β-catenin-responsive transcription (CRT; IC50 = 40.3 nM)
β-catenin/TCF4 transcription complex
iCRT14 is an inhibitor of catenin responsive transcription (CRT). It disrupts the interaction between β-catenin and TCF4, and may also interfere with TCF binding to DNA.[1]

除了影响 TCF-β-cat 反应外，iCRT 14 还能够阻碍 TCF 的 DNA 结合 [1]。尽管仍低于 iCRT 3，但 iCRT 14（10、25、50 μM）可有效且呈时间和剂量依赖性地减少 BT-549 细胞的生长 [2]。

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在果蝇Cl8细胞中，当通过dsRNA介导的Axin敲低激活Wnt信号时，iCRT14抑制Wnt响应性荧光素酶报告基因dTF12的活性。
在HEK293细胞中，iCRT14抑制Wnt响应性STF16荧光素酶报告基因的活性，其IC50在纳摩尔到微摩尔范围内（具体数值未提供）。
iCRT14在体外降低了纯化的重组His标记β-连环蛋白与GST标记的TCF4 N端结构域之间的相互作用。
在转染了抗降解β-连环蛋白（S37Aβ-cat）的HEK293细胞中，iCRT14处理显著抑制了免疫共沉淀实验中β-连环蛋白与TCF的相互作用。
热熔分析显示，iCRT14使β-连环蛋白-TCF4复合物的熔解温度改变高达2°C，表明其直接结合该复合物。
用50 µM iCRT14处理HCT116结肠癌细胞（携带β-连环蛋白S45缺失）导致Axin2和细胞周期蛋白D1 mRNA水平显著降低，效果与β-连环蛋白 siRNA相似。
Western印迹分析显示，经iCRT14处理的HCT116细胞中细胞周期蛋白D1蛋白水平相应降低。
流式细胞术分析显示，用50 µM iCRT14处理HCT116细胞导致细胞周期显著停滞在G0/G1期。
在用50 µM iCRT14处理的HT29结肠癌细胞（具有APC突变）中也观察到类似的G0/G1期阻滞。
这种细胞周期阻滞与HCT116细胞中磷酸化组蛋白H3阳性细胞数量和BrdU掺入的急剧减少相关。
在基质胶包被的Boyden小室实验中，iCRT14降低了稳定表达S37Aβ-cat-HA的MCF7乳腺癌细胞的侵袭能力。
iCRT14抑制了MCF7-S37Aβ-cat-HA细胞中膜性E-钙粘蛋白的下调。
在C57MG小鼠乳腺上皮细胞中，iCRT14抑制了Wnt3a诱导的形态学转化，并降低了β-连环蛋白靶基因WISP1的mRNA水平。[1]

在 HCT116 异种移植物中，iCRT 14（50 mg/kg，腹腔注射）显着降低 CycD1（肿瘤肿胀）[1]。

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在无胸腺裸鼠中建立的HCT116和HT29异种移植模型中，腹腔注射iCRT14（50 mg/kg，每周三次，持续3周）导致与DMSO对照组相比，肿瘤切片中细胞周期蛋白D1染色显著减少，磷酸化组蛋白H3阳性细胞数量减少。
这与治疗最初3周（约第19天）内肿瘤初始生长率降低约50%相吻合。第19天后，生长率与对照组相当。
通过微型泵给予较低浓度（20 mg/kg）的iCRT14在HCT116异种移植模型中产生了相同的效果。
在整个研究过程中，未观察到小鼠出现全身毒性迹象或体重减轻。[1]

热稳定性分析。[1]
在室温下，在不同浓度的iCRTs（如iCRT-14）和0.5×SYPRO Orange的存在下，将纯化的β-cat-His和TCF4-N-GST以1:2的摩尔比在1×PBS中混合。使用LightCycler 480系统在96孔板中以0.06°C/s的升温速率将样品从20°C加热到95°C。在加热阶段，以0.1s的间隔采集荧光读数。通过绘制荧光强度读数的负导数与温度的关系来计算熔化温度。负导数曲线上的拐点被认为是熔化温度（Tm）。

对于每种细胞系的细胞增殖测定，细胞用DMSO作为载体或不同浓度的每种Wnt抑制剂处理：iCRT-3（25、50、75μM）、iCRT-5（50、100、200μM），iCRT-14（10、25、50μM）；IWP-4（1、2.5、5μM）和XAV-939（5、10μM）。对于SOX4敲除的BT549细胞的细胞增殖、迁移和侵袭试验，用DMSO或25μM iCRT-3处理细胞。CIM板16的上腔涂有Matrigel（1:40稀释），用于细胞侵袭试验。此外，在实验时用50μM染料木素和25μM iCRT-3处理6天的SOX4敲除的BT-549细胞中测量了细胞增殖。每个样品都进行了三次分析，并进行了三个独立的实验。细胞增殖试验进行48小时，细胞迁移和侵袭实验进行24小时。RTCA软件包1.2计算了每个样本的细胞指数值，用于测量电阻抗的相对变化，以表示细胞形态、粘附或存活率。[2]

