Estrogen Receptor Beta (ERβ) - selective agonist [2]

Prinaberel (ERB-041) (0-60 µM; 24 hours) treatment of human SCC cells results in reduced colony formation, cell differentiation, and cell cycle arrest [2]. In A431 cells, prinaberel significantly decreased the expression of inflammatory regulatory proteins as p-NFκBp65, iNOS, and COX-2. Phosphorylated PI3K and AKT are decreased by prinaberel, which is linked to increased E-cadherin expression and decreased A431 cell motility [2]. Cell growth is inhibited by prinaberel (0.01-10 µM) in a dose- and time-dependent manner [3]. Prinaberel (10 µM; 48 hours) induces ovarian cancer (SKOV-3 cells) to undergo apoptosis [3].

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In A431 and SCC13 human squamous cell carcinoma cells, Erb-041 treatment (0, 20, 40, 60 μM for 24h) induced G1 phase cell cycle arrest, which was associated with reduced expression of cyclin D1, D2, D3 and CDK4 [2].
Erb-041 treatment dramatically reduced the number and size of colonies formed by A431 and SCC13 cells in a colony formation assay [2].
In A431 and SCC13 cells, Erb-041 treatment reduced cell migration in a wound healing assay, with approximately 55% inhibition in A431 cells and 71% inhibition in SCC13 cells [2].
Erb-041 treatment induced the expression of cytokeratin 10, a differentiation marker, in HaCaT, A431, and SCC13 cells [2].
In HaCaT, A431, and SCC13 cells, Erb-041 treatment enhanced the expression of ERβ at the protein level [2].
In A431 cells, Erb-041 treatment reduced the expression of p-c-Jun and SP-1, which are associated with increased ERβ expression [2].
In A431 cells, Erb-041 treatment reduced the expression of pro-inflammatory proteins including p-NFκBp65, iNOS, and COX-2 [2].
In A431 cells, Erb-041 treatment diminished the phosphorylation of PI3K and AKT, while enhancing the expression of E-cadherin [2].
Erb-041 treatment reduced the expression of WNT signaling pathway components, including WNT7b, β-catenin, and p-GSK3β, and diminished nuclear localization of β-catenin in A431 cells [2].
Erb-041 treatment reduced the expression of WNT target proteins c-Myc and cyclin D1 in A431 cells [2].

In SKH-1 hairless mice, the topical drug prinaberel (2 mg/mouse; 30 minutes prior to UVB irradiation for 30 weeks) prevents the growth of squamous cell carcinoma [2]. Prinaberel causes apoptosis and inhibits UVB-induced proliferation and angiogenesis in skin cancers. In skin cancers caused by UVB radiation, prinaberel suppresses pro-inflammatory signaling pathways. Plinabiber inhibits WNT signaling and the PI3K-AKT pathway to reduce tumor invasiveness [2].

