β-catenin-regulated transcription (CRT)

iCRT-5and PNU-75654 had no detrimental effects on the immuno-phenotype of stimulated DCs. Hence, DCs treated with iCRT5 in the course of stimulation exerted comparably strong T cell proliferation as did control DCs. In contrast, DCs stimulated in the presence of PNU-75654 induced less T cell proliferation than the control population despite enhanced uptake and processing of OVA. Our findings suggest that the differential effects of β-catenin inhibitors on stimulated DCs reflect off target effects. Concerning potential application of β-catenin inhibitors for tumor therapy, iCRT-5 may be most beneficial, since it did not exert detrimental effects on stimulated DCs.[1]

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Researchers demonstrated that iCRTs (such as iCRT5) block Wnt/β-cat reporter activity, down regulate β-cat expression and inhibit cell proliferation in a dose-dependent manner with an optimal dose closer to 15 μM. Our data further indicate that iCRTs do not influence the expression of the upstream components of the Wnt pathway DKK1 at the optimal dose, suggesting that iCRTs may specifically target β-cat in MM cells. Additionally, iCRT-treatment of MM cells, co-cultured with BMSC, showed an inhibitory effect on VEGF and cell migration. Conclusion: This study provides the first in vitro data evaluation of newly-described iCRTs as potential Wnt-β-cat/VEGF pathway antagonists in multiple myeloma.[2]

VEGF analysis by Enzyme-linked Immunosorbent Assay (ELISA)[2]
MM cells (U266) were grown as described in the earlier section. The primary BMSCs used in this study were obtained from patients and cultured in Iscove’s modified Dulbecco’s medium containing 20% FBS, 2 mM L-glutamine and 5mg/ml penicillin/streptomycin. Cell culture medium collected from U266 cells and co-cultured with adherent BMSCs (grown in 6 well plates) and treated with iCRT-5 were used for VEGF analysis. ELISA assays were performed using Human VEGF Quantikine ELISA Kits by following the manufacturer’s protocol. These assays employ the quantitative sandwich enzyme immunoassay technique. The resultant color was read at 450 nm using an ELISA plate reader Max-M2-. The concentrations of VEGF in the samples were determined by interpolation from a standard curve made out of the standard provided by the manufacturer. The experiments were performed in triplicate, repeated at least twice.

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RNA isolation and quantitative real-time PCR[2]
Total RNA was extracted from the MM cells treated with iCRT-5 (15 µM) using Trizol reagent as described earlier (20, ). A two-step RT-PCR was carried out with total RNA (5 µg) extracted from U266 and BMSC treated with 50 µM iCRT-5 was used for initial denaturing for 2 minutes at 95°C and continued the amplification with an extension at 72°C, 7 minutes for 33 cycles using VEGF gene-specific primer sequences upper 5’atttacaacgtctgc gcatctt 3’ lower, 5’ctcgccttgctgctctacctc3’ along with the amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), upper 5’ ggatgaccttgcccacagcct 3’ lower, 5’catctctg ccccctctgctga 3’ as the internal control (IDT). Real-time quantitative PCR was performed in triplicate with a Smart Cycler using SYBR-Green mix as described earlier by us. Results were normalized to amplification of GAPDH and to determine the fold change based on 2ΔΔCt.

Cell proliferation analysis[2]
Actively growing cells were plated in triplicate in 96-well plates at a density of 5×105 per well with 100 µl medium containing 5–50 µM iCRT-3 or iCRT-5, and cells treated with 1% DMSO served as the control. After 48h of treatment, cell proliferation analysis was performed using MTS kit. Absorbance was read after at 490nm using a 96 well plate reader. Cells pre-treated with LiCl (20 µM) a GSK3β inhibitor and served as the control for Wnt activation. Cell growth was calculated from the mean relative decrease or increase in the optical density at 490 nm compared to the DMSO treated cells; Inhibition of cell proliferation was calculated based on the mean values of three repeated assays.

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Transmigration migration assay[2]
Rate of migration of MM cells was assessed using a 24-well BD FluoroBlok Transwell Inserts with 8 µM pore size. Briefly, MM cells (50,000) pretreated with the iCRTs-3 and iCRT-5 (15 µM) were seeded (in 200 µl) into the inserts with RPMI medium containing 0.25% serum. The bottom well contained RPMI with 10% FBS. After 48 h of incubation the bottom well was filled with 500 µl of Calcein fluorescent dye prepared according to the directions of the manufacturer. Calcein AM is the most suitable indicator for staining viable cells due to its low cytotoxicity property. The fluorescence intensity emitted by the migrated cells was measured at 540 nm using a plate reader Max-M2-. The experiments were repeated three times.

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Western blot analysis[2]
Total protein lysate (30 µg/lane) of MM cells treated without or with iCRT-5 (15 µM) for 48h was prepared in RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate and 0.1% SDS) containing protease inhibitor cocktail as described earlier. Immunobloting was done by standard SDS-PAGE (12%) using antibodies against β-catenin and DKK1. In addition, protein samples from HEK 293 cells were used to confirm β-cat in nonmyeloma cells. Reactive protein bands for β-cat were developed using an enhanced ECL chemiluminescence detection kit. All the blots were stripped and re-probed with α-tubulin to normalize protein loading. Each experiment was repeated three times using same sets of samples. Quantification of reactive protein bands were performed by densitometric analysis and the fold change was calculated by normalizing with α-tubulin.