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将细胞以20000个细胞/孔的速度接种到96孔板中。孵育过夜后，用DMSO或每种Wnt抑制剂（iCRT-3，75μM；iCRT-5，200μM；iCRT-14，50μM；IWP-4，5μM和XAV-939，10μM）处理细胞48小时。根据制造商的说明，使用Cell Titer Glo发光细胞存活率测定试剂盒测定细胞存活率。使用FLUOstar微孔板读数器测量发光。所有治疗均进行三次，每次实验重复三次[2]。

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初筛（Wnt报告基因实验）： 将果蝇Cl8细胞转染Wnt响应性dTF12-荧光素酶报告基因和靶向Axin的dsRNA以激活信号通路。4天后，加入小分子化合物库中的化合物（终浓度9 ng/mL）。约16小时后，使用双荧光素酶系统测量荧光素酶活性。该实验的Z因子为0.77。
次级上位性分析： 将Cl8细胞转染靶向Slimb/βTrCP（位于Axin/APC/GSK-3β复合物下游）的dsRNA和dTF12报告基因。评估化合物对报告基因活性的影响。
β-连环蛋白-TCF相互作用实验（免疫共沉淀）： 将HEK293细胞转染S37Aβ-cat，并用iCRT14处理过夜。用抗β-连环蛋白抗体对全细胞裂解液进行免疫沉淀，沉淀物用内源性TCF4抗体进行免疫印迹分析。
体外蛋白质结合实验： 将纯化的重组His标记β-连环蛋白与不同浓度的iCRT14预孵育，评估其与纯化的GST标记TCF4 N端结构域的结合能力。
热熔分析： 将纯化的β-连环蛋白-His和TCF4-N-GST以1:2的摩尔比在PBS中与iCRT14和SYPRO Orange染料混合。样品在热循环仪中从20°C加热到95°C，升温速率为每秒0.06°C。监测荧光，并根据荧光曲线的负导数计算熔解温度。
定量RT-PCR（qRT-PCR）： 用iCRT14处理细胞指定时间（MCF7/HCT116细胞1天，C57MG细胞5天），裂解细胞并合成cDNA。使用SYBR Green化学法和特异性引物分析基因表达（如Axin2、Cyclin D1、WISP1、c-Myc、GAPDH）。
细胞周期分析（流式细胞术）： 固定处理后的细胞，用碘化丙啶染色，通过流式细胞术分析DNA含量和细胞周期分布。
侵袭实验（Boyden小室）： 将MCF7-S37Aβ-cat-HA细胞接种于基质胶包被的Transwell上室。下室含有含或不含iCRT14的培养基。孵育后，计数穿过基质胶迁移到膜下侧的细胞。
免疫荧光/免疫组织化学： 固定细胞或肿瘤切片，透化，封闭，用一抗（如抗E-钙粘蛋白、抗磷酸化组蛋白H3、抗细胞周期蛋白D1）染色，然后用荧光或酶标二抗染色并成像。[1]

Xenograft Model: HCT116 or HT29 colon cancer cells were implanted subcutaneously in athymic nude mice. When tumors reached 80-120 mm³, mice were treated with iCRT14 dissolved in DMSO.
Dosing: iCRT14 was administered via intraperitoneal injection at a dose of 50 mg/kg, three times a week for 3 weeks. In a separate experiment, a lower dose of 20 mg/kg was administered via minipump.
Control: Control animals received DMSO vehicle.
Monitoring: Tumor volume was measured periodically. Mice were monitored for signs of systemic toxicity and body weight changes.[1]

In the mouse xenograft study, administration of iCRT14 at 50 mg/kg (i.p., 3 times/week for 3 weeks) did not cause any signs of systemic toxicity or weight loss in the animals.[1]

[1]. An RNAi-based chemical genetic screen identifies three small-molecule inhibitors of the Wnt/wingless signaling pathway. Proc Natl Acad Sci USA. 2011 Apr 12;108(15):5954-63.

[2]. Wnt signaling blockage inhibits cell proliferation and migration, and induces apoptosis in triple-negative breast cancer cells. J Transl Med. 2013 Nov 4;11:280.

