Specific Inhibition of Estrogen Receptor Alpha Function by Antisense Oligodeoxyribonucleotides

Abstract

We have tested the effect of a range of antisense oligodeoxyribonucleotides (ODN) directed against the human estrogen receptor alpha (ERa) on ERa protein expression and function. Antisense ERa ODN transfected into the ERa-positive human breast carcinoma cell line MCF7-K2 showed variable responses dependent on the oligo used. The most active antisense ODN (oligo 7) decreased the levels of ERa protein by 61% as measured by Western blot analysis. Exogenous 17b-estradiol (17b-E 2 ), but not 17a-E 2 , augmented this effect, with a threshold effect at 10 28 M 17b-E 2 . The inhibitory effect of antisense ERa oligo 7 was confirmed by measurement of functional ERa protein. 3H-17b-E 2 binding to MCF7 cell extracts was inhibited to approximately 40% of control values in the presence of oligo 7. Antisense-transfected MCF7-K2 cell cultures produced a further 30% binding reduction in the presence of exogenous 17b-E 2 . An inhibitory effect on 17b-E 2 -dependent cell function was confirmed by the demonstration that ERa oligo 7-transfected MCF7-K2 cells failed to exhibit 17b-E 2 -stimulated cell proliferation. Exogenous 17b-E 2 enhanced the inhibitory effect of the antisense ODN by increasing ODN transfection efficiency but without ERa catabolism via the proteosomal pathway, suggesting an effect of 17b-E 2 on the plasma membrane and the existence of different ERa degradation pathways in the MCF7-K2 cell subclone. As 17b-E 2 had no effect on ERa protein degradation, we conclude that the observed reduction of ERa protein levels is due solely to the presence of the antisense ERa ODN. Antisense ERa ODN molecules, therefore, may form the basis of effective therapies against ERa-dependent malignancies.

The Modulation of Aromatase and Estrogen Receptor Alpha in Cultured Human Dermal Papilla Cells by Dexamethasone: A Novel Mechanism for Selective Action of Estrogen via Estrogen Receptor Beta?

“…prostatic stroma without altering ERb levels (Prins et al, 1998) and in cancer cell lines 17b-estradiol causes a decrease in ERa protein expression (Read et al, 1989;Taylor et al, 2001). Likewise androgens have also been reported to alter the ratio of ERa to ERb in the mammary gland (Dimitrakakis et al, 2003).…”

The G Protein-coupled Receptor GPR30 Mediates c-fos Up-regulation by 17β-Estradiol and Phytoestrogens in Breast Cancer Cells

“…Antisense Oligodeoxynucleotide Experiments-Antisense oligonucleotides were synthesized as described (63)(64)(65) and purchased from MWG (Florence, Italy). The oligonucleotides used were: 5Ј-TTGGGAAGTCA-CATCCAT-3Ј for GPR30; and 5Ј-GATCTCAGCACGGCAAAT-3Ј for the scrambled control (66).…”

17β-Estradiol Enhances the Production of Nerve Growth Factor in THP-1-Derived Macrophages or Peripheral Blood Monocyte-Derived Macrophages

“…Treatment with anti-sense oligodeoxynucleotides Anti-sense oligonucleotides were synthesized as described (O'Dowd et al, 1998;Lau et al, 2000;Taylor et al, 2001). The oligonucleotides were ERa, 5 0 -GACCATGACCATGACCCT-3 0 ; ERb, 5 0 -CATCACAGCAGGGCT-ATA-3 0 ; GPR30, 5 0 -TTGGGAAGTCACATCCAT-3 0 ; random control, 5 0 -GATCTCAGCACGGCAAAT-3 0 .…”