20a

Amino Acid Sequence

3D Structure (/structures/AF-P03372-F1-model_v6.cif)

Function

Nuclear hormone receptor. The steroid hormones and their receptors are involved in the regulation of eukaryotic gene expression and affect cellular proliferation and differentiation in target tissues. Ligand-dependent nuclear transactivation involves either direct homodimer binding to a palindromic estrogen response element (ERE) sequence or association with other DNA-binding transcription factors, such as AP-1/c-Jun, c-Fos, ATF-2, Sp1 and Sp3, to mediate ERE-independent signaling. Ligand binding induces a conformational change allowing subsequent or combinatorial association with multiprotein coactivator complexes through LXXLL motifs of their respective components. Mutual transrepression occurs between the estrogen receptor (ER) and NF-kappa-B in a cell-type specific manner. Decreases NF-kappa-B DNA-binding activity and inhibits NF-kappa-B-mediated transcription from the IL6 promoter and displace RELA/p65 and associated coregulators from the promoter. Recruited to the NF-kappa-B response element of the CCL2 and IL8 promoters and can displace CREBBP. Present with NF-kappa-B components RELA/p65 and NFKB1/p50 on ERE sequences. Can also act synergistically with NF-kappa-B to activate transcription involving respective recruitment adjacent response elements; the function involves CREBBP. Can activate the transcriptional activity of TFF1. Also mediates membrane-initiated estrogen signaling involving various kinase cascades. Essential for MTA1-mediated transcriptional regulation of BRCA1 and BCAS3. Maintains neuronal survival in response to ischemic reperfusion injury when in the presence of circulating estradiol (17-beta-estradiol/E2).

Mechanism

The ubiquitin-proteasome pathway (UPP) constitutes the principal pathway for protein degradation in eukaryotic cells, mediating the breakdown of target proteins through ubiquitin tagging and subsequent proteasomal degradation. In an ATP-dependent manner, the ubiquitin-activating enzyme (E1) activates ubiquitin (Ub) and conjugates it to a ubiquitin-conjugating enzyme (E2). E3 ligase subsequently catalyzes the transfer of Ub to the target protein, leading to the formation of a polyubiquitin chain that completes the ubiquitination mark. The 19S regulatory particle of the 26S proteasome recognizes this ubiquitination signal and, after deubiquitination, mediates the ATP-dependent delivery of the substrate into the 20S proteasome for degradation.

Description

To further investigate how 20a degrades ERα in LCC2 cells, we performed rescue experiments using proteasome inhibitor MG132 and autophagy inhibitor CQ, respectively. MG132 reversed 20a-mediated ERα degradation, whereas CQ did not, indicating 20a degrades ERα via the UPS pathway (Fig. 5.E). In the UPS pathway, target proteins are ubiquitinated and degraded by the proteasome. Immunoprecipitation (IP) assays showed that with MG132 inhibiting proteasomal degradation, 20a significantly increased ubiquitination of both total proteins and ERα, confirming it promotes ubiquitin-dependent ERα degradation (Fig. 5.F)

Description

To explore its mechanism, we first investigated 20a's ability to degrade ERα and the underlying molecular mechanism. Treating MCF-7 and T47D cells with escalating concentrations of 20a or for varying durations revealed it reduced ERα protein levels in a dose-dependent and time-dependent manner (Fig. 3.A, B) (A) Western blot detected downregulated ERα levels in MCF-7 (upper) and T47D (lower) after 24-h treatment with varying 20a doses β-actin as loading control, while fulvestrant (5 μM) as positive control.

Description

To explore its mechanism, we first investigated 20a's ability to degrade ERα and the underlying molecular mechanism. Treating MCF-7 and T47D cells with escalating concentrations of 20a or for varying durations revealed it reduced ERα protein levels in a dose-dependent and time-dependent manner (Fig. 3.A, B) (A) Western blot detected downregulated ERα levels in MCF-7 (upper) and T47D (lower) after 24-h treatment with varying 20a doses β-actin as loading control, while fulvestrant (5 μM) as positive control.

Description

Cell viability assays showed 20a significantly inhibited LCC2 cell proliferation with an IC50 value of 7.727 μM, while fulvestrant exhibited a weaker effect, and LCC2 cells were far less sensitive to tamoxifen, with an IC50 exceeding fourfold that of 20a (Fig. 4.A).

Description

Subsequently, using OBHSA, 4-hydroxytamoxifen (4-OHT), and fulvestrant (Ful) as positive controls, we evaluated the anti-proliferative activity and cytotoxicity of compounds 20a-d (linker length n = 1, 3, 5, 7) against ERα-positive BC cell lines via the CCK8 assay, with results summarized in Table 1. The data demonstrated that 20a-d exhibited potent inhibitory effects on MCF-7, T47D, and the tamoxifen-resistant LCC2 cell line.

Description

Subsequently, using OBHSA, 4-hydroxytamoxifen (4-OHT), and fulvestrant (Ful) as positive controls, we evaluated the anti-proliferative activity and cytotoxicity of compounds 20a-d (linker length n = 1, 3, 5, 7) against ERα-positive BC cell lines via the CCK8 assay, with results summarized in Table 1. The data demonstrated that 20a-d exhibited potent inhibitory effects on MCF-7, T47D, and the tamoxifen-resistant LCC2 cell line.

Description

Subsequently, using OBHSA, 4-hydroxytamoxifen (4-OHT), and fulvestrant (Ful) as positive controls, we evaluated the anti-proliferative activity and cytotoxicity of compounds 20a-d (linker length n = 1, 3, 5, 7) against ERα-positive BC cell lines via the CCK8 assay, with results summarized in Table 1. The data demonstrated that 20a-d exhibited potent inhibitory effects on MCF-7, T47D, and the tamoxifen-resistant LCC2 cell line.