87 897-904
  PubMedSearch in Google Scholar
• 2 Krishnan V, Heath H, Bryant H U. Mechanism of action of estrogens and selective estrogen receptor modulators.  Vitam Horm. 2000;  60 123-47
  PubMedSearch in Google Scholar
• 3 O’Lone R, Frith M C, Karlsson E K, Hansen U. Genomic targets of nuclear estrogen receptors.  Mol Endocrinol. 2004;  18 1859-75
  CrossrefPubMedSearch in Google Scholar
• 4 McDonnell D P. The molecular determinants of estrogen receptor pharmacology.  Maturitas. 2004;  48 (Suppl 1) 7-12
  CrossrefPubMedSearch in Google Scholar
• 5 McKay L I, Cidlowski J A. Molecular control of immune/inflammatory responses: interactions between nuclear factor-kappa B and steroid receptor-signaling pathways.  Endocr Rev. 1999;  20 435-59
  CrossrefPubMedSearch in Google Scholar
• 6 Kurebayashi S, Miyashita Y, Hirose T, Kasayama S, Akira S, Kishimoto T. Characterization of mechanisms of interleukin-6 gene repression by estrogen receptor.  J Steroid Biochem Mol Biol. 1997;  60 11-7
  CrossrefPubMedSearch in Google Scholar
• 7 Cos P, De Bruyne T, Apers S, Vanden Berghe D, Pieters L, Vlietinck A J. Phytoestrogens: recent developments.  Planta Med. 2003;  69 589-99
  Thieme ConnectPubMedSearch in Google Scholar
• 8 Krebs E E, Ensrud K E, MacDonald R, Wilt T J. Phytoestrogens for treatment of menopausal symptoms: a systematic review.  Obstet Gynecol. 2004;  104 824-36
  CrossrefPubMedSearch in Google Scholar
• 9 Pons M, Gagne D, Nicolas J C, Mehtali M. A new cellular model of response to estrogens: a bioluminescent test to characterize (anti) estrogen molecules.  Biotechniques. 1990;  9 450-9
  PubMedSearch in Google Scholar
• 10 Demirpence E, Duchesne M J, Badia E, Gagne D, Pons M. MVLN cells: a bioluminescent MCE-7-derived cell line to study the modulation of estrogenic activity.  Steroid Biochem Mol Biol. 1993;  46 355-64
  PubMedSearch in Google Scholar
• 11 Pottratz S T, Bellido T, Mocharla H, Crabb D, Manolagas S C. 17beta-Estradiol inhibits expression of human interleukin-6 promoter-reporter constructs by a receptor-dependent mechanism.  J Clin Invest. 1994;  93 944-50
  PubMedSearch in Google Scholar
• 12 Tora L, White J, Brou C, Tasset D, Webster N, Scheer E. et al . The human estrogen receptor has two independent nonacidic transcriptional activation functions.  Cell. 1989;  59 477-87
  CrossrefPubMedSearch in Google Scholar

Dr. Oliver Zierau

Institut für Zoologie

Technische Universität Dresden

Zellescher Weg 20

01217 Dresden

Phone: +49-351-4633-7841

Fax: +49-351-4633-1923

Email: Oliver.Zierau@mailbox.tu-dresden.de