β-catenin-responsive transcription (CRT)
The target of iCRT3 is the β-catenin/T-cell factor (TCF) complex, specifically inhibiting the interaction between β-catenin and TCF1; [2]
The target of iCRT3 is the Wnt/β-catenin signaling pathway, acting as a small-molecule inhibitor of β-catenin-responsive transcription; [3]
The target of iCRT3 is the Wnt/β-catenin signaling pathway; [1]

iCRT3 inhibits transcription that is sensitive to both Wnt and β-catenin. Both TOP Flash activity and NTSR1 level are markedly lowered by iCRT3. iCRT3 can significantly counteract the anti-apoptotic effects of neurotensin (NTS) and Wnt3a[1]. Long-term iCRT3-maintained cells exhibit increased expression of classic pluripotency compared to the DMSO control, but concurrently decreased expression of differentiation markers and T-cell factor (TCF) target genes[2]. iCRT3 treatment reduces TNF-α levels by 14.7%, 18.5%, 44.9%, and 61.3% at dosages of 12.5, 25, 50, and 75 μM, respectively. IκB levels rise dose-dependently with iCRT3 therapy in contrast to the vehicle[3].

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1. Inhibition of Wnt/β-catenin signaling in glioblastoma cells: Pharmacological inhibition of iCRT3 on the Wnt/β-catenin pathway reduced the mRNA and protein expression levels of NTSR1 in glioblastoma cells [1]
2. Inhibition of β-catenin/TCF1 interaction in mouse embryonic stem cells (mESCs): iCRT3 specifically inhibited the interaction between β-catenin and TCF1 without affecting the binding of β-catenin to TCF3. This inhibition reduced the transcriptional activity of TCF-dependent target genes (e.g., Cdx1, Axin2) and delayed mESC differentiation. Treatment with iCRT3 for 14 days enhanced the self-renewal ability of mESCs, maintaining higher Nanog and Rex1 expression, and reducing spontaneous differentiation. In RA-induced differentiation models, iCRT3 enabled mESCs to resist differentiation, increasing the proportion of Nanog-GFP-positive cells and enhancing colony-forming efficiency. Additionally, iCRT3 increased the formation of the β-catenin/Oct4 complex, which is associated with ground-state pluripotency [2]
3. Regulation of inflammatory responses in LPS-stimulated RAW264.7 macrophages: iCRT3 pre-treated (for 50 min) RAW264.7 macrophages at different concentrations, followed by LPS (1 ng/ml) stimulation. It dose-dependently inhibited LPS-induced Wnt/β-catenin pathway activation (measured by TOP-TK-Luc reporter activity) and TNF-α production (detected by ELISA). iCRT3 also dose-dependently suppressed LPS-induced IκB degradation (analyzed by Western blotting) without affecting cell viability (determined by MTS assay, with viability maintained at 100% compared to untreated cells) [3]

Treatment with iCRT3 significantly reduces tumor growth rates. The tumor-suppressive function of iCRT3 is consistently correlated with a decrease in the proliferation marker Ki67 index[1]. Compared to the vehicle group, the IL-6 levels in the 10 mg/kg iCRT3 therapy group are 82.9% lower. In the sham, IL-1β levels are not detectable; however, in septic mice, they reach 371 pg/mL and decrease by 30.2% and 53.2%, respectively, when given 5 and 10 mg/kg iCRT3. The AST levels of these septic mice treated with iCRT3 at doses of 5 and 10 mg/kg are, respectively, 15.4% and 44.2% lower than those of the animals treated with vehicle. In comparison to the vehicle group, lung morphology improves with much less microscopic degradation following therapy with 10 mg/kg iCRT3. When comparing the lung tissues of the iCRT3-treated animals to the vehicle group, there are 92.7% fewer apoptotic cells[3].

