Clomiphene citrate down-regulates estrogen receptor-α through the ubiquitin-proteasome pathway in a human endometrial cancer cell line

We examined how clomiphene citrate (CC) reduces estrogen receptor-alpha (ER alpha) in a human endometrial cancer cell line. Ishikawa human endometrial cancer cells were treated with ER alpha. ligands such as 17 beta-estradiol (E2), CC, and the pure antiestrogen, ICI 182,780 (ICI). Thereafter, the expression levels of ER alpha protein and mRNA were analyzed by western blot and real-time quantitative PCR, respectively, and those of ubiquitinated ER alpha were analyzed by immunoprecipitation of ER alpha followed by immunoblotting with an anti-ubiquitin antibody. The expression levels of ER alpha protein after treatment with E2, CC, and ICI were significantly decreased compared to pre-treatment levels without a corresponding decrease in ER alpha mRNA. These ligands significantly increased the levels of ubiquitinated ER alpha compared to vehicle treatment. Co-treatment with the proteasome inhibitor, MG132, abrogated the decrease in ER alpha levels caused by treatment with the ligands only. We demonstrated, for the first time, a CC-induced decrease in ER alpha mediated by the ubiquitin-proteasome pathway in human endometrial cancer cells. (C) 2016 Elsevier Ireland Ltd. All rights reserved.