Abstract
Several lines of evidence attest to the existence of alternative ligand binding sites on the oestrogen receptor (ER), including non-competitive inhibition by trilostane or tamoxifen. It is possible that the inhibitory action of conventional oestrogen agonists at high concentrations may indicate that they too interact at alternative ER sites, albeit at low affinity. To test this possibility an oestrogen reporter assay was used to compare the activity of different oestrogens and antagonists in breast cancer and prostate cell lines. All four cell lines tested contained different amounts of oestrogen receptor α (ERα), ERβ, progesterone receptor and coregulator mRNA. Though differences were observed in response to stimulation and inhibition, these correlated only with the presence or absence of ERα, and not with the other components. Thus stimulation of the reporter by oestradiol and oestrone was biphasic in the breast cancer cells, while prostate cells were unable to respond. Only T47D cells were stimulated by oestriol or diethylstilboestrol, however reporter activity of all the cell lines was repressed by 10μM diethylstilboestrol. Reporter activity of MCF-7 cells was inhibited by tamoxifen, raloxifene and ICI 182,780, but stimulated by trilostane, yet all these antioestrogens inhibited agonist-stimulated activity. Trilostane also inhibited the agonism seen in cells co-treated with E2 and tamoxifen. It is clear that several of the compounds tested may have either agonist or antagonist effects under different conditions and at different concentrations, acting through ERα alone. Though biphasic dose response curves, or hormesis, have been attributed to various mechanisms, we here provide evidence that alternative ligand binding sites may contribute to this phenomenon.
Highlights
The steroid hormone oestradiol (E2) and the nuclear oestrogen receptors (ERα and ERβ) are involved in the regulation of many biological processes [1]
In agreement with [42], the present results show that ligand-binding affinity does not necessarily predict gene transactivation and that reporter activity is regulated in a cell-specific manner
The results presented here may be explained by the presence of a second binding site because: The dose response curves to oestrogens were biphasic such that stimulation of the reporter became auto-inhibitory at high concentrations (Figure 2), consistent with theoretical considerations (Figure 6)
Summary
The steroid hormone oestradiol (E2) and the nuclear oestrogen receptors (ERα and ERβ) are involved in the regulation of many biological processes [1]. The polynuclear hydrocarbon tetrahydrochrysene (THC) and derivatives stimulated gene transcription by ERα in endometrial cancer cells, but depending on size and position of substituents, were antagonistic to ERβ [32,33], THC is known to bind to the ligand binding domain [34,35,36]. To test the plausibility of these modes of action, the activities of various oestrogens were examined in ER positive breast and prostate cell lines, and the actions of trilostane and other SERMs were compared using an ERE-regulated reporter system In this way subtle differences in the interactions between oestrogens and antioestrogens could be determined and compared to cellular levels of ERα, ERβ and PR and to variations in coregulator proteins
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