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1. Inhibition of tumor growth in glioblastoma models: Pharmacological inhibition of the Wnt/β-catenin signaling pathway by iCRT3 suppressed tumor growth in glioblastoma in vivo [1]
2. Mitigation of sepsis-induced inflammatory responses and organ injury in C57BL/6 mice: Male C57BL/6 mice were subjected to cecal ligation and puncture (CLP)-induced sepsis, followed by intraperitoneal injection of iCRT3 (5 mg/kg or 10 mg/kg body weight) at 5 h post-CLP. At 20 h post-CLP, iCRT3 dose-dependently reduced plasma levels of proinflammatory cytokines (IL-6, TNF-α, IL-1β) and organ injury markers (AST, ALT, LDH). Histological analysis showed that iCRT3 (10 mg/kg) improved lung tissue integrity, reduced lung injury scores, decreased lung collagen deposition (stained by Masson’s Trichrome), and inhibited lung cell apoptosis (detected by TUNEL staining). Additionally, iCRT3 downregulated the mRNA expression of IL-6, TNF-α, IL-1β, neutrophil chemoattractants (MIP-2, KC) in lung tissues, and reduced lung myeloperoxidase (MPO) activity, thereby alleviating neutrophil infiltration [3]

β-catenin-TCF reporter activity assay[3]
RAW264.7 cells were seeded the day before transfection at a density of 1.24 × 105 cells per ml. Cells were transiently co-transfected with 250 ng of TOP-TK-Luc or FOP-TK-Luc and 25 ng pRL-TK reporter plasmids, using the Lipofectamine 3000 Reagent according to the manufacturer’s instructions. At 24 h after transfection, cells were pre-treated with iCRT3 or vehicle for 50 min and then stimulated with LPS (1 ng/ml) for another 24 h. The cells were lysed 48 h post-transfection and luciferase activity was measured with a Dual-Luciferase reporter assay system according to the manufacturer’s instructions. TOP-TK-Luc contains optimal and FOP-TK-Luc contains mutated TCF-binding sites placed upstream of a firefly luciferase reporter gene. The TOP and FOP firefly luciferase activity was normalized to Renilla luciferase activity from the cotransfected pRL-TK plasmid used as an internal control for transfection efficiency. All experiments were performed in triplicate at least twice.

Luciferase reporter assay[1]
Cells were plated at 4 × 105 cells/wells in 24-well plates and transiently transfected with the TopFlash (0.5 µg) and the Renilla reporter (0.05 µg) using Lipofectamine 2000. The NTS, Wnt3a, SR48692 and iCRT3 treatment were added to A172 or U87 cells for 24 h after plating. The cells were harvested and luciferase activity was measured two days after transfection. The luciferase activity was measured by using the Dual Luciferase Reporter Assay System.

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Cell proliferation and cell apoptosis assay[1]
Cells were seeded into 96-well plates to a density of 5 × 103 cells per well and incubated in the culture medium with indicated treatment for an additional 48 h. Cell viability and cell apoptosis assays were carried out using a Cell Counting kit-8 and a Caspase-Glo 3/7 assay kit according to the manufacturer’s instructions, respectively.

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For long-term cultures, cells were plated in limiting dilutions in 6- or 96-well plates for multiple passages (14 d) in stem conditions (serum plus LIF) with DMSO or iCRT3, with daily media changes. AP staining was performed for every passage to monitor the relative pluripotency levels. Small molecules used include 10 µM iCRT3 and 1 µM XAV939, which were diluted with DMSO. L-Wnt3a and control L cells were gifts from R.T. Moon. [2]

