LF3

β-Catenin/TCF4 (IC50 = 1.65 μM by AlphaScreen; and 1.82 μM by ELISA)[1]

在含有外源报告基因的细胞和具有高内源性 Wnt 活性的结肠癌细胞中，LF3 抑制 Wnt/β-连环蛋白信号。此外，LF3 还能抑制与 Wnt 信号传导相关的癌细胞中 Wnt 靶基因的过度表达、高细胞运动性和细胞周期进展。然而，LF3 不会损害钙粘蛋白介导的细胞间粘附或导致细胞死亡。令人惊讶的是，LF3 以依赖于浓度的方式抑制癌症干细胞的自我更新能力[1]。

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- 抑制含有外源性报告基因的细胞和内源性Wnt活性高的结肠癌细胞中的Wnt/β - 连环蛋白信号[1]
- 抑制与Wnt信号相关的癌细胞中Wnt靶基因的过表达、高细胞迁移能力和细胞周期进程，且不会导致细胞死亡或干扰钙黏蛋白介导的细胞间黏附[1]
- 以浓度依赖的方式阻断癌症干细胞的自我更新能力，通过在非贴壁条件下结肠和头颈部癌症干细胞的成球实验来验证[1]

在结肠癌小鼠异种移植模型中抑制肿瘤生长并诱导分化。以50 mg/kg的剂量给予高GFP细胞的小鼠时，肿瘤生长明显受到抑制，且小鼠肠道的正常组织学未受影响[1]
LF3 在结肠癌小鼠异种移植模型中抑制肿瘤生长并促进分化。当 LF3 以 50 mg/kg 的剂量给予 GFPhigh 细胞的小鼠时，肿瘤生长受到显着抑制。小鼠肠道的正常组织学不受 LF3 治疗的影响[1]。

将LF3的类似物溶解在DMSO中至浓度为50 mmol/L。在AlphaScreen中，纯化的GST-hβ-catenin首先与镍螯合受体珠一起孵育，His-hTCF4与谷胱甘肽供体珠（Perkelmer）一起在PBS中孵育。然后在20μmol/L的测试化合物存在下，将它们合并成384孔的AlphaPlates。滴定法用于测定AlphaScreen中使用的纯化蛋白质的浓度，产生线性范围内的信号。孵育1小时后，蛋白质-蛋白质相互作用诱导发光，如EnVision多标记平板阅读器所测[1]。

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在AlphaScreen实验中，将纯化的GST - hβ - 连环蛋白与镍螯合受体珠子孵育，将His - hTCF4与谷胱甘肽供体珠子在PBS中孵育。然后将它们在20 μmol/L测试化合物（LF3及其类似物）存在的情况下组合到384孔Alpha板中。孵育1小时后，蛋白质 - 蛋白质相互作用会诱导发光，通过Envision多功能酶标仪测量发光情况，以确定LF3对β - 连环蛋白和TCF4相互作用的抑制效果[1]

24孔细胞培养板预先涂上250μL聚血（在95%乙醇中为12mg/mL；Sigma），以促进非附着球体的生长。将SW480报告细胞和小鼠唾液腺CSC胰蛋白酶消化，并作为单细胞接种到球形培养基（F12:DMEM 1:1，1×B-27补充，20 ng/mL EGF，20 ng/mL FGF，0.5%甲基纤维素）中，进行或不进行处理。10天后，在相差显微镜下计数球体，并用AF6000和DFC350FX拍摄照片。[1]

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- 对于含有外源性报告基因的细胞和内源性Wnt活性高的结肠癌细胞，进行体外培养，加入不同浓度的LF3，然后通过相应方法检测Wnt/β - 连环蛋白信号通路的活性，如Wnt靶基因表达水平、细胞迁移能力和细胞周期进程等（文献未具体描述具体检测方法）[1]
- 对于结肠和头颈部癌症干细胞，在非贴壁条件下培养，加入不同浓度的LF3，观察成球情况以评估癌症干细胞的自我更新能力[1]