Misregulated β-catenin responsive transcription (CRT) has been implicated in the genesis of various malignancies, including colorectal carcinomas, and it is a key therapeutic target in combating various cancers. Despite significant effort, successful clinical implementation of CRT inhibitory therapeutics remains a challenging goal. This is, in part, because of the challenge of identifying inhibitory compounds that specifically modulate the nuclear transcriptional activity of β-catenin while not affecting its cytoskeletal function in stabilizing adherens junctions at the cell membrane. Here, we report an RNAi-based modifier screening strategy for the identification of CRT inhibitors. Our data provide support for the specificity of these inhibitory compounds in antagonizing the transcriptional function of nuclear β-catenin. We show that these inhibitors efficiently block Wnt/β-catenin-induced target genes and phenotypes in various mammalian and cancer cell lines. Importantly, these Wnt inhibitors are specifically cytotoxic to human colon tumor biopsy cultures as well as colon cancer cell lines that exhibit deregulated Wnt signaling.
[1]

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Background: Triple-negative breast cancer (TNBC) is an aggressive clinical subtype of breast cancer that is characterized by the lack of estrogen receptor (ER) and progesterone receptor (PR) expression as well as human epidermal growth factor receptor 2 (HER2) overexpression. The TNBC subtype constitutes approximately 10%-20% of all breast cancers, but has no effective molecular targeted therapies. Previous meta-analysis of gene expression profiles of 587 TNBC cases from 21 studies demonstrated high expression of Wnt signaling pathway-associated genes in basal-like 2 and mesenchymal subtypes of TNBC. In this study, we investigated the potential of Wnt pathway inhibitors in effective treatment of TNBC.
Methods: Activation of Wnt pathway was assessed in four TNBC cell lines (BT-549, MDA-MB-231, HCC-1143 and HCC-1937), and the ER+ cell line MCF-7 using confocal microscopy and Western blot analysis of pathway components. Effectiveness of five different Wnt pathway inhibitors (iCRT-3, iCRT-5, iCRT-14, IWP-4 and XAV-939) on cell proliferation and apoptosis were tested in vitro. The inhibitory effects of iCRT-3 on canonical Wnt signaling in TNBC was evaluated by quantitative real-time RT-PCR analysis of Axin2 and dual-luciferase reporter assays. The effects of shRNA knockdown of SOX4 in combination with iCRT-3 and/or genistein treatments on cell proliferation, migration and invasion on BT-549 cells were also evaluated.
Results: Immunofluorescence staining of β-catenin in TNBC cell lines showed both nuclear and cytoplasmic localization, indicating activation of Wnt pathway in TNBC cells. iCRT-3 was the most effective compound for inhibiting proliferation and antagonizing Wnt signaling in TNBC cells. In addition, treatment with iCRT-3 resulted in increased apoptosis in vitro. Knockdown of the Wnt pathway transcription factor, SOX4 in triple negative BT-549 cells resulted in decreased cell proliferation and migration, and combination treatment of iCRT-3 with SOX4 knockdown had a synergistic effect on inhibition of cell proliferation and induction of apoptosis.
Conclusions: These data suggest that targeting SOX4 and/or the Wnt pathway could have therapeutic benefit for TNBC patients.[2]

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iCRT14 is a thiazolidinedione-class small molecule identified from an RNAi-based chemical genetic screen as an inhibitor of catenin responsive transcription (CRT).
It functions by disrupting the β-catenin/TCF4 transcription complex and may also interfere with TCF binding to DNA.
It shows specific cytotoxicity towards cancer cell lines with constitutively active Wnt/β-catenin signaling (e.g., HCT116, HT29) but not towards normal cells or cell lines without such addiction.
It demonstrates modest but consistent antitumor efficacy in xenograft models without significant toxicity, highlighting its potential as a lead compound for targeting Wnt-driven cancers.[1]

C21H17N3O2S

375.44358

375.104

C, 67.18; H, 4.56; N, 11.19; O, 8.52; S, 8.54

677331-12-3

901751-47-1(iCRT3); 18623-44-4 (iCRT5)

5967294

Light yellow to yellow solid powder

4.795

80.5

0

4

3

27

617

0

CC1=CC(=C(N1C2=CN=CC=C2)C)/C=C\3/C(=O)N(C(=O)S3)C4=CC=CC=C4

NCSHZXNGQYSKLR-XDHOZWIPSA-N

InChI=1S/C21H17N3O2S/c1-14-11-16(15(2)23(14)18-9-6-10-22-13-18)12-19-20(25)24(21(26)27-19)17-7-4-3-5-8-17/h3-13H,1-2H3/b19-12+

5-[[2,5-dimethyl-1-(3-pyridinyl)-1H-pyrrol-3-yl]methylene]-3-phenyl-2,4-thiazolidinedione

iCRT14; iCRT-14; 677331-12-3; iCRT-14; iCRT14; (5Z)-5-[(2,5-dimethyl-1-pyridin-3-ylpyrrol-3-yl)methylidene]-3-phenyl-1,3-thiazolidine-2,4-dione; CHEMBL3589010; (5Z)-5-{[2,5-Dimethyl-1-(3-pyridinyl)-1H-pyrrol-3-yl]methylene}-3-phenyl-1,3-thiazolidine-2,4-dione; (5Z)-5-{[2,5-dimethyl-1-(pyridin-3-yl)-1H-pyrrol-3-yl]methylidene}-3-phenyl-1,3-thiazolidine-2,4-dione; iCRT 14

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 : ≥ 29 mg/mL (~77.24 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.66 mM) (饱和度未知) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80+，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
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