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1. Glioblastoma cell experiment: Glioblastoma cells were treated with iCRT3 (concentration not specified) to inhibit the Wnt/β-catenin pathway. After treatment, total RNA and protein were extracted from the cells. qPCR was used to detect the mRNA expression level of NTSR1, and Western blotting was used to analyze the protein expression level of NTSR1 [1]
2. Mouse embryonic stem cell (mESC) experiments:
- β-catenin/TCF transcriptional activity assay: mESCs (NG4-TOPluc) were transfected with TOPFlash reporter plasmid and internal control pRL-TK plasmid. After transfection, cells were treated with iCRT3 (concentration not specified) and cultured in serum plus LIF (S+LIF) or serum plus RA (S+RA) medium. Luciferase activity was measured to evaluate the inhibitory effect on β-catenin/TCF-dependent transcriptional activity [2]
- Flow cytometry analysis: TNGA (Nanog reporter) and Rex1-GFP mESCs were treated with iCRT3 for 14 days in S+LIF medium. Flow cytometry was used to detect the expression levels of Nanog-GFP and Rex1-GFP to assess self-renewal ability. For differentiation resistance experiments, mESCs were treated with iCRT3 for 6 days without LIF or for 48 h with RA, and flow cytometry was used to analyze the proportion of Nanog-GFP-positive cells [2]
- qPCR analysis: mESCs treated with iCRT3 for four passages or during RA-induced differentiation were collected. Total RNA was extracted and reverse-transcribed into cDNA. qPCR was performed to detect the mRNA expression levels of pluripotency markers (Nanog, Oct4, Sox2), differentiation markers, and TCF target genes (Cdx1, Axin2) with actin as an internal reference [2]
- Colony-forming efficiency (CFE) assay: mESCs were pre-cultured with RA plus iCRT3 or DMSO for 48 h, then plated in limiting dilutions in S+LIF medium without inhibitors and cultured for another 48 h. The number of colonies, colony area, and alkaline phosphatase (AP) activity were quantified to evaluate colony-forming efficiency [2]
- Co-immunoprecipitation (CoIP) assay: Total cell lysates of mESCs treated with iCRT3 during RA-induced differentiation were prepared. Antibodies against β-catenin, TCF1, and TCF3 were used for CoIP experiments to detect the interaction between β-catenin and TCF1/TCF3. Western blotting was used for detection and densitometric analysis [2]
3. RAW264.7 macrophage experiments:
- Reporter gene assay: RAW264.7 cells were co-transfected with β-catenin/TCF response reporter TOP-TK-Luc (or control FOP-TK-Luc) and internal control pRL-TK. After transfection, cells were pre-treated with iCRT3 at different concentrations for 50 min, then stimulated with LPS (1 ng/ml) for 24 h. Luciferase activity was measured to assess Wnt/β-catenin pathway activation [3]
- TNF-α ELISA assay: RAW264.7 cells were pre-treated with iCRT3 at different concentrations for 50 min, then stimulated with LPS (1 ng/ml) for 4 h. Cell supernatants were collected, and TNF-α levels were measured by ELISA [3]
- MTS cell viability assay: RAW264.7 cells were treated with iCRT3 at different concentrations, and cell viability was detected by MTS assay, with the viability of untreated cells set as 100% [3]
- Western blotting for IκB: RAW264.7 cells were pre-treated with iCRT3 at different concentrations for 50 min, then stimulated with LPS (1 ng/ml) for 15 min. Total cell lysates were prepared, and Western blotting was performed using antibodies against IκB and actin to detect IκB degradation [3]

Dissolved in 5% DMSO in saline; 5 and 10 mg/kg; i.p.
C57BL/6 mice A172 cells were used to establish a subcutaneous xenograft and to determine the anti-tumor effects of SR48692 and iCRT3. NOD-SCID BALB/c mice were inoculated subcutaneously in the right back with 2 × 106 A172 cells. The growth of the primary tumors was recorded every 4 days. SR48692 (10 mg/kg) and iCRT3 (5 mg/kg) was diluted in PBS i.p. triweekly when tumors grew to ∼200 mm3. The control mice were treated with blank PBS containing 5% (v/v) DMSO. Tumor volume was evaluated with the following formula: volume = tumor length × width2/2. The mice were sacrificed 24 days after pharmaceutical treatment. The tumors were resected and embedded in paraffin, and the Ki67 staining was analyzed by immunohistochemistry.[1]

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Mice were randomly allocated to three groups: sham (n = 5 mice), vehicle and treatment (n = 8 mice per group). iCRT3 was reconstituted with cell culture grade 100% DMSO as 50 mg/ml stock. 5 and 10 mg/kg body weight (BW) concentrations of iCRT3 were made by diluting stock in sterile normal saline with 5% DMSO. At 5 h after CLP, 5% DMSO in normal saline (vehicle) or iCRT3 at 5 or 10 mg/kg BW doses in 200 μl volume was delivered by intraperitoneal injection using 25 G × 7/8″ hypodermic needle. The investigator performing the animal experiments was blinded to the treatment assignment to eliminate any bias.[3]

