Elsevier

Maturitas

Volume 54, Issue 1, 20 April 2006, Pages 72-77
Maturitas

Comparison of possible carcinogenic estradiol metabolites: Effects on proliferation, apoptosis and metastasis of human breast cancer cells

https://doi.org/10.1016/j.maturitas.2005.08.010Get rights and content

Abstract

Objectives

Certain estradiol metabolites may play a pivotal role in breast carcinogenesis. Of special interest are the metabolites 2-hydroxyestradiol (2-OHE2), which can react anti-carcinogenically, and 4-hydroxyestradiol (4-OHE1) and 16a-hydroxyestrone (16-OHE1), which may have procarcinogenic potential. In the present study, we have compared for the first time the effect of these metabolites and their parent substance 17β-estradiol (E2) on proliferation, apoptosis, apoptosis markers and markers of metastatic property of human breast cancer cells.

Methods

MCF-7 cells (human estrogen-receptor positive metastatic breast cancer cell line) were incubated with the estrogens at concentrations of 0.1–100 nM. Cell proliferation rate was measured by the ATP-assay. Apoptosis was measured by cell death assay and the apoptosis markers cytochrome C, Bcl-2, Fasl and p53 were determined in cell lysates by ELISAs. The markers of metastatic property of the cell line, VEGF and MCP-1 were measured in the cell supernatant by ELISAs.

Results

The estrogens E2, 4-OHE2 and 16-OHE1 display a proliferative effect on MCF-7 cells which is accompanied by a down-regulation of apoptosis. Various markers of apoptosis such as Bcl-2, cytochrome C and p53 appear to be involved. No significant effect was found for the metabolite 2-OHE2. VEGF and MCP-1 were up-regulated by E2 and 16-OHE1, whereas 2-OHE2 and 4-OHE2 did not show any effect.

Conclusions

The most potent estrogen regarding proliferation, apoptosis and metastasis of breast cancer cells seems to be estradiol. However, the estradiol metabolites 4-OHE2 and 16-OHE1 elicit similar properties on cell proliferation, apoptosis and metastasis as compared to estradiol but only at higher concentrations. In contrast 2-OHE2 did not show any significant effect on these parameters. Thus, intracellular estradiol metabolism may determine an individual's risk for breast carcinogenesis.

Introduction

Estrogens are mainly responsible for the proliferation of human breast epithelial cells. A possible association between estrogens and breast carcinogenesis is given by the increased mitotic rate which may accelerate the possibility of genomic mutations and thus may influence breast cancer risk. In vivo, 17β-estradiol, the most potent human estrogen, is mainly metabolized by hydroxylation at the A- or the D-ring [1]. Evidence is accumulating that certain estradiol metabolites may play a more important role in enhancing breast cancer risk than their parent substance, 17β-estradiol [2]. In this context the 4-hydroxylated and 16a-hydroxylated estradiol metabolites are of special interest. In the present experimental work, we have directly compared the effects of the main estradiol metabolites on proliferation, apoptosis and markers of metastasis of human breast cancer cells.

Section snippets

Material and methods

17β-Estradiol (E2), 2-hydroxyestradiol (2-OHE2), 4-hydroxyestradiol (4-OHE2) and 16a-hydroxyestrone (16-OHE1) were purchased from Steraloids, USA. The test substances were dissolved in ethanol and diluted with an ethanol/buffer mixture to the appropriate test concentrations.

MCF-7, a human estrogen-receptor positive breast cancer cell line, was used for the experiments. The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 5% (v/v) fetal calf serum supplemented with

Results

The results of the proliferation of MCF-7 cells are depicted in Fig. 1. E2 elicited a significant increase in cell number over the entire concentration range tested. The highest proliferation rate was induced at the concentrations 0.1 and 1 nM with about a 40% increase as compared to the control value. The metabolite 2-OHE2 did not show any significant increase over the concentration range. 4-OHE2 significantly increased cell proliferation at the concentrations of 1 and 10 nM with values of about

Discussion

Two main hypotheses are currently discussed concerning estradiol metabolites and breast carcinogenesis. The first one favours the idea that overproduction of 16a-hydroxyestrone as compared to 2-hydroxyestrone may be responsible for increased breast carcinogenesis. This metabolite has been shown to have a potent estrogenic activity by binding irreversibly to the estrogen-receptor [4]. Thus, 16-OHE1 may activate estrogen-receptor mediated oncogene expression and growth stimulation for a prolonged

References (25)

  • F.J. Geske et al.

    The biology of apoptosis

    Hum Pathol

    (2001)
  • G. Mor et al.

    Regulation of Fas ligand expression in breast cancer cells by estrogen: functional differences between estradiol and tamoxifen

    J Steroid Biochem Mol Biology

    (2000)
  • S. Ikegawa et al.

    Concentration of 16α-hydroxyestrone in human plasma as measured by a specific RIA

    J Steroid Biochem

    (1983)
  • T.H. Lippert et al.

    Metabolism of endogenous estrogens

  • B.T. Zhu et al.

    Functional role of estrogen metabolism in target cells: review and perspectives

    Carcinogenesis

    (1998)
  • P.E. Andreotti et al.

    ATP tumor chemosensitivity assay

  • G.E. Swaneck et al.

    Covalent binding of the endogenous estrogen 16α-hydroxyestrone to estradiol receptor in human breast cancer cells: characterization and intranuclear localization

    Proc Natl Acad Sci USA

    (1988)
  • H.L. Bradlow et al.

    Estradiol 16α-hydroxylation in the mouse correlates with tumor incidence and presence of murine mammary tumor virus: a possible model for the hormonal etiology of breast cancer in humans

    Proc Natl Acad Sci USA

    (1985)
  • N.T. Telang et al.

    In vitro biotransformation of estradiol by explant cultures of murine mammary tissues

    Breast Cancer Res Treat

    (1989)
  • J.D. Yager et al.

    Molecular mechanisms of estrogen carcinogenesis

    Annu Rev Pharmacol Toxicol

    (1996)
  • J.G. Liehr et al.

    4-Hydroxylation of estrogens as marker of human mammary tumors

    Proc Natl Acad Sci USA

    (1996)
  • E. Cavalieri et al.

    Estrogens as endogenous genotoxic agents—DNA adducts and mutations

    J Natl Cancer Inst Monogr

    (2000)
  • Cited by (74)

    • CYP1B1 prevents proteasome-mediated XIAP degradation by inducing PKCε activation and phosphorylation of XIAP

      2019, Biochimica et Biophysica Acta - Molecular Cell Research
      Citation Excerpt :

      Several studies have shown that 4-OHE2 is a potential carcinogen for animals and humans [9–11]. Mueck et al. [12] showed that 4-OHE2 induces Bcl-2 and reduces cytochrome c levels in MCF-7 cells. However, 2-OHE2 was not found to have any significant effects.

    • Association of Catechol-O-methyltransferase polymorphism Val158Met and mammographic density: A meta-analysis

      2017, Gene
      Citation Excerpt :

      This may suggest that estrogen receptor-mediated regulation affects MD. Catechol-O-methyltransferase (COMT) catalyzes the methylation of catechol estrogens (Dawling et al., 2001) that have been reported to be genotoxic (Zahid et al., 2006) and to enhance cell proliferation (Seeger et al., 2006). A product of COMT, 2-methoxyestradiol, has been suggested to exert anti-tumorigenic activity (Zhu and Conney, 1998).

    View all citing articles on Scopus
    View full text