NOD/SCID mice
Unsorted GFPlow and GFPhigh SW480 cells (1 × 104) were subcutaneously injected into the back skin of NOD/SCID mice. Tumor growth was monitored over a period of 45 days. For therapy, LF3 was administered i.v. at 50 mg/kg body weight for three rounds over 5 consecutive days, with 2-day breaks.[1]

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Dissolve LF3 in an appropriate solvent, and administer it to mice with colon cancer xenografts at a dose of 50 mg/kg, but the administration route and frequency are not mentioned. Observe the growth of xenografts and the histology of mice's guts [1]

[1]. A Small-Molecule Antagonist of the β-Catenin/TCF4 Interaction Blocks the Self-Renewal of Cancer Stem Cells and Suppresses Tumorigenesis. Cancer Res. 2016 Feb 15;76(4):891-901.

Wnt/β-catenin signaling is a highly conserved pathway essential for embryogenesis and tissue homeostasis. However, deregulation of this pathway can initiate and promote human malignancies, especially of the colon and head and neck. Therefore, Wnt/β-catenin signaling represents an attractive target for cancer therapy. We performed high-throughput screening using AlphaScreen and ELISA techniques to identify small molecules that disrupt the critical interaction between β-catenin and the transcription factor TCF4 required for signal transduction. We found that compound LF3, a 4-thioureido-benzenesulfonamide derivative, robustly inhibited this interaction. Biochemical assays revealed clues that the core structure of LF3 was essential for inhibition. LF3 inhibited Wnt/β-catenin signals in cells with exogenous reporters and in colon cancer cells with endogenously high Wnt activity. LF3 also suppressed features of cancer cells related to Wnt signaling, including high cell motility, cell-cycle progression, and the overexpression of Wnt target genes. However, LF3 did not cause cell death or interfere with cadherin-mediated cell-cell adhesion. Remarkably, the self-renewal capacity of cancer stem cells was blocked by LF3 in concentration-dependent manners, as examined by sphere formation of colon and head and neck cancer stem cells under nonadherent conditions. Finally, LF3 reduced tumor growth and induced differentiation in a mouse xenograft model of colon cancer. Collectively, our results strongly suggest that LF3 is a specific inhibitor of canonical Wnt signaling with anticancer activity that warrants further development for preclinical and clinical studies as a novel cancer therapy.[1]

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Wnt/β - catenin signaling is a highly conserved pathway essential for embryogenesis and tissue homeostasis, and its deregulation can initiate and promote human malignancies, especially of the colon and head and neck. LF3 is a 4 - thioureido - benzenesulfonamide derivative, which acts as a specific antagonist of the β - catenin/TCF4 interaction and has anticancer activity, worthy of further pre - clinical and clinical research as a new cancer therapy [1]

C20H24N4O2S2

416.56

416.134

C, 57.67; H, 5.81; N, 13.45; O, 7.68; S, 15.39

664969-54-4

1213452

White to off-white solid powder

1.3±0.1 g/cm3

609.6±65.0 °C at 760 mmHg

322.5±34.3 °C

0.0±1.7 mmHg at 25°C

1.680

1.97

119Ų

2

5

5

28

625

0

S=C(N([H])C1C([H])=C([H])C(=C([H])C=1[H])S(N([H])[H])(=O)=O)N1C([H])([H])C([H])([H])N(C([H])([H])/C(/[H])=C(\[H])/C2C([H])=C([H])C([H])=C([H])C=2[H])C([H])([H])C1([H])[H]

ZUQIFHLBPBLRRM-QPJJXVBHSA-N

InChI=1S/C20H24N4O2S2/c21-28(25,26)19-10-8-18(9-11-19)22-20(27)24-15-13-23(14-16-24)12-4-7-17-5-2-1-3-6-17/h1-11H,12-16H2,(H,22,27)(H2,21,25,26)/b7-4+

4-[(E)-3-phenylprop-2-enyl]-N-(4-sulfamoylphenyl)piperazine-1-carbothioamide

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.00 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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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配方 2 中的溶解度: ≥ 2.08 mg/mL (4.99 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.08 mg/mL (4.99 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (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 20.8 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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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);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。