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1. Glioblastoma in vivo experiment: Specific details such as animal model establishment (e.g., cell inoculation method, number of cells), iCRT3 dosage, administration route, and frequency were not explicitly stated. Pharmacological inhibition of the Wnt/β-catenin pathway by iCRT3 was used to observe the inhibitory effect on tumor growth [1]
2. Sepsis model experiment in C57BL/6 mice:
- Animal preparation: Male C57BL/6 mice were selected as experimental animals and randomly divided into sham-operated group, CLP + vehicle group, and CLP + iCRT3 treatment group (5 mg/kg and 10 mg/kg body weight, n = 5–8 mice per group) [3]
- Sepsis model establishment: Mice in the CLP groups were subjected to cecal ligation and puncture to induce sepsis, while the sham-operated group only underwent laparotomy without cecal ligation and puncture [3]
- Drug administration: At 5 h after CLP, mice in the treatment groups were intraperitoneally injected with iCRT3 dissolved in 5% DMSO in normal saline at the specified doses. Mice in the vehicle group were injected with the same volume of 5% DMSO in normal saline [3]
- Sample collection and detection: At 20 h after CLP, blood samples were collected from mice to detect plasma cytokine levels and organ injury markers. Lung tissues were harvested for histological analysis (H&E staining, Masson’s Trichrome staining), TUNEL staining, qPCR analysis of cytokine and chemokine mRNA expression, and MPO activity detection [3]

[1]. A Novel Positive Feedback Loop Between NTSR1 and Wnt/β-Catenin Contributes to Tumor Growth of Glioblastoma. Cell Physiol Biochem. 2017 Oct 24;43(5):2133-2142.

[2]. Inhibition of β-catenin-TCF1 interaction delays differentiation of mouse embryonic stem cells. J Cell Biol. 2015 Oct 12;211(1):39-51.

[3]. Mitigation of sepsis-induced inflammatory responses and organ injury through targeting Wnt/β-catenin signaling. doi: 10.1038/s41598-017-08711-6.

Background/Objective: Neurotensin (NTS) is an intestinal hormone that plays a crucial role in cancer progression by binding to its major receptor, NTSR1. The conserved Wnt/β-catenin signaling pathway regulates cell proliferation and differentiation by activating the β-catenin/T cytokine (TCF) complex and subsequently modulating a series of target genes. This study aimed to reveal the potential link between the NTS/NTSR1 and Wnt/β-catenin signaling pathways. Methods: We employed gene silencing, pharmacological inhibition, gain-of-function experiments, and bioinformatics analysis to elucidate the connection between the NTS/NTSR1 and Wnt/β-catenin signaling pathways. This study evaluated the efficacy of targeting the NTS/NTSR1 or Wnt/β-catenin pathways in vivo using two inhibitors. Results showed that NTS/NTSR1 induces activation of the mitogen-activated protein kinase (MAPK) and NF-κB pathways, thereby promoting the expression of Wnt proteins (including Wnt1, Wnt3a, and Wnt5a). Meanwhile, in glioblastoma cells, the Wnt pathway activator Wnt3a upregulated the mRNA and protein expression levels of NTSR1, while the Wnt inhibitor iCRT3 downregulated the mRNA and protein expression levels of NTSR1. In addition, pharmacological inhibition of the NTS/NTSR1 or Wnt/β-catenin signaling pathways inhibited tumor growth in vitro and in vivo. Conclusion: These results reveal a positive feedback loop between the NTS/NTSR1 and Wnt/β-catenin signaling pathways in glioblastoma cells, which may be crucial for tumor development and provide potential targets for the treatment of glioblastoma. [1]