[1]. Inhibitors of β-catenin affect the immuno-phenotype and functions of dendritic cells in an inhibitor-specific manner. Int Immunopharmacol. 2016 Mar;32:118-124.

[2]. Antagonistic effect of small-molecule inhibitors of Wnt/β-catenin in multiple myeloma. Anticancer Res. 2012 Nov;32(11):4697-707.

Many tumors are characterized by constitutive activation induced by mutations in β-catenin, thereby promoting tumor growth and survival. Therefore, the development of specific β-catenin inhibitors for tumor therapy has become a key focus of drug development. β-catenin has also been shown to contribute to the immune tolerance-promoting function of unstimulated dendritic cells (DCs). Upon activation, DCs acquire potent T-cell stimulation and induce strong tumor antigen-specific immune responses. This study investigated the effects of preclinically established β-catenin inhibitors (CCT-031374, iCRT-5, and PNU-75654) on mouse bone marrow-derived (BM) DCs. All three inhibitors moderately increased the expression of MHCII, CD80, and CD86 on the surface of unstimulated DCs, but did not enhance their ability to stimulate ovalbumin (OVA)-specific CD4+ T cell proliferation. CCT-031374 interfered with the upregulation of co-stimulatory molecules (CD40, CD86) and cytokines (IL-1β, TNF-α, IL-6, IL-10, IL-12) in LPS-stimulated dendritic cells (DCs). Consequently, the CD4+ T cell stimulatory activity of this DC population was impaired. iCRT-5 and PNU-75654 had no adverse effects on the immunophenotype of stimulated DCs. Therefore, DCs treated with iCRT-5 during stimulation exhibited comparable T cell proliferation capacity to control DCs. Conversely, although PNU-75654 enhanced OVA uptake and processing, the T cell proliferation capacity induced by DCs stimulated in the presence of PNU-75654 was lower than that in the control group. Our results suggest that the different effects of β-catenin inhibitors on stimulated DCs reflect off-target effects. Regarding the potential application of β-catenin inhibitors in tumor therapy, iCRT-5 may be the most advantageous because it does not have detrimental effects on stimulated dendritic cells (DCs). [1]

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Background: The development and progression of multiple myeloma depend on the bone marrow (BM) microenvironment, which secretes a variety of factors, including Wnt ligands. Wnt ligands secreted by bone marrow stromal cells (BMSCs) can activate the Wnt signaling pathway in multiple myeloma. The classical Wnt pathway is mediated by the transcriptional effector β-catenin (β-cat) and is dysregulated in a variety of cancers. Cells with active β-cat-regulated transcription (CRT) are protected from apoptosis; conversely, inhibition of CRT may prevent cell proliferation. Materials and Methods: This study tested the selective antagonism of the Wnt-β-cat signaling pathway in recently reported CRT inhibitors (iCRTs; oxazoles and thiazoles) in MM cell lines MM.1, U266, bone marrow mesenchymal stem cells (BMSCs), and primary bone marrow mononuclear cells (BMMCs) isolated from patient samples (n=16). Results: We confirmed that the iCRTs used could block the activity of Wnt/β-cat reporter genes, downregulate β-cat expression, and inhibit cell proliferation in a dose-dependent manner, with the optimal dose being close to 15 μM. Our data further showed that at the optimal dose, iCRTs did not affect the expression of DKK1, an upstream component of the Wnt pathway, suggesting that iCRTs may specifically target β-cat in MM cells. In addition, iCRT treatment of MM cells co-cultured with BMSCs showed inhibitory effects on vascular endothelial growth factor (VEGF) and cell migration. Conclusion: This study provides the first in vitro data evaluation of the newly discovered iCRT as a potential Wnt-β-cat/VEGF pathway antagonist in multiple myeloma. [2]

C16H17NO5S2

367.43

367.055

C, 52.30; H, 4.66; N, 3.81; O, 21.77; S, 17.45

18623-44-4

1416325

Light yellow to yellow solid powder

2.707

1

7

7

24

537

0

COC1=C(C=C(C=C1)/C=C\2/C(=O)N(C(=S)S2)CCCC(=O)O)OC

IJWKSBPTJQMUHJ-LCYFTJDESA-N

InChI=1S/C16H17NO5S2/c1-21-11-6-5-10(8-12(11)22-2)9-13-15(20)17(16(23)24-13)7-3-4-14(18)19/h5-6,8-9H,3-4,7H2,1-2H3,(H,18,19)/b13-9-

4-[(5Z)-5-[(3,4-dimethoxyphenyl)methylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]butanoic acid

iCRT 5; iCRT-5; iCRT5; 18623-44-4; iCRT-5; 4-[5-(3,4-Dimethoxy-benzylidene)-4-oxo-2-thioxo-thiazolidin-3-yl]-butyric acid; 4-(5-(3,4-Dimethoxybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)butanoic acid; 4-[(5Z)-5-[(3,4-dimethoxyphenyl)methylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]butanoic acid; (Z)-4-(5-(3,4-dimethoxybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)butanoic acid; CRT Inhibitor iCRT5; iCRT5

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 : ~36 mg/mL (~97.98 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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