β-catenin-responsive transcription (CRT)

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].

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].

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]

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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Dissolved in 5% DMSO in saline; 5 and 10 mg/kg; i.p.

C57BL/6 mice

[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/aims: Neurotensin (NTS), an intestinal hormone, is profoundly implicated in cancer progression through binding its primary receptor NTSR1. The conserved Wnt/β-Catenin pathway regulates cell proliferation and differentiation via activation of the β-catenin/T-cell factor (TCF) complex and subsequent modulation of a set of target genes. In this study, we aimed to uncover the potential connection between NTS/NTSR1 signaling and Wnt/β-Catenin pathway. Methods: Genetic silencing, pharmacological inhibition and gain-of-function studies as well as bioinformatic analysis were performed to uncover the link between NTS/ NTSR1 signaling and Wnt/β-Catenin pathway. Two inhibitors were used in vivo to evaluate the efficiency of targeting NTS/NTSR1 signaling or Wnt/β-Catenin pathway. Results: We found that NTS/NTSR1 induced the activation of mitogen-activated protein kinase (MAPK) and the NF-κB pathway, which further promoted the expression of Wnt proteins, including Wnt1, Wnt3a and Wnt5a. Meanwhile, the mRNA and protein expression levels of NTSR1 were increased by the Wnt pathway activator Wnt3a and decreased by the Wnt inhibitor iCRT3 in glioblastoma cells. Furthermore, pharmacological inhibition of NTS/NTSR1 or Wnt/β-Catenin signaling suppressed tumor growth in vitro and in vivo. Conclusion: These results reveal a positive feedback loop between NTS/NTSR1 and Wnt/β-Catenin signaling in glioblastoma cells that might be important for tumor development and provide potential therapeutic targets for 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 a core pluripotency transcriptional network (PTN) comprising Nanog, Oct4, and Sox2. The Wnt/β-catenin pathway promotes pluripotency by alleviating T cell factor TCF3-mediated repression of the PTN. However, it has remained unclear how β-catenin's function as a transcriptional activator with TCF1 influences mESC fate. Here, we show that TCF1-mediated transcription is up-regulated in differentiating mESCs and that chemical inhibition of β-catenin/TCF1 interaction improves long-term self-renewal and enhances functional pluripotency. Genetic loss of TCF1 inhibited differentiation by delaying exit from pluripotency and conferred a transcriptional profile strikingly reminiscent of self-renewing mESCs with high Nanog expression. Together, our data suggest that β-catenin's function in regulating mESCs is highly context specific and that its interaction with TCF1 promotes differentiation, further highlighting the need for understanding how its individual protein-protein interactions drive stem cell fate. [2]

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The Wnt/β-catenin pathway has been involved in regulating inflammation in various infectious and inflammatory diseases. Sepsis is a life-threatening condition caused by dysregulated inflammatory response to infection with no effective therapy available. Recently elevated Wnt/β-catenin signaling has been detected in sepsis. However, its contribution to sepsis-associated inflammatory response remains to be explored. In this study, we show that inhibition of Wnt/β-catenin signaling reduces inflammation and mitigates sepsis-induced organ injury. Using in vitro LPS-stimulated RAW264.7 macrophages, we demonstrate that a small-molecule inhibitor of β-catenin responsive transcription, iCRT3, significantly reduces the LPS-induced Wnt/β-catenin activity and also inhibits TNF-α production and IκB degradation in a dose-dependent manner. Intraperitoneal administration of iCRT3 to C57BL/6 mice, subjected to cecal ligation and puncture-induced sepsis, decreases the plasma levels of proinflammatory cytokines and organ injury markers in a dose-dependent manner. The histological integrity of the lungs is improved with iCRT3 treatment, along with reduced lung collagen deposition and apoptosis. In addition, iCRT3 treatment also decreases the expression of the cytokines, neutrophil chemoattractants, as well as the MPO activity 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 treatment of 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.)