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The ability of mouse embryonic stem cells (mESCs) to self-renew or differentiate into various cell lineages is regulated by signaling pathways and the core pluripotency transcriptional network (PTN) composed of Nanog, Oct4 and Sox2. The Wnt/β-catenin pathway promotes pluripotency by alleviating T cell cytokine TCF3-mediated PTN inhibition. However, it remains unclear how β-catenin, as a transcriptional activator, works in synergy with TCF1 to affect the fate of mESCs. This study shows that TCF1-mediated transcription is upregulated in differentiated mouse embryonic stem cells (mESCs), and chemical inhibition of the β-catenin/TCF1 interaction improves long-term self-renewal capacity and enhances functional pluripotency. TCF1 gene deletion inhibits differentiation by delaying pluripotency exit and confers a transcriptional profile in mESCs that is very similar to that of self-renewing mESCs with high Nanog expression. In summary, our data suggest that the function of β-catenin in regulating mESCs is highly context-specific and that its interaction with TCF1 promotes differentiation, which further highlights the need to understand how its single protein-protein interactions drive stem cell fate. [2] The Wnt/β-catenin pathway is involved in regulating inflammatory responses in a variety of infectious and inflammatory diseases. Sepsis is a life-threatening disease caused by dysregulation of the inflammatory response to infection, and there is currently no effective treatment. Recent studies have found that the activity of the Wnt/β-catenin signaling pathway is increased in sepsis. However, its mechanism of action in sepsis-related inflammatory responses remains to be explored. This study demonstrates that inhibiting the Wnt/β-catenin signaling pathway can alleviate inflammation and reduce sepsis-induced organ damage. Using LPS-stimulated RAW264.7 macrophages in vitro, we confirmed that the small molecule inhibitor iCRT3, a reactive β-catenin transcription inhibitor, significantly reduced LPS-induced Wnt/β-catenin activity and inhibited TNF-α production and IκB degradation in a dose-dependent manner. Intraperitoneal injection of iCRT3 into C57BL/6 mice with cecal ligation-induced sepsis dose-dependently reduced plasma levels of pro-inflammatory cytokines and organ damage markers. iCRT3 treatment improved lung tissue integrity and reduced pulmonary collagen deposition and apoptosis. Furthermore, iCRT3 treatment also reduced the expression of cytokines, neutrophil chemokines, and myeloperoxidase (MPO) in the lungs of septic mice. Based on these findings, we conclude that targeting the Wnt/β-catenin pathway may provide a potential therapeutic approach for sepsis. [3]

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1. Background: Neurotensin (NTS) binds to its major receptor NTSR1, promoting cancer progression. The Wnt/β-catenin pathway regulates cell proliferation and differentiation by activating the β-catenin/TCF complex. In glioblastoma cells, there is a positive feedback loop between the NTS/NTSR1 and Wnt/β-catenin signaling pathways, which is involved in tumorigenesis and development. [1]
2. Mechanism of action: iCRT3 inhibits the Wnt/β-catenin signaling pathway, thereby reducing NTSR1 expression and inhibiting glioblastoma growth. [1]
3. Background: The Wnt/β-catenin signaling pathway regulates the self-renewal and differentiation of mouse embryonic stem cells (mESCs). β-catenin can alleviate TCF3-mediated inhibition of the pluripotency transcription network (PTN), thereby promoting pluripotency; at the same time, the interaction between β-catenin and TCF1 can promote mESC differentiation[2]
4. Mechanism of action: iCRT3 specifically inhibits the interaction between β-catenin and TCF1, reducing the transcriptional activity of differentiation-promoting target genes. It enhances the self-renewal of mESCs, delays differentiation, and improves functional pluripotency, without affecting the interaction between β-catenin and TCF3[2]
5. Background: The Wnt/β-catenin signaling pathway is involved in regulating inflammatory responses in infectious and inflammatory diseases. Sepsis is caused by an inflammatory response disorder caused by infection. Activation of the Wnt/β-catenin signaling pathway has been detected in sepsis [3]
6. Mechanism of action: iCRT3 blocks the Wnt/β-catenin signaling pathway, thereby inhibiting the NF-κB pathway, reducing the production of pro-inflammatory cytokines and chemokines, alleviating pulmonary collagen deposition, apoptosis, and neutrophil infiltration, and ultimately reducing the inflammatory response and organ damage caused by sepsis [3]
7. Therapeutic potential: iCRT3 has potential therapeutic value in treating glioblastoma by targeting the Wnt/β-catenin pathway [1]
8. Therapeutic potential: iCRT3 provides a tool for studying the role of β-catenin/TCF1 interaction in the regulation of mESC fate and may have potential applications in stem cell research [2] 9. Therapeutic potential: Targeting the Wnt/β-catenin pathway with iCRT3 may be a potential treatment for sepsis [3]

C23H26N2O2S

394.53

394.171

C, 70.02; H, 6.64; N, 7.10; O, 8.11; S, 8.13

901751-47-1

6622273

White to off-white solid powder

5.195

1

4

9

28

462

0

QTDYVSIBWGVBKU-UHFFFAOYSA-N

InChI=1S/C23H26N2O2S/c1-3-18-9-11-20(12-10-18)23-25-21(17(2)27-23)15-28-16-22(26)24-14-13-19-7-5-4-6-8-19/h4-12H,3,13-16H2,1-2H3,(H,24,26)

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.34 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 25.0 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: ≥ 2.5 mg/mL (6.34 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 25.0 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.)