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. 2016 May;7(5-6):209-17.
doi: 10.18632/genesandcancer.107.

Melatonin decreases estrogen receptor binding to estrogen response elements sites on the OCT4 gene in human breast cancer stem cells

Juliana Lopes  1 , David Arnosti  2 , James E Trosko  3 , Mei-Hui Tai  3 , Debora Zuccari  4
Affiliations

Melatonin decreases estrogen receptor binding to estrogen response elements sites on the OCT4 gene in human breast cancer stem cells

Juliana Lopes et al. Genes Cancer. 2016 May.

Abstract

Cancer stem cells (CSCs) pose a challenge in cancer treatment, as these cells can drive tumor growth and are resistant to chemotherapy. Melatonin exerts its oncostatic effects through the estrogen receptor (ER) pathway in cancer cells, however its action in CSCs is unclear. Here, we evaluated the effect of melatonin on the regulation of the transcription factor OCT4 (Octamer Binding 4) by estrogen receptor alpha (ERα) in breast cancer stem cells (BCSCs). The cells were grown as a cell suspension or as anchorage independent growth, for the mammospheres growth, representing the CSCs population and treated with 10 nM estrogen (E2) or 10 μM of the environmental estrogen Bisphenol A (BPA) and 1 mM of melatonin. At the end, the cell growth as well as OCT4 and ERα expression and the binding activity of ERα to the OCT4 was assessed. The increase in number and size of mammospheres induced by E2 or BPA was reduced by melatonin treatment. Furthermore, binding of the ERα to OCT4 was reduced, accompanied by a reduction of OCT4 and ERα expression. Thus, melatonin treatment is effective against proliferation of BCSCs in vitro and impacts the ER pathway, demonstrating its potential therapeutic use in breast cancer.

Keywords: chromatin immunoprecipitation; estrogen receptor; mammospheres; melatonin; three-dimensional growth.

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Conflict of interest statement

CONFLICTS OF INTEREST

None declared.

Figures

Figure 1
Figure 1. MCF-7 cells grown in 3-dimensional method of mammospheres, treated with E2 or BPA with or without melatonin
(A) Cell suspension treated for 6 days. (B) Anchorage Independent Growth (AIG) treated for 14 days. The magnification was 40 X.
Figure 2
Figure 2. Effect of E2 or BPA with or without melatonin on MCF-7 mammospheres
(A) Number of mammospheres. (B) Size of mammospheres. Significant value in ANOVA followed by Bonferroni's test (*P ≤ 0.05).
Figure 3
Figure 3. Chromatin immunoprecipitation to verify the binding activity of ER to the putative ERE sequences in OCT4 transcription site (OCT4 -1999 and OCT4 -3544) after treatment with E2 or BPA with or without melatonin in mammospheres
Significant value in ANOVA followed by Bonferroni's test (*P ≤ 0.05).
Figure 4
Figure 4. Analysis of OCT4 and ER gene expression after treatment with melatonin, E2 and BPA
(A) OCT4 gene expression. (B) ER gene expression. The gene expression values were log10 represented. Significant value in ANOVA followed by Bonferroni's test (*P ≤ 0.05).
Figure 5
Figure 5. Analysis of OCT4 and ER protein expression after treatment with melatonin, E2 and BPA
(A) OCT4 protein expression. (B) ER protein expression. Protein expression values were represented in relative levels. OCT4 and ER protein levels were normalized to β-actin protein, shown in the boxes. Significant value in ANOVA followed by Bonferroni's test (*P ≤ 0.05).
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