Synthesis and characterization of subtype-selective estrogen receptor ligands and their application as pharmacological tools - Cross-talk between estrogen and NPY Y1 receptors in human breast cancer cells

摘要

The estrogen receptor (ER), a member of the nuclear receptor family, is the classical target of hormonal treatment of breast cancer. Recently, also individual membrane bound receptors have gained interest in both, diagnosis and possibe treatment of mammary carcinoma. Especially neuropeptide Y (NPY) receptors, belonging to the superfamily of G-protein coupled receptors, have become a topic in breast cancer research, as the Y1 receptor (Y1R) subtype was found to be expressed by the majority (85 %) of human mammary carcinomas. This thesis aimed at the investigation of the functional expression and cross-talk between ERs and Y1Rs in human breast cancer cells.udSelective �pure antagonists� of the ER alpha (ERa) and beta (ERb) subtypes are considered useful pharmacological tools for the characterization of ER subtype specific cellular and physiological effects. 2-Phenylbenzofurans, in particular those bearing substituents in position 7 of the benzofuran core, are known as ERb subtype selective agonists. Aiming at ERb-selective �pure antagonists�, functionalized aliphatic side chains were introduced into position C7 of the benzofuran core within the scope of this thesis. A benzofuran, bearing a small 1-propenyl substituent in the C7 position, revealed high affinity to ERb (RBA = 34) and >30-fold selectivity over ERa. This compound was shown to be an ER agonist in the luciferase gene reporter assay. Benzofurans, substituted with long functionalized aliphatic side chains revealed strongly decreased receptor affinities and selectivities. In the luciferase assay, these compounds were either weak antagonists or inactive.udTo obtain ERa-selective antagonists, a library of 2-aryl-tetrahydroisoquinolin-6-ols (THIQs), substituted with different functionalized aliphatic side chains in position C1, was synthesized. ER affinities of synthesized THIQs strongly depended on the nature of the side chain, whereas a 3�-hydroxy function at the N-phenyl ring was favourable for ERa-subtype selectivity. THIQs, containing side chains bearing a tertiary amine group, revealed binding affinities to the ER in the same order of magnitude (RBA ≈ 10) as determined for the potent antiestrogens fulvestrant (ICI 182.780) and 4-hydroxytamoxifen, respectively. ERa-selectivities of 13- and 17-fold over ERb were observed for compounds with an amine and thioether containing chain or a side chain bearing a sulfoxy function in combination with a 3�-hydroxy group. Both compounds exerted full antagonism in the luciferase assay with IC50 values in the sub-micromolar range, becoming appropriate pharmacological tools for blocking ERa-subtype specific cellular effects. Furthermore, the THIQ containing a sulfoxide side chain, was a potent inhibitor of the proliferation of estrogen responsive MCF-7 breast cancer cells, suggesting further investigation of its value with respect to the hormonal therapy of breast cancer. udER and Y1R protein expression by different subclones of MCF-7 breast cancer cells showing differential sensitivities against antiestrogen treatment were quantified by radioligand binding assays. For this purpose, Y1Rs were labeled with the selective, high-affinity radioligand [3H]-UR-MK114, recently developed in our work group. Basal expression of Y1Rs by MCF-7 cells varied between 40,000 and 100,000 receptors per cell, and inversely correlated with ER expression in vitro. In agreement with published results at the mRNA level, the Y1R protein was up-regulated by 100 % after treatment of the cells with estradiol at physiological concentrations (EC50 = 20 pM).udThe potent, highly ERa-selective agonist 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole up-regulated the Y1R by 100 % with an EC50 value of 0.25 nM, indicating a predominant role of ERa in Y1R induction. The �pure ER antagonist� fulvestrant abrogated estradiol-induced Y1R expression in a concentration-dependent manner (IC50 = 5 nM) to 25 % of the basal level. Two THIQ-based moderately ERa-selective antagonists, synthesized within the scope of this thesis, down-regulated the Y1R expression to the same extent as fulvestrant. udFunctional coupling of the Y1R to both, mobilization of intracellular calcium and inhibition of adenylyl cyclase activity, was demonstrated in MCF-7 cells by triggering intracellular calcium transients and suppression of forskolin-stimulated cAMP synthesis with NPY, respectively. However, there was neither an effect of NPY treatment on the proliferation of MCF-7 cells nor on ER-mediated transcriptional activity. udAccording to the results of this thesis, the Y1R-mediated signaling cascade does not regulate downstream processes involved in tumor growth, which might be addressed in the therapy of breast cancer. Nonetheless, the Y1R is a useful endogenous gene reporter for the quantification of ER alpha specific (anti)estrogenic effects in whole-cell radioligand binding assays and a potential target for diagnostic imaging of breast cancer metastases.