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In SKH-1 hairless mice subjected to UVB-induced photocarcinogenesis (180 mJ/cm², twice weekly for 30 weeks), topical treatment with Erb-041 (2 mg/mouse in 200 μl ethanol, applied 30 min prior to UVB) significantly reduced tumor development. Tumor incidence was reduced to 75% compared to 100% in UVB-alone group [2].
Erb-041 treatment reduced the number of tumors per mouse by >60% (3.3 ± 0.62 vs. 8.95 ± 0.94 in UVB-alone group) and reduced tumor volume by approximately 84% [2].
The latency period for tumor induction was increased from 17 weeks to 21 weeks by Erb-041 treatment [2].
Erb-041 treatment reduced the number of squamous cell carcinomas per mouse by 86% [2].
Histological analysis showed that tumors from Erb-041-treated animals were less invasive and better differentiated, with fewer poorly differentiated SCCs compared to the UVB-alone group [2].
Erb-041 treatment reduced the expression of proliferation markers (PCNA, cyclin D1, Ki67) and angiogenesis markers (CD31, VEGF) in UVB-induced skin tumors, as assessed by immunohistochemistry and western blot [2].
Erb-041 treatment increased apoptosis in UVB-induced tumors, as evidenced by increased TUNEL-positive cells and an increased Bax/Bcl-2 ratio [2].
Erb-041 treatment restored or enhanced ERβ expression in UVB-induced murine SCCs at both protein and mRNA levels [2].
Erb-041 treatment reduced UVB-induced inflammatory responses, including decreased epidermal hyperplasia, dermal leukocyte infiltration, myeloperoxidase activity, and reduced levels of pro-inflammatory cytokines (IL1β, IL6, IL10) [2].
Erb-041 treatment decreased the number of tumor-associated inflammatory cells, including GR-1+/CD11b+ myeloid cells and F4/80+ macrophages [2].
Erb-041 treatment reduced the phosphorylation of ERK1/2, p38 MAPK, and IκBα, and decreased the expression of iNOS and COX-2, as well as nuclear p-NFκBp65 in UVB-induced tumors [2].
Erb-041 treatment reduced epithelial-mesenchymal transition in UVB-induced tumors, as evidenced by increased E-cadherin expression and decreased expression of N-cadherin, Snail, Slug, Twist, MMP-2, and MMP-9 [2].
Erb-041 treatment reduced the phosphorylation of PI3K and AKT in UVB-induced tumors [2].
Erb-041 treatment down-regulated the WNT/β-catenin signaling pathway in UVB-induced tumors, reducing the expression of WNT3a, WNT7b, FZD1, and β-catenin, and diminishing nuclear localization of β-catenin [2].

Western Blot Analysis[2]
Cell Types: A431 Cell
Tested Concentrations: 0, 20, 40 and 60 µM
Incubation Duration: 24 hrs (hours)
Experimental Results: diminished expression of G1 cyclins (D1, D2 and D3) and CDK4.

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Cell proliferation assay[3]
Cell Types: SKOV-3, A2780CP or OVCAR-3 Cell
Tested Concentrations: 0.01, 0.1 and 10 µM
Incubation Duration: 24-48 hrs (hours)
Experimental Results: Significant inhibitory effect on cell proliferation.

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Cell Cycle Analysis by Flow Cytometry: A431 and SCC13 cells were treated with or without Erb-041 for 0, 24, 36, and 48 hours. Cells were trypsinized, washed, and fixed in ice-cold 70% ethanol overnight at -20°C. Fixed cells were washed and incubated with RNase A and propidium iodide in PBS for 30 minutes at room temperature. Cell cycle distribution was analyzed using a flow cytometer, and the percentage of cells in G1, S, and G2/M phases was determined [2].
Colony Formation Assay: A431 and SCC13 cells were seeded into 6-well plates at 500 cells per well and allowed to grow overnight. Cells were treated with or without Erb-041 for 24 hours and then incubated for an additional 10 days at 37°C in a humidified chamber. Colonies were fixed with 4% paraformaldehyde for 5 minutes, stained with 0.5% crystal violet for 30 seconds, and counted [2].
Wound Healing Assay: A431 and SCC13 cells were grown to 90-100% confluence in culture dishes. A scratch was made using a sterile pipette tip. Cells were then treated with or without Erb-041 and incubated at 37°C. Cell motility was observed at 12 and 24 hours using a microscope, and images were captured to assess wound closure [2].
Immunocytostaining: HaCaT, A431, and SCC13 cells were grown on round glass coverslips in 24-well plates and treated with or without Erb-041. Cells were fixed with 4% paraformaldehyde, permeabilized, and blocked. They were then incubated with primary antibodies (e.g., anti-ERβ, anti-cytokeratin 10), followed by appropriate fluorescent secondary antibodies. Coverslips were mounted with DAPI and observed under a fluorescence microscope [2].
Western Blot Analysis: Cells or tissue samples were lysed in ice-cold lysis buffer containing protease and phosphatase inhibitors. Proteins (60-80 μg) were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were probed with specific primary antibodies (e.g., anti-PCNA, anti-cyclin D1, anti-ERβ, anti-E-cadherin, anti-N-cadherin, anti-Snail, anti-Twist, anti-p-PI3K, anti-p-AKT, anti-WNT7b, anti-β-catenin, anti-p-NFκBp65, anti-COX-2, etc.) and appropriate secondary antibodies. Protein bands were visualized and quantified. β-actin was used as a loading control [2].
RT-PCR Analysis: Total RNA was extracted from cells or tissues. cDNA was synthesized, and PCR was performed using specific primers for genes of interest (e.g., ERβ, IL1β, IL6, IL10). PCR products were analyzed by gel electrophoresis [2].
Real-Time PCR Analysis: Quantitative real-time PCR was performed to assess the expression levels of cytokines (IL1β, IL6, IL10). Relative quantification of target mRNA levels was calculated after normalization to GAPDH [2].

