The anti-estrogenic activity of indole-3-carbinol in neonatal rat osteoblasts is associated with the estrogen receptor antagonist 2-hydroxyestradiol

Abstract

Purpose

To gain new insight into the roles of cruciferous vegetable-derived bioactive phytochemicals in bone cells, we investigated the effects of indole-3-carbinol (I3C) on cell proliferation and differentiation in estradiol (E2)-exposed calvarial osteoblasts that were obtained from neonatal rats.

Methods

Osteoblast activity was assessed by analyzing cellular DNA, cell-associated osteocalcin (OC) levels and alkaline phosphatase (AP) activity. We also examined [3H]-estrone (E1) metabolism and estrogen-agonistic and estrogen-antagonistic activities of 2-hydroxy (OH) E1 and 2-OHE2 and their capacity to displace [3H]-E2 at ER binding sites using competition studies.

Results

I3C did not affect on cellular DNA, OC levels or AP activity. However, I3C completely inhibited E2-induced increases in cell proliferation and differentiation in neonatal rat osteoblasts. Metabolic studies demonstrated that I3C promoted the conversion of [3H]-E1 to 2-OHE1 and 2-OHE2 and those higher rates of conversion (twofold–threefold) were archived when a higher dose of I3C was applied. Proliferation and differentiation studies showed that 2-OHE2 but not 2-OHE1 inhibited E2-induced increases in cell proliferation and differentiation via an ER-mediated mechanism. Likewise, Esr1 was expressed at high level than Esr2. 2-OHE1 showed no activity or affinity for ER.

Conclusions

This study is the first to show that a bioactive compound derived from cruciferous vegetables, I3C, abolishes the E2-mediated stimulation of cell activities including, proliferation and differentiation, in rat osteoblasts and increases the 2-hydroxylation of E1, resulting in the formation of inactive and anti-estrogenic metabolites. These results suggest that in neonatal rat osteoblasts, the anti-estrogenic effect of I3C is mediated by 2-OHE2 through ER-α.

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Acknowledgments

This study was fully supported by the Department of Reproductive Biology, INCMNSZ. The authors thank Dr. Verónica Navarro for technical assistance.

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Correspondence to J. Enríquez.

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All authors declare that there are no actual or potential conflicts of interest, including financial, personal or other relationships, with other people or organizations that could inappropriately influence this work.

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All procedures were performed in accordance with the institutional guidelines, and approved by the animal research ethical committee at INCMNSZ.

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This article does not contain any studies with human participants performed by any of the authors.

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Enríquez, J., Velázquez-Cruz, R., Parra‐Torres, A. et al. The anti-estrogenic activity of indole-3-carbinol in neonatal rat osteoblasts is associated with the estrogen receptor antagonist 2-hydroxyestradiol. J Endocrinol Invest 39, 1149–1158 (2016). https://doi.org/10.1007/s40618-016-0494-9

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Keywords

  • Osteoblast 2-hydroxylation
  • Estrogen metabolites
  • Indole-3-carbinol
  • Osteoblasts differentiation
  • Osteoblasts proliferation