Animal/Disease Models: Six to eight week old SKH-1 hairless female mice [2]
Doses: 2 mg/mouse in 200 µl ethanol UVB (180mJ/cm2) 30 minutes before irradiation for 30 weeks
Experimental Results: SKH -1 diminished UVB-induced skin tumor development in hairless mice.

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Photocarcinogenesis Study:** Six- to eight-week-old female SKH-1 hairless mice were randomly divided into three groups (n=20 per group). Group I (negative control) received topical ethanol. Groups II and III were irradiated with UVB (180 mJ/cm², twice weekly) for 30 weeks. Group II received topical vehicle (ethanol), while Group III received topical Erb-041 (2 mg/mouse in 200 μl ethanol) 30 minutes prior to each UVB irradiation. Tumor number and size were recorded weekly using an electronic Vernier caliper. After 30 weeks, mice were euthanized, and skin and tumor tissues were harvested for histological and biochemical analyses [2].
* **Histology and Immunohistochemistry:** Formalin-fixed tissues were embedded in paraffin and sectioned at 5 μm. For histological evaluation, sections were stained with hematoxylin and eosin (H&E). For immunohistochemistry, sections were incubated with primary antibodies (e.g., anti-PCNA, anti-cyclin D1, anti-Ki67, anti-CD31, anti-VEGF, anti-ERβ) followed by appropriate detection systems and chromogens. Stained sections were observed under a microscope [2].
* **Immunofluorescence Staining:** Tissue sections or cells were incubated with primary antibodies (e.g., anti-GR-1, anti-CD11b, anti-F4/80, anti-E-cadherin, anti-N-cadherin, anti-Snail, anti-Slug, anti-Twist, anti-WNT7b, anti-β-catenin, anti-p-NFκBp65, anti-COX-2) followed by fluorescently labeled secondary antibodies. Slides were mounted with DAPI and observed under a fluorescence microscope [2].
* **TUNEL Assay:** Apoptosis in tumor tissues was detected using an in situ cell death detection kit (fluorescein) according to the manufacturer's guidelines. Paraffin-embedded tissue sections were processed and incubated with the TUNEL reaction mixture. TUNEL-positive cells were visualized under a fluorescence microscope [2].
* **Myeloperoxidase Activity Assay:** Skin samples were homogenized, and MPO activity was determined by measuring the change in absorbance at 460 nm. Data were expressed as mean MPO units per mg protein per minute [2].

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Photocarcinogenesis Study: Six- to eight-week-old female SKH-1 hairless mice were randomly divided into three groups (n=20 per group). Group I (negative control) received topical ethanol. Groups II and III were irradiated with UVB (180 mJ/cm², twice weekly) for 30 weeks. Group II received topical vehicle (ethanol), while Group III received topical Erb-041 (2 mg/mouse in 200 μl ethanol) 30 minutes prior to each UVB irradiation. Tumor number and size were recorded weekly using an electronic Vernier caliper. After 30 weeks, mice were euthanized, and skin and tumor tissues were harvested for histological and biochemical analyses [2].
Histology and Immunohistochemistry: Formalin-fixed tissues were embedded in paraffin and sectioned at 5 μm. For histological evaluation, sections were stained with hematoxylin and eosin (H&E). For immunohistochemistry, sections were incubated with primary antibodies (e.g., anti-PCNA, anti-cyclin D1, anti-Ki67, anti-CD31, anti-VEGF, anti-ERβ) followed by appropriate detection systems and chromogens. Stained sections were observed under a microscope [2].
Immunofluorescence Staining: Tissue sections or cells were incubated with primary antibodies (e.g., anti-GR-1, anti-CD11b, anti-F4/80, anti-E-cadherin, anti-N-cadherin, anti-Snail, anti-Slug, anti-Twist, anti-WNT7b, anti-β-catenin, anti-p-NFκBp65, anti-COX-2) followed by fluorescently labeled secondary antibodies. Slides were mounted with DAPI and observed under a fluorescence microscope [2].
TUNEL Assay: Apoptosis in tumor tissues was detected using an in situ cell death detection kit (fluorescein) according to the manufacturer's guidelines. Paraffin-embedded tissue sections were processed and incubated with the TUNEL reaction mixture. TUNEL-positive cells were visualized under a fluorescence microscope [2].
Myeloperoxidase Activity Assay: Skin samples were homogenized, and MPO activity was determined by measuring the change in absorbance at 460 nm. Data were expressed as mean MPO units per mg protein per minute [2].

[1]. Design and synthesis of aryl diphenolic azoles as potent and selective estrogen receptor-beta ligands. J Med Chem. 2004;47(21):5021-5040.

[2]. Erb-041, an estrogen receptor-β agonist, inhibits skin photocarcinogenesis in SKH-1 hairless mice by downregulating the WNT signaling pathway. Cancer Prev Res (Phila). 2014;7(2):186-198.

[3]. Estrogen receptor modulators genistein, daidzein and ERB-041 inhibit cell migration, invasion, proliferation and sphere formation via modulation of FAK and PI3K/AKT signaling in ovarian cancer. Cancer Cell Int. 2018;18:65. Published 2018.

Prinaberel is an estrogen receptor beta agonist.

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Erb-041 (C₁₅H₁₀FNO₃) is a selective estrogen receptor beta (ERβ) agonist with reported strong anti-inflammatory activity [2].
At the time of this publication, Erb-041 was under clinical trial for its potential use in rheumatoid arthritis [2].
This study demonstrates that Erb-041 is a potent skin cancer chemopreventive agent in UVB-induced photocarcinogenesis in SKH-1 hairless mice [2].
The chemopreventive mechanism of Erb-041 involves down-regulation of the WNT/β-catenin signaling pathway, which underlies the pathogenesis of skin cancer [2].
Erb-041 treatment restored or enhanced ERβ expression in murine SCCs and human SCC cells, whereas ERβ expression is lost during skin cancer pathogenesis [2].
The anti-inflammatory effects of Erb-041 include reduced cytokine production (IL1β, IL6, IL10), decreased infiltration of inflammatory cells (GR-1+/CD11b+ myeloid cells, F4/80+ macrophages, neutrophils), and inhibition of NFκB signaling [2].
Erb-041 treatment reduced epithelial-mesenchymal transition and tumor invasiveness, resulting in better-differentiated, less aggressive tumors [2].
The effects of Erb-041 on EMT and inflammation were similar to those observed with the WNT signaling inhibitor XAV939, suggesting that WNT pathway inhibition is a key mechanism of action [2].

C15H10FNO3

271.24

271.064

524684-52-4

524684-52-4;

656954

White to gray solid powder

1.4±0.1 g/cm3

451.6±45.0 °C at 760 mmHg

250-252ºC

226.9±28.7 °C

0.0±1.1 mmHg at 25°C

1.695

3.97

2

5

2

20

367

0

MQIMZDXIAHJKQP-UHFFFAOYSA-N

InChI=1S/C15H10FNO3/c1-2-8-5-10(18)7-12-14(8)20-15(17-12)9-3-4-13(19)11(16)6-9/h2-7,18-19H,1H2

2-(3-fluoro-4-hydroxyphenyl)-7-vinylbenzo[d]oxazol-5-ol

ERB041 ERB 041 ERB041 Prinaberel WAY-202041 WAY202041 WAY 202041

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 40 mg/mL (~147.47 mM)

Solubility in Formulation 1: ≥ 5 mg/mL (18.43 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 5 mg/mL (18.43 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 5 mg/mL